KR101768280B1 - A composition comprising Carpinus pubescens extracts having anti-oxidation or anti-inflammation activity - Google Patents

Abstract

The present invention relates to a method for the treatment of carpinus spp. The present invention relates to a food composition, a cosmetic composition and a pharmaceutical composition for antioxidant or antiinflammatory activity, which comprises, as an active ingredient, Since the composition containing the Carpinus fubesense extract of the present invention exhibits antioxidant or anti-inflammatory activity, it can be used for preventing, ameliorating or treating diseases induced by oxidative and inflammatory action, or for use in foods, cosmetics and pharmaceuticals .

Description

[0001] The present invention relates to an antioxidant or anti-inflammatory composition comprising Carpinus fubesense extract,

The present invention relates to a method for the treatment of carpinus spp. The present invention relates to a composition for antioxidant or antiinflammatory activity, which comprises an extract of Carpinus fubesense as an active ingredient, a cosmetic composition and a pharmaceutical composition.

Although the human body has a balance of the oxidation promoting substance and the oxidation inhibiting substance, when the balance state is lost due to various factors and is inclined to promote the oxidation, oxidative stress is induced in the living body, Leading to pathological disease. Reactive oxygen species (ROS), which is a direct cause of oxidative stress, are chemically unstable and highly reactive, and can easily react with various biomaterials such as DNA, protein, lipid and carbohydrate. Attacks, causing irreversible damage to cells and tissues, or causing mutations, cytotoxicity, and cancer.

In the human body, macrophages respond to pathogens and cause inflammation such as tumor necrotic factor-α, TNF-α, interleukin-6 (IL-6), and interleukin-1β (NO) and prostaglandins (prostaglandin E2, PGE2) by synthesizing inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) ). Physiologically, NO plays a variety of roles such as removing bacteria and tumors, regulating blood pressure, or mediating neurotransmission. However, when the inflammatory reaction occurs, the expression of iNOS is increased in related cells, and a large amount of NO is produced. Excessively produced NO causes tissue damage, gene mutation, nerve damage, etc., And promote the inflammatory response.

In the inflammatory reaction, various free radicals are produced along with various inflammation inducers. Normal free radicals are involved in maintaining the homeostasis of cells and affect cell differentiation, growth, survival, and aging. These reactive oxygen species (ROS) in the free radicals are produced constantly through oxidation and reduction of oxygen by respiration and immune reaction in intracellular mitochondria. Harmful ROS are usually caused by superoxide dismutase, catalase, glutathione peroxidase, glutamine reductase, vitamin C, vitamin E, uric acid, bilirubin, It is eliminated by the action of the same antioxidant system. However, when the balance of generation and elimination of free radicals is collapsed, oxidative stress occurs, which causes various pathological changes such as inflammation, aging, or cancer. In fact, it has been confirmed that oxidative stress is increased in many clinical diseases. Various studies on new antioxidants have been made to reduce such oxidative stress.

On the other hand, Korean Patent Laid-Open No. 10-2009-0111991 entitled " Cosmetic Composition Containing Extract of Dogwood with Anti-inflammatory Effect "is known about the anti-inflammatory effect of the same birch and Pseudomonas sp. , The antioxidant and anti-inflammatory effects of Carpinus fubescens were not known.

Under these circumstances, the inventors of the present invention have made efforts to search for natural products having antioxidative or anti-inflammatory effects and less side effects, and as a result, it has been found that an extract of Carpinus fubescens, which is one of native plants derived from China, removes active oxygen species and inhibits iNOS , A strong antioxidant and anti-inflammatory effect, and completed the present invention.

One object of the present invention is to provide a process for the preparation of carpinus < RTI ID = 0.0 > Pubescens ) extract as an active ingredient.

Another object of the present invention is to provide a cosmetic composition for antioxidant or antiinflammation comprising the above Carpinus fubesense extract as an active ingredient.

It is still another object of the present invention to provide a pharmaceutical composition for antioxidant or antiinflammation comprising the Carpinus fubesense extract as an active ingredient.

In one aspect to achieve the above object, the present invention provides a pharmaceutical composition comprising Carpinus Pubescens ) extract as an active ingredient.

The term " Carpinus " pubescens "belongs to the horsetail of Betulaceae, a leafy tree, with alternate phyllotaxis, with serrate on the edge, with one male and one inflorescence, and the fruit is overlaid with nuts. The present inventors confirmed that the extract of Carpinus fubesens has antioxidative and antiinflammatory effects and applied it to foods or cosmetics and then to medicines It can be done.

