WO2012026771A2 - Composition comprising wercklea insignis extracts and fractions thereof for preventing and treating inflammatory diseases or asthma - Google Patents

Abstract

The present invention relates to a composition comprising Wercklea insignis extracts and fractions thereof for preventing and treating inflammatory diseases or asthma. The Wercklea insignis extracts and fractions thereof according to the present invention inhibit the generation of NO and PGE₂ TNF-α which are rapidly increased by the artificial induction of inflammation in macrophages, inhibit the expression of iNOS and COX-2, inhibit MAPK protein phosphorylation, inhibit the nuclear transfer of NF-κ Β, and reduce the quantities of cytokines IL-6 and IL-1beta, thereby inhibiting inflammation, inhibiting podedema in a carrageenan-induced mouse model, and significantly reducing the quantities of Th2-type cytokines IL-4 and IL-13 which increase in asthma or allergy. Therefore, the Wercklea insignis extracts and fractions thereof according to the present invention may be valuably used in a composition which is intended for medicine or health-promoting foods and which may require the Wercklea insignis extracts and fractions thereof as active ingredients for preventing and treating inflammatory diseases, allergies, and asthma.

Description

 【Specification】

 [Name of invention]

 Composition for the prophylaxis and treatment of inflammatory diseases or asthma containing Walkria insignia extract or fractions thereof

Technical Field

The present invention is a walklia insigny

Insignis) relates to a composition for the prevention and treatment of inflammatory diseases or asthma containing extracts or fractions thereof. Background Art

 In general, inflammatory reactions are biological defense reactions that attempt to repair and repair the damaged area when an invasion that results in any organic change in cells or tissues. Therefore, the series of reactions include local blood vessels, various tissue cells of body fluids, immune-related cells, and the like. Recently, with the development of molecular biology, an understanding at the molecular level that inflammatory diseases are cytokines (cytokine) has been attempted, and factors affecting these diseases have been identified one by one.

 Signal transduction pathways from the cell membrane to the nucleus follow a MAPK (Mitogen-activated protein kinase) cascade, and well known MAPKs include p38 MAPK and JNK jun N-terminal kinase (Erk) in addition to Erk. Genes mostly produce hormones that promote cell division or growth necessary to grow cells and are involved in inflammatory reactions and apoptosis (Vanden Berghe W et al., J. Biol.

Chem. , 273: 3285-3290, 1998; Yang M et al. , Am. J. Physiol. Heart. Circ.

Physiol. 294: H994-1001). Inflammatory cytokines and mediators are regulated by elements of the nucleus. For example, NF-icB (nuclear factor-kappa B) is a nuclear protein of the Rel gene family, and there are seven kinds of nuclear proteins. In the cytoplasm, I κΒ (inhibitor kappa) B) is present in an inactive form in combination with it, but after ΙκΒ kinase is activated by various stimuli such as reactive oxygen, chemokines like TNF-α (tumor necrosis factor-alpha), and LPS Phosphorylation causes the ΙκΒ to fall off. NF-κΒ, composed of ρ50 and ρ65 heterodimers, is known to promote the expression of genes (tumor necrosis factor or cyclooxide synthase) that, after being activated, move to the nucleus and induce inflammatory reactions (Oh GT et al., Artherosclerosis, 159 (1): 17-26, 2001; Epstein FH et al. 'The New England Journal of Medicine, 336 (15): 1066-1071, 1997; Zhang WJ et al., FASEB J, 15 (130): 2423-2431, 2001; Denk A et al., J. Biol. Chew., 276 (30): 28451-28458, 2001; Sahnoun Z et al., Phsiology, 53 (4): 315- 339, 1998; Lindner V Pathobiology, 66 (6): 311-320, 1998; Landry DB et al., Am. J. Pathol., 151 (4): 1085-1095, 1997 ;; Gerritsen ME et al., Am. J. Pathol., 7 (2): _P 278-292, 1995).

 Nitrogen oxides (NO) are produced by oxidation of L-arginine by nitrogen oxide synthase (N0S), which plays a role in maintaining homeostasis by acting as a mediator of the inflammatory process by damaging pathogenic DNA. (Kou and Schroder, Annuals of Surgery 221, 220-235, 1995). Among NOs, inducible nitric oxide synthase (iNOS) is known to be closely related to the overproduction of NO in cells.

Prostaglandin E ₂ (PGE ₂ ) and leukotriene are also inflammatory mediators produced from arachidonic acid, especially PGE ₂ , which is produced by cyclooxygenase-2 enzyme (C0X-2) and is primarily found in macrophages and monocytes. It was found that macrophages are rapidly induced by inflammatory agents such as LPS.

Substances used in animal models for provoking inflammation include adjuvant, collagen II, carrageenan, which is extracted from Chondrus cr // s, a kind of algae, has various effects such as acute inflammation and chronic inflammation, DIC induction, tumor growth, activation of Hageman factor and kinin, and inactivation of complement. It is known to have a biological action (Chan WY et al., J. Pharmacol. Exptl. Therap., 147: 48, 1965). The most potent anti-inflammatory drug of all time has been developed by steroids. However, long-term use must involve side effects. Therefore, in the treatment of asthma, steroids exert a surprising effect on the first use and completely disappear the symptoms, but this is only for a while, and the symptoms reappear with stopping use of steroids. It is getting worse. Steroids have many side effects, including back-blooded face, retention of body fluids, adrenal suppression, increased susceptibility to infections, and other psychosis, cataracts, glaucoma, peptic ulcers, delayed wound healing, and reactivation of early infections.

Therefore, there is a need for a study on a drug containing a natural plant extract as an active ingredient or a substance that can be safely consumed without a separate purification process, has an effect of inhibiting inflammation, and can be easily used in food. According to a recent Asian economy article published on June 14, 2010, "As a result of SK Chemicals' development of new drugs, asthma and dementia are currently undergoing Phase II clinical trials. According to an article published on June 17, 2010, according to an article published on June 17, 2010, Dr. Hyung-Kyu Lee, a team of immunological control laboratories of the Korea Research Institute of Bioscience and Biotechnology, developed NDC-052, extracted from Shin-Ki (flower buds), as an asthma treatment. ， It is being carried out in Phase III clinical trial under the brand name 'Asmang', which was transferred to Korea New Drug, and is released in the first half of this year. ”According to the article on January 22, 2006 Researchers found that extracts of triticale and leather tree are effective in treating asthma and allergies On the 19th, Korea advanced pharmacies have signed a technology transfer contract with Korea advanced pharmacies of 150 million won and 4% of ordinary fee sales. ”As reported, natural plant extracts are being actively researched.

Walkria Insigny

insignis Pittier & Standi.) is a plant belonging to the mallow family Mallow, native to Costa Rica. Although there is a prior art document that mallow and mallow plants belong to this plant for the treatment of inflammation and allergy (KR 10-2007- 0001359, KR 10-2008-0079489, US 2004/019429 Al, US 1989/0346104 Al, WO 03/033007 Al), the literature on workhorses and homologues and homologues have not been reported yet. Accordingly, the present inventors have prepared extracts and extracts thereof of Waria insignia, the extracts and their fractions inhibited the production of nitrogen oxides increased by inflammation, inhibited iNOS expression, inhibited prostaglandin production, inhibited C0X-2 expression , Inhibition of phosphorylation of MAPK protein, inhibition of migration to NF-κΒ nuclei, inhibition of IL-6 and IL-lbeta cytokine production, inhibition of TNF-α production, IL-4 and IL-, Th2 type cytokines in spleen cells 13 Inhibition of production and carrageenan-induced mouse model has excellent anti-inflammatory and anti-asthmatic effects such as foot edema suppression and is a safe, non-toxic material that can be used as a composition for the prevention and treatment of inflammatory diseases or asthma. The present invention was completed by confirming.

[Detailed Description of the Invention]

 [Technical problem]

The object of the present invention is a walkria insignificance

insignis) composition for the prevention and treatment of inflammatory diseases or asthma containing extracts or fractions thereof, health food It is providing a composition for cosmetics and a cosmetic composition.

Technical Solution

 In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention and treatment of inflammatory diseases or asthma containing as an active ingredient extract er ^ / ea ins ignis.

 In addition, the present invention provides a pharmaceutical composition for the prevention and treatment of inflammatory diseases or asthma containing the present invention prepared by adding an organic solvent to the extract of the workria insignificant as an active ingredient.

 In addition, the present invention provides a composition for the prevention and improvement of inflammatory diseases or asthma, the health food containing the workria insignificant extract as an active ingredient.

