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    • 专利摘要:
    The present invention relates to functional foods for antioxidant and anti-aging, and more particularly, after solvent fractionation of crude alcohol extract of Park Ki-gi, which is easy to secure resources and has not been previously reported on antioxidant activity and anti-aging activity of skin cells. Antioxidant and Anti-aging Functionality Including Antioxidant Activity, Isolated from Ethyl Acetate Fraction and Butanol Fraction Exhibiting Antioxidant Activity, and Extracted from Strawberries Containing Compounds of Formula 1 to Formula 20 Having Aging Inhibitory Activity as Active Ingredients It is about food. The extract of Park Tae-gi is not only excellent in inhibiting oxidative stress, but also extends the lifespan of cells by slowing the shortening of the telomeres associated with aging. And the preparation of functional foods for preventing or treating diseases caused by free radicals.
    公开号:KR20040060730A
    申请号:KR1020030085838
    申请日:2003-11-28
    公开日:2004-07-06
    发明作者:나민균；유재국；이찬복；김진표
    申请人:주식회사한국신약；
    IPC主号:
    专利说明:

    Anti-oxidative and skin-aging protective functional food containing the extract of Cercis chinensis}
    [13] The present invention relates to an antioxidant and anti-aging functional food comprising a plant extract having antioxidant and anti-aging activity as an active ingredient.
    [14] Aging is the collective term for all physiological changes in the body that occur over time, and is a life phenomenon that is caused by a number of factors that vary from person to person. There are many difficulties in studying aging at the individual level because of different aspects. 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).
    [15] 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 Drosophila increased life span by more than 30%, which is closely related to ROS 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.
    [16] 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, a lot of research is being conducted to develop a safer and high free radical scavenging material as an important task not only in the pharmaceutical and food sectors but also in the cosmetics industry.
    [17] 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. Plays an important role in the protection (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).
    [18] 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. Thus, some candidate plants with antioxidant activity were selected by examining the radical scavenging activity and lipid peroxidation inhibitory activity in 350 plant species. Considering the literature review and the availability of resources, Cercis chinensis was selected as the final candidate plant.
    [19] 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 Korean cabbage are divided into the bark of the bark, the bark of the bark, the bark, the bark, and the bark. It is called 花, and it is used for wind, rain, pain, dysmenorrhea, fertilization, gonorrhea, and blood circulation (金昌玟 外., 完 譯 中藥 大 辭典, 圖書 出版) Wong, Seoul, 1997, 3631-3634).
    [20] Thus, the inventors of the present invention, unlike the synthetic antioxidants, the extract is harmless to the human body, and compared to other natural antioxidants, as well as excellent cytoprotective activity against oxidative stress as well as slow the shortening of the telomeres length By extending the life of the cell, the present invention has been completed by revealing that the extract isolated from the baktaegi tree can be usefully used in the pharmaceutical composition and functional food for inhibiting antioxidant and skin aging.
    [21] It is an object of the present invention to provide a functional food for antioxidant and anti-aging, which contains the extract of P. chinensis as an active ingredient.
    [1] 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.
    [2] Figure 2 is a graph measuring the DPPH radical scavenging activity of ethanol crude extract extracted from 0% ethanol from 10% ethanol from 100% to 100% concentration.
    [3] 3 is a graph measuring DPPH radical scavenging activity of hexane, ethyl acetate, butanol, water fraction and ethanol extract.
    [4] Figure 4 is a schematic diagram showing the process of separating the compound showing the antioxidant activity from the ethyl acetate fraction.
    [5] 5 is a schematic diagram illustrating a process of separating a compound exhibiting antioxidant activity from a butanol fraction.
    [6] Figure 6a is a cell photograph showing the degree of DNA damage showing the cell protective effect on UV irradiation of the extract of Park Tae Ki and compounds isolated therefrom.
    [7] 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.
    [8] 7silver 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.
    [9] 8silver This is a graph showing that the extract of Park Tae Ki of the present invention and the compound isolated therefrom prolong cell life.
    [10] 9silver It is a photograph of the southern blot of the telomeres length showing that the extract of the extract of the present invention and the compound separated therefrom have a telomere length extension action.