The term "extract " of the present invention means a preparation which is obtained by squeezing a herbal medicine with an appropriate leaching solution and concentrating the liquid by evaporating the leaching solution, and is not limited thereto. The extract is obtained by drying the extract, diluted solution, Dried products, controlled products thereof or purified products thereof. The extract of the present invention can be prepared by extracting with an extraction solvent or extracting with an extraction solvent and fractionating the extract with a fraction solvent. The extraction solvent may be, but is not limited to, water, an organic solvent or a mixed solvent thereof. The organic solvent may be an alcohol having 1 to 4 carbon atoms, a polar solvent such as ethyl acetate or acetone, hexane or dichloromethane Non-polar solvents or mixed solvents thereof may be used. Further, water, an alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof may be preferably used. In one embodiment of the present invention, an extract using 95% ethanol as the solvent was prepared.

The Carpinus fubesense extract can be prepared by using common extraction, isolation and purification methods known in the art. The extraction method may be, but not limited to, hot water extraction, hot water extraction, cold extraction, reflux cooling extraction, or ultrasonic extraction.

The term "antioxidant" of the present invention means an antioxidant action. The human body has a balance between prooxidant and antioxidant, but due to various factors, And leaning towards oxidative stimulation leads to oxidative stress leading to potential cellular damage and pathological disease. Reactive oxygen species (ROS), which is a direct cause of oxidative stress, is unstable and highly reactive. It reacts easily with various bio-materials, attacks irreversible damage to cells and tissues, Cytotoxicity and carcinogenesis. Active nitrogen species (RNS) such as NO, HNO ₂ , and ONOO ^- are produced by the immune response of macrophage neutrophils and other immune cells during inflammatory reaction, and ROS is also produced. Such active oxygen oxidizes and destroys cells in the body, thereby exposing them to various diseases. Therefore, by incorporating the Carpinus fubesense extract of the present invention into a food, the antioxidative effect can be achieved, thereby contributing to health promotion.

In one embodiment of the present invention, it was confirmed that DPPH radical scavenging activity (Table 1) and reactive oxygen species scavenging ability (Fig. 2) of Carpinus fubesense extract were strongly antioxidant, Confirming that the antioxidant ability is due to the induction of HO-1 expression through the Nrf2 signaling pathway (FIG. 3). Thus, the antioxidant effect of the present invention may be achieved by removal of active oxygen species.

The term "anti-inflammation" of the present invention means an action to suppress inflammation. It is known that the control of the inflammatory reaction is extremely complicated, which is known to reduce the enhancement and damage of the in vivo restoration system. However, if the inflammatory response is sustained by repeated tissue damage or regeneration, ROS and RNS are overproduced in inflammation-related cells, resulting in permanent gene deformation. Thus, ROS and RNS are deeply involved in the inflammatory response that regulates the action of various cells in vivo. During inflammation, large amounts of proinflammatory cytokines, nitric oxide (NO), and prostaglandin (prostaglandin E2, PGE2) are induced by inducible nitric oxide synthase (iNOS) and cyclooxygenase (cyclooxygenase-2, COX-2). Inflammation is a cause of various inflammatory diseases. The composition containing the Carpinus fubesense extract of the present invention may have various inflammatory diseases prevention and improvement effects through anti-inflammatory action.

In one embodiment of the present invention, an analysis of iNOS protein expression (Fig. 6), which is a key protein for NO production inhibition (Fig. 5) and NO production as well as active oxygen species scavenging ability (Fig. 2) of Carpinus fubesense extract Dependent effect, indicating that it has a strong anti-inflammatory effect. Thus, the anti-inflammatory effect of the present invention may be achieved by iNOS inhibition.

When the composition of the present invention is used as a food additive, the above Carpinus fubesense extract can be directly added or used in combination with other food or food ingredients, and can be suitably used according to ordinary methods. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment), and may further include a food-acceptable food-aid additive. Since the composition of the present invention contains an extract derived from a natural substance as an active ingredient, there is no problem in terms of stability, so there is no great limitation on the amount of the mixture.

The food composition of the present invention may include all foods having a conventional meaning, and may be mixed with terms known in the art such as functional foods, health functional foods, and the like.

