KR100511561B1 - Extract of Acer okamotoanum having anti-oxidant activity and antioxidative cosmetics comprising the same - Google Patents

Abstract

The present invention relates to an anti-oxidant cosmetics containing an extract of Umbrella and the Umbrella extract as an active ingredient, which is extracted with water or an organic solvent and has an antioxidant activity. Umbrella extract according to the present invention can be usefully used not only for the treatment or prevention of diseases caused by free radicals, but also for preventing the aging of the skin due to the maintenance and oxidative damage of food.

Description

Extract of Acer okamotoanum having anti-oxidant activity and antioxidative cosmetics comprising the same}

The present invention relates to an antioxidant cosmetic comprising an extract of water or an organic solvent and having an antioxidant activity, and Acer okamotoanum extract and the umbrella extract as an active ingredient.

Oxygen, which was present in a stable state, was exposed to environmental and biochemical factors such as enzymes, reduction metabolism, chemicals, pollutants, and photochemical reactions, resulting in superoxide anion radicals (O _{2 '} ^- ) and hydroxyl radicals (hydroxyl radicals). Cellular components of the human body when converted to reactive oxygen species (ROS) such as radicals, OH _' ), hydrogen peroxide (H ₂ O ₂ ), singlet oxygen ( ¹ O ₂ ), etc. Phosphorus protein, lipids and DNA can be irreversibly destroyed. The action of free radicals is the antioxidant mechanisms such as superoxide dismutase (SOD), catalase, peroxidase, glutathione and vitamin C (vitamin C, ascorbic). acid), vitamin E (tocopherol) and the like 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, Parkinson's disease, heart disease, ischemia, arteriosclerosis, skin disease, gastrointestinal disease, inflammation, rheumatism, autoimmune disease, and so on by inducing destruction. (Cross et al . , Ann. Intern. Med ., 1987, 107 , 526; Alderson et al . , Proc. Natl. Acad. Sci. USA , 1988, 85 , 2706).

As such, as oxidative stress is found to be an important cause of various diseases including aging, studies are being actively conducted to develop active oxygen scavenger which effectively removes free radicals in vivo. In particular, antioxidants such as superoxide dismutase, catalase, peroxidase, and glutathione that can regulate active oxygen species, and low molecular weight active oxygen scavengers derived from natural products such as vitimin C, vitamin E, and tocopherol Much research is being done on this. In addition, as a synthetic active oxygen scavenger, butylated hydroxy anosole (BHA), butylated hydroxy toluene (BHT) and nordihydroguaiaretic acid (NDGA) have been developed and used in medicine and food fields.

However, the synthesized reactive oxygen scavenger is toxic in vivo and has the disadvantage of causing allergy and tumors. Therefore, the development of natural reactive oxygen scavenger is more demanded, but the natural active oxygen scavenger known to date is safer than synthetic active oxygen scavenger, but in terms of its effect and production cost, it does not surpass that of synthetic active oxygen scavenger. It is true. Therefore, there is an urgent need for the development of a new natural active oxygen scavenger which is excellent in active oxygen scavenging activity and is safer and has low production cost.

Meanwhile, Acer okamotoanum is a native plant of Ulleungdo, which grows native to Ulleungdo in Korea, and recently by the present inventors, quercetin 3- O- (2 ", 6" -O -digaloyl) -β-D The first separation of -galactopyranoside [quercetin 3- O- (2 ", 6" -O -digalloyl) -β-D-galactopyranoside] from the leaves of Umbrella, revealing its structure and flavonoids It has been reported that there is an antiviral effect on lycoside- and tannin-based compounds (Hyungja et al., Journal of Natural Products , 1998, 61 , 145). However, there have been no reports on the antioxidant activity of the Umbrella extract and the components contained in the Umbrella.

Accordingly, the present inventors have completed the present invention by revealing that the alcohol extract of Umbrella is excellent in antioxidant activity, and thus the extract can be usefully used for the prevention or treatment of various diseases caused by oxidative stress.

It is an object of the present invention to provide an umbrella cosmetic extract containing an extract of water or an organic solvent and having an antioxidant activity, and the extract as an active ingredient.

In order to achieve the above object, the present invention provides an extract of Umbrella, characterized in that extracted with water or an organic solvent and has an antioxidant activity.

The present invention also provides an antioxidant pharmaceutical composition, food additives or cosmetics containing the Umbrella extract as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention provides an umbrella croquette extract, which is extracted with water or an organic solvent and has antioxidant activity.

In the above, the organic solvent may be a compound selected from the group consisting of alcohol, ethyl acetate, dichloromethane and acetone or mixtures thereof, preferably alcohol. The alcohol may also be selected from the group consisting of methanol, ethanol, propanol, ethylene glycol, propylene glycol, glycerol and butanol, more preferably methanol or ethanol. In preparing the Umbrella crocus extract of the present invention, the umbrella crocus may be used in its entirety, but it is preferable to use leaves, and more preferably, it is dried and used in the shade.

