KR102254895B1 - Composition for Anti-inflammation Using Carex glabrescens - Google Patents

Abstract

The present invention inhibits NO production in a concentration-dependent manner in a mouse macrophage cell line (RAW 264.7 cells) stimulated with PM (particulate matters), inhibits the production of inflammatory cytokines TNF-α and IL-6 and the production of PGE ₂ , Disclosed is a composition for anti-inflammatory using a curly sedge extract having an activity of inhibiting the expression of COX-2, which is an inflammatory mediator.

Description

Composition for Anti-inflammation Using Carex glabrescens}

The present invention relates to a composition for anti-inflammatory using a curly sedge (Carex glabrescens) extract.

Inflammation is a local protective reaction in the living body in response to physical trauma, infection by harmful chemicals, bacteria, fungi, viruses, or pathological conditions caused by irritants in metabolites in the living body. Inflammation is triggered by various inflammatory mediators produced from damaged tissue and migrating cells. During an inflammatory reaction, plasma accumulates in the inflamed area, diluting the toxicity secreted by bacteria, increasing blood flow, and accompanying symptoms such as erythema, pain, swelling, and fever. In a normal case, the living body neutralizes or removes pathogenic factors through an inflammatory reaction and regenerates damaged tissue to restore normal structure and function, but otherwise, it may progress to a disease state such as chronic inflammation.

With the recent development of molecular biology, many studies have been made on the inflammatory response at the molecular level.

Although various biochemical phenomena are involved in the inflammatory response, it is known that macrophages in particular play an important role in the inflammatory response by producing nitric oxide (NO) and various inflammatory cytokines by chemical stimulation (Ito T., et al. al., Curr Drug Traget Inflamm Allergy, 2(3):257-265, 2003). Nitric oxide is synthesized from L-arginine by the action of nitric oxide synthase (NOS), and NOS has several isoforms. BNOS (brain NOS) present in the brain, nNOS (neuronal NOS) present in the nervous system, eNOS (endothelial NOS) present in the vascular endothelial system are always expressed in the body at a certain level, and a small amount of monoxide is produced by them. Nitrogen (NO) plays an important role in maintaining homeostasis of the human body by performing various physiological reactions related to blood pressure regulation, neurotransmission, learning, and memory. On the other hand, iNOS (induced NOS), whose expression is induced by a certain stimulus, produces excessive NO, and nitrogen oxide generated excessively by iNOS reacts with superoxide to form peroxynitrite. It acts as a potent oxidant, damaging cells, and is involved in various pathological processes including inflammation and cancer (Gupta SC et al., Exp Biol Med., 236:658-671, 2011; Riehemann et al., FEBS. Lett., 442:89-94, 1999; Stamler et al., Science, 258:1898-1902, 1992).

_{Cyclooxygenase (COX) synthesizes the intermediates PGG 2} and PGH ₂ from arachidonic acid with the function of COX and hydroperoxidase (HOX), and these compounds are PGE ₂ , PGF ₂ , PGD ₂ , prostacyclin and thromboxane A ₂ (thromboxane A2, TxA2) are produced, and there are two types of isoforms in COX. COX-1 is always expressed in most tissues to synthesize prostaglandins (PGs), which are required for cytoprotective action, whereas COX-2 rapidly induces its expression during inflammatory reactions _{and generates PGE 2} to induce an inflammatory reaction. Plays an important role (Weisz A., Biochem. J., 316:209-215, 1996; (Miller MJ et al., Mediators of inflammation, 4:387-396, 1995: Appleton L. et al., Adv) Pharmacol., 35:27-28, 1996).

The expression of iNOS and COX-2, which induces overproduction of NO and PGs, is regulated by the nuclear transcription factor NF-κB. NF-κB is a nuclear protein of the Rel gene family. In the cytoplasm, it is combined with I-κB and exists in an inactive form. However, when I-κB kinase is activated, I-κB is released through phosphorylation. It is activated by falling apart. NF-κB, which is composed of a heterodimer of p50 and p65, is activated and then moves to the nucleus to induce gene expression of iNOS and COX-2, which induces an inflammatory response (Oh, GT et al., Atherosclerosis, 159). (1):17-26, 2001).

