KR100600249B1 - Extract of Cercis chinensis having anti-oxidant activity and anti-aging activity, and cosmetical composition containing the extract for anti-oxidation, skin-aging protection and wrinkle improvement - Google Patents

Abstract

The present invention relates to an antioxidant, skin aging inhibitory, skin elasticity maintenance or wrinkle suppression Cercis chinensis extract and a cosmetic composition for inhibiting skin aging and wrinkles containing the same as an active ingredient, more specifically, resources are secured After solvent fractionation of alcohol extract of Park Tae-gi, which was easy and had no previous report on antioxidant activity and skin cell aging inhibitory activity, it had antioxidant activity and anti-aging activity separated from ethyl acetate fraction and butanol fraction showing antioxidant activity. The present invention relates to a extract of Park Tae Ki containing the compound represented by Formula 1 to Formula 20 and a cosmetic composition for inhibiting skin aging comprising the same. Park Tae-gi extract of the present invention not only has an excellent function of suppressing the oxidative stress causing aging of the skin, but also extends the lifespan of skin cells by slowing the shortening rate of telomeres associated with aging. The cosmetic composition comprising a can be usefully used as a cosmetic composition of the external preparation for skin to prevent skin aging, maintain skin elasticity or reduce wrinkles.

Description

Extract of Cercis chinensis Extract with Antioxidant and Anti-aging Activity, and Anti-oxidant Activity and Anti-aging Activity, and Cosmetical Composition Containing Extract for Anti- oxidation, skin-aging protection and wrinkle improvement}             

Figure 1 is a schematic diagram showing a process of extracting the crude ethanol extract from Park Tae-gi, hexane, ethyl acetate, butanol in order.

Figure 2 is a graph measuring the DPPH radical scavenging activity of the ethanol crude extract extracted from 0% ethanol from 0% to 10% in sequential increments.

3 is a graph measuring DPPH radical scavenging activity of hexane, ethyl acetate, butanol, water fraction and ethanol extract.

Figure 4 is a schematic diagram showing the process of separating the compound showing the antioxidant activity from the ethyl acetate fraction.

5 is a schematic diagram illustrating a process of separating a compound exhibiting antioxidant activity from a butanol fraction.

Figure 6a is a cell photograph showing the degree of DNA damage showing the effect of protecting the cells against UV irradiation of the extract of Park Tae Ki and compounds isolated therefrom of the present invention.

Figure 6b is a graph showing the fluorescence intensity showing the degree of DNA damage showing the effect of protecting the cells against UV irradiation of the extract of Park Tae Ki and compounds of the present invention.

7 is It is a photograph of skin damage of hairless rats, which confirmed that the extract of Park Tae Ki of the present invention and the compound isolated therefrom show a protective effect against UV irradiation.

8 is This is a graph showing that the extract of Park Tae-gi and the compounds isolated therefrom prolong cell life.

9 is The extract of Park Tae-gi and the compounds isolated therefrom are photographs of southern blots of telomeres that inhibit the shortening rate of telomeres.

10 is It is a telomeres shortening rate graph showing that the extract of the extract of the present invention and the compound separated therefrom have a telomere length extension action.

11 is a photograph showing flowers, leaves, stems, and fruit of Park Tae-gi.

The present invention relates to a plant extract having a skin aging inhibitory activity and a cosmetic composition for inhibiting aging comprising the same as an active ingredient, specifically, the extract of Park Tae-gi, which has an excellent inhibitory activity against oxidative stress and a long life effect of skin cells. It relates to a cosmetic composition for inhibiting skin aging.

Aging refers to all the physiological changes in the body that occur over time, and is a life phenomenon that is caused by a variety of factors depending on the individual. Aging differs in its aspect and speed from individual to individual, and even in an individual, its pattern is different, and thus, aging at the individual level has many difficulties. On the other hand, if you look specifically at the aging phenomenon, changes in the function of each component organ and tissue occurs, which is due to the change in the function of the cells as a structural unit. In other words, the aging of the individual may be caused by the loss of nerve cells in the brain, the loss of cognitive function, the loss of elasticity of the skin due to the loss of subcutaneous fat cells, or the whitening of hair root melanocytes due to loss of melanin pigment production. This is due to the aging of the cells that make up the individual. Therefore, in recent years, many studies of aging have been made at the cellular level. Numerous scientists have studied cell aging over the last few decades, but the exact mechanisms are still unknown due to the various phenomena and complexities of aging. However, numerous phenomenological studies have raised various hypotheses about aging, among which oxidative stress caused by free radicals generated during normal metabolic processes accumulates and causes aging. The telomeres of aging that the telomere at the end of the chromosome end up dividing and gradually cease to cease cell division and eventually die. There are many other hypotheses, but these hypotheses do not contradict each other but complement each other and explain aging. Active oxygen theory of aging means that reactive oxygen, which is generated as a result of normal metabolism, is highly reactive and thus irreversibly destroys cell components such as lipids, proteins, sugars, or DNA, resulting in oxidative stress of cells or tissues. the cause by, including cancer, stroke, and cardiovascular diseases such as atherosclerosis, rheumatoid, such as chronic inflammatory diseases, respiratory diseases, or self, as well as cause a variety of diseases, including autoimmune diseases (Halliwell, B and Gutteridge, JMC , Biochem. J. , 1984, 219, 1-14; Freeman , BA and Grapo, JD, Lab Invest, 1982, 47, 412-426; Ames, BN, Science, 1983, 221, 1256-1264;. Fridovich, I., Arch Biochem Biophys ., 1986, 247, 1-11; Vishwanath, MS, Nutrition in Clinical Practice , 1995, 10, 19-25). These oxidative damages accumulate over time, leading to aging and death. This active oxygen theory of aging was first proposed by Harman in 1956 (Harman, D., Free radical theory of aging , Alan R Liss, New York , 1986, 3-49). It supports the hypothesis. It has been observed that lifespan is extended by reducing basal metabolic rate, i.e. oxygen consumption, by limiting diet or reducing momentum under different experimental conditions (Medvedev, ZA, Biol. Rev. , 1990, 65, 375-398; Loe , J., Northrop, JH, J. Biol. Chem ., 1971, 32, 103-121; Sohal, RS, Insect aging , Springer-Verlag, Heidelberg , 1986, 23-44; Sohal, RS, Aging , 1982, 5, 21-24).

Meanwhile, antioxidants such as superoxide dismutase (hereinafter referred to as SOD), catalase or peroxidase to protect against oxidative damage are found in vivo. It has been reported that the protective ability against free radicals decreases with age (Orr, WC and Sohal, RS, Science , 1994, 263, 1128-1130; Sohal, RS et al ., J . Biol. Chem., 1995, 270, 15671-15674). In other words, SOD isolated from livers of old rats was lower than that of young rats. Especially, increasing the activity of antioxidant enzymes SOD and catalase in fruit flies increased lifespan by more than 30%, which is closely related to active oxygen and aging. It can be seen that. Therefore, antioxidants such as substances capable of scavenging active oxygen and lipid peroxidation inhibitors are expected to be used as therapeutic agents for preventing various diseases caused by active oxygen and as inhibitors for aging prevention.

In addition, continuous exposure to oxidative stress from harmful environments such as air pollution, UV exposure, stress, or disease causes increased radicals in the body and collagen, elastin, hyaluronic acid, which are the connective tissues of the dermis. It can destroy hyaluronic aicd and cause subsidence of the skin (wrinkle), and also oxidize the lipid part of the cell membrane and cause cell destruction, leading to diseases such as dermatitis, acne or skin cancer. In addition, radicals are involved in the process of melanin formation, causing spots, freckles and wrinkles. Conventionally, ascorbic acid, α-tocopherol, or SOD has been used as a free radical scavenging substance in cosmetics and medicines to prevent wrinkles and other skin diseases, but they are expensive and have poor chemical stability when formulated. There was a problem that it was difficult to expect a practical effect. For this reason, the development of safer and more free radical scavenging effects has become an important task not only in the pharmaceutical and food sectors, but also in the cosmetics industry.

Another theory for understanding aging is the telomeres hypothesis of aging. Human normal cells divide only a certain number of times in vitro and no longer divide. This is called replication aging, and it is the telomer hypothesis that explains why this phenomenon occurs (Kim, SH, et al ., Oncogene 21: 503-511 (2002); Harley, CB, et al ., Nature 345: 458). -460 (1990); Olovnikov, AM J. Theoret. Biol. 41: 181-190 (1973); Harley, CB, Exp. Gerontol. 27: 375-382 (1992); Allsopp, RC, Weissman, IL, Oncogene , 21: 3270-3273 (2002)). Telomeres consist of a unique structure in which the TTAGGG sequence is repeated as a terminal part of a eukaryotic linear chromosome. In particular, guanine (G) forms a very stable G-quartet structure through hydrogen bonding to stabilize the chromosome. (Moyzis, RK, et al ., Proc. Natl. Acad. Sci. 85: 6622-6626 (1988)). However, human somatic cells have been found to decrease in telomere length with each cell division (Harley, CB, Futcher, AB, Greider, CW, Nature 345: 458-460 (1990); Harley, CB et al . , Exp. Gerontol. 27: 375-382 (1992); Allsopp, RC, Weissman, IL, Oncogene 21: 3270-3273 (2002)). This phenomenon occurs because of the so-called "end replication problem" where the 3'-terminal primer portion is not replicated when DNA is replicated (Olovnikov, AM J. Theoret. Biol. 41: 181-190 (1973). )). Therefore, every time a cell divides, DNA that is as short as the primer is replicated. As the cell divides, the telomeres of chromosomes become shorter, and when the critical length is less than the critical length, single- and double-stranded DNAs are disconnected. Induction of inhibitors of cyclin dependent kinase causes cell division to stagnate in G1 phase (Harley, CB, et al ., Exp. Gerontol, 27: 375-382 (1992)). Recent studies have shown that telomere length is affected by oxidative stress. That is, oxidative stress increases the rate of telomere shortening, which is thought to be due to oxidative damage that the telomeric DNA portion is less repaired than other parts of the chromosome (Saretzki, G., von Zglinicki, T. , Ann.New York Acad. Sci. 959: 24-29 (2002); von Zglinicki, T., Ann.New York Acad.Sci . 908: 99-110 (2000); von Zglinicki, T., TRENDS Biochem. Sci. 27: 339-344 (2002); Lorenz, M., et al ., Free Radic. Biol. Med. 31: 824-831 (2001).

Based on this theory of aging, we tried to find a substance that can suppress skin aging from plants. Plants are thought to have well developed defenses to protect themselves from these oxidative stresses, since many free radicals, including superoxide radicals, are produced as a by-product of photosynthesis. Therefore, plants can themselves be an important source of antioxidants. Therefore, some candidate plants with antioxidant activity were selected from 350 species of plants by examining their radical scavenging activity and lipid peroxidation inhibitory activity. Considering the literature review and the availability of resources, Cercis chinensis was selected as the final candidate plant.

