KR20100081627A - A composition comprising the compound isolated from the extract of rubiae radix for preventing and treating inflammatory disease - Google Patents

Abstract

PURPOSE: A composition containing a compound isolated from Rubiae Radix is provided to reduce expression of protein which causes inflammation and pro-inflammatory cytokine expression. CONSTITUTION: A pharmaceutical composition for preventing and treating inflammatory disease contains a compound isolated from Rubiae Radix extract or pharmaceutically acceptable salt as an active ingredient. The compound is 2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone, 3'-bis(3,4-dihydro-4-hydroxy-6-methoxy-2H-1-benzopyran), Epoxymollugin, Soranjidiol, Oleanolic acid, 1-acetoxy-3-methoxy-9,10-anthraquinone, Alizarin 2-methyl ether, Furonollugin, or mollugin. The Rubiae Radix extract is crude extract, polar solvent soluble extract, or non-polar solvent soluble extract.

Description

A composition comprising the compound isolated from the extract of Rubiae Radix for preventing and treating inflammatory disease}

The present invention provides a composition for the prophylaxis and treatment of inflammatory diseases containing a compound isolated from Cheoncho root extract as an active ingredient.

Hawiger J., Innate Immunity and Inflammation: A Transcriptional Paradigm, Immunologic Research, 23 , pp. 99-109, 2001

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Graeme B, et al., Acute Inflammation, American Journal of Pathology, 86 (1) , pp.185-274, 1977

Yamaki K, et al., Characteristics of histamine-induced leukocyte rolling in the undisturbed microcirculation of the rat mesentry, British J. Pharmacol., 123 , pp . 390-399, 1998

Document 5 Brocklehurst WE, Role of Kinins and Prostaglandins in Inflammation, Proc. Roy. Soc. Med., 64 , pp . 4-6, 1971

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Huang AL, et al., Effects of Systemic Inflammation on Endothelium-Dependent Vasodilation, Trends Cardiovasc. Med ., 16 , pp. 15-20, 2006

Renauld JC, New insights into the role of cytokines in asthma, J. Clin. Pathol, 54 , pp. 577-589, 2001

9 Blake GJ, et al., Tumor necrosis factor-α, inflammatory biomarkers, and atherogenesis, Eur. Heart J., 23 , pp. 345-347, 2002

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Mordan LJ, et al., Inhibitors of endogenous nitrogen oxide formation block the promotion of neoplastic transformation in C3H10T1 / 2 fibroblasts, Carcinogenesis, 14 , pp. 1555-1559, 1993

Lirk P, et al., Inducible nitric oxide synthase. Time for reappraisal, Curr. Drug. Targets. Inflamm. Allergy., 1 , pp.89-108, 2002

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Inflammatory reactions are biological defenses caused by various factors, such as infection by tissues or damage to tissues, and perform an initial protective action to limit the damage to only infected or damaged areas. In most cases, this inflammatory response leads to the elimination of pathogenic factors using intrinsic immune components and induction of specific adaptive immunity (Hawiger J., Innate Immunity and Inflammation: A Transcriptional Paradigm, Immunologic Research, 23 , pp. 99-109, 2001). Rubs, tumors, heat, and pain, known as inflammation-associated features, are associated with the expansion of local blood flow due to the expansion of blood vessels by the action of inflammatory mediators and cytokines. Increased extravascular outflow of plasma components due to increased and decreased local blood flow rate, increased permeability of blood vessels, increased extravascular outflow of immune cells due to increased adhesion of vascular endothelial cells to circulating immune cells, and infection due to chemotaxis This is the result of successive immune responses such as increased migration to (Gallo RL, et al., Biology and clinical relevance of naturally occurring antimicrobial peptides, J. Allergy. Clin. Immunol, 110 , pp. 823-831. 2002; Graeme B, et al., Acute Inflammation, American Journal of Pathology, 86 (1) , pp. 185-274, 1977. Histamine produced in some cells as a response to tissue damage, and low molecular peptide kinin, which is inactivated in the blood and is activated by tissue damage, also contributes to inflammation-associated vasodilation and increased blood vessel permeability (Yamaki K, et al., Characteristics of histamine-induced leukocyte rolling in the undisturbed microcirculation of the rat mesentry, British J. Pharmacol, 123 , pp. 390-399, 1998; Brocklehurst WE, Role of Kinins and Prostaglandins in Inflammation, Proc. Roy. Soc. Med., 64 , pp. 4-6, 1971). Especially, bradykinin, a kind of kinin, works to stimulate the pain of the skin. On the other hand, dilation of blood vessels and increased permeability of blood vessels in damaged tissues also cause blood coagulation enzymes to leak into the tissues, followed by the chain reaction of these enzymes to produce insoluble fibrous strands, which is a major component of blood coagulation. Coagulated fibrous strands act as a barrier against the spread of infection through damaged tissue (Esmon CT., The interactions between inflammation and coagulation. British Journal of Haematology. 131 , pp . 417-430, 2005 ). In general, acute inflammatory reactions progress rapidly and are maintained for a short period of time. Acute inflammation is accompanied by a systemic reaction called an acute phase reaction. On the other hand, chronic inflammation can be induced as a result of continuous immune activation associated with some diseases such as infections and autoimmune diseases, and the accumulation and activation of macrophages is evidence of chronic inflammatory response (Huang AL, et al. ., Effects of Systemic Inflammation on Endothelium-Dependent Vasodilation, Trends, Cardiovasc.Med ., 16 , pp. 15-20, 2006). However, persistent chronic inflammatory reactions can cause serious damage to host cells or tissues.