    [11] 10silver Telomere shortening rate graph showing that the extract of the present invention and the compound isolated therefrom has a telomere length extension action.
    [12] 11 is a photograph showing flowers, leaves, stems, and fruit of Park Tae-gi.
    [22] In order to achieve the above object, the present invention provides a functional food for antioxidant and anti-aging containing the extract of P. loccus as an active ingredient.
    [23] Hereinafter, the present invention will be described in detail.
    [24] The present invention provides a functional food for antioxidant and anti-aging, containing the extract of Park Seonggi as an active ingredient.
    [25] The present inventors selected several candidate plants by searching for antioxidant activity based on 140 kinds of herbal medicines and 250 kinds of plants collected from all over the country, based on the oxidative oxygen aging 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.
    [26] In the extract of Park Tae Ki included in the functional food of the present invention, the extract is characterized in that the extraction using an aqueous alcohol solution. The alcohol aqueous solution is preferably selected from the group consisting of an aqueous methanol solution, an aqueous ethanol solution, an aqueous propanol solution and an aqueous butanol solution. In addition, the aqueous alcohol 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.
    [27] In the present invention, the extract of the extract from the above-mentioned alcoholic crude extract, preferably ethanol (EtOH) crude extract of the above extract with hexane (hexane), ethyl acetate (ethyl acetate, EtOAc) and butanol (butanol, BuOH) After the respective fractions were obtained, a chromatographic process was prepared from the ethyl acetate fraction and the butanol fraction showing antioxidant activity, and the extract comprising the compounds represented by the following Chemical Formulas 1 to 20 from the ethyl acetate fraction and the butanol fraction was 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.
    [28]
    [29]
    [30]
    [31]
    [32]
    [33]
    [34]
    [35]
    [36]
    [37]
    [38]
    [39]
    [40]
    [41]
    [42]
    [43]
    [44]
    [45]
    [46]
    [47]
    [48] 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%.
    [49] 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.
    [50] Aerobic organisms use oxygen to metabolize energy, but when oxygen is subjected to various physical, chemical and biological stresses, superoxide anion radicals, hydrogen peroxide (H 2 O 2 ), and hydroxy radicals It turns into harmful active oxygen species such as radicals and causes fatal physiological disorders in the human body. 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.
    [51] In addition, the compounds of the present invention represented by Formula 1 to Formula 20 have cytoprotective activity against oxidative damage induced by t-butanol (see Table 7), cytoprotective effect against UV irradiation (see FIGS. 6A and 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 (FIG. 9) And FIG. 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.
    [52] Accordingly, the extract of Park Tae-Ki in the functional food for antioxidant and anti-aging of the present invention is formula 1 (isoliquiritigenin), formula 2 (2 ', 4'-dihydroxy-4-methoxychalcone), formula 3 (liquiritigenin), formula 4 ( resveratrol), 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) It is preferable to include.
    [53] In the functional food of the present invention, the extract of Park Tae-gi or the active ingredient isolated 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 sample of functional food for maintaining skin elasticity or improving wrinkles.
    [54] When the extract of the present invention is used as a food additive, the extract can be added as it is or used together with other food or food ingredients, and can be appropriately used according to a conventional method. The blending amount of the active ingredient can be suitably determined according to the purpose of its use (prevention, health or therapeutic treatment). In general, during the preparation of food or beverage, the extract of the present invention is added in an amount of 0.1 to 15% by weight, preferably 0.2 to 10% by weight based on the raw material. However, in the case of long-term intake for health and hygiene or health control, the amount may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety. Is sure.
    [55] There is no particular limitation on the kind of food. Examples of foods to which the above substances can be added include dairy products including drinks, meat, sausages, breads, chocolates, candy, snacks, confectionery, pizza, ramen, other noodles, gums, ice creams, various soups, beverages, alcoholic beverages. And vitamin complexes and the like, and include all of the functional foods in the conventional sense.
    [56] Hereinafter, the present invention will be described in detail by way of examples.
    [57] However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.
    [58] Example 1 Extraction of Active Ingredient from Park Tae-Ki
    [59] <1-1> Primary Antioxidant Activity Fractions
    [60] 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 dry leaves and stems of the dried Paktaegi tree were ground with a mill, and powdered and extracted twice with ethanol (EtOH) for 2 weeks at room temperature. 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 .