Umbrella extract of the present invention is characterized in that it comprises one or more compounds selected from the group consisting of compounds represented by the formula (1) to (9) as the active ingredient.

In Chemical Formulas 1, 2, 7 and 8, G is a galloyl group, and is represented by the following Chemical Formula 10.

Umbrella extract of the present invention having an antioxidant activity may be prepared by extracting the leaves and stems of the Umbrella with organic solvents such as alcohol, hydrous alcohol or ethyl acetate, for example, may be prepared by the following method. That is, after cutting or powdering the umbrella leaf and stem finely used or lyophilized, here is a solution of 60 to 100% lower alcohol, such as methanol (MeOH) or ethanol, or acetone per kilogram of umbrella It is added in an amount of 0.1 to 5 L, preferably 0.5 to 1.0 L and left at room temperature for 4 to 5 days. The process may be repeated three or more times as necessary. The obtained extract is filtered and evaporated under reduced pressure to obtain an alcohol extract of Umbrella. Ethyl acetate extract can be obtained by adding 1 to 5 L, preferably 1.5 to 2.0 L of water per kg of alcohol extract, and then sufficiently extracting with 0.1 to 5 L of ethyl acetate (EtOAc), preferably 1.0 to 1.5 L. have. The ethyl acetate extract may be prepared by omitting the alcohol extraction process and directly extracting the umbrella leaf with ethyl acetate.

As a result of confirming the antioxidant activity of the Umbrella extract obtained by the above method, the methanol extract of the Umbrella leaf and stem showed a strong antioxidant activity (see Table 1 to Table 4). In addition, the antioxidative activity of the extracts of methanol extracts from the leaves and stems of Umbrella and dichloromethane, ethyl acetate, butanol and water was the strongest in the ethyl acetate extract, and the antioxidant activity was excellent in the butanol fraction. (See Tables 1-2). Therefore, in order to use the umbrella extract of the present invention as an antioxidant, alcohol solvent extracts such as methanol, ethanol, hydrous ethanol, ethylene glycol, propylene glycol, and glycerol may be used, and an ethyl acetate fraction or a butanol fraction may be very effective. Pharmacological action can be expected.

The present invention also provides a method for separating the compound represented by Formula 1 to Formula 9 from the Umbrella extract.

The compounds can be purified by performing column chromatography on ethyl acetate extract of the umbrella leaf with filler selected from the group consisting of silica gel, Sephadex, RP-18, polyamide, Toyopearl and XAD resin. . Column chromatography may be carried out several times by selecting an appropriate filler as necessary, and it is most preferable to perform appropriate combination of column chromatography using Sephadex, RP-18 and silica gel as fillers.

Quercetin 3- O- (2 ", 6" -O -digalloyl) -β-D-galactopyranoside [quercetin 3- O from the ethyl acetate extract of the leaves of Usangorod through the column chromatography process - (2 ", 6" - O -digalloyl) -β-D-galactopyranoside] ( compound of formula (1)), paroxetine Qasr 3- O - (2 "- galloyl) -β-D- galactosyl side pyrano [ quercetin 3- O - (2 "-galloyl ) -β-D-galactopyranoside] ( compound of formula (2)), paroxetine Qasr 3- O- (2" - galloyl) -α-L- arabino-side-pyrano [quercetin 3- O - (2 "-galloyl) -α-L-arabinopyranoside] ( compound of formula (3)), paroxetine Qasr 3- O- β-D- galactosyl side pyrano [quercetin 3- O -β-D- galactopyranoside (hyperoside)] (compound of formula 4), paroxetine Qasr 3- O -α-L- person nopi side pyrano [quercetin 3- O -α-L- rhamnopyranoside (quercitrin)] ( compound of formula (5)), Kam Ferrol 3 - O -α-L- person nopi side pyrano [kaempferol 3- O -α-L- rhamnopyranoside (afzelin)] ( compound of formula (6)), to go 1,2,3,4,6-penta Llano glue nose side (1,2,3,4,6-pentagalloyl glucopyranoside) (compound of formula (VII)), 1,2,6-tree - O - galloyl -β-D- gluconic nose side pyrano (1,2 Tannins and flavonoid glycoside compounds such as, 6-tri- O- galloyl-β-D-glucopyranoside (compound 8), gallic acid methyl ester (compound 9) Compounds of the family could be separated, and among them, the quercetin-based compounds represented by the formulas (1) to (5) of the flavonoid glycoside-based compounds showed strong antioxidant activity. In addition, the compounds represented by the formula (7) and formula (9) which is a tannin-based compound showed excellent antioxidant activity. Therefore, the active ingredients for antioxidant activity of the extract of the Umbrella leaf of the present invention are tannin-based compounds, quercetin-based flavonoid glycoside compounds, and flavonoid glycoside compounds substituted with galloyl groups. It can be seen that this is to further demonstrate the results of the present invention that the extract of the umbrella leaf can be effectively used for antioxidant action.

