KR101955961B1 - Composition including v. amurensis root extracts for antioxidant activation - Google Patents

Abstract

The present invention relates to a composition for antioxidant activation, which contains an extract of Vitis amurensis root as an active component and contains the extract of Vitis amurensis root having an antioxidative effect and an effect of preventing skin aging by oxidative stress as the active component. The composition for antioxidant activation can solve a problem of cytotoxicity, has no side effects, and also can prevent and control skin diseases, caused by oxidative stress, by protecting skin cells. In addition, it is possible to provide the composition for antioxidant activation which contains the extract of Vitis amurensis root having excellent radical scavenging ability and antioxidation activity, and an excellent protecting effect of skin cells exposed to oxidative stress.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for an antioxidant activity,

The present invention relates to a composition for an antioxidative activity, comprising an extract of a horse radish root as an active ingredient. More specifically, the present invention relates to a composition for antioxidant activity, which comprises, as an active ingredient, an antioxidant effect using radish scavenging ability of a horse radish root extract and an antioxidant extract which is excellent in skin exfoliation protection effect exposed to oxidative stress.

Signs of aging affecting the face and body's skin include, for example, wrinkles, thin wrinkles, thinned skin, stubborn skin, dry skin and itchy skin.

With regard to skin aging, the so-called "endogenous" aging is distinguished from the "exogenous" aging, and the crucial factor for the latter is the effect of exogenous influences, especially ultraviolet (UV) radiation ("photoaging"). Mechanically, reactive oxygen species (ROS) are produced in the course of normal cell metabolism or by physiological processes, in particular by the UVA and UVB components of UV radiation, so that oxidative stress is an endogenous and exogenous It plays a major role in both aging. ROS has a profound effect on the integrity of all cellular biological molecules, such as DNA, proteins and lipids, in connection with UV induced aging of the skin if not inhibited during formation. In particular, overexpression of ROS can cause lipid peroxidation, damage to biomolecules, and effects on cell viability. An immediate consequence, particularly important in photoaging, is the ROS induction of substrate metalloproteinase, which increases the degradation of collagen proteins and creates an imbalance between collagen synthesis and degradation, resulting in skin thinning. So far, strategies for preventing photoaging have been made by reduction of UV exposure, physical protection from UV exposure, and / or application of certain vitamins such as vitamin C or vitamin E.

A major risk factor for skin damage when overexposed to ultraviolet radiation from sunlight is the production of abnormal reactive oxygen species (ROS). Although a certain level of ROS acts as a physiologically important intracellular messenger in the redox signal, the uncontrolled overproduction of ROS induces damage to lipids, proteins and DNA and is involved in the pathogenesis of many human skin diseases . As a result, the damaged DNA contributes to the initiation of apoptosis of skin cells and causes destruction of skin epithelium.

Medical procedures, such as dermal injection and restoration surgery, are also available to reduce the signs of aging. Beauty surgery has been growing in popularity recently to aesthetically increase the appearance of the skin. However, because procedures can be expensive, painful and very invasive, these procedures are not always a desirable option.

Thus, the discovery of antioxidants for safer and more effective skin protection against UV exposure is an important concern for the prevention and control of skin diseases due to oxidative stress.

Under these circumstances, it is urgent to develop a composition of natural materials for prevention and control of skin diseases caused by oxidative stress.

(Patent Document 1) KR 10-2016-0143793 A1

It is an object of the present invention to provide a composition for antioxidative activity containing an extract of Aspergillus oryzae as an active ingredient which can solve the problem of cytotoxicity and which can prevent and control skin diseases through protection of skin cells by oxidative stress without side effects .

Another object of the present invention is to provide a composition for antioxidative activity, which comprises an extract of a hairy root which has excellent ladder clearing ability and antioxidative action and is excellent in protecting skin cells exposed to oxidative stress.

In order to achieve the above object, the composition for antioxidative activity according to one embodiment of the present invention is characterized by comprising an extract of Aspergillus oryzae as an active ingredient, and an extract of Aspergillus oryzae having excellent antioxidative and antioxidative effects against oxidative stress Or a pharmaceutically acceptable salt thereof.

The horse radish root extract may be extracted with a solvent selected from the group consisting of water, alcohols having 1 to 6 carbon atoms, and mixtures thereof.

The skin aging caused by the oxidative stress is at least one selected from the group consisting of skin wrinkles, thinned skin, drooping skin, dry skin and itchy skin.

The antioxidant activity composition may further comprise a silk-tree extract.

The antioxidant activity composition may further comprise a Chinese horned Holly extract.

The pharmaceutical composition according to one embodiment of the present invention may include a composition for antioxidant activity, which comprises the above-mentioned mulberry root extract as an active ingredient.

The food composition according to one embodiment of the present invention may include a composition for antioxidant activity, which comprises the above mulberry root extract as an active ingredient.

Hereinafter, the present invention will be described in more detail.

The composition for an antioxidant activity according to an embodiment of the present invention contains an antioxidant extract as an active ingredient and has excellent antioxidative and antioxidative effects against oxidative stress.

Vitis amurensis Rupr. Is a wild grape belonging to the Vitaceae family, which is mainly distributed in Korea, Japan, and China. Roots and stems have long been used for the prevention and treatment of various diseases in East Asia including Korea and Japan. In recent studies, it has been known that extracts or effective ingredients of roots of hairy loins have various pharmacological effects such as anti-inflammatory, antitumor, anti-aging and neuroprotective effects. In particular, amyloid-induced oxidative stress-blocking effects have been reported, thus providing experimental evidence for the protective effect against Alzheimer's disease. In addition, the seed extracts of Bambalu increased the expression of Nrf2 (nuclear transcription factor erythroid-2-like factor 2), an important regulator of cell defense against oxidative stress, and inducible nitric oxide synthase ) And cyclooxygenase 2, and thus have strong anti-inflammatory effects as well as antioxidant activity. However, there have been few studies on the radical scavenging effect and antioxidant effect on the extracts.

