KR101817053B1 - Composition for anti-oxidant or anti-cancer or anti-obesity or immune-enhancing containing Codonopsis lanceolata extract - Google Patents
Composition for anti-oxidant or anti-cancer or anti-obesity or immune-enhancing containing Codonopsis lanceolata extract Download PDFInfo
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- KR101817053B1 KR101817053B1 KR1020160022424A KR20160022424A KR101817053B1 KR 101817053 B1 KR101817053 B1 KR 101817053B1 KR 1020160022424 A KR1020160022424 A KR 1020160022424A KR 20160022424 A KR20160022424 A KR 20160022424A KR 101817053 B1 KR101817053 B1 KR 101817053B1
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- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
- A23V2200/308—Foods, ingredients or supplements having a functional effect on health having an effect on cancer prevention
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
- A23V2200/332—Promoters of weight control and weight loss
Abstract
The present invention relates to an antioxidant, anti-cancer, anti-obesity or immunomodulating composition comprising roots extract as an active ingredient. The dodok extract according to the present invention is excellent in antioxidant, anti-cancer, anti-obesity or immunity enhancing activity, and is free from toxicity and side effects due to natural products, and can be used safely even when taken for a long time.
Description
The present invention relates to a composition for antioxidant, anti-cancer, anti-obesity or immunity enhancement, which comprises rosacea extract as an active ingredient.
Dodok is a perennial plant of the dicotyledonous plant, the podocarpus, and is a plant indigenous to humid grasslands, forests, or valleys such as Korea, Japan, China, and the Far East of Russia . It is mainly used for edible roots and also called ginseng or white ginseng. The roots of dodok are thick like bellflower, and when cutting plants, white juice comes out. Leaves are alternate and at the end of short branch, four leaves are facing each other and facing each other. They are long oval with length of 3-10cm and width of 1.5-4cm. Leaf edges are flat, green on the front, white on the back. In August-September, bell-shaped flowers often run from the end of the short branch to the bottom. Calyx is green, length 2-5cm, and butterfly 6-10cm. The corolla is 2.7 ~ 3.5cm long and the end is divided into 5 pieces and dried backward. The surface is light green and the inside has purple spots. The fruit ripens in September. Young leaves in spring, edible roots in autumn. The raw ginseng (沙 蔘) is a term referring to dried root.
Obesity is one of the most prevalent diseases in which the pattern of disease is rapidly changing to a developed country type as the hygiene environment is improved due to the improvement of the living standard and the average life span is extended due to the westernization of the diet. Thus, adult diseases have emerged as the biggest medical problem today, and the obesity, which is a major cause of these adult diseases, is increasing rapidly. Obesity refers to the phenomenon that extra calories are accumulated in the body in the form of fat by consuming excessive calories compared to the calories consumed. Obesity is thought to be caused by various causes such as genetic influences, environmental influences due to westernized diet, and psychological effects due to stress. However, the precise cause and mechanism of obesity has not been established yet. However, since obesity can act not only as a problem of obesity itself, but also as a cause of diseases such as cardiovascular disease or diabetes (Manson et al., New England J. Med., 333, pp677-685, 1995; JAMA, 282, pp1523-1529, 1999). There is a growing interest in the treatment of obesity worldwide.
In addition, studies on the regulation of the human immune system against the natural extract have been actively conducted because the immune system abnormality breaks health and causes various diseases. Therefore, the development of immunomodulators for safe natural products that can minimize side effects can be a very important material for the development of food, cosmetics and pharmaceuticals. For example, mulberry bark and shiitake mushrooms are used as immunostimulants, starfish, longevity, and mushroom as anti-cancer adjuvants. Licorice, licorice glycyrrhizin, sea cucumber, Research is underway on cell differentiation. The development of immunomodulatory agents using these natural materials has attracted many people's interest and interest because they can safely treat cancer and chronic inflammatory diseases without side effects.
In order to solve such a problem, it is urgently required to develop a composition having antioxidant, anti-cancer, anti-obesity or immunity-enhancing effect derived from natural materials that does not cause side effects and is safe.
Accordingly, the present inventors have completed the present invention by confirming that it has antioxidant, anti-cancer, anti-obesity or immunity enhancing activity.
Accordingly, an object of the present invention is to provide a pharmaceutical composition having antioxidant, anti-cancer, anti-obesity or immunity-enhancing activity, comprising the extract of Rootgrass as an active ingredient.
Another object of the present invention is to provide a health functional food having antioxidant, anti-cancer, anti-obesity or immuno-promoting activity comprising roeae extract as an active ingredient.