    [61] <Equation 1>
    [62]
    [63] A control : Absorbance of the control without added sample
    [64] A sample : absorbance of the reaction sphere
    [65] 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). ).
    [66] Ethanol concentrationDPPH radical scavenging activity (%)IC 50 (μg / ml) 3.125 μg / ml6.25 μg / ml12.5 μg / ml25 μg / ml50 μg / ml 0%2.42.9816.83375.2 10%2.96.112.62545.354.3 20%3.78.416.332.256.143.3 30%5.412.322.442.469.733.8 40%6.513.225.947.477.929.8 50%6.71427.150.181.928.2 60%715.628.553.685.326.6 70%3.510.921.94272.833.0 80%4.211.423.146.978.930.2 80%38.917.635.359.540.4 100%4.110.721.747.470.732.7
    [67] 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 ).
    [68] 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.
    [69] 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 ).
    [70] <1-2> Secondary Antioxidant Active Fraction Separation
    [71] 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.
    [72] 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.1 to -1). 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. .
    [73] 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.
    [74] 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'.
    [75] 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'.
    [76] 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'.
    [77] 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'.
    [78] Next, silica gel chromatography was performed on mobile phase chloroform: methanol (15: 1) with respect to Fr.9 (2.8 g), and divided into two small fractions (Fr.9.1 to Fr.9-2). 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 ).
    [79] 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.
    [80] 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'.
    [81] 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'.
    [82] 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 '.
    [83] 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 ).
    [84] <1-3> Structural Analysis of Isolated Compound
    [85] 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.
    [86] 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.
    [87] <Characteristics of Formula 15>
    [88] Pale yellow powder,
    [89] FeCl 3 , Mg-HCl, Zn-HCl test: positive,
    [90] Positive FAB-MS: m / z 823 [M + H] +
    [91] [α] D -80 ( c 0.1, MeOH)
    [92] IR ν max cm -1 : 3400 (-OH), 1650 (C = O), 1610, 1500, 1455 (aromatic C = C), 1200, 1020 (glycosidic CO)
    [93] UV λ max nm (log ε): 257 (3.60), 360 (3.82)
    [94] 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)
    [95] 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).
    [96] ClassificationFractioncompound ChalconesCCEA82CCEA83CCEA913Formula 1 (isoliquiritigenin) Formula 2 (luquiritigenin) Formula 3 (2 ', 4'-dihydroxy-4-methoxychalcone) StilbenesCCEA622CCEA442Piceatannol Formula 5 resveratrol PhenolicsCCBt231CCEA1212CCEA33Formula 6 (gallic acid) Formula 7 (methyl gallate) Formula 8 (ethyl gallate) FlavonolsCCEA413CCBt722CCBt623CCBt622CCBt641CCBt533CCBt724Formula 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) FlavanolsCCEA1211CCEA111CCEA112Formula 16 ((+)-cathechin) Formula 17 ((-)-epicatechin-3-O-gallate) Formula 18 ((-)-epigallocatechin-3-O-gallate) LignansCCBt521CCBt522Formula 19 ((-)-lyoniresiol 3a-O-β-D-xylopyranoside) Formula 20 ((+)-lyoniresiol 3a-O-β-D-glucopyranoside)
    [97] Example 2 Measurement of DPPH radical scavenging activity
    [98] 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.
    [99] 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-rhamnopyranoside IC 50 values of 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, but all of these compounds were positive. 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 control ( Table 3 ).
    [100] 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. Radical scavenging activity was shown, which is thought to be due to the presence of phenolic substituents 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)).
    [101] ClassificationcompoundDPPH radical scavenging activity IC 50 (㎍ / ㎖) ChalconesFormula 1 Formula 2 Formula 3> 50> 50> 50 StilbenesFormula 520.9 ± 1.339.5 ± 2.8 PhenolicsFormula 75.1 ± 0.45.3 ± 0.37.0 ± 1.0 FlavonolsFormula 9 Formula 10 Formula 11 Formula 12 Formula 13 Formula 14 Formula 157.3 ± 0.333.4 ± 1.612.4 ± 0.611.6 ± 0.48.6 ± 0.735.1 ± 1.529.2 ± 1.8 FlavanolsChemical Formula 16 Chemical Formula 1715.6 ± 0.86.8 ± 0.56.7 ± 0.4 LignansFormula 2045.7 ± 4.042.6 ± 3.1 Positive control groupα-tocopherol BHA25.4 ± 0.915.3 ± 0.6
    [102] Example 3 Measurement of Lipid Peroxidation Inhibitor Activity
    [103] 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.
