KR101833011B1 - Composition comprising Rhamnetin or as active ingredients for preventing or treating of Tuberculosis - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating tuberculosis and a health functional food composition for preventing or alleviating tuberculosis comprising rhamnetin, rhamnetin derivatives or pharmaceutically allowable salts thereof as active ingredients.

Description

The present invention relates to a composition for preventing or treating tuberculosis comprising rhamnetin and a pharmaceutically acceptable salt thereof as an active ingredient,

The present invention relates to a composition for preventing or treating tuberculosis comprising rhametin, a rhamethine derivative or a pharmaceutically acceptable salt thereof as an active ingredient.

Tuberculosis (TB) is transmitted through the air by Mycobacterium tuberculosis