NO, Inflammatory cytokines such as TNF-α and IL-6, PGE _2, etc. are arthritis (Jang CH et al., Rheumatology, 2006, 45(6):703-710), fibromyalgia (Hernandez ME et. al., BMC Res). Notes., 2010, 3(1):156), Sjogren's syndrome (Baturone R. et. al., Scand J Rheumatol., 2009, 38(5):386-389) as an important factor inducing inflammatory response. It has been reported (Jang CH et al., Rheumatology, 45(6):703-710, 2006; Hernandez ME et. al., BMC Res. Notes., 3(1):156, 2010; Baturone R. et. al., Scand J Rheumatol., 38(5):386-389, 2009).

On the other hand, particulate matters (PM) are substances that are suspended in the atmosphere with very complex components, and are mostly generated from automobile exhaust gas and road dust. The mechanisms for the effects of fine dust on the human body are currently explained in various ways, including inflammatory reactions, secretion of cytokines and chemokines in the lungs (Toxicol Sci. 2010. 113(2):422-33), increase in the number of white blood cells, and in the lungs. The production of reactive oxygen species and the reaction of cells and tissues by endotoxin are typical. In particular, as a result of treatment with urban fine dust in RAW264.7 cells, a mouse peritoneal macrophage cell line, the secretion of TNF-alpha and IL-6 was increased (Toxicology. 2003. 183(1-3):243-54) in humans. Treatment of normal bronchial epithelial cells, NHBE cells, showed that the secretion of pro-inflammatory cytokines such as IL-6 and IL-8 and production of ROS increased (Environ Health Perspect. 2005. 113(8):1032-8). ) Has been reported, research on fine dust and inflammation is steadily progressing.

Non-steroidal anti-inflammatory drugs (NSAIDS), which are currently widely used as anti-inflammatory drugs, are known to cause serious side effects such as gastrointestinal disorders, liver disorders, and renal disorders (Rainsford KD., Subcell biochem., 42:3). -27, 2007; Guruprasad P. Aithal., Rheumatology., 7:139-150, 2011; Praveen PN Rao et al., Pharmaceuticals., 3:1530-1549, 2010).

Therefore, it can be said that it is still necessary to develop a new drug that has anti-inflammatory activity, has few side effects, and is effective.

It is an object of the present invention to provide an anti-inflammatory composition using a curly sedge extract.

Other or specific objects of the present invention will be presented below.

The present invention is a concentration-dependent inhibition of NO production in mouse macrophage cell line (RAW 264.7 cells) stimulated with fine dust (particulate matters; PM), as confirmed in the following Examples and Experimental Examples, and inflammatory It was completed by confirming that it suppresses the production of cytokines TNF-α and IL-6 and the production of PGE ₂ , and suppresses the expression of COX-2, an inflammation mediator.

In consideration of the above, the present invention relates to an anti-inflammatory composition comprising a curly sedge extract as an active ingredient.

In the present specification, "curly sedge extract" refers to stems, leaves, fruits, flowers, roots, etc. of curly sedges to be extracted, water, lower alcohols having 1 to 4 carbon atoms (methanol, ethanol, butanol, etc.), methylene chloride, ethylene, acetone , Hexane, ether, chloroform, ethyl acetate, butyl acetate, N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1,3-butylene glycol, propylene glycol, or leaching using a mixed solvent thereof It refers to an extract obtained by using a supercritical extraction solvent such as carbon dioxide, pentane, etc., or a fraction obtained by fractionating the extract, and the extraction method is cold sedimentation, reflux, and warming in consideration of the polarity of the active substance, the degree of extraction, and the degree of preservation. , Ultrasonic radiation, supercritical extraction, etc. can be applied. In the case of a fractionated extract, a fraction obtained by suspending the extract in a specific solvent and then mixing and policing it with a solvent having a different polarity, the crude extract is adsorbed on a column filled with silica gel, etc., and then a hydrophobic solvent, a hydrophilic solvent, or a mixed solvent thereof is added. It means including the fraction obtained as a mobile phase. In addition, the meaning of the extract includes a concentrated liquid extract or a solid extract from which the extraction solvent has been removed by a method such as freeze drying, vacuum drying, hot air drying, spray drying, or the like. Preferably, it means obtained by extraction (immersion or distillation) with water, ethanol, or a mixed solvent thereof, particularly 60% to 90% of an aqueous ethanol solution as an extraction solvent.

