KR20110108686A

KR20110108686A - Antioxidant containing extracts of natural materials and composition of feed, food or cosmetic

Info

Publication number: KR20110108686A
Application number: KR1020100028035A
Authority: KR
Inventors: 김동명
Original assignee: 주식회사 케이씨아이
Priority date: 2010-03-29
Filing date: 2010-03-29
Publication date: 2011-10-06

Abstract

The antioxidant according to the present invention consists of processed extracts obtained by bioconverting the extracts extracted from natural raw materials into enzyme-treated or enzyme-treated extracts into lactic acid bacteria or specific microorganisms, which are subjected to antioxidant activity, anti-aging activity, and oxidative stress. Has a regenerative effect on cells. In addition, it is extracted from natural raw materials and is safe for human skin and has no acute oral toxicity. In addition, antioxidants, which are decolorized and deodorized by enzyme treatment and processed extracts which are bioconverted by a specific lactic acid bacterium or a specific microorganism as an active ingredient, can be easily prepared in solid powder form, liquid form, or emulsion form during the manufacturing process. Do. In addition, the complex natural antioxidant according to the present invention is excellent in compatibility with other ingredients, and is very stable in formulation when preparing a cosmetic composition, a food composition, or a feed composition.

Description

Antioxidant containing natural raw material extract and feed, food, or cosmetic composition comprising same {Antioxidant containing extracts of natural materials and composition of feed, food or cosmetic}

The present invention relates to an antioxidant and its use, and more particularly, to an antioxidant including a processed extract obtained by fermentation of an enzyme-treated or an enzyme-treated extract extracted from a natural raw material selected by bioscreening and feed thereof It relates to the use as an additive, a food additive, or a cosmetic additive.

It is a known fact that our human bodies are in close contact with the external environment and are easily sensitized due to exposure to ultraviolet rays, environmental pollution, chemicals, detergents and pollen. Skin exposed by such an external environment is easily exposed to reactive oxygen species, and the generated reactive oxygen species attack normal cellular tissues or cell membranes, and the skin eventually causes inflammation. Inflammated tissues cause skin aging such as wrinkles and the like by breaking down proteins and genetic material on the skin.

In general, there are many theories explaining skin aging, but among them, the free radical theory that the skin ages due to free radicals caused by various factors in and out of the body is accepted as the most valid theory. Free radicals are chemically highly reactive chemicals that occur when the skin is irradiated with UV light or during the respiration of cells in the skin. Most of the free radicals associated with aging of the skin are Reactive Oxygen Species (ROS), which include superoxide radicals, hydrogen peroxide (H ₂ O ₂ ), and hydroxy radicals (Hydroxy Radical). , Singlet oxygen, and the like.

These reactive oxygen species are generated during ultraviolet irradiation or respiratory processes to cause peroxidation of lipids, which are components of cell membranes, through a multi-step chain reaction. Lipid peroxidation generates various active species such as radicals, peroxides, aldehydes, and epoxides and damages DNA, RNA, proteins, cell membranes, and cellular structures. Toxicity of these reactive oxygen species is considered to be a major cause of cancer, tissue damage, and aging (Black HS, Photochem photobiol, 46 (2), 213,1987).

Antioxidants such as tocopherol, polyphenols, Coenzyme Q 10, BHT, and BHA are widely used for the purpose of neutralizing these active oxygen species, but most of them are synthetic products, and their use is limited due to safety issues when applied to human skin for a long time. . In particular, butylated hydroxy anisol (BHA) and butylated hydroxy tolune (BHT), which are widely used as phenolic synthetic antioxidants, have been widely used due to their effectiveness, economy, and safety, but they have been used as a general avoidance of synthetic additives. It is known to cause serious toxic effects on the kidneys, circulatory system.

Since BHA and BHT have the same problems as described above, they are mainly used as preservatives. Until now, synthetic vitamin C is most frequently used as an antioxidant added to cosmetics. In that sense, there was a problem that it can not be used relatively safely compared to natural extracts. In addition, there have been many efforts to obtain skin aging prevention effects by adding superoxide dismutase (SOD) and vitamin E derivatives to cosmetics, but due to the limitation of concentration in actual product application due to difficulty in stabilization or poor solubility. Antioxidants known to date, such as difficult to achieve a substantial effect has been most of the problems in terms of skin safety and stability when formulating cosmetics is limited in its use.

The present invention has been made to solve the conventional problems, one object of the present invention is to reduce the side effects of the conventional synthetic antioxidant is safe for the human body, while at the same time having a high natural antioxidant properties, formulation stability and reproducibility It is to provide antioxidants. In addition, the present invention has another object to provide a use of the antioxidant in feed, food, cosmetics.

In order to solve the above object, the inventors of the present invention through bioscreening for antioxidant activity against about 2500 kinds of natural products of plant origin, as a candidate substance 칡, bean sprouts, green onions, shiitake mushrooms, mushrooms, arc Knotweed, golden, yellowish white, hwangyeon, cedar leaf, broccoli, sancho, triticale, eoseongcho, sari, sesin, shrill, pine needles, knee roots, jinjin mugwort, buttercup, ginger, venom, yacon, omija, indongcho, grapefruit, jangsaeng bellflower, Seonji, red vegetable, plantain, persimmon, sesame, bark skin, let's eat, licorice, incense, red ginseng, Seokgok, cilantro, spectators, guk gu, gujeolcho, kkujippong, golden ruler, golden cherry tree, gilkyung The present invention has been completed through the anti-oxidant effect of the combination of processed extracts obtained by screening garlic, Ecklonia cava, dandelion, white paper, and Bokryeong and enzymatically treating the extracts or fermenting the extracts.

In order to achieve the above object of the present invention, the present invention is the extract of 칡, bean sprouts extract, green onion extract, shiitake mushroom extract, chopped mushroom extract, kojang-gun extract, golden extract, baekbaek extract, sulfur extract, cedar leaf extract, brokerage extract, Sancho extract, Triticale extract, Echo extract, Prunus extract, Secinin extract, Root extract, Pine needle extract, Knee root extract, Injin mugwort extract, Buttercup extract, Ginger extract, Poison extract, Yacon extract, Schisandra extract, Indongcho extract, Grapefruit extract, One or more extracts selected from the group consisting of Jangsaeng Bellflower Extract, Geranium Extract, Red Vegetable Extract, Plantain Extract, Perilla Extract, and Sesame Extract, and Bark Extract, Garza Extract, Licorice Extract, Ginseng Extract, Red Ginseng Extract, Seokgok Extract, Coriander Extract, spectral extract, gut extract, gujeolcho extract, Selected from the group consisting of kkujippong extract, Geumjinja extract, Geumjakja extract, Gilgyeong extract, Mandang extract, Dansam extract, sugar citron extract, lavender extract, rosemary extract, garlic extract, Ecklonia cava extract, dandelion extract, white paper extract and Bokyeong extract Provided is an antioxidant comprising a second complex extract obtained by glycosidase treatment of the first complex extract comprising one or more extracts as an active ingredient. The present invention also provides an antioxidant comprising the third complex extract obtained by bioconversion of the second complex extract into a lactic acid bacterium or a specific microorganism as an active ingredient. Antioxidants according to the present invention, as well as antioxidant activity, functions to inhibit the expression of proteins known to cause oxidative stress to increase the immune activity (regenerative effect) and senescence (or apoptosis) of cells in the environment. Has The antioxidant according to the present invention may exist in various forms such as solid powder form, emulsion form, or liquid form, and is preferably in emulsion form in consideration of storage stability and ease of handling. Looking at the method of producing an antioxidant in the emulsion form according to the present invention, for example, adding an oily medium and an emulsion stabilizer to the second complex extract or the third complex extract, and emulsifying and nano-dispersing by applying a shear force.

In order to achieve the above object of the present invention, the present invention, 칡, bean sprouts, green, shiitake mushrooms, red mushrooms, Ho Jang-geun, golden, yellow white, yellow yeon, yangyangyeop, broccoli, Sancho, three hundred seconds, eoseongcho, sari, Sesin, sound At least one selected from the group consisting of bamboo shoots, pine needles, knee roots, jinjin mugwort, buttercup, ginger, venom, yacon, schisandra chinensis, indongcho, grapefruit, Jangsaeng bellflower, ghost, red pepper, plantain, perilla, and wormwood From the group consisting of licorice, incense, ginseng, Seokgok, cilantro, spectators, guk, gujeolcho, KUJIGON, golden man, golden cherry tree, Gilkyung, mandang, sweet ginseng, sugar citron, lavender, rosemary, garlic, Ecklonia cava, dandelion, white paper Obtaining an extract containing an antioxidant active ingredient by a solvent extraction method or a supercritical extraction method from an extraction raw material comprising at least one selected; Enzymatic treatment by adding and reacting glycosidase to the extract comprising the antioxidant active ingredient; Filtering the enzyme-treated extract into an extract and a residual extract raw solid; Concentrating the separated extract to obtain a second complex extract; It provides a method for producing an antioxidant comprising a. In addition, the present invention comprises the steps of forming a mixture of the second complex extract with a lactic acid bacteria medium or a specific microbial medium; Bioconverting the second complex extract by inoculating and fermenting the mixture with a lactic acid bacterium or a specific microorganism; And filtering the biologically converted second composite extract to obtain a third composite extract.