In the present invention, tongue refers to tongue roasted without adding water and roasted as it is, and the roasted roots have a deeper flavor than the roots of general roots, so that the flavor can be enhanced. In addition, Since radical scavenging and antioxidant activity are enhanced, it is much more effective in increasing the antioxidant activity.

The above-mentioned mugwort muscle extract refers to an extract obtained by extracting the mugwort roots using a solvent.

The mulberry root extract is extracted with a solvent selected from the group consisting of water, an alcohol having 1 to 6 carbon atoms, and a mixture thereof, and more particularly, it can be extracted using water, but the present invention is not limited to the above examples.

The extraction solvent may be used in an amount of 2 to 50 times, preferably 2 to 20 times, based on the weight of the sample. For extraction, the sample may be left for 1 to 72 hours for leaching in the extraction solvent, and specifically for 1 to 24 hours. The extraction temperature may be from 10 캜 to 100 캜.

After extraction, the extract can be fractionated by sequentially applying a fresh fraction solvent. As the fraction solvent used for fractionation, the solvent is at least one selected from the group consisting of water, hexane, butanol, ethyl acetic acid, ethyl acetate, methylene chloride, and mixtures thereof, preferably ethyl acetate or methylene chloride.

After the extract or fraction is obtained, a method such as concentration or freeze-drying may be further used.

On the other hand, the mugwort muscle extract may be a fermented product obtained by preparing an extract using a mugwort muscle. The fermentation of the mulberry root extract has the advantage that it can be used stably at a relatively high concentration due to low cytotoxicity.

Examples of the microorganism used for the fermentation include Lactobacillus salivarius, Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus rhamnosus, Lactobacillus plantarum Lactobacillus plantus, Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus fermentum, Lactobacillus paracasei, Lactobacillus casei, Lactobacillus delbrueckii, Such as Lactobacillus reuteri, Lactobacillus buchneri, Lactobacillus gasseri, Lactobacillus johonsonii, Lactobacillus kefir, and the like, lactic acid bacteria such as lactobacillus kefir, Lactococcus lactis, Lactococcus plantarum, Lactococcus raffinolactis, Enterococcus faecalis, Enterococcus faecium, Streptococcus thermophilus, Streptococcus spp. Bifidobacterium bifidum, Bifidobacterium bifidum, Bifidobacterium bifidum, and Bifidobacterium bifidum, such as Leuconostoc lactis, Leuconostoc mesenteroides and the like, Bifidobacterium species such as Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium longum, Bifidobacterium pseudolongum, Bifidobacterium themophilum, Bifidobacteria such as Bifidobacterium adolescentis, and the like, Lactobacillus casei, Lactobacillus rhamnosus, Bifidobacterium bifidum bifidobacterium, Bifidobacterium breve bifidobacterium, and Lactobacillus species such as Lactobacillus casei, Lactobacillus rhamnosus, And Lactobacillus acidophilus. ≪ Desc / Clms Page number 2 >

The composition for antioxidative activity may further comprise a silk-tree extract.

The silk tree is a tree symbolizing a couple's gold thread. It is also called a joining tree, a sum-comer, Yaesu, and Yu Jung-soo. Because of this, I planted a lot of trees growing in mountains and fields in the garden as a garden. Because it was a tree used to make the handle of a swordfish, it is called a silkworm tree.

Also, the stem of the tree is bent or slightly leaned. It is 3 ~ 5m in height, large branches spread sparsely and small branches have ridges. There are 2 or 3 scars of winter snow, but it is hardly visible. Leaves are alternate and double leafy leaves. The small leaf is like a sickle and has a long oval shape with uneven left and right sides and flat edges. The small leaf has a length of 6 to 15 mm and a width of 2.5 to 4.0 mm. There are no hairs on both sides or hairs on the back vein.

The flower is pink to bloom in June or July, and it runs in 15-20 squares on the tip of a small branch. Calyx and corolla are shallowly divided into 5 pieces and turn green. Surgery is about 25, it comes out long and the upper part is scarlet. It is because of the color of the operation that the flower looks red. Fruits are ripened in late September to early October and are flat pods, about 15cm long and contain 5 or 6 seeds. The unusual thing is that the nerves and Mimosa are attached to the external stimuli, but when they are sunny, the leaves are folded facing each other.

In the case of using the extract of the Japanese apricot fruit, the radical scavenging effect is increased according to the synergistic effect of the mixture. Therefore, it is possible to obtain an excellent effect on the antioxidant activity with a smaller content.

The antioxidant activity composition may further comprise a Chinese horned Holly extract.

It is also called aphids. It grows on the shore of a low mountain on the beach. It is 2 ~ 3m in height and has no branches. Leaves are slanting, thick, shiny, oval hexagonal, and each point is sharp. Flowers bloom in 4 or May, and there are fragrance, and 5 or 6 of them hang on a leaf axil. The pistil has no style, and the stigma is slightly elevated to divide into 4 pieces and become black. Fruits are round and have a diameter of 8 to 10 mm and ripen red in 9 or October. There are four seeds, ovate and mature.

Leaves are used for strong winds, tangles, etc. Fruits are used for consonants, The breeding takes the seeds ripe in autumn and sows in spring. It is distributed in South Korea (South Jeolla Province) and South China.

In the case of using the above extracts of rape seeds and Rhododendron thunbergii in combination, the radical scavenging effect is increased due to the synergistic action by the mutual reaction of the respective extracts, and the synergistic effect Lt; / RTI >

Preferably, the composition for antioxidant activity comprises 1 to 15 parts by weight of a Japanese apricot fruit extract and 10 to 40 parts by weight of a rhododendron extract in 100 parts by weight of the extract. In the above range, a synergistic effect with a critical significance is expressed by the synergistic effect due to the interaction of the respective extracts, and when the concentration is outside the above range, the synergistic effect is significantly lowered or hardly occurs.

More preferably, the composition for antioxidative activity may comprise 1 to 15 parts by weight of the extract of Zaragoza japonica, 10 to 40 parts by weight of the rhododendron extract and 1 to 15 parts by weight of the extract of Zygomycota .