Cercis chinensis is a deciduous shrub belonging to legumes (Leguminosae), native to China. Its height is 3 to 5 m and the twigs have no hairs and several shell eyes. The leaves are regenerated, single-leafed, shaped like a round heart, 6-11 cm in diameter, without hair, and flat at the edges. The upper side of the leaf is dark green, glossy, and the back side is light green. Chin leaves are square and lose early. Flowers are 1 to 2 cm in length, leaf axilla is attached several times, there is no flower stalk, only peduncle. Calyxes are bell-shaped, with five dull teeth on the upper edge. Corollas are butterfly-shaped, magenta, with 5 petals, but the size is not constant. The stamens are 10, separated, the base is attached to the calyx, and the flower thread is thin and long. There is one pistil, the ovary is glossy, there is no hair, and there is a bag. The top of the style is bent, the style is short, small and pressed flat. Blooms bloom in April before leaves. Fruits are stalks, flat, band-shaped, with a slightly rounded tip, with a short snout. The pod is 7-12cm long and ripens in August and September, and the seed is round, flat and close to black (李 永 魯, 原色 韓國 植物 圖鑑, 敎 學 社, Seoul, 1996, 362-363). In oriental medicine, bark, root bark, woody part, fruit, flower, etc. of Paktaegi tree are divided into the bark of the bark, the bark of the bark, the bark, the bark, and the bark. It is called 등에 花, and it is used for abundant rain, wind, dysmenorrhea, fertilization, gonorrhea, and blood circulation (金昌玟 外., 完 譯 中藥 大 辭典, 圖書)出版 서울, Seoul, 1997, 3631-3634).

Unlike the synthetic antioxidants, the present inventors have found that the extracts are harmless to the human body, unlike synthetic antioxidants, and have superior cell protective activity against oxidative stress as well as other natural antioxidants. The present invention was completed by confirming that it can be usefully used as a cosmetic composition for preventing aging, maintaining skin elasticity or improving wrinkles, since it can prolong the life.

It is an object of the present invention to provide an antioxidant, skin aging inhibitory, skin elasticity maintenance or anti-wrinkle baktaegi extract and an anti-oxidant, skin aging inhibitor or wrinkle cosmetic composition containing the same as an active ingredient.

In order to achieve the above object, the present invention provides an extract for antioxidant, skin aging inhibition, skin elasticity maintenance or wrinkle suppression extracted using water or an aqueous alcohol solution as an extraction solvent.

In addition, the present invention is for antioxidant, skin aging inhibition, skin elasticity maintenance or wrinkle improvement containing a compound selected from the group consisting of the extract or a compound represented by Formula 1 to Formula 20 isolated therefrom as an active ingredient It provides a cosmetic composition.

The present invention also provides a pharmaceutical composition containing the extract as an active ingredient.

The present invention also provides a method of preparing the extract.

Hereinafter, the present invention will be described in detail.

The present invention provides an extract for antioxidant, skin aging inhibition, skin elasticity maintenance or wrinkle suppression extracted using water or an aqueous alcohol solution as an extraction solvent.

The present inventors selected several candidate plants by searching for antioxidant activity based on 140 herbal medicines and 210 plant species collected from all over the country based on the aging of free radicals of aging, and among them, it is easy to secure resources and antioxidant Antioxidant activity was confirmed from the extract of Park Tae-gi, which had no report on the activity. The Park Tae-gi tree used in the present invention was confirmed in mid-September 2001 from Daedeok Research Complex and Chungnam National University compared to the plant illustration, and the confirmed sample is stored in the Korea New Drug Research Institute.

The solvent used to prepare the extract of Paktaegi of the present invention is an aqueous alcohol solution, the aqueous alcohol solution is preferably selected from the group consisting of aqueous methanol solution, aqueous ethanol, aqueous solution of propanol and aqueous butanol. In addition, the alcohol aqueous solution is preferably an ethanol aqueous solution, the ethanol aqueous solution is preferably 50 to 80% ethanol aqueous solution, more preferably 60% ethanol aqueous solution.

In the present invention, the extract of the extract from the above-mentioned alcoholic crude extract, preferably ethanol (ethanol, EtOH) crude extract of ethanol (hexane), ethyl acetate (ethyl acetate, EtOAc) and butanol (butanol, BuOH) After extracting each fraction to obtain a fraction, and prepared by a chromatographic process from the ethyl acetate fraction and butanol fraction showing the antioxidant activity, comprising a compound represented by the following formula 1 to formula 20 from the ethyl acetate fraction and butanol fraction An extract is obtained (see FIGS . 4 and 5 ). In addition, as a result of the structural analysis, it was confirmed that the compound represented by the general formula (15) obtained in the present invention (syringetin-3-O- (2 "-O-galloyl) -rutinoside) is a novel compound which is not known until now.

In the case of the compound of the present invention represented by Formula 1 to Formula 20, the compound represented by Formula 6 is 0.01 to 1.00 wt%, the compound represented by Formula 12 is 0.01 to 1.00 wt%, based on the total weight of the extract It is preferable to contain the compound represented by 0.01 to 0.5 weight%.

Compounds of the present invention represented by Formula 1 to Formula 20 are DPPH radical scavenging activity (1,1-Diphenyl-2-Pycryl-Hydrazyl radical scavenging activity) (see Table 3 ), lipid peroxidation inhibitory activity (Lipid peroxidation inhibitory activity) (See Table 4 ), hydroxyl radical scavenging activity and nitric oxide scavenging activity (see Table 6 ) and Superoxide radical scavenging activity (see Table 5 ) ) And antioxidant activity.

Aerobic organisms use oxygen to metabolize energy, but when oxygen in vivo is subjected to various physical, chemical and biological stresses, superoxide anion radicals, hydrogen peroxide (H ₂ O ₂ ), and hydroxy radicals (hydroxy) It turns into harmful active oxygen species such as radicals and causes fatal physiological disorders. The reactive oxygen species as described above attack the unsaturated fatty acid which is a component of the cell biofilm and cause a peroxidation reaction, and the lipid peroxide accumulated in the living body may cause aging and various diseases. In the present invention, by examining the scavenging function and the ability to inhibit lipid peroxidation of the reactive oxygen species, the antioxidant activity of the extract of Park Tae-gi was measured. As a result, the extract was similar or higher in activity than vitamin E and BHA (tert-butyl-4-hydroxyanisole), a synthetic antioxidant known as an antioxidant, and from the results, the extract was It can be confirmed that it is an excellent extract.

In addition, the compounds of the present invention represented by formulas (1) to (20) have cytoprotective activity against oxidative damage induced by t-butylhydroperoxide (t-BuOOH) (see Table 7 ), cytoprotective effect against UV irradiation (See FIG . 6A and FIG. 6B ), protective effect against UV irradiation in hairless mice (see FIG. 7 ), inhibitory activity of production of lipid peroxidation by UV irradiation (see Table 8 ), effect of prolonging cell life (see FIG. 8 ) , Telomere length extension effect (see FIGS . 9 and 10 ). That is, it can be seen that the extract of Park Tae-gi and the active ingredient separated therefrom exhibits antioxidant activity as well as excellent cell aging inhibitory effect.

In addition, the present invention is an antioxidant, skin aging inhibition, skin elasticity maintenance or anti-wrinkle cosmetic containing the compound selected from the extract of the taegi tree or the compound represented by the formula 1 to formula 20 separated therefrom as an active ingredient To provide a composition.

The active ingredient isolated from the extract of Park Tae-Ki in the cosmetic composition of the present invention is formula 1 (isoliquiritigenin), formula 2 (2 ', 4'-dihydroxy-4-methoxychalcone), formula 3 (liquiritigenin), formula 4 (resveratrol) , Formula 5 (piceatannol), formula 6 (gallic acid), formula 7 (methyl gallate), formula 8 (ethyl gallate), formula 9 (myricetin), formula 10 (afzelin), formula 11 (quercitrin), formula 12 (myricitrin) ), Formula 13 (myricetin-3-O- (2 "-O-galloyl) -α-L-rhamnopyranoside), formula 14 (syringetin-3-O-rutinoside), formula 15 (syringetin-3-O-2" -O-galloyl) -rutinoside), formula 16 ((+)-catechin), formula 17 ((-)-epicatechin-3-O-gallate), formula 18 ((-)-epigallocatechin-3-O-gallate) , A compound selected from the group consisting of compounds represented by formula (19) ((-)-lyoniresinol 3a-O-β-D-xylopyranoside) and formula (20) ((+)-lyoniresiol 3a-O-β-D-glucopyranoside) desirable.

In the cosmetic composition of the present invention, the extract of Park Tae Ki or the active ingredient separated therefrom has high antioxidant activity due to its excellent peroxidation inhibitory activity and radical scavenging activity, and has excellent cell protective effect, cell life extension effect, inhibiting skin aging, It can be used as a cosmetic sample for maintaining skin elasticity or improving wrinkles.

The cosmetic composition of the present invention may be used as a basic cosmetic sample or skin external preparation of a flexible lotion, nourishing lotion, nourishing cream, essence, pack or bath powder.

The content of the oil phase component in the preparation of the cosmetic composition is selected in consideration of the emulsification capacity enhancement and economical efficiency, and as the oil which can be mainly used as the oil phase component, one or more selected from vegetable oil, mineral oil, silicone oil and synthetic oil can be used. have. In addition, 0.1 to 5% by weight of a surfactant, a higher alcohol, and the like may be added to reinforce the emulsifying ability. As the surfactant, a conventional surfactant such as a nonionic surfactant may be used, and as the higher alcohol, an alcohol having 12 to 20 carbon atoms may be used alone or in combination of one or more thereof.

In addition, in the preparation of the cosmetic composition, the aqueous phase component may further add 0.001 to 5% by weight of one or more thickeners such as carbomer, xanthan gum, bentonite, etc. to adjust the viscosity or hardness.

In addition, to the cosmetic composition of the present invention, if necessary, active ingredients such as higher fatty acids and vitamins, and ingredients used in conventional cosmetics such as sunscreens, antioxidants, preservatives, fragrances, colorants, and pH adjusters may be further added.

In a preferred embodiment of the present invention using the extract of the baktaegi of the present invention to prepare a flexible cosmetic water, viscous liquid, emulsion, lotion and cream (see Table 9 to Table 11 ).

In the cosmetic composition containing the extract of the present invention, the extract of the present invention is added to an amount of 1 to 15% by weight, preferably 2 to 10% by weight.