 In another aspect, the present invention provides a composition for preventing and improving inflammatory diseases or asthma containing the present invention prepared by adding the organic solvent to the extract of the workria insignificant as an active ingredient.

 In another aspect, the present invention provides a cosmetic composition for preventing and improving inflammatory diseases containing a walkia insignificant extract or a fraction thereof as an active ingredient.

 The present invention also provides a method of treating inflammatory disease or asthma, comprising administering a pharmaceutically effective amount of a walkia insignia extract or a fraction thereof to an individual suffering from an inflammatory disease or asthma.

 The present invention also provides a method for preventing inflammatory disease or asthma, comprising administering to a subject a pharmaceutically effective amount of a walkia insignia extract or a fraction thereof.

The present invention also provides a workria insignificant extract or a fraction thereof for use as a pharmaceutical composition for the prevention and treatment of inflammatory diseases or asthma. In addition, the present invention is for the prevention of inflammatory diseases or asthma, and for improving the food for health food Workria insignificant extract or fraction thereof is provided for use as a holy material. In addition, the present invention provides a workia insignia extract or a fraction thereof for use as a cosmetic composition for preventing and improving inflammatory diseases. Hereinafter, the present invention will be described in detail. The present invention provides a pharmaceutical composition for the prophylaxis and treatment of inflammatory diseases or asthma, containing the extract of Walkria insignia ¾rc ± / ea insignis) or a fraction thereof. Walkria insignificant extract of the present invention

 1) extracting the walkria insignia with water, alcohol or a combination thereof; And

 2) The extract of step 1) is preferably prepared by a manufacturing method comprising the step of concentrating and drying under reduced pressure to obtain a dry powder, but is not limited to the above method.

 In the above production method, in step 1), the workria insignals may be used without limitation, those grown or marketed.

In the production method, the alcohol of step 1) is characterized in that using d to c ₂ lower alcohol, the lower alcohol is characterized in that the ethane or methane, extraction using methane desirable. In addition, since organic substances are better eluted in 100% alcohol and glycosides are better eluted in aqueous alcohol solution, it can be used as an alcohol or an aqueous alcohol solution as needed. Extraction at room temperature is preferred, but is not limited thereto. Arler extraction frequency

It is preferably 1 to 5 times, it is more preferable to extract three times, but is not limited thereto.

In the above production method, the decompression concentration and drying process of step 2) in the art It may be carried out by the conventional method used. The organic solvent fraction of the walkia insignificant extract of the present invention

 1) fractionating the water suspension of Walkria insignificant extract into nucleic acid, chloroform, ethyl acetate, butane, and

 2) It is preferable that the fraction of step 1) is prepared by a manufacturing method comprising the step of concentrating and drying under reduced pressure to obtain a dry powder, but is not limited to the above method.

 In the above production method, the walkia insignificant extract of step 1) may be prepared according to the extract preparation method described above, but is not limited thereto.

 The inflammatory diseases include dermatitis, allergy, atopy, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, hemorrhoids, gout, ankylosing spondylitis, rheumatic fever, lupus, fibromyalgia ( fibromyalgia, psoriatic arthritis, osteoarthritis, osteoarthritis, rheumatoid arthritis, periarthritis 건초 tendinitis, hay salt, peritonitis, myositis, hepatitis, cystitis, nephritis, Sjogren's syndrome, multiple sclerosis, and acute and chronic inflammatory diseases It is preferably one selected from the group but is not limited thereto. In another aspect, the present invention provides a composition for the prevention and improvement of inflammatory diseases or asthma containing the workria insignificant extract or fractions thereof.

 Walkria insignificant extract of the present invention

 1) extracting the workria insignia with water, ethanol or a combination thereof; And

2) is preferably prepared by a manufacturing method comprising the step of concentrating and drying the extract of step 1) under reduced pressure to obtain a dry powder, but not limited to the above method. All.

 In the above production method, in step 1), the workria insignals may be used without limitation, those grown or marketed.

 Organic substances are better eluted in 100% alcohol and glycosides are better eluted in aqueous alcohol solution, so ethanol or ethane can be used as an aqueous solution if necessary. Extraction at room temperature is preferred, but is not limited thereto. In addition, the number of extraction is preferably 1 to 5 times, more preferably three times repeated extraction is not limited thereto.

 In the above production method, the vacuum concentration and drying process of step 2) may be performed by a conventional method used in the art. The organic solvent fraction of the walkia insignificant extract of the present invention

 1) fractionating the water suspension of the walkia insignia extract into nucleic acid, chloroform, ethyl acetate, butane, and

 2) Preferably, the fraction of 1) is prepared by a manufacturing method comprising the step of concentrating under reduced pressure and drying to obtain a dry powder, but not limited to the above method. In the above production method, the walkia insignificant extract of step 1) may be prepared according to the extract preparation method described above, but is not limited thereto.

The inflammatory diseases include dermatitis, allergy, atopy, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, hemorrhoids, gout, ankylosing spondylitis, rheumatic fever, lupus, fibromyalgia ( fibromyalgia), psoriatic arthritis, osteoarthritis, rheumatoid arthritis, periarthritis, tendonitis, hay salt, peritonitis myositis, hepatitis, cystitis, nephritis, Sjogren's syndrome, multiple sclerosis, and acute and chronic inflammatory diseases It is preferably any one selected from, but is not limited thereto. The present inventors treated the control group (DMS0), the extract of the present invention and then cultured the macrophages to determine whether the workia insignificant extract or fractions thereof are cytotoxic, and then measured the survival rate of the cells. As a result, it was confirmed that the extract of walkia insignificant methane and the butanol fraction were almost non-toxic up to a concentration of 40 / g / m «. However, chloroform and ethyl acetate fractions were found to increase rapidly cytotoxicity at a concentration of 40. Accordingly, it can be seen that the extract or fractions thereof of the walkia insignificant methane of the present invention is a cytotoxic substance (see Table 1). The present inventors measured the amount of inducible nitric oxide (NO), iNOS expression, and IL-6 inhibitory effect in order to investigate the inhibitory effect of WORKIA insignificant extract or fractions thereof on inflammatory diseases or asthma. As a result, the WPS-induced Methanol extract reduced concentrations of nitrogen oxides and IL-6 in a concentration-dependent manner and inhibited LPS-induced inflammation in all fractions except water fraction. 2). In addition, the concentration of iNOS nucleic acid expression (see FIG. 2A) and protein expression (see FIG. 2B and FIG. 2C) were significantly decreased according to the treatment concentration in the group treated with the Walkria insignificant methanol extract.

The present inventors measured the change of prostaglandin E ₂ production and C0X-2 expression in order to investigate the inhibitory effect of the wkria insignificant extract on inflammatory diseases or asthma. As a result, the concentration of prostaglandin E ₂ decreased in a concentration-dependent manner with respect to the LPS-induced inflammation, especially in the group treated with the extract 40 / ^ of the workria insignificant methane. 0.69) (see Table 3). In addition, the concentration of C0X-2 nucleic acid expression (see FIG. 3A) and protein expression (see FIG. 3B and FIG. 3C) were significantly decreased according to the treatment concentration in the group treated with the Walkria insignificant methanol extract.

The present inventors inhibited the expression of the signaling protein of the Walkria insignificant extract. As a result, when the LPS and the walkria insignificant methanol extract of the present invention were simultaneously treated with macrophages, the expression of all signaling proteins in the same form was the same, whereas the phosphorylated proteins were found in the walkia insignificant methanol extract. It was confirmed that the expression is reduced depending on the concentration of (see FIG. 4).

 The present inventors measured whether the walkia insignificant extract migrates to the nucleus of the NF-κΒ protein to investigate the inhibitory effect on inflammatory diseases or asthma. As a result, it was confirmed that less p65 was observed in the nucleus in the cells treated with the Walkria insignificant methanol extract compared to the cells treated with only LPS (see FIG. 5). The present inventors measured the change in the production of cytokines IL-6 and IL-lbeta in order to investigate the inhibitory effect of the Walkria insignificant extract against inflammatory diseases or asthma. As a result, the amount of IL-6 and IL-lbeta was increased in the group treated with LPS, and the amount of cytokine increased by LPS decreased with the concentration of extract. (See FIG. 6).

 The present inventors measured the change in the amount of production of TNF-α in order to investigate the inhibitory effect of the Walkria insignificant extract against inflammatory diseases or asthma. As a result, the amount of TNF-a was increased in the LPS-treated group, and the amount of TNF-a increased by LPS decreased with the concentration of the extract. Reference) .