By the above range, the radical scavenging effect can be activated by a higher synergistic effect due to the mixing action of each extract, so that the antioxidative activity effect can be enhanced.

The Kirilow indigo (Kirilow indigo) grows frequently on the shore, about 1m high and has many shoots from its roots. Several stalks are rising and there are line-shaped projections on the branches. Leaves are alternate and odd numbered haploid leaf. Small leaves are 7 ~ 11 thick, round, elliptical or inverted egg-shaped, with hairs on both sides. A red-purple flower about 2cm in length blooms in May or June, and it forms a flower-like flower on the axilla. On the other hand, calyx is about 3mm long and there is hairs on the outer surface of the board (旗.). The fruit is in the shape of a string with a cone and ripens in October. It is distributed all over the country excluding Manchuria, China and North Hamgyong Province. It is also used for beekeeping and farming. It is called "coreana". It is called "coreana." It is a species similar to a flower, and its ear is twice as long as a leaf.

Preferably, the antioxidant activity composition further comprises an extract of Yellow melilot, a extract of Pachysandra terminalis and a extract of Hosta longipes. When the extract is further included, the strong flavor of the root is neutralized and the flavor is improved by mixing with other extracts, so that it can be provided as a food composition having more favorable taste.

Yellow melilot is also called Mitsutosugi or Melitoseus. It is the northern part of China. It grows in low-lying meadows. It is 60 to 90 cm high and branches are split and smelled when dried. There are hairs in my childhood, but they disappear later. Leaves are alternate phyllotaxis of three small leaves. Leaves are long oval or inverted, and have small sawtooth edges. The flower blooms in July or August and it is yellow, and it runs tightly with gunshot at the tip of the branch or the axilla. Calyx has hairy hairs. The fruit is egg-shaped, has no hair, and is black. The entire paste is used for heat, heat, nephritis, etc. It is called white algae (M. alba) that flowers are smaller and white. Everything that grows as a grass resource grows wild and grows.

The Pachysandra terminalis grows in the shade of the tree, with the main stem hanging sideways, the tip is straight, green, and initially there is hairy hair but gradually disappears. It grows up to about 30cm in height. Leaves are alternate but they are gathered in the upper part and look like inverted egg, with sawtooth on the upper part. Leaf hairs on leaf vein and narrowed lower part to become petiole. Flowers also bloom in April or May and are white and run on aquatic inflorescences. The female flower runs slightly under the flower head and the male flower runs on the upper part. Calyx is divided into 4 pieces and there are no petals. The stamen is divided into 3 to 5 and the style is divided into 2 pieces. The fruit is ovate, egg-shaped and has no hairs on the surface. It is distributed in Korea, Japan, Sakhalin Island, China.

Hosta longipes grow well in mountain streams or wet places and are 30 to 40 cm high. All leaves grow from the roots and grow at an angle. Leaves are elliptical, pointed end, and have 8 or 9 veins. The flower is light purple and blooms in July or August. It is biased to one side and runs on a gun. The flower stem is 30 to 40cm long. The bract is thin, purplish white and almost similar to the peduncle. The corolla is divided into 6 pieces, and the pieces are bent back a little, and 6 stamens and 1 pistil come out of the flower for a long time. Fruit is long oval with capsule. Seeds are black with wings on the edge. It is edible for soft season and planted for ornamental purposes. Wild species are distributed in Korea, Japan, and China. Vivicula is a kind of horticultural variety and has been popular as a garden plant in foreign countries.

Preferably, the composition for antioxidant activity comprises 1 to 3 parts by weight of an electric sphagnum extract, 5 to 15 parts by weight of a preservative extract, and 5 to 15 parts by weight of a non-vegitable extract, based on 100 parts by weight of the extract.

According to the above range, it is possible to provide a functional food composition which is improved in radical scavenging effect and antioxidative action of the mulberry root extract and has an improved antioxidative activity effect and excellent palatability, It is highly advantageous to be widely distributed.

More preferably, the composition for antioxidative activity comprises 1 to 15 parts by weight of a rhododendron japonica extract, 10 to 40 parts by weight of a rhododendron extract and 1 to 15 parts by weight of a root extract, relative to 100 parts by weight of the horse root extract, 1 to 3 parts by weight of the extract, 5 to 15 parts by weight of the succulent herb extract, and 5 to 15 parts by weight of the non-vegetable extract.

In the above range, the antioxidant activity is remarkably enhanced due to the synergistic effect due to the mixing action of the respective extracts, and the flavor according to each combination is very high, and thus, it can be provided as a food composition having high functionality and palatability.

The pharmaceutical compositions of the present invention may comprise a pharmaceutically acceptable carrier. Compositions comprising a pharmaceutically acceptable carrier can be of various oral or parenteral formulations. In the case of formulation, it can be prepared using diluents or excipients such as fillers, extenders, binders, humectants, disintegrants, surfactants and the like which are usually used. Solid formulations for oral administration may include tablet pills, powders, granules, capsules and the like, which may contain one or more excipients such as starch, calcium carbonate, sucrose or lactose lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like may also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the non-aqueous solvent and the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

In addition, the pharmaceutical composition of the present invention may be formulated into tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations and suppositories And can have any one of the formulations selected.

The body lotion muscle extract of the present invention has been used for edible and medicinal purposes from the past, and there is no particular limitation on its administration dose, and there is no particular limitation on its absorption, body weight, age, sex, health condition, diet, Rate, severity of the disease, and the like. In general, the extracts of horse mackerel can be administered in an amount of about 10 to 1000 mg per 1 kg of body weight, more specifically about 50 to 500 mg per 1 kg of body weight. The pharmaceutical composition comprising the extract of the present invention as an active ingredient is prepared in consideration of an effective dose range. The formulated unit dosage form is prepared by the judgment of an expert who observes or observes the administration of a drug, Depending on your needs, you may use a specialized dosage regimen or several doses at regular intervals.

The present invention also provides a quasi-drug composition for antioxidant activity, which comprises an extract of a horse mackerel as an active ingredient. The production and composition of the above-mentioned flesh root extract are as described above. More specifically, the mulberry root extract of the present invention can be added to the quasi-drug composition for the purpose of antioxidative activity.

