KR101889431B1 - Pharmaceutical composition for antioxidation comprising black burdock root extracts as an effective component - Google Patents

Abstract

The present invention relates to a method for producing health functional foods and cosmetic compositions containing an extract of black burdock as an active ingredient, which is prepared by heat-treating the ground portion of the lappa under high temperature for a long time and aging at a low temperature . The black burdock extract having excellent antioxidative effect of the present invention has a strong eradication ability against various active radicals as compared with fresh burdock, as demonstrated by the examples of the present specification. The active ingredient of the present invention can be processed into various forms such as extract, powder, ring, tablet and the like and can be applied to a pharmaceutical composition for antioxidation, a health functional food or a cosmetic composition, so that it is very useful in the pharmaceutical industry, food industry and cosmetics industry It is an invention.

Description

TECHNICAL FIELD The present invention relates to a pharmaceutical composition for antioxidant containing a black burdock root extract as an active ingredient,

The present invention relates to a method for producing health functional foods and cosmetic compositions containing an extract of black burdock as an active ingredient, which is prepared by heat-treating the ground portion of the lappa under high temperature for a long time and aging at a low temperature .

Oxidative stress can not be avoided by oxygen breathing, and this oxidative stress is caused by the accumulation of reactive oxygen species. That is, when the final electron acceptor reacts with oxygen to convert to water (H ₂ O) during the metabolism of the organic matter, cytotoxicity does not appear, but hydrogen peroxide (H ₂ O ₂ ), superoxide (O ₂ . , Hydroxy radicals (OH ·), etc., are generated, the activated oxygen species maintains a high radical concentration in the cell, causing damage to the protein or DNA, inducing various oxidative stresses, (Choi et al .. 2008. J. Korean Soc. Food Sci. Nutr. 37: 276-281). In addition, recent studies have shown that oxidative stress is directly related to cardiovascular disease-related thrombogenesis (Bijak M et al., 2012, Thrombosis Res. 130, 123-128), intravascular fibrinogen fibrinogen) is known to promote thrombus formation, thereby inhibiting blood flow and causing thrombosis (Martinez M et al., 2013. Free Radical Biol. Med. 65, 411-418). In fact, recent reports have shown that oxidative stress in vivo is associated with carcinogenesis, aging, dementia, inflammation, and various cardiovascular diseases (Zhang et al., 2013. Chines J. Cancer biotheraphy, 18: 7-12: Shin, Jeong-hee et al., 2012. Korea Academy of Science and Technology 13: 3561-3569). Therefore, it can be expected that the natural ingredients capable of removing various active radicals can be expected to provide considerable health benefits as the stability is secured.

To date, various substances capable of eliminating various active oxygen species and radical substances have been developed as antioxidants. Typically, vitamin C, vitamin E, butylated hydroxy toluene (BHT) and butylated hydroxy anisole (BHA) It is used as health functional foods and medicines. However, the side effects of BHT and BHA, which are chemical synthetic antioxidants, have been reported (Hyeonju et al., 2014. Korean J. Culinary Res. 20: 16-26; Choe and Yang, 1982. Korean J. Food Sci. Technol. 14: 283-288), it has become necessary to develop a powerful antioxidant capable of replacing chemical synthetic antioxidants. In particular, the development of a natural antioxidant having excellent heat resistance such as vitamin C, which is capable of replacing weak and unstable substances, (Ahn et al., 2009. Korean J. Microbiol. Biotechnol. 37: 266-272; Kim et al., 2009. Korean J. Microbiol. Biotechnol. 37: 133-139).

On the other hand, burdock (Arctium lappa ) is a biennial plant of the dicotyledonous plant, Lycopersicona, and is widely distributed in Siberia, Manchuria, and Korea. In Korea, it is cultivated mainly in Gyeongnam, and it is a root vegetable that boasts unique flavor and chewiness and boils young seeds and eats roots. It is usually not eaten raw, it is added to salt, it is added to vinegar, or it is consumed in soy sauce. In one room, the roots are referred to as the right roots, the fruits as the right roots, and the roots as the bad roots. The undergrowth of the burdock, which is mainly used for edible purposes, is viscous and has a bitter taste. It is mainly consumed in soy sauce. It is inedible by inulin, vitamin B1, acetic acid, arctiazenine, beta-carotene, butyric acid, coffee acid, chlorogenic acid, It is known to contain a large amount of minerals such as lixic acid, lauric acid, lignin, myristic acid, propionic acid, sitosterol, stigmasterol and copper, chromium, manganese, phosphorus, selenium, silicon and zinc.