In another aspect, the present invention provides an antioxidant and anti-aging pharmaceutical composition containing the extract of the extract as an active ingredient.

Antioxidant and anti-aging pharmaceutical composition containing the extract of Paktaegi as an active ingredient can be usefully used for the treatment or prevention of diseases caused by oxidation of cellular components by free radicals. Such diseases include, for example, cancer, aging, coronary heart disease, hyperlipidemia, arteriosclerosis, multiple sclerosis, autoimmune encephalomyelitis, stroke, Alzheimer's disease and enteritis, but are not necessarily limited thereto.

The pharmaceutical composition containing the extract of P. chinensis as an active ingredient may further include commonly used excipients, disintegrants, sweeteners, lubricants, flavoring agents, etc., and tablets, capsules, powders, granules by conventional methods. , Suspensions, emulsions, syrups, and other liquids.

Specifically, the pharmaceutical composition containing the extract of the extract of the present invention as an active ingredient is an oral dosage form, for example, tablets, troches, lozenges, water-soluble or oily suspensions, preparation powders or granules. , Emulsions, hard or soft capsules, syrups or elixirs. Lactose, saccharose, sorbitol, mannitol, starch, amylopectin, binders such as cellulose or gelatin, excipients such as dicalcium phosphate, disintegrants such as corn starch or sweet potato starch, magnesium stearate, for formulation into formulations such as tablets and capsules Lubricants such as calcium stearate, sodium stearyl fumarate or polyethylene glycol wax. Capsules contain liquid carriers, such as fatty oils, in addition to the substances mentioned above.

In addition, the pharmaceutical composition containing the extract of the extract of the present invention as an active ingredient can be administered parenterally, parenteral administration is by subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection injection method. To formulate into parenteral formulations, the extract of the present invention is prepared as a solution or suspension by mixing in water with a stabilizer or buffer and formulated in a unit dosage form of ampoules or vials.

The dosage of the active ingredient according to the present invention is appropriately selected according to the absorption rate, inactivation rate and excretion rate of the active ingredient in the body, the age, sex and condition of the patient, the severity of the disease to be treated, but in the case of oral administration in general The adult Gaza extract of the present invention per kilogram of body weight per day may be administered once or several times in an amount of 2-200 mg, preferably in an amount of 10-100 mg.

In addition, the present invention provides a method for producing the extract.

Park Tae-gi extract of the present invention

1) crude extraction of the pulverized powder of baktaegi with alcohol;

2) extracting the crude alcohol extract of step 1 in the order of hexane, ethyl acetate, butanol;

3) obtaining the ethyl acetate fraction or butanol fraction of step 2 and performing column chromatography with a methanol: water gradient; And

4) obtaining the antioxidant active fraction of step 3 to perform column chromatography, TLC or HPLC to obtain a final antioxidant extract.

In step 1, the alcohol is preferably methanol, ethanol, propanol or butanol, of which ethanol is preferred. In the above ethanol is most preferably 60% ethanol. In a preferred embodiment of the present invention, DPPH radical scavenging activity was measured using a crude extract of Bacteria ethanol of 0% to 100%, the highest 60% ethanol crude extract activity (see Fig. 2 ).

Hereinafter, the present invention will be described in detail by the following 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 Extraction of Active Ingredient from Park Tae-Ki

<1-1> Primary Antioxidant Activity Fractions

To extract the effective ingredients having the antioxidant activity from baktaegi tree was an experiment in the order shown in the schematic diagram of FIG. Specifically, 1 kg of the leaves and stems of the dried Paktaegi tree were ground with a mill and powdered and extracted twice with ethanol (EtOH) at room temperature for 2 weeks. At this time, the concentration of ethanol was sequentially increased from 0% to 10%, and finally 100% ethanol was prepared and extracted using the same. In order to confirm the antioxidant activity of the extract according to the concentration of ethanol, DPPH (1,1-Diphenyl-2-Pycryl-Hydrazyl, hereinafter referred to as 'DPPH') method was performed to measure the antioxidant activity (Taco, T. et al. , Biosci. Biotech.Biochem. , 1994, 58, 1780-1783; Na, MK et al ., Nat. Prod. Sci ., 2002, 8, 26-29). DPPH is a relatively stable free radical, which exhibits maximum absorbance at 517 nm when it is in radical state, and loses absorbance when it is eliminated. Specifically, the ethanol extracts of the persimmons were taken, diluted to 3.125, 6.25, 12.25, 25, and 50 ㎍ / ml using DMSO (Sigma), and then each 10 μl of the solution was added to a 96 well plate and 2 × 190 μl of a DPPH (Sigma, St. Louis, Mo, USA) solution at a concentration of 10 ⁻⁴ M / ml ethanol was added thereto, and allowed to stand at room temperature for 30 minutes. Then, the OD value was measured at 517 nm. As a control, DMSO was added instead of the sample to investigate the degree of absorbance reduction of the sample. DPPH radical scavenging activity was calculated according to Equation 1 below, and the concentration of the 50% scavenging DPPH radical was set to IC ₅₀ .

<Equation 1>

A _control : Absorbance of the control without added sample

A _sample : absorbance of the reaction sphere

As a result, DPPH radical scavenging activity increased as the concentration of ethanol used for extraction increased, while 0%, 10%, 20% and 90% ethanol extracts had low radical scavenging activity, but the degree was 30%, 40 %, 50%, 60%, 70%, 80% and 100% ethanol extracts were generally high in radical scavenging activity. In particular, in the case of 60% ethanol extract, the radical scavenging activity was the highest with increasing the concentration of the extract, and the IC ₅₀ value was 26.6, which was the lowest, showing the highest antioxidant activity among the ethanol extracts ( Table 1 and FIG. 2). ).

Ethanol concentration DPPH radical scavenging activity (%) IC ₅₀ (μg / ml) 3.125 μg / ml 6.25 μg / ml 12.5 μg / ml 25 μg / ml 50 μg / ml 0% 2.4 2.9 8 16.8 33 75.2 10% 2.9 6.1 12.6 25 45.3 54.3 20% 3.7 8.4 16.3 32.2 56.1 43.3 30% 5.4 12.3 22.4 42.4 69.7 33.8 40% 6.5 13.2 25.9 47.4 77.9 29.8 50% 6.7 14 27.1 50.1 81.9 28.2 60% 7 15.6 28.5 53.6 85.3 26.6 70% 3.5 10.9 21.9 42 72.8 33.0 80% 4.2 11.4 23.1 46.9 78.9 30.2 80% 3 8.9 17.6 35.3 59.5 40.4 100% 4.1 10.7 21.7 47.4 70.7 32.7

After confirming that the DPPH radical scavenging activity of the 60% ethanol extract is the highest, an extract for each solvent was obtained therefrom. Specifically, 60% ethanol extract was suspended in distilled water, extracted three times with hexane and concentrated under reduced pressure to obtain 11 g of a hexane fraction (hereinafter referred to as 'Fr'), and the remaining suspension was ethyl acetate ( ethyl acetate, EtOAc) was extracted three times and concentrated under reduced pressure to give 25 g of ethyl acetate fraction (hereinafter referred to as 'EtOAc Fr'). The remaining suspension was extracted three times with saturated butanol (BuOH) and decompressed. Concentration gave 19 g of butanol fraction (hereinafter referred to as 'BuOH Fr'). And the remaining fraction 20 g was considered to be the water fraction ( FIG. 1 ).

In order to find the fractions having antioxidant activity from the solvent-extracted hexane Fr, EtOAc Fr and BuOH Fr, DPPH radical scavenging activity was measured using the extract of each fraction in the same manner as described above. At this time, vitamin E which is known to have high DPPH radical scavenging activity was used as a comparison group.

As a result, EtOAc Fr and for BuOH Fr or IC ₅₀ values are showed, respectively 24.0 and a strong activity of a similar degree and 27.0 ㎍ / ㎖ as vitamin _{E (IC 50 24.9 ㎍ / ㎖} ) was used as a control group shown, the other fraction Was poorly active ( FIG. 3 ).

<1-2> Secondary Antioxidant Active Fraction Separation

According to the result of Example <1-1>, column chromatography was performed on the highly active EtOAc Fr and BuOH Fr as follows, and the antioxidant activity experiment was performed on each of the fractions obtained as a result. Was performed to select only the fractions showing high activity.

First, EtOAC Fr (25 g) was subjected to YMC column chromatography (column size: 5 x 30 cm) with methanol: water (1: 4-&gt; 1: 0) as a mobile phase to eleven small fractions (Fr. Fr.11). Fr. 1 (3.6 g) in the 11 small fractions was further subjected to YMC column chromatography (column size: 3 × 30 cm) to obtain 5 small fractions (Fr.1-1 to Fr.1-5). . Among them, Fr.1-1 (450 mg) was purified by HPLC [column: μBondapak ^TM C ₁₈ (3.9 x 300 mm, Waters), mobile phase: ACN: 0.1% TCA (16:18), flow rate: 1 ml / min, UV 280 nm] was used to obtain compounds of 18 mg and 21 mg having a retention time (hereinafter referred to as 't _R ') of 11.1 and 6.2 minutes, respectively, and were named 'CCEA111' and 'CCEA112', respectively. .

Next, preparative HPLC [YMC-Pack ODS-A column (20 × 250 mm), mobile phase: methanol: water (3: 7), flow rate: 6 ml / min was performed on Fr. 1-2 (500 mg). UV: 254 nm] to give 77 mg of a compound having t _R of 16 minutes and 19 mg of a compound having t _R of 24 minutes, respectively, and named 'CCEA1211' and 'CCEA1212', respectively.

Next, preparative HPLC [YMC-Pack ODS-A column (20 × 250 mm), mobile phase: ACN: 0.1% TCA (25:75), flow rate: 6 mL / min on Fr. 3 (4.8 g), detector: UV (280 nm)] to give 19 mg of a compound having a t _R of 24.5 minutes was named 'CCEA33'.

Next, Fr.4 (4.0 g) was subjected to silica gel column chromatography (4 × 25 cm, 230-400 mesh) with mobile phase chloroform: methanol (85:15), and further 6 small fractions (Fr.4-1) were used. ~ Fr.4-6), of which preparative HPLC [Mobile phase: methanol: water (35:65), flow rate: 6 ml / min, UV: 254 nm was used for preparative HPLC. ] To obtain 30 mg of a compound having a t _R of 25 minutes, named 'CCEA413', and Fr.4-4 (1 g) was also prepared for preparative HPLC [mobile phase: methanol: water (1: 1), flow rate: 6 Ml / min, UV: 254 nm] to obtain 57 mg of a compound having a t _R of 15 minutes, and name it 'CCEA442'.