As described above, because the Umbrella extract according to the present invention has excellent antioxidant activity, it can be usefully used for the treatment or prevention of diseases caused by free radicals, maintenance of food and prevention of damage caused by oxidation of the skin.

The present invention also provides a pharmaceutical composition for antioxidant containing at least one compound selected from the group consisting of the Umbrella extract or the compound represented by Formula 1 to Formula 9 as an active ingredient.

The pharmaceutical composition of the present invention contains as an active ingredient an extract of the umbrella umbrella or a compound selected from the group consisting of Formulas 1 to 9 purified therefrom, and may contain conventional pharmacologically acceptable excipients. The pharmaceutical compositions of the present invention may comprise alone or one or more pharmaceutically acceptable carriers, which carriers are compatible with the active ingredients of the present invention and should not be harmful to the receptor. Pharmaceutical compositions of the invention can be determined by one of ordinary skill in the art based on common patient symptoms and disease severity. It may also be formulated in various forms, such as powders, tablets, capsules, solutions, injections, ointments, syrups, and the like, and may also be provided in unit-dose or multi-dose containers, such as sealed ampoules and bottles. .

The pharmaceutical composition of the present invention can be administered orally or parenterally. Routes of administration of the pharmaceutical compositions according to the invention are not limited to these, for example, oral, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intestinal Sublingual, or topical administration is possible.

For such clinical administration, the pharmaceutical compositions of the present invention can be formulated into suitable formulations using known techniques. For example, during oral administration, it may be mixed with an inert diluent or an edible carrier, sealed in hard or soft gelatin capsules, or pressed into tablets. For oral administration, the active compound may be mixed with excipients and used in the form of intake tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers and the like. In addition, various formulations, such as for injection and parenteral administration, can be prepared according to techniques known in the art or commonly used techniques.

The pharmaceutical composition of the present invention can be usefully used for the treatment or prevention of diseases caused by oxidation of cellular components by free radicals. Such diseases include, but are not limited to, aging, cancer, multiple atherosclerosis, arthritis, Parkinson's disease, stroke, concussion, Alzheimer's disease, vascular disorders, Hyperlipidemia, myocardial infarction or cerebral infarction.

The pharmaceutical compositions of the present invention can be administered in various dosage forms, oral or parenteral, and can be used in the form of general pharmaceutical preparations. The pharmaceutical compositions of the present invention can be used to prepare pharmaceutical formulations according to conventional methods. In the preparation of the formulation, it is preferred to mix the active ingredient with the carrier, to dilute it with the carrier, or to enclose it in a carrier in the form of a capsule, assay or other container. Thus, the formulation may be a tablet, pill, powder, sasay, elixir, suspension, emulsion, liquid, syrup, aerosol, soft or hard gelatin capsule, injectable solution or suspension, ointment, cream, gel or lotion, etc. It may be in the form of. Examples of suitable carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, Propylhydroxybenzoate, talc, magnesium stearate and mineral oil. The formulation may further comprise fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives and the like. Compositions of the invention can be formulated using methods well known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal.

An effective amount of the extract of the umbrella umbrella contained in the pharmaceutical composition of the present invention or the compound represented by Formula 1 to Formula 9 separated therefrom is 0.1 mg / kg to 500 mg / kg. Preferably a range of 500 to 5,000 mg is common, and some diseases may require higher daily dosages. In addition, the specific dose level for a particular patient may vary depending on the particular compound to be used, weight, age, sex, health condition, diet, time of administration, method of administration, rate of excretion, drug mixing and the severity of the disease.

In addition, the present invention provides an antioxidant food additive containing at least one compound selected from the group consisting of the Umbrella extract or the compound represented by Formula 1 to Formula 9 as an active ingredient.

Umbrella extract according to the present invention and the active ingredient separated therefrom is excellent in antioxidant activity can effectively prevent the smell or flavor of food caused by oxidation, rancidity of oil and fat, and discoloration of food. Therefore, by combining the Umbrella extract and the compound separated therefrom to various conventional foods can be used to preserve these foods or to maintain the freshness and quality of foods for a long time.

In another aspect, the present invention provides a cosmetic for antioxidant containing at least one compound selected from the group consisting of the Umbrella extract or the compound represented by Formula 1 to Formula 9.

Cosmetics of the present invention can be used as it is or as diluted as needed or the compound separated from the Umbrella. The umbrella croquette extract and compounds isolated therefrom may be prepared in liquid or solid form using bases, adjuvants and additives commonly used in the cosmetic field. Cosmetics in liquid or solid form may include, but are not limited to, for example, cosmetics, creams, lotions, baths and the like. Bases, auxiliaries and additives commonly used in the cosmetic field are not particularly limited and may include, for example, water, alcohols, propylene glycol, stearic acid, glycerol, cetyl alcohol and liquid paraffin.