To date, the major researches on burdock include cultivation, root dressing and cleaning (Jung, Jung-Woong, 2003. Korean Journal of Food Science and Technology, International Symposium Rice Fair, P-23, p145: P-100, p202) (Kim JH, et al., 2005, Kor J. Food Preserv. 12: 489-495), and urine kimchi (Choi, Mi Jung et al., 1999, J. Kor. Soc. Food Sci. Nutr. 27: 618-624) and (Kim, Mi-Kyung et al., 2010, Kor, J. Food Cookery Sci. 26: 575-585) have been reported to be limited.

Recently, the useful physiological activity of burdock has been actively studied, and the anti-inflammatory effect of burdock root extract (Kim, Yeonjin et al., 2012, Korea Resources Plant Biology, 25: 1-6) 2012) and cancer cell killing activity (Susanti et al., 2012, J. Nat. Med. 66: 614-621), the antioxidative effect of caffeoylquinic acids in burdock (Liu et al. Agric. Food Chem. 60: 4067-4075), stress relieving effect of burdock arctiin on vesicle stress (Gu et al., 2012, Acta Pharmacol. Sin. 33: 941-952), enhancing sexual performance and eliminating infertility And the prevention of gastric ulcers (de Almeida et al., 2012. J. Med. Food. 15: 378-383) have recently been reported. . In addition, in Korea, anti-inflammatory activity of burdock seeds (Park, Soo Young et al., 2005, Nat. Product Sci. 11: 85-88), rod heat oxidation and auto oxidation prevention effect of ethyl acetate and hexene extract of burdock (Kim, Antioxidative and antimutagenic effects of burdock ethanol extracts on the antioxidative effects of burdock sprout vegetables (Lee, Moo-yeol et al., 2009, Korea Resources and Bioscience Biotechnol., 22: 1304-311) Korean Journal of Food Science and Nutrition, 24: 713-719). According to a recent report by Kim Hyunsoo et al., The antioxidative activities (DPPH anion scavenging activity) of methanol extracts of burdock, burdock and burdock roots were 80.5%, 26.8% and 19.3%, respectively. The roots of burdock were burdock root, (Kim, Hyun-Soo et al., 2014. Korean Journal of Food Storage and Distribution, 21: 593-599)

On the other hand, patent documents related to burdock include cosmetics containing natural products effective for the treatment and prevention of acne (Korea Patent No. 10-0357824, registered Oct. 10.9, 2002), cosmetic compositions for reducing pores using burdock extract fermentation broth (Korean Registered Patent No. 10-0874113, registered on Dec. 9, 2008), a cosmetic composition containing burdock extract and cold sensation (Korean Registered Patent No. 10-0767974, Registration Date Oct. 11, 2007), a cosmetic composition containing a mixed extract of a natural plant having anti-inflammation and skin irritation alleviating effect 10-1000695, Registered on Dec. 6, 2010), a shampoo composition having an anti-hair-loss function, which comprises Angelica edulis and Burdock extract as main components (Korean Patent No. 10-0609210, ), A cosmetic composition for removing blackhead (Korean Patent Registration No. 10-0962344, registered on June 1, 2010), herbal medicine extracts, especially oriental herbal cosmetic compositions comprising at least a burdock extract as a main ingredient (Korean Patent No. 10-0905386 (Patent Registration No. 10-0960305, registered on May 20, 2010), and a composition for the removal of nicotine (Korean Patent Registration No. 10-0960305, filed on May 20, 2010) A composition for hemostasis comprising burdock roots as an active ingredient (Patent No. 10-0628429) is known. Open Patent No. 10-2013-0013950 discloses a composition for a detergent for antibacterial activity using a fermented burdock extract obtained by fermenting a hot water extract of burdock with yeast, Bacillus subtilis, and lactic acid bacteria in a detergent composition containing burdock fermented extract Is disclosed. However, there is no known antioxidant composition containing black burdock extract as an active ingredient, which is prepared by long-time heat treatment in a sealed container at the bottom of burdock root and aging at low temperature.

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DISCLOSURE Technical Problem Accordingly, the present invention has been made in order to solve the problems of the prior art as described above, and it is an object of the present invention to provide a pharmaceutical composition for antioxidation, a health functional food and a cosmetic composition containing black burdock extract as an active ingredient .

In order to solve the above-mentioned problems, the present invention provides a pharmaceutical composition for antioxidant containing a black burdock extract as an active ingredient.

Preferably, the black burdock root extract is a black burdock hydrothermal extract or an ethanol extract.

Preferably, the black bean curd hydrothermal extract or the ethanol extract is an ethylacetate fraction of black shallower heat or an ethanol extract.

