KR101539977B1 - Pharmaceutical Composition for the prevention or treatment of inflammatory disease comprising terrein - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the prophylaxis and treatment of inflammatory diseases comprising terrine as an active ingredient, wherein the terenine inhibits IL-6 (interleukin-6) activity and p65 protein phosphorylation in macrophages . Therefore, the terenine can be effectively used as an active ingredient of a pharmaceutical composition for the prevention and treatment of inflammatory diseases.

Description

TECHNICAL FIELD The present invention relates to a pharmaceutical composition for the prevention and treatment of inflammatory diseases comprising terenine as an active ingredient,

The present invention relates to a pharmaceutical composition for preventing and treating inflammatory diseases comprising terrein as an active ingredient.

Inflammation refers to the pathological condition of abscesses formed by the infiltration of external infectious agents (bacteria, fungi, viruses, various allergens). Specifically, when external bacteria invade a specific tissue and proliferate, the leukocyte of the living body recognizes it and actively attacks the proliferated foreign germs. In this process, the dead cells of the leukocyte are accumulated in the invaded tissue At the same time, the cell debris of invading microorganisms killed by leukocytes melts into the invading tissues and abscess forms. The treatment of abscess due to inflammation can be promoted by the anti-inflammatory action. The anti-inflammatory action is to inhibit the growth of invading microorganisms by using an antibacterial agent or to activate the macrophage that digests foreign substances accumulated in the abscess, And enhancing the function of excretory macrophages.

In general, an inflammatory reaction is a defensive reaction process of a living body that attempts to repair and regenerate a damaged region when an invasion of biological changes occurs in the cell or tissue of the living body. Therefore, these series of reactions include local blood vessels, various tissue cells of body fluids, and immune-mediated cells. With the recent development of molecular biology, inflammatory diseases have been attempted to be understood at the molecular level of cytokine, and factors affecting these diseases are also being clarified one by one.

The inflammatory cytokines and mediators are regulated by nuclear elements. For example, NF-κB (nuclear factor-kappa B) is a nuclear protein of the Rel gene family. In the cytoplasm, NF-κB is associated with IκB (inhibitory kappa B) IκB kinase is activated by various stimuli such as chemokines and lipopolysaccharide (LPS) such as reactive oxygen and tumor necrosis factor-alpha (TNF-alpha) Out. NF-κB, composed of a heterodimer of p50 and p65, is known to promote the expression of genes (tumor necrosis factor or cyclooxide synthase) that are activated and then migrate to the nucleus to induce an inflammatory response (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; A Denk et al . , J. Biol . Chem. , 276 (30): 28451-28458, 2001; Sahnoun Z et al . , & Lt ; / RTI > Phsiology , 53 (4): 315-339,1998; Lindner V Pathobiology , 66 (6): 311-320, 1998; Landry DB meat al ., Am . J. Pathol ., 151 (4): 1085-1095,1997; ; Gerritsen ME et al . , Am . J. Pathol ., 147 (2): p278-292, 1995).

Interleukin-6 is a factor that regulates the growth, differentiation and activation of various cells that constitute the human immune system. It is produced in various cells and plays an important role in the defense mechanism of the human body (Hirano, T., et al., Immunol (Muraguchi, A., et al., Immunol., 1996). It is known that human interleukin-6 is a factor found in the culture medium of mononuclear cells and has a function of inducing antibody production of B cells (Muraguchi, A., et al., Immunol. , 1987). In 1986, the cDNA of human interleukin-6 has been cloned (Hirano, t., Et al., Nature, 324, 73 (1986)).

Interleukin-6 functions as a (1) growth factor for B-cell hybridoma and plasmacytoma (Snick, VJ, et al., Proc. Nacl. Acad Sci USA, 83, 9679 (1986), (2) activation and growth of T cells (Lotz, M., et al., J. Exp. Med., 167, 1253 Induces an acute phase response (Geiger, T., et al., Eur. J. Immunol., 18, 718 (1988)), (4) acts as a factor involved in hematopoiesis Cell differentiation of nervous system cells (Satoh, T., Mol. Cell. Biol. 8, 3546 (1988)), ), (6) keratinocyte growth, (7) regulation of bone metabolism, (8) development of mesangial cells in the kidney, (9) malignant melanoma ) And to inhibit the growth of breast cancer cells.