In the present invention, the term 'quasi-drug' means a fiber, a rubber product or the like used for the purpose of treating, alleviating, treating or preventing a disease of a human or an animal, a weak action against the human body, Used for the purpose of diagnosing, curing, alleviating, treating or preventing diseases of human or animal, which are not machinery, similar products, products for sterilization, insecticide and similar uses for the prevention of infectious diseases. Machinery, or apparatus, and that is not an apparatus, machine or apparatus of an article used for the purpose of causing pharmacological effects on the structure or function of a person or animal.

When the mugwort extract of the present invention is used as a quasi-drug additive, the mugwort extract can be used as it is, or it can be used in combination with other quasi-drugs or quasi-drugs, and can be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be appropriately determined depending on the purpose of use.

The quasi-drug composition of the present invention is not limited thereto, but may be a disinfectant cleaner, shower foam, gagrin, wet tissue, detergent soap, hand wash, humidifier filler, mask, ointment or filter filler.

The present invention also provides a health functional food composition for antioxidative activity comprising an extract of a horse mackerel as an active ingredient. The production and composition of the above-mentioned flesh root extract are as described above. More specifically, the hair follicle extract of the present invention can be added to a health functional food composition for the purpose of antioxidant activity.

When the mugwort muscle extract of the present invention is used as a health functional food additive, the mugwort muscle extract may be directly added or used in combination with other health functional foods or health functional food ingredients, and may be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be appropriately determined depending on the purpose of use.

There is no particular limitation on the kind of the health functional food of the present invention. Examples of the health functional food to which the above extraction mixture can be added include meat products, sausages, breads, chocolates, candies, snacks, confectionery, pizza, ramen noodles, other noodles, gums, dairy products including ice cream, , A drink, an alcoholic beverage, and a vitamin complex, and may include foods used as food for animals, which may include all health functional foods in the conventional sense. In addition to the above, the health functional food composition of the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, , Alcohols, carbonating agents used in carbonated drinks, and the like. It may also contain flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks.

According to the composition for antioxidant activity of the present invention, the problem of cytotoxicity can be solved, and skin diseases can be prevented and controlled through protection of skin cells due to oxidative stress, without side effects.

In addition, it is possible to provide a composition for antioxidant activity, which comprises radish root extract having excellent radar scavenging ability and antioxidation activity and excellent protecting effect on skin cells exposed to oxidative stress.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 relates to the effect of the carrot muscle extract on DPPH and ABTS radical scavenging activity.
Fig. 2 relates to the effect of the carrot muscle extract on cell viability of H ₂ O ₂ -treated HaCaT human dermal keratinocytes.
Figure 3 relates to preventing H ₂ O ₂ -induced cell apoptosis by Haematoderm extract from HaCaT human dermal keratinocytes.
Fig. 4 relates to the expression of bax and Bcl-2 in HaCaT human dermal keratinocytes treated with H ₂ O ₂ and the effect of the bark extract on the MMP levels.
FIG. 5 relates to the protection of H ₂ O ₂ -induced DNA damage and ROS production by the carrot muscle extract in HaCaT human dermal keratinocytes.
Figure 6 relates to the induction of NQO-1, HO-1 and Nrf2 expression by Haematoderma root extract in HaCaT human dermal keratinocytes.

Hereinafter, embodiments of the present invention will be described in detail so that those skilled in the art can easily carry out the present invention. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

[Preparation Example 1: Preparation of a muscle root extract]

Mud roots were obtained from CANA Co., Ltd. (Busan, Republic of Korea) and extracted with distilled water at 100 ° C for 3 hours to obtain mud roots extract. The extract was filtered through filter paper (Whatman No. 3 filter paper, Whatman International Ltd., Maidstone, England), concentrated and the concentrate was lyophilized and powdered. Dissolve the extracts in sterile distilled water at a concentration of 100 mg / ml (stock solution), filter with a Minisart® syringe filter (0.2 μm, Sartorius AG, Weender Landstr. Germany) and store at -20 ° C until use And diluted appropriately according to the method of experiment.

[Preparation Example 2: Cell culture]

HaCaT human dermal keratinocytes were obtained from Dulbecco-modified eagle medium (Dulbecco's Modified Eagle Medium) containing 10% fetal bovine serum (FBS), 100 U / ml penicillin and 100 mg / ml streptomycin DMEM, Gibco-BRL, Grand Island, NY, USA) at 37 ° C and 5% CO ₂ .

[Experimental Example 1: Determination of Phenol Content and Antioxidant Potential of Angora Root Extract]

1. Experimental Method

Total phenol content

The total phenol content in the water extract of V. amurensis root (WEVA) was determined according to the Folin-Ciocalteu method and was determined by adding gallic acid (Sigma-Aldrich Chemical Co., St. Louis, MO, USA) was used as a reference material. To this end, 1 ml of 2% Na 2 CO 3 was added to 50 μl of the extract of Muriru diluted to the appropriate experimental concentration, and the mixture was allowed to stand at room temperature for 2 minutes. Then, 50% Folin-Ciocalteu reagent (Sigma-Aldrich Chemical Co.) (Molecular Devices, Sunnyvale, Calif., USA), and the absorbance at 750 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader Respectively. Gallic acid was used as a calibration curve and total phenol content was expressed as mg gallic acid equivalents (GAE) / g of sample dry weight (DW).

Measurement of FRAP activity

In this method, the ferric tripyridyltriazine (Fe ³⁺ -TPTZ) complex is reduced to a blue ferrous tripyridytriazine (Fe ²⁺ -TPTZ) by a reducing substance in the acidic pH range , Which is based on the principle that the absorbance increases at 593 nm, and it is a test method designed considering the fact that most antioxidants have a reducing power. In order to measure the antioxidative activity of FRAP extract, 300 mM acetate buffer (pH 3.6), 10 mM 2,4,6-tripyridyl-s-triazine dissolved in 40 mM HCl (2, 1: 1 (v / v / v) were mixed with 20 mM FeCl ₃ .6H ₂ O to prepare FRAP reagent . Next, 10 μl of the diluted sample solution and 240 μl of the FRAP reagent were mixed and incubated at 37 ° C for 5 minutes, and the absorbance was measured at 593 nm. Positive controls were prepared using standard trolox and 2,3-butyl-4-hydroxyanisole (BHA, Sigma-Aldrich Chemical Co.) The results were expressed as mg trolox / g of sample dry weight (DW).