The present invention also provides an antioxidant health food containing the above-mentioned active ingredient of the present invention.

The present invention also provides a cosmetic composition for antioxidation comprising the above-mentioned active ingredient of the present invention.

The black burdock extract having excellent antioxidative effect of the present invention has a strong eradication ability against various active radicals as compared with fresh burdock, as demonstrated by the examples of the present specification. The active ingredient of the present invention can be processed into various forms such as extract, powder, ring, tablet and the like and can be applied to a pharmaceutical composition for antioxidation, a health functional food or a cosmetic composition, so that it is very useful in the pharmaceutical industry, food industry and cosmetics industry It is an invention.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a photographic view of a burdock root (right skeletal muscle) used in the present invention. FIG.
FIG. 2 is a photograph of a black burdock produced by heat-treating burial undergrowth at a high temperature for a long time.

Hereinafter, the present invention will be described in detail.

In order to test the antioxidant activity of burdock, the inventors of the present invention prepared black burdock from burdock by a certain method, prepared ethanol and hot water extract of burdock and black burdock, , And the excellent antioxidative activity of ethanol and hot water extract of black burdock was confirmed. Then, the sequential organic solvent fractions of the black and white burdock ethanol extract and the hot water extract were prepared to evaluate the antioxidant activity. As a result, the ethyl acetate fraction was recovered as an active ingredient. The active extract showed no hemolytic activity on human erythrocytes , Heat stability and acid stability. Thus, the black burdock extract was used as an antioxidant composition.

In order to develop antioxidant compositions using burdock, which is known to have various physiological activities in the private sector, the present inventors prepared dark brown burdocks by heat-treating burdock at a high temperature for a long time at a high temperature under airtight conditions, Respectively. As a result, the antioxidant activity was significantly increased in ethanol and hot water extract of black burdock root and ethyl acetate fraction thereof, compared with burdock, And no hemolytic activity was observed.

Hereinafter, the method for producing the black burdock root extract of the present invention and the efficacy experiments will be described in more detail.

The present invention relates to a process for producing black burdock; Preparing an extract from burdock and black burdock; Preparing a sequential organic solvent fraction of the extract; The step of evaluating the antioxidative activity of the extract and the fractions and the step of examining the stability of the active substance of the black burdock extract.

The "black burdock extract" contained in the composition of the present invention is a step of collecting and washing the bottom part of mature burdock at the beginning of September to October and washing it in a closed container at 70 to 80 ° C. for 10 days or more, And the mixture is aged at a temperature of 1 to 10 ° C, preferably 4 ° C for 1 to 2 days to prepare black burdock, which is extracted with a solvent, and the extract is filtered using a filter net of 0.06 mm or less, And then concentrating it under reduced pressure.

The solvent to be used in the present invention may be selected from the group consisting of water (cold water, hot water), alcohol, anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, alcohol, propanol, butanol), a mixed solvent of the lower alcohol and water Most preferred is hot water, or 95% ethanol extraction.

In addition, the extract of the present invention may be fractionated by an additional organic solvent to obtain each organic solvent fraction. For example, the organic solvent may be hexene, ethyl acetate, butanol, and the like.

In the present invention, various radical scavenging ability and reducing power were evaluated at a concentration of 0.5 mg / ml after preparing ethanol extract and hot water extract of burdock and black burdock, and the ethanol extract of black burdock showed 1.9 ~ 3.6 times stronger antioxidative power than the ethanol extract of burdock And DPPH anion scavenging activity and nitrite scavenging activity were increased by 2.66 and 1.56 times, respectively, in the hot - water extract of black burdock. Particularly, the ethyl acetate fraction of the sequential organic solvent fractions of black burdock root extract showed DPPH anion scavenging ability of 83-85%, ABTS cation scavenging activity of 88% or more, nitrite scavenging ability of 75-79%, reducing power of 0.845 ~ 0.994 Respectively. The ethyl acetate fraction of black burdock showed no hemolytic activity against human erythrocytes up to a concentration of 1 mg / ml.

The black burdock extract of the present invention may be made into powders through conventional powdering processes such as vacuum drying and freeze drying, spray drying and the like. They are not degraded by various degradation enzymes in the plasma, and maintain their activity even at 100 ° C heat treatment and pH 2 in human body.

The black burdock extract of the present invention exhibits a strong antioxidative activity and can be used as a material for pharmaceutical compositions, health functional foods and cosmetic compositions for preventing or treating various diseases related to oxidative stress.