In order to achieve the above object, the present invention provides a pharmaceutical composition having antioxidant, anti-cancer, anti-obesity or immunostimulating activity, comprising the extract of Rootgrass as an active ingredient.
In one embodiment of the present invention, the doduck may be a Korean three-year roots grown in Hwasun, Muju, Jecheon, Hoengseong or Uljin.
In one embodiment of the present invention, the extract may be extracted with at least one solvent selected from the group consisting of lower alcohols having 1 to 4 carbon atoms, ethyl acetate, acetone, water and hexane.
In one embodiment of the present invention, the extract may be one that increases DPPH, nitrite or ABTS cation scavenging activity.
The present invention provides a health functional food having antioxidant, anti-cancer, anti-obesity or immuno-promoting activity, comprising the extract of Rootgrass as an active ingredient.
In one embodiment of the present invention, the doduck may be a Korean three-year roots grown in Hwasun, Muju, Jecheon, Hoengseong or Uljin.
In one embodiment of the present invention, the food is selected from the group consisting of beverage, meat, chocolate, foods, confectionery, pizza, ram noodles, gums, candy, ice cream, alcoholic beverages, .
The present invention relates to a composition composition having antioxidant, anti-cancer, anti-obesity or immuno-promoting activity, which comprises roots extract as an active ingredient. The extract according to the present invention is excellent in antioxidant, anti-cancer, anti-obesity or immuno-stimulating activity and thus can be usefully used as a pharmaceutical composition. In addition, there is no cytotoxicity, there is no toxicity to the drug, and no side effects, so that it can be safely used even when taken for a long period of time.
Fig. 1 shows the kind of dodec used in the present invention.
Figure 2 shows the roots of Duck Root Extract and its fractions.
FIG. 3 is a graph showing the results of SOD enzyme activity assay of the fractions of Fusarium oxysporum extract according to the present invention.
nH: n-hexane
MC: Methylene chloride
EA: Ethyl acetate
BA: Butyl alcohol
DW: distilled water
FIG. 4 is a graph showing the results of SOD enzyme activity assay of roeae extract according to the ethanol content according to the present invention.
FIG. 5 is a graph showing the results of the CAT activity assay of the fractions of Fusarium oxysporum according to the present invention.
nH: n-hexane
MC: Methylene chloride
EA: Ethyl acetate
BA: Butyl alcohol
DW: distilled water
FIG. 6 is a graph showing the results of CAT activity assay for roeae extract according to the ethanol content according to the present invention.
FIG. 7 is a graph showing the results of assaying the APX activity of the fractions of Rootgrass extract according to the present invention.
nH: n-hexane
MC: Methylene chloride
EA: Ethyl acetate
BA: Butyl alcohol
DW: distilled water
FIG. 8 is a graph showing the results of assaying APX activity of roeae extract according to the ethanol content according to the present invention.
FIG. 9 is a graph showing the results of POD activity assay of the extract of Fusarium exudata according to the present invention.
nH: n-hexane
MC: Methylene chloride
EA: Ethyl acetate
BA: Butyl alcohol
DW: distilled water
FIG. 10 is a graph showing the results of POD activity assay of Root Root Extract according to ethanol content according to the present invention.
FIG. 11 shows the results of cell viability measurement of the Hwasun daetuck extract using the solvent according to the ethanol content of 3T3-L1 cells.
FIG. 12 shows the results of cell viability measurement of Uljinchoduk extract using a solvent according to ethanol content of 3T3-L1 cells.
FIG. 13 shows the results of measurement of cell survival rate of the solvent-use transversal roots extract according to the ethanol content of 3T3-L1 cells.
14 shows the results of measurement of cell viability of Jeju dodok extract using a solvent according to the ethanol content of 3T3-L1 cells.
15 shows the results of cell viability measurement of Jecheon duck extract using a solvent according to the ethanol content of 3T3-L1 cells.
FIG. 16 shows the results of measurement of cell viability of the extract of Mujoo-dodok extract using a solvent according to the ethanol content of 3T3-L1 cells.
Fig. 17 shows the results of measurement of cell growth of Hwasun dodok extract using a solvent according to the ethanol content of human T cells.
18 shows the results of measurement of cell growth of Uljinchuk extract using a solvent according to the ethanol content of human T cells.
FIG. 19 shows the results of measurement of cell growth of the transversal sextet leaf extract using the solvent according to the ethanol content of human T cells.
20 shows the results of measurement of cell growth of Jeju Dodok extract using a solvent according to the ethanol content of human T cells.