Interleukin-6, which regulates various functions, is secreted from various cows in various organs of the human body, and misregulation of these substances causes various diseases. These diseases include rheumatoid arthritis (Hirano, T., et al., Eur. J. Immunol., 18, 1797 (1988)), cirrhosis (Deviere, J., et al., Clin. Exp. Immunol, 77 , 221 (1989)), psoriasis dermatosis (Grossman, RM, et al., Proc. Natl. Acad. Sci.USA 86,6367 (1989)), multiple myeloma (Bataille, R., et al., J. Clin Cardiac myxoma, AIDS (Miles, SA, et al., Proc. Natl. Acad Sci, USA, 87, 4068 (1990)), and many others Diseases are known to occur in the face. Controlling the function and production of interleukin-6, which plays an important role in various organs of the human body, is also very important for maintaining the homeostasis of the immune system in the human body and for the treatment and prevention of diseases. For example, it has been reported that an antibody of interleukin-6 or interleukin-6 receptor can be used to inhibit myeloma cell proliferation in a myeloma patient caused by excessive secretion of interleukin-6 (Suzuki, H., Eur. J Immuno., 22, 1989 (1992)). On the other hand, it has been reported that the levels of interleukin-6 in amniotic fluid are high in the case of premature labor and premature rupture of membranes (Romero, R., et al., J. Clin. Invest., 85, 1392 (1990) ; Romero, R., et al., AJRI., 30, 169 (1993)), and it has been reported that interleukin-6 can be used as an index for the diagnosis of preterm birth.

The most powerful anti-inflammatory drug developed by humankind is steroids. However, long-term use is accompanied by side effects. Therefore, in the treatment of inflammation, steroids exert an astonishing effect on initial use, completely eliminating the symptoms, but this is only a brief period. Symptoms of steroid use are reversed with the discontinuation of steroid use. . Side effects of steroids include side effects such as round polyhedronic face, retention of fluid, adrenal suppression, increased susceptibility to infection, and other psychoses, cataracts, glaucoma, peptic ulcer, delayed wound healing, Have. Therefore, there is a need to study a substance derived from a natural product, which can be safely ingested safely without any separate purification process, has an effect of inhibiting inflammation, and can be easily used. However, the therapeutic agent for antiinflammatory diseases using terenine has not been developed so far.

Thus, the present inventors have made efforts to find a natural-substance-having substance having anti-inflammatory activity. As a result, it has been found that terenine isolated from Aspergillus terrius extract significantly inhibits IL-6 (interleukin-6) activity and p65 protein phosphorylation in macrophages The present inventors have completed the present invention by confirming that the terenine can be effectively used as an active ingredient of a pharmaceutical composition for the prevention and treatment of inflammatory diseases.

It is an object of the present invention to provide a composition for the prevention and treatment of inflammatory diseases containing terrein and a pharmaceutically acceptable salt thereof as an active ingredient.

In order to achieve the above object,

The present invention provides a pharmaceutical composition for the prevention and treatment of inflammatory diseases comprising terrein or a pharmaceutically acceptable salt thereof as an active ingredient.

The present invention also provides a health food for the prevention and / or amelioration of inflammatory diseases comprising terenine or a pharmaceutically acceptable salt thereof as an active ingredient.

The present invention relates to a pharmaceutical composition for the prevention and treatment of inflammatory diseases comprising terrein as an active ingredient, wherein the terenine isolated from Aspergillus terreus extract is an IL-6 (interleukin -6) activity and p65 protein phosphorylation, it is possible to use the terenine effectively as an active ingredient of a pharmaceutical composition for the prophylaxis and treatment of inflammatory diseases.

1 is a graph showing inhibition of IL-6 expression activity induced by lipopolysaccharides in macrophage RAW 264.7 by terrein.
FIG. 2 is a graph showing the inhibition of IL-6 mRNA expression induced by terpene in lipopolysaccharides in macrophage RAW 264.7.
Figure 3 shows the inhibition of p65 phosphorylation induced by terpene in Lipopolysaccharides in macrophage RAW 264.7.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for the prevention and treatment of inflammatory diseases comprising terrein or a pharmaceutically acceptable salt thereof as an active ingredient.

The terrain is teriwooseu Aspergillus (Aspergillus terreus extract, but is not limited thereto.

 The terenes are preferably represented by the following formula (1), but are not limited thereto.

[Chemical Formula 1]

.

.