The present inventors confirmed the inhibitory effect of the extract of Warriag insignificant methane on carrageenan-induced mouse foot edema, the negative control group induced inflammation by carrageenan, but increased the thickness of the foot. In the group pre-treated with the extract it was confirmed that the thickness of the foot decreases in a concentration-dependent (see Figure 8). The present inventors measured the change in the production of cytokines IL-4 and IL-13 in splenocytes in order to investigate the inhibitory effect of the Walkria insignificant extract on inflammatory diseases or asthma. As a result, cytokines IL-4 and IL-13 induced by ConA The amount was reduced concentration-dependently in the group treated with the Walkria insignificant methanol extract (see FIG. 9).

 Accordingly, the walkia insignificant methanol extract and fractions thereof of the present invention inhibit the production of nitrogen oxides, inhibit the production of prostaglandins, inhibit the phosphorylation of MAPK proteins, inhibit the migration of NF-κΒ to the nucleus, cytokines IL-6 and IL- It can be used to prevent or treat inflammatory diseases or asthma by inhibiting lbeta production, inhibiting TNF-α production, inhibiting foot edema in carrageenan-induced mouse models, and inhibiting the production of cytokines IL-4 and IL-13. Able to know. The composition for the prevention and treatment of inflammatory diseases or asthma of the present invention may contain the Walkria in signature extract or fractions thereof, and may further contain one or more active ingredients exhibiting the same or similar functions in addition to the above components. .

The compositions of the present invention can be administered orally or parenterally during clinical administration and can be used in the form of general pharmaceutical formulations. That is, the composition of the present invention can be administered in various oral and parenteral formulations during actual clinical administration, and when formulated, it is a diluent such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactants that are commonly used. Or using excipients. Solid preparations for oral administration include tablets, pills, powders, granules and capsules, and the like, which may be used in the compositions of the present invention at least one excipient such as starch, calcium carbonate, sucrose, lactose. And gelatin etc. are mixed and prepared. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions and syrups. Water, liquid paraffin, which is a commonly used simple diluent, includes various excipients, for example wetting agents, sweeteners, fragrances and preservatives. Can be. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories. Non-aqueous and suspending solvents Ropelene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyloleate and the like can be used. As a basis for suppositories

Oitepsol), macrogol, tween 61, cacao butter, laurin, glycerol and gelatin dung can be used. The composition of the present invention may be via subcutaneous injection, intravenous injection or intramuscular injection during parenteral administration.

 Dosage units may contain, for example, 1, 2, 3 or 4 times the individual dosage or 1/2, 1/3 or 1/4 times. The individual dosage preferably contains the amount in which the effective drug is administered at one time, which usually corresponds to the total, 1/2, 1/3 or 1/4 times the daily dosage. The effective dose of the composition of the present invention is 0.0001-10 g / kg, preferably 0.0001 g-5 g / kg, may be administered 1 to 6 times a day.

 The composition of the present invention can be used alone or in combination with methods using surgery, hormonal therapy, chemotherapy and biological response modifiers for the prevention and treatment of inflammatory diseases or asthma. In addition, the present invention provides a cosmetic composition for preventing and improving inflammatory diseases containing a walkia insignificant extract or a fraction thereof.

 Walkria insignificant extract of the present invention

 1) extracting the walkria insignia with water, alcohol or a combination thereof; And

 2) The extract of step 1) is preferably prepared by a manufacturing method comprising the step of concentrating and drying under reduced pressure to obtain a dry powder, but is not limited to the above method.

 In the above production method, in step 1), the workria insignals may be used without limitation, those grown or marketed.

In the production method, the alcohol of step 1) is Ci to C ₂ lower alcohol It is characterized in that the use, the lower alcohol is characterized in that the ethane or methane, it is preferable to extract using methane. In addition, organic matter

Elution is better in 100% alcohol and glycosides are better in elution in an aqueous alcohol solution. Extraction at room temperature is preferred, but is not limited thereto. In addition, the number of extraction is preferably 1 to 5 times, more preferably three times repeated extraction is not limited thereto.

 In the above production method, the vacuum concentration and drying process of step 2) may be carried out by conventional methods used in the art. The organic solvent fraction of the walkia insignificant extract of the present invention

 1) fractionating the water suspension of Walkria insignificant extract with nucleic acid, chloroform ᅳ ethyl acetate, butanol, and

 2) It is preferable that the fraction of step 1) is prepared by a manufacturing method comprising the step of concentrating and drying under reduced pressure to obtain a dry powder, but is not limited to the above method.

 In the above production method, the walkia insignificant extract of step 1) may be prepared according to the extract preparation method described above, but is not limited thereto.

The inflammatory diseases include dermatitis, allergy, atopy, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, hemorrhoids, gout, ankylosing spondylitis, rheumatic fever, lupus, fibromyalgia ( fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, periarthritis, tendinitis, hay salt, periarthritis, myositis, hepatitis, cystitis, nephritis, Sjogren's syndrome, multiple sclerosis, and acute and chronic inflammatory diseases It is preferably one selected from the group but is not limited thereto. The walkia insignificant extract and fractions thereof of the present invention inhibit the production of nitrogen oxides, inhibit the production of prostaglandins, inhibit the phosphorylation of MAPK proteins, inhibit the migration of NF-κΒ to the nucleus, and the cytokines IL-6 and IL-lbeta. Inhibition of TNF-α production, inhibition of foot edema in carrageenan-induced mouse models, and inhibition of the production of cytokines IL-4 and IL-13 may be useful in preventing or treating inflammatory diseases. Able to know. The cosmetic composition is, for example, an emulsion, suspension, microemulsion, microcapsules, fine granules or ionic (liposomes), nonionic, obtained by dispersing an oil phase in a solution, gel, solid or pasty anhydride, aqueous phase. It may be provided in the form of a vesicle dispersant, cream, skin, lotion, powder, ointment, spray or cone stick. Also, foam

It may also be prepared in the form of a foam or in the form of an aerosol composition further containing a compressed propellant.

 In addition, the cosmetic composition is in addition to the fraction of the present invention, fatty substances, organic solvents, solubilizers, thickeners and gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water Commonly used in ionic or nonionic emulsifiers, layering agents, metal ion sequestrants and chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or cosmetics It may contain adjuvants conventionally used in the cosmetic field, such as any other ingredients employed. In another aspect, the present invention provides a composition for the prevention and improvement of inflammatory diseases or asthma containing the workria insignificant extract or fractions thereof.

When using the workria insignia extract of the present invention or a fraction thereof as a food additive, the workria insignia extract or a fraction thereof is added as it is. Or other food or food ingredients, and may be appropriately used according to conventional methods. The walkia insignificant extract or fractions thereof is preferably extracted using hot water and ethane. The combined amount of active ingredient can be suitably determined depending on the purpose of use (prevention, health or therapeutic treatment). In general, in the preparation of food or beverages, the workria insignificant extract or fractions thereof of the present invention is added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less with respect to the raw material. However, in the case of long-term intake for health and hygiene or health control, the amount may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety. There is no particular limitation on the kind of food. Examples of the food to which the walkia incidence extract or fractions thereof of the present invention may be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, and ice cream. , Various soups, beverages, teas, drinks, alcoholic beverages and vitamin complexes, and includes all healthy foods in the usual sense.

 The health beverage composition of the present invention may contain various flavors or natural carbohydrates, etc. as additional components, as in the general beverage. Natural carbohydrates are sugars such as glucose, monosaccharides such as fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrins and cyclodextrins, xylides, sorbates and erythrides. As the sweetener, natural sweeteners such as tautin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like can be used. The proportion of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g, per 100 compositions of the present invention.

In addition to the above, the walkia insignificant extract or fractions thereof of the present invention may be various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acids and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH regulators, Stabilizers, preservatives, glycerin, Alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the walkia insignificant extract or fractions thereof of the present invention may contain pulp for producing natural fruit juice, fruit juice beverage and vegetable beverage. These components can be used independently or in combination.

The present invention also provides a method of treating inflammatory disease or asthma, comprising administering to a subject with an inflammatory disease or asthma a pharmaceutically effective amount of the above-mentioned workria insignificant extract or fraction thereof.

 In addition, the present invention provides a method for preventing inflammatory disease or asthma, comprising administering to a subject a pharmaceutically effective amount of the walkia insignia extract or a fraction thereof.

 The pharmaceutically effective amount means an amount sufficient to treat the disease at a reasonable benefit or risk ratio applicable to the medical treatment, which means the type, severity, activity of the drug, sensitivity to the drug, and administration of the individual's disease. It can be determined by time, route of administration and rate of release, duration of treatment, factors including the drug being used simultaneously, and other factors well known in the medical field.