21 shows the results of measurement of cell growth of Jecheon duck extract using a solvent according to ethanol content of human T cells.
22 shows the results of measurement of cell growth of Mujootak extract using a solvent according to the ethanol content of human T cells.
23 shows the results of measurement of cell growth of Hwasun daetuck extract using the solvent according to the ethanol content of human B cells.
24 shows the results of measurement of cell growth of Uljinchuk extract using a solvent according to the ethanol content of human B cells.
FIG. 25 shows the results of cell growth measurement of the solvent-induced Hoechst etudes extract according to the ethanol content of human B cells.
26 shows the results of measurement of cell growth of Jeju Dodok extract using a solvent according to the ethanol content of human B cells.
FIG. 27 shows the results of measurement of cell growth of Jecheon duck extract using a solvent according to the ethanol content of human B cells.
FIG. 28 shows the results of cell growth measurement of the extract of Mujoochuk extract using a solvent according to the ethanol content of human B cells.
The present invention is characterized by providing a pharmaceutical composition for antioxidant, anti-cancer, anti-obesity or immunity enhancement, which comprises the extract of Rootgrass as an active ingredient.
The dodec according to the present invention can be obtained by extracting and isolating from nature using extraction and isolation methods known in the art, and the 'extract' defined in the present invention is extracted from dodec using an appropriate solvent , For example, hot water extracts of rosemary, polar solvent-soluble extracts, or non-polar solvent-soluble extracts.
As a suitable solvent for extracting the extract from roeae, any solvent acceptable in the art may be used, and water or an organic solvent may be used. Examples of the solvent include alcohols having 1 to 4 carbon atoms, acetone, ether, and the like, including purified water, methanol, ethanol, propanol, isopropanol, butanol, Various solvents such as benzene, chloroform, ethyl acetate, methylene chloride, hexane and cyclohexane may be used alone or in combination. But is not limited to.
As the extraction method, any one of the methods such as hot water extraction method, cold extraction method, reflux cooling extraction method, solvent extraction method, steam distillation method, ultrasonic extraction method, elution method and compression method can be selected and used. In addition, the desired extract may be further subjected to a conventional fractionation process or may be purified using a conventional purification method. There is no limitation on the method for producing the roeae extract of the present invention, and any known method can be used.
For example, the roots extract contained in the composition of the present invention can be prepared into a powdery state by an additional process such as vacuum distillation, freeze-drying or spray-drying, or the like, with the primary extract extracted by the hot water extraction or solvent extraction method described above . Further, the primary extract can be further purified by using various chromatographies such as silica gel column chromatography, thin layer chromatography, high performance liquid chromatography and the like, You can get it.
Therefore, in the present invention, roeae extract is a concept including all the extracts, fractions and tablets obtained in each step of extraction, fractionation or purification, their diluted solutions, concentrates or dried products.
The pharmaceutical composition of the present invention may be prepared by using pharmaceutically acceptable and physiologically acceptable adjuvants in addition to the above-mentioned active ingredients. Examples of the adjuvants include excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, A lubricant or a flavoring agent can be used.
The pharmaceutical composition may be formulated into a pharmaceutical composition containing at least one pharmaceutically acceptable carrier in addition to the above-described active ingredients for administration.
The pharmaceutical composition may be in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, solutions, aerosols, excipients, injections, transdermal drugs, suppositories and the like. For example, for formulation into tablets or capsules, the active ingredient may be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Also, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included as a mixture. Suitable binders include, but are not limited to, natural sugars such as starch, gelatin, glucose or beta-lactose, natural and synthetic gums such as corn sweeteners, acacia, tracker candles or sodium oleate, sodium stearate, magnesium stearate, Sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum and the like. Acceptable pharmaceutical carriers for compositions that are formulated with a liquid include sterilized and sterile water, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, And other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added as needed. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Further, it can be suitably formulated according to each disease or ingredient, using the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA as an appropriate method in the field.
In one embodiment of the present invention, the roots extract of the present invention may be contained at a concentration of 0.1 μg / ml to 500 μg / ml based on the total weight of the composition.
In the meantime, 'dodok' of the present invention is a perennial plant of dicotyledonous plants and lobsters, and is distributed in Korea, Japan, China and Russian Far East, Or a plant native to the valley. It is mainly used for edible roots and also called ginseng or white ginseng. The roots of dodok are thick like bellflower, and when cutting plants, white juice comes out. Leaves are alternate and at the end of short branch, four leaves are facing each other and facing each other. They are long oval with length of 3-10cm and width of 1.5-4cm. Leaf edges are flat, green on the front, white on the back. In August-September, bell-shaped flowers often run from the end of the short branch to the bottom. Calyx is green, length 2-5cm, and butterfly 6-10cm. The corolla is 2.7 ~ 3.5cm long and the end is divided into 5 pieces and dried backward. The surface is light green and the inside has purple spots. The fruit ripens in September. Young leaves in spring, edible roots in autumn.