Inflammatory responses at the site of infection are initiated by the response of macrophages to pathogens. Reactive reactive oxygen species and reactive nitrogen species produced by macrophages activated by pathogens, inflammatory mediators such as prostaglandins, leukotrienes, and inflammatory cytokines such as TNF-α, IL-6 and IL-1β. (Renauld JC, New insights into the role of cytokines in asthma, J. Clin. Pathol ., 54 , pp. 577-589, 2001; Blake GJ, et al., Tumour necrosis factor -α, inflammatory biomarkers, and atherogenesis, Eur. Heart J., 23 , pp.345-347, 2002). In the inflammation-related action of such macrophages, the activation of NF-κB, a transcription factor that induces the expression of these inflammatory mediator genes, is very important. Inflammatory genes such as inducible nitric oxide synthase (iNOS), cyclooxygenase (COX-2), TNF-α, IL-6 and IL-1β in macrophages is transcribed by κB.

Nitric oxide (NO) is a short-lasting free radical that is derived from L-arginine when the oxidation reaction of L-arginine occurs by catalysis of nitric oxide synthase (NOS). It is well known that nitric oxide is produced with L-citrulline. Nitric oxide exhibits various immune-related functions in vivo (Moncada S, et al., Nitric oxide: Physiology, pathophysiology, and pharmacology.Pharmacol. Rev. , 43 , pp.109-142, 1991) Production may be cytotoxic and may lead to damage to host cells and tissues, as well as to various pathological phenomena such as atherosclerosis and carcinogenesis (Mordan LJ, et al., Inhibitors of endogenous nitrogen oxide formation block the promotion of neoplastic transformation in C3H10T1 / 2 fibroblasts, Carcinogenesis, 14 , pp. 1555-1559, 1993; Lirk P, et al., Inducible nitric oxide synthase, Time for reappraisal, Curr. Drug.Targets.Inflamm.Allergy., 1 , pp.89-108, 2002). Three types of nitric oxide synthase (NOS) are known to catalyze NO production, including neuronal nitric oxide synthase (neuronal NOS, nNOS, NOS1) and endothelial nitric oxide synthase (endothelial NOS, eNOS, NOS3). While constitutively expressed at all times, iNOS is inducible to be transcribed by the transcription factor NF-κB, which is activated when phagocytes are stimulated by the bacterial endotoxin lipopolysaccharide (LPS).

In macrophages, the transcription factor NF-κB is present in the cytoplasm in an inactive state by binding to its inhibitor, IκB, but is a signaling process induced when bacterial LPS binds to Toll-like receptors on the cell surface. NF-κB finally becomes activated as IκB is phosphorylated to dissociate from NF-κB / IκB and removed by proteasome digestion. NF-κB then migrates to the nucleus to induce transcription of various inflammation related genes. Such activation of NF-κB includes Akt signaling (Hattori Y, et al., Lipopolysaccharide activates Akt in vascular smooth muscle cells resulting in induction of inducible nitric oxide synthase through nuclear factor-kappa B activation, Eur. J. Pharmacol , 481 , 153-158, 2003) and ERK, c-jun- and p38-MAPK signaling pathways have been reported to be involved (Kim SH, et al., Selenium attenuates lipopolysaccharide-induced oxidative stress responses through modulation of p38 MAPK and NF-κB signaling pathways, Exp.Biol.Medicine ., Pp.565-701, 2004; Robinson MJ, et al., Mitogen-activated protein kinase pathways, Cur.Opi.Cell Biol., 9 , pp.180-186 , 1997).

The expression regulation of iNOS in the transcriptional phase of the inflammatory response is of great importance in determining the duration and production of NO. Therefore, iNOS expression control mechanism and iNOS enzymatic activity have been used as a target of drug action in the study to isolate new anti-inflammatory drugs for the improvement of chronic inflammation-related diseases.

Rubiae Radix is the dried root of Rubia akane Nakai (= R. Cordifolia L. var.mangista Miq.) And Rake cordifolia ( Rbia cordifolia L.). (草草), Hongcheongeun, Jisobon, Cheongeun, Tosukboncho, Blood Bran Number, Live Blood Troupes, Jisomok It is called 木, Cheoncho, Parasitic, Yeoryo, Yeombicho, Saengchocho, Wind Jarcho, Persanyong, and Uman. Its origin is Korea and China, and its characteristic odor is a little sweet and cold. Its ingredients are alizarin and rubierythric acid (= alizarin-2- primeveroside) belonging to anthraquinones. ), Purpurin (purpurin), xanthopurpurin (xanthopurpurin), munjistin (munjistin), pseudopurpurin (pseudopurpurin). It is used as a dyeing material and has the effects of hemostatic action, menstrual disorder, blood circulation disorder, and blood bleeding (Park Jong Hee, Encyclopedia of Herbal Medicine (B), Book Publishing Shinil Corp., p.796-797, 2002). The compounds isolated from the myofibrillar root extracts of do not teach or disclose that they can be used as compositions for the prevention and treatment of inflammatory diseases by inhibiting NO production and proinflammatory cytokines production induced by the treatment of LPS.

Therefore, the present inventors found a new drug that inhibits the production of NO and proinflammatory cytokines produced by the treatment of mouse macrophage RAW264.7 with bacterial LPS, the root of Cheoncho ( Rubia cordifolia L.) Inhibition of NO production and pro-inflammatory cytokine production in RAW264.7 cells was investigated in the purified single-substances from 2-carbomethoxy-2,3-epoxy-3. Transdermal isolated compounds, such as prenyl-1,4-naphthoquinone (hereinafter referred to as 'RA-MC-AA'), are NO-producing and proinflammatory cytokines induced by LPS treatment in RAW264.7 cells. For the first time, it has been shown to have an anti-inflammatory function that inhibits the production of LPS-induced NO production, p-IkBα, iNOS and COX-2 protein expression in mouse macrophage RAW264.7 cells as well as iNOS, COX-2, TNF-α, IL-6 and The present invention was completed by confirming that the expression content of proinflammatory cytokines such as mRNA expression of IL-1β was significantly reduced.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention and treatment of inflammatory diseases containing a compound or a pharmaceutically acceptable salt thereof isolated from Cheoncho root extract as an active ingredient.

In addition, the present invention provides a health functional food for the prevention and improvement of inflammatory diseases containing a compound or a pharmaceutically acceptable salt thereof isolated from Cheoncho root extract as an active ingredient.