Next, Fr. 6 (2.2 g) was subjected to silica gel chromatography (3 × 30 cm, 230-400 mesh) with mobile phase chloroform: methanol (10: 1) to obtain three small fractions (Fr. 6-1 to Fr). .6-3), preparative HPLC [mobile phase: methanol: water (1: 1), flow rate: 6 ml / min, UV: 254 nm] was performed on Fr.6-2 (200 mg). Thus, 25 mg of a compound having a t _R of 20 minutes was named 'CCEA622'.

Next, Fr. 8 (2.1 g) was subjected to silica gel chromatography using chloroform: methanol (10: 1) as a mobile phase solvent to obtain 20 mg of the compound, which was named 'CCEA82'. CCEA82 was separated and the remaining fractions were dissolved in methanol and recrystallized, and 10 mg of the compound obtained was named 'CCEA83'.

Next, Fr. 9 (2.8 g) was subjected to silica gel chromatography with mobile phase chloroform: methanol (15: 1), and divided into two small fractions (Fr.9.1 to Fr.9-2), of which Fr. 9-1 (220 mg) was subjected to preparative HPLC [mobile phase: methanol: water (4: 1), flow rate: 6 ml / min, UV: 254 nm] to obtain 32 mg of a compound having a t _R of 25 minutes. It was named 'CCEA913' ( FIG. 4 ).

For BuOH Fr (19 g), YMC gel column chromatography (column size: 5 x 30 cm) was carried out using methanol: water (0: 1 → 1: 0) as a mobile phase, and eight small fractions (Fr. Fr.8) was obtained.

Fr.2 (3.8 g) was subjected to silica gel column chromatography (3 x 30 cm, 230-400 mesh) with mobile phase chloroform: methanol: water (70: 30: 5). 1 to Fr.2-5), of which preparative HPLC [mobile phase: acetonitrile: water (18:82), flow rate: 6 ml / min, UV: was compared to Fr.2-3 (420 mg). 254 nm] to give Fr.2-3-1 and 2-3-2, again preparative HPLC [mobile phase: acetonitrile: water (10:90). Flow rate: 6 mL / min, UV: 254 nm] to give 32 mg of a compound having a t _R of 15 minutes, and name it 'CCBt231'.

Subsequently, Fr. 5 (3 g) was subjected to silica gel column chromatography (3 × 30 cm, 230-400 mesh) with mobile phase chloroform: methanol: water (70: 30: 5), and further four small fractions (Fr) were obtained. .5-1 to Fr.5-4), preparative HPLC [Mobile phase: methanol: water (35:65), flow rate: 6 ml / min, for Fr.5-2 (300 mg); UV: 254 nm] to give 20 mg of a compound having a t _R of 25 minutes and 13 mg of a compound having a t _R of 30 minutes, which were named 'CCBt521' and 'CCBt522', respectively. Further, Fr. 5-3 (600 mg) was subjected to YMC gel column chromatography (column size: 3 x 30 cm) using 30% methanol as the mobile phase, and further four small fractions (Fr. 5-3- 1 to Fr. 5-3-4), of which the precipitate-producing material was purified to obtain 29 mg of the compound, which was named 'CCBt533'.

Next, Fr. 6 (3.2 g) was further subjected to YMC column chromatography (column size: 3 x 30 cm) to obtain four small fractions (Fr. 6-1 to Fr. 6-4). Preparative HPLC [Mobile phase: methanol: water (3: 7), flow rate: 6 mL / min, UV: 254 nm] was carried out on Fr. 6-2 (370 mg) to 9 mg of a compound having a t _R of 26 minutes; and 16 mg of a compound having a t _R of 28 minutes were obtained and named as 'CCBt622' and 'CCBt623', respectively. In addition, preparative HPLC (mobile phase: methanol: water (4: 6), flow rate: 6 ml / min) was performed on Fr.6-4 (200 mg) to obtain 5 mg of a compound having a t _R of 18 minutes. CCBt641 '.

Next, Fr.7 (1.9 g) was subjected to silica gel chromatography (column size: 3 x 30 cm) with mobile phase chloroform: methanol (4: 1) to give four small fractions (Fr.7-1 to Fr.7). -4) was obtained. Fr.7-2 (50 mg) was subjected to Sephadex LH-20 column chromatography (column size: 3 x 30 cm) again with 80% methanol in mobile phase to give five small fractions (Fr.7-2-1 to Fr). .7-2-5), of which Fr.7-2-2 was recrystallized from methanol to obtain 9 mg of compound, which was named 'CCBt722'. In addition, the purified Fr.7-2-4 precipitated to obtain a 10 mg compound was named 'CCBt724' ( Fig. 5 ).

<1-3> Structural Analysis of Isolated Compound

The structure of 21 fractions separated in Example <1-2> was analyzed. Specifically, the melting point of the separated compound is used by an electrothermal melting point apparatus (Electrothermal Eng. Ltd., AZ 9003), and the optical intensity is used by a DIP-370 digital polarimeter (JASCO). IR spectra are used for the IR Report-100 Spectrophotometer (JASCO), mass spectrometry uses a Tandem Mass Spectrometer (Jeol, JMS HX-110 / 110A), and NMR analysis is performed using an NMR spectrophotometer (Bruker, NMR AMX-600 spectrometer) and gas chromatography were analyzed according to the manufacturer's policy using STAR 3400CX GC (Varian). The structure of each compound was determined using the analyzed melting point, optical intensity, IR spectrum, mass, and NMR analysis results.

As a result, the structurally analyzed compound was specified as a group represented by a chalcone-based, stilbene-based, phenolic, flavonol-based, flavanol-based and lignan-based, as shown in Table 2 , each of the formulas (1) to (20) It was found that the compound represented by. In addition, the compound represented by Formula 15 (syringetin-3-O- (2 "-O-galloyl) -rutinoside) in the analyzed compound has the following characteristics, and it was found that the compound was a novel compound.

<Characteristics of Formula 15>

Pale yellow powder,

FeCl ₃ , Mg-HCl, Zn-HCl test: positive,

Positive FAB-MS: m / z 823 [M + H] ⁺

[α] _D -80 ( c 0.1, MeOH)

IR ν _max cm ^-1 : 3400 (-OH), 1650 (C = O), 1610, 1500, 1455 (aromatic C = C), 1200, 1020 (glycosidic CO)

UV λ _max nm (log ε): 257 (3.60), 360 (3.82)

¹ H-NMR (600 MHz, DMSO- d6 ): 7.46 (2H, s, H-2, 6), 6.91 (2H, s, galloyl-2, 6), 6.48 (1H, d, J = 1.8 Hz, H-8), 6.21 (1H, d, J = 1.8 Hz, H-6), 5.48 (1H, d, J = 7.2 Hz, glc-1), 4.49 (1H, s, rham-1), 3.84 ( 6H, s, OMe-3, 5), 3.70 (1H, d, J = 10.2 Hz, glc-6), 3.38 (1H, d, J = 10.2 Hz, glc-6), 1.00 (3H, d, J = 5.0 Hz, rham-6)

¹³ C-NMR (150 MHz, DMSO- d6 ): 156.3 (C-2), 133.1 (C-3), 177.3 (C-4), 161.1 (C-5), 98.6 (C-6), 164.0 ( C-7), 93.9 (C-8), 156.4 (C-9), 104.0 (C-10), 119.7 (C-1), 106.9 (C-2, 6), 147.4 (C-3, 5) , 138.6 (C-4), 100.8 (glc-1), 76.4 (glc-2), 74.9 (glc-3), 70.1 (glc-4), 74.3 (glc-5), 66.7 (glc-6), 101.0 (rha-1), 70.2 (rha-2), 70.5 (rha-3), 71.7 (rha-4), 68.3 (rha-5), 17.6 (rha-6), 165.3 (C = O), 120.4 (galloyl-1), 108.7 (galloyl-2, 6), 145.3 (galloyl-3, 5), 137.9 (galloyl-4).

Classification Fraction compound Chalcones CCEA82 CCEA83 CCEA913 Formula 1 (isoliquiritigenin) Formula 2 (luquiritigenin) Formula 3 (2 ', 4'-dihydroxy-4-methoxychalcone) Stilbenes CCEA622 CCEA442 Piceatannol formula 5 resveratrol Phenolics CCBt231 CCEA1212 CCEA33 Formula 6 (gallic acid) Formula 7 (methyl gallate) Formula 8 (ethyl gallate) Flavonols CCEA413 CCBt722 CCBt623 CCBt622 CCBt641 CCBt533 CCBt724 Formula 9 (myricetin) Formula 10 (afzelin) Formula 11 (quercitrin) Formula 12 (myricitrin) Formula 13 (myricetin-3-O- (2 "-O-galloyl) -α-L-rhamnopyranoside) Formula 14 (syringetin-3 -O-rutinoside) Formula 15 (syringetin-3-O- (2 "-O-gallate) Flavanols CCEA1211 CCEA111 CCEA112 Formula 16 ((+)-cathechin) Formula 17 ((-)-epicatechin-3-O-gallate) Formula 18 ((-)-epigallocatechin-3-O-gallate) Lignans CCBt521 CCBt522 Formula 19 ((-)-lyoniresiol 3a-O-β-D-xylopyranoside) Formula 20 ((+)-lyoniresiol 3a-O-β-D-glucopyranoside)

Example 2 Measurement of DPPH radical scavenging activity

The present inventors measured the DPPH radical scavenging activity in the same manner as in Example 1 to determine the antioxidant activity of the extracts containing the compounds represented by Formula 1 to Formula 20 isolated in Example 1. At this time, synthetic antioxidants BHA (tert-butyl-4-hydroxyanisole) and α-tocopherol were used as positive controls.

As a result, phenolic acids and flavonoid compounds showed strong radical scavenging activity in a concentration-dependent manner, and stilbene compounds also showed relatively strong radical scavenging activity. In particular, galloyl esters including gallic acid showed strong activity. Specifically, gallic acid, methyl 7, methyl gallate, 17 ((-)-epicatechin-3-O-gallate), formula 18 ((-)-epigallocatechin-3-O-gallate) and formula 13 (myricetin-3-O- (2-O-galloyl) -aL- The IC ₅₀ values of rhamnopyranoside were 5.1 ± 0.4, 5.3 ± 0.3, 7.0 ± 1.1, 6.8 ± 0.5, 6.7 ± 0.4 and 8.6 ± 0.7 g / ml, respectively, but there was no significant difference in activity between the compounds. It showed significantly stronger radical scavenging activity than α-tocopherol (IC ₅₀ 25.4 ± 0.9 g / ml) and BHA (IC ₅₀ 15.3 ± 0.6 g / ml) used as a positive control ( Table 3 ).