The term "functional food" in the present invention means a food prepared and processed by using a raw material or ingredient having functionality useful to the human body according to Law No. 6727 on health functional foods, and " And function of the nutrient for the purpose of obtaining a beneficial effect in health use such as controlling the nutrient or physiological action.

The term "health functional food" of the present invention refers to a food prepared by processing a specific ingredient as a raw material for the purpose of health assisting or by extracting, concentrating, refining, mixing, or the like a specific ingredient contained in a food raw material, Refers to a food which is designed and processed so that the body control function such as bio-defense, regulation of biorhythm, prevention and recovery of disease and the like can be sufficiently exhibited to the living body by the above components. Recovery, and so on.

There is no limitation on the kind of food in which the composition of the present invention can be used. In addition, the composition comprising the Carninus fubesense extract of the present invention as an active ingredient can be prepared by mixing other suitable auxiliary ingredients that can be contained in foods and known additives, according to the selection of a person skilled in the art. Examples of foods that can be added include dairy products, such as meat, sausage, bread, chocolates, candies, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, Vitamin complex, and the like, and can be prepared by adding to the juice, tea, jelly, and juice prepared from the extract of the present invention as a main component.

Examples of foods that can be used in the present invention include special nutritional foods such as crude oil, milk, baby food, meat products, fish meat products, tofu, mushrooms, noodles such as ramen noodles, (Such as soy sauce, doenjang, kochujang, mixed potatoes), sauces, confectionery (eg snacks), dairy products (eg fermented milk, cheese), other processed foods, kimchi, pickles ), Beverages (such as fruit, vegetable beverages, beverages, fermented beverages, etc.), and natural seasonings (eg, ramen soup, etc.).

When the health functional food composition of the present invention is used in the form of a drink, it may contain various sweeteners, flavors, or natural carbohydrates as an additional ingredient such as ordinary beverages. 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.

In another aspect, the present invention provides a cosmetic composition for antioxidant or anti-inflammation comprising an extract of Carpinus fubesense as an active ingredient. Since the extract of the present invention has antioxidative and anti-inflammatory effects, the extract of the present invention can be added to cosmetic compositions for the purpose of antioxidant or anti-inflammation .

The cosmetic composition of the present invention can be produced in the form of a general emulsified formulation and a solubilized formulation. Examples of the emulsified formulations include nutritive lotions, creams, essences, and the like, and the solubilization formulations include softening longevity. Suitable formulations include, but are not limited to, solutions, gels, solid or paste anhydrous products, emulsions obtained by dispersing the oil phase in water, suspensions, microemulsions, microcapsules, microgranules or ionic (liposomes) A cream, a skin, a lotion, a powder, an ointment, a spray, or a conical stick. It may also be in the form of a foam or in the form of an aerosol composition further containing a compressed propellant.

The cosmetic composition may further contain at least one selected from the group consisting of fatty substances, organic solvents, solubilizers, thickening and gelling agents, softening agents, antioxidants, suspending agents, stabilizers, foaming agents, perfumes, surfactants, water, ionic or nonionic emulsifiers, Auxiliaries such as ionic sequestering agents, chelating agents, preservatives, vitamins, blocking agents, moisturizers, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or any other ingredients conventionally used in cosmetic compositions ≪ / RTI >

In another aspect, the present invention provides a pharmaceutical composition for antioxidant or antiinflammation, which comprises Carpinus fubesense extract as an active ingredient. Since the extract of the present invention has antioxidative and antiinflammatory effects for removing active oxygen, it can be used as a pharmaceutical composition to treat diseases caused by active oxygen and inflammatory And can be included in medicines for preventing, improving and treating diseases.

The above-mentioned antioxidation is as described above, and thus can be included in a pharmaceutical composition for the purpose of antioxidation, and can have a preventive or therapeutic effect on a disease caused by active oxygen. The diseases caused by the active oxygen include, but are not limited to, degenerative neurological diseases including arteriosclerosis, Lou Gehrig's disease, Parkinson's disease, Alzheimer's disease, proximal axillary sclerosis and Huntington's disease, myocardial infarction, angina pectoris, coronary artery disease, May include various diseases such as cardiovascular diseases including diseases, ischemic brain diseases including stroke, digestive system diseases including diabetes, gastritis and gastric cancer, cancer, leukemia, aging, rheumatoid arthritis, hepatitis, atopic dermatitis, Preferably aging caused by active oxygen.