The inflammatory disease may be selected from the group consisting of allergies, dermatitis, atopy, conjunctivitis, periodontitis, rhinitis, otitis, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, gout, ankylosing spondylitis, rheumatic fever, lupus, fibromyalgia, Wherein the disease is any one selected from the group consisting of osteoarthritis, rheumatoid arthritis, shoulder inflammation, tendinitis, hay fever, perianal inflammation, myositis, hepatitis, cystitis, nephritis, sjogren's syndrome, multiple sclerosis and acute and chronic inflammatory diseases But is not limited thereto.

The terenine preferably inhibits the activity of IL-6, but is not limited thereto.

The terenine preferably inhibits phosphorylation of NF-kB p65 (Nuclear factor-kB p65), but is not limited thereto.

In a specific example of the present invention, the present inventors treated IL-6 (interleukin-6) expression with lipopolysaccharides (LPS) in mouse macrophage RAW 264.7 (TIB-71, ATCC) In order to confirm the effect of phosphorus, ELISA method was used to examine the effect of terenine. As a result, it was confirmed that RAW 264.7 cells were treated with lipopolysaccharide to increase IL-6 expression, It was confirmed that IL-6 expression was inhibited in a concentration-dependent manner in the treated cells (see FIG. 1).

In order to examine the effect of terenine during the activation of IL-6 (interleukin-6) mRNA expression by treating lipopolysaccharides (LPS) in mouse macrophage line RAW 264.7 (TIB-71, ATCC) The effect of terenine was confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR). As a result, lipopolysaccharides (LPS) , And it was confirmed that IL-6 expression was inhibited in a concentration-dependent manner in the cells treated with terenine (see FIG. 2).

In addition, Nuclear factor-kB p65 protein was induced by treating lipopolysaccharides (LPS) with mouse macrophage line RAW 264.7 (TIB-71, ATCC) In order to confirm the effect of terenine, the effect of terenine was confirmed using enhanced chemiluminescent (ECL) method. As a result, 15 minutes after treatment of lipopolysaccharide in macrophage RAW 264.7, p65 phosphorylation , And it was confirmed that the expression of p65 was greatly suppressed in the cells pretreated with terenine (see FIG. 3).

Therefore, the terrein of the present invention significantly inhibits IL-6 (interleukin-6) activity and p65 protein phosphorylation in macrophages, and thus the terenin is useful for the prophylactic and therapeutic treatment of inflammatory diseases And can be usefully used as an effective ingredient of the composition.

The composition containing terrane of the present invention may further contain one or more active ingredients showing the same or similar functions in addition to the above components.

The composition of the present invention may further comprise a pharmaceutically acceptable additive, wherein pharmaceutically acceptable additives include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose Starch glycolate, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, calcium stearate, , White sugar, dextrose, sorbitol and talc. The pharmaceutically acceptable additives according to the present invention are preferably included in the composition in an amount of 0.1 to 90 parts by weight, but are not limited thereto.

That is, the composition of the present invention can be administered in various formulations of oral and parenteral administration at the time of actual clinical administration. In the case of formulation, a diluent such as a filler, an extender, a binder, a wetting agent, a disintegrant, . ≪ / RTI > Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose ), Lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate talc may also be used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions and syrups, and various excipients such as wetting agents, sweetening agents, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used . Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used as the non-aqueous solvent and suspension agent. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The composition of the present invention may be administered orally or parenterally in accordance with the intended method, and may be administered orally, parenterally or intraperitoneally, rectally, subcutaneously, intravenously, intramuscularly, . The dosage varies depending on the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and disease severity.

The dosage of the composition of the present invention varies depending on the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of disease, and the daily dosage is Aspergillus therius extract , Preferably 0.001 to 10 mg / kg, and 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, radiation therapy, hormone therapy, chemotherapy and biological response modifiers for the prevention and treatment of inflammatory diseases.

The terenes of the present invention can be used in the form of pharmaceutically acceptable salts, and as salts, acid addition salts formed by pharmaceutically acceptable free acids are useful. Acid addition salts include those derived from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, and aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates, Derived from organic acids such as acetic acid, benzoic acid, citric acid, lactic acid, maleic acid, gluconic acid, methanesulfonic acid, 4-toluenesulfonic acid, tartaric acid, fumaric acid, and the like. Such pharmaceutically innocuous salts include, but are not limited to, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate chloride, bromide, Butyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, succinate, maleic anhydride, maleic anhydride, , Sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, Methoxybenzoate, phthalate, terephthalate, benzene sulfonate, toluene sulfonate, chlorobenzene sulfide Propyl sulphonate, naphthalene-1-yne, xylenesulfonate, phenylsulfate, phenylbutyrate, citrate, lactate,? -Hydroxybutyrate, glycolate, maleate, Sulfonate, naphthalene-2-sulfonate or mandelate.