Cosmetics according to the present invention are very useful in preventing skin oxidative damage, for example, spots (brown spots), freckles, skin cracks, UV damage (sunburn), etc. It can be very useful to maintain the quality of cosmetics.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Preparation of Extract of Umbrella Croquette Leaf

It was collected from Ulleungdo and 2.1 kg of the dried Umbrella leaf was immersed in 12 L of methanol and extracted at room temperature for 4-5 days. The above procedure was repeated three times, and then concentrated at a rotary concentrator at 40 ° C. to obtain 296 g of methanol extract. The methanol extract was suspended in 2,500 ml of water and then extracted with 2,000 ml of dichloromethane (CH ₂ Cl ₂ , 2,000 ml × 3), the water layer was extracted with ethyl acetate (EtOAc, 2,000 ml × 3) and the water layer again Fully extracted with butanol (BuOH, 2,000 mL × 3) and partitioned into each solvent fraction.

Example 2 Preparation of Extract of Umbrella Croquette Leaf

150.2 g of the dried Umbrella stem was immersed in 2 L of methanol, which was collected from Ulleungdo, and extracted at room temperature for 4-5 days. The same procedure was repeated three times, and then concentrated at a rotary concentrator at 40 ° C. to obtain 4.63 g of methanol extract. The methanol extract was suspended in 250 ml of water and then extracted with 200 ml of dichloromethane (CH ₂ Cl ₂ , 200 ml × 3), the water layer was extracted with ethyl acetate (EtOAc, 200 ml × 3) and the water layer was again Extracted sufficiently with butanol (BuOH, 200 mL × 3) and partitioned into each solvent fraction.

Example 3 Isolation of Antioxidant Active Substances from Ethyl Acetate Fraction of Umbrella Leaf

75 g of an ethyl acetate fraction of an umbrella leaf was subjected to column chromatography (7 × 50 cm) using Sephadex. As a developing solvent, a mixed solvent of dichloromethane-methanol (60:40) was used. Finally, the column was washed with methanol. The obtained fractions were observed by normal phase silica gel TLC (developing solvent: dichloromethane-methanol-water = 70: 10: 1), and the compounds having similar polarities were collected and divided into six small fractions (fractions 1 to 6). Small fraction 5 (7.2 g) was subjected to column chromatography (4 × 40 cm) using Sephadex LH-20 using a mixed solvent of dichloromethane-methanol (60:40) as a developing solvent, and was represented by Formula 7. Compound (879 mg) was obtained. Small fraction 2 (28.9 g) was subjected to column chromatography (4 × 35 cm) using silica gel (230-400 mesh). The solvent used as the developing solvent was started with a mixed solvent of dichloromethane-ethyl acetate-methanol-water (21: 4.5: 3.5: 0.3) and gradually increased in polarity to dichloromethane-ethyl acetate-methanol-water (18: 6: 6: 0.5) was used. The obtained fractions were observed by normal phase silica gel TLC (developing solvent: dichloromethane-methanol-water = 70: 10: 1), and then the compounds having similar polarities were collected into nine small fractions (fractions 2-1 to 2-9). Divided by). Small fraction 2-2 (14.7 g) was recrystallized in a mixed solvent of dichloromethane-methanol (20: 1) to obtain the compound represented by the formula (9.9 g), and small fraction 2-3 (1.7 g). ) Was subjected to reverse phase column chromatography (Cat.No .: 13900, Merck, 50% methanol, 3 × 30 cm) and Toyopearl HW 40 chromatography (methanol, 2.5 × 30.5 cm) to obtain a compound represented by the formula (13.0) Mg), the compound represented by the formula (306.5 mg) and the compound represented by the formula (32.0 mg) were obtained. Column fractionation (2 × 35 cm) and reversed phase column using a small fraction 2-4 (1.4 g) as a developing solvent using a mixed solvent of dichloromethane-methanol-water (7: 1: 0.1) as silica gel Chromatography (50% methanol, 2 × 30 cm) was carried out to obtain a compound represented by the formula (6) (59.2 mg) and a compound represented by the formula (80.0 mg). Small fraction 2-5 (2.1 g) was subjected to reverse phase column chromatography (50% methanol, 3 × 30 cm) to obtain a compound (90.1 mg) represented by the formula (4). Small fraction 2-7 (6.3 g) was subjected to reverse phase column chromatography (50% methanol, 3 × 40 cm) to obtain a compound (818.2 mg) represented by Chemical Formula 5.

Example 4 Free Radical Scavenging Effects of Extracts from Umbrella Stem and Leaves and Compounds Isolated from Leaves

1) Experiment Method

Free radical scavenging action was carried out in 190 μl of 100 μM 1,1-diphenyl-2-picryl hydrazyl (1,1-diphenyl-2-picryl hydrazyl; DPPH) ethanol solution. 10 μl of an ethanol solution was added to each of the extracts of the Umbrella leaf and stem and the compounds isolated in Example 3 and reacted at 37 ° C. for 30 minutes, and then the absorbance was measured at 515 nm for free radical scavenging effect. Obtained as IC ₅₀ value (Blois et al ., Nature , 1958, 181 , 1199). IC ₅₀ means the concentration (IC ₅₀ ) which shows a 50% inhibitory effect by calculating the free radical scavenging effect.