The inventors of the present invention have experimentally confirmed that the above extract of Dusty rosemary has antioxidant, anti-cancer, anti-obesity or immunity enhancing activity.
More specifically, according to one embodiment of the present invention, the antioxidative activity of Duckweed extract was examined, and the effect of DPPH, ABTS, and nitrite scavenging activity of Duckweed extract was investigated.
The present inventors conducted an MTT assay to measure the anticancer effect of Duckweed extract according to another embodiment. As a result of the test using the human cancer cell line, the duck dog extract of the present invention was found to have anticancer effect Respectively.
In addition, the present inventors tried to verify the effect of enhancing the immune function of Duckweed extract according to another embodiment. As a result of the test using T cells and B cells, which are human immune cells, Indicating the promotion.
Furthermore, the present inventors evaluated the anti-obesity effect of the extract of Dulcidae using 3T3-L1 cell. As a result, the cell survival rate was decreased depending on the treatment concentration of Dulcidae extract. Showed efficacy.
Therefore, the inventors of the present invention have found that a composition comprising the extract of the present invention as an active ingredient can be used as a composition for antioxidant, anti-cancer, anti-obesity, or immunity enhancement based on the experimental results obtained in the following examples.
Further, the present invention provides a health functional food having antioxidant, anti-cancer, anti-obesity or immunity-enhancing properties, comprising the extract of Rootgrass as an active ingredient.
In the present invention, the term 'immunological enhancement' or 'immunity enhancement' refers to a cell-growth promoting action of T cells and B cells, and may include immune cells other than the above-described immune cells.
In the present invention, 'anti-obesity' refers to the ability of cells to inhibit α-amylase, α-glucose, pancreatic lipase inhibition and adipocyte differentiation, and the concentration of the composition for anti-obesity may be 10 to 1000 μg / ml .
In the present invention, 'obesity' means that the amount of fat in the body is excessively increased. Obesity is caused by an imbalance in the synthesis and degradation of fat in adipocytes. Obesity, which is caused by excess fat accumulation, increases the risk of developing chronic degenerative diseases and increases the risk of coronary artery disease, hypertension, stroke, hyperlipemia and diabetes .
In the present invention, " prevention " means any disease or disease for which the term is applied by administration of the composition of the present invention, or any action that inhibits or delays the progress of one or more symptoms of the disease or condition.
In the present invention, " improvement " means any disease or disease to which the term applies as a result of administration of the composition of the present invention, or any action in which one or more symptoms of the disease or disease are alleviated or beneficially altered.
In the present invention, " treatment " means, unless otherwise indicated, reversing, alleviating, inhibiting or preventing the disease or disorder to which the term applies, or one or more symptoms of the disease or disorder And the term " treatment " as used herein refers to an act of treating when " treating " is defined as described above.
The term " administration " as used herein is meant to provide any desired composition of the invention to a subject in any suitable manner.
The composition of the present invention may be a food composition. In addition to containing the active ingredient doodak extract, such a food composition may contain various flavors or natural carbohydrates as an additional ingredient such as a conventional food composition.
Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. The above-described flavors can be advantageously used as natural flavorings (tau martin), stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.).
The food composition of the present invention can be formulated in the same manner as the above pharmaceutical composition and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, meat, chocolates, foods, confectionery, pizza, ram noodles, other noodles, gums, candy, ice cream, alcoholic beverages, vitamin complexes, .
In addition, the food composition may contain various additives such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and flavors such as natural flavors, coloring agents and aggravating agents (such as cheese and chocolate), pectic acid and its salts , Alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks and the like. In addition, the food composition of the present invention may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks.
Dodok extract, which is an effective ingredient of the present invention, is a natural substance and has little toxicity and side effects. Therefore, it can be safely used for long-term use for antioxidant, anti-cancer, anti-obesity or immunity enhancement.
The health functional food of the present invention can be manufactured and processed in the form of tablet, capsule, powder, granule, liquid, ring and the like for the purpose of antioxidation, anti-cancer, anti-obesity or immunity enhancement.