Accordingly, the present invention provides a pharmaceutical composition for antioxidant containing a black burdock extract as an active ingredient. The black burdock extract is preferably a black or white burdock hydrothermal extract or an ethanol extract. The black burdock hydrothermal extract or ethanol extract is preferably an ethylacetate fraction of a black shallower heat or an ethanol extract.

The above-mentioned "antioxidant pharmaceutical composition" may be used for the prevention or treatment of various diseases related to oxidative stress, and the diseases include, for example, aging, cancer, stroke, Parkinson's disease, heart disease, ischemia, Spots, freckles, skin diseases, gastrointestinal diseases, inflammation, rheumatism, autoimmune diseases, and the like.

The pharmaceutical composition of the present invention may be formulated into various forms such as oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, injections of sterilized injection solutions, And can be administered by various routes including oral administration or intravenous, intraperitoneal, subcutaneous, rectal, topical administration, and the like.

Such pharmaceutical compositions may further comprise carriers, excipients or diluents, and examples of suitable carriers, excipients or diluents that may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, But are not limited to, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, amorphous cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, And the like. In addition, the pharmaceutical composition of the present invention may further include a filler, an anti-coagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent, an antiseptic, and the like.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level will depend on the type of disease, severity, The sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts.

The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiply. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by those skilled in the art.

In a preferred embodiment, the effective amount of the extract of black burdock root extract or ethyl acetate fraction having excellent antioxidative activity in the pharmaceutical composition of the present invention may vary depending on the age, sex and body weight of the patient, and is generally 1 to 5,000 mg per body weight, preferably 100 to 3,000 mg may be administered daily or every other day or one to three times a day. However, the dosage may not be limited in any way because it may be increased or decreased depending on route of administration, severity of disease, sex, weight, age, and the like. The pharmaceutical composition of the present invention can be administered to a subject through various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

In the present invention, "administration" means providing a predetermined substance to a patient by any suitable method, and the administration route of the pharmaceutical composition of the present invention is either oral or non-oral May be administered orally. The composition of the present invention may also be administered using any device capable of delivering an effective ingredient to a target cell. In the present invention, the term "object" includes, but is not limited to, human, monkey, cow, horse, sheep, pig, chicken, turkey, quail, cat, dog, mouse, rat, rabbit or guinea pig , Preferably a mammal, more preferably a human.

In addition to the above medicinal uses, the present invention provides an antioxidant health functional food containing the active ingredient of the pharmaceutical composition of the present invention.

The health functional food of the present invention can be variously used for foods and beverages effective for the improvement of various diseases described above related to oxidative stress. Examples of the foods containing the black rye extract or ethyl acetate fraction having excellent antioxidant activity of the present invention include various foods, beverages, gums, tea, vitamin complexes, health supplement foods, etc., Capsule or beverage. The active fraction of the present invention may generally be added in an amount of 0.01 to 15% by weight of the total food, and the health beverage composition may be added in a proportion of 0.02 to 10 g, preferably 0.3 to 1 g, based on 100 ml.

The health functional food of the present invention may contain, as an additional ingredient, a food-acceptable food-aid additive such as natural carbohydrates and various flavors, in addition to containing the above-mentioned compound as an essential ingredient in the indicated ratio.

Examples of the natural carbohydrate include sugar sugars such as glucose, monosaccharides such as fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol.

Examples of the flavor agent include tau martin, rebaudioside A, glycyrrhizin, saccharin, and aspartame. The proportion of the above-mentioned flavoring agent is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the health functional food of the present invention.

In addition to the above, the health functional food of the present invention may contain various kinds of nutrients, vitamins, minerals, flavors such as synthetic flavors and natural flavors, colorants and heavy stabilizers, pectic acid and its salts, alginic acid and its salts, Thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like.

In addition, the health functional food of the present invention may contain flesh for producing natural fruit juice, fruit juice drink, vegetable drink and the like. These components may be used independently or in combination. The ratio of such additives is generally selected in the range of 0.01 to about 20 parts by weight per 100 parts by weight of the active fraction of the present invention.

In addition to the pharmaceutical use and the functional food use of the active ingredient of the present invention, the present invention provides an antioxidative cosmetic composition comprising the active ingredient of the pharmaceutical composition of the present invention.

The above-mentioned effect of "antioxidation" may mean, for example, skin aging improvement, skin wrinkle improvement, skin elasticity improvement, skin regeneration effect and the like.

The cosmetic composition of the present invention can be used as a cosmetic composition in the form of a black rye extract or ethyl acetate fraction having excellent antioxidant activity of the present invention in the form of an essence, a nutritional cream, a body lotion, a rinse, a shampoo, a body cleanser, a lotion, But is not limited thereto. The preparation in this form may be easily carried out according to various production processes known to those skilled in the art.