In addition, in the present specification, the term "active ingredient" refers to an ingredient that can exhibit a desired activity alone or exhibit activity with a carrier that is not itself active.

In addition, in the present specification, "anti-inflammatory" is meant to include improvement (reduction of symptoms), treatment, and inhibition or delay of the onset of inflammatory diseases as defined below.

In addition, in the present specification, the term "inflammatory disease" refers to an inflammatory reaction specified by an external physical or chemical stimulus or an infection of an external infectious agent such as bacteria, fungi, viruses, various allergens, or a local or systemic biological defense response against autoimmunity. It can be defined as the pathological condition that causes it. These inflammatory reactions include activation of various inflammatory mediators and enzymes related to immune cells (eg iNOS, COX-2, etc.), secretion of inflammatory mediators (eg, secretion of NO, TNF-α, IL-6, etc.), body fluid infiltration, It involves a series of complex physiological reactions such as cell migration and tissue destruction, and is externally manifested by symptoms such as erythema, pain, swelling, fever, deterioration or loss of specific functions of the body. Since the inflammatory disease may be acute, chronic, ulcerative, allergic or necrotic, so long as any disease is included in the definition of such an inflammatory disease, whether it is acute, chronic, ulcerative, allergic, or Regardless of whether it is necrotic or not. Specifically, the inflammatory diseases include asthma, allergic and non-allergic rhinitis, chronic and acute rhinitis, chronic and acute gastritis or enteritis, ulcerative gastritis, acute and chronic nephritis, acute and chronic hepatitis, chronic obstructive pulmonary disease, pulmonary fibroids. , Irritable bowel syndrome, inflammatory pain, migraine, headache, back pain, fibromyalgia, myofascial disease, viral infection (e.g., type C infection), bacterial infection, fungal infection, burn, surgical or dental surgery wound, pro Hyperstagladin E syndrome, atherosclerosis, gout, arthritis, rheumatoid arthritis, ankylosing spondylitis, Hodgkin's disease, pancreatitis, conjunctivitis, iritis, scleritis, uveitis, dermatitis (including atopic dermatitis), eczema, multiple sclerosis, etc. Will be included.

The anti-inflammatory composition of the present invention may contain the active ingredient in any amount (effective amount) as long as it can exhibit the improvement activity of the inflammatory disease intended for treatment depending on the use, formulation, and combination purpose, etc. Silver will be determined within the range of 0.001% to 15% by weight based on the total weight of the composition. Herein, the term "effective amount" refers to an improvement, treatment, or suppression of the onset of such pathological symptoms when the composition of the present invention is administered to a mammal, preferably a human, to which the composition of the present invention is administered during the administration period according to the advice of a medical professional, etc. / It refers to the amount of active ingredient that can exhibit the intended medical and pharmacological effects such as delay. Such effective amounts can be determined empirically within the range of ordinary skill in the art.

In addition to the active ingredient, the anti-inflammatory composition of the present invention may be administered through addition of similar activities such as anti-allergic activity and skin protection activity (suppression of skin damage by ultraviolet rays, skin moisturization, etc.), in addition to the active ingredient, for enhancement or reinforcement of the anti-inflammatory effect. In order to enhance the convenience of ingestion, it may further include any compound or natural extract, which has already been verified for safety in the art and is known to have the corresponding activity.

These compounds or extracts include compounds or extracts, pharmaceuticals, etc. listed in the pharmacopoeia of each country (“Korean Pharmacopoeia” in Korea), health functional food code in each country (“health functional food standards and standards” notified by the Ministry of Food and Drug Safety in Korea), etc. In accordance with the laws of each country governing the manufacture and sale of products (“Pharmaceutical Affairs Act” in Korea), laws governing the manufacture and sale of compounds, extracts, and health functional foods that have been licensed as items (in Korea, “The Act on Health Functional Foods”) 」), and the compounds or extracts individually recognized for their functionality are included. For example, dimethylsulfonylmethane (MSM) with'arthritis improvement' function of the Korean Health Functional Food Code, N-acetylglucosamine with'arthritis improvement' function and'skin moisturizing' function, etc. Enterococcus faecalis heat-treated dry powder, complexes such as guava leaf extract, complexes such as stalk extract, lobule extract, picaopreto powder, etc., individually recognized for their functionality as'relieving irritable immune response', PLAG (1-palmitoyl-2-linoleoyl- 3-acetyl-rac-glycerol), etc., L. sakei Probio 65, which has been individually recognized for its functionality for'improving sensitive skin condition', oil containing gammarinolenic acid, L. plantarum CJLP133, a lactic acid bacteria derived from fruits and vegetables, and probiotics ATP are these compounds or It will correspond to the extract.