    [104] 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 to stop the reaction, and then 250 μl of 1% TBA (Sigma) was added thereto, followed by reaction 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.
    [105]
    [106] A control : Absorbance of control group without sample
    [107] A sample : Absorbance of reaction group with sample added
    [108] A blank : Absorbance without adding sample, TCA and TBA solution
    [109] 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.
    [110] ClassificationcompoundLipid peroxidation inhibitory activity IC 50 (㎍ / ㎖) ChalconesFormula 1 Formula 2 Formula 33.33 ± 0.5012.81 ± 1.866.05 ± 0.99 StilbenesFormula 50.89 ± 0.100.09 ± 0.01 PhenolicsFormula 76.80 ± 0.637.05 ± 0.877.01 ± 0.62 FlavonolsFormula 9 Formula 10 Formula 11 Formula 12 Formula 13 Formula 14 Formula 150.95 ± 0.0610.25 ± 0.916.21 ± 0.405.27 ± 0.324.73 ± 0.4119.0 ± 1.5210.10 ± 1.02 FlavanolsChemical Formula 16 Chemical Formula 174.71 ± 0.262.90 ± 0.061.00 ± 0.08 LignansFormula 2037.42 ± 2.0639.10 ± 3.11 Positive control groupα-tocopherol BHA6.61 ± 0.950.11 ± 0.02
    [111] <Example 4> Measurement of hydroxyl radical scavenging activity and nitric oxide scavenging activity
    [112] 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.
    [113]
    [114] A control : absorbance of control wells
    [115] A sample : Absorbance of reaction wells with sample added
    [116] A blank : Absorbance of wells without addition of sample, TCA and TBA solution
    [117] 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 ).
    [118] 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 ).
    [119] ClassificationcompoundHydroxyl radical scavenging activity%Nitrile oxide scavenging activity% ChalconesFormula 1 Formula 2 Formula 36.14.53.2StilbenesFormula 59.412.726.5 ± 1.430.8 ± 1.9 PhenolicsFormula 733.643.844.025.0 ± 2.032.6 ± 2.833.4 ± 2.1 FlavonolsFormula 9 Formula 10 Formula 11 Formula 12 Formula 13 Formula 14 Formula 158.73.016.619.545.410.216.425.9 ± 0.85.9 ± 0.510.2 ± 1.011.9 ± 0.923.0 ± 0.66.0 ± 0.814.1 ± 1.4 FlavanolsChemical Formula 16 Chemical Formula 1712.739.954.016.9 ± 1.125.4 ± 1.226.2 ± 0.9 LignansFormula 207.38.10.8 ± 0.60.8 ± 0.8 Positive control groupα-tocopherol BHA0.815.6
    [120] Example 5 Measurement of Superoxide Radical Scavenging Activity
    [121] 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.
    [122]
    [123] A control : Absorbance of control well without sample
    [124] A sample : Absorbance of reaction wells with sample added
    [125] A blank : absorbance of sample and wells without NBT solution
    [126] 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 ).
    [127] ClassificationcompoundLipid peroxidation inhibitory activity IC 50 (㎍ / ㎖) ChalconesFormula 1 Formula 2 Formula 347.6 ± 4.049.8 ± 3.159.2 ± 3.8 StilbenesFormula 545.3 ± 1.912.1 ± 1.7 PhenolicsFormula 734.1 ± 2.416.5 ± 1.415.8 ± 1.6 FlavonolsFormula 9 Formula 10 Formula 11 Formula 12 Formula 13 Formula 14 Formula 1512.1 ± 1.150.2 ± 3.432.1 ± 2.029.7 ± 1.813.2 ± 2.545.2 ± 2.237.1 ± 2.0 FlavanolsChemical Formula 16 Chemical Formula 1716.5 ± 2.011.9 ± 2.124.0 ± 3.5 LignansFormula 20> 100> 100 Positive control groupCaffeic Acid BHA11.0 ± 1.848.8 ± 2.5
    [128] <Example 6> t Cytoprotective Activity Against Oxidative Damage Induced by -Butylhydroperoxide
    [129] 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.