The substitution of an electron donating group at the ortho -position of the benzene ring is known to increase the radical scavenging activity because it can easily stabilize the phenoxy radical (Cuvelier, ME, Richard). , H., Berset, C., Biosci. Biotechnol.Biochem. 56: 324-325 (1992); Kikuzaki, H., et al., J. Agric.Food Chem. 50: 2161-2168 (2002)). In the case of the galloyl group, three hydroxyl groups are substituted at adjacent positions, which can effectively stabilize phenoxy radicals. As shown in Table 3, in addition to galloyl esters, flavonoid compounds such as Formula 16 ((+)-catechin), Formula 9 (myricetin), Formula 10 (afzelin), Formula 11 (quercitrin) and Formula 12 (myricitrin) are also strong. It showed radical scavenging activity, which can be thought of as having a phenolic substituent that can effectively stabilize the phenoxy radicals generated in the flavonoid structure. In other words, the compound having a substituent having a large electron-donating ability was shown to increase the radical scavenging activity, which is consistent with the report of Pekarinen et al. (Pekkarinen, SS, et al., J. Agric. Food Chem. 47: 3036-3043 (1999)).

Classification compound DPPH radical scavenging activity IC ₅₀ (㎍ / ㎖) Chalcones Formula 1 Formula 2 Formula 3 > 50> 50> 50 Stilbenes Formula 4 Formula 5 20.9 ± 1.3 39.5 ± 2.8 Phenolics Formula 6 Formula 7 Formula 8 5.1 ± 0.4 5.3 ± 0.3 7.0 ± 1.0 Flavonols Chemical Formula 9 Chemical Formula 10 Chemical Formula 11 Chemical Formula 12 Chemical Formula 13 Chemical Formula 14 Chemical Formula 15 7.3 ± 0.3 33.4 ± 1.6 12.4 ± 0.6 11.6 ± 0.4 8.6 ± 0.7 35.1 ± 1.5 29.2 ± 1.8 Flavanols Chemical Formula 16 Chemical Formula 17 Chemical Formula 18 15.6 ± 0.8 6.8 ± 0.5 6.7 ± 0.4 Lignans Formula 19 Formula 20 45.7 ± 4.0 42.6 ± 3.1 Positive control group α-tocopherol BHA 25.4 ± 0.9 15.3 ± 0.6

Example 3 Measurement of Lipid Peroxidation Inhibitor Activity

The present inventors measured the lipid peroxidation inhibitory activity in order to measure the antioxidant activity of the compound represented by Formula 1 to Formula 20 isolated in Example 1. Oxidation of lipids can include lipid hydroperoxides, conjugated fatty acids (HODEs), epoxy fatty acids, malondialdehyde (MDA), and 4-hydroxynonenal (4). -hydroxynonenal (4-HNE), which is known to be an important cause of many degenerative diseases and aging processes by directly damaging the biofilms that make up cells or by causing secondary reactions with other cellular components (Esterbauer, H. et al ., Free Radic. Biol. Med ., 1991, 11: 81-128; Esterbauer, H., Cheeseman, KH, Methods Enzymol ., 1990, 186: 407-421; Jira, W., et al , Chem. Phys. Lipids , 1996, 84: 165-173; Jira, W. et al., Biosci , 1998, 53: 1061-1071). In general, superoxide radicals generated in intracellular electron transport systems, such as mitocoderia and microsomes, are converted to H ₂ O ₂ through enzymatic reactions in the body, which are then converted into non-toxic water by enzymes such as catalase and glutathione peroxidase. do. However, when a number of factors vivo active oxygen is much generated by the H ₂ O ₂ is Fenton (Fenton) reaction ^{_{(Fe 2+ + H 2 O 2}} → Fe 3+ + HO · + HO -) or a metal-grease the Catalyst harbor-Weiss reaction (metal-catalysed Haber-Weiss reaction ) (O 2- + H 2 O 2 → O 2 + HO · + HO -) to generate the hydroxyl radical is reactive with the (HO ·) the lipid peroxide It is well known to induce lipid peroxidation chain reaction by inducing lipid radical (L ·) or lipid peroxy radical (LOO ·) by reaction with metal ions in the body (Halliwell, B., Gutteridge, JMC, Free radicals). in biology and medinene, 3rd Edition, Oxford University Press, Oxford, 1999; Halliwell, B., Gutteridge, JMC, Biochem.J. , 1999, 219: 1-14; Harman, D., Free radical theory of aging.Alan R Liss, New York, 1986, 3-49; Stadtman, ER, Levine, RL, Ann. NY Acad. Sci ., 2000, 899: 191-208). Therefore, a compound having an electron donating ability is consequently accepted as capable of inhibiting lipid peroxidation.

To measure the lipid peroxidation inhibitory activity, malondialdehyde (MDA) formed by oxidizing lipids of hydroxyl radicals produced by Fe ²⁺ / ascorbic acid reaction system was used to determine thiobarbituric acid. And hereinafter referred to as 'TBA') to quantify by spectroscopic method. Specifically, 10 μl of the compound samples represented by Formulas 1 to 20 and 50 μl of Rat brain homogenate and 740 μl of 50 mM phosphate buffer (pH 7.4), respectively, at a concentration of 10 μl and 10 mg protein / ml. After mixing, 200 μl of a 0.1 mM FeSO ₄ · 7H ₂ O and 1 mM ascorbic acid mixture was added and reacted at 37 ° C. for 30 minutes. 250 μl of 20% trichloroacetic acid (hereinafter referred to as “TCA”) (Sigma) was added to the reaction solution, and the reaction was stopped. Then, 250 μl of 1% TBA (Sigma) was added thereto and reacted at 100 ° C. for 10 minutes. The reaction solution was centrifuged at 10,000 rpm for 10 minutes and the absorbance was measured at 532 nm. The lipid peroxidation inhibitory activity of each sample was calculated according to Equation 2 below, and the concentration for inhibiting the lipid peroxidation reaction by 50% was set to IC ₅₀ . As a control group, α-tocopherol and BHA were used, and as the control group, no sample and solution were added.

A _control : Absorbance of control group without sample

A _sample : Absorbance of reaction group with sample added

A _blank : Absorbance without adding sample, TCA and TBA solution

As a result, flavonoid-based, stilbene-based and phenolic acid-based compounds showed a concentration-dependent strong lipid peroxidation inhibitory activity. In particular, the stilbene formula (piceatannol) showed a strong activity similar to that of BHA (IC ₅₀ 0.11 ± 0.02 g / ml), which is known as a potent lipid peroxidation inhibitor with an IC ₅₀ value of 0.09 ± 0.01 g / ml. In addition, most of the flavonoid compounds that detected the activity showed a strong lipid peroxidation inhibitory activity, specifically, the formula (myricetin), Formula 17 ((-)-epicatechin-3-O-gallate), Formula 18 (( IC ₅₀ values of-)-epigallocatechin-3-O-gallate, formula 11 (quercitrin), formula 12 (myricitrin) and formula 13 (myricetin-3-O- (2-O-galloyl) -aL-rhamnopyranoside) 0.95 ± 0.06, 2.9 ± 0.06, 1.0 ± 0.08, 6.21 ± 0.40, 5.27 ± 0.32 and 4.73 ± 0.41 g / ml, respectively, indicating a significantly stronger lipid peroxidation inhibitory activity than α-tocopherol used as a positive control ( Table 4 ). In the case of lipid peroxidation inhibitory activity, as in the DPPH radical scavenging activity of Example 2, the more the electron-donating group is substituted in the benzene ring, the more the phenoxy radicals are stabilized, so the activity increases. In particular, the structure of the flavonoid-based compound can be effectively stabilized phenoxy radicals and chelate the metal ions, it is considered to exhibit a strong lipid peroxidation inhibitory activity.

Classification compound Lipid peroxidation inhibitory activity IC ₅₀ (㎍ / ㎖) Chalcones Formula 1 Formula 2 Formula 3 3.33 ± 0.50 12.81 ± 1.86 6.05 ± 0.99 Stilbenes Formula 4 Formula 5 0.89 ± 0.10 0.09 ± 0.01 Phenolics Formula 6 Formula 7 Formula 8 6.80 ± 0.63 7.05 ± 0.87 7.01 ± 0.62 Flavonols Chemical Formula 9 Chemical Formula 10 Chemical Formula 11 Chemical Formula 12 Chemical Formula 13 Chemical Formula 14 Chemical Formula 15 0.95 ± 0.06 10.25 ± 0.91 6.21 ± 0.40 5.27 ± 0.32 4.73 ± 0.41 19.0 ± 1.52 10.10 ± 1.02 Flavanols Chemical Formula 16 Chemical Formula 17 Chemical Formula 18 4.71 ± 0.26 2.90 ± 0.06 1.00 ± 0.08 Lignans Formula 19 Formula 20 37.42 ± 2.06 39.10 ± 3.11 Positive control group α-tocopherol BHA 6.61 ± 0.95 0.11 ± 0.02

<Example 4> Measurement of hydroxyl radical scavenging activity and nitric oxide scavenging activity

Hydroxyl radicals (HO.) Are produced by reaction with metal ions or superoxide radicals in the body, and are known to be highly reactive and directly cause oxidative damage to proteins, lipids and DNA in the body. Nitric oxide (NO ·) is also known to exhibit a similar action to hydroxyl radicals by reacting with superoxide radicals to produce highly reactive peroxy nitrite (ONOO ⁻ ) (Yan, LJ, Sohal, RS). , Free Radic. Biol. Med ., 2000, 29: 1143-1150). Accordingly, the inventors of the present invention, Holleywell's method (Halliwell, B. et al., Anal Biochem., 1987, 165, 215-) to determine the antioxidant activity of the compound represented by Formula 1 to Formula 20 isolated in Example 1 219), the hydroxyl radical scavenging activity was measured. Specifically, 10 μl of the sample dissolved in DMSO, phosphate buffer (20 mM, pH 7.4), deoxyribose 5.6 mM, FeCl ₃ 0.1 mM, H ₂ O ₂ 1 mM and ascorbic acid were mixed by mixing It was made to 1 ml and reacted at 37 degreeC for 60 minutes. 250 µl of 20% TCA and 250 µl of 1% TBA solution (dissolved in 50 mM NaOH) were added to the reaction solution, and the mixture was thermally reacted at 95 ° C for 5 minutes. After the reaction, absorbance was measured at 532 nm. The hydroxyl radical scavenging activity was calculated according to the following equation. As a positive control, BHA and α-tocopherol were used.