Compounds isolated from the herbaceous root as defined herein may be 2-carbomethoxy-2,3-epoxy-3-prenyl- (2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone). 1,4-naphthoquinone), 3,3'-bis (3,4-dihydro-4-hydroxy-6-methoxy-2H-1-benzopyran) (3,3'-bis (3,4- dihydro-4-hydroxy-6-methoxy-2H-1-benzopyran), Epoxymollugin, Soranjidiol, Oleanolic acid, 1-acetoxy-3-methoxy- Group consisting of 9,10-anthraquinone (1-acetoxy-3-methoxy-9,10-anthraquinone), alizarin 2-methyl ether, furonollugin, mollugin It is a compound selected from, preferably 2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone (2-carbomethoxy-2,3-epoxy-3-prenyl-1,4 -naphthoquinone) compound.

The compounds of the present invention can be prepared with pharmaceutically acceptable salts and solvates according to methods conventional in the art.

As the pharmaceutically acceptable salt, acid addition salts formed by free acid are useful. Acid addition salts are prepared by conventional methods, for example by dissolving a compound in an excess of aqueous acid solution and precipitating the salt using a water miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. Equal molar amounts of the compound and acid or alcohol (eg, glycol monomethylether) in water can be heated and the mixture can then be evaporated to dryness or the precipitated salts can be suction filtered.

In this case, organic acids and inorganic acids may be used as the free acid, hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, tartaric acid, etc. may be used as the inorganic acid, and methanesulfonic acid, p -toluenesulfonic acid, acetic acid, trifluoroacetic acid, etc. may be used as the organic acid. , Citric acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, manderic acid, propionic acid, citric acid, lactic acid, glycolic acid, gluconic acid (gluconic acid), galacturonic acid, glutamic acid, glutaric acid (glutaric acid), glucuronic acid (glucuronic acid), aspartic acid, ascorbic acid, carbonic acid, vanic acid and hydroiodic acid may be used.

In addition, bases can be used to make pharmaceutically acceptable metal salts. An alkali metal or alkaline earth metal salt is obtained by, for example, dissolving a compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the non-soluble compound salt, and then evaporating and drying the filtrate. At this time, as the metal salt, it is particularly suitable to prepare sodium, potassium or calcium salt, and the corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (for example, silver nitrate).

Pharmaceutically acceptable salts of the compounds of the invention include salts of acidic or basic groups which may be present in the compounds of the invention, unless otherwise indicated. For example, pharmaceutically acceptable salts include sodium, calcium and potassium salts of the hydroxy group, and other pharmaceutically acceptable salts of the amino group include hydrobromide, sulfate, hydrogen sulphate, phosphate, hydrogen phosphate, dihydrogen Phosphate, acetate, succinate, citrate, tartrate, lactate, mandelate, methanesulfonate (mesylate) and p -toluenesulfonate (tosylate) salts. It can be prepared through.

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

The compounds of the present invention can be obtained by the preparation method as follows.

For example, first, the method of obtaining the crude extract of Cheoncho root of the present invention will be described. First, the Cheoncho root is washed and shaded, and then finely pulverized into a crusher and then finely powdered. Pear, preferably about 1 to 7 times the volume of water and C1 to C4 lower alcohols such as methanol, ethanol, butanol and the like or a mixed solvent thereof, preferably methanol, more preferably 50 to 100% methanol Extraction method such as hot water extraction, cold needle extraction, reflux cooling extraction or ultrasonic extraction at 10 ° C. to 100 ° C., preferably 50 ° C. to 90 ° C. for 1 to 20 hours, preferably 5 to 15 hours, preferably reflux. Using the extraction method, the supernatant was recovered by vacuum filtration, and the above procedure was repeated 2 to 7 times, preferably four times, to collect the supernatant and concentrated under reduced pressure to obtain the supernatural root methane of the present invention. Crude extract can be obtained.

The methanol crude extract obtained above was mixed with a mixed solvent of water and methylene chloride, and then fractionated. The mixture was concentrated under reduced pressure to obtain a cheoncho root water soluble fraction and a methylene chloride soluble fraction of the present invention, respectively. And methylene chloride mixed solvent using a method of increasing polarity to selectively purify the chromatography using chromatography, such as flash column chromatography, RP C18 column chromatography, or silica gel open column chromatography, to selectively perform the compounds of the present invention. That is, 2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone (3, carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone), 3, 3′-bis (3,4-dihydro-4-hydroxy-6-methoxy-2H-1-benzopyran) (3,3′-bis (3,4-dihydro-4-hydroxy-6-methoxy -2H-1-benzopyran)), epoxymollugin, small Soranjidiol, Oleanolic acid, 1-acetoxy-3-methoxy-9,10-anthraquinone, alizarin 2-methyl Alizarin 2-methyl ether, furonollugin, mollugin can be obtained.

The present invention provides a pharmaceutical composition for the prevention and treatment of inflammatory diseases containing the compound or a pharmaceutically acceptable salt thereof isolated from the cheoncho root extract obtained by the above method as an active ingredient.

In addition, cheoncho root is a medicine that has been used for a long time as an edible or herbal medicine isolated from the cheoncho root extract of the present invention also has no problems such as toxicity and side effects.

The extract as defined herein is characterized in that crude extract of cheoncho root, polar solvent soluble extract or nonpolar solvent soluble extract.

The crude extract as defined herein is a solvent selected from water containing purified water, lower alcohols having 1 to 4 carbon atoms such as methanol, ethanol, butanol, or a mixed solvent thereof, preferably methanol, more preferably 50 to 100% methanol Contains extracts available for use.

The nonpolar solvent soluble extract as defined herein includes extracts soluble in methylene chloride, hexane, chloroform, dichloromethane or ethyl acetate, preferably methylene chloride.