Extracts of the leaves and stems of Umbrella, which were fractionated in Examples 1 and 2, and the respective compounds isolated in Example 3 and conventional scavengers [ascorbic acid (vitamin C), α-tocopherol (vitamin E), resveratrol ( resveratrol)] and free radical scavenging effect is compared with the results shown in Table 1. Data presented are averages of three runs.

As a result, as shown in Table 1, since the high activity in the ethyl acetate and butanol fraction of the umbrella, the compounds separated from the ethyl acetate showing a high activity was found to show mostly free radical scavenging activity. It was.

Test substance Free radical scavenging effect (IC ₅₀ ) Methanol Extract of Leaf 13.48 ± 0.85 Dichloromethane Fraction of Leaves > 50 Ethyl Acetate Fraction of Leaves 3.13 ± 0.14 Butanol fraction of leaves 18.48 ± 0.79 Water fraction of leaves > 50 Methanol Extract of Stem 32.97 ± 3.5 Dichloromethane Fraction of Stems > 50 Ethyl Acetate Fraction of Stems 8.41 ± 0.23 Butanol fraction of stem 19.12 ± 0.43 Water fraction of stem > 50 Formula 1 3.35 ± 0.30 Formula 2 4.30 ± 0.20 Formula 3 4.38 ± 0.15 Formula 4 10.38 ± 0.26 Formula 5 11.59 ± 0.93 Formula 6 > 50 Formula 7 2.32 ± 0.24 Formula 8 2.73 ± 0.05 Formula 9 2.09 ± 0.15 Ascorbic acid (vitamin C) 5.53 ± 0.13 α-tocopherol (vitamin E) 9.42 ± 0.26 Resveratrol 17.05 ± 1.08

As a result, as can be seen in Table 1, it showed a high activity in the methanol extract of the Umbrella gourd, it can be seen that this activity is transferred to the ethyl acetate fraction and butanol fraction. Compounds isolated from ethyl acetate, which exhibits high activity, show most activity, but among the flavonoid glycosides, compounds having a substituted galloyl group showed high antioxidant activity. In addition, tannin-based compounds also show high activity, which can also be seen that the compound substituted with a galloyl group.

Example 5 Xanthine / Xanthine Oxidase-Induced Superoxide Anion (O) of Extracts from Umbrella Leaf and Stem and Compounds Isolated from Leaves _2' ^- ) Elimination effect

1) Experiment Method

Toda et al., Planta, Med. 1991, 57 , 8 to evaluate the efficacy of inhibiting the production of superoxide anions produced by the reaction of Xanthine and xanthine oxidase (XOD) . ) Was carried out as follows. Specifically, 0.1 mM xanthine, 0.1 mM EDTA, 50 μg / ml bovine serum albumin (BSA), 25 mM nitroblue tetrazolium (NBT) and 40 mM Na ₂ CO ₃ solution, for each concentration. The diluted test compound solution and the solution containing 200 μl of the final volume containing 1.4 × 10 ⁻³ unit XOD were mixed and reacted at 25 ° C. for 20 minutes. After the reaction was stopped by adding 6.6 μl of 6 mM CuCl ₂ to the reaction solution, the absorbance of the resulting formazan at 560 nm was measured to compare the xanthine / xanthine oxidase-induced superoxide anion scavenging effect. Obtained _by IC ₅₀ value. IC ₅₀ means the concentration (IC ₅₀ ) which shows a 50% inhibitory effect by calculating the xanthine / xanthine oxidase induced superoxide radical (O _{2 ′} ⁻ ) scavenging effect.

Extracts of the leaves and stems of the Umbrella leaf and stem fractionated in Examples 1 and 2 and the xanthine / xanthine oxidase-induced superoxide anion (O _{2 ′} ^- ) scavenging effect of the compounds isolated in Example 3 Table 2 shows the results compared with the macrophages (ascorbic acid (vitamin C), α-tocopherol (vitamin E), resveratrol). Data presented are averages of three runs.

Test substance Xanthine / Xanthine oxidase-induced superoxide radical (O _{2 '} ^- ) scavenging effect (IC ₅₀ ) Methanol Extract of Leaf 8.92 ± 0.87 Dichloromethane Fraction of Leaves > 50 Ethyl Acetate Fraction of Leaves 2.04 ± 0.16 Butanol fraction of leaves 11.81 ± 0.43 Water fraction of leaves > 50 Methanol Extract of Stem 8.85 ± 1.78 Dichloromethane Fraction of Stems 24.21 ± 0.45 Ethyl Acetate Fraction of Stems 1.07 ± 0.28 Butanol fraction of stem 4.96 ± 0.65 Water fraction of stem 42.02 ± 1.01 Formula 1 6.57 ± 0.26 Formula 2 7.95 ± 0.21 Formula 3 8.19 ± 0.19 Formula 4 12.95 ± 1.02 Formula 5 18.19 ± 0.66 Formula 6 > 50 Formula 7 4.61 ± 0.22 Formula 8 7.54 ± 0.16 Formula 9 0.84 ± 0.12 Ascorbic acid (vitamin C) > 50 α-tocopherol (vitamin E) > 50 Resveratrol > 50