DPPH radical scavenging ability

The DPPH radical scavenging ability, which is a method of measuring radical scavenging effect by donating electrons to DPPH radical scavenging, was investigated according to the method of Guo et al. To this end, 100 μl of a diluted sample of ruminant muscle extract was added to 150 μl of 0.4 mM DPPH (Sigma-Aldrich Chemical Co.) solution, reacted at 37 ° C. for 30 minutes, and then analyzed by ELISA reader at 518 nm Absorbance was measured. The antioxidant activity of the DPPH radical scavenging activity of the prescription extract samples of the control group was shown by the absorbance value of the reactants, trolox was compared with the positive control group, and the negative control group was methanol Was calculated based on the following equation using the following equation.

DPPH radical scavenging activity (%) = 100 - [(OD of sample / OD of control) 100]

ABTS · + radical cation abatement

Extracts of ABTS free radicals produced by reaction with potassium persulfate and using ABTS radical cation-scavenging ability based on the principle of decolorizing with a radical-specific index cyan color The antioxidant activity was measured by Re method. Namely, 7 mM ABTS (Sigma-Aldrich Chemical Co.) and 2.45 mM potassium persulfate were mixed at a ratio of 1: 1 and reacted at room temperature for 24 hours to form a radical. The ABTS solution was diluted with phosphate buffered saline (PBS, pH 7.4) to an absorbance of 0.7 ± 0.02 at 734 nm. A total of 10 μl of the sample diluted to the experimental concentration was added to 190 μl of ABTS radical cationic solution and reacted for 6 minutes at room temperature. The absorbance was measured at 734 nm. Antioxidant activity was measured using trolox as a positive control. Respectively.

ABTS radical scavenging activity (%) = 100 - [(OD of sample / OD of control) 100]

2. Results

Total phenolic content
(Total phenolic content) FRAP Sample mg GAE / g Sample mgTrolox / g Root Extract 56.37 + - 6.57 * Root Extract 49.03 + - 5.61 BHA 860.46 + - 128.63 BHA 878.61 + - 128.03

In general, many of the plant-derived physiologically active components are phenolic compounds, which are general compounds of aromatic compounds having a phenolic hydroxyl group as a secondary metabolite (22, 23). Therefore, quantitative evaluation of phenol compounds as a comparative index for comparing the antioxidant capacity of certain plant extracts is the most basic process. The total phenolic content of the mulberry root extract prepared as a basic method for the standardization of the mulberry root extract used in this study was investigated. The value obtained from the calibration curve obtained by selecting gallic acid as the standard solution was 56.37 mg mg GAE / g (Table 1). In addition, the reducing power of the mulberry root extract by the PRAP method was 49.03 mgTrolox / g, and the results of DPPH and ABTS radical scavenging activities were as shown in FIG.

[Experimental Example 2: Evaluation of the efficacy of the extracts from the carrot root on the cytotoxicity caused by oxidative stress]

1. Experimental Method

Cell viability measurement

To investigate the effect of the extract on the cytotoxicity caused by oxidative stress, HaCaT cells were treated with H ₂ O ₂ (Sigma-Aldrich Chemical Co.) at an appropriate concentration for 24 hours, H ₂ O ₂ was treated for 24 hours. The treated cells were treated with 0.5 mg / ml of 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (3- -yl) -2,5-diphenyltetrazolium bromide, MTT, Sigma-Aldrich Chemical Co.), the formed formazan was dissolved in dimethyl sulfoxide (DMSO) And the absorbance at 540 nm was measured. The cell viability of the control group was expressed as a percentage, and N-acetyl cysteine (NAC, Sigma-Aldrich Chemical Co.) was used as a control.

Morphological Observation of Apoptosis Induction

In order to observe morphologic changes of nuclei that are specific to apoptosis induced by oxidative stress, HaCaT cells cultured under various conditions were treated twice with phosphate-buffered saline (PBS) After washing with water, 3.7% paraformaldehyde fixing solution (Sigma-Aldrich Chemical Co.) diluted in 0.1 M phosphate buffer (pH 7.2) was treated for 1 hour. After fixed cells were washed with 4,6-diamidino-2-phenylindole diluted to a concentration of 2.5 μg / ml in 0.05 M phosphate buffer (pH 7.2) DAPI, Sigma-Aldrich Chemical Co.) solution at room temperature for 20 minutes. The cells were washed with PBS, dried, and then examined for their nuclear morphology under a fluorescence microscope (Carl Zeiss, Oberkochen, Germany).

Quantitative analysis of induction of apoptosis by flow cytometry

Cycle TEST PLUS DNA REAGENT Kit (Becton Dickinson, San Jose, CA, USA) was used for the quantitative analysis of the effect of the extracts on the induction of apoptosis of HaCaT cells by oxidative stress. The prepared cells were fixed according to a protocol and reacted with propidium iodide (PI) solution at 4 ° C for 30 minutes in a dark room. These cells were separated into single cells using a 35-mm mesh and at least 10,000 cells per test group were applied to a flow cytometer (Becton Dickinson) to determine the histogram according to intracellular DNA content Cells belonging to the sub-G1 phase were counted as apoptosis-induced cells.

Analysis of changes in mitochondrial membrane potential (MMP, Δψm)

To determine whether mitochondrial function was impaired by oxidative stress, the degree of MMP changes was measured. To this end, HaCaT cells cultured under various conditions were supplemented with 10 μM of 5,5 ', 6,6'-tetrachloro-1,1', 3,3'-tetraethyl-imidacaborous iodide , 6,6'-tetrachloro-1,1 ', 3,3'-tetraethyl-imidacarbocyanine iodide, JC-1, Sigma-Aldrich Chemical Co.) was reacted for 20 minutes at room temperature. After the reaction was completed, the supernatant was removed, the cells were suspended by adding PBS, and the changes of MMP were measured by applying a flow cytometer.