Inflammatory diseases defined herein are inflammatory diseases include atopic dermatitis, arthritis, urethritis, cystitis, arteriosclerosis, allergic diseases, rhinitis, asthma, acute pain, chronic pain, periodontitis, gingivitis, inflammatory bowel disease, gout, myocardial infarction, congestion Sexual heart failure, hypertension, angina pectoris, gastric ulcer, cerebral infarction, Down syndrome, multiple sclerosis, obesity, diabetes, dementia, depression, schizophrenia, tuberculosis, sleep disorders, sepsis, burns or pancreatitis, preferably atopic dermatitis, arthritis, urethritis , Cystitis or periodontitis, more preferably periodontitis, urethritis or cystitis.

The composition for preventing and treating inflammatory diseases of the present invention comprises 0.1 to 50% by weight of the compound based on the total weight of the composition.

The pharmaceutical composition comprising the compound of the present invention may further include appropriate carriers, excipients and diluents commonly used in the preparation of pharmaceutical compositions.

Carriers, excipients and diluents that may be included in the compositions of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

Pharmaceutical dosage forms of the compounds of the present invention may be used in the form of their pharmaceutically acceptable salts, or may be used alone or in combination with other pharmaceutically active compounds, as well as in a suitable collection.

Compositions comprising the compounds of the invention are each formulated in the form of oral dosage forms, external preparations, suppositories, and sterile injectable solutions, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, according to conventional methods Can be used.

More specifically, when formulating the composition, it can be prepared using a diluent or an excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, and the like. Solid form preparations for oral administration include tablets, pills, powders, granules and capsules, and the like form at least one excipient such as starch, calcium carbonate, sucrose in the compound. ), Lactose, gelatin and the like can be mixed. In addition to the simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral use include suspensions, solvents, emulsions and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances and preservatives, in addition to the commonly used simple diluents, water and liquid paraffin. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations and suppositories. As the non-aqueous solvent and suspending agent, vegetable oils such as propylene glycol, polyethylene glycol and olive oil, and injectable esters such as ethyl oleate may be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter and glycerogelatin may be used.

Preferred dosages of the compounds of the present invention depend on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, but may be appropriately selected by those skilled in the art. However, for the desired effect, the compound of the present invention may be administered in an amount of 0.0001 to 100 mg / kg, preferably in an amount of 0.00 1 to 100 mg / kg once or several times a day. The compound of the present invention in the composition may be formulated in an amount of 0.0001 to 50% by weight based on the total weight of the total composition.

The pharmaceutical composition of the present invention can be administered to mammals such as mice, mice, livestock, humans, etc. by various routes. All modes of administration can be expected, for example by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural and intracere broventricular injections.

The present invention also provides a dietary supplement for the prevention and improvement of an inflammatory disease, which contains the mycelial root separating compound or a pharmaceutically acceptable salt thereof as an active ingredient.

The compounds of the present invention can be used in various ways, such as drugs, food and beverages for the prevention and treatment of inflammatory diseases. Foods to which the compound of the present invention may be added include, for example, various foods, beverages, gums, teas, vitamin complexes and dietary supplements, and may be used in the form of powders, granules, tablets and capsules or beverages. have.

Since the compound of the present invention has little toxicity and side effects, it is a drug that can be used safely even for long-term administration for the purpose of prevention.

The compounds of the present invention may be added to food or beverages for the purpose of preventing and treating inflammatory diseases. At this time, the amount of the compound in the food or beverage is generally added to the health food composition of the present invention to 0.01 to 15% by weight of the total food weight, the health beverage composition is 0.02 to 30 g based on 100 ml, preferably Can be added at a rate of 0.3 to 10 g.

The health beverage composition of the present invention, in addition to containing the compound as an essential ingredient in the indicated proportions, has no particular limitation on the liquid component and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. Examples of the above-mentioned natural carbohydrates include monosaccharides such as disaccharides such as glucose and fructose, for example polysaccharides such as maltose and sucrose, and conventional sugars such as dextrin and cyclodextrin. Sugar alcohols such as xylitol, sorbitol and erythritol. As flavoring agents other than those described above, natural flavoring agents (tauumatin, stevia extract (e.g., Rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of said natural carbohydrates is generally about 1-20 g, preferably about 5-12 g per 100 ml of the composition of the present invention.

In addition to the above, the compounds of the present invention include various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like. In addition, the compounds of the present invention may contain flesh for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components can be used independently or in combination. The proportion of such additives is not so critical but is usually selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

As mentioned above, the compounds isolated from the mycorrhizal extract of the present invention may reduce LPS-induced NO production, p-IkBα, iNOS, and COX-2 protein expression in RAW264.7 cells, which are mouse macrophages. In addition, by confirming that the expression content of pro-inflammatory cytokines such as mRNA expression of iNOS, COX-2, TNF-α, IL-6, and IL-1β significantly decreased, the composition prevents and prevents inflammatory diseases. It can be usefully used as a therapeutic pharmaceutical composition or health functional food.

Hereinafter, the present invention will be described in detail with reference to the following examples and experimental examples.

However, the following Examples and Experimental Examples are only illustrative of the present invention, the contents of the present invention is not limited by the following Examples, Reference Examples and Experimental Examples.

Example 1. Preparation of Cultivated Root Extract

1-1. Preparation of Cheoncho-Geun Crude Extract

After washing Cheoncho root purchased from Yangnyeong Market, Bongsan-dong, Jung-gu, Daegu Metropolitan City, dry it with a grinding machine and finely powder it. The supernatant was recovered by vacuum filtration. This process was repeated four times to collect the supernatant, and then concentrated under reduced pressure to obtain 1048.4 g of crude chondroitin extract.

1-2. Preparation of Crude Soluble Extracts

1048.4 g of Cheoncho root methanol crude extract obtained in Example 1-1 was suspended in 2.2 L of water, followed by solvent fractionation with methylene chloride (CH ₂ Cl ₂ ). First, 2 liters of methylene chloride were added to the solubilized extract of Cheonchogeun methanol suspended in 2.2 liters of water, and then vacuum-dried by separating only the components dissolved in the methylene chloride layer in the fractionated filter. This process was repeated five times to give 360 g of methylene chloride soluble extract.