The results of comparing the xanthine / xanthine oxidase-induced superoxide radical (O _{2 '} ^- ) scavenging effects of methanol extracts from the leaves and stems of the umbrella and the ethyl acetate fractions from the methanol extracts with conventional scavengers It was found that the methanol extracts from the leaves and stems, the ethyl acetate fractions and the butanol fractions from the methanol extracts showed very high activity, and the tannin-based compounds also exhibited high activity. In addition, it was found that most of the compounds isolated from the ethyl acetate extract of the umbrella leaf showed high antioxidant activity. However, most of the compounds known as antioxidants were ineffective.

Example 6 Inhibition of Hydrogen Peroxide-Induced Cytotoxicity of Extracts from Umbrella Leaf and Stem and Compounds Isolated from Leaves

1) Experiment Method

Inhibitory effects of extracts from the leaves and stems of Umbrella and stems and compounds isolated from the leaves on hydrogen peroxide-induced cytotoxicity were tested using mouse-derived fibroblasts (NIH / 3T3). Hydrogen peroxide produces hydroxyl radicals in the cell, causing fatal damage to the cell. In order to test the inhibitory effect of hydrogen peroxide-induced cytotoxicity of the extract of the Umbrella leaf and stem and the compounds isolated from the leaves of the present invention, the following experiment was performed.

Mouse-derived skin fibroblasts were inoculated in a 96 well plate at 5,000 wells per well and incubated in 100 μl of DMEM medium for 24 hours, followed by the extract of Umbrella leaf (Example 1) and the extract of Umbrella stem (Example) 2) and 50 μl of a dimethyl sulfoxide (DMSO) solution were added to the compounds isolated from the extract of the umbrella leaf (Example 3), and 50 μl of hydrogen peroxide diluted to a final concentration of 200 μM in the medium was added. Time incubation. 20 μl of MTT solution [3- (4,5-dimethyl-2-thiazolyl) 2,5-diphenyl-2H-tetrazolium bromide: 2 mg / ml] was added per well, and the culture medium was removed after 4 hours of incubation. 150 μl of dimethyl sulfoxide solution was added to each well, followed by agitation for 20 minutes, followed by measurement of absorbance at 540 nm using a microplate reader. Inhibitory effect was measured. The results of comparing the numerical values with IC ₅₀ values are shown in Table 3, where IC ₅₀ means a concentration (IC ₅₀ ) indicating a 50% inhibitory effect by calculating an inhibitory effect on hydrogen peroxide-induced cytotoxicity. The values presented are the average of three times.

Test substance Inhibitory effect on hydrogen peroxide-induced cytotoxicity (IC ₅₀ ) Methanol Extract of Leaf 22.60 ± 1.89 Dichloromethane Fraction of Leaves > 50 Ethyl Acetate Fraction of Leaves 8.49 ± 0.92 Butanol fraction of leaves > 50 Water fraction of leaves > 50 Methanol Extract of Stem > 50 Dichloromethane Fraction of Stems > 50 Ethyl Acetate Fraction of Stems 21.44 ± 1.37 Butanol fraction of stem > 50 Water fraction of stem > 50 Formula 1 10.68 ± 0.56 Formula 2 4.08 ± 0.97 Formula 3 > 50 Formula 4 > 50 Formula 5 > 50 Formula 6 23.68 ± 0.87 Formula 7 6.43 ± 0.90 Formula 8 21.09 ± 1.58 Formula 9 20.55 ± 1.63 Ascorbic acid (vitamin C) > 50 α-tocopherol (vitamin E) > 50 Resveratrol > 50

As a result, as can be seen in Table 3, it was found that the ethyl acetate fraction fractionated from the methanol extract and the methanol extract of the umbrella leaf and stem showed very high activity. In addition, among the compounds isolated from ethyl acetate of the leaves, the compound substituted with the galloyl group showed high activity in the flavonoid glycoside series, which was superior to various compounds well known as antioxidants. Tannin-based compounds also have a galloyl group it can be seen that shows a high activity. Therefore, it was confirmed that the extracts of the leaves and stems and the compounds separated from the leaves of the umbrellas suppress cytotoxicity by scavenging hydroxyl radicals generated in skin fibroblasts.