The acid addition salt according to the present invention can be obtained by a conventional method, for example, by dissolving a derivative of Chemical Formula 1 in an organic solvent such as methanol, ethanol, acetone, methylene chloride, acetonitrile, Or by drying, or by distillation of the solvent and excess acid under reduced pressure, followed by drying or crystallization in an organic solvent.

Can be manufactured.

In addition, bases can be used to make pharmaceutically acceptable metal salts. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess amount of an alkali metal hydroxide or an alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate. At this time, it is preferable for the metal salt to produce sodium, potassium or calcium salt. The corresponding silver salt is also obtained by reacting an alkali metal or alkaline earth metal salt with a suitable salt (such as silver nitrate).

The present invention also provides a health food for preventing and improving inflammatory diseases containing terrein or a pharmaceutically acceptable salt thereof as an active ingredient.

The terrain is teriwooseu Aspergillus (Aspergillus terreus extract, but is not limited thereto.

 The terenes are preferably represented by the following formula (1), but are not limited thereto.

[Chemical Formula 1]

.

.

The inflammatory disease may be selected from the group consisting of allergies, dermatitis, atopy, conjunctivitis, periodontitis, rhinitis, otitis, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, gout, ankylosing spondylitis, rheumatic fever, lupus, fibromyalgia, Wherein the disease is any one selected from the group consisting of osteoarthritis, rheumatoid arthritis, shoulder inflammation, tendinitis, hay fever, perianal inflammation, myositis, hepatitis, cystitis, nephritis, sjogren's syndrome, multiple sclerosis and acute and chronic inflammatory diseases But is not limited thereto.

The terenine preferably inhibits the activity of IL-6, but is not limited thereto.

The terenine preferably inhibits phosphorylation of NF-kB p65 (Nuclear factor-kB p65), but is not limited thereto.

Since the teren of the present invention has an effect of significantly inhibiting IL-6 (interleukin-6) activity and p65 protein phosphorylation in macrophages, the teren is useful as an effective ingredient of health foods for the prevention and treatment of inflammatory diseases Can be used.

There is no particular limitation on the kind of the food. Examples of the food to which the above substance can be added include various foods such as confectionery, bread, and noodles, drinks such as water, soft drinks and fruit drinks, gum, tea, vitamin complex, seasoning, Health functional foods in a conventional sense.

The terenes of the present invention may be added directly to food or used together with other food or food ingredients, and may be suitably used according to conventional methods. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (for prevention or improvement). Generally, the amount of the compound in the health food may be from 0.01 to 15% by weight, preferably from 0.1 to 5% by weight of the total food, and 0.01 to 5.0 g, preferably 0.01 to 5% by weight, 1.0 g. However, in the case of long-term intake for the purpose of health control, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

The health functional beverage composition of the present invention is not particularly limited to other components other than the above-mentioned compounds as essential components in the indicated ratios, and may contain various flavors or natural carbohydrates as additional components such as ordinary beverages. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like and polysaccharides such as dextrins, cyclodextrins and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavorings (taumakin, stevia extract, etc.) and synthetic flavorings (saccharin, aspartan, etc.) can be advantageously used as flavorings other than those described above. The ratio of the natural carbohydrate is generally about 0.1 to 2.0 g, preferably about 0.1 to 1.0 g per 100 of the composition of the present invention.

In addition to the above, terenes of the present invention can be used as flavorings such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, colorants and heavies (cheese, chocolate etc.), pectic acid and its salts, A salt thereof, an organic acid, a protective colloid concentrating agent, a pH adjusting agent, a stabilizer, a preservative, a glycerin, an alcohol, and a carbonating agent used in a carbonated beverage. In addition, the Aspergillus oryzae extract of the present invention or its fractions may contain flesh for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components may be used independently or in combination. The proportion of such additives is not so critical, but is generally selected in the range of about 20 parts by weight per 100 parts by weight of the Aspergillus oryzae extract of the present invention.