In addition, the anti-inflammatory activity is as described above, and thus can be included in a pharmaceutical composition for the prophylaxis or treatment of anti-inflammatory, i.e., inflammatory diseases. The inflammatory diseases include allergic diseases including allergic asthma, allergic rhinitis, allergic mucositis, urticaria, and anaphylax, although it is not limited thereto. Arthritis, atopic dermatitis, psoriasis, asthma, multiple sclerosis, ssRNA and dsRNA viral infection, sepsis, multiple chondritis, scleroderma, including systemic sclerosis, dermatomyositis and inclusion body myositis , Eczema, gout, periodontal disease, Behcet's syndrome, edema, vasculitis, Kawasaki disease, diabetic retinitis, autoimmune pancreatitis, vasculitis, glomerulonephritis, acute and chronic bronchitis, and influenza infection.

The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" of the present invention means that it exhibits properties that are not toxic to the cells or humans exposed to the composition. Such carriers may be used without limitation as long as they are known in the art such as buffers, preservatives, wetting agents, solubilizers, isotonic agents, stabilizers, bases, excipients and lubricants.

In addition, the pharmaceutical composition of the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterilized injection solutions according to a conventional method have. Furthermore, it can be used in the form of an external preparation for skin in the form of ointments, lotions, spray agents, patches, creams, powders, suspensions, gels or gels. Examples of carriers, excipients and diluents that can be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used.

Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, Sucrose, lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use may include various excipients such as wetting agents, sweetening agents, fragrances, preservatives, etc. in addition to water and liquid paraffin, which are simple diluents commonly used in suspension, liquid solutions, emulsions and syrups have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of suppository bases include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like.

Meanwhile, the pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The term "administering" of the present invention means introducing a predetermined substance into an individual by an appropriate method, and the administration route of the composition can be administered through any conventional route so long as it can reach the target tissue. But are not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, intrathecal, rectal.

The term "individual" refers to all animals, including humans, including mice, mice, and livestock. Preferably, it may be a mammal, including a human.

The term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment and not causing side effects, and the effective dose level is determined by the patient's sex, Including, but not limited to, medicaments and other medical fields that are used in combination, or in combination with, or in combination with, a pharmaceutically acceptable carrier, excipient, Can be readily determined by those skilled in the art according to known factors. The administration may be carried out once per day, or divided into several doses.

The Carpinus fubesense extract of the present invention contains a natural medicinal herb as a raw material, so that it can be used safely and usefully when it is used as a food composition, a cosmetic composition, or a pharmaceutical composition because it has less side effects than a synthetic compound.

Since the composition containing the Carpinus fubesense extract of the present invention exhibits antioxidant or anti-inflammatory activity, it can be used for preventing, ameliorating or treating diseases induced by oxidative and inflammatory action, or for use in foods, cosmetics and pharmaceuticals .

Figure 1 shows the effect of Carpinus fubesense extract on RAW 264.7 cell viability.
Fig. 2 shows the active oxygen species-removing activity of the extract of Carpinus fubesens.
Figure 3 shows the expression of the antioxidant enzyme HO-1 and its parent transcription factor Nrf2 protein by the extract of Carpinus fubesense in RAW 264.7 cells.
Figure 4 shows the effect of Carpinus fubesens extract on LPS-induced RAW 264.7 cell viability.
Figure 5 shows LPS-induced NO production regulation by Carninus fubesense extract in RAW 264.7 cells.
Figure 6 shows the regulation of LPS-induced iNOS protein expression by Carpinus fubesense extract in RAW 264.7 cells.

Hereinafter, the constitution and effects of the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Example  One: Carpinus Fubesense  Preparation of extract

One of the Chinese native plants, Carpinus The extracts were purchased from Korea Biotechnology Research Center Overseas Biomaterials Hub Center. The Carpinus fubesense was extracted using a 95% ethanol solvent. Specifically, 95% ethanol was added to the sample, followed by sonication for 15 minutes and then for 2 hours. The procedure was repeated 10 times a day for 3 days at room temperature. After completion of the extraction, the extract was filtered, concentrated, dried and stored in a cold room at minus 4 ° C until used in the experiment.

Example  2: RAW  264.7 Culture of Mouse Macrophages

A major murine macrophage cell line, RAW 264.7, which is used as an experimental model for antiinflammatory activity, was purchased from the American Type Tissue Collection (ATCC), and 10% fetal bovine serum (FBS) and penicillin / streptomycin Were cultured in DMEM medium.