 The subject can be any animal, including humans.

 Methanol extracts and fractions thereof of the present invention may be used to inhibit the production of nitrogen oxides, inhibit the production of prostaglandins, inhibit the phosphorylation of MAPK proteins, inhibit the migration of NF-κΒ to the nucleus, and the cytokines of IL-6 and IL-lbeta. Inhibition of TNF-α production, inhibition of foot edema in carrageenan-induced mouse models, and inhibition of the production of cytokines IL-4 and IL-13, which are useful for preventing or treating inflammatory diseases or asthma. It can be seen that.

The walkia insignificant extract or fractions thereof is further the same or similar It may contain one or more active ingredients exhibiting dead-end function.

 The administration may be administered orally, or parenterally by subcutaneous injection, intravenous injection or intramuscular injection, and may be used in the form of a general pharmaceutical preparation.

 The dosage unit may contain 1, 2, 3 or 4 times the individual dose or may contain 1/2, 1/3 or 1/4 times. The individual dosage preferably contains the amount in which the effective drug is administered at one time, which usually corresponds to the total, 1/2, 1/3 or 1/4 times the daily dosage. An effective dose is 0.0001 to 10 g / kg, preferably 0.0001 g to 5 g / kg, and may be administered 1 to 6 times a day. The present invention also provides a workria insignificant extract or a fraction thereof for use as a pharmaceutical composition for the prevention and treatment of inflammatory diseases or asthma.

 The present invention also provides a workria insignificant extract or a fraction thereof for use as a composition for the prevention of inflammatory diseases or asthma, and for improving health food. In addition, the present invention provides a walkia insignificant extract or a fraction thereof for use as a cosmetic composition for preventing and improving inflammatory diseases.

 The inflammatory diseases include dermatitis, allergy, atopic dermatitis, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, hemorrhoids, gout, ankylosing spondylitis, rheumatic fever, lupus, fibromyalgia ( fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, periarthritis, tendonitis, hay salt, peritonitis myositis, hepatitis, cystitis, nephritis, Sjogren's syndrome, multiple sclerosis, and acute and chronic inflammatory diseases It is preferably any one selected from, but is not limited thereto.

The extracts and fractions thereof of the present invention were treated with inhibitors of the production of nitrogen oxides, inhibition of prostaglandin production, inhibition of phosphorylation of ΜΑΡΚ protein, inhibition of migration of NF-κΒ to the nucleus, cytokines IL-6 and IL ᅳ lbeta. Inhibition of the production of, billion generation of T F-α First, the carrageenan-induced mouse model exhibits the effect of inhibiting foot edema and the production of cytokines IL-4 and IL-13, and thus is an effective ingredient in pharmaceutical compositions, health food compositions and cosmetic compositions for the prevention or treatment of inflammatory diseases or asthma. It can be seen that it can be usefully used.

Advantageous Effects

 Walkia insignificant extract or fractions thereof of the present invention is a non-toxic safe substance, inhibiting the production of nitrogen oxides increased by inflammation, iNOS expression inhibition, prostaglandin production inhibition, C0X-2 expression inhibition, MAPK protein Inhibition of phosphorylation, inhibition of NF—κΒ migration, inhibition of IL-6 and IL-lbeta cytokine production, inhibition of TNF-α production, inhibition of IL-4 and IL-13 production in splenocytes and a carrageenan-induced mouse model By inhibiting foot edema in can be usefully used as a pharmaceutical composition, a health food composition or a cosmetic composition for the prevention and treatment of inflammatory diseases or asthma. [Brief Description of Drawings]

 1 is a graph showing a method of preparing a walkia insignis fer ± / ea insignis extract or a fraction thereof.

 FIG. 2 is a graph showing the inhibitory effect of Walkria insignificant methanol extract on the expression of lipopolysaccharide (LPS, 1 ipopolysacchar ides) induced iNOS in RAW264.7 cells:

 Negative control group: 0.2% DMS0 only;

 LPS: positive control induced inflammation by treatment with LPS of l // g / m £; And

 LPS + WI extract: the group was induced with LPS after 4 (g / i treatment of the extract of the workria insignificant methane.

2A: nucleic acid amplification analysis; 2B: western blotting; And

 2C: immunofluorescence assay

 FIG. 3 is a graph showing the inhibitory effect of the Walkria insignificant methanol extract on lipopolysaccharide (LPS) induced C0X-2 expression in RAW264.7 cells: FIG. 3A: nucleic acid amplification assay;

 3B: western blotting; And

 3C: immunofluorescence assay.

 Figure 4 is a diagram showing the inhibitory effect of the workria insignia methanol extract on the phosphorylation of lipopolysaccharide-induced signaling protein in RAW264.7 cells. FIG. 5 is a graph showing the inhibitory effect of extracts from the workria insignificant methane on the migration of lipopolysaccharide-induced NF-κΒ protein to the nucleus in RAW264.7 cells.

 FIG. 6 is a graph showing the inhibitory effect of the extract of Warriag insignificant methane on the production of lipopolysaccharide-induced cytokines in RAW264.7 cells:

 #: P value for the negative control group was 0.005 or less;

 *: P value for the positive control group was 0.05 or less; And

 **: P value for a positive control group is 0.005 or less.

 Figure 7 is a graph showing the inhibitory effect of the Walkria insignificant methanol extract on the production of lipopolysaccharide-induced TNF-alpha in RAW264.7 cells. FIG. 8 is a graph showing the inhibitory effect of Walkria insignificant methanol extract on carrageenan-induced mouse foot edema:

 *: P value for the negative control group was 0.15 or less; And

 **: P value for a negative control group is 0.05 or less.

FIG. 9 is a graph showing the inhibitory effect of extracts of W. insignia methane on the production of ConA-induced cytokines in spleen cells: #: P value for the negative control group was 0.005 or less;

 *: P value for the positive control group was 0.05 or less; And

 **: P value for a positive control group is 0.005 or less. [Best form for implementation of the invention]

 Hereinafter, the present invention will be described in detail by Examples, Experimental Examples and Preparation Examples. However, the following Examples, Experimental Examples and Preparation Examples are specifically illustrated in the present invention, and the content of the present invention is not limited to Examples, Experimental Examples, and Preparation Examples. Example 1 Preparation of Extracts or Fractions thereof from Warcia Insignis Methane

 <1-1> Preparation of Worcia insignificant methane extract

 Extract 0.6 kg of dried Dyrcria insignia leaves from 100% methane three times at room temperature at 24 hour intervals (hereinafter referred to as "Worthria in Signis Extract"). 29.5 g were obtained.

<1-2> Preparation of a fraction of the extract of Warriak insignificant methane

The workria insignificant methanol extract was suspended in 1 L of water and shaken for 3 times by adding the same amount of 100% nucleic acid to remove the water layer to obtain 8.27 g of the nucleic acid extract. After removing the nucleic acid extract and adding the same amount of chloroform to the remaining water layer, the mixture was left to shake three times to obtain 4.28 g of the chloroform extract. The same amount of ethyl acetate was added to the remaining water layer to obtain 1.44 g of the ethyl acetate extract in the same manner. Again, the same amount of butanol was added to the remaining water layer to obtain 3.31 g of butanol extract in the same manner, and the remaining water layer was concentrated to obtain 4.68 g of water extract (FIG. 1). Experimental Example 1 Extract and Fraction of Walkria Insignificant Methane from Raw264.7 Cells Cytotoxicity Test on Water

 <1-1> cell culture

Macrophage line R 264.7 cells (ATCC ^® No. ΒΒ-71) were 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 units penicillin and 100 g / m streptomycin. Incubated in 37 ^° C, 5% C0 ₂ wet incubator using the contained DMEM medium.

<1-2> cytotoxicity measurement

Rat macrophage R _a w264.7 cells were suspended in DMEM (D ^ becco's Modified Eagle Medium, Gibco) medium containing ⁵ % Fetal Bovine Serum at a concentration of lxlO ⁵ / ^. Inoculated in well plate (96 well plate). After 4 hours the extract and its fractions were treated by concentration. After incubation for 24 hours, 5 ^ / MTT solution was added 10 ^ per well, followed by 4 hours of incubation. After incubation, the supernatant was removed, and the absorbance at 570 nm after 100 ^ addition of DMSO. The cell survival rate was calculated according to the following equation with 100% of the negative control group treated with 0.1% of DMS0.