A _control : absorbance of control wells

A _sample : Absorbance of reaction wells with sample added

A _blank : Absorbance of wells without addition of sample, TCA and TBA solution

As a result of searching for hydroxyl radical scavenging activity, the flavonoid-based, stilbene-based and phenolic acid-based compounds showed scavenging activity of about 12.7 to 54.0% compared to the control group. In particular, Formula 17 ((-)-epicatechin-3- O- gallate), Formula 18 ((-)-epigallocatechin-3- O -gallate), Formula 7 (methyl gallate), Formula 8 (ethyl gallate), and Formula 13 Compounds substituted with a galloyl group, such as (myricetin-3- O- (2- O- galloyl) -aL-rhamnopyranoside), exhibited strong hydroxyl radical scavenging activity ( Table 5 ).

In addition, as a result of searching for nitric oxide scavenging activity, Chemical Formula 18 ((-)-epigallocatechin-3- O -gallate), Chemical Formula 17 ((-)-epicatechin-3- O -gallate), Chemical Formula 16 ((+) -catechin), formula 9 (myricetin) and the like showed a relatively strong activity ( Table 5 ).

Classification compound Hydroxyl radical scavenging activity% Nitrile oxide scavenging activity% Chalcones Formula 1 Formula 2 Formula 3 6.1 4.5 3.2 Stilbenes Formula 4 Formula 5 9.4 12.7 26.5 ± 1.4 30.8 ± 1.9 Phenolics Formula 6 Formula 7 Formula 8 33.6 43.8 44.0 25.0 ± 2.0 32.6 ± 2.8 33.4 ± 2.1 Flavonols Chemical Formula 9 Chemical Formula 10 Chemical Formula 11 Chemical Formula 12 Chemical Formula 13 Chemical Formula 14 Chemical Formula 15 8.7 3.0 16.6 19.5 45.4 10.2 16.4 25.9 ± 0.8 5.9 ± 0.5 10.2 ± 1.0 11.9 ± 0.9 23.0 ± 0.6 6.0 ± 0.8 14.1 ± 1.4 Flavanols Chemical Formula 16 Chemical Formula 17 Chemical Formula 18 12.7 39.9 54.0 16.9 ± 1.1 25.4 ± 1.2 26.2 ± 0.9 Lignans Formula 19 Formula 20 7.3 8.1 0.8 ± 0.6 0.8 ± 0.8 Positive control group α-tocopherol BHA 0.8 15.6

Example 5 Measurement of Superoxide Radical Scavenging Activity

Superoxide radicals (O ^2- ) themselves are relatively weak, but are easily converted to H ₂ O ₂ , resulting in highly reactive hydroxyl radicals, or nitric oxides (NO ·) reaction by a strong reactive peroxidase nitrite ^- is caused by creating a cause such as the oxidation of SH- groups, protein tyrosine nitride (nitration), lipid peroxidation, DNA damage (peroxy nitrite, ONOO). Thus, in order to confirm the antioxidant activity of the compounds represented by Formula 1 to Formula 20 isolated in Example 1, the scavenging activity of Superoxide radical (O ₂ ⁻ ) acting as a precursor of other harmful active oxygen species Was measured. Superoxide radical scavenging activity can be detected by measuring the activity of superoxide dismutase (SOD), an enzyme that eliminates superoxide by dismutating superoxide, and in the present invention, xanthine / xanthine oxidation Superoxide generation system by enzyme reaction of enzyme (xanthine / xanthine oxidase) and nitro blue tetrazolium (hereinafter referred to as 'NBT') is reduced to formmazan (formazan) reaction (NBT + 2O ₂ ^- > NBTH ₂ + 2O ₂ ) the degree of inhibition of the sample was measured. Specifically, 50 μl of 4 mM xanthine (Sigma), 50 μl of 250 mM NBT (Sigma), 50 μl of 50 mM phosphate buffer (pH 7.8, 1 mM EDTA) and 10 μl of sample were added to a 96 well plate. 40 μl of oxidase (Sigma) was added and reacted. After the reaction, each reaction solution was collected by time, and the absorbance was measured at 550 nm with an ELISA meter. Superoxide radical scavenging activity of each sample was calculated as the reduction of NBT reduction for the control as shown in Equation 4 below, the concentration of the sample to remove the superoxide radical 50% was set to IC ₅₀ . As a positive control, α-tocopherol and caffeic acid were used to compare the antioxidant effects.

A _control : Absorbance of control well without sample

A _sample : Absorbance of reaction wells with sample added

A _blank : absorbance of sample and wells without NBT solution

As a result, flavonoid-based, stilbene-based and phenolic acid-based compounds showed a concentration-dependently strong superoxide radical scavenging activity. In particular, the formula 17 ((-)-epicatechin-3-O-gallate), formula 18 ((-)-epigallocatechin-3-O-gallate), and formula 15 of flavan-3-ol The IC ₅₀ values of (myricetin-3-O- (2-O-galloyl) -aL-rhamnopyranoside) are 11.9 ± 2.1, 24.0 ± 3.5, and 13.2 ± 2.5 g / ml, respectively, caffeic acid, known as a potent superoxide radical scavenger. It showed strong activity similar to (caffeic acid) (IC ₅₀ 11.0 ± 1.8 g / mL). In addition, the flavonoid-based and phenolic acid-based compounds showed a strong superoxide radical scavenging activity, the formula (myricetin), 16 ((+)-catechin), 7 (methyl gallate) and 8 (ethyl gallate) IC ₅₀ values showed strong activity as 12.1 ± 1.1, 16.5 ± 2.0, 16.5 ± 1.4, and 15.8 ± 1.6 g / ml, respectively. However, in the case of the chalcone-based compound, the activity was relatively weak. In the case of the superoxide radical scavenging activity, compounds having a structure capable of easily stabilizing the phenoxy radicals generated by reacting with the superoxide showed good activity. In particular, it was confirmed that the compound substituted with the galloyl group showed strong activity ( Table 6 ).

Classification compound Lipid peroxidation inhibitory activity IC ₅₀ (㎍ / ㎖) Chalcones Formula 1 Formula 2 Formula 3 47.6 ± 4.0 49.8 ± 3.1 59.2 ± 3.8 Stilbenes Formula 4 Formula 5 45.3 ± 1.9 12.1 ± 1.7 Phenolics Formula 6 Formula 7 Formula 8 34.1 ± 2.4 16.5 ± 1.4 15.8 ± 1.6 Flavonols Chemical Formula 9 Chemical Formula 10 Chemical Formula 11 Chemical Formula 12 Chemical Formula 13 Chemical Formula 14 Chemical Formula 15 12.1 ± 1.1 50.2 ± 3.4 32.1 ± 2.0 29.7 ± 1.8 13.2 ± 2.5 45.2 ± 2.2 37.1 ± 2.0 Flavanols Chemical Formula 16 Chemical Formula 17 Chemical Formula 18 16.5 ± 2.0 11.9 ± 2.1 24.0 ± 3.5 Lignans Formula 19 Formula 20 > 100> 100 Positive control group Caffeic Acid BHA 11.0 ± 1.8 48.8 ± 2.5

<Example 6> t -Protective activity against oxidative damage induced by -butylhydroperoxide

t -Butylhydroperoxide is known to enter cells and metabolize into free radical intermediates, causing lipid peroxidation and eventually cell damage. Since this phenomenon is similar to the oxidative stress occurring in cells and tissues, it is a useful method to evaluate the inhibition of aging by oxidative stress in a practical sense. Treatment of neonatal epidermal cells, HEK-N / F cells, with 1.5 mM of t -butylhydroperoxide for 3 hours resulted in a significant decrease in cell viability of 11.2 ± 1.2%, whereas 50.0 g / ml When treated together at a concentration, it showed significant cytoprotective activity. In particular, the combination of Formula 5 (piceatannol) and the combination showed the strongest cell survival rate of 84.7 ± 6.9%, and the flavone 3-ol compounds of Formula 9 (myricetin), Formula 11 (quercetin), respectively 61.0 ± 4.5 Cell viability of 4% and 48.1 ± 5.7% was shown. In addition, Formula 6 (gallic acid), Formula 7 (methyl gallate), Formula 8 (ethyl gallate) and the like also showed a cytoprotective activity of about 41.5 ± 3.1%, 43.0 ± 5.6%, 43.1 ± 4.1%, respectively. Through the above results, it was confirmed that the compound obtained in the extract of Paktaegi of the present invention can effectively inhibit the oxidative stress of the cell and thereby inhibit aging caused by the oxidative stress by inhibiting cell death by peroxidation.

Classification compound Cell survival rate (%) TBARS (pmol / mg protein) Chalcones Formula 1 Formula 2 Formula 3 13.9 ± 2.6 16.2 ± 3.2 12.1 ± 1.0 5118.1 ± 410.5 3449.7 ± 305.8 8255.1 ± 576.4 Stilbenes Formula 4 Formula 5 18.1 ± 1.6 84.7 ± 6.9 2792.4 ± 259.1 520.2 ± 60.7 Phenolics Formula 6 Formula 7 Formula 8 41.5 ± 3.1 43.0 ± 5.6 43.1 ± 4.1 1245.7 ± 112.4 1024.4 ± 91.9 1115.6 ± 96.2 Flavonols Chemical Formula 9 Chemical Formula 10 Chemical Formula 11 Chemical Formula 12 Chemical Formula 13 Chemical Formula 14 Chemical Formula 15 61.0 ± 4.5 16.8 ± 1.3 21.4 ± 1.9 25.4 ± 1.7 45.1 ± 3.6 20.0 ± 2.6                                              810.7 ± 77.0 3295.1 ± 304.5 2217.2 ± 200.3 1852.6 ± 126.8 1019.8 ± 154.0 2928.1 ± 209.9                                              Flavanols Chemical Formula 16 Chemical Formula 17 Chemical Formula 18 24.5 ± 1.9 49.4 ± 6.4 46.6 ± 5.9 1912.4 ± 112.0 1007.3 ± 95.2 1032.6 ± 103.5 Lignans Formula 19 Formula 20 13.5 ± 2.0 12.9 ± 2.1 5981.1 ± 412.6 7635.7 ± 631.2 Control t-butanol 11.2 ± 1.2 984.0 ± 95.2

Example 7 Protective Activity Against Oxidative Stress Induced by UV Irradiation

<7-1> Cytoprotective effect on UV irradiation ( in vitro )

UV irradiation significantly increases free radicals in cells, causing DNA damage, DNA-protein linkages, and the like. Irradiation of 35 mJ / cm ² UVB to HEK-N / F cells breaks the DNA chain of the nucleus, which binds to the ethidium homodimer (Et2), a DNA strand intercalating fluorescent dye. Quantification by measuring the intensity. Specifically, HEK-N / F cells were inoculated in serum-free KGM medium (Clonetics) and incubated in a 24-well plate so as to be 0.5 to 4 × 10 ⁴ cells / 2 cm ² . Cells were incubated for 18 hours and then sampled for 2 hours. After washing with PBS, the cells were irradiated with UV using a UV detector (Spectronics UV transilluminator EBF-260, the maximal wavelength, 312 nm; a half-peak intensity range, 297-328 nm). UV irradiated cells were incubated for 1-7 hours and administered with 5 M ethidium homodimer (Et2, Millipore). After 30 minutes, fluorescence was measured using a Millipore microplate fluorometer Cytofluor 2350 (excitation: 485 nm, emission: 645 nm).