Example 7 Measurement of Cytotoxicity of Extracts from Umbrella Leaf and Stem and Compounds Isolated from Leaves

Cytotoxicity of the extracts of the leaves and stems and the compounds themselves isolated from the leaves of the umbrellas were measured using mouse-derived skin fibroblasts in the following manner. Specifically, mouse-derived dermal fibroblasts were inoculated into a 96-well plate at 5,000 cells per well and incubated for 24 hours in 100 μl of DMEM medium, followed by an extract of an umbrella leaf (Example 1) and an extract of a stem (Example 2). And to the compounds isolated from the umbrella leaf (Example 3) was added so that the final concentration of the dimethyl sulfoxide solution to 3.125, 6.25, 12.5, 25 ㎍ / ㎖ and incubated for 24 hours. 20 μl of MTT solution (2 mg / ml) per well was added and left for 4 hours to remove the media portion. 150 μl of dimethyl sulfoxide solution was added to each well, followed by agitation for 20 minutes, followed by measurement of absorbance at 540 nm using a microplate reader. It was. The cytotoxicity was expressed as cell viability (A,%) and was calculated based on the following formula. The calculated results are shown in Table 4 below, and the values presented represent three average values.

A (%) = absorbance at treatment of test substance / absorbance of control × 100

Test substance Cell viability (A,%) Experimental concentration (µg / ml) 3.125 6.25 12.5 25 Methanol Extract of Leaf 89.24 86.40 81.54 78.92 Dichloromethane Fraction of Leaves 83.14 78.37 75.89 66.16 Ethyl Acetate Fraction of Leaves 93.99 94.17 95.78 87.47 Butanol fraction of leaves 100.88 100.45 100.60 101.58 Water fraction of leaves 100.22 101.48 100.27 98.71 Methanol Extract of Stem 99.38 98.97 100.52 100.28 Dichloromethane Fraction of Stems 98.36 96.27 96.59 97.95 Ethyl Acetate Fraction of Stems 100.98 100.19 95.65 93.70 Butanol fraction of stem 102.52 103.85 103.83 105.69 Water fraction of stem 99.43 100.44 99.58 103.26 Formula 1 99.34 102.00 104.09 103.15 Formula 2 96.47 96.20 97.67 91.78 Formula 3 99.56 100.04 103.03 100.73 Formula 4 90.62 89.62 88.67 88.23 Formula 5 95.86 97.93 99.25 93.19 Formula 6 97.19 99.13 96.45 98.46 Formula 7 99.78 101.36 92.42 95.80 Formula 8 98.68 99.20 102.22 102.54 Formula 9 101.65 101.72 105.03 104.22 Ascorbic acid 105.08 111.59 113.32 114.83 α-tocopherol 109.91 112.69 108.71 113.65 Resveratrol 76.27 71.75 60.65 58.91

As a result, it was found that the extracts of the leaves and stems and the compounds separated from the leaves of the umbrella leaf were safe substances with little toxicity to cells even at high concentrations as shown in Table 4. However, resveratrol, which is widely known as an antioxidant, did not grow more than 41% of cells at high concentrations and showed some cytotoxicity at low concentrations. Therefore, the extracts from the leaves and stems and the compounds separated from the leaves are useful as a safe substance for the treatment or prevention of diseases caused by free radicals, as well as the maintenance of foods and the prevention of skin oxidative damage. It was confirmed that it can be used.

Hereinafter, a specific disease to which the pharmaceutical composition comprising the umbrella extract of the present invention or the compound of Formula 1 to Formula 9 may be applied.

However, the following application examples are only for illustrating the present invention, and the content of the present invention is not limited to the following application examples.

<Application example 1> Cancer

Cancer is caused by a myriad of causes, but the most fundamental cause is the destruction of cells by free radicals and their inability to repair them, leading to cell dysfunction, which has been shown to metastasize to cancer (Ames, BN, Science , 1983, 221, 1256-1264). On the other hand, the effects of phenolic acid in fruits on liver cancer (Sun J et al ., J Agric Food Chem , 2002, 4; 50 (25), 7449-7454), and the effect of lycopene on tomatoes on breast cancer (Hadley CW et al ., Exp Biol Med , 2002, 227 (10), 869-80), effect of isoverbascoside on gastric cancer (Chen RC et al ., Acta Pharmacol Sin , 2002, 23 ( 11), 997-1001) and several known antioxidants are also effective against other cancers. Therefore, it can be seen that the antioxidant can be effectively used for the treatment and prevention of various types of cancer, the antioxidant pharmaceutical composition of the present invention excellent in antioxidant activity can be effectively used for the treatment and prevention of cancer.

Application Example 2 Aging

Free radicals, which are generated by the metabolic process in the normal course of metabolism, cause oxidative damage by non-selective and irreversible destruction of cell components such as lipids, proteins, sugars, or DNA. Harman, D, Free radical theory of aging , 1986, 3-49). In addition, it has been observed that lifespan is extended by reducing basal metabolic rate, ie oxygen consumption, such as by limiting diet or reducing exercise by varying experimental conditions (Medvedev, ZA, Biol Rev. , 1990, 65, 375-398; Loe , J. et al ., J. Biol. Chem ., 1971, 32, 103-121; Sohal, RS, Aging , 1982, 5, 21-24). In other words, the removal of free radicals is a way to delay aging and there are many antioxidants are currently developed to remove free radicals. Therefore, the antioxidant pharmaceutical composition of the present invention excellent in antioxidant activity may delay aging.