[Equation 11

 Table 1

 Walkria insignificant cell viability (%)

 Negative Control 100.00 土 1.53

 Methanol Extract 5 101.81 土 0.10

 Methane extract 10 95.59 士 0.35

Methane extract 20 94.77 土 0.21 Extraction of methane 40 92.25 土 0.36 nucleic acid fraction 5 99.33 土 0.83 nucleic acid fraction 10 86.26 10 4. 13 nucleic acid fraction 20 86.12 土 4.74 nucleic acid fraction 40 78.89 土 0.76 chloroform fraction 5 95. 19 土 1.85 chloroform fraction 10 87.70 土 0.56 chloroform fraction 20 92.54 土 1.45 Chloroform fraction 40 59.93 土 0.44 Ethyl acetate fraction 5 90.37 士 1.44 Ethyl acetate fraction 10 93. 13 土 2.23 Ethyl acetate fraction 20 98.96 土 3.07 Ethyl acetate fraction 40 55.81 士 3.74

Butane fraction 5 94.11 土 0.75 Butane fraction 10 97.09 土 5.50 Butane fraction 20 91.03 土 2.62 Butanol fraction 40 84. 14 土 1.29 Water fraction 5 93. 14 土 1.07 Water fraction 10 94.27 土 0.31 Water fraction 20 98.82 土 5.81 Water Fraction 40 94.06 土 0.62 Table 1 shows the results of toxicological studies on the Warlia insignia extract and fractions thereof. It was confirmed that there is little toxicity up to the concentration. However, chloroform and ethyl acetate fractions were found to increase rapidly cytotoxicity at a concentration of 40 / zg /.

Experimental Example 2 Inhibitory Effect of the Walkria Insignificant Methanol Extract on the Production of Nitrogen Oxide (NO) and Cytokine IL-6

 <2-1> Inhibition of NOx and Cytokine IL-6 Production in Raw264.7 Cells

 The amount of inducible nitrogen oxides was measured to investigate the inhibitory effect on artificially induced inflammation by treating LPS to Ra 264.7 cells.

Specifically, 5% Fetal Bovine Serum was added to DMEM (D / ibecco's Modified Eagle Medium, Gibco Co., Ltd.) containing no phenol-Red, and the cells were suspended by lxlO ⁵ cells. 96 well plates were inoculated. After attaching for 4 hours, the samples were treated at 0, 5, 10, 20 or 40 ^ and incubated for 1 hour, followed by l ^ g / me lipopolysaccharide (LPS, lipopolysaccharide, Sigma) Was incubated for 24 hours. After that, 100 μl of the supernatant was collected and placed in a new 96-well plate, and the same amount of grease reagent (Griess reagent, Sigma) was added and reacted at room temperature for 10 minutes, followed by a microplate reader (Bio-Rad). The absorbance was measured at 540 nm. A calibration curve was prepared by using sodium nitrite, and the amount of nitrogen oxides produced in the culture solution was determined based on this. The amount of nitrogen oxides produced in the lipopolysaccharide treated group was 100%. Represented by. In addition, in order to confirm the inhibitory effect of cytokine production of the lipopolysaccharide-derived Warria insignificant extract and its fractions in Raw264.7 cells, lipopolysaccharide-induced IL-6 production was analyzed. It was measured using an immunological assay kit (mouse IL-6 Enzyme Immunometric Assay Kit, Assay designs, USA). [Table 2]

Workria insignificant NO production IL-6 production IL-6 inhibition Sample / g / m (uM) (pg / n) () negative control 0.29 土 0.07 -18.00 土 1.57

 LPS 21.55 土 0.14 1341.63 士 28.28

 LPS + 19.84 士 0.12 1206.82 士 62.85 10.05 土 4.68 Methanol Extract 5

 LPS + 19.09 土 0.24 954.96 士 104.76 28.82 土 7.81 Methanol Extract 10

 LPS + 16.85 土 0.08 790.15 土 26.71 41.11 土 1.99 Methanol Extract 20

 LPS + 11.61 土 0.20 194.59 土 47.14 85.50 土 3.51 Methanol Extract 40

 LPS + 21.00 土 0.19 1225.89 66 66.26 8.63 士 4.94 Nucleic Acid Fraction 5

 LPS + 21.24 0.1 0.19 1181.63 土 42.43 11.93 士 3.16 Nucleic acid fractions 10

 LPS + 17.91 0.8 0.86 908.48 土 86.69 32.29 土 6.46 Nucleic Acid Fraction 20

 LPS + 13.21 土 0.85 149.96 土 80.40 88.82 土 5.99 Nucleic Acid Fraction 40

LPS + 20.44 土 0.76 1158.67 土 17.81 13.64 土 1.33 Chloroform fraction 5 LPS + 18.26 土 0.23 1083.67 28 28.02 19.23 土 2.09 Chloroform fraction 10

 LPS + 14.58 土 1.06 736.07 土 80.14 45.14 土 5.97 Chloroform fraction 20

 LPS + 1.32 土 0.03 7.74 士 2.36 99.42 土 0.18 Chloroform Fraction 40

 LPS + 20.79 土 0.34 1228.11 土 11.26 8.46 土 0.84 Ethyl Acetate

 Fraction 5

 LPS + 18.65 土 0.15 1159.59 土 3.40 13.57 土 0.25 Ethyl Acetate

 Fraction 10

 LPS + 12.74 土 1.72 684.78 土 57.35 48.96 土 4.27 ethyl acetate

 Fraction 20

 LPS + 1.36 土 0.15 -11.15 土 2.36 100.83 土 0.18 ethyl acetate

 Fraction 40

 LPS + 19.78 土 1.09 1021.44 土 23.83 23.87 土 1.78 Butanol fraction 5

 LPS + 19.13 + 0.44 910.52 土 81.19 32.13 土 6.05 Butanol fraction 10

LPS + 14.77 + 0.05 569.41 土 74.90 57.56 土 5.58 Butane fraction 20 LPS + 2.20 土 0.02 67.93 土 24.09 94.94 士 1.80 Butanol fraction 40

 LPS + Water Fraction 5 22.38 土 0.23 1296.07 土 33.52 3.40 土 2.50

LPS + Water fraction 10 21.83 0.1 0.18 1276.63 土 10.21 4.84 土 0.76

LPS + water fraction 20 19.69 土 1.42 1249.59 土 97.69 6.86 土 7.28

LPS + water fraction 40 21.80 土 0.82 1312.19 土 8.64 2.19 土 0.64 Table 2 shows the inhibitory effects of NOx production and cytokine production of the Waria insignis extract and its fractions. Compared with the treated group, it was confirmed that the production of NOx and IL-6 decreased in the concentration-dependent group in the treated group of the WYC in methanol extract, and showed the inhibitory effect in all fractions except the water fraction. (Table 2). Therefore, the subsequent experiment was conducted with a methanol extract easy to obtain a sample.

<2-2> Expression inhibition experiment of iNOS gene (RT-PCR)

Experimental Example <2-1> by dispensing lx 10 ⁶ cells in 100 mm Petri dish, treated with the concentration of walkia insignificant methanol extract, induction of inflammation by LPS for 24 hours culture, After removal of the cells, the cells were removed from the culture vessel and the cells were homogenized using ribo nucleic acid extraction solution (Invitrogen, CA, USA). After 5 minutes, the cells were collected, transferred to a centrifuge tube, 200 ^ chloroform was added, mixed for 15 seconds, left for 3 minutes, and centrifuged at 14000 rpm for 15 minutes. The supernatant containing ribonucleic acid was transferred to a new tube and mixed with isopropyl alcohol 500 ^. After 10 minutes, the supernatant was discarded again, and the supernatant was discarded. Then, 75¾> ethane was added to the precipitate and centrifuged at 10000 rpm for 5 minutes. The supernatant was discarded and the precipitated ribonucleic acid was dried at room temperature for 20 minutes. Dried ribonucleic acid is produced by DEPCX Diethylpyrocarbonate, Sigma) was suspended in treated distilled water. Ribonucleic acid was synthesized after complementary nucleic acid (complementary DNA) using RT-PreMix (AccuPower RT PreMix, Bioneer, Korea). The synthesized cDNA was used as a template, iNOS primers were mixed, and the expression level of ribonucleic acid was confirmed using PCR Preraix (AccuPower PCR PreMix, Bioneer, Korea).

 As a result, as shown in FIG. 2A, iNOS expression of the positive control group treated with lipopolysaccharide was increased as compared to the negative control group. It was confirmed that the amount of nucleic acid expression is significantly reduced (Fig. 2a).