In the present invention, the term "health functional food" refers to a food prepared and processed by using raw materials or ingredients having useful functions in accordance with Law No. 6727 on Health Functional Foods, and the nutritional control on the structure and function of the human body Or for the purpose of obtaining a beneficial effect for health use such as physiological action.
The health functional foods of the present invention may contain conventional food additives and, unless otherwise specified, whether or not they are suitable as food additives are classified according to the General Rules for Food Additives approved by the Food and Drug Administration, Standards and standards.
Examples of the food items included in the above food additives include chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; Natural additives such as persimmon extract, licorice extract, crystalline cellulose, high color pigment and guar gum; L-glutamic acid sodium preparations, noodle-added alkalis, preservative preparations, tar coloring preparations and the like.
For example, the health functional food in the form of tablets may be prepared by granulating a mixture of the active ingredient of the present invention, Mallinberry extract, with an excipient, a binder, a disintegrant and other additives in a conventional manner, Alternatively, the mixture can be directly compression molded. In addition, the health functional food of the tablet form may contain a mating agent or the like if necessary.
The hard capsule of the capsule type health functional food can be prepared by filling a normal hard capsule with a mixture of an effective ingredient of the present invention dodok extract and an additive such as an excipient and the soft capsule may contain additives such as excipients And filling the mixture with a capsule base such as gelatin. The soft capsule may contain a plasticizer such as glycerin or sorbitol, a coloring agent, a preservative and the like, if necessary.
The health functional food of the ring form can be prepared by molding a mixture of the extract of Dodok, the active ingredient of the present invention, excipient, binder, disintegrant and the like by a conventionally known method, and if necessary, Or the surface may be coated with a material such as starch, talc.
The granular health functional food may be prepared by granulating a mixture of the active ingredient of the present invention, Mallinberry extract, excipient, binder, disintegrant and the like, into granules by a known method, and if necessary, ≪ / RTI >
The health functional food may be a beverage, a meat, a chocolate, a food, a confectionery, a pizza, a ramen, a noodle, a gum, a candy, an ice cream, an alcoholic beverage, a vitamin complex and a health supplement food.
Hereinafter, the present invention will be further described by way of examples. These examples are for further illustrating the present invention, and the scope of the present invention is not limited to these examples.
<Preparation Example 1> Preparation of Doduk
The material used in the present invention was purchased from a farmhouse and purchased from 6 farms (Jeju Island, Chunnam Province, Hwasun, Jeonbuk Province, Jecheon, Chungcheongbuk-do, Gyeongbuk, Uljin). The above materials were used in experiments for establishing optimum extraction conditions for developing a health functional food having blood pressure lowering effect using rosewood.
≪ Example 1 >
In order to elucidate the optimal extraction conditions according to the conditions of hot water extraction, two kinds of conditions were used to obtain a hot water extract. First, 10 times of distilled water was added to 50 g of freeze-dried dodok sample, and the whole was extracted three times at 100 ° C for 20 minutes to obtain a hot-water extract. In another condition, 50 g of freeze- And repeatedly extracted three times to obtain a hot water extract. Each of the hot-water extracts obtained above was cooled and filtered at room temperature, and the extract was concentrated using a rotary evaporator. As a result of measuring the yield by lyophilizing the concentrated extracts, the yields of 100 째 C extracts from whole roots were higher than those obtained from 70 째 C, and their antioxidant activities were also higher in 100 째 C hot water extracts (See Tables 1 to 3).
On the other hand, for the uniformity of raw materials collected from each region, freeze-dried dodok samples were sorted and removed by using a brush, washed with a brush, cut into a suitable size, and freeze-dried Dried and then powdered and used as an experimental sample.
Culture region
< Example 2> Room temperature extraction
For the extraction at room temperature, 50 g of freeze-dried Deodorant powder was extracted three times at a ratio of 30%, 50%, 70%, and 100% of ethanol (EtOH), and the extract was completely concentrated by using a reduced pressure concentrator, Recovery rate.
As a result, it was found that ethanol extracts of 50% ethanol showed higher recovery than those of ethanol extracts of 30% and 70%.
< Example 3> Reflux cooling extraction
In the reflux cooling extraction, 10 times of distilled water was added to 50 g of freeze-dried dodder sample in a flask equipped with a vertical reflux condenser, and then extracted three times for 12 hours at 100 ° C. Ethanol extraction was carried out in a vertical reflux condenser- Of 70% alcohol and then extracted three times at 70 ° C for 3 hours. The extract was cooled and filtered at room temperature, and the extract was concentrated by a rotary evaporator. The extract was lyophilized and then the weight was measured.