<Application Example 3> Coronary heart disease, hyperlipidemia and arteriosclerosis

Treatment with cholesterol biosynthetic enzyme inhibitors in patients with coronary heart disease and hypercholesterolemia reduces cholesterol content in low-density fats, but inhibits the biosynthesis of ubiquinone Q10 for low-density lipoproteins against peroxidation by free radicals. Because of its protective effect, it is not effective in the treatment of the disease, so the administration of the antioxidant cerivastatin or probucol to the patients rapidly reduced the content of low density lipoprotein (Lankin VZ et al . , Bull Exp Biol Med , 2002, 134 (1), 39-42). In addition, in clinical trials in which the antioxidant dihydropyridine calcium antagonist lacidipine was administered to atherosclerosis patients, blood pressure was lowered and cholesterol levels in the blood vessel wall were lowered, thereby reducing the size of atherosclerotic lesions (Haller H et. al ., Drugs RD , 2002, 3 (5), 311-23). That is, the antioxidant substance can be seen that it is an excellent substance in the treatment and prevention of vascular diseases related to cholesterol, such as coronary heart disease, hyperlipidemia and arteriosclerosis, the antioxidant pharmaceutical composition of the present invention excellent in antioxidant activity is coronary heart disease It can be used for the treatment or prevention of vascular diseases such as hyperlipidemia, arteriosclerosis.

Application Example 4 Multiple Sclerosis and Autoimmune Encephalomyelitis

In patients with multiple sclerosis and autoimmune encephalomyelitis disease models, a weakness was observed in clinical trials in which ALA (alpha lipoic acid), a type of antioxidant, was administered to mice. In other words, oxidative stress is a major cause of multiple sclerosis and autoimmune encephalomyelitis, and antioxidants can be used to treat such neurological disorders (Marracci GH et al ., J Neuroimmunol , 2002, 131 (1-2)). , 104-14). Therefore, the antioxidant pharmaceutical composition of the present invention having excellent antioxidant activity can be used for the treatment or prevention of neurological diseases such as multiple sclerosis and autoimmune encephalomyelitis.

Application Example 5 Stroke and Alzheimer's Disease

Oxidative stress, or free radicals, oxidizes the components of cells, leading to their dysfunction, and their dysfunction also impairs the function of nerve cells. This neuronal dysfunction causes stroke, shock, and the like. However, when these symptoms are not treated and given continuous oxidative stress, serious brain diseases such as stroke and Alzheimer's disease are caused. In addition, antioxidants that can remove free radicals can be used to treat brain diseases such as stroke and Alzheimer's disease (Perry G et al ., Comp Biochem Physiol C Toxicol Phaarmacol , 2002, 133 (4), 507-13; Cecchi C et al ., Free Radic Biol Med , 2002, 15:33 (10), 1372-9; Smith MA et al ., Free Radic Biol Med , 2002, 1:33 (9), 1194-9). Therefore, the antioxidant pharmaceutical composition of the present invention excellent in antioxidant activity can be used for the treatment or prevention of stroke and brain diseases such as Alzheimer's disease.

<Application Example 6> Enteritis

Excess peroxide by-products accumulate in leukocytes of enteritis patients, and cell damage by accumulated peroxide by-products is the cause of primary or secondary pathological mechanisms in the intestinal infection. That is, oxidative stress is a major cause of inflammatory enteritis by inducing intestinal infections (Kruidenier L et al ., Aliment Pharmacol Ther , 2002, 16 (12), 1997-2015). Therefore, the antioxidant pharmaceutical composition of the present invention excellent in antioxidant activity can be used for the treatment or prevention of diseases related to inflammation such as inflammatory enteritis.

As described above, the Umbrella extract of the present invention and the compound isolated therefrom may be useful for the treatment or prevention of diseases caused by free radicals, maintenance of food and prevention of damage caused by oxidation of the skin.

Claims (9)

Umbrella extract, characterized in that extracted with water, C ₁ ~ C ₄ alcohol, dichloromethane, ethyl acetate or a mixed solvent thereof and has an antioxidant activity. delete The Umbrella extract according to claim 1, wherein the alcohol is methanol. According to claim 1, Umbrella extract, characterized in that it comprises one or more compounds selected from the group consisting of compounds represented by the formula (1) to (9) as an active ingredient. <Formula 1> <Formula 2> <Formula 3> <Formula 4> <Formula 5> <Formula 6> <Formula 7> <Formula 8> <Formula 9> In Chemical Formulas 1, 2, 7 and 8, G is a galloyl group having the following structure: According to claim 4, wherein the compounds of Formula 1 to Formula 9 using a filler selected from the group consisting of silica gel, Sephadex, RP-18, polyamide, Toyopearl and XAD resin from the ethyl acetate extract of Umbrella Umbrella extract, characterized in that separated by performing column chromatography. delete delete delete Antioxidant cosmetics for preventing the aging of the skin by oxidative damage containing the extract of Umbrella.