In order to achieve the above object of the present invention, the present invention provides a feed composition, a food composition or a cosmetic composition comprising the antioxidant. Since the antioxidant of the present invention has antioxidant activity, anti-aging activity, and regenerative effect of cells subjected to oxidative stress, when used as a feed additive, a food additive, or a cosmetic additive, each product has a health function due to the antioxidant activity. You can give it.

1 shows the results of high performance liquid chromatography (HPLC) analysis of the second complex extract (top) and the third complex extract (bottom).
Figure 2 shows the antioxidant efficacy of the composite natural antioxidant prepared in Preparation Example 9 of the present invention.
Figure 3 is a fluorescence micrograph showing the cell regeneration effect by the composite natural antioxidant prepared in Preparation Example 9 of the present invention.
Figure 4 is a Western blot picture showing the effect of inhibiting the expression of aging-induced protein by the complex natural antioxidant prepared in Preparation Example 9 of the present invention.

One aspect of the present invention relates to an antioxidant and a method for producing the same, including extracts extracted from natural raw materials.

Antioxidant agent of the present invention is a second complex extract obtained by treating the first complex extract comprising an extract extracted from two or more natural raw materials with glycosidase or the agent obtained by bioconversion of the second complex extract to lactic acid bacteria or specific microorganisms 3 contains a complex extract as an active ingredient. Hereinafter, the antioxidant according to the present invention and a manufacturing method thereof will be described by dividing into a first complex extract, a second complex extract and a third complex extract.

First Complex Extract

The first complex extract according to the present invention, for example, 칡 extract, bean sprout extract, green onion extract, shiitake mushroom extract, chopped mushroom extract, kojang-geun extract, golden extract, baekbaek extract, sulfur extract, cedar leaf extract, broccoli extract, sancho extract , Triticale Extract, Estuary Extract, Pear Extract, Sessin Extract, Root Extract, Pine Needle Extract, Knee Root Extract, Root Worm Extract, Buttercup Extract, Ginger Extract, Poison Extract, Yacon Extract, Schisandra Extract, Indongcho Extract, Grapefruit Extract, Jangsaeng Bellflower At least one extract selected from the group consisting of extracts, germ extracts, red vegetable extracts, plantain extracts, perilla extracts, and wormwood extracts, bark skin extracts, Gaza extracts, licorice extracts, nectarine extracts, red ginseng extracts, stone extracts, coriander extracts, Spectrum extract, gut extract, gujeolcho extract, kkujippong 1, selected from the group consisting of extract, Geumja extract, Geumjakja extract, Giltyeong extract, Mandang extract, Dansam extract, Sacrose extract, Lavender extract, Rosemary extract, Garlic extract, Ecklonia cava extract, Dandelion extract, White paper extract and Bokryeong extract Extracts of species or more. In addition, the first complex extract includes cedar leaf extract, triticale extract, Eungchocho extract, Injin mugwort extract, and optionally Ecklonia cava extract.

In addition, the first complex extract includes a cedar leaf extract, three hundred vine extract, Eungchocho extract, and jinjin mugwort extract, preferably the first composite extract further comprises Ecklonia cava extract. When the first complex extract includes Ecklonia cava extract, the antioxidant activity of the antioxidant is further increased. The content in the complex extract of each extract extracted from each natural raw material is not particularly limited, generally 20 to 200 parts by weight, 300 to 200 parts by weight, 20 to 200 parts by weight of Echo extract, Injin mugwort extract 20 ˜200 parts by weight and optionally 20-200 parts by weight of Ecklonia cava extract.

Description of the main raw material of the first complex extract according to the present invention is as follows. Fish herbaceous perennial herbaceous species belonging to more than three hundred, mainly distributed in Korea and Japan, grow in the mountains, or in shady and wet land. Eoseongcho is named after its fishy fishy smell. The three hundred perennial herbaceous perennial plant is a perennial herb of more than three hundred dicotyledonous plants. Three hundred seconds is the name given because the roots, leaves, and flowers are white. Injin mugwort is a perennial plant of the dicotyledon plant Lantern Asteraceae, mainly distributed in Korea, Japan, China, etc., and often grows in sandy lands near streams. It is an evergreen juniper tree of the dicotyledon azalea, Rhododendron, which is distributed mainly in Mt. Baekdu, Korea and eastern Siberia, and grows under the forest of high mountains. Ecklonia cava is a perennial seaweed with brown seaweed kelp, seaweed, distributed mainly in the south coast of Korea and Japan, and grows deep in the lunch. Other raw materials are omitted from the description.

The composite extract according to the present invention includes various antioxidant active substances, antimicrobial active substances and the like contained in the extract raw material as the active substance. The active substances are mainly flavonoids, polyphenols or terpenes, specifically quercitrin, isoquercitrin, quercetin, rutin, myrcene, myrcene, Mircetin, Seanol, methyl-N-nonene, bacalin, bacicalein, and the like, but are not limited thereto.

The method for producing the first composite extract according to the present invention is 칡, bean sprouts, green onion, shiitake mushrooms, red mushrooms, Ho Jang Geun, golden, yellow white, yellow yeon, yang hyang leaf, broccoli, Sancho, three hundred seconds, Eoseongcho, sari, Sesin, sound At least one selected from the group consisting of bamboo shoots, pine needles, knee roots, jinjin mugwort, buttercup, ginger, venom, yacon, schisandra chinensis, indongcho, grapefruit, Jangsaeng bellflower, ghost, red pepper, plantain, perilla, and wormwood From the group consisting of licorice, incense, ginseng, Seokgok, cilantro, spectators, guk, gujeolcho, KUJIGON, golden man, golden cherry tree, Gilkyung, mandang, sweet ginseng, sugar citron, lavender, rosemary, garlic, Ecklonia cava, dandelion, white paper Obtaining an extract containing an antioxidant active ingredient by a solvent extraction method or a supercritical extraction method from an extraction raw material comprising at least one selected; And filtering the extract comprising the antioxidant active ingredient into an extract and a residual extract raw solid; and concentrating the separated extract to obtain a first complex extract.

In addition, the method for producing a first composite extract according to the present invention is an extract containing an antioxidant active ingredient by a solvent extraction method, or a supercritical extraction method from the extract raw material containing the cedar leaf, three hundred vine, Eochocho, phosphorus mugwort, and optionally Ecklonia cava Obtaining a; And filtering the extract comprising the antioxidant active ingredient into an extract and a residual extract raw solid; and concentrating the separated extract to obtain a first complex extract.

Looking at the solvent extraction method, the solvent used may be water, alcohol having 1 to 5 carbon atoms, ethyl acetate, chloroform, acetone, 1,2-hexanediol, 1,3-butanediol, butylene glycol, water It is also possible to use a mixed solvent in which at least one selected from the remaining solvents is mixed. Of these, considering the hazards to the human body, it is preferable that the solvent is a mixed solvent of water, an alcohol having 1 to 5 carbon atoms or a mixture of water and an alcohol having 1 to 5 carbon atoms. The extraction temperature of the solvent extraction method is not particularly limited, and is preferably characterized in that 40 ~ 80 ℃. If the extraction temperature is less than 40 ℃ extraction rate to extraction yield is low, and if the extraction temperature exceeds 80 ℃ there is a fear that the extracted active ingredient is deformed by heat. In addition, the use ratio of the extraction raw material to the extraction solvent is not particularly limited, and typically, about 300 to 1000 parts by weight of the extraction solvent is used based on 100 parts by weight of the extraction raw material. Extraction time typically requires about 1 to 3 hours, but is not limited thereto.