Example  3: Cytotoxicity analysis

To determine the effect of the extract of Carpinus fubescens on RAW 264.7 cell viability, the cytotoxicity test was carried out.

RAW 264.7 cells were plated at 3.0 × 10 ⁵ cells per well in a 24-well tissue culture vessel and the presence or absence of cytotoxicity of the Carpinus fubesense extract was confirmed by WST analysis.

The water soluble tetrazolium (WST) assay is a representative method for measuring cell viability using water-soluble tetrazolium. After 24 hours of sample treatment, the cells were incubated at 37 ° C. for one hour. Absorbance was measured at 450 nm using a multi-plate reader, and the value was expressed as the average value of three repeated experiments.

As a result of examining the effect of the extract of Carpinus fubescens on the cell viability of RAW 264.7, it was confirmed that it did not induce cytotoxicity up to 75 袖 g / mL and caused almost no cytotoxicity even at 200 袖 g / mL (Fig. 1).

Example  4: DPPH Radical  Scavenging activity analysis

The electron donating ability is used as an index of antioxidant activity and is widely used for measuring the antioxidant activity of plant extracts. The electron donating action is used to donate electrons of free radicals generated in the human body, thereby suppressing aging and disease by free radicals. The electron donating ability was measured by the DPPH radical scavenging method.

DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity (DPPH radical scavenging activity) measurement was performed to determine the electron donating ability of the Carpinus fubesense extract prepared by the method of Example 1 above. Specifically, Carpinus fubesense extract was dissolved in methanol at various concentrations (2.56 to 320 / / ml) and prepared. Then, 40 쨉 l of DPPH at a concentration of 1.5 × 10 ^-4 M dissolved in methanol was added to 96 well plates at various concentrations 160 μl of the prepared Carpinus fubesense extract solution was dispensed, and the mixture was reacted at room temperature for 30 minutes, and the absorbance was measured at 520 nm using a microplate reader. The free radical scavenging activity was expressed as a percentage of the negative control to which no sample was added, and a 50% inhibitory concentration (IC ₅₀ ) was calculated. As a positive control, ascorbic acid (vitamin C), a representative antioxidant, was used. The formula for calculating the percentage of the free radical scavenging ability is as follows.

DPPH radical scavenging activity (%) = {1- (AB) / C} 100

A is the absorbance of the sample at 520 nm, B is the color control absorbance at 520 nm, and C is the control absorbance at 520 nm.

The analytical results of the DPPH radical scavenging activity of the Carninus fubesense extract of the present invention and the calculated IC ₅₀ values are shown in Table 1 below. The calculated IC ₅₀ value for DPPH radical scavenging activity of Carpinus fubesens extract was 1.04 / / ㎖, which is stronger than that of ascorbic acid (1.53 ㎍ / ㎖) used as a positive control. .

Concentration (/ / ml) Erase rate (%) DPPH (IC _{50 ,}占 퐂 / ml) Carpinus pubescens ( Carpinus pubescens ) 0.1024 21.85 ± 0.72 1.04 0.512 37.04 + - 0.76 2.56 86.99 + - 0.53 12.8 99.85 + 0.27 Ascorbic acid (= vitamin C, positive control) 0.512 19.79 ± 0.23 1.53 2.56 80.38 ± 0.21 12.8 97.59 ± 0.16

Example  5: active Oxygen species ( reactive oxygen species , ROS ) Scatters  analysis

The effects of hydrogen peroxide (H ₂ O ₂ ) - induced ROS on the antioxidative capacity of Carpinus fubesens extract were investigated. RAW 264.7 cells were pretreated with 50 μM DCFH-DA, a cell permeable fluorescent dye, for 2 hours, and then treated with 500 μM H ₂ O ₂ and extracts. The inhibition was analyzed by fluorescence measurement using a multiplate reader.

As a result, it was confirmed that ROS production induced by H ₂ O ₂ was effectively inhibited by Carpinus fubesense extract in a concentration-dependent manner, confirming that the extract had a strong antioxidant ability (FIG. 2).

Example  6: HO -1 and Nrf2  Expression analysis

In order to examine the antioxidative activity of the extract of Carpinus fubesense, the expression of HO-1, a representative antioxidant enzyme, and Nrf2, a transcription factor, was examined by western blot hybridization Western blot hybridization). Proteins were extracted from the cultured cells after the sample treatment, and the protein concentration was determined by Bradford assay. Then, 50 μg of the protein was electrophoresed on 10% SDS-PAGE, blotted onto a nitrocellulose membrane, Lt; / RTI > After washing the membranes, HRP-labeled secondary antibodies were reacted for one hour and protein expression was analyzed using a chemiluminescence detection system.