One or more of these compounds or natural extracts may be included in the anti-inflammatory composition of the present invention together with the active ingredient.

The anti-inflammatory composition of the present invention can be grasped as a food composition in a specific aspect.

The food composition of the present invention may be prepared in any form, such as beverages such as tea, juice, carbonated drinks, ion drinks, processed oils such as milk, yogurt, gums, rice cakes, Korean sweets, bread, snacks, noodles, etc. Foods, tablets, capsules, pills, granules, liquids, powders, flakes, pastes, syrup, gels, jelly, can be prepared in health functional food preparations such as bars.

In addition, the food composition of the present invention can be classified as a product as long as it conforms to the enforcement regulations at the time of manufacture and distribution in terms of legal and functional classification. For example, it is a health functional food according to the Korean 「Health Functional Food Act」, or confectionery, bean, tea, and beverages according to each food type according to the food code of the Korean 「Food Sanitation Act」 (``Food Standards and Standards'' notified by the Ministry of Food and Drug Safety). , Special use food, etc.

The food composition of the present invention may contain food additives in addition to the active ingredients. Food additives can generally be understood as substances that are added to food and mixed or infiltrated in manufacturing, processing, or preserving food. Since they are consumed daily and for a long time with food, their safety must be ensured. Food additives with guaranteed safety are limited in terms of ingredients or functions in the Food Additives Code according to the laws of each country governing the manufacture and distribution of food (the “Food Sanitation Act” in Korea). In the Korean Food Additives Code (“Food Additive Standards and Standards” notified by the Ministry of Food and Drug Safety), food additives are classified into chemical synthetic products, natural additives, and mixed preparations in terms of ingredients.These food additives are sweeteners and flavors in terms of function. It is categorized into agents, preservatives, emulsifiers, acidulants, and thickeners.

Sweeteners are used to impart an appropriate sweet taste to foods, and both natural and synthetic can be used in the composition of the present invention. Preferably, a natural sweetener is used, and examples of the natural sweetener include sugar sweeteners such as corn syrup solids, honey, sucrose, fructose, lactose, and maltose.

Flavoring agents can be used to enhance taste or aroma, and both natural and synthetic can be used. Preferably, it is the case of using a natural one. In the case of using natural ones, the purpose of nutrient enhancement can be combined in addition to flavor. As a natural flavoring agent, it may be obtained from apples, lemons, tangerines, grapes, strawberries, peaches, or the like, or from green tea leaves, roundtails, bamboo leaves, cinnamon, chrysanthemum leaves, jasmine, and the like. In addition, you can use those obtained from ginseng (red ginseng), bamboo shoots, aloe vera, and ginkgo. The natural flavoring agent may be a liquid concentrate or a solid extract. In some cases, synthetic flavoring agents may be used, and synthetic flavoring agents may include esters, alcohols, aldehydes, terpenes, and the like.

As a preservative, sodium calcium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate, EDTA (ethylenediaminetetraacetic acid), etc. can be used, and as an emulsifier, acacia gum, carboxymethylcellulose, xanthan gum, Pectin, etc. may be mentioned, and as the acidulant, arithmetic, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, phosphoric acid, and the like can be used. In addition to the purpose of enhancing taste, the acidulant may be added so that the food composition has an appropriate acidity for the purpose of inhibiting the growth of microorganisms.

As the thickening agent, a suspending agent, a settling agent, a gel-forming agent, a swelling agent, and the like may be used.

In addition to the food additives described above, the food composition of the present invention may include a physiologically active substance or minerals known in the art for the purpose of supplementing and reinforcing functionality and nutritional properties and ensuring stability as a food additive.