As a result, as shown in Figures 6a and 6b , when treated with the extract of Paktaegi, Formula 9 (myricetin) and Formula 5 (piceatannol) showed a fluorescence of 54.7% and 66.7% lower than the fluorescence of the control, respectively. In other words, the extract of Park Tae-gi and the active ingredients separated from them significantly inhibited the oxidative stress caused by UV and significantly reduced DNA damage.

<7-2> Skin tissue protection effect against UV irradiation ( in vitro )

5-6 week old female hairless mice (hairless mouse, SKH-hr-1) were used in the experiment after adapting the temperature 24 ± 2 ℃, relative humidity 50 ± 10%, 12 hours day / night cycle for about 14 days. Thirty-60 minutes prior to the experiment, the samples were subcutaneously injected at five sites of hairless rats and then irradiated with UVB on the rats' backs. Next, 5 rats per group in a 20 × 15 × 5 cm cage were placed at a distance of 15 cm per square meter with UV lamps (HP-15M, 280-400 nm, max. 312 nm; Atto Co., Japan). UVB was irradiated to 15 kJ. After irradiating UVB for 24 hours, the dorsal skin was cut and stored at -70 ° C until the peroxidation measurement.

Since UVB irradiation causes significant lipid peroxidation in skin tissue, the extent of damage to the skin can be measured by measuring the content of peroxidized lipids. Lipid peroxidation was measured. Specifically, UVB was irradiated on the back surface of SKH-1 hairless rat, and after 48 hours, the skin tissue of the back was cut and lipid peroxidation was measured by thiobarbituric acid (TBA) method. Dorsal skin of hairless rats was placed in 10-fold 50 mM K-P buffer and then homogenized.

Next, hydrogen peroxide was observed. Frozen sections of skin were placed in 0.1 M Tris-HCl buffer (pH 7.5) containing 1 mg / ml glucose and 1 mg / ml diaminobenzidine (DAB) and incubated at 37 ° C. for 5-6 hours. . After washing with distilled water and stained with 2% methyl green (methyl green) for 60 minutes. Microscopically observed brown DAB-peroxidase and nuclei stained blue.

UV irradiation is known to induce the generation of reactive oxygen species in the skin tissues, causing DNA damage, protein oxidation, lipid peroxidation, and eventually cause skin damage such as inflammation, cancer, and skin aging. As shown in FIG. 7 , when irradiated with UVB at 90 mJ / cm ² , significant skin damage was observed. However, the skin damage of SKH-1 hairless rats was significantly inhibited when the extract of Park Tae Ki and Formula 5 (piceatannol) were pretreated at a concentration of 50 mg / kg. As can be seen from the results of the above example, the extract of Park Tae-gi and its active ingredient, formula (piceatannol), showed strong radical scavenging activity, lipid peroxidation inhibitory activity and protective activity of skin cells against oxidative stress, and thus, UVB irradiation The protective effect against skin damage induced by H. pylori was thought to be due to this antioxidant activity. Diaminobenzidine (DAB) reacted with tissue peroxidase to produce dark brown DAB-peroxidase, and thus observed the H ₂ O ₂ produced by UVB irradiation. As shown in FIG. 7 , dark brown DAB-peroxidase was observed when UVB was irradiated at 90 mJ / cm ² , but when the extract of P. chinensis and Formula 5 (piceatannol) was pretreated at a concentration of 50 mg / kg H ₂ O ₂ production was markedly inhibited in skin tissues of SKH-1 hairless mice.

As a result of measuring lipid peroxidation, the average thibarbituric acid reactive substances (TBARS) content of the UVB-irradiated group was about 0.68 nmol / mg protein, which was twice as much as the control group (0.32 ± 0.05 nmol / mg protein) without UVB irradiation. Peroxidation occurred ( Table 8 ). The group treated with the extract of Park Tae-Ki and Formula 5 (piceatannol) reduced the lipid peroxidation of the skin in a concentration-dependent manner. In particular, Formula 5 showed higher activity than magnesium-L-ascorbyl-2-phosphate (MAP) used as a positive control at the same concentration.

Treatment group Throughput (mg / kg) TBARS (nmol / mg protein) Control 0 0.32 ± 0.05 UVB probe 0 0.68 ± 0.1 Park Ki-Tee Extract + UVB Irradiation 50 30 10 0.21 ± 0.10 0.34 ± 0.09 0.64 ± 0.10 Formula 5 + UVB irradiation 30 10 0.07 ± 0.03 0.16 ± 0.08 MAP + UVB irradiation 50 30 0.16 ± 0.05 0.28 ± 0.06

Example 8 Prolonged Effect on Cell Lifespan

HEK-N / F cells were treated with uridine bromide, another major component of skin, and then cultured in DMEM medium containing 10% FBS obtained from human neonatal foreskin. HEK-N / F cells were cultured continuously at a dilution ratio of 1: 4. The samples were grown to a population doubling level (PDL) of about 3 before treatment, and then each sample was administered at a concentration of 3 g / ml during incubation. Medium exchange was performed every 3 days with the addition of a culture solution containing the same concentration of sample. After cell culture, cell division was observed by treating the extract of Park Tae-Ki, Formula 5 (piceatannol) and Formula 9 (myricetin) of the present invention.

As a result, it was found that the extract and active ingredient of taegi tree extends the life of skin cells. The control group which was not treated with anything had an average lifespan of about 35 days, whereas the life expectancy was extended by 1.2 times as compared to the control group when the average lifespan was 42 days when incubated with 3 ㎍ / ml of the extract. When the cells were cultured with the active ingredients of Formula 9 (Myricetin) and Formula 5 (piceatannol), the average lifespan was 56 days and 76 days, respectively, and the lifespan of the cells was extended by 1.6 and 2.1 times, respectively ( Fig. 8 ). Therefore, it was found that the extract of Park Tae-Ki of the present invention and the active ingredient isolated therefrom have the effect of prolonging cell life.

Example 9 Extension Effect on Telomere Length and Cell Lifetime

In Example 8, it was confirmed that the extract of Park Tae Ki of the present invention and the active ingredient separated therefrom have an effect of prolonging cell life. The aim of this study was to investigate the relationship between cell life extension effect and telomere length. Specifically, genomic DNA was extracted from cells of each age using an IsoQuick Nucleic Acid Extraction kit (ORCA Research Inc.), and Tris-EDTA (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). ) And stored at 4 ° C. 2 μl of 10 × H buffer (TaKaRa) and 2 μl of the genomic DNA solution extracted above were added to a 1.5 ml tube, followed by tertiary distilled water to make 19 μl of the total restriction enzyme, Hinf I (6 U / l, TaKaRa). 1 μl was added. The mixture was reacted at 37 ° C. for 3 to 4 hours, followed by electrophoresis. In electrophoresis, agarose (type I, Sigma) gel was prepared by adjusting the concentration of the gel so that the bridge part was 1% and the bed part was 0.8% (Marisol KS-8405, 20 × 14 cm), 1 × Boy Buffer (50 mM Tris-HCl, 20 mM sodium acetate, 2 mM EDTA, 18 mM NaCl, pH 8.0) was used. As a marker, 0.5 μg was loaded using 1 Kb DNA ladder (GIBCO), and all samples were subjected to electrophoresis for 20 hours at 35 V / cm in 3 μl of loading buffer. After electrophoresis was completed, stained with ethidium bromide (2 ㎍ / ㎖) for 15 minutes, placed in UV and confirmed. The gel was then shaken for 15 minutes in 0.25 N HCl and washed twice with distilled water. The gel was immersed in a denatured solution (0.2 N NaOH, 0.6 M NaCl), shaken for 25 minutes at room temperature, and washed three times with distilled water. 6 x SSC-filled blotting device in order of nitrocellulose membrane Optitran BA-S 85, Schleicher & Schuel, 3 mm noji, paper towel, glass plate, weight (2 kg) Blotting while. After blotting was complete the membrane filter was immersed in 3 x SSC, lightly dehydrated and the wells positioned in UV. Baked at 80 ° C. overnight at 80 ° C., prehybridized with denatured salmon sperm DNA (Wako) at 65 ° C., and then mixed buffer (1 × Denhan solution, 1 M at 50 ° C.). NaCl, 50 mM Tris-HCl, 10 mM EDTA, 0.1% SDS, 50 g / ml denatured salmon sperm DNA) and 5'-terminal [ ³² P] -labeled (TTAGGG) ₄ . The hybridized membrane was immersed in a cleaning solution (4 × SSC / 0.1% SDS), shaken at 55 ° C. for 15 minutes, dried, covered with a wrap, and placed in a sensitized cassette together with an X-ray film (Scientific Imaging Film, Kodak). Autoradiography was performed overnight at &lt; RTI ID = 0.0 &gt; The position of the well was marked by magic by combining the developed film and the position of the film. The density peaks of TRFs were detected with a laser densitometer UltroScan XL, Pharmacia to calculate their mobility.

As a result, it was observed that the length of telomeres gradually decreased with cell division. As shown in FIG . 9 , the control group splits 14.8 times, and the telomeres decrease to about 8.0 kbp in length, and no longer divide. On the other hand, in the group treated with the extract or active ingredient, the length of telomeres reached a critical value (about 8.0 kbp in the present invention) after much more cleavage than the control group. Cell division could continue as long as telomeric DNA remained above the threshold (presumably human skin keratinocytes were estimated to be 7.9-8.4 kb). In this regard, it has been shown that by delaying the rate at which telomeres are shortened, the rate at which the cell division capacity is terminated can be extended by slowing the rate of reaching the threshold. In other words, the maximum PDL of the control group was 14.1, whereas the maximum PDL was significantly increased when the extract was administered to 17.2, formula 9 (myricetin) 23.1, formula 5 (piceatannol) 29.9.

In addition, as a result of calculating the telomere shortening rate from the relationship between the number of cell division and the measured telomere length, the telomere shortening rate of the group administered the extract of Park Tae-gi, extracts (myricetin) and formula (piceatannol) were respectively compared to the control group. It was confirmed that the delayed by 1.2, 1.6 and 2.1 times ( Fig. 10 ). Therefore, the effect of prolonging the skin cell lifespan of the extract of Park Tae Ki and the active ingredient was considered to be the result of slowing down the telomeres shortening rate.