As a result, it was found that the water extraction reflux extraction showed higher recovery than the ethanol reflux cooling extraction, and that the antioxidant activity showed high efficacy in the ethanol extraction from all regions.
< Example 4> Enzymatic decomposition extraction
(W: w: w) of enzyme cellulase: pectinase: amyloglucosidase at a ratio of 3: 2: 1, 3: 2: 2, 3: 1: 2 (2% w / w) were added to each of these ratios, and the mixture was extracted at 45 ° C for 10 hours. Each of the enzyme extracts was cooled at room temperature, filtered, concentrated by rotary evaporator, and the yield was determined on the basis of 30 brix concentrate yield. As a result, relatively high yield was obtained at the ratio of 3: 2: 2 enzyme. However, in order to investigate the optimum ratio more closely, it is necessary to carry out a supplementary experiment by treating the enzyme mixture ratio more variously. Antioxidant activity also showed a generally high efficacy at the 3: 2: 2 ratio.
(W / W / W)
The control was value by hot-water (100 ° C) extraction method.
Culture region
(W / W / W)
(W / W / W)
< Example 4> By extraction solvent , By fraction Extract preparation
4-1. Extraction and solvent fraction
The cultivated doducks of each region were selected, washed, freeze dried, and then extracted three times with 70% alcohol at room temperature and concentrated under reduced pressure to obtain an extract. The concentrated extracts were lyophilized and then suspended in distilled water to obtain n-hexane, methylene chloride, ethyl acetate, butyl alcohol, and D.W. Followed by sequential extraction and fractionation (see Fig. 2).
4-2. Yield by solvent fraction
The lyophilized frozen dough was repeatedly extracted with 70% alcohol at room temperature three times, and then the extract was concentrated under reduced pressure to obtain lyophilized powder. 50 g of the lyophilized powder was fractionated by repeating three times for each solvent such as n-hexane, methylene chloride, ethyl acetate, butyl alcohol and DW (distilled water), and then the extract was completely concentrated with a vacuum concentrator and then weighed And the recovery of each fraction was investigated. Regardless of region, all samples showed the highest recoveries in water extraction and very low yields in the ethyl acetate layer (see Table 11).
4-3. By extraction solvent yield
10 g of lyophilized powder obtained by lyophilization was repeated three times for each solvent such as EtOH, MeOH, Ether, Acetone, and distilled water, and the extract was completely concentrated by using a reduced pressure concentrator, Recovery rate. As a result, water extraction showed the highest recovery rate in all six cultivars and yield was relatively low in ether and acetone layers.
< Experimental Example 1> By region Cultivated Antioxidant activity test according to extraction condition
Experiments were carried out to establish the criteria for the preparation of optimal extracts by verifying differences in the physiological functions of each of the extracts according to each extraction condition.
1-1. DPPH radical scavenging ability
Antioxidant activity of each extract was measured by hydrogen electron donating ability by the method of Choi et al. (2003). Dissolve samples of various concentrations in a methanol (or DMSO) solvent, mix 900 μL of DPPH solution (100 μM) with 100 μL of each sample and stir. This mixture was reacted in a dark place for 30 minutes and absorbance was measured at 517 nm. The hydrogen electron donating ability was evaluated by repeating each experiment three times and then calculating the degree of decrease in absorbance of the control by the following equation.
An = (A0-A) /
An: Antioxidant activity against DPPH radical scavenging activity (%)
A0: Absorbance of DPPH solution without added sample
A: The absorbance of DPPH in the reaction solution and the absorbance of the sample
Cuture region
The DPPH scavenging activity of the extracts at 1, 2.5, 5, 10, and 20 mg / mL and DPPH removal efficiencies by EtOH extract concentration were not significantly different between the two cultivars. However, , And the higher the concentration of EtOH, the higher the efficacy. Overall, the increase in DPPH scavenging activity showed a concentration-dependent tendency that was proportional to the concentration.
1-2. ABTS Cation ( ABTS +) Scatters
A solution of 7.4 mM ABTS (2,2'-azinbis- (3-ethyl-benzothiazoline-6-sulfonic acid) and 2.6 mM potassium persulphate was added and reacted in the dark for about 15 hours. The absorbance at 414 nm 1.5, 150 μl of each sample was added to each diluted solution, and the mixture was shaken for 10 seconds with a vortex mixer, allowed to stand at room temperature for 90 minutes, and the absorbance was measured at 414 nm. And the absorbance was measured by the same method. The cationic scavenging activity was expressed as AEAC (relative ascorbic acid equivalent antioxidant capacity), which was calculated as 1.000 when the scavenging activity of ascorbic acid was 1.000, Which was calculated by the following equation.