Example 2. Isolation of Compound Isolated from Cheoncho Geun Extract

The methylene chloride soluble extract obtained in Example 1-2 was subjected to flash column chromatography (YMC Gel ODS-A, 60A, 220 mesh), that is, 230 to 400 in a column having a length of 75 cm and a diameter of 9 cm. After filling the mesh silica gel and eluting with 100% hexane to make the stationary phase uniform, the methylene chloride soluble extract obtained in Example 1-2 was obtained. 360 g was adsorbed onto 720 g of silica gel (70-230 mesh) and loaded onto the column. Then, increase the polarity of the mobile phase in the order of hexane: methylene chloride = 98: 2, 95: 5, 90: 10, 85: 15, 80: 20, 60: 40, 50: 50, and then methylene chloride: methanol Elution was performed at 99: 1, 98: 2, and 96: 4 to fractionate fractions of RA-MC-1 to RA-MC-34 based on the TLC pattern.

The fifteenth fraction, RA-MC-15, was subjected to RP C18 column chromatography and RP C18 (40-63 μm) was added to the column (ID 57 cm × 3.5 cm). After filling in cm, eluting with a mixture of CH ₃ CN: H ₂ O = 3: 7 to make a uniform solution, 6.2 g of RA-MC-15 fraction was completely dissolved in 100% CH ₃ CN and loaded, followed by the ratio of CH ₃ CN. a step-by-step to the seventh fraction is RA-MC-15-7 fractions, about 1.2 g of silicon again open column chromatography (SiO ₂ open column chromatography) obtained by the elution was carried out while increasing. Fill the column (ID 53 cm x 2.3 cm) with silica gel (less than 70 mesh) about 15 cm, elute with 100% hexane to make a homogeneous state, and sample is adsorbed to about 0.72 g of silica gel (less than 70 mesh) and loaded onto the column And eluting with a Hexane: EtOAc = 18: 0.3 mixed solution, as shown in Table 1 below, the compound 2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone ( 754 mg of 2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone) was obtained by separation and purification. In addition, 3'-bis (3,4-dihydro-4-hydroxy-6-methoxy-2H-1-benzopyran) of the compounds of the present invention in the same manner as described above using the RA-MC fraction in the fraction (3,3'-bis (3,4-dihydro-4-hydroxy-6-methoxy-2H-1-benzopyran)) 51 mg, Epoxymollugin 36.4 mg, Soranjidiol 30 mg, 28 mg oleanolic acid, 31 mg 1-acetoxy-3-methoxy-9,10-anthraquinone, alizarin 2-methyl ether 42 mg of Alizarin 2-methyl ether), 76 mg of furomollugin, and 670 mg of molugin were obtained by sequentially separating and purifying them (see Table 1).

16 Compounds Isolated from Cheoncho Geun Extract Plant Fraction Chemical compound name Cheoncho Geun
(Rubiae Radix) RA-MC-AA 2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone RA-MC-BB 3,3′-bis (3,4-dihydro-4-hydroxy-6-methoxy-2H-1-benzopyran) RA-MC-CC Epoxymollugin RA-MC-DD Soranjidiol RA-MC-EE Oleanolic acid RA-MC-FF 1-acetoxy-3-methoxy-9,10-anthraquinone RA-MC-GG Alizarin 2-methyl ether RA-MC-Ha Furonollugin RA-MC-Hb mollugin

Reference Example 1. Preparation of Experimental Materials

DMEM (Dulbecco's Modified Eagle Medium) and FBS (fetal bovine serum) were purchased from Hyclone (Gaithersbug, MD, USA), LPS (Lipopolysaccharide), Griess reagent, MTT (3- (4,5-dimethylthiazol-2-yl) ) -2,5-diphenyl-tetrazolium bromide) was purchased from Sigma (St. Louis, Mo, USA). BCA Protein assay reagent was purchased from Pierce (Rockford, IL, USA), and ECL Western Blot reagent was purchased from Amersham (Arlington Heights, IL, USA). Other primary anti-IkBα and anti-p-Ikbα monoclonal antibodies used in other experiments were purchased from Cell signaling, mAb (iNOS monoclonal antibody) from BD Science, and COX-2 antibody from Santa Cruz Biotechnology. A secondary antibody, anti-mouse or anti-goat IgG horseradish peroxidase (HRP) -conjugated antibody, was purchased from Amersharm (Arlington Heights, IL, USA). All reagents used in the experiments were used above the analytical grade.

Reference Example 2. Cell Culture

RAW264.7 cells, a macrophage line in mice, were distributed from the American Type Culture Collection (NOCC.TIB-71) and contained Dulbecco's Modified Eagle Medium containing 10% FBS and 100 μg / ml gentamycin. ) Was cultured in a medium at 37 ℃, 5% CO ₂ conditions were used in the following experiment.

Experimental Example 1. Measurement of cytotoxicity

In order to measure the cytotoxicity of the compounds obtained in Example 2 was performed using the MTT assay method (Jun DY, et al., Apoptogenic activity of Zanthoxylum schinifolium toward human acute leukemia Jurkat T cells is associated with ER stress-mediated caspase-8 activation that stimulates mitochondria-dependent or -independent caspase cascade, Carcinogenesis, 28 , pp.1303-1313, 2007).

For cytotoxicity analysis, the cell concentration was adjusted to a 96-well plate (96-well plate) at 2 × 10 ⁵ cells / well, and the RAW264.7 cells were dispensed and cultured for 16 hours. After pretreatment for 1 hour, treatment with 0.1 μg / ml LPS, incubation for 12 hours, and then 50 μl of MTT [3- (4,5-dimethylthiazol-2-yl) -2,5-dip henyl tetrazolium bromide] was added. After reaction for 4 hours to form formazan (formazan), centrifugation at 2300 rpm for 15 minutes to remove the supernatant, and dissolved formazan by adding 150 μl / well DMSO and 540 using ELISA reader (Variokan, Thermo Electron) Measured at nm wavelength.