In a preferred embodiment, the active ingredient of the cosmetic composition of the present invention may be contained in an amount of 0.01 to 50% by weight, preferably 0.1 to 20% by weight, more preferably 1 to 10% by weight based on the total weight of the composition. Proper formulation stability can be ensured within such a content range, and a desired antioxidative effect can be expected.

The composition of the present invention can be used in addition to the active ingredient of the present invention in addition to known natural extracts or other ingredients for moisturizing and promoting skin regeneration within the range not inhibiting the active activity of the present invention.

In addition, the composition may also contain components commonly added to cosmetic compositions, such as fatty substances, organic solvents, solubilizers, thickeners, gelling agents, softeners, antioxidants, suspending agents, stabilizers, foaming agents, A cosmetic or dermatological composition such as a perfume, a perfume, a surfactant, water, an ionic or nonionic type emulsifier, a filler, a sequestering and chelating agent, a preservative, a vitamin, a barrier, a wetting agent, an essential oil, a dye, a pigment, a hydrophilic or lipophilic active agent May be formulated into a particular formulation, including adjuvants or carriers commonly used in the field of dermatology.

The cosmetic composition of the present invention may be prepared in any form conventionally produced in the art and may be formulated into various forms such as solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, Containing cleansing, oil and spray, but are not limited thereto. More specifically, the present invention relates to a lotion, a lotion, a flexible lotion, a nutritional lotion, a cream, a nutritional cream, a massage cream, an essence, an eye cream, a powder foundation, an emulsion foundation, a wax foundation, a cleansing cream, a foam, a cleansing foam, , Spray, powder, fact, lip gloss, lipstick, shadow, shampoo and rinse.

When the formulation of the present invention is a paste, cream or gel, an animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as the carrier component .

When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as the carrier component. In particular, in the case of a spray, a mixture of chlorofluorohydrocarbons, Propane / butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, a solubilizing agent or an emulsifying agent is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid esters of sorbitan.

In the case where the formulation of the present invention is a suspension, a carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Cellulose, aluminum metahydroxide, bentonite, agar or tracant, etc. may be used.

When the formulation of the present invention is an interfacial active agent-containing cleansing, the carrier component may include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters.

Hereinafter, the specific method of the present invention will be described in more detail by way of examples. The following examples are only a preferred embodiment of the present invention, and the scope of the present invention is not limited to the scope of the following examples.

[Example]

Example  1: High temperature treatment of burdock Black burdock  pharmacy

In the case of edible natural products, which are usually poor in functionality or have side effects, it is known that when heated and boiled at high temperature, the physiologically active ingredient increases with the increase of the sensory properties, and the side effect decreases. This is related to the increase of the flavor due to the browning reaction product at high temperature, the increase of the physiologically active ingredient and the generation of new substance. Representative examples include red ginseng, black garlic, and blackberry production. Actually, red ginseng, black garlic, and black bellflower have excellent antioxidant activity, brain function enhancement and immunity enhancing activity compared to the original ginseng, garlic, and bellflower. In the present embodiment, in order to prepare black burdock root by heating the burdock at a high temperature, the buried burdock is heated in a sealed container at 70 to 80 ° C for 10 days or more and then aged at 4 ° C for 1 to 2 days at low temperature To produce a darkened burdock. As the most preferable treatment, black burdock having the highest sensibility was produced when it was put in a stainless steel sealed container at 75 캜 for 11 days and then aged at 4 캜 for 1 day at low temperature. 1 and 2, the water content of the burdock was 80%, while that of the black burdock was about 70% in the case of the heat treatment under the closed condition, Partial water loss was observed during low temperature aging.

Example  2: Burdock, Black burdock  Preparation of extracts and component analysis of extracts