Examples of such bioactive substances include catechins contained in green tea, vitamins such as vitamin B1, vitamin C, vitamin E, and vitamin B12, tocopherol, dibenzoyl thiamine, and the like, and minerals include calcium preparations such as calcium citrate, magnesium stearate. Magnesium preparations such as, iron preparations such as iron citrate, chromium chloride, potassium iodide, selenium, germanium, vanadium, and zinc.

In the food composition of the present invention, the food additive described above may be included in an appropriate amount to achieve the purpose of addition according to the product type.

Regarding other food additives that may be included in the food composition of the present invention, reference may be made to the food code of each country or the food additive code.

The composition of the present invention may be understood as a pharmaceutical composition in other specific embodiments.

The pharmaceutical composition of the present invention may be prepared in an oral dosage form or a parenteral dosage form according to an administration route by a conventional method known in the art, including a pharmaceutically acceptable carrier in addition to the active ingredient. Here, the route of administration may be any suitable route including a local route, an oral route, an intravenous route, an intramuscular route, and direct absorption through mucosal tissue, and two or more routes may be used in combination. An example of a combination of two or more routes is a case in which two or more formulations of drugs are combined according to the route of administration, for example, when one drug is first administered by an intravenous route and the second drug is administered by a local route.

Pharmaceutically acceptable carriers are well known in the art depending on the route of administration or formulation, and specifically, reference may be made to the pharmacopoeia of each country, including the “Korean Pharmacopoeia”.

When the pharmaceutical composition of the present invention is prepared in an oral dosage form, powders, granules, tablets, pills, dragees, capsules, solutions, gels, syrups, suspensions, wafers according to a method known in the art together with a suitable carrier It can be prepared in a formulation such as. Examples of suitable carriers at this time include sugars such as lactose, glucose, sucrose, dextrose, sorbitol, mannitol, and xylitol, starches such as corn starch, potato starch, wheat starch, cellulose, methylcellulose, ethylcellulose, sodium carboxymethylcellulose, Celluloses such as hydroxypropylmethylcellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, polyol, vegetable oil, ethanol, grease Serol, etc. are mentioned. In the case of formulation, appropriate binders, lubricants, disintegrants, colorants, and diluents may be included as needed. Suitable binders include starch, magnesium aluminum silicate, starch ferryst, gelatin, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone, glucose, corn sweetener, sodium alginate, polyethylene glycol, wax, etc., and lubricants include oleic acid. Sodium, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, silica, talcum, stearic acid, magnesium salts and calcium salts thereof, polydecylene glycol, etc., and as disintegrating agents starch, methyl cellulose , Agar, bentonite, xanthan gum, starch, alginic acid, or a sodium salt thereof. Moreover, lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, glycine, etc. are mentioned as a diluent.

When the pharmaceutical composition of the present invention is prepared in a parenteral dosage form, it may be formulated in the form of an injection, a transdermal administration, a nasal inhalation and a suppository according to a method known in the art together with a suitable carrier. When formulated as an injection, an aqueous isotonic solution or suspension may be used as a suitable carrier, and specifically, triethanol amine-containing PBS (phosphate buffered saline), sterile water for injection, an isotonic solution such as 5% dextrose, etc. may be used. . When formulated as a transdermal administration, it can be formulated in the form of an ointment, cream, lotion, gel, external solution, pasta, liniment, air roll, and the like. In the case of nasal inhalants, suitable propellants such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, etc. can be used to form an aerosol spray.When formulated as a suppository, the carrier is Withepsol ( witepsol), tween 61, polyethylene glycols, cacao butter, laurin paper, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, sorbitan fatty acid esters, and the like.

The specific formulation of pharmaceutical compositions is known in the art, and for example, Remington's Pharmaceutical Sciences (19th ed., 1995) may be referred to. This document is considered as part of this specification.

The preferred dosage of the pharmaceutical composition of the present invention is in the range of 0.001 mg/kg to 10 g/kg per day, preferably 0.001 mg/kg to 1 g, depending on the patient's condition, weight, sex, age, patient severity, and route of administration. May be in the /kg range. Administration can be made once a day or divided into several times. Such dosage should not be construed as limiting the scope of the invention in any aspect.

The composition of the present invention can be understood as a cosmetic composition in another specific aspect. When the composition of the present invention is regarded as a cosmetic composition, its use can be understood as a use for suppressing inflammatory skin problems and alleviating inflammatory skin irritation.