In addition, the first complex extract according to the present invention may be preferably extracted by a supercritical extraction method. When the supercritical extraction method is used, the extraction yield is higher than that of the solvent extraction method, and the first composite extract extracted by the supercritical extraction method reduces odor and increases the transparency of the liquid form (or the size of the color) than that extracted by the solvent extraction method. Is reduced). The supercritical extraction method uses a mixture of 1 to 20 parts by weight of an auxiliary solvent with respect to 100 parts by weight of carbon dioxide as a main solvent as a reaction medium, wherein diethylamine or triethylamine is methanol, ethanol, water, 1, It is composed of an alkaline cosolvent dissolved in 2 to 20% (vol%, v / v) in 2-hexanediol, 1,3-butanediol, butylene glycol, or a mixed solvent thereof. In supercritical extraction, extraction temperature is about 70 ~ 90 ℃ and extraction pressure is about 4000 ~ 6000 PSI.

Extracts extracted by solvent extraction or supercritical extraction are separated into extracts and residual extract raw solids by a filter cloth or filter paper having a pore size of micrometers, wherein the extract is concentrated to form a first complex extract in liquid form. In addition, the concentrated first complex extract may be prepared as a first complex extract in powder form by drying. At this time, the drying method is not limited in kind, but is preferably lyophilized in order to minimize the thermal deformation of the active ingredient. On the other hand, the residual extract raw material solid may be used to extract the active ingredient again by solvent extraction or supercritical extraction.

2nd complex extract

The second composite extract according to the present invention, for example, 칡 extract, bean sprout extract, green onion extract, shiitake mushroom extract, chopped mushroom extract, kojang-geun extract, golden extract, baekbaek extract, sulfur extract, cedar leaf extract, brokerage extract, Sancho extract , Triticale Extract, Estuary Extract, Pear Extract, Sessin Extract, Root Extract, Pine Needle Extract, Knee Root Extract, Root Worm Extract, Buttercup Extract, Ginger Extract, Poison Extract, Yacon Extract, Schisandra Extract, Indongcho Extract, Grapefruit Extract, Jangsaeng Bellflower At least one extract selected from the group consisting of extracts, germ extracts, red vegetable extracts, plantain extracts, perilla extracts, and wormwood extracts, bark skin extracts, Gaza extracts, licorice extracts, nectarine extracts, red ginseng extracts, stone extracts, coriander extracts, Spectrum extract, gut extract, gujeolcho extract, kkujippong 1, selected from the group consisting of extract, Geumja extract, Geumjakja extract, Giltyeong extract, Mandang extract, Dansam extract, Sacrose extract, Lavender extract, Rosemary extract, Garlic extract, Ecklonia cava extract, Dandelion extract, White paper extract and Bokryeong extract A first complex extract comprising extracts of at least one species is obtained by treatment with glycosidase.

In addition, the second complex extract according to the present invention is obtained by enzymatic treatment of the first complex extract, including the cedar leaf extract, three hundred vine extract, Echo vinegar extract, Injin mugwort extract, and optionally Ecklonia cava extract.

In this case, the first complex extract provided for the enzyme treatment may use a concentrated form in the manufacturing process or may use a solid powder form. An enzyme used in enzyme treatment is glycosidase, and glycosidase is also called glycoside hydrolase, and has a function of hydrolyzing glycoside bonds of glycosides or small sugars. In the present invention, when the glycosidase treatment, the extraction raw material itself can be decomposed to make the extraction easier and the extraction yield can be increased, and the extraction of various active ingredients can be performed to increase the antioxidant activity of the complex extract. have. In addition, glycosidase treatment serves to deodorize and decolorize the extract by decomposing off-flavor or color components contained in the extract extracted by solvent extraction or supercritical extraction. Moreover, the removal of impurities by the membrane filter which mentions glycosidase treatment later is made smooth.

Method for producing a second complex extract according to the present invention, 칡, bean sprouts, green onion, shiitake mushrooms, red mushrooms, Ho Jang Geun, golden, yellow white, yellow yeon, yangyangyeop, brokerage, Sancho, three hundred vine, Eoseongcho, sari, Sesin, sound At least one selected from the group consisting of bamboo shoots, pine needles, knee roots, jinjin mugwort, buttercup, ginger, venom, yacon, schisandra chinensis, indongcho, grapefruit, Jangsaeng bellflower, ghost, red pepper, plantain, perilla, and wormwood From the group consisting of licorice, incense, ginseng, Seokgok, cilantro, spectators, guk, gujeolcho, KUJIGON, golden man, golden cherry tree, Gilkyung, mandang, sweet ginseng, sugar citron, lavender, rosemary, garlic, Ecklonia cava, dandelion, white paper Obtaining an extract containing an antioxidant active ingredient by a solvent extraction method or a supercritical extraction method from an extraction raw material comprising at least one selected; Enzymatic treatment by adding and reacting glycosidase to the extract comprising the antioxidant active ingredient; Filtering the enzyme-treated extract into an extract and a residual extract raw solid; And concentrating the separated extract to obtain a second complex extract.

In addition, the method for producing a second complex extract according to the present invention is an extract containing an antioxidant active ingredient by a solvent extraction method, or a supercritical extraction method from the extract raw material including cedar leaf, three hundred vine, Eochocho, phosphorus mugwort, and optionally Ecklonia cava Obtaining a; Enzymatic treatment by adding and reacting glycosidase to the extract comprising the antioxidant active ingredient; Filtering the enzyme-treated extract into an extract and a residual extract raw solid; And concentrating the separated extract to obtain a second complex extract.

The type of glucosidase used is not particularly limited, and specifically, amylae, cellulase, xylanase, galactose, lactase, maltase (maltase) or mixed enzymes thereof, and the like, and are preferably amylases. Commercially available glucosidases include Viscozyme ^® , Econase ^® , Fungamyl ^® , Sebamyl ^® , and Clariceb ^® .

The reaction temperature, reaction pH, and reaction time of the first complex extract for glucosidase treatment vary depending on the enzyme used. Typically, the reaction temperature is 40-60 ° C., the reaction pH is 4-6, and the reaction time is 1-. 3 hours. In addition, the amount of enzyme is about 0.1 ~ 1 parts by weight based on 100 parts by weight of the extraction raw materials in consideration of economics such as enzyme processing time, enzyme cost.

Extracts treated with glucosidase are separated into extracts and residual extract solids by filtration, where filtration separates the extracts into extracts and residual extract solids by filter cloth or filter paper with a pore size of micrometers. And the primary filtered extract is filtered with a membrane filter having a pore size of nanometers to remove impurities (odor component or color component). Glucosidase is difficult to perform due to the large load when filtration of the extract passed through the filter cloth or filter paper from the extract raw material by solvent extraction method or supercritical extraction method with a membrane filter having a pore size of nanometers. Enzymatically treated and primary filtered extract is easily filtered by the membrane filter, the odor or components that contribute to the color can be removed. The filtrate obtained by filtration in the glycosidase treated extract is concentrated to form a second complex extract in liquid form. In addition, the concentrated second complex extract may be prepared in powder form by drying. At this time, the drying method is not particularly limited, but is preferably lyophilized in order to minimize thermal deformation of the active ingredient. On the other hand, the residual extract raw material solid may be used to extract the active ingredient again by solvent extraction or supercritical extraction.

Third Complex Extract

The third complex extract according to the present invention, for example, 칡 extract, bean sprouts extract, green onion extract, shiitake mushroom extract, chopped mushroom extract, kojang-gun extract, golden extract, baekbaek extract, sulfur extract, cedar leaf extract, brokerage extract, Sancho extract , Triticale Extract, Estuary Extract, Pear Extract, Sessin Extract, Root Extract, Pine Needle Extract, Knee Root Extract, Root Worm Extract, Buttercup Extract, Ginger Extract, Poison Extract, Yacon Extract, Schisandra Extract, Indongcho Extract, Grapefruit Extract, Jangsaeng Bellflower At least one extract selected from the group consisting of extracts, germ extracts, red vegetable extracts, plantain extracts, perilla extracts, and wormwood extracts, bark skin extracts, Gaza extracts, licorice extracts, nectarine extracts, red ginseng extracts, stone extracts, coriander extracts, Spectrum extract, gut extract, gujeolcho extract, kkujippong 1, selected from the group consisting of extract, Geumja extract, Geumjakja extract, Giltyeong extract, Mandang extract, Dansam extract, Sacrose extract, Lavender extract, Rosemary extract, Garlic extract, Ecklonia cava extract, Dandelion extract, White paper extract and Bokryeong extract The second complex extract obtained by treating the first complex extract including the extract of species or more with glycosidase is obtained by bioconversion by lactic acid bacteria or a specific microorganism.