Commercially available burdocks were purchased, washed with flowing water to remove foreign matter, and then used for the production of ethanol extract and hot water extract. To prepare the ethanol extract, 10 times of ethanol was added to the burdock samples, and the extracts were collected twice at room temperature. The extracts were collected and filtered, and concentrated under reduced pressure to give powder. In the case of the hydrothermal extract, 10 times of distilled water was added to the burdock samples, and the mixture was heated at 100 ° C. for 1 hour and then cooled. The resultant mixture was cooled once, and the above procedure was repeated once. The combined extracts were filtered and concentrated under reduced pressure to give powder Respectively. On the other hand, commercially available burdock was prepared in the same manner as in Example 1, and black burdock was prepared. Ethanol and hot water extracts were prepared in the same manner as above. Total polyphenols, total flavonoids, total sugars and reducing sugar content were determined by analysis of components of the prepared burdock and black burdock extracts. Total polyphenol content was determined by adding 50 μl of Folin-ciocalteau and 100 μl of saturated Na 2 CO 3 solution to 400 μl of the extract solution, leaving it at room temperature for 1 hour and measuring the absorbance at 725 nm. Tannic acid was used as a standard reagent. The total flavonoid content of each sample was measured by stirring for 18 hours in methanol. To the 400 μl of the filtered extract, 4 ml of 90% diethylene glycol was added and 40 μl of 1N NaOH was added. The reaction was carried out at 37 ° C. for 1 hour and then absorbance was measured at 420 nm. As a standard reagent, rutin was used. Reducing sugar was quantified by DNS method and total sugar was quantified by phenol-sulfuric acid method.

As a result, as shown in Table 1, the extraction efficiency of black burdock increased sharply compared to burdock, and the ethanol extraction efficiency was increased by 1.8 times or more and the hot water extraction efficiency was increased by 2.54 times or more. Total polyphenol content was also increased in black burdock compared to burdock, 5.64 times in ethanol extract and 1.46 times in hot water extract. However, the total flavonoid content was increased 3.8 fold only in the ethanol extract of black burdock. However, the total sugar content of black burdock was reduced by 0.51 times compared with that of burdock, while that of hot water extract was 0.76 times higher than that of burdock. Reduced sugar content of black burdock was 2.35 times higher than that of burdock, while that of hot - water extract was 5.45 times higher than that of burdock. These changes mean that the long - term heat treatment of burdock increased the amount of useful components such as polyphenol component, and decreased the total sugar content but increased the sugar content and increased the sugar content.

Example  3: burdock and Black burdock  Evaluation of Antioxidative Activity of Extracts

The antioxidant activity of the ethanol and hot water extracts of burdock and black burdock prepared in Example 2 was evaluated. The extract was dissolved in dimethylsulfoxide (DMSO), diluted to an appropriate concentration, and DPPH (1,1-diphenyl-2-picryl hydrazyl) anion Radical scavenging ability, ABTS [2,2-azobis (3-ethylbenzo thiazoline-6-sulfonate)] cation radical scavenging ability, nitrite scavenging ability and reducing power. For DPPH removal, 380 μl of 2 × 10 ^-4 M DPPH solution in 99.5% ethanol was added to 20 μl of diluted samples at various concentrations, and the mixture was reacted at 37 ° C. for 30 minutes. Then, microplate reader (Asys Hitech, Expert 96, Asys Co., Austria). DPPH radical scavenging activity was expressed as a percentage of sample addition and non-addition. For the ABTS scavenging ability, 5 ml of 7 mM ABTS (Sigma Co., USA) and 88 ml of 140 mM potassium persulfate were mixed, and light was blocked for 16 hours at room temperature to form ABTS cations. The solution was then diluted with ethanol Lt; / RTI > After mixing 190 ml of the prepared diluted solution and 10 ml of the samples prepared at various concentrations, the reaction was allowed to proceed at room temperature for 6 minutes. Absorbance was measured at 734 nm and the ABTS radical scavenging activity was calculated by the following equation.

ABTS radical scavenging ability (%) = [(C-S) / C] x 100,

C: absorbance at the time of addition of solvent control DMSO,

S: Absorbance at the time of sample addition.

For the measurement of nitrite scavenging ability, a sample solution was added to a nitrite solution (1 mM), and 0.1 N HCl was added thereto to adjust the pH to 1.2. Thereafter, the mixture was incubated at 37 ° C for 1 hour and then reacted with Griess reagent (Sigma Co., USA) Were added and mixed. After incubation for 15 minutes at room temperature, absorbance was measured at 520 nm and the amount of residual nitrite was measured. The nitrite scavenging activity (%) was calculated by the following equation.

NSA (%) = [1- (A-C) / B] x100,

A: Absorbance after 1 hour reaction with 1 mM nitrite solution,

B: absorbance of 1 mM nitrite solution,

C: Black burdock  Absorbance of extract samples.

In the above antioxidant test, vitamin C (Sigma Co., USA) was used as a control and DMSO was used as a solvent control. Each activity evaluation was expressed as the mean and deviation of each experiment repeated three times. Finally, each antioxidant ability was expressed as the concentration of the extract (RC ₅₀ ) required for 50% radical scavenging ability.