Even if the composition of the present invention is identified as a cosmetic composition, the cosmetic composition can be classified as a product for its use and legally, and specifically, functional cosmetics with uses such as improving skin troubles and improving atopic dermatitis, non-functional It may be a general cosmetic or the like. The product form can also take any product form, specifically solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansing, oils, powder foundations, emulsion foundations, waxes. It can take the form of a product such as a foundation or spray. In a specific product form, it may be a flexible lotion, a nutritional lotion, a nutritional cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray, or a powder formulation.

In addition to the active ingredient, the cosmetic composition of the present invention may include components commonly used in the cosmetic composition, such as stabilizers, solubilizers, surfactants, vitamins, conventional adjuvants such as pigments and perfumes, and carriers.

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

When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder may be used as a carrier component. In particular, in the case of a spray, additionally chlorofluorohydrocarbon, propane / May contain propellants such as butane or dimethyl ether.

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

When the formulation of the present invention is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, an ethoxylated isostearyl alcohol, a suspending agent such as polyoxyethylene sorbitol ester, polyoxyethylene sorbitan ester, and crystallites Castle cellulose, aluminum metahydroxide, bentonite, agar, and the like can be used.

When the formulation of the present invention is a surfactant containing cleansing, as a carrier component, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters may be used.

The cosmetic composition of the present invention can be prepared according to a method for preparing a cosmetic composition commonly used in the art, except that the active ingredient exhibiting anti-inflammatory activity is included.

As described above, according to the present invention, it is possible to provide an anti-inflammatory composition using a curly sedge extract.

The anti-inflammatory composition of the present invention may be commercialized as food, cosmetics, medicines, etc. for use in improving inflammatory diseases, alleviating inflammatory skin irritation, and the like.

1 is a result of Raw264.7 cytotoxicity measurement and a result showing NO production inhibitory activity in PM-stimulated macrophages.
2 is a result showing the _{PGE 2} production inhibitory activity in PM-stimulated macrophages.
3 is a result showing the inhibitory activity of the production of TNF-α, an inflammatory cytokine, in PM-stimulated macrophages.
4 is a result showing the inhibitory activity of the production of IL-6, an inflammatory cytokine, in PM-stimulated macrophages.
5 is a result showing the inhibitory activity of COX-2 production in PM-stimulated macrophages.

Hereinafter, the present invention will be described with reference to Examples and Experimental Examples. However, the scope of the present invention is not limited to these Examples and Experimental Examples.

<Example> Curly sedge extract preparation

Curly sedge (outpost) used in this experiment was collected and identified in Yeongyang-gun, Gyeongsangbuk-do, and dried in the shade. The shaded plant was shredded and pulverized, and used as an experimental material.

After extracting 10 times the weight of 70% ethanol to the dried curly sedge powder three times for 24 hours at room temperature, then the filtrate filtered (Whatman No.2) was concentrated with a rotary vacuum evaporator (EYELA. Japan) and lyophilized until the experiment- It was stored and used at 20°C.

< Experimental example > Evaluation of anti-inflammatory activity

1. Experimental method

Cell culture

Murine macrophage cell line RAW 264.7 cells were purchased from Korean Cell Line Bank, Dulbecco's containing 1% antibiotic (Gibco, USA) and 10% fetal bovine serum (FBS; Gibco, Grand Island, USA) It was cultured in a 37°C, 5% CO ₂ incubator using modified Eagle's medium (DMEM) medium, and passage culture was performed once every 2 days.

Cytotoxicity test (LDH assay)

^{RAW 264.7 cells were added to a 24 well plate at 1.8×10 5} cells/mL using DMEM medium added with 10% FBS, and cultured for 18 hours, followed by simultaneous treatment of the sample and fine dust (250 μg/mL) for 24 hours. . Thereafter, the culture medium is centrifuged at 3,000 rpm for 5 minutes. Lactate dehydrogenase (LDH) assay was measured using a non-radioactive cytotoxicity assay kit (Promega, WI, USA), 50 μL of culture medium obtained by centrifugation and 50 μL of reconstituted substrate mix were added to a 96 well plate, and reacted for 30 minutes at room temperature. After that, 50 μL of stop solution was added and the absorbance was measured at 490 nm using a microplate reader (Bio-TEK Instruments Inc., Vermont, WI, USA). The average absorbance value was obtained for each sample group, and cytotoxicity was evaluated by comparing it with the absorbance value of the control group.