Preparation Example 1 Preparation of Cosmetic Composition Containing Extraction

The present inventors have prepared a cosmetic composition (the formulation 1 to the formulation 6) containing the extract of the extract as an active ingredient as shown in Table 9 to Table 11 . In addition, other extracts that can be used to prepare cosmetics, in addition to the extract of baktaegi tree included in the preparation examples may be further included. For example, baekbaekpi, brown alga, donkey, uiyiin, bark skin, machi, persimmon leaves, witch hazel and centella extract.

First, the softening longevity and viscous liquid containing the extract of Paktaegi of the present invention was prepared according to the composition described in Table 9 .

Preparation of Soft Cosmetics and Viscous Liquid Raw material name (% by weight) Formulation 1 Formulation 2 1,3-butylene glycol 3.7 3.7 glycerin 3.6 5.5 PEG-60 Hydrogenated Castor Oil 0.3 0.1 D-panthenol 0.3 0.5 Allantoin 0.1 0.1 Plant extracts 5.6 5.9 ethanol 4.0 3.0 Carbomer 0.1 - Pellets - 0.5 Disodium ID a very small amount a very small amount Dipotassium glycylizate - a very small amount Sodium hyaluronate a very small amount a very small amount Triethanolamine Quantity Quantity antiseptic Quantity Quantity Combination spices Quantity Quantity Cedar extract 2.0 4.0 Purified water to 100 to 100

Next, the emulsion and lotion containing the extract of Paktaegi according to the present invention was prepared according to the composition described in Table 10 below.

Preparation of Latex and Lotion Raw material name (% by weight) Formulation 3 Formulation 4 1,3-butylene glycol 6.5 4.5 glycerin 1.2 3.0 D-panthenol 0.2 0.1 Pellets - 0.5 Plant extracts 6.2 6.3 Magnesium Aluminum Silicate - 0.3 ethanol 3.0 - PEG-60 Hydrogenated Castor Oil - 0.2 Carbomer 0.1 0.1 Stearic acid 1.5 - Polysorbate 60 0.7 0.2 Lipophilic glyceryl stearate 0.6 - Sorbitan sesquioleate 0.3 - Cetearyl Alcohol 0.6 - Mineral oil 5.0 - Squalane 3.5 1.0 Carlylic / Capric Triglycerides 3.0 0.7 vegetable oil 2.0 2.3 Dimethicone 0.4 Quantity Dipotassium glycylizate a very small amount a very small amount Allantoin a very small amount a very small amount Sodium hyaluronate a very small amount a very small amount Tocopheryl Acetate Quantity Quantity Triethanolamine Quantity Quantity antiseptic Quantity Quantity Combination spices Quantity Quantity Cedar extract 5.0 6.0 Purified water to 100 to 100

Next, according to the composition shown in Table 11 to prepare a cream containing the extract of the extract of the present invention.

Manufacture of cream Raw material name (% by weight) Formulation 5 Formulation 6 1,3-butylene glycol 7.0 5.0 glycerin 1.0 5.0 D-panthenol 0.1 0.1 Plant extracts 3.2 2.7 Magnesium Aluminum Silicate 0.3 - PEG-40 Stearate 1.2 0.8 Stearic acid 2.0 3.2 Polysorbate 60 1.5 0.9 Lipophilic glyceryl stearate 2.0 2.0 Sorbitan sesquioleate 1.5 1.0 Cetearyl Alcohol 3.0 2.9 Microcrystalline Wax - 1.0 Mineral oil 4.0 2.0 Petrolatum - 0.5 Squalane 3.8 4.5 Carlylic / Capric Triglycerides 2.8 2.5 vegetable oil 1.8 2.3 Dimethicone 0.4 0.4 Dipotassium glycylizate a very small amount a very small amount Allantoin a very small amount a very small amount Sodium hyaluronate a very small amount a very small amount Pyridoxine Dipalmitate - a very small amount Pellets Quantity - Carbomer - Quantity Tocopheryl Acetate Quantity Quantity Triethanolamine Quantity Quantity antiseptic Quantity Quantity Combination spices Quantity Quantity Cedar extract 5.0 3.0 Purified water to 100 to 100

Preparation Example 2 Preparation of Antioxidant and Anti-aging Pharmaceutical Composition

The present inventors prepared an antioxidant and anti-aging pharmaceutical composition containing the extract of Paktaegi as an active ingredient as follows.

<2-1> Manufacturing method of syrup

Syrup containing 2% (weight / volume) of the extract of Paktaegi tree of the present invention as an active ingredient is prepared by the following method.

The extract of Saccharin, saccharin, and sugar were dissolved in 80 g of warm water. After the solution was cooled, a solution consisting of glycerin, saccharin, spices, ethanol, sorbic acid and distilled water was prepared and mixed thereto. Water was added to this mixture to make 100 mL ( Table 12 ).

Raw material name Quantity (g) Cedar extract 2 saccharin 0.8 Party 25.4 glycerin 8.0 Spice 0.04 ethanol 4.0 Sorbic acid 0.4 Distilled water dose

<2-2> Manufacturing method of tablet

A tablet containing 15 mg of active ingredient is prepared by the following method.

It was mixed with 250 g of the extract of Strawberries, 175.9 g of lactose, 180 g of potato starch, and 32 g of colloidal silicic acid. 10% gelatin solution was added to the mixture, which was then ground and passed through a 14 mesh sieve. It was dried and the mixture obtained by adding 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate was made into a tablet ( Table 13 ).

Raw material name Quantity (g) Cedar extract 250 Lactose 175.9 Potato starch 180 Colloidal silicic acid 32 Gelatin solution 10% Potato starch 160 talc 50 Magnesium stearate 5

As described above, the extract of Park Tae Ki of the present invention has high antioxidant activity, and thus the pharmaceutical composition containing the extract of Park Tae Ki as an active ingredient may be used for the treatment and prevention of diseases related to peroxidation.

Hereinafter, a specific disease to which the pharmaceutical composition comprising the extract of Paktaegi of the present invention can be applied will be described.

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

It is caused by a myriad of cancers, but the most fundamental cause is the destruction of cells by free radicals and their inability to repair them, leading to impairment of cell function, which has progressed to cancer (Ames, BN, Science , 1983, 221). , 1256-1264). On the other hand, the effect of phenolic acid in fruits on liver cancer (Sun J et al ., J Agric Food Chem , 2002, 4; 50 (25), 7449-7454), and lycopene in tomato against breast cancer Effect (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) A variety of currently 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 of 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 peroxidation by free radicals, thereby reducing low-density lipoproteins. The protective effect was not effective in the treatment of the disease, and the administration of the antioxidant cerivastatin or probucol to the patients resulted in a sharp decrease in the content of low density lipoproteins (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, the size of atherosclerotic lesions was reduced by lowering blood pressure and lowering cholesterol levels in the blood vessel walls (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, if these symptoms are not treated and given constant oxidative stress, serious brain diseases such as stroke and Alzheimer's disease will occur. 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 brain diseases such as stroke and 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 extract of Park Tae Ki and the compounds represented by Formula 1 to Formula 20 isolated therefrom are harmless to human body, unlike synthetic antioxidants, and have excellent antioxidant activity compared to other natural antioxidants. In addition to effectively inhibiting oxidative stress and inhibiting the shortening of the telomere length associated with cell aging, the life span of the skin cells can be extended, and the cosmetic composition of the present invention comprising the same as an active ingredient inhibits skin aging and skin elasticity. It can be usefully used for maintenance and wrinkle improvement.

Claims (14)

Extract of antioxidant and skin aging extracts using water or alcohol aqueous solution as extractant. The extract according to claim 1, wherein the aqueous alcohol solution is selected from the group consisting of aqueous methanol solution, aqueous ethanol solution, aqueous propanol solution and aqueous butanol solution. The extract of claim 2, wherein the aqueous alcohol solution is 50-80% aqueous ethanol. 4. The extract of claim 3, wherein the aqueous alcohol solution is an aqueous 60% ethanol solution. The method of claim 1, wherein the extract is formula 1 (isoliquiritigenin), formula 2 (2 ', 4'-dihydroxy-4-methoxychalcone), formula 3 (liquiritigenin), formula 4 (resveratrol), formula 5 (piceatannol), formula 6 (gallic acid), formula 7 (methyl gallate), formula 8 (ethyl gallate), formula 9 (myricetin), formula 10 (afzelin), formula 11 (quercitrin), formula 12 (myricitrin), formula 13 (myricetin-3) -O- (2 "-O-galloyl) -α-L-rhamnopyranoside), formula 14 (syringetin-3-O-rutinoside), formula 15 (syringetin-3-O-2" -O-galloyl) -rutinoside , Formula 16 ((+)-catechin), Formula 17 ((-)-epicatechin-3-O-gallate), Formula 18 ((-)-epigallocatechin-3-O-gallate), Formula 19 ((-)- lyoniresinol 3a-O-β-D-xylopyranoside) and an extract characterized in that it comprises a compound selected from the group consisting of compounds represented by the formula (20) ((+)-lyoniresiol 3a-O-β-D-glucopyranoside). The method according to claim 5, wherein the extract is 0.01 to 1.00% by weight of the compound represented by the formula (6), 0.01 to 1.00% by weight of the compound represented by the formula 12, containing 0.01 to 0.5% by weight of the compound represented by the formula (5) Characterized in that the extract. Antioxidant, anti-aging skin cosmetic composition containing the extract of claim 1 as an active ingredient. An anti-oxidant and anti-aging skin cosmetic composition containing a compound selected from the compounds represented by Formula 1 to Formula 20 separated from the extract of claim 1 as an active ingredient. The cosmetic composition according to claim 7 or 8, wherein the cosmetic composition is selected from the group consisting of skin external preparations based cosmetics such as flexible lotion, nourishing lotion, nourishing cream, essence, pack and bath powder. Anti-oxidant and anti-aging pharmaceutical composition containing the extract of the extract of claim 1 as an active ingredient. The pharmaceutical composition of claim 10, wherein the pharmaceutical composition is prepared from the group consisting of injections, tablets and syrups. 1) crude extraction of the pulverized powder of baktaegi with alcohol; 2) extracting the crude alcohol extract of step 1 in the order of hexane, ethyl acetate, butanol; 3) obtaining the ethyl acetate fraction or butanol fraction of step 2 and performing column chromatography with a concentration gradient of ethanol: water; And, 4) The method of claim 1, comprising the step of obtaining the antioxidant active fraction of step 3 to obtain a final antioxidant extract by column chromatography, TLC or HPLC. 13. A process according to claim 12 wherein the alcohol is 60% ethanol. A syringetin-3-O- (2 "-O-galloyl) -rutinoside of Formula 15 or a pharmaceutically acceptable salt thereof. Formula 15

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