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

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

< Example  1> Aspergillus  Terius ( Aspergillus terreus ) &Lt; / RTI &gt; terrein ) detach

Terrine, isolated from Aspergillus terreus extract, was purchased from Adipogen (Cat #: BVT-0193-M010).

< Example  2> Terein ( terrein )of IL -6 Confirmation of inhibitory effect

<2-1> Cell culture

Mouse macrophage cell line RAW 264.7 (TIB-71, ATCC) was inoculated into DMEM (Dulbecco's) containing 10% fetal bovine serum (FBS), 100 U / ml penicilin and 100 μg / ml streptomycin modified Eagle &apos; s medium) (Invitrogen Life Tehchnologies). The cells were cultured in an incubator at 37 ° C., 95% humidity, 5% CO ₂ , and subcultured in a fresh culture medium every 2 to 3 days.

<2-2> Lipopolysaccharide ( Lipopolysaccharides ) Activated by IL -6 expression inhibition confirmation

IL-6 (interleukin-6) expression was activated by treating lipopolysaccharides (LPS) in macrophage RAW 264.7 (TIB-71, ATCC) cultured by the method described in Example <2-1> , The effect of terenine was confirmed using an ELISA method to confirm the effect of terenine.

Specifically, the macrophage cell line RAW 264.7 cultured by the method described in Example <2-1> was dispensed into a 96-well microplate at a concentration of 5 × 10 ⁵ cells / ml at a rate of 200 μl / well , And 1, 3, 10, and 30 μM terrein, respectively, were added and pretreated for 1 hour. Lipopolysaccharides (LPS) were then added to the cells and supernatants were obtained after 24 hours. The obtained supernatant was measured for IL-6 expression using an ELISA kit (R & D systems INC.) According to the protocol provided by the manufacturer.

As a result, as shown in Fig. 1, RAW 264.7 cells were treated with lipopolysaccharide to increase the expression of IL-6, and the expression of IL-6 was inhibited in a concentration-dependent manner in the cells treated with terenine (Fig. 1).

<2-3> Lipopolysaccharide ( Lipopolysaccharides ) Activated by IL -6 mRNA  Expression Check for inhibition

IL-6 (interleukin-6) mRNA expression was activated by treating lipopolysaccharides (LPS) to the macrophage RAW 264.7 (TIB-71, ATCC) cultured by the method described in Example <2-1> The effect of terenine was confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR) in order to confirm the effect of terenine.

Specifically, the macrophage cell line RAW 264.7 cultured by the method described in Example <2-1> was cultured at a concentration of 5 × 10 ⁵ cells / ml in a 6-well tissue culture plate. After incubation, the cells were washed with PBS stored at 4 ° C and cells were separated using a scraper. The separated cells were centrifuged at 1,200 rpm at 4 DEG C for 2 minutes to discard the supernatant to obtain a pellet. Total RNA was then extracted using the RNeasy Plus Mini Kit (Qiagen, Valencia, CA) in a manner known in the art. DEPC-treated RNA was dissolved in distilled water and its absorbance was measured by A260 / 280 ratio (Versa Max microplate reader, Molecular Devices). Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to confirm the amount of transcribed mRNA expression. Equal amounts of RNA were amplified (dt) ₁₅ Primer And then PCR was carried out. The PCR conditions were: heating the sample at 50 캜 for 20 seconds at 95 캜 for 10 minutes, repeating 25 times at 95 캜 for 15 seconds at 56 캜 for 30 seconds at 72 캜 for 1 minute. The primers used for the reverse transcriptase-polymerase chain reaction (RT-PCR) are as follows.

As a result, as shown in Fig. 2, it was confirmed that IL-6 (interleukin-6) expression was increased by treating lipopolysaccharides (LPS) in macrophage RAW 264.7, It was confirmed that IL-6 expression was inhibited in a concentration-dependent manner (Fig. 2).

order SEQ ID NO: TNF-α: sence (SEQ ID NO: 1) 5'-CCT GTA GCC CAC GTC GTA GC-3 ' TNF-α: anti-sence (SEQ ID NO: 2) 5'-TTG ACC TCA GCG CTG AGT TG-3 ' β-actin: sence (SEQ ID NO: 3) 5'-TGG AAT CCT GTGGCA TCC ATG AAA C-3 ' β-actin: anti-sence (SEQ ID NO: 4) 5'-TAA AAC GCA GCT CAG TAA CAG TCC G-3 '

< Example  3> Terein ( terrein )On by Nuclear factor - kB p65  Confirm protein phosphorylation inhibition

The Nuclear factor-kB p65 protein was induced by treating lipopolysaccharides (LPS) to the macrophage cell line RAW 264.7 (TIB-71, ATCC) cultured by the method described in Example <2-1> To confirm the effect of terenine, the effect of terenine was confirmed by enhanced chemiluminescence (ECL).