Measurement of ROS generation change

To confirm the extent of ROS production in HaCaT cells due to oxidative stress and the degree of blocking effect by the extract of cornucopia, the cells were incubated with 10 μM of fluorescent probe, 2 ', 7'-di-chlorodihydrofuro (2 ', 7'-di-chlorodihydrofluorescein diacetate, DCF-DA, Molecular Probes, Leiden, Netherlands) for 20 min and examined for changes in the production of ROS under fluorescence microscopy. In order to quantitatively compare the production of ROS in cells prepared under the same conditions, the changes of the ROS value were analyzed by applying a flow cytometer to the cells after the completion of the reaction.

Protein isolation and Western blot analysis

In order to observe the expression of specific genes according to the oxidative stress protection of the extracts, the cells were treated with an appropriate amount of a lysis buffer (25 mM Tris-Cl (pH 7.5), 250 mM NaCl, 5 mM ethylenediamine tetra (1% Nonidet-P40, 1 mM phenymethylsulfonyl fluoride, 5 mM dithiothreitol) was added to the reaction mixture, followed by reaction at 4 ° C for 1 hour or longer. Proteins in the supernatant were separated and the same amount of protein was electrophoresed using sodium dodecyl sulfate (SDS) - polyacrylamide gel. The protein contained in the gel was transferred to a polyvinylidene difluoride membrane (Schleicher and Schuell, Keene, NH, USA) and the membrane was immersed in 5% skim milk solution Blocking was performed on nonspecific proteins by time treatment. And Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) and Cell Signaling Technology, Inc. (Danvers, MA, USA) and incubated at room temperature for 2 hours or longer. The cells were washed with PBS-T solution (PBS with Tween 20) and reacted at room temperature for 1 hour . After the reaction was completed, enhanced chemiluminescence (ECL) solution (Santa Cruz Biotechnology Inc.) was applied in the dark room, and then exposed to X-ray film to analyze changes in specific protein expression.

Statistical analysis

In order to test the significance of the test results, ANOVA was performed and Duncan's multiple range tests were performed at p <0.05. The results were expressed as mean ± standard deviation (SD).

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, Of the right.

2. Experimental results

In HaCaT keratinocytes, H ₂ O ₂ Protective Effects of the Antioxidative Stress on the Antioxidant-Induced Oxidative Stress

In order to establish the experimental conditions for the evaluation of in vitro antioxidative capacity using HaCaT cells, the effect of H ₂ O ₂ and the mulberry root extract on the survival of HaCaT cells was investigated. Various concentrations of the HaCaT cells To this _{H 2 O 2 (10 ~ 500} μM) , and wild grape root extract (0.25 ~ 2.0 mg / ml) after 24 hours incubation at the processing conditions result subjected to the MTT assay, H ₂ O ₂ treatment The survival rate of HaCaT cells was significantly decreased with increasing concentration, and showed about 50% viability in 500 μM treatment group (data not shown). In the case of the mulberry root extract, the survival rate was not significantly changed at the concentration of 1.5 mg / ml or less, but the survival rate was about 85% in the 2.0 mg / ml treated group (FIG. 2A). Therefore, 500 μM of H ₂ O ₂ was set as the oxidative stress condition, and the cytotoxic protective effect of the extract of the duckweed was investigated. If sikyeoteul exposing wild grape root extract before to process the H ₂ O ₂ as 1 hour as in 2B, H ₂ O ₂ is increased wild grape cell viability in concentration dependent manner the muscle extract as compared to the only treatment group (0.75 mg / ml And about 67% and 87%, respectively, in the 1.0 mg / ml bark extract extract group), indicating that the bark extract was able to improve the cell viability by H ₂ O ₂ treatment. In the pretreatment group of 10 mM NAC used as a control group, the effect was similar to the treatment concentration of 1.0 mg / ml of the myrtle muscle extract treatment. Therefore, all subsequent experiments were carried out on HaCaT cells pretreated with 1.0 mg / ml bovine muscle extract for 1 hour and exposed to 500 μM H ₂ O ₂ for 24 hours.

The Effect of Radix Root Extract on the Induction of Apoptosis of HaCaT keratinocytes by Oxidative Stress

We investigated whether the protective effect of Haemaphylococcus aureus extract against oxidative stress was related to inhibition of apoptosis induction. For this purpose, the effect of chromatin condensation on the formation of apoptotic bodies, specifically observed in the nuclei of apoptotic cells, was investigated through DAPI staining. As shown in FIG. 3A, a typical nucleus morphological change indicating that apoptosis was induced in HaCaT cells treated with H ₂ O ₂ was observed, but this phenomenon was hardly observed in the pretreatment group .

On the other hand, the death of cells by oxidative stress is related to the activity of caspase cascade, and in the induction of caspase activity-dependent apoptosis, the activity of effector caspase (ADP-ribose) polymerase (PARP). Therefore, in order to investigate whether the oxidative stress inhibitory effect of mulberry muscle extract was due to the inhibition of caspase activation-induced apoptosis induction, the expression of PARP was observed. As a result, only H ₂ O ₂ In a HaCaT cell cultured under typical conditions, a typical PARP fragmentation phenomenon was observed, and this phenomenon was completely suppressed in the pretreatment conditions of the horse radish root extract (FIG. 3B). In addition, caspase-3, which is a typical effector caspase associated with PARP fragmentation, and caspase-9, an initiator caspase of caspase-3, Type expression showed a marked decrease in H ₂ O ₂ alone treatment group, but remained at the control level in the pretreatment group of duck muscle extract (FIG. 3B). In order to quantitatively analyze the inhibition of induction of apoptosis by HaCaT cells induced by oxidative stress in these mulberry root extracts, flow cytometry was carried out. In the cells treated with H ₂ O ₂ alone, The frequency of cells belonging to the sub-G1 phase corresponding to the apoptosis-inducing group was about 28.7%, whereas that of the control group of the mulberry muscle extract was about 6.3% (FIG. 3C). Therefore, if the decrease in the survival rate of HaCaT cells induced by oxidative stress is related to the induction of apoptosis, the hairy root extract blocks the caspase activity pathway, which is a key to induce apoptosis, Respectively.