RAEAC = Caa x? As
Aaa Cs
ΔAaa: change in absorbance when ascorbic acid is added, Caa: concentration of ascorbic acid
ΔAs: Absorbance change when sample is loaded, Cs: Sample concentration
region
The ABTS cationic scavenging activity of each solvent was found to be proportional to the concentration of the extract, but there was a significant difference between the extraction solvents such as EtOH, MeOH, DW, Aceton and Ether. And showed relatively high scavenging activity at high concentration of EtOH.
1-3. nitrite Scatters
To measure the nitrite scavenging effect of the sample extract, a volume of 200 μl was adjusted to 20 μl of 1
N (%) = [1- (A-C) / B] 100
N: nitrite scavenging ability
A: absorbance of 1 mM NaNO2 added sample after standing for 1 hour
B: absorbance of 1NaNO2
C: absorbance of control
Most of the extracts were able to decompose more than 60% of nitrite when the pH of the reaction solution was 1.2. However, 100% of nitrite was decomposed by the EtOH extract concentration at pH 1.2, 4.2 and 6.0. The resolution of EtOH was 40% or less. At pH 4.2, the nitrite scavenging activity was almost 30% or less, but almost no activity was observed at pH 6.0. Therefore, the nitrite scavenging activity of extracts of Ettenucine by EtOH extract concentration was the highest at pH 1.2 and the activity gradually decreased or decreased with increasing pH.
< Experimental Example 2> By region Cultivated Antioxidant enzyme activity assay according to extraction conditions
2-1. Enzyme solution pharmacy
To 0.5 g of the sample was homogenized with 2 ml of Extract Buffer [100 mM K-PO4 buffer (pH 7.5), 100 mM EDTA, 1% PVP, 100 mM PMSF] and centrifuged at 15.000 g for 20 minutes. . For ascorbate peroxidase (APX), 10 mM was added to the above extraction solution in extraction buffer. Protein quantification was performed according to the Bradford (1976) method using BSA as a standard.
2-2. SOD ( SuperOxide Dismutase )
SOD enzyme activity assays were performed using an assay kit (Sigma, 19160). That is, the photochemical NBT method was used to test the ability of SOD enzyme to inhibit the reduction of NBT (Nitroblue tetrazolium). The reaction solution was adjusted to 50 mM carbonic buffer (pH 10.2), 0.1 mM EDTA, 0.1 mM Xanthine and 0.025 mM NBT, and the inhibition of NBT reduction was measured at 450 nm. Each sample was repeated three times, and SOD enzyme activity was converted by the following equation (see FIGS. 3 and 4).
SOD activity (NBT reduction inhibition rate,%) = [(Ablank1-Ablank3) - (Asample-Ablank2)] / (Ablank1-Ablank3)
As a result, the antioxidant enzyme activities of the extracts of Etodol and EtOH extracts were higher in the order of EA> BA> MC> nH> DW in the order of SOD activity, There was no significant difference between the two groups.
2-3. CAT ( Catalase )
CAT activity was measured by the method of Aebi et al. (1984). The reaction solution was 50 mM potassium phosphate buffer (pH 7.0) and 10 mM H 2
As a result, CAT activity showed the highest activity in EA fraction, and 50% and 100% of EtOH extract and Jeju cultivation showed relatively high activity.
2-4. APX ( Ascorbate Peroxidase )
The activity of APX was measured by the Nacano and Asada (1981) method. The degree of oxidation of ascorbate was measured at 290 nm for 2 minutes. The reaction solution of APX was 100 mM potassium phosphate buffer (pH 7.5) containing 0.5 mM ascorbate and 0.2 mM H 2 O 2 (see FIGS. 7 and 8).
As a result, APX activity was higher in order of EA> MC> nH> BA> DW, but BA and DW fractions showed relatively low activity. The APO activity of EtOH extract was relatively high at the 30% extract concentration and the Jeju cultivated doduck was higher in the cultivation area.
2-5. POD ( Peroxidase )
POD activity was measured by the method of Egley et al. (1983). The reaction solution was adjusted to a final concentration of 40 mM K-PO4 buffer (pH 6.9), 1.5 mM guaiacol, and 6.5 mM H2O2. The POD activity was measured by measuring the absorbance at 470 nm for 2 minutes using a spectrophotometer by mixing the sample with the reaction solution (see FIGS. 9 and 10).
As a result, POD activity showed relatively high activity in EA fraction and no significant difference in other fractions. Also, EtOH showed slightly higher activity at 50% and 100% extraction concentration.