    [130] ClassificationcompoundCell survival rate (%)TBARS (pmol / mg protein) ChalconesFormula 1 Formula 2 Formula 313.9 ± 2.616.2 ± 3.212.1 ± 1.05118.1 ± 410.53449.7 ± 305.88255.1 ± 576.4 StilbenesFormula 518.1 ± 1.684.7 ± 6.92792.4 ± 259.1520.2 ± 60.7 PhenolicsFormula 741.5 ± 3.143.0 ± 5.643.1 ± 4.11245.7 ± 112.41024.4 ± 91.91115.6 ± 96.2 FlavonolsFormula 9 Formula 10 Formula 11 Formula 12 Formula 13 Formula 14 Formula 1561.0 ± 4.516.8 ± 1.321.4 ± 1.925.4 ± 1.745.1 ± 3.620.0 ± 2.6810.7 ± 77.03295.1 ± 304.52217.2 ± 200.31852.6 ± 126.81019.8 ± 154.02928.1 ± 209.9 FlavanolsChemical Formula 16 Chemical Formula 1724.5 ± 1.949.4 ± 6.446.6 ± 5.91912.4 ± 112.0 1007.3 ± 95.21032.6 ± 103.5 LignansFormula 2013.5 ± 2.012.9 ± 2.15981.1 ± 412.67635.7 ± 631.2 Controlt-butanol11.2 ± 1.2984.0 ± 95.2
    [131] Example 7 Protective Activity Against Oxidative Stress Induced by UV Irradiation
    [132] <7-1> Cytoprotective effect on UV irradiation ( in vitro )
    [133] 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 UVtransilluminator 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).
    [134] 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.
    [135] <7-2> Skin tissue protection effect against UV irradiation ( in vitro )
    [136] 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.
    [137] 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.
    [138] 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.
    [139] 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. The protective effect against induced skin damage is 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.
    [140] 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.
    [141] Treatment groupThroughput (mg / kg)TBARS (nmol / mg protein) Control00.32 ± 0.05 UVB probe00.68 ± 0.1 Park Tae Ki Extract + UVB Irradiation5030100.21 ± 0.100.34 ± 0.090.64 ± 0.10 Formula 5+ UVB irradiation30100.07 ± 0.030.16 ± 0.08 MAP + UVB irradiation50300.16 ± 0.050.28 ± 0.06
    [142] Example 8 Prolonged Effect on Cell Lifespan
    [143] 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.
    [144] 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 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, compared to the control group ( 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.
    [145] Example 9 Extension Effect on Telomere Length and Cell Lifetime
    [146] 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. Ethidium bromide (2) was performed. [Mu] g / ml) was stained for 15 minutes, placed in UV and checked. 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.
    [147] 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.
    [148] 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.
    [149] Preparation Example 1 Preparation of Functional Food Containing Extract of Park Tae Ki
    [150] The present inventors prepared the food containing the extract of Paktaegi as an active ingredient as follows.
    [151] <1-1> Beverage Production
    [152] 522 mg of honey
    [153] Chioctosanamide 5 mg
    [154] Nicotinamide 10 mg
    [155] Riboflavin Sodium Hydrochloride 3 mg
    [156] Pyridoxine hydrochloride 2 mg
    [157] Inositol 30 mg
    [158] Orthoic acid 50 mg
    [159] Park Tae-gi Extract 0.48-1.28 mg
    [160] 200 ml of water
    [161] With the above composition and content, drinks were prepared using conventional methods.
    [162] <1-2> Preparation of Chewing Gum
    [163] Gum base 20%
    [164] Sugar 76.36-76.76%
    [165] Park Tae-gi Extract 0.24 ~ 0.64%
    [166] 1% fruit flavor
    [167] Water 2%
    [168] Chewing gum was prepared using conventional methods using the above composition and content.