In addition, the third complex extract according to the present invention is characterized in that the second complex extract obtained by treating the first complex extract, including cedar leaf extract, triticale extract, Echochocho extract, Injin mugwort extract, and optionally Ecklonia cava extract with glycosidase Obtained by bioconversion by lactic acid bacteria or specific microorganisms.

In this case, lactic acid bacteria or the second complex extract provided for a specific microorganism fermentation may use a concentrated form in the manufacturing process or may use a solid powder form. The lactic acid bacteria or specific microorganisms used for bioconversion of the second complex extract are not particularly limited as long as they are harmless to the human body, and specifically, Lactobacillus and Streptococcus , Pediococcus, Leuconostoc and the like.

When the second complex extract is bioconverted by lactic acid bacteria or specific microbial fermentation, various active substances are produced to increase the antioxidant activity of the complex extract. In addition, compatibility with other ingredients is increased when preparing a feed composition, a food composition, or a cosmetic composition including an antioxidant in the form of an emulsion or a complex natural antioxidant, which will be described later, thereby improving stability of the formulation.

Looking at the method for producing a third complex extract according to the present invention, forming a mixture of the second complex extract with lactic acid bacteria medium or a specific microbial medium; Bioconverting the second complex extract by inoculating and fermenting the mixture with a lactic acid bacterium or a specific microorganism; And filtering the bioconverted second composite extract to obtain a third composite extract. In this case, when the second complex extract used to prepare the third complex extract is subjected to nanofiltration by a membrane filter in the manufacturing process, the filtration of the step of obtaining the third complex extract is a filter cloth or filter paper having a pore size in micrometers. Filtration by is sufficient. On the other hand, when the second complex extract used to prepare the third complex extract is not subjected to nanofiltration by the membrane filter in the manufacturing process, the filtration of the step of obtaining the third complex extract is a filter cloth having a pore size of micrometer unit or It is preferred to consist of primary filtration with filter paper and secondary filtration with membrane filter with pore size in nanometers. The third complex extract obtained by filtration can then be prepared as a third complex extract in concentrated liquid form, and the concentrated third complex extract can also be prepared in powder form by drying. At this time, the drying method is not limited in kind, but is preferably lyophilized in order to minimize the thermal deformation of the active ingredient.

The complex natural antioxidant according to the present invention may also be composed of the second complex extract or the third complex extract itself, and preferably the storage stability, processability into the formulation, other ingredients in the product, so long as the antioxidant activity is not inhibited. Various auxiliary additives may be further included in order to improve compatibility, and the like. In addition, the complex natural antioxidant may be present in liquid form, in solid powder form, or in emulsion form, and is preferably in emulsion form in view of storage stability and ease of handling. Method of producing a complex natural antioxidant in the form of an emulsion includes, for example, adding an oily medium and an emulsion stabilizer to the second complex extract or the third complex extract and emulsifying by applying shear force.

There are various types of auxiliary additives to be used depending on the purpose of the auxiliary additives, specifically glycerin (Clycerin), caster oil (Gaster), glycine (Glycine), Tween (Dextrin), Lactic acid (Lactic acid) ).

Another aspect of the invention relates to the use of said complex natural antioxidant.

Complex natural antioxidant according to the present invention is composed of extracts of various natural raw materials, has a high antioxidant effect, anti-aging effect, safety for human skin, and non-toxic during acute oral administration, and other whitening effects derived from natural products It has effects such as wrinkle improvement, anti-inflammatory action, atopy improvement, acne healing. Therefore, the antioxidant according to the present invention can be used for the purpose of improving immune function or enhancing cell regeneration, anti-aging, etc. in various products such as pharmaceutical compositions, cosmetic compositions, food compositions, or feed compositions. When the complex natural antioxidant according to the present invention is applied to a pharmaceutical composition, a cosmetic composition, a food composition, or a feed composition, the content thereof is not particularly limited, and the content may be appropriately selected in consideration of toxicity. For example, when the complex natural antioxidant according to the present invention is applied to the cosmetic composition, the amount thereof is preferably about 0.05 to 5.0 wt% based on the total weight of the product, and when used in a food composition, the amount thereof is used as the total amount of the product. 0.01 to 2.0% by weight is preferred based on the weight.

Hereinafter, the case in which the complex natural antioxidant according to the present invention is applied to the cosmetic composition will be described in more detail. Those skilled in the art to which the present invention pertains will be able to produce a cosmetic composition comprising the complex natural antioxidant of the present invention by selecting suitable formulations and additives as is well known. Specific examples of the cosmetic composition that can be prepared include various creams, lotions, skins, shampoos, rinses, cleansing agents, face washes, soaps, treatments, essences, and the like, and specific examples of face wash creams, face wash foams, cleansing creams, cleansing milks, and cleansing lotions. , Massage cream, cold cream, moisturizing cream, latex, lotion, pack, aftershave cream, sunburn prevention cream, suntan oil, soap, body shampoo, hair shampoo, hair rinse, hair treatment, wool, hair care, hair Cream, hair liquid, set lotion, hair spray, hair bridge, coloring, color spray, permanent wave liquid, press powder, loose powder, eye shadow, hand cream, lipstick and the like.

The cosmetic composition including the complex natural antioxidant of the present invention includes an aqueous vitamin, an oily vitamin, a polymer peptide, a polymer polysaccharide, a sphingolipid, and the like.

The aqueous vitamin may be any compound that can be incorporated into the cosmetic composition, but preferably vitamin B1, vitamin B2, vitamin B6, pyridoxine, pyridoxine hydrochloride, vitamin B12, pantothenic acid, nicotinic acid, nicotinic acid amide, folic acid, vitamin C, vitamin H and the like. And salts thereof (thiamine hydrochloride, sodium ascorbate salt, etc.) and derivatives (ascorbic acid-2-sodium phosphate salt, ascorbic acid-2-magnesium phosphate salt, etc.) can also be used in the present invention. Included in

The oil-based vitamin may be any compound that can be blended into the cosmetic composition, but preferably vitamin A, carotene, vitamin D2, vitamin D3, vitamin E (d1-α-tocopherol, d-α-tocopherol, d-δ-tocopherol) and the like. And derivatives thereof (ascorbic palmitate, ascorbic stearate, ascorbic palmitate, acetic acid dl-α-tocopherol, dl-α-tocopherol vitamin E, DL-pantothenyl alcohol, D-pantothenyl). Alcohols, pantotenylethyl ether, etc.) are also included in the oil-soluble vitamins used in the present invention.

The polymer peptide may be any compound as long as it can be incorporated into the cosmetic composition. Preferably, collagen, hydrolyzed collagen, gelatin, elastin, hydrolyzed elastin, keratin, and the like can be given.

The polymer polysaccharide may be any compound as long as it can be blended into the cosmetic composition. Preferably, hydroxyethyl cellulose, xanthan gum, sodium hyaluronate, chondroitin sulfate or a salt thereof (sodium salt, etc.) may be mentioned. For example, chondroitin sulfate or its salt can be normally purified from a mammal or fish.

The sphingolipid may be any compound as long as it can be blended into the cosmetic composition. Preferably, ceramide, pit spingosine, sphingoglycolipid and the like can be given.

Moreover, you may mix | blend with the said cosmetic composition the other component normally mix | blended with a cosmetics as needed with the said component. Examples of the blending component that may be added include oils and fats, moisturizers, emollients, surfactants, organic and inorganic pigments, organic powders, ultraviolet absorbers, antioxidants, plant extracts, pH adjusters, alcohols, pigments, flavors, and the like. .

Examples of the fat or oil component include ester fats, hydrocarbon fats, silicone fats, fluorine fats, animal fats, and vegetable fats and oils.

Examples of the moisturizing agent include a water-soluble low molecular moisturizer, a fat-soluble molecular moisturizer, a water-soluble polymer, and a fat-soluble polymer.

Examples of the emollient include long-chain acyl glutamic acid cholesteryl esters, hydroxy stearic acid cholesterol, 12-hydroxystearic acid, stearic acid, rosin acid, lanolin fatty acid cholesteryl esters, and the like.