For the evaluation of the reducing power of black burdock extract, the method of Oyaizu et al. Was modified (Anseonmi et al., 2011. J. Life Sci. 21: 576-583). 2.5 ml of 0.2 M sodium phosphate buffer (pH 6.6) and 2.5 ml of 10% potassium ferricyanide were added to 2.5 ml of the sample dissolved in ethanol. The mixture was reacted at 50 ° C for 20 minutes and then 2.5 ml of 10% trichloroacetic acid was added to terminate the reaction And centrifuged at 4,000 rpm for 10 minutes to recover the supernatant. The recovered supernatant was diluted 2-fold with distilled water, mixed with freshly prepared 0.1% ferric chloride solution at a ratio of 5: 1 (v / v), and absorbance was measured at 700 nm. The results of the respective antioxidant power measurement and the concentration (RC ₅₀ ) of the extract required for 50% radical scavenging ability are shown in Tables 2 and 3.

As shown in Table 2, the ethanol and hot water extracts of burdock showed weak antioxidative activities as a whole, but the hot water extract (0.5 mg / ml) of burdock showed 66% of ABTS cationic scavenging ability and 0.358 of reducing power. On the other hand, the hydrothermal and ethanol extracts of black burdock showed excellent antioxidative activity. In particular, ethanol extract of black burdock showed 1.9 ~ 3.6 times more antioxidative activity than burdock. In the hydrothermal extract of black burdock, the DPPH anion scavenging activity increased 2.6 times and nitrite scavenging activity 1.56 times compared with the hot water extract of burdock. Therefore, it was found that black burdock prepared by heating and cooking burdock increased total polyphenol content, flavonoid content and antioxidant ability by several times.

In order to compare the actual antioxidant capacity, we calculated the concentration (RC ₅₀ ) required to eliminate 50% of the active radicals of burdock and black burdock extract. As a result, the black burdock showed a low RC ₅₀ (Table 3). However, black burdock extract showed low antioxidant ability compared to vitamin C.

Example  4: Black burdock  Ethanol extract and Heat number  Extract Organic solvent  Sequential Fraction  Preparation and analysis of their components

The ethanol extract and hot water extract of black burdock prepared in Example 2 were fractionated with hexane, ethyl acetate and butanol sequentially, and finally the water residue was recovered. Table 4 shows the extraction efficiency, the organic solvent fraction efficiency, and the component analysis results of the extract / fraction.

As a result, as shown in Table 4, the fraction of the ethanol extract of black burdock had a large amount of 72.16% of the butanol fraction and 25.18% of the water residue, while the hexane fraction and the ethyl acetate fraction were 2.16% and 3.92% It took up a small amount. On the other hand, the fraction of water extract of black burdock showed 98.24% of water residue, 0.05% of hexene fraction and 3.43% of ethyl acetate fraction. This means that 0.77 g of the ethyl acetate fraction of the ethanol extract and 0.084 g of the ethyl acetate fraction of the hot water extract can be recovered from the black burdock root (average moisture content 65%). Considering the fact that the ethyl acetate fraction of the ethanol extract of green burdock can recover 0.046 g per 100 g of burdock, the present inventors have reported (Korean Patent No. KR 10-1602190) that ethanol extract , And more than 1.8 times in the case of the hot-water extract, can be recovered.

Total polyphenol content and total flavonoid content were 26.5 ~ 28.2mg / g in hot water extract and ethanol extract of black burdock, and total flavonoid content was 3.5 ~ 3.8mg / g Respectively. Among the sequential organic solvent fractions, total polyphenol and total flavonoid contents were 267.5 ~ 252.0 mg / g and 28.5 ~ 18.6 mg / g in the hot water extract of black burdock and ethylacetate fraction of ethanol extract, respectively. The total polyphenol content and total flavonoid content were 1.28 times and 0.42 times, respectively, as compared with the ethyl acetate fraction of the raw burdock hydrothermal extract. The total polyphenol content and the total polyphenol content were 5.63 times and 0.4 times Total flavonoid content. That is, the total polyphenol content was increased and the total flavonoid content was decreased in the ethyl acetate fraction of black burdock. Total sugar and reducing sugar contents were higher in the butanol fraction and water residue of the extract.

Example  5: Black burdock  Ethanol extract and Heat number  Sequence of extract Organic solvent Fraction Evaluation of antioxidative activity of water

Antioxidant activity was measured in the same manner as in Example 3, using the fractions of the black burdock extract extract of Example 4, in the same manner as in the measurement of the active radical scavenging ability and reducing power. The results are shown in Table 5 and Table 6, and a very strong antioxidative ability was confirmed in the ethyl acetate fraction of the fractions. In the active radical scavenging ability, both the ethanol extract of black burdock and the ethyl acetate fraction (0.5 mg / ml) of hot water extract showed almost the same 85% DPPH scavenging ability, 88% ABTS scavenging ability and 75% or more nitrite scavenging ability, The ethyl acetate fraction of the ethanol extract showed 0.845, while the ethyl acetate fraction of the hot-water extract had 0.994, indicating that the ethyl acetate fraction of the hot-water extract had a stronger reducing power (Table 5).