<2-3> Western blotting inhibition of expression of iNOS protein

Experimental Example <2-1> by dispensing IX 10 ⁶ cells in 100 瞧 Petri dish, treatment with the concentration of the Warlia insignificant methanol extract, induction of inflammation by LPS for 24 hours culture, After removal of the cells, the cells were removed from the culture vessel and homogenized using a protein eluate (CelLyticTM-MT Tissue Lysis Reagent, Sigma, USA) containing a protease inhibitor cocktail (Roche, USA). The extract was centrifuged at 14000 rpm for 20 minutes to separate the supernatant and the insoluble agglomerates. Protein concentration of the isolated supernatant was measured using a Bio-Rad protein assay kit (Bio-Rad, USA). In addition, the supernatant was mixed with 5 × SDSC0.156M Tris-HCl, pH 6.8, 2.5% SDS, 37.5% glycerol, 37.5 mM DTT) and 1: 4 and boiled at 100 ^° C. for 10 minutes. 40 proteins from the boiled samples were loaded on SDS 4-12% SDS-PAGE gel and electrophoresed at 125 V for 2 hours to separate them according to molecular weight, and the proteins were subjected to electrophoresis for 1 hour under conditions of 50 mA per gel. Was transferred to PVDF membrane. Block the protein-free portion of the membrane from which the protein was transferred with skim milk powder, and then remove the primary antibody [anti-iNOS antibody (1: 1000, Santa Cruz Biotechnology, USA)] and secondary antibodies (anti-rabbit -IgG-HRP; Amersham Biosciences, UK), followed by luminescence reaction with ECL detection kit (Amersham Biosciences, UK) and exposure to X-ray film It was made photosensitive. The relative density of the protein bands can be determined using an image analysis system to detect the degree of photosensitivity, and the Tina 2.0 software (// 1 inux.softpedia.com/get/System/Operating-Systems/Linux-Distributions/TINA-KN0PPIX- Quantification was performed using Live-CD-5172.sht O.

 As a result, as shown in Fig. 2b, when the LPS and the workria insignificant methanol extract of the present invention were simultaneously treated with macrophages, protein expression of iNOS was reduced (Fig. 2b).

<2-4> Immunofluoresence of iNOS protein (Immunofluoresence)

About 2xi0 ⁵ / m £ cells were incubated on Permanox chambered plastic slides (Nunc, USA) and then the supernatant was removed. After ethanol was fixed at 4 ^° C for 30 minutes, washed with phosphate complete solution (PBS), and then blocked with 3% bovine serum albumin (bovine serum albumin) at room temperature for 30 minutes. After blocking the primary antibody [anti-iNOS antibody (1: 100)] was reacted at 4 ^° C. for one day. After washing three times with a phosphate complete solution, a secondary antibody (Santa Cruz Biotechnology, USA) to which Texas red (Santa Cruz Biotechnology, USA) was connected was reacted for 2 hours at room temperature in dark conditions. The phosphate supernatant was mounted three times with Proton Gold Antifade solution (Invitrogen, USA) and photographed by confocal microscope (LSM510m Carl Zeiss, Germany).

As a result, as shown in Figure 2c it was confirmed that the expression of iNOS was reduced in the cells treated with the extract with the workria in signature methane compared to the cells treated only LPS (Fig. 2c). Experimental Example 3 Inhibitory Effect of Walkria insignificant Methanol Extract on Prostaglandin Production

 <3-1> Prostaglandin production inhibition test in Raw264.7 cells

The experiment was to verify the work man-hours that Ria varnish methane extracts of prostaglandin E ₂ (Prostaglandin E2) production-inhibiting effect. Experimental Example <2-1> was taken from the culture supernatant of Raw264.7 cells treated with the Walkria insignificant methanol extract and lipopolysaccharide, and the prostaglandin measurement kit (PGE ₂ assay kit; R & D systems, Minneapolis, USA) was used to determine the effect of inhibiting the production of prostaglandin ^ (Prostaglandin E2).

Table 3

Table 3 shows the effect of inhibiting the production of prostaglandin E ₂ produced by the extract of workria insignificant methanol, and the production amount of prostaglandin E ₂ was reduced depending on the concentration of the extracts treated with workria insignificant methane, 40; / ι showed prostaglandin E ₂ production inhibition rate of 85.63 土 0.69% (Table 3).

<3-2> Expression inhibition experiment of C0X-2 gene (RT-PCR) RT-PCR was performed to confirm the effect of the extracts of Warria insignis methane on the expression of cyclooxygenase (C0X-2), which is involved in the production of prostaglandins. The cDNA obtained in the same manner as in Experimental Example <2-2> was used as a template, the C0X-2 primer was mixed, and the expression level of ribonucleic acid was confirmed by using a PCR premix.

 As a result, as shown in Figure 3a, it was confirmed that the expression of C0X-2 increased in the LPS treatment group, the nucleic acid expression of C0X-2 in accordance with the treatment concentration in the group treated with the Walkria insignificant methanol extract It was confirmed to be inhibited (FIG. 3A). <3-3> Western blotting inhibition of expression of C0X-2 protein

 Experimental Example <2-3 after binding an anti-C0X-2 antibody (1: 1000, Santa Cruz Biotechnology, USA) with a primary antibody to a PVDF membrane to which a protein was obtained in the same manner as in Experimental Example <2-3> The photosensitizing method was analyzed in the same manner as>.

 As a result, as shown in Fig. 3b, the expression of C0X-2 protein was reduced in a concentration-dependent manner when LPS and extracts of the present invention werria insignificant methane were simultaneously treated with macrophages (Fig. 3b).

<3-4> Immunofluorescence staining of COX-2 protein (I 隠 unofhioresence)

 Experimental Example <2-4> After the cells were fixed and blocked, the primary antibody [anti-Cox-2 antibody (1; 100)] was bound and confirmed by the same method.

As a result, as shown in FIG. 3c, it was confirmed that the expression of C0X-2 was reduced in the cells treated with the extract of Walkria in signature methane as compared to the cells treated with LPS only (FIG. 3c). Experimental Example 4 Signaling Step of Walkria Insignificant Methanol Extract in Raw264.7 Cells Phosphorylation Inhibitory Effect of White Matter

Dispense the cells by the method of Experimental Example <2-3>, treat the extracts with the concentration of Warria insignificant methane, induce inflammation with LPS, incubate for 15 or 30 minutes, remove the medium, and then remove the cells. Was removed from the culture vessel and homogenized using a protein eluate containing a protease inhibitor and phosphatase inhibitor (Roche, Germany). The extract was centrifuged at 14000 rpm for 20 minutes, and then the supernatant and the insoluble aggregate were separated. Proteins of the separated supernatants were quantified and electrophoresed on 20 SDS 10% SDS-PAGE gels per sample and transferred to PVDF membranes. The protein-free portion of the membrane to which the protein has been transferred is blocked with skim milk powder, and then the anti-ERK, anti-p38 MAP, anti-JNK, anti-IkBa (l: 1000, Santa Cruz Biotechnology, USA) or the anti- _p 38 MAPK and anti-JNK (1: 1000, Enzo, Rarmingdale, NY) or anti-myo 1 (, anti-IkBaCl: 1000, Cell Signaling Technology, USA) After attaching the phosphorylated form of antibody, the secondary antibody was bound sequentially.

 As a result, as shown in Figure 4 when the LPS and the extract treated with the workia insignia methane of the present invention to the macrophage at the same time the expression of the signaling proteins of the overall form, while the phosphorylated forms of the protein It was confirmed that the expression decreases in a concentration-dependent manner of ria insignificant methanol extract (FIG. 4). Experimental Example 5 Effect of Inhibition of NF-κΒ Protein Transfer on Warlia Insignis Methane Extracts After the cells were fixed and blocked in the same manner as in Experimental Example <2-4>, the primary antibody [anti-p65 antibody ( 1; 100)] and confirmed in the same manner.

As a result, as shown in Figure 5 it was confirmed that less p65 in the nucleus in the cells treated with the Walkria in signature methanol extract compared to the cells treated with only LPS (Fig. 5). Experimental Example 6 Cytokine Production Inhibitory Effect of the Extract of Walkria insignis methane in Raw264.7 Cells

 In order to confirm the cytokine production inhibitory effect of the extract of Walkria insignificant methanol in lipopolysaccharide-treated Raw264.7 cells, the production of lipopolysaccharide-induced IL-6 and IL-lbeta was analyzed. The immunological assay kit (mouse IL-6 Enzyme Immunometr Assay Kit and mouse IL-lbeta Enzyme Immunometr Assay Kit; Assay designs, USA) was used. Experimental Example <2-1> 50 μί was recovered from the cell culture solution treated in the same manner and dispensed into a 96 well plate coated with mouse immunoglobulin and stirred for 2 hours. Then, the solution was washed four times with a washing solution, and the anti-IL-6 or anti-IL-lbeta antibody, which is the primary antibody, was dispensed by 50 ^ and reacted for 2 hours. After the reaction, the cells were washed, the second antibody was aliquoted and reacted for 30 minutes. After washing again, the substrate was added and reacted for 30 minutes, and the color development was measured at 450 nm with a microplate measuring instrument.