As a result, it was confirmed that HO-1 and Nrf2 induce significant expression depending on the concentration of Carpinus fubesense extract, and it was confirmed that the antioxidant ability of the extract was due to the induction of HO-1 expression through the Nrf2 signaling pathway 3).

Example  7: presence or absence of cytotoxicity and nitrogen monoxide ( NO ) produce Inhibition  analysis

In order to confirm the anti-inflammatory activity of the extract of Carpinus fubesense prepared in the examples, the present inventors tried to analyze the ability to inhibit the production of nitrogen monoxide (NO), one of inflammatory substances.

RAW 264.7 cells were plated at 3.0 × 10 ⁵ cells per well in a 24-well tissue culture vessel and treated with 1 μg / mL of lipopolysaccharide (LPS) to induce NO production. In order to confirm the inhibitory effect of NO production on NO production, the presence or absence of cytotoxicity of the extract of Carpinus fubesense was confirmed by WST analysis and the inhibition of NO production by Griess reaction was analyzed.

The water soluble tetrazolium (WST) assay is a representative method for measuring cell viability using water-soluble tetrazolium. After 24 hours of sample treatment, the cells were incubated at 37 ° C. for one hour. Absorbance was measured at 450 nm using a multi-plate reader, and the value was expressed as the average value of three repeated experiments.

The grease reaction is a method for quantitatively analyzing the amount of released NO produced from the cultured cells. After 24 hours of sample treatment, 100 μl of the cell culture medium is dispensed into a 96-well plate, and then the grease reaction solution A (0.1% naphthylenediamine dihydrochloride) buffer solution (1%) was dispensed into a 96-well plate in an amount of 100 쨉 l per well, The absorbance at 540 nm was measured using a plate reader, and the value was expressed as an average value of three repeated experiments.

First, the effect of the extract of Carpinus fubescens on LPS-induced RAW 264.7 cell survival rate was examined. As shown in FIG. 4, it did not induce cytotoxicity up to 100 μg / mL, In the case of the present invention.

In addition, the anti-inflammatory activity of the extract of Carpinus fubesense was analyzed by RAW 264.7 cells in the rat macrophage cell line induced by LPS. As a result, it was confirmed that the extract showed inhibitory effect on NO production as shown in FIG. 5, It was confirmed that the compound had a strong anti-inflammatory activity.

As a result, it was confirmed that the extract of Carpinus fubesense did not cause toxicity to RAW 264.7 cells induced by LPS, and thus, it had an anti-inflammatory effect.

Example  8: Expression of anti-inflammatory activity-related proteins in plant extracts Control ability  analysis

In order to clarify the anti-inflammatory activity of the extract of Carpinus fubesense, the expression of iNOS protein, a key protein for NO production, was analyzed by Western blot hybridization. After extracting proteins from the cultured cells, 50 μg of protein was electrophoresed on SDS-PAGE, blotted onto a nitrocellulose membrane, and then hybridized with the antibody of the target protein. After washing the membrane, HRP was reacted with a tagged secondary antibody for one hour, and protein expression was analyzed using a chemiluminescence detection system.

As a result, it was confirmed that the expression of iNOS protein induced by LPS was decreased dependently on the extract concentration, and thus it was confirmed that the extract had a strong anti-inflammatory activity (FIG. 6).

Thus, the above results indicate that Carpinus fubesense extract has strong antioxidative ability through DPPH radical scavenging activity and induction of HO-1 expression through Nrf2 signal transduction pathway, and iNOS expression Inhibitory effect on NO production, and thus can be used for prevention, improvement or treatment of diseases caused by oxidative and inflammatory action, or for foods, cosmetics and medicines.

Claims (7)

A cosmetic composition for antioxidation comprising an extract of Carpinus pubescens as an active ingredient.
The method according to claim 1,
Wherein the extract is extracted with water, an alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof.
The method according to claim 1,
Wherein said antioxidant effect is achieved by removal of reactive oxygen species.
A cosmetic composition for antiinflammation comprising an extract of Carpinus pubescens as an active ingredient.
5. The method of claim 4,
Wherein said anti-inflammatory effect is achieved by inhibition of iNOS (inducible nitric oxide synthases).
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