As surfactant, nonionic surfactant, anionic surfactant, cationic surfactant, amphoteric surfactant, etc. are mentioned. Nonionic surfactants include self-emulsifying glycerin monostearate, propylene glycol fatty acid esters, glycerin fatty acid esters, polyglycerol fatty acid esters, sorbitan fatty acid esters, POE (polyoxyethylene) sorbitan fatty acid esters, POE sorbitan fatty acid esters, POE Glycerin fatty acid ester, POE alkyl ether, POE fatty acid ester, POE hardened castor oil, POE castor oil, POE-POP (polyoxyethylene-polyoxypropylene) copolymer, POE-POP alkyl ether, polyether modified silicone, lauric acid Alkanolamide, alkylamine oxide, hydrogenated soybean phospholipid, etc. are mentioned. As anionic surfactant, fatty acid soap, (alpha)-acyl sulfonate, alkyl sulfonate, alkyl allyl sulfonate, alkyl naphthalene sulfonate, alkyl sulfate, POE alkyl ether sulfate, alkylamide sulfate, alkyl phosphate, POE alkyl phosphorus salt, alkylamide Phosphates, alkyloylalkyltaurine salts, N-acylamino acid salts, POE alkyl ether carboxylate salts, alkyl sulfosuccinate salts, sodium alkyl sulfo acetates, acylated hydrolyzed collagen peptide salts, perfluoroalkyl phosphate esters, and the like. have. As cationic surfactant, alkyl trimethylammonium chloride, stearyl trimethyl ammonium chloride, stearyl trimethyl ammonium chloride, cetostearyl trimethyl ammonium chloride, distearyl dimethyl ammonium chloride, stearyl dimethyl benzyl ammonium bromide, behenyl trimethyl ammonium chloride, chloride Benzalkonium, diethylaminoethyl stearate, dimethylaminopropyl stearate, lanolin derivatives, quaternary ammonium salts, and the like. Examples of amphoteric surfactants include carboxybetaine type, amidebetaine type, sulfobetaine type, hydroxysulfobetaine type, amide sulfobetaine type, phosphobetaine type, aminocarboxylate type, imidazoline derivative type and amideamine type. An amphoteric surfactant etc. are mentioned.

Organic and inorganic pigments include silicic acid, silicic anhydride, magnesium silicate, talc, sericite, mica, kaolin, bengala, clay, bentonite, titanium film mica, bismuth oxychloride, zirconium oxide, magnesium oxide, zinc oxide, titanium oxide, aluminum oxide Inorganic pigments such as calcium sulfate, barium sulfate, magnesium sulfate, calcium carbonate, magnesium carbonate, iron oxide, ultramarine blue, chromium oxide, chromium hydroxide, calamine, carbon black, and composites thereof; Polyamide, polyester, polypropylene, polystyrene, polyurethane, vinyl resin, urea resin, phenol resin, fluorine resin, silicon resin, acrylic resin, melamine resin, epoxy resin, polycarbonate resin, divinylbenzene, styrene copolymer, Organic pigments such as silk powder, cellulose, CI pigment yellow, CI pigment orange, and composite pigments of these inorganic pigments and organic pigments;

As organic powder, Metal soaps, such as a calcium stearate; Alkyl phosphate metal salts such as sodium cetylinate, zinc lauryl acid and calcium laurate; Acylamino acid polyvalent metal salts such as N-lauroyl-β-alanine calcium, N-lauroyl-β-alanine zinc, and N-lauroylglycine calcium; Amide sulfonic acid polyvalent metal salts, such as N-lauroyl-taurine calcium and N-palmitoyl-taurine calcium; N-acyl basic amino acids, such as N (epsilon) -lauroyl-L- lysine, N (epsilon) -palmitolysine, N (alpha)-partoylol nitin, N (alpha)-lauroyl arginine, and N (alpha) -cured fatty acid acyl arginine; N-acyl polypeptides, such as N-lauroyl glycyl glycine; α-amino fatty acids such as α-aminocaprylic acid and α-aminolauric acid; Polyethylene, polypropylene, nylon, polymethyl methacrylate, polystyrene, divinylbenzene-styrene copolymer, ethylene tetrafluoride and the like.

Examples of the ultraviolet absorber include paraaminobenzoic acid, ethyl paraaminobenzoate, amyl paraaminobenzoic acid, octyl paraaminobenzoate, ethylene glycol salicylate, phenyl salicylate, octyl salicylate, benzyl salicylate, butylphenyl salicylate, homomentyl salicylate and cinnamic acid benzyl , Paramethoxy cinnamic acid 2-ethoxyethyl, paramethoxy cinnamic acid octyl, diparamethoxy cinnamic acid mono 2-ethylhexane glyceryl, paramethoxy cinnamic acid isopropyl, diisopropyl diisopropyl cinnamic acid ester mixture, urokanoic acid Ethyl urocanate, hydroxymethoxybenzophenone, hydroxymethoxybenzophenonesulfonic acid and salts thereof, dihydroxymethoxybenzophenone, dihydroxymethoxybenzophenonedisulfonate, dihydroxybenzophenone, tetra Hydroxybenzophenone, 4-tert-butyl-4'-methoxydibenzoylmethane, 2,4,6-trianilino-p- (carbo-2'-ethylhexyl-1'-oxy) -1,3 , 5-triazine, 2- (2-hydroxy 5-methylphenyl) benzotriazole and the like.

Examples of the alcohol include higher alcohols such as cetyl alcohol.

In addition, the complex natural antioxidant of the present invention can be applied to a functional food composition because it can anticipate the effects of biological defense, prevention of disease, recovery of disease or inhibition of aging by providing antioxidant efficacy. The application to the functional food composition is not particularly limited in form, and may be specifically applied to ice cream, powdered milk, yogurt, health drinks and alcohol (vodka and other alcoholic beverages). In addition, the complex natural antioxidant of the present invention can be applied to feed compositions, pharmaceutical compositions, etc., wherein each composition can be prepared with ingredients and formulations commonly used in the art.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are only intended to more clearly describe the present invention, and do not limit the protection scope of the present invention.

1. Preparation of Complex Extracts and Characteristics of Complex Extracts

(1) Preparation of the first composite extract from natural raw materials

Production Example 1

50 kg of dried fruit, 50 kg of Schisandra chinensis, 50 kg of Gujeolcho and 50 kg of Dandelion were added to the extraction reactor, and 1000 kg of water was added to the extraction solvent. The temperature of the extraction reactor was maintained at about 80 ℃ for 3 hours to extract the antioxidant active ingredient of natural raw materials. The extract was filtered with a filter cloth to separate the extract and the remaining extract raw solids, and the filtered extract was concentrated to a concentration of about 2.0 Brix using a reduced pressure evaporator to obtain 140 kg of the first complex extract in the form of a concentrate. In addition, the first complex extract in the form of a concentrate was lyophilized to obtain a first complex extract in the form of a powder.

Production Example 2

40 kg of dried cedar leaves, 40 kg of dried trichophyte, 40 kg of dried fish herb, 40 kg of dried mulberry leaf, and 40 kg of dried Ecklonia cava were added thereto, and 1000 kg of water was added thereto as an extraction solvent. The temperature of the extraction reactor was maintained at about 80 ℃ for 3 hours to extract the antioxidant active ingredient of natural raw materials. The extract was filtered with a filter cloth to separate the extract and the remaining extract raw solid, and the filtered extract was concentrated to a concentration of about 2 Brix using a reduced pressure evaporator to obtain 140 kg of the first complex extract in the form of a concentrate. In addition, the first complex extract in the form of a concentrate was lyophilized to obtain a first complex extract in the form of a powder.

(2) Preparation of Second Composite Extract from Natural Raw Materials

Preparation Example 3.

50 kg of dried fruit, 50 kg of Schisandra chinensis, 50 kg of Gujeolcho and 50 kg of Dandelion were added to the extraction reactor, and 1000 kg of water was added to the extraction solvent. The temperature of the extraction reactor was maintained at about 80 ℃ for 3 hours to extract the antioxidant active ingredient of natural raw materials. After lowering the temperature of the extraction reactor to about 55 ℃ and adjusting the pH of the extract to about 5.5, the extract is charged with about 200 g of glycosidase (Fungamyl ^® , Sebamyl ^® , and Clariseb ^® ; Special Enzyme, USA) for 3 hours After the reaction, the temperature of the extract was raised to about 80 ° C. to inactivate the enzyme. The enzyme-treated extract was first filtered through a filter cloth to separate the extract and the remaining extract raw material solids, and then the filtered extract was passed through a membrane filter (pore size: 0.45 μm) and filtered secondly to remove impurities using a reduced pressure evaporator. Components that contribute to color, odor, etc.) were removed. The extract passed through the membrane filter was concentrated to a concentration of about 2.5 Brix to give 140 kg of the second complex extract in the form of a concentrate. In addition, the second complex extract in the form of a concentrate was lyophilized to obtain a second complex extract in the form of a powder.

Preparation Example 4.