The Effect of Antioxidant Extract on the Mitochondrial Damage of HaCaT Keratinocyte by Oxidative Stress

Previous studies have shown that induction of apoptosis by oxidative stress is directly related to intrinsic apoptosis pathway caused by functional impairment of mitochondria. The Bcl-2 family is the most important gene to control apoptosis induced by these mitochondrial dysfunctions. Bcl-2 family proteins induce apoptosis Or inhibiting factors. Currently, the most representative method of measuring mitochondrial function is to examine whether the MMP value changes, and the decrease in the MMP value is an indicator that mitochondrial function is impaired. Therefore, we investigated whether mitochondrial dysfunction is linked to induction of apoptosis by HaCaT cells induced by oxidative stress, and whether or not it can block it. As shown in FIG. 4A, the disappearance of MMP in HaCaT cells cultured in the medium containing H ₂ O- ₂ was 41.9%, which was about 2.9 times higher than that of the control group , And 18.3% in the pretreatment group. In addition, the expression of pro-apoptotic Bax inducing mitochondrial dysfunction, which was increased by treatment with H ₂ O ₂ , was increased while that of anti-apoptotic Bcl-2 , But the expression of the two Bcl-2 family proteins remained at the control level by pretreatment of the bark extract (Fig. 4B). These results demonstrate that the extracts of horseshoe muscle inhibit the induction of apoptosis by inhibiting mitochondrial dysfunction by inhibiting the expression of Bcl-2 family members by H ₂ O ₂ .

The Effect of Hairy Root Extract on DNA Damage of HaCaT Keratinocyte by Oxidative Stress

Next, we investigated whether the cell death protection effect of oxidative stress of the duckweed extracts directly related to the production of ROS and DNA damage inhibition was observed. The reason for this investigation is that most of the ROS produced in the cell is associated with mitochondrial dysfunction, and the resulting apoptosis is linked to DNA damage. By targeting HaCaT cells with H ₂ O ₂ is processed through the DCF-DA staining For this investigation whether to generate ROS result starts to the generation of ROS increase within handle 30 minutes H ₂ O _2, 6 hours after the peak (Data not shown). However, when H ₂ O ₂ was pretreated for 1 hour and then treated for 6 hours, the extracts showed a strong antioxidative effect, but not to the level of the control group. However, fluorescence microscopy and flow cytometry (Figs. 5A and B). Therefore, we investigated the effect of bark extract on the phosphorylation of γH2AX protein (serine 139), a marker of DNA double helix damage, in order to confirm whether the antioxidative effect of the bark extract was related to the DNA damage block by oxidative stress. Immunoblotting of FIG. 5C showed that phosphorylation was greatly increased in HaCaT cells exposed to H ₂ O ₂ without significant change in the overall expression of γH2AX protein, but this phenomenon was observed in pretreated cells I could not. Therefore, it was found that the protective effect of DNA damage caused by the mulberry root extract was directly related to the blocking of ROS production.

Influence of Extracts on the Expression of Nrf2 and Nrf2 Related Gene in HaCaT Keratinocyte

Nrf2 is a redox-sensitive transcription factor and is known to regulate the expression of various antioxidant enzymes. In the absence of an oxidative defense signal, Nrf2 is complexed with Kelch-like ECH-associated protein 1 (Keap1), an inhibitor of Nrf2, in the cytoplasm. However, when Nrf2 is separated from Keap1 by activation signal of Nrf2, (HO-1) and NAD (P) H-quinone oxidoreductase-1 (NQO-1) by binding to the antioxidant response element (ARE) site of the corresponding gene promoter Increases the transcriptional activity of antioxidant enzymes. Therefore, the discovery of novel substances for activating this signal system can be applied not only to oxidative skin damage but also to prevent and treat various degenerative and metabolic diseases associated with oxidative stress. In order to investigate whether the protective effect of oxidative stress on the oxidative stress on the Nrf2 signal transduction system was examined, the HaCaT cells cultured on the medium supplemented with the extracts of Nilfu, Nrf2, HO-1 and NQO-1 were examined. As shown in Western blot analysis shown in FIG. 6, the expression of Nrf2 was increased with the decrease of Keap1 expression and the expression of HO-1 and NQO-1 was increased . Therefore, it was found that at least Nrf2 pathway was involved in the antioxidative activity of Haemaphylococcus aureus extracts in HaCaT cells.

3. Experimental Conclusion

It was found that the water extract of mulberry root, preferably water-soluble extract of mulberry juice, significantly inhibited the survival rate of HaCaT cells due to H ₂ O ₂ , and that this was due to inhibition of induction of apoptosis. In addition, the water - soluble extracts of myrrh inhibited mitochondrial dysfunction caused by oxidative stress and inhibited excessive ROS production. The water - soluble extracts from the mulberry juice also inhibited DNA damage caused by oxidative stress, and the antioxidant activity of the mugwort water - soluble extracts was at least related to the increase of Nrf2 signaling activity. In view of the results of the above experiment, it was found that the extract of Myrtle muscle has an excellent protective function of keratinocytes and that the antioxidant activity of Myrtle muscle extract is due to the antioxidant activity.

[Preparation Example 3: Preparation of mixed extract]

1. Preparation of rape tree extract (AF)

The silkworms were washed, dried and then ground. The pulverized material was mixed with purified water and maintained at 98-100 ° C for 2 hours. After cooling, it was filtered with Watman filter paper to obtain a filtrate.

2. Manufacture of other plant extracts

(BF), CF, DF, EF and FF were prepared by the same method as that of A. japonica extract.