As a result, as shown in Table 2 and FIG. 1, the cell viability of RAW264.7 cells was not significantly affected by the compound treatment of 6.25 μg / ml concentration, but in the case of RA-MC-AA compound, 12.5 μg / ml Cell viability was reduced by 23.2% compared to the control at ml concentration, it was confirmed that there is a cytotoxic effect on macrophage RAW264.7 cells at 12.5 μg / ml concentration (see Table 2 and Figure 1).

Fraction Cell viability (%) 6.25 μg / ml 12.5 μg / ml RA-MC-AA 85 23 RA-MC-BB 93 112 RA-MC-CC 108 111 RA-MC-DD 112 110 RA-MC-EE 110 93 RA-MC-FF 106 110 RA-MC-GG 108 102 RA-MC-Ha 108 109 RA-MC-Hb 120 115 LPS-only group 100 Control (No treatment) 114

Experimental Example 2 Measurement of NO Production

In order to determine the amount of NO produced, experiments were conducted using the method described in Wang S et al., J. Ethnopharmacol. , 114 (3) , pp. 458-462, 2007.

In order to measure NO production, the cells were diluted in a 96-well plate with a cell concentration of 2 × 10 ⁵ cells / well, and the RAW264.7 cells were dispensed and incubated for 16 hours, and the compound of Example 2 was prepared by concentration. After pretreatment for 1 hour, incubated for 16 hours after treatment with 0.1 μg / ml LPS, 100 μl of the supernatant was collected, 100 μl of Greases Reagent was added, and reacted with light at room temperature for 15 minutes. Absorbance was measured at 540 nm using an Automatic ELISA reader (precision microplate reader, Molecular Devices, BIO-TEX EL800uv-PC, USA). NO concentration in the culture was determined using a standard curve of concentration of sodium nitrite.

Experimental results, as shown in Table 3 and Figure 2 RA-MC-AA compound showed a significant inhibitory effect of the NO production of the mouse macrophage RAW264.7 almost 99% at 6.25 μg / ml concentration conditions (Table 3 and FIG. 2), adding RA-MC-AA compounds at a concentration of 1 μg / ml, 2 μg / ml or 4 μg / ml to identify the minimum concentration that can inhibit NO production in RAW264.7 cells. In addition, it was confirmed whether NO production of RAW264.7 cells by LPS was inhibited.

As a result, as shown in Fig. 3-A, NO production of RAW264.7 cells by LPS was inhibited at a concentration of 1 μg / ml, 2 μg / ml or 4 μg / ml, resulting in 58%, 82% and It was confirmed that each inhibited by 99% (see Figure 3-A), RA-MC-AA compound confirmed by the MTT assay under the same concentration conditions was confirmed that no cytotoxicity as shown in Figure 3-B (Fig. 3-B).

Fraction NO synthesis inhibition (%) 6.25 μg / ml 12.5 μg / ml RA-MC-AA 99 100 RA-MC-BB 8 10 RA-MC-CC 3 7 RA-MC-DD 6 4 RA-MC-EE 19 40 RA-MC-FF 7 5 RA-MC-GG 7 6 RA-MC-Ha 10 6 RA-MC-Hb 43 92 LPS-only group 0

Experimental Example 3. Western blot analysis

To determine the expression of Ik Bα, p-IkBα, COX-2, or iNOS proteins, Western using the method of Hu et al. (Heo et al., J. Immunol., 179 (9) , pp.6305-6310, 2007) Blot analysis was performed as follows.

PBS (ice-cold phosphate buffered saline: 2.7 mM KCl, 137 mM NaCl, 10 mM Na ₂ HPO ₄ , 2 mM KH ₂ PO ₄ , pH 7.4) was added to 4 × 10 ⁶ cells cultured by the method of Reference Example 2. After washing several times, with lysis buffer (lysis buffer: 137 mM NaCl, 15 mM EGTA, 1 mM sodium orthovanadate, 15 mM MgCl2, 0.1% Triton X-100, 25 mM MOPS, 2.5 μg / ml proteinase inhibitor E64, pH 7.2) The suspension was crushed with an ultrasonic mill, and then reacted at 4 ° C. for 30 minutes and centrifuged at 14000 rpm for 20 minutes to collect the supernatant. The same amount of protein was separated by 4-12% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), and then the protein was transferred to an Immobilon-P membrane (membrane). The membrane was reacted for one hour in a blocking buffer (TBS solution containing 3% non-fat milk and 0.1% Tween 20) to block nonspecific binding of the antibody, followed by antibody to each protein (anti- iNOS and anti-COX2) were added and reacted for 1-2 hours. Subsequently, the cells were washed three times for 10 minutes with a TBST solution containing 0.1% Tween 20, and then reacted with a secondary antibody (anti-mouse or anti-rabbit IgG horseradish peroxidase-conjugated antibody) for 90 minutes to 2 hours. The protein was then identified on X-ray film (AGFA, Belgium) after reaction with the ECL system. Protein quantification of each sample was performed by measuring the absorbance at 562 nm using a Micro BCA protein assay kit.

For reference, macrophages play an important role in the initiation and expansion of inflammatory responses. Macrophages that stimulate Toll-like receptors on the cell surface by bacterial LPS are expressed through intracellular signaling. It activates the transcription factor NF-κB, which in turn increases the expression of inducible nitric oxide synthase (iNOS) by the action of activated NF-κB. As a result, it can be seen that the production of NO, one of the inflammatory mediators from L-arginine, is enhanced by catalysis of increased iNOS in cells (Haiqi He, et al., Molecular Immunology, 43 , p. .783, 2006).

As shown in FIG. 4, the expression of iNOS and the level of COX-2 associated with activation of the transcription factor NF-κB by LPS treatment tended to decrease concentration-dependently by RA-MC-AA compounds. (See FIG. 4),

In addition, as shown in FIG. 5, phosphorylation of IκB protein associated with NF-κB activation was significantly increased under the same conditions, but it was confirmed that phosphorylation of IκB was significantly reduced in the presence of RA-MC-AA compound. (See Figure 5).