40 kg of dried cedar leaves, 40 kg of dried trichophyte, 40 kg of dried fish herb, 40 kg of dried mulberry leaf, and 40 kg of dried Ecklonia cava were added thereto, and 1000 kg of water was added thereto as an extraction solvent. The temperature of the extraction reactor was maintained at about 80 ℃ for 3 hours to extract the antioxidant active ingredient of natural raw materials. After lowering the temperature of the extraction reactor to about 55 ℃ and adjusting the pH of the extract to about 5.5, the extract is charged with about 200 g of glycosidase (Fungamyl ^® , Sebamyl ^® , and Clariseb ^® ; Special Enzyme, USA) for 3 hours After the reaction, the temperature of the extract was raised to about 80 ° C. to inactivate the enzyme. The enzyme-treated extract was first filtered through a filter cloth to separate the extract and the remaining extract raw material solids, and then the filtered extract was passed through a membrane filter (pore size: 0.45 μm) and filtered secondly to remove impurities using a reduced pressure evaporator. Components that contribute to color, odor, etc.) were removed. The extract passed through the membrane filter was concentrated to a concentration of about 2.5 Brix to give 140 kg of the second complex extract in the form of a concentrate. In addition, the second complex extract in the form of a concentrate was lyophilized to obtain a second complex extract in the form of a powder.

(3) Preparation of the third composite extract

Preparation Example 5.

1 L of LB variable medium (Luria-Bertani media) and 94 L of the second complex extract in the form of a concentrate obtained in Preparation Example 3 were added thereto, and the fermenter was sterilized by steam. The temperature of the sterilized fermenter was maintained at 40 ° C, and 5 l of lactic acid bacteria culture solution was added to the fermenter to inoculate. The inoculated lactic acid culture medium was precultured with Lactobacillus bacteria in LB variable media (Luria-Bertani media), and the cell count concentration of lactic acid bacteria was 1 × 10 ⁷ cfu (Colony Forming Unit) / ml. LB medium was used consisting of 10 g / L tryptone, 10 g / L sodium chloride (NaCl), 5 g / l yeast extract on the distilled water base.

The temperature of the fermenter inoculated with the lactic acid bacteria culture medium was adjusted to about 35 ° C. and fermented for about 12 hours to bioconvert the second composite extract. Thereafter, the fermentation broth of the fermenter was filtered sequentially using a filter cloth and a membrane filter to obtain 100 kg of the third complex extract in liquid form. In addition, it was concentrated and dried to obtain a third complex extract in powder form.

Preparation Example 6.

1 L of LB variable medium (Luria-Bertani media) and 94 L of the second complex extract in the form of a concentrate obtained in Preparation Example 4 were added, and the fermenter was sterilized by steam. The temperature of the sterilized fermenter was maintained at 40 ° C, and 5 l of lactic acid bacteria culture solution was added to the fermenter to inoculate. At this time, the inoculated lactic acid bacteria cultured Lactobacillus (Lactobacillus) bacteria in LB medium (Luria-Bertani media) preculture (Preculture), the number of cells of lactic acid bacteria concentration of 1 × 10 ⁷ cfu (Colony Forming Unit) / ㎖ The LB variable medium was composed of 10 g / l tryptone, 10 g / l sodium chloride (NaCl) and 5 g / l yeast extract on distilled water base.

(4) Characteristics of Complex Extract

1) Color and Odor Evaluation of Complex Extracts

The color and smell of the solvent extract of the natural raw material, the enzyme-treated extract treated with the enzyme extract and the enzyme-treated extract, and the extract obtained by bioconversion of the enzyme-treated extract by lactic acid bacteria fermentation were observed. The composite extract in powder form prepared by Preparation Example 2, Preparation Example 4, and Preparation Example 6 was dissolved in distilled water to a concentration of 2.5% (w / v), and color and smell were observed. In the case of color, Preparation Example 2 had a deep navy blue, Preparation Example 4 had a dark yellow color, Preparation Example 6 had a light yellow color. In addition, in the case of the smell, in Preparation Example 2 was strong off odor inherent in the extraction raw material, in the case of Preparation Example 4 and Preparation Example 6 almost no off-flavor. From the above results, it can be seen that the complex extract extracted with the solvent is decolorized and deodorized through the enzyme treatment and the fermentation treatment.

2) Changes in Active Substances of the Third Complex Extract Bioconverted by Lactic Acid Bacteria or Specific Microbial Fermentation

High performance liquid chromatography (HPLC) analysis was carried out to observe the active substances of the second and third complex extracts in powder form obtained in Preparation Examples 4 and 6. As a mobile phase solvent, acetonitrile and 0.5% of phosphoric acid (Phosphoric acid) were mixed at 27:73 (v / v). The column was a u-Bondapak C18 silica column. It was used to dissolve the extract in the form of distilled water. 1 shows the results of high performance liquid chromatography (HPLC) analysis of the second complex extract (top) and the third complex extract (bottom). As shown in Figure 1 it can be seen that the rich active material is produced by lactic acid bacteria fermentation.

3) Evaluation of antioxidant efficacy of the second complex extract and the third complex extract

Antioxidant efficacy of the second complex extract and the third complex extract in powder form obtained in Preparation Example 4 and Preparation Example 6 was measured using the DPPH (1,1-diphenyl-2-picrylhydrazyl) method. DPPH is a colored radical that can be used to directly determine the radical removal capacity of a sample. The second complex extract and the third complex extract in powder form obtained in Preparation Example 4 and Preparation Example 6 were dissolved in a solvent (ethanol), respectively, to prepare a sample having 100 µg / ml (converted at w / v% concentration of 0.01%). 1 ml of 100 uM DPPH was mixed with 1 ml of the sample, followed by reaction at room temperature for 30 minutes. Absorbance was measured at 517 nm to determine the amount of DPPH remaining, and the relative antioxidant effect of each sample was expressed as a percentage of radical scavenging activity relative to the control group (control group: test group consisting of DPPH and solvent only, without sample). In addition, as a comparative example, the radical scavenging capacity of ascorbic acid was measured, and the experimental results are shown in Table 1. As shown in Table 1, the second complex extract and the third complex extract according to the present invention showed a high antioxidant effect over ascorbic acid, and particularly, the highest antioxidant activity in the third complex extract in which abundant active substances were produced by lactic acid bacteria fermentation. It showed an effect.

sample Radical scavenging ability (%) Preparation Example 4 89.9% Preparation Example 6 95.2% Ascorbic acid 89.3%

2. Preparation and Characteristics of Complex Natural Antioxidants Containing Third Complex Extract

(1) Preparation of Complex Natural Antioxidants

Preparation Example 7 (Preparation of Natural Antioxidant in Powder Form)

A small amount of glycine (Glycine) was added to the third complex extract of the liquid form obtained in Preparation Example 6 as a buffering agent, concentrated, and concentrated by drying with a spray dryer or a lyophilizer. Natural antioxidants were prepared.

Preparation Example 8 (Preparation of Natural Antioxidant in Liquid Form)

A small amount of glycerin and dextrin was added to the third complex extract of the liquid form obtained in Preparation Example 6, dissolved, and then the pH was adjusted to 5.0 with lactic acid to prepare a complex natural antioxidant in liquid form. Prepared.

Preparation Example 9 (Preparation of Natural Antioxidant in Emulsion Form)

The third complex extract in liquid form obtained in preparation 6 was concentrated to about 2.5 Brix. 18 kg of the third complex extract concentrated in the emulsification tank. 60 kg of glycerin and 22 kg of castor oil were added thereto, and the temperature of the emulsifying tank was increased to about 70 to 75 ° C., and homogenized by stirring with a homogenizer (about 220 to 250 rpm). . Thereafter, the temperature of the emulsion tank was lowered to about 32 to 35 ° C., and the pH of the content homogenized with lactic acid was adjusted to about 3 to 5 to prepare a complex natural antioxidant in the form of an emulsion.

(2) Evaluation of Antioxidant Efficacy of Complex Natural Antioxidants

DPPH (1,1-diphenyl-2-picrylhydrazyl) method was used to confirm the antioxidant effect of the composite natural antioxidant prepared in Preparation Example 9. DPPH is a colored radical that can be used to directly determine the radical removal capacity of a sample. Samples (complex natural antioxidants and comparative samples prepared in Preparation Example 9) were dissolved in a solvent (ethanol) and prepared for each concentration. 1 ml of each sample was mixed with 1 ml of 100 uM DPPH, followed by 30 minutes at room temperature. Reacted. Absorbance was measured at 517 nm to determine the amount of DPPH remaining, and the relative antioxidant effect of each sample was expressed as a percentage of radical scavenging activity relative to the control group (control group: test group consisting of DPPH and solvent only, without sample).