3. Preparation of mixed extract

(DF), succulent extract (EF), and non-vivax extract (FF) were prepared by the following method. The extracts of NF, AF, BF, CF, Of Table 2 were mixed to prepare a mixed extract.

MX1 MX2 MX3 MX4 MX5 MX6 MX7 MX8 MX9 MX10 NF 100 100 100 100 100 100 100 100 100 100 AF 0.5 One 10 15 20 10 10 10 10 10 BF 5 10 25 40 50 25 25 25 25 25 CF 0.5 One 10 15 20 10 10 10 10 10 DF - - - - - 0.5 One 2 3 5 EF - - - - - One 5 10 15 20 FF - - - - - One 5 10 15 20

(Unit: parts by weight)

[Experimental Example 3: Confirmation of anti-inflammatory effect of mixed extract]

1. Arthritis inhibitory effect experiment

In order to confirm the synergistic effect due to the use of the above-mentioned mixed extracts, the above-mentioned antioxidative activity-inhibiting effect of NF-κB on DNA damage and Nrf2 signaling activity was compared.

In the same manner as described above, the DNA damage inhibition and the degree of Nrf2 signaling activity increase were evaluated in the experimental group administered with each of the mixed extracts (MX 1 to MX 5) for 3 weeks, and the results were compared with the results of the above extracts. In order to directly reveal the effects of each of the mixed extracts, the extracts and each of the mixed extracts were administered at a dose of 10 mg / kg once a day by reducing the dose to 10% of the previous dose.

 In addition, the inhibition of the DNA damage of the above-mentioned mulberry root extract (NF) and the increase effect of the Nrf2 signal system activity were fixed at an index of 5, and the results of the use of the above mixed extracts were expressed as an index of 1 to 10 in comparison. The higher the number, the better the inhibitory activity and the better the antioxidant activity.

The results are shown in Table 3 below.

MF MX1 MX2 MX3 MX4 MX5 DNA damage prevention 5 4 9 9 9 5 Nrf2 signaling activity 5 4 8 9 9 6

(Unit: index)

Referring to Table 3, it can be seen that a synergistic effect is exhibited depending on the use of the mixed extract. Especially, the effect of antioxidant activity is greatly increased by MX 2 to MX 4.

Therefore, it can be seen that the synergistic effect of the combination of the extracts in the range of MX 2 to MX 4 is higher than that of the simple combination due to the synergistic action by mixing the respective extracts.

Thus, it can be seen that the present invention can exert an excellent effect on blocking of DNA damage and increase of Nrf2 signaling activity by using less amount of the mixed extract. Therefore, the antioxidative activity effect can be obtained at a relatively low concentration, so that a product containing a composition for antioxidative activity that can be taken for a long time with few side effects can be produced.

2. Sensory Evaluation

The sensory properties of the flavor and taste of each combination were evaluated so that the composition containing the noodle root extract (NF) and each of the mixed extracts could be provided as a functional food composition.

Therefore, the extracts of Neroderma root (NF) and mixed extracts MX 3 and MX 6 to MX 10 were prepared as tea beverages, and the sensory evaluation of 20 adult male and female persons was carried out. The sensory evaluation was evaluated by an index of 1 to 10 according to palatability on the basis of flavor and taste and classified by an average index. The results are shown in Table 4.

On the other hand, the higher the number is, the higher the palatability.

MF MX3 MX6 MX7 MX8 MX9 MX10 incense 5 One 2 7 9 9 5 flavor 4 One 3 7 8 9 4 Purity 4.5 One 2.5 7.0 8.5 9.0 4.5

(Unit: index)

[Table 4] Referring to the above Table 4, because the flavor and taste of the mulberry muscle extract are increased during the cooking process, the mulberry flavor of the mulberry muscle is unique and the overall flavor of the mulberry muscle is higher. .

However, when the results of mixed extracts according to MX 3 are included, it can be seen that the antioxidant activity is enhanced when the extracts of Zagyubi extract, Vermicelli tree extract and Zygomycorrhizae extract are contained, but flavor and taste are significantly lowered depending on the combination of them.

However, as shown in the above Table 4, in the case of mixing the extracts of Panax ginseng extract, Panax ginseng extract and Radix Salviae extract in a certain range, even though the mixture of Zagyubi extract, It can be seen that the taste is improved.

Especially, referring to MX 7 to MX 9, when a certain range of Zygomyceta japonica extract, Turmeric root extract and Zygomycorrhizae extract is mixed, the fragrance of the mulberry root is maintained, and the mixture of Zagyubi extract, It can be seen that flavor and taste of tea beverage greatly improves according to mutual combination of each extracts while eliminating flavor and rough taste.

Therefore, when mixed extracts of the above range are used, it is possible to greatly enhance the inhibitory effect on the degenerative arthritis, including the mugwort or mugwort extract, Taste can be widely used as a functional food or a functional tea with high palatability.

Claims (8)

Mugwort muscle extract, silkworm tree extract and rhododendron tree extract as active ingredients,
The above-mentioned mugwort extract is a solvent-extracted mugwort,
Through skin protection, you can prevent skin aging caused by oxidative stress
Composition for antioxidative activity comprising an extract of Aspergillus oryzae as an active ingredient The method according to claim 1,
Wherein said horse radish root extract is extracted with a solvent selected from the group consisting of water, alcohols having 1 to 6 carbon atoms, and mixtures thereof
A composition for an antioxidant activity, comprising an extract of Aspergillus oryzae as an active ingredient. The method according to claim 1,
The skin aging caused by the oxidative stress may be at least one selected from the group consisting of skin wrinkles, thinned skin, drooping skin, dry skin and itchy skin
A composition for an antioxidant activity, comprising an extract of Aspergillus oryzae as an active ingredient. delete delete A composition for antioxidative activity, which comprises the composition as claimed in any one of claims 1 to 3 as an active ingredient,
A pharmaceutical composition. A composition for antioxidative activity, which comprises the composition as claimed in any one of claims 1 to 3 as an active ingredient,
Food composition. A composition for antioxidative activity, which comprises the composition as claimed in any one of claims 1 to 3 as an active ingredient,
Tea (tea).

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