On the other hand, as shown in Table 6, the concentrations required for 50% scavenging the active radicals of the ethyl acetate fraction of the black burdock ethanol extract and the hot-water extract were calculated. As a result, DPPH scavenging ability was 90-112 μg / ml, 54 μg / ml, and nitrite scavenging activity was 43 ~ 55 μg / ml. In addition, antioxidative active substances derived from black burdock produced by heat treatment at high temperature for a long time are resistant to heat, unlike vitamin C, and can be developed as foods for prevention and treatment of antioxidant stress related diseases, health functional foods, pharmaceuticals and cosmetic materials Respectively.

Example  6: Black burdock  Extract and its Fraction  Evaluation of hemolytic activity of human erythrocytes

The burdock root has been used as a food, and it is an edible plant registered as a food ingredient and secured. In addition, even in the case of black burdock, since there are no additives or harmful substances, safety is secured. However, in order to evaluate the potential acute toxicity during the conversion of burdock to black burdock, human erythrocyte hemolytic activity of black burdock extract and its fractions was evaluated, and the results are shown in Table 7. The hemolytic activity was assessed in accordance with the existing reports (Jung In-chang, Son Ho Yong, 2014, Korean J. Microbiol. Biotechnol. 42: 285 ~ 292). In brief, 100 μl of human red blood cells, , 100 μl of various concentrations of sample solution was added and the reaction was allowed to proceed at 37 ° C for 30 minutes. Then, the reaction solution was centrifuged for 10 minutes at 1,500 rpm, and 100 μl of the supernatant was transferred to a new microtiter plate. The hemoglobin efflux 414 nm. Triton X-100 (1 mg / ml) was used as an experimental control for erythrocyte hemolysis. DMSO (2%) was used as a solvent control of the sample. Hemolytic activity was calculated using the following formula.

(%) Hemolysis = [(Abs.S-Abs.C) / (Abs.T-Abs.C)] 100

Abs. S: absorbance of sample added sphere,

Abs . C: DMSO Of furniture  Absorbance,

Abs . T: Triton X-100 Additive  Absorbance.

First, DMSO and distilled water used as control did not have erythrocyte hemolytic activity, and Triton X-100 showed 100% hemolysis of red blood cells at a concentration of 1 mg / ml. On the other hand, there was no hemolytic activity in the hydrolyzate of black burdock hydrothermal extract and ethanol extract, and the hemolytic activity was exclusively shown in the hexane fraction of the black oats' ethanol extract. On the other hand, the ethylacetate fractions of black rumberry extract, which had excellent antioxidant activity, showed no erythrocyte hemolysis up to a concentration of 1 mg / ml and thus did not show acute toxicity and erythrocyte hemolytic activity. These results suggest that ethyl acetate fraction of black burdock extract can be effectively used as an antioxidant and it can be used for various foods, medicines and cosmetics.

Example  7: Black burdock  The ethyl acetate extract Fraction  Plasma, acid and thermal stability assessment

The plasma stability, thermal stability and acid stability of the ethyl acetate fraction of the black ocher extract obtained in Example 4 were confirmed. The ethyl acetate active fraction remained in good activity without heat treatment at 100 ° C for 1 hour, 1 hour treatment in pH 2 (0.01M HCl), and 1 hour treatment in plasma, without scavenging DPPH and ABTS scavenging ability. Therefore, considering the component analysis results of the fractions of Example 4, the organic solvent fraction characteristics and the above stability results, the active substance of the black burdock ethyl acetate fraction is the phenolic compound produced in the process of high temperature heating in the closed container of burdock for a long period of time Of glycosides.

Claims (5)

A method for increasing the yield of a black ocher ethyl acetate fraction having antioxidative activity, comprising the steps of:
(1) placing the burdock root in a closed container and heating at 70 to 80 ° C for 10 to 20 days;
(2) aging the result of step (1) at 1 to 10 ° C for 1 to 2 days;
(3) extracting the result of step (2) with hot water or ethanol; And
(4) fractionating the product of step (3) with ethyl acetate. The method according to claim 1,
Wherein the step (3) is carried out by fractionating the resultant product with hexene to remove the hexane fraction, and then carrying out the step (4). delete delete delete

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