Figure 2 shows the antioxidant efficacy of the composite natural antioxidant prepared in Preparation Example 9 of the present invention. As shown in FIG. 2, the composite natural antioxidant prepared in Preparation Example 9 of the present invention showed higher antioxidant activity than polyphenol, green butylated hydroxyanisole (BHA), and butylated hydroxytoluene (BHT) of green tea powder. From these antioxidant activity results, it can be seen that the complex natural antioxidant prepared in Preparation Example 9 of the present invention can be used as a food additive, a cosmetic additive, a feed additive, a pharmaceutical additive, and the like.

(3) Cell Regeneration Effect of Complex Natural Antioxidants (Cell Recovery Test)

1) Cell Culture

Incubate Bovine aortic endothelial cells (BAEC; supplied by Kyushu University, Japan) with Dulbecco's Modified Eagle medium (DMEM, GIBCO BRL, Grand Island, NY) containing 10% fetal calf serum (FCS, GIBCO BRL) (37 ° C., 5% CO ₂ ).

2) Treatment of Cultured Cells

BAEC cells cultured for 24 hours were treated with 0.2 μM 4-HNE (4-Hydroxynonenal, oxidant) and incubated for 12 hours. After 12 hours of incubation with 4-HNE, the cells were washed well to remove the oxidant, and then cultured again. The complex natural antioxidant prepared in Preparation Example 9 (dissolved in ethanol was adjusted to 10 μg / ml) was added thereto, and the cells were harvested after 12 hours or 24 hours of incubation.

3) Cell state change measurement

Harvested cells were stained with 100 μg / ml acridine orange (Sigma Chemical Co.) and 100 μg / ml ethidium bromide (Sigma Chemical Co.) in 35 mm Acas dishes (Mat-Tek, USA). It was observed under a microscope (reflected fluorescence microscopy; Olympus, Japan). The cells that appear green when viewed under a fluorescence microscope are living cells and those cells that are observed in red are cells that have apoptosis.

Figure 3 is a fluorescence micrograph showing the cell regeneration effect by the composite natural antioxidant prepared in Preparation Example 9 of the present invention. In FIG. 3, "A" was cultured without 4-HNE treatment and the complex natural antioxidant treatment prepared in Preparation Example 9, and "B" was cultured with 4-HNE for 12 hours, and "C". Was treated for 12 hours with 4-HNE and then cultured for 12 hours with the complex natural antioxidant prepared in Preparation Example 9, "D" is a complex natural prepared in Preparation Example 9 after 12 hours treatment with 4-HNE. It was incubated for 24 hours with antioxidants. As shown in FIG. 3, when the complex natural antioxidant prepared in Preparation Example 9 was treated to cells subjected to oxidative stress with 4-HNE, the immune activity of the cells was increased, such as the cells of the control group not treated with oxidative stress. It can be seen that is recovered. Therefore, when the complex natural antioxidant prepared in Preparation Example 9 of the present invention is used as feed additives, functional food additives, etc. of livestock, the immune activity of the livestock or human body is increased to increase disease resistance, and in the case of livestock, the increase of the individual is expected. Could be.

(4) Anti-aging Effect of Complex Natural Antioxidants

1) Cell Culture

Bovine pulmonary artery endothelial cells [BPAE; Korean Cell Line Bank (supplied from KCLB0, Korea) was incubated with Dulbecco's Modified Eagle medium (DMEM, GIBCO BRL, Grand Island, NY) containing 10% fetal calf serum (FCS, GIBCO BRL) (37 ° C). , 5% CO ₂ ).

2) Treatment of Cultured Cells

BPAE cells cultured for 24 hours were treated with 0.2 μM 4-HNE (4-Hydroxynonenal, oxidant) and incubated for 6 hours. After 12 hours of incubation with 4-HNE, the cells were washed well to remove the oxidant, and then cultured again. The complex natural antioxidant prepared in Preparation Example 9 (dissolved in ethanol was adjusted to 10 μg / ml) was added thereto, and the cells were harvested after 12 hours or 24 hours of incubation.

3) Western blot anaylsis

Western blot was used to analyze the expression level of PARP cleaved form of harvested cells. PARP fragment proteins are known as protein forms that increase expression upon induction of apoptosis. After culturing, the cells were removed from the incubator, washed twice with cold PBS, 1 ml of PBS was added to the culture dish, and the cells were removed with a scraper. The cells were transferred to a centrifuge tube and centrifuged (3000 rpm, 3 min, 4 ° C.), and then the supernatant was discarded and 1 μg / ml aprotinin and 100 μg / ml phenylmethylmethylsulfonyl fluoride (PMSF) lysis buffer (1%). Protein was extracted with Triton X-100.50 mM Tris-HCL, 150 mM NaCl, 0.2% sodium azide) and centrifuged at 12,000 rpm for 10 minutes to separate the protein extract. The concentration of the isolated protein was measured by BSA assay (Bicinichonic acid solution), and 20-40 µg of protein was electrophoresed on 12% SDS-polyacrylamide gel. The protein on the electrophoretic gel was transferred to a polyvinylidene difluoride (PVDF) membrane at 280 mA for 90 minutes, and the membrane was washed with TBST (0.1% Tween-Tris-buffered saline) and blocked at room temperature for 1 hour with 5% skim milk. The primary antibody (dilution ratio 1: 2000) was reacted at room temperature for 2 hours, washed three times with TBST for 10 minutes, and then reacted with HRP-conjugated secondary antibody (dilution ratio: 10000) for 1 hour. After the antibody reaction was completed, the membrane was washed 4 times with TBST for 1 minute, and the formed immune complexes were identified using ECL (enhanced chemiluminescence) coloring reagent.

Figure 4 is a Western blot picture showing the effect of inhibiting the expression of aging-induced protein by the complex natural antioxidant prepared in Preparation Example 9 of the present invention. In FIG. 4, "A" is treated for 6 hours with 4-HNE and then cultured for 12 hours with the complex natural antioxidant prepared in Preparation Example 9, and "B" is prepared after 6 hours with culture for 4HNE. 24 hours treatment culture with the complex natural antioxidant prepared in 9, "C" is 6 hours treatment culture with 4-HNE, "D" is the 4-HNE treatment and the complex natural antioxidant prepared in Preparation Example 9 as a control It was cultured without first treatment. As shown in FIG. 4, treatment of the complex natural antioxidant prepared in Preparation Example 9 to cells subjected to oxidative stress with 4-HNE inhibited the intracellular expression of PARP fragment protein known as a protein associated with aging. Can be. On the other hand, it can be seen that a large amount of PARP fragment protein is expressed in cells subjected to oxidative stress with 4-HNE. Therefore, when the composite natural antioxidant prepared in Preparation Example 9 of the present invention is used as feed additives, functional food additives, functional cosmetic additives, etc. of livestock, it may be expected to not only increase immunity due to antioxidant of livestock or human body but also inhibit aging. .

(5) Acute Oral Toxicity Evaluation of Complex Natural Antioxidants

An acute oral toxicity test was conducted to confirm whether the complex natural antioxidant prepared in Preparation Example 9 can be safely applied to food, medicine, and the like. Twenty five to six week old SPF SD rats were used as an experimental animal under the acute toxicity test under the following conditions.

ㆍ Temperature and humidity conditions: Temperature 22 ± 2 ℃. Relative Humidity 50 ± 10%

ㆍ Contrast cycle: Fluorescent lamp lighting (lights up at 08, lights out at 20)

ㆍ Roughness: 200 to 300 Lux

ㆍ Free drinking water treated with ultraviolet rays

ㆍ Dilute the test substance in sterile distilled water to prepare a sample for administration.

On the day of administration, the general condition is observed every hour up to 4 hours, and once a day from the day after the administration, the general condition changes, poisoning symptoms, mobility, appearance, autonomic nerves, and the presence of dead animals are carefully observed. Pathological anatomical verification was performed to determine the toxicity. The half lethal dose (LD ₅₀ ) was higher than 20ml / kg body weight (BW), and it was determined that there was no toxicity.

(6) Safety evaluation of human natural skin of complex natural antioxidants

In order to confirm whether the complex natural antioxidant prepared in Preparation Example 9 is safe for human skin, skin safety verification experiment was performed. As a test method, a skin cumulative stimulation test was performed. 0.1% (w / v), 1% (w / v), 5% (w / v) and 10% (w / v) were added to the composite natural antioxidant prepared in Preparation Example 9 based on squalene, respectively. A skin external preparation was prepared and used to determine whether complex natural antioxidants irritated the skin by subjecting 30 healthy adults to cumulative patching on the upper forearm for a total of nine 24 hours every other day. . The patching method used a fin chamber (Finn chamber, Epitest Ltd, Finland). 15 µl of each of the external preparations for skin was added dropwise to the chamber, followed by patching. As a result of the measurement, the composite natural antioxidant prepared in Preparation Example 9 of the present invention did not show a distinct cumulative stimulation pattern, and was found to be safe for human skin.