Evaluation of inhibition of nitric oxide formation

^{RAW 264.7 cells were adjusted to 2.0×10 5} cells/mL using DMEM medium added with 10% FBS, inoculated into a 24 well plate, and treated with extract sample and fine dust (250 μg/mL) at the same time for 24 hours. Cultured. The amount of NO produced is measured in the form of ^NO2- present in the cell culture solution using Griess reagent [1% (w/v) sulfanilamide, 0.1% (w/v) naphylethylenediamine in 2.5% (v/v) phosphoric acid]. I did. 100 μL of the cell culture supernatant and 100 μL of Griess reagent were mixed and reacted in 96 well plates for 10 minutes, and the absorbance was measured at 540 nm. The amount of NO produced was compared with sodium nitrite (NaNO2) as standard.

Prostaglandin E ₂ (PGE ₂ ) Generate evaluation

RAW 264.7 cells were adjusted to 2.0×10 ⁵ cells/mL using DMEM medium, inoculated into a 24 well plate, and cultured 18 hours before in a _{5% CO 2 incubator.} After removing the medium, 50 μL of an extract sample prepared at a 10-fold concentration (1 mg/mL) and a new medium containing 450 μL of fine dust (250 μg/mL) were simultaneously treated and cultured under the same conditions as the pre-culture. . After 24 hours, the culture medium was centrifuged (12,000 rpm, 3 min) to measure _{prostaglandin E 2} (PGE _{2) to obtain a supernatant.} The measurement of PGE ₂ was quantified using the PGE2 ELISA kit (R&D Systems Inc., Minneapolis, MN, USA), and the r2 value of the standard curve for the standard was 0.99 or higher.

Evaluation of inhibition of the production of pro-inflammatory cytokines (TNF-α, IL-6)

RAW 264.7 cells (2.0×10 ⁵ cells/mL) were inoculated into a 24 well plate using DMEM medium, and cultured before 18 hours in a _{5% CO 2 incubator.} Thereafter, the medium was removed, and a new medium containing 50 µl of the test substance prepared at a 10-fold concentration (1 mg/mL) and 450 µl of fine dust (250 μg/mL) were simultaneously treated and cultured under the same conditions as the previous culture. After 24 hours, the culture medium was centrifuged (12,000 rpm, 3 minutes), and the content of pro-inflammatory cytokines produced in the obtained supernatant was measured. All samples were stored at -20 ℃ or less until quantification. Pro-inflammatory cytokines were quantified using a mouse enzyme-linked immnunosorbent assay (ELISA) kit (R&D Systems Inc., Minneapolis, MN, USA), and the r2 value of the standard curve for the standard was 0.99 or higher.

Evaluation of COX-2 expression inhibition efficacy (western-blotting)

After collecting the cultured cells and washing them with PBS 2-3 times, cell lysis buffer ([50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 2 mM EDTA, 1 mM EGTA, 1 mM NaVO3, 10 mM NaF, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 25 μg/mL aprotinin, 25 μg/mL leupeptin) was added and dissolved at 4°C for 30 minutes, followed by centrifugation at 15,000 rpm for 15 minutes. The back was removed. Protein concentration was quantified using the Bio-Rad Protein Assay Kit by standardizing bovine serum albumin (BSA). The separated protein 20 μg of lysate was denatured and separated by 8-12% mini gel SDS-PAGE, and this was transferred to a PVDF (polyvinylidene difluoride) membrane (BIO-RAD, Richmond, CA, USA) at 200 mA for 2 hours. And blocking of the membrane was carried out for 2 hours at room temperature in TTBS (0.1% Tween 20 + TBS) solution containing 5% skim milk. As an antibody for examining the expression level of COX-2, anti-rabbit COX-2 (1:2000) (cell signaling, USA) was diluted in TTBS solution, reacted at room temperature for 2 hours, and washed 3 times with TTBS. As a secondary antibody, HRP (horse radish peroxidase) conjugated anti-rabbit IgG (Amersham Pharmacia Biotech, Little Chalfont, UK) was diluted 1:5000 and reacted at room temperature for 1 hour, then washed 4 times with TTBS and ECL After reacting with the substrate (Amersham Biosciences, Piscataway, NJ, USA) for 1 to 3 minutes, it was sensitized on an X-ray film.