    [169] <1-3> Preparation of Candy
    [170] 50 to 60% sugar
    [171] Starch syrup 39.26-49.66%
    [172] Park Tae-gi Extract 0.24 ~ 0.64%
    [173] Orange flavor 0.1%
    [174] Candy was prepared using conventional methods with the above composition and content.
    [175] <1-4> Preparation of Biscuits
    [176] Force Class 1 88 kg
    [177] Gravity First Class 76.4 ㎏
    [178] 16.5 kg per white
    [179] 2.5 kg of salt
    [180] 2.7 kg of glucose
    [181] Palm shortening 40.5 kg
    [182] 5.3 kg of ammo
    [183] Medium kg 0.6 kg
    [184] 0.55 kg sodium bisulfite
    [185] Rice flour 5.0 kg
    [186] Vitamin B1 0.003 kg
    [187] 0.003 kg of vitamin B2
    [188] Milk Flavor 0.16 ㎏
    [189] 71.1 kg of water
    [190] Whole milk powder 4 kg
    [191] Substitute powder 1 kg
    [192] 0.1 kg of calcium phosphate
    [193] Spray salt 1 kg
    [194] 25 kg of spray oil
    [195] 0.2 ~ 0.5 kg
    [196] Biscuits were prepared using conventional methods with the above composition and content.
    [197] <1-5> Preparation of Ice Cream
    [198] Milkfat 10.0%
    [199] Non-fat solids 10.8%
    [200] Sugar 12.0%
    [201] Starch syrup 3.0%
    [202] Emulsifying stabilizer (span) 0.5%
    [203] Spices (Strawberries) 0.15%
    [204] Water 63.31-62.91%
    [205] Park Tae-gi Extract 0.24 ~ 0.64%
    [206] Ice cream was prepared using conventional methods using the above composition and content.
    [207] <1-6> Preparation of Chocolate
    [208] Sugar 34.36-34.76%
    [209] Cocoa Butter 34%
    [210] Cocoa Mass 15%
    [211] Cocoa Powder 15%
    [212] Lecithin 0.5%
    [213] 0.5% vanilla
    [214] Park Tae-gi Extract 0.24 ~ 0.64%
    [215] With the above composition and content, chocolate was prepared using conventional methods.
    [216] As described above, the extract of Park Tae-gi and the compounds represented by Chemical Formulas 1 to 21 isolated therefrom are harmless to the human body, unlike synthetic antioxidants, and have excellent activity as compared to other natural antioxidants. Since the lifespan of skin cells can be prolonged by suppressing the shortening of the telomere length associated with aging, it can be usefully used as a functional food for the prevention or treatment of diseases caused by aging or free radicals.
    权利要求:
    Claims (5)
    [1" claim-type="Currently amended] Antioxidant and anti-aging functional foods containing extracts of Korean cauliflower as active ingredients.
    [2" claim-type="Currently amended] The functional food according to claim 1, wherein the extract is extracted using an aqueous alcohol solution selected from the group consisting of an aqueous methanol solution, an ethanol ethanol solution, an aqueous propanol solution, and an aqueous butanol solution as a solvent.
    [3" claim-type="Currently amended] The functional food according to claim 2, wherein the extract is extracted using 60% ethanol aqueous solution as a solvent.
    [4" claim-type="Currently amended] The extract of claim 1, wherein the extract of P. chinensis is 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 a compound selected from the group consisting of compounds of formula 20 ((+)-lyoniresiol 3a-O-β-D-glucopyranoside) food.
    [5" claim-type="Currently amended] According to claim 1, wherein the functional food is a drink, meat, sausage, bread, chocolate, candy, snacks, confectionary, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, alcoholic beverages and A functional food selected from the group consisting of vitamin complexes.
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    同族专利:
    公开号 | 公开日
    KR100825038B1|2008-04-24|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    法律状态:
    2002-12-27|Priority to KR1020020085383
    2002-12-27|Priority to KR20020085383
    2003-11-28|Application filed by 주식회사한국신약
    2004-07-06|Publication of KR20040060730A
    2008-04-24|Application granted
    2008-04-24|Publication of KR100825038B1
    优先权:
    申请号 | 申请日 | 专利标题
    KR1020020085383|2002-12-27|
    KR20020085383|2002-12-27|
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