Experimental Example 4 Reverse Transcriptase Chain Reaction (RT-PCR)

The method described in the existing literature to determine whether the inhibition of NO production of RA-MC-AA compounds is due to the inhibition of the expression of iNOS mRNA and proinflammatory cytokines (Lee et al., J. Ethnopharmacol., 97 , pp.561- 566, 2005) was performed as follows.

RAW264.7 cells were treated with compound RA-MC-AA (2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone) of Example 2 at a concentration of 1 to 4 μg / ml for 1 hour. After the reaction, 0.1 μg / ml of LPS was pretreated for 4 hours to induce NO production. After 4 hours of LPS treatment, cells were recovered by treatment with Trypsin-EDTA and total RNA was isolated using Trizole: Invitrogen, Carlsbad, CA, USA. The first strand cDNA was synthesized from 2 μg of total RNA using MMLV-reverse transcriptase (Gibco BRL). The base sequence of the primer (Primer) is shown in Table 4 below.

genes oligonucleotide sequence size iNOS sense 5-ATGTCCGAAGCAAACATCAC-3 449 bp antisense 5-TAATGTCCAGGAAGTAGGTG-3 COX-2 sense 5-CAGCAAATCCTTGCTGTTCC-3 ′ 490 bp antisense 5-TGGGCAAAGAATGCAAACATC-3 TNF-α sense 5-TACTGAACTTCGGGGTGATCGGTCC-3 295 bp antisense 5-CAGCCTTGTCCCTTGAAGAGAACC-3 IL-6 sense 5-GAAATGATGGATGCTTCCAAACTGG-3 414 bp antisense 5-GGATATATTTTCTGACCACAGTGAGG-3 IL-1β sense 5-CAAGGAGAACCAAGCAAC-3 350 bp antisense 5-GGGGAAGGCATTAGAAAC-3 GAPDH sense 5-CCACTGGCGTCTTCACCAC-3 349 bp antisense 5-CCTGCTTCACCACCTTTTG-3

PCR was repeated 20 times in order of heating at 98 ° C. for 2 minutes and then reacting at 98 ° C. for 10 seconds, at 50 ° C. for 30 seconds, and at 72 ° C. for 1 minute, and then at 5 ° C. for 5 minutes. PCR products were confirmed by electrophoresis on 1.2% agarose gel and stained with EtBr (ethidium bromide: 0.5 μg / ml).

As a result of the experiment, RAW264 processed LPS as shown in FIG. INOS mRNA expression level was significantly increased in 7 cells. However, when RA-MC-AA compound and LPS were treated together, iNOS and COX-2 mRNA levels decreased depending on the concentration of RA-MC-AA compound. Inflammatory cytokines TNF-α, IL-6 and IL-1β mRNA levels were also treated with LPS treated RAW264. Although significantly increased in 7 cells, when the RA-MC-AA compound and LPS were treated together, it was confirmed that the concentration was decreased depending on the RA-MC-AA compound (see FIG. 6).

Therefore, the inhibitory effect of RA-MC-AA compound on NO production induced by LPS treatment in RAW264.7 cells, a mouse macrophage, was achieved through the inhibition of activation of the transcription factor NF-κB associated with iNOS gene expression. It is suggested that this inhibition of NF-κB occurs by interfering with its phosphorylation mechanism, which is an inactivation mechanism of IkB protein, an inhibitor of NF-κB. The inhibitory effect of the RA-MC-AA compound on the activation of the transcription factor NF-κB induced in macrophages by LPS treatment is characterized by TNF-α, a variety of proinflammatory cytokines induced by the transcriptional activity of NF-κB. It was also confirmed by the reduction of IL-6 and IL-1β mRNA levels.

As shown in FIG. 7, 2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone (RA-MC-AA), which is a single substance isolated from Cheoncho root extract, showed phosphorylation of IκB. Reduction inhibits the activation of NF-κB, a transcription factor in RAW264.7 cells induced by LPS, through which iNOS, COX-2 and proinflammatory cytokines, TNF-α, IL-6, IL, are induced by LPS. By inhibiting the gene transcription of -1β, it is possible to lower the levels of iNOS, COX-2 and proinflammatory cytokines TNF-α, IL-6, IL-1β protein, thus inhibiting NO production by LPS treatment, i.e. It can be shown to have an anti-inflammatory effect (see FIG. 7).

Examples of the pharmaceutical compositions containing the compounds of the present invention will be described, but the present invention is not intended to be limited thereto, but is intended to be described in detail.

Formulation Example 1 Preparation of Powder

RA-MC-AA Compound 300 mg

Lactose 100 mg

Talc 10 mg

The above ingredients are mixed and filled in an airtight cloth to prepare a powder.

Example 2 Preparation of Tablet

RA-MC-BB Compound 300 mg

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, tablets are prepared by tableting according to a conventional method for preparing tablets.

Formulation Example 3 Preparation of Capsule

RA-MC-CC Compound 300 mg

3 mg of crystalline cellulose

Lactose 14.8 mg

Magnesium Stearate 0.2 mg

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare capsules.

Formulation Example 4 Preparation of Injection

RA-MC-DD Compound 300 mg

Mannitol 180 mg

Sterile distilled water for injection 2974 mg

Na ₂ HPO ₄ 12H ₂ O 26 mg

According to the conventional method for preparing an injection, the amount of the above ingredient is prepared per ampoule (2 ml).

Formulation Example 5 Preparation of Liquid

RA-MC-EE Compound 300 mg

10 g of isomerized sugar

5 g of mannitol

Purified water

According to the conventional method of preparing a liquid solution, each component is added to the purified water to dissolve, the lemon flavor is added appropriately, the above components are mixed, purified water is added, the whole is adjusted to 100 ml by the addition of purified water, and then filled into a brown bottle. The solution is prepared by sterilization.