<Experimental Example 3> Analysis of antitumor activity of doduk on human cancer cell lines according to extraction conditions of cultivated doduck
(MTT) (3- (4,5-dimethyl-thiazol-2-yl) -2,5-diphenyltetrazoliumbromide) assay was performed to determine the anticancer effect of 6 dermal cultures on human cancer cell lines. Human uterine cancer cell line HeLa, breast cancer cell MCF-7, lung cancer cell Calu-6, human-derived kidney normal cell HEK293 cell line were plated on a 96-well plate and cultured for 24 hours at 37 ° C in a 5% CO2 incubator. The cultured cells were treated with 10 μl of each sample to a concentration of 50, 100, 200, 400, 800 μg / ml, and cultured for 24 hours. 10 μl of 0.2 mg / ml MTT solution was added to each well, Lt; / RTI > After the incubation, the supernatant was removed and 100 μl of DMSO was added to each well. After incubation at room temperature for 10 minutes, the absorbance was measured at 540 nm using a microplate reader. The cytotoxicity was expressed as a percentage of the absorbance of the control .
The cytotoxicity of human dendritic cell extracts by MTT method showed that the cytotoxicity of human dendritic cells was lower than that of human cancer cells even at the concentration of 800 ug / mL. We need to verify it. In addition, the EtOH extracts showed good effects on both the Calu-6 and MCF-7 cell lines in Jeju, and the 70% EtOH extract showed relatively high toxicity.
< Experimental Example 4> By region Cultivated An anti-obesity effect of 3T3-L1 cell extract
3T3-L1 cells were cultured in KCLB No. 10092.1 from Korean Cell Line Bank. Cells were cultured in DMEM containing 10% FBS in a 5
4-1. Cytotoxicity measurement
MTT (methylthiazolyldiphenyltetrazolium bromide) assay for measuring cell viability was performed according to Sladowski et al. The cultured 3T3-L1 cells were cultured in a 96-well plate at 1105 cells / well and cultured for 16-18 hours. The diluted sample extracts were treated for 24 hours. MTT solution dissolved at 5mg / ml was added to 10uL / 990ul medium and incubated at 37 ° C for 3 hours in 5% CO2. After the culture was completed, the culture solution was removed, and the mixture was added to 200 μL / well of DMSO, stirred for 20 minutes, and absorbance was measured at 595 nm using an ELISA reader.
4-2. Oil red O dyeing
Cell culture medium was discarded and 50 μL of 10% formaldehyde was added to each well to fix the cells, followed by incubation at 4 ° C. for 1 hour. After that, the formaldehyde was discarded and washed three times with PBS. The oil red O staining solution (0.25 g of oil red O dissolved in 50 mL of isopropyl alcohol was mixed with distilled water at a ratio of 3: 2 and filtered with a 0.45 μm filter) Were added, and the cells were stained again at room temperature for 1 hour and then washed three times with PBS. The stained cells were observed under a microscope. After observation, the stained dye in the adipocyte was extracted with 500 μL of isoprophyl alcohol per well and the absorbance was measured at 520 nm by a spectrophotometer (see FIGS. 11 to 16).
The effect of 3T3-L1 cells on cytotoxicity was investigated. The cell survival rate was decreased depending on the treatment concentration of each sample, and the overall efficacy was relatively higher at the extract concentration of Jeju cultivated etude and EtOH.
<Experimental Example 5> Effect of extraction conditions on growth of immune cells
The immune function enhancing effect of extractive EtOH was verified by using T cell and B cell (RPMI 8226, KCLB No.10155) which are human immune cells. The cells were cultured in RPMI 1640 medium supplemented with 10% FBS at 5
The results of T cell and B cell growth stimulation of human immune cells, T cell and B cell, showed that the concentration of EtOH extract stimulated the growth in both T cell and B cell, I did not see it.
The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.
Claims (7)
Separating the extract of step 1 with hexane and separating into a hexane fraction and an aqueous layer (step 2);
Separating the aqueous layer obtained in the step 2 into methylene chloride and separating into a methylene chloride fraction and an aqueous layer (step 3); And
And separating the aqueous layer obtained in the step 3 into ethyl acetate and ethyl acetate fractions (step 4).
The above-mentioned doduck is a three-year-old root which was cultivated in Korea, Hwasun, Muju, Jecheon, Hoengseong or Uljin.
Wherein the health food is selected from the group consisting of beverage, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gums, candy, ice cream, alcoholic beverages, vitamin complexes and health supplement foods.
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