In addition, 30 subjects were subjected to a patch test on the upper arm using an IQ chamber. Psoriasis, eczema, and other skin lesion holders, or those who are pregnant, nursing or contraceptives, and antihistamines, were excluded from this study. Wipe the test site with 70% ethanol and dry it, and then prepare samples of the composite natural antioxidant 1% (w / v) and Gly 20% (w / v) prepared in Preparation Example 9 on the upper arms of 30 prepared subjects. This was added dropwise to the IQ Chamber 25μl each and fixed on the upper arm area, which is a test site. After the patch was applied for 24 hours and the patch was removed, the test sites were marked with a marking pen. After 30 minutes, 24 hours, and 48 hours, the test sites were observed. After 30 minutes, 24 hours, and 48 hours after removal of the patch, skin reactions were classified according to the criteria of the International Contact Dermatitis Research Group (ICDRG). In addition, after calculating the mean score according to the average skin response calculation formula, the presence or absence of irritation was determined according to the patch test result determination table. Table 2 shows the patch test results. As shown in Table 2, for the samples containing 1% (w / v) and 20% (w / v) of the complex natural antioxidants prepared in Preparation Example 9, the average skin reactivity was determined to be 0.19 as irritant-free.

division Time lapse 30 minutes 24 hours 48 hours Responsiveness - 29 30 30 ± One 0 0 + 0 0 0 ++ 0 0 0 +++ 0 0 0 Mean score 0.19 Judgment No stimulation

※ Criteria for Determination of Skin Response in Table 2

1) Negative (-): No irritation

2) Doubtful or slight reaction and erythema (±): Mild erythema that can be detected as unstimulated faint or barely

3) Erythema + Induration (+): Mild erythema, edema and papules with pronounced hard stimulus boundaries

4) Erythema + Induration + Vesicle (++): Measles, erythema, papules and vesicles

5) Erythema + Induration + Bullae (+ + +): Strong stimulation Severe erythema, Alveolar vesicles, Skin formation

In addition, if the mean score is 0.00-0.75, it is non-irritating, 0.76-1.50 is non-irritating, 1.51-2.50 is light stimulation, 2.51-4.00 is medium stimulation, and 4.01 or more is determined as strong stimulation.

3. Feed compositions, food compositions, including complex natural antioxidants, and Cosmetics Preparation of the composition

Production Example 10. (Animal Feed Composition Production Example)

Animal feed composition comprising a composite natural antioxidant prepared in Preparation Example 9 was prepared in the composition of Table 3.

ingredient Component Content (% by weight) Preparation Example 9 One casein 20 Sucrose 40 Corn starch 12 Cocoa butter 8 Corn oil 8 cholesterol One Sodium cholate 0.5 α-cellulose 5 vitamin One mineral 3 Cholinebitartrate 0.5

Preparation Example 11 (Drinking Food Composition Preparation Example)

The beverage food composition containing the composite natural antioxidant prepared in Preparation Example 9 was prepared in the composition of Table 4.

ingredient Component Content (% by weight) Preparation Example 9 0.1 Citric acid 0.1 oligosaccharide 10 Plum concentrate 0.2 Taurine 0.1 Purified water Balance

Production Example 10. (Care cream cosmetic composition production example)

Care cream containing a composite natural antioxidant prepared in Preparation Example 9 was prepared in the composition of Table 5.

ingredient Component Content (% by weight) Preparation Example 9 0.3 Cetostearyl alcohol) 2.7 G.M.S. # 105 2.6 Aracel 165 1.2 Dermofeel BGC 5.0 Meadowfoam seed oil 2.0 Cosmacol EMI 1.0 DC 200 Fluid 100 CST 1.0 Tween # 60 1.0 Aracel 83 1.0 Vitamin E acetate 0.2 Shea butter 0.5 DC 345 Fluid 3.0 E.D.T.A.-2Na 0.02 Urea 10.0 Glycerin 2.0 Butylene Glycol (1,3) 4.0 SC-Glucan Solution 1.0 Na-Lactate 60% 2.0 Sepigel 305 0.7 Spices 0.08 Purified water Balance

Preparation Example 11 (Toner Cosmetic Composition Preparation Example)

Toner including the composite natural antioxidant prepared in Preparation Example 9 was prepared in the composition shown in Table 6.

ingredient Component Content (wt%) Preparation Example 9 0.3 Propylene glycol 3.5 Phenyl trimecicone 0.7 Fiji-40 Hydrogenated Castor Oil 0.4 Fiji-8 Dimethicone 1.0 ethanol 5.0 Disodium ID 0.1 Butylene Glycol 2.5 Triethanolamine 0.15 Spices 0.08 Purified water Balance

In addition to the feed composition, food composition, and cosmetic composition, the antioxidant according to the present invention may be applied to a pharmaceutical composition and the like to exhibit a high antioxidant effect, an increase in immunity, and an anti-aging function.

In FIG. 3, "A" was cultured without 4-HNE treatment and the complex natural antioxidant treatment prepared in Preparation Example 9, and "B" was cultured with 4-HNE for 12 hours, and "C". Is treated with 12 hours of 4-HNE incubation culture and then 12 hours of incubation with the complex natural antioxidant prepared in Preparation Example 9, "D" is 12 hours of incubation treatment with the 4-HNE complex natural prepared in Preparation Example 9 It was incubated for 24 hours with antioxidants. In FIG. 4, "A" is treated for 6 hours with 4-HNE and then cultured for 12 hours with the complex natural antioxidant prepared in Preparation Example 9, and "B" is prepared after 6 hours with culture for 4HNE. 24 hours treatment culture with the complex natural antioxidant prepared in 9, "C" is 6 hours treatment culture with 4-HNE, "D" is the 4-HNE treatment and the complex natural antioxidant prepared in Preparation Example 9 as a control It was cultured without first treatment.

Claims

칡 extract, bean sprout extract, green onion extract, shiitake mushroom extract, oyster mushroom extract, rhododendron extract, golden extract, yellow baek extract, yellow lotus extract, cedar leaf extract, broccoli extract, vinegar extract, triticale extract, eochocho extract, sari extract, sexin Extract, Blackberry Extract, Pine Needle Extract, Knee Root Extract, Injin mugwort Extract, Buttercup Extract, Ginger Extract, Venom Extract, Yacon Extract, Schisandra Extract, Honeysuckle Extract, Grapefruit Extract, Jangsaeng Bellflower Extract, Spirit Extract, Red Vegetable Extract, Plantain Extract, Perilla extract, and one or more extracts selected from the group consisting of wormwood extract, bark skin extract, Gaza extract, licorice extract, nectarine extract, red ginseng extract, Seokgok extract, cilantro extract, spectral extract, gut extract, gujeolcho extract, kkujippong extract , Golden Horse Extract, Golden Primrose Extract, Gilt First complex extract comprising one or more extracts selected from the group consisting of extracts, mandrel extracts, dansam extract, sugar extract, lavender extract, rosemary extract, garlic extract, Ecklonia cava extract, dandelion extract, white paper extract and Bokyeong extract An antioxidant comprising the second complex extract obtained by treating with glycosidase as an active ingredient.

An antioxidant comprising a third complex extract obtained by bioconversion of the second complex extract of claim 1 to a lactic acid bacterium or a specific microorganism as an active ingredient, and characterized by increasing immune activity and suppressing expression of aging-inducing proteins.

The antioxidant according to claim 1 or 2, wherein the first complex extract is extracted by a solvent extraction method or a supercritical extraction method.

The antioxidant according to claim 3, wherein the solvent of the solvent extraction method is any one selected from water, an alcohol having 1 to 5 carbon atoms, and a mixed solvent of water and an alcohol having 1 to 5 carbon atoms.

The antioxidant according to claim 1 or 2, wherein the glycosidase is amylase.

The antioxidant according to claim 1 or 2, wherein the antioxidant is in the form of an emulsion.

The antioxidant of claim 6, wherein the antioxidant in the emulsion form comprises any one of an active ingredient selected from the second or third complex extract, an oily medium, and an emulsion stabilizer.

A feed composition comprising the antioxidant of claim 1.

A food composition comprising the antioxidant of claim 1.

A cosmetic composition comprising the antioxidant of claim 1.