Formulation Example 6 Preparation of Healthy Food

RA-MC-FF Compound 1000 mg

Vitamin mixture proper amount

70 μg of Vitamin A Acetate

Vitamin E 1.0 mg

Vitamin B ₁ 0.13 mg

0.15 mg of vitamin B ₂

Vitamin B ₆ 0.5 mg

Vitamin B ₁₂ 0.2 g

Vitamin C 10 mg

10 μg biotin

Nicotinic Acid 1.7 mg

50 μg folic acid

Calcium Pantothenate 0.5mg

Mineral mixture

Ferrous Sulfate 1.75 mg

Zinc Oxide 0.82 mg

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Dibasic calcium phosphate 55 mg

Potassium Citrate 90 mg

Calcium Carbonate 100 mg

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional method for producing healthy foods , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods.

Formulation Example 7 Preparation of Healthy Drink

RA-MC-Hb Compound 300 mg

15 g of vitamin C

100 g of vitamin E (powder)

Iron lactate 19.75 g

3.5 g of zinc oxide

Nicotinamide 3.5 g

0.2 g of vitamin A

Vitamin B ₁ 0.25 g

0.3 g of vitamin B ₂

Water quantification

After mixing the above components in accordance with the conventional healthy beverage production method, and stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed sterilization and refrigerated It is used to prepare a healthy beverage composition of the present invention.

Although the compositional ratio is relatively mixed with a component suitable for a favorite drink, it is also possible to arbitrarily modify the compounding ratio according to the regional or national preference such as the demand class, the demanding country, and the use purpose.

[National R & D project supporting this invention]

[Task unique number]

       B0009-008

[Name of Buddha]

       Ministry of Knowledge Economy

[Name of research project]

       Regional Innovation Center Project

[Name of Research Project]

       Mechanism of immunomodulation and development of industrialization technology

[Host]

        Daegu Haany University Industry-Academic Cooperation Foundation

[Research period]

        March 01, 2008 to February 28, 2010

1 is a diagram showing the cytotoxicity of the compounds isolated from Cheoncho root extract (0, 6.25, 12.5 ug / ml),

Figure 2 is a diagram showing the reduced NO production of compounds isolated from Cheoncho root extract (0, 6.25, 12.5 ug / ml),

Figure 3-A is a diagram showing the NO production reduction amount of RA-MC-AA compound (0, 1, 2, 4 ug / ml),

Figure 3-B shows cytotoxicity of RA-MC-AA compounds (0, 1, 2, 4 ug / ml),

Figure 4 is a diagram showing the reduced production of inflammatory related proteins of RA-MC-AA compound (iNOS, COX-2),

Figure 5 is a diagram showing the reduced production of inflammatory related proteins of RA-MC-AA compound (p-IkBα, IkBα),

6 is a diagram showing the inhibition of iNOS, COX-2, TNF-α, IL-6, IL-1β mRNA expression of RA-MC-AA compound (0, 1, 2, 4 ug / ml),

7 is a diagram schematically illustrating the inflammatory signaling of macrophage line Raw264.7.

Claims (9)

2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone isolated from Cheoncho Geun extract (2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone ), 3,3′-bis (3,4-dihydro-4-hydroxy-6-methoxy-2H-1-benzopyran) (3,3′-bis (3,4-dihydro-4-hydroxy -6-methoxy-2H-1-benzopyran), Epoxymollugin, Soranjidiol, Oleanolic acid, 1-acetoxy-3-methoxy-9,10-anthra A compound selected from the group consisting of 1-acetoxy-3-methoxy-9,10-anthraquinone, alizarin 2-methyl ether, furonollugin, and mollugin; or A pharmaceutical composition for the prevention and treatment of inflammatory diseases containing a pharmaceutically acceptable salt thereof as an active ingredient. The compound of claim 1, wherein the compound isolated from the myelin extract is 2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone (2-carbomethoxy-2,3-epoxy- 3-prenyl-1,4-naphthoquinone) compound. According to claim 1, wherein the extract is a pharmaceutical composition, characterized in that the crude extract of cheoncho root, polar solvent soluble extract or non-polar solvent soluble extract. The pharmaceutical composition according to claim 3, wherein the crude extract comprises an extract available in a solvent selected from water including purified water, a lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof. The pharmaceutical composition according to claim 3, wherein the nonpolar solvent soluble extract comprises an extract soluble in methylene chloride, hexane, chloroform, dichloromethane or ethyl acetate. The method of claim 1, wherein the inflammatory disease is atopic dermatitis, arthritis, urethritis, cystitis, arteriosclerosis, allergic diseases, rhinitis, asthma, acute pain, chronic pain, periodontitis, gingivitis, inflammatory bowel disease, gout, myocardial infarction, congestive A pharmaceutical composition characterized in that heart failure, hypertension, angina pectoris, gastric ulcer, cerebral infarction, Down syndrome, multiple sclerosis, obesity, diabetes, dementia, depression, schizophrenia, tuberculosis, sleep disorders, sepsis, burns or pancreatitis. The pharmaceutical composition according to claim 1, wherein the extract is included in an amount of 0.1 to 50% by weight based on the total weight of the composition. 2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone isolated from Cheoncho Geun extract (2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone ), 3,3′-bis (3,4-dihydro-4-hydroxy-6-methoxy-2H-1-benzopyran) (3,3′-bis (3,4-dihydro-4-hydroxy -6-methoxy-2H-1-benzopyran), Epoxymollugin, Soranjidiol, Oleanolic acid, 1-acetoxy-3-methoxy-9,10-anthra Compound selected from the group consisting of quinone (1-acetoxy-3-methoxy-9,10-anthraquinone), alizarin 2-methyl ether, furonollugin, and mollugin, Or health functional food for the prevention and improvement of inflammatory diseases containing a pharmaceutically acceptable salt thereof as an active ingredient. The dietary supplement of claim 8 which is a powder, granule, tablet, capsule or beverage.

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