2. Experiment result

Cytotoxicity assessment

RAW 264.7 cells, macrophages, were cultured in a 24 well plate at 2.0×10 ⁵ cells/well for 24 hours, treated with a concentration of 200 μg/mL or less, and cultured for 24 hours, followed by cell viability using LDH analysis. Was confirmed.

As shown in FIG. 1, it was confirmed that the extract of curly sedge showed a cell viability of 90 to 100% in a concentration range of 200 μg/mL or less, and did not exhibit cytotoxicity. Therefore, subsequent experiments were conducted at a concentration that did not show cytotoxicity in all of the extracts.

Evaluation of inhibition of nitric oxide formation

RAW 264.7 cells were cultured for 24 hours by simultaneously treating extracts (50, 100, and 200 μg/mL) and fine dust (250 μg/mL) for each concentration of curly sedge. The amount of NO produced was measured in the form of NO2- present in the cell culture medium using the Griess reagent.

As shown in Figure 1, as a result of measuring the NO production inhibitory activity of the extract of curly sedge, the group treated with fine dust alone induces the production of NO, and 50, 100, and 200 μg of both the 70% ethanol extract and the water extract of curly sedge It was confirmed that NO production was suppressed in a concentration-dependent manner at /mL. In particular, it was confirmed that the 70% ethanol extract 200 μg/mL inhibited NO production by 93.7%.

Prostaglandin E ₂ (PGE ₂ ) Production inhibitory activity

Prostaglandin E2 (PGE2) is a well-known inflammatory reaction inducer and is known to be involved in the movement of immune cells, such as pain and blood vessels, and macrophages to the inflammatory site. Accordingly, the inhibitory effect on the generation of PGE2 increased induced by fine dust with respect to the frangipani extract was confirmed.

As shown in Figure 2, the fine dust alone treatment group induced the expression of PGE2, and the 70% ethanol extract of curly sedge inhibited 6.7%, 38.47%, and 77.56% PGE2 production at 50, 100 and 200 μg/mL, respectively. I could confirm.

Inhibitory activity of the production of pro-inflammatory cytokines (TNF-α, IL-6)

Cytokine is a protein secreted by immune cells and is a factor that mediates the inflammatory response by regulating the activity, proliferation and differentiation of immune cells. The role of cytokine is very important to identify the cause and treatment of inflammatory diseases, and TNF-α, IL-1β, and IL-6 are substances that regulate inflammatory responses in both in vitro and in vivo, and are representative pro-inflammatory cytokines. Is known.

3 and 4, as a result of checking how the 70% ethanol extract of curly sedge has an effect on the inhibitory activity of TNF-α and IL-6 production increased by fine dust, the 70% ethanol extract of curly sedge is IL-6 It was confirmed that the production of and TNF-α was inhibited in a concentration-dependent manner.

Evaluation of COX-2 expression inhibition efficacy (western-blotting)

As a result of protein expression analysis, it was confirmed that COX-2 expression was induced by the fine dust treatment when compared with the fine dust-free treatment group. In addition, it was confirmed that the 70% ethanol extract of curly sedge inhibited the expression of COX-2 protein in a concentration-dependent manner (FIG. 5). This suggests that the results of the _{intracellular PGE 2} inhibitory activity test in the previous experiment are supported.

Claims (6)

Anti-inflammatory composition using the extract of frangipani sedge as an active ingredient.
The method of claim 1,
The extract is anti-inflammatory composition, characterized in that obtained by extraction with water, ethanol or a mixed solvent thereof.
The method of claim 1,
The anti-inflammatory composition for anti-inflammatory, characterized in that it means the improvement, treatment, suppression of the onset or delay of the onset of inflammatory diseases.
The method according to any one of claims 1 to 3,
The composition is an anti-inflammatory composition, characterized in that the pharmaceutical composition.
The method according to any one of claims 1 to 3,
The composition is an anti-inflammatory composition, characterized in that the food composition.
The method according to any one of claims 1 to 3,
The composition is an anti-inflammatory composition, characterized in that the cosmetic composition.

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