 As a result, as shown in Figure 6 in the LPS-treated group, the amount of IL-6 and IL-lbeta increased, the concentration of the extract of the workia insignia methane treated with the amount of cytokine increased by LPS It was confirmed that the decrease according to (Fig. 6).

Experimental Example 7 Inhibition of TNF-α Production by Warriac insignificant Methane Extract in Raw264.7 Cells

Tumor necrosis f actor- α and TNF-α induced lipopolysaccharide-induced tumor necrosis in order to confirm the inflammatory inhibition effect of the walkia insignificant extract in Lipopolysaccharide-treated Raw264.7 cells. ) Production was measured using a mouse tumor necrosis factor Grouse TNF-A Enzymen Immunometric Assay Kit (Assay designs, USA). After culturing Raw264.7 cells by the method of Experimental Example <1-1>, 50 βΐ of the supernatant was collected, dispensed into 96 well plates coated with mouse immunoglobulin, and reacted with stirring for 2 hours. Then, after washing four times with the washing solution, the anti-TNF-α antibody as the primary antibody was dispensed 50 ^ each and reacted for 2 hours. After reaction, washing was performed, the secondary antibody was dispensed, and reaction was carried out for 30 minutes. After washing again, the substrate was added and reacted for 30 minutes, and the color development was measured at 450 ran using a microplate meter.

 As a result, as shown in FIG. 7, the amount of TNF-a in the LPS-treated group was increased, and the amount of TNF-a increased by LPS was increased in accordance with the concentration of extract. It was confirmed that the decrease (Fig. 7).

Experimental Example 8 Inhibitory Effect of Walkria incisence Methanol Extract on Carrageenan-Induced Mouse Foot Edema

 Since the extract of Walkria insignificant had an excellent anti-inflammatory effect on the animal cells, the anti-inflammatory effect was confirmed through animal experiments.

 Specifically, BALB / c female mice weighing 20-25 g (Cortech, South Korea) were randomly selected and divided into groups. Workia extract was solubilized in phosphate buffer (PBS) ultrasonically, and 40 black was administered at a concentration of 80 mg / kg. Administration was at a concentration of 5 nig / kg. After 30 minutes, carrageenan (1% v / v) solution dissolved in PBS was injected 25 ul into the left foot of the mouse to induce inflammation. The thickness of the foot was measured by caliper (Digital caliper, Niigata Seiki, Japan) before carrageenan injection.

As a result, as shown in FIG. 8, the negative control group induced inflammation by carrageenan, and the thickness of the foot increased to 0.66 ± 0.05 画. In the pretreatment group, the thickness of the foot was reduced (40 mg / kg; 0.43 土 0.04, 80 mg / kg; 0.32 sul 0.06) in a concentration-dependent manner (FIG. 8).

Experimental Example 9 Inhibitory Effects of Walkria insignificant Methanol Extracts on Cytokine Production in Spleen Cells

Aseptic detachment of the spleen from BALB / c mice (Orient, Korea), 10% fetal bovine serum (FBS), 25 mM HEPES, 2 mM glutamine, penicillin (100 U / mi) and straptomycin (100 / gzg /) Was ground in RPMI 1640 medium (GibcoBRL, USA) was prepared as a single cell suspension (single cell suspension). The prepared spleen cell suspension was centrifuged at 1500 rpm for 10 minutes at room temperature, the supernatant was discarded, and red blood cell lysis buffer 1 ^ was added and digested at 37 ^° C for 10 minutes. After adding 10 PBS to the solution, the mixture was centrifuged at 1500 rpm for 5 minutes at room temperature, and the supernatant was discarded twice. Finally, RPMI 1640 medium with 1 fetal calf serum (GIBC0 BRL, USA) added instead of PBS was suspended. After adjusting the cell number to 1 10 ⁶ cells / m £, the cell suspension 200 ^ was dispensed into 96-well plates, respectively, and the concentration of Walkria insignificant methanol extract was adjusted (0, 5, 10, 20, 40 zg /). After incubation for 1 hour at 5% C0 ₂₎ 37 ^° C in a wet incubator, concanavalin to induce interleukin-4 (IL-4) and interleukin -13 (IL-13) in cells A (Concanaval in A, Sigma, USA) was added to l ^ g / ra concentration, and incubated for 3 days at 37 ^° C in 5¾ CO ₂ in a wet incubator. In order to measure the levels of interleukin-4 and 13, ELISA kits (R & D Systems, Minneapolis, USA) that responded to each cytokine were used, and the content of each cytokine was measured according to the manufacturer's method. The control group was not treated with Con A.

As a result, as shown in Fig. 9, the concentrations of the cytokines IL-4 and IL-13 induced by ConA were treated in the group treated with the Walkria insignificant methanol extract. Dependently reduced (FIG. 9). The preparation examples for the compositions of the present invention are illustrated below. Production Example 1 Preparation of Pharmaceutical Formulation

 <1-1> Preparation of Powder

 Walkia insignificance fe / "c ± / ea insignis) ^ methanol extract of the present invention 2 g Lactose 1 g The above ingredients were mixed and filled in an airtight cloth to prepare a powder.

<1-2> Preparation of Tablet

 The methane of the walkia insignia of the present invention was extracted 100 rag corn starch 100 mg lactose 100 mg magnesium stearate 2 mg The above components were mixed and then tableted according to the manufacturing method of a conventional tablet to prepare a tablet.

<1-3> Preparation of Capsule

100 mg corn starch 100 mg lactose 100 rag magnesium stearate 2 rag After mixing the above components, the capsules are filled into gelatin capsules according to the conventional method for preparing capsules. Was prepared. <l-4> Preparation of the ring

 Methanol extract 1 g Lactose 1.5 g Glycerin 1 g Xylitol of the workia insignia of the present invention was prepared by mixing 0.5 g of the above components, 4 g per ring according to a conventional method.

<1-5> Preparation of Granules

150 mg soy extract 50 mg glucose 200 rag starch 600 rag After mixing the above components, add 100 mg 30 ¾ ethanol and dried at 60 ^° C to form granules After filling the gun.

Preparation Example 2 Manufacture of Food

 <2-1> Preparation of Flour Food

 Promote health by adding 0.5-5.0 parts by weight of hot water extract (^ / ea insignis) ^ \ hot water extract of the present invention to flour, and preparing breads, cakes, cookies, crackers and noodles using this mixture Food was prepared.

<2-2> Preparation of Soups and Gravys

Soupia insignia of the hot water extract of 0.01 to 5.0 parts by weight of the soup and Addition to gravy was made for health promoting meat products, noodle soup and gravy.

<2-3> Preparation of ground beef

 Health portion ground beef was prepared by adding 10 parts by weight of hot water extract of Walkria insignificant of the present invention to ground beef.

<2-4> Manufacture of dairy products

 5 to 10 parts by weight of hot water extract of Walkria insignificant of the present invention was added to milk, and various dairy products such as butter and ice cream were prepared using the milk.

<2-5> Preparation of wire

 Brown rice, barley, glutinous rice, and yulmu were alphanized by a known method and dried, and then roasted to prepare a powder having a particle size of 60 mesh.

 Black beans, black sesame seeds, and sesame seeds were also steamed and dried by a known method, and then ground to a powder having a particle size of 60 mesh.

 The water extract of Walkia insignia of the present invention was concentrated under reduced pressure in a vacuum concentrator, dried by spraying and drying with a hot air dryer, and ground to a particle size of 60 mesh by a grinder to obtain a dry powder.

 The dry powders of the hot water extracts of the grains, seeds, and walkria insignificant prepared above were prepared in the following ratio.

 Cereals (30 parts by weight brown rice, 15 parts by weight of radish, 20 parts by weight of barley),

 Seeds (7 parts by weight perilla, 8 parts by weight black beans, 7 parts by weight black sesame seeds),

 Extract of Example <1-2> (3 parts by weight),

Manor (0,5 parts by weight) ， Foxglove (0.5 part by weight)

Preparation Example 3 Manufacture of Drinks

 <3-1> Preparation of health drink

 Homogenous components such as liquid fructose (0.5%), oligosaccharide (2%), sugar (2%), salt (0.5%), water (75%) and homogenous 5 g of hot water extracts of the walkia insignia of the present invention After sterilization and instantaneous sterilization, it was packaged in small packaging containers such as glass bottles and plastic bottles to prepare healthy drinks.