Specifically, the macrophage cell line RAW 264.7 cultured by the method described in Example <2-1> was dispensed into a 6-well microplate at a concentration of 5 x 10 ⁵ cells / ml at a rate of 3 ml / well, And the cells were pretreated for 1 hour, and the cells were obtained after 0, 15, 30 and 60 minutes by adding lipopolysaccharide. The cells were washed with 50 mM Tris-Cl (pH 7.4), 150 mM NaCl, 1 mM EGTA, 5 mM EDTA, 0.5% Triton X-100, 0.25% sodium deoxycholate, A buffer solution containing sodium fluoride, 1 mM sodium orthovanadate, 5 mg / ml leupeptin, 0.2 mM phenylmethylsulfonyl fluoride, 10 mg / ml aprotinin, 0.5 mM DTT (lysis buffer). The amount of the separated proteins was measured using a Bio-Rad protein assay kit (Bio-Rad Laboratories, Inc., Hercules, Calif., USA). The quantified protein was separated by SDS-polyacrylamide gel electrophoresis (12% SDS-polyacrylamide gel electrophoresis), and then electrophoretically transferred to a nitrocellulose membrane. The cells were blocked with 5% skim milk at room temperature for 1 hour. Then, a specific primary antibody (anti-phospho-p65) was added to Tris-buffered saline (TBS) containing 0.1% Tween-20 at 4 캜. To remove the specific primary antibody used, the cells were washed 3 times with Tris-buffer (TBS-T), diluted with 1: 1000 to 1: 2000 peroxidase (HRP) Lt; / RTI &gt; Then, expression of p65 phosphorylation was confirmed using enhanced chemiluminescence (Amersham Biosciences, Little Chalfont, United Kingdom).

As a result, as shown in Fig. 3, it was confirmed that the expression of p65 phosphorylation increased gradually after 15 minutes of treatment with lipopolysaccharide in macrophage RAW 264.7, and that the cells pretreated with terenine showed a decrease in p65 phosphorylation (Fig. 3).

<110> Korea Research Institute of Bioscience and Biotechnology <120> Pharmaceutical Composition for the prevention or treatment of          inflammatory disease comprising terrein <130> 13P-10-76 <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TNF ALPHA SENCE PRIMER 1 <400> 1 cctgtagccc acgtcgtagc 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TNF ALPHA ANTI-SENCE PRIMER 2 <400> 2 ttgacctcag cgctgagttg 20 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Beta actin SENCE PRIMER 3 <400> 3 tggaatcctg tggcatccat gaaac 25 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Beta actin ANTI SENCE PRIMER 4 <400> 4 taaaacgcag ctcagtaaca gtccg 25

Claims (7)

A pharmaceutical composition for the prevention and treatment of any one or more diseases selected from the group consisting of Crohn's disease, rheumatoid arthritis, and rheumatoid arthritis comprising terrein or a pharmaceutically acceptable salt thereof as an active ingredient.
The pharmaceutical composition according to claim 1, wherein the terenine is separated from Aspergillus terreus extract, and a pharmaceutical composition for the prevention and treatment of any one or more diseases selected from the group consisting of Crohn's disease, lupus and rheumatoid arthritis .
[Claim 2] The pharmaceutical composition according to claim 1, wherein the terenine is represented by the following formula (1): wherein R &lt; 1 &gt; is selected from the group consisting of Crohn's disease, lupus and rheumatoid arthritis.
[Chemical Formula 1]

.
delete The pharmaceutical composition according to claim 1, wherein the terenine inhibits the activity of IL-6. The pharmaceutical composition for the prevention and treatment of any one or more diseases selected from the group consisting of Crohn's disease, lupus and rheumatoid arthritis.
2. The method according to claim 1, wherein the terenine inhibits phosphorylation of NF-kB p65 (Nuclear factor-kB p65). A pharmaceutical composition.
A health food for preventing or ameliorating any one or more diseases selected from the group consisting of Crohn's disease, lupus and rheumatoid arthritis, comprising terenine or a pharmaceutically acceptable salt thereof as an active ingredient.

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