<3-2> production of vegetable juice

 5 g of hot water extract of Walkria insignia of the present invention was added to tomato or carrot juice 1,000 m £ to prepare a vegetable juice for health promotion.

<3-3> production of fruit juice

 1 g of hot water extract of Walkria insignia of the present invention was added to 1,000 apple or grape juices to prepare fruit juice for health promotion.

Preparation Example Preparation of Cosmetic Composition

 <4-1> Preparation of cream

 0.1 parts by weight of hydrothermal extract of Walkria insignificant of the present invention

 2.8 parts by weight of cetostearyl alcohol

 Milnan 2.6 parts by weight

 Stearic acid 1.4 parts by weight

 2 parts by weight of glycerol phosphate monostearate

Sebum-100 stearate 1 part by weight Sesquiolein sorbate 1.4 parts by weight Jojoba oil 4 parts by weight squalane 3.8 parts by weight polysorbate 60 1. 1 parts by weight macadamia oil 2 parts by weight 0.2 parts by weight of tocopherate acetate 0.4 parts by weight methyl pullisiloxane 0.4 parts by weight ethyl paraben 0.1 parts by weight 0.1 parts by weight of propylparaben

Euxyl K-400 0.1 parts by weight 1,3-butylene glycol 7 parts by weight methylparaben 0.05 parts by weight glycerin 6 parts by weight d-pandenol 0.2 parts by weight triethanolamine 0.2 parts by weight pt 41891 0.2 parts by weight purified water 50.55 parts by weight

<4-2> Preparation of Lotion

0.1 part by weight of cestostearyl alcohol 1.6 parts by weight of stearic acid 1.4 parts by weight of lipophilic monostearic acid 1.8 parts by weight of phosphine -100 stearate 2.6 parts by weight of sesquiurerate sorbate part Squalene 4.8 parts by weight Macadamia oil 2 parts by weight Jojoba oil 2 parts by weight 0.4 parts by weight of tocoacetic acid methyl polysiloxane 0.2 parts by weight Ethyl paraben 0.1 parts by weight propyl paraben 0.1 parts by weight

1,3-butylene glycol 4 parts by weight methylparaban 0.1 parts by weight xanthan gum 0.1 parts by weight glycerin 4 parts by weight d-pandenol 0.15 parts by weight allantoin 0.1 parts by weight carbone (2¾ aq. Sol) 4 parts by weight triethanolamine 0.15 Parts by weight ethane 3 parts by weight pt 41891 0.1 parts by weight 51.7 parts by weight of purified water <Preparation Example 5> Preparation of detergent in cosmetic composition

 <5-1> Preparation of soap

4.0 parts by weight of hot water extract of the Walkria insignia of the present invention 94.25 parts by weight of soap base 0.3 parts by weight of acetate to glycerin 0.2 parts by weight of glycerin PEG 4000 (polyethylene glycol 4000) 0.8 part by weight Jojoba oil 0.3 part by weight

 Cetanol0.15 parts by weight

 The components were mixed well in a mixer and then extruded in a soap making machine, cut and molded to a uniform size to prepare a soap composition. The soap base may be a sodium salt of palm oil, a natural fat or oil commonly used in the art.

<5-2> Preparation of Body Cleanser

 4.0 parts by weight of hydrothermal extract of Walkria insignia of the present invention

 Ammonium Lauryl Sulfate 42 parts by weight

 Cocamidopropyl Betaine 12 parts by weight

 0.5 parts by weight of glycol distearate

 Propylene glycol 1.0 part by weight ¬ small amount of fragrance

 Proper amount of citric acid

 Drain (50% concentrate) 1.0 part by weight

 100 parts by weight of purified water is added.

 Ammonium Lauryl Sulfate, Cocamidopropyl Betaine, Glycol Dis

And the propylene glycol was heated to 70 ^° C and the heated purified water was added to emulsify while adding the remaining ingredients and cooled to room temperature to prepare a body cleanser.

Claims

[Range of request]

 [Claim 1]

 Pharmaceutical composition for the prevention and treatment of inflammatory diseases or asthma containing the extract of Walkria insignis (e c ^ s insignis) as an active ingredient.

[Claim 2]

 According to claim 1, wherein the extract is a pharmaceutical composition for the prevention and treatment of inflammatory diseases or asthma, characterized in that the extract is extracted with water, alcohol or a mixture thereof as a solvent.

[Claim 3]

The pharmaceutical composition for preventing and treating inflammatory diseases or asthma according to claim _2, wherein the alcohol is d to C ₂ lower alcohol.

[Claim 4]

 4. The pharmaceutical composition for preventing and treating inflammatory disease or asthma according to claim 3, wherein the lower alcohol is ethane or methane.

[Claim 5]

The method of claim 1, wherein the inflammatory disease is dermatitis, allergy, atopic dermatitis, conjunctivitis, periodontitis, rhinitis, otitis media, sore throat, tonsillitis, pneumonia, stomach itch, gastritis, Crohn's disease, colitis, hemorrhoids, gout, ankylosing spondylitis, rheumatic fever , Lupus, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, periarthritis, tendonitis, hay, peritonitis, myositis, hepatitis, cystitis, nephritis, Sjogren's syndrome, multiple sclerosis, and acute and chronic It is any one selected from the group consisting of inflammatory diseases Pharmaceutical composition for the prevention and treatment of inflammatory diseases or asthma.

[Claim 6]

 Containing any one or more of the nucleic acid fraction, chloroform fraction, ethyl acetate fraction, butanol fraction, and water fraction as active ingredients obtained by sequentially fractionating the workia insignificant extract using nucleic acid, chloroform, ethyl acetate and butanol Pharmaceutical composition for the prevention and treatment of inflammatory diseases or asthma.

[Claim 7]

 Health food composition for the prevention and improvement of inflammatory diseases or asthma containing the workia insignia extract as an active ingredient.

[Claim 8]

 According to claim 7, The extract is a health food composition for the prevention and improvement of inflammatory diseases or asthma, characterized in that the extract is extracted with water, ethanol or a mixture thereof as a solvent.

[Claim 9]

The method of claim 7, wherein the inflammatory disease is dermatitis, allergy, atopic dermatitis, conjunctivitis, periodontitis, rhinitis, diarrhea, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, hemorrhoids, gout, ankylosing spondylitis, rheumatic fever , Lupus, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, periarthritis, tendonitis, hay, peritonitis, myositis, hepatitis, cystitis, nephritis, Sjogren's syndrome, multiple sclerosis, and acute and chronic Health food composition for the prevention and improvement of inflammatory diseases or asthma, characterized in that any one selected from the group consisting of inflammatory diseases.

[Claim 10]

 Containing any one or more of the nucleic acid fraction, chloroform fraction, ethyl acetate fraction, butanol fraction, and water fraction as active ingredients, which is obtained by sequentially fractionating the workia insignificant extract using nucleic acid, chloroform, ethyl acetate and butanol Health food composition for the prevention and improvement of inflammatory diseases or asthma.

[Claim 111]

 Cosmetic composition for the prevention and improvement of inflammatory diseases containing a walkia insignificant extract or a fraction thereof as an active ingredient.

[Claim 12]

 The method of claim 11, wherein the inflammatory disease is dermatitis, allergy, atopic dermatitis, conjunctivitis, periodontitis, rhinitis, otitis media, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, hemorrhoids, gout, ankylosing spondylitis, rheumatic fever, Lupus, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, periarthritis, tendonitis, hay, peritonitis, myositis, hepatitis, cystitis, nephritis, Sjogren's syndrome, multiple sclerosis, and acute and chronic inflammation Inflammatory disease prevention and improvement cosmetic composition, characterized in that any one selected from the group consisting of diseases.

[Claim 13]

A method of treating inflammatory disease or asthma comprising administering a pharmaceutically effective amount of a walkia insignificant extract or a fraction thereof to an individual suffering from an inflammatory disease or asthma.

[Claim 14]

 A method for preventing an inflammatory disease or asthma comprising administering to a subject a pharmaceutically effective amount of a walkia insignia extract or a fraction thereof.

[Claim 15]

 Walkria insignia extract or fraction thereof for use as a pharmaceutical composition for the prevention and treatment of inflammatory diseases or asthma.

[Claim 16]

 Walkria insignificant extract or fractions thereof for use as a composition for the prevention and improvement of inflammatory diseases or asthma, and for health foods.

[Claim 17]

 Walkria in signature extract or fraction thereof for use as a cosmetic composition for preventing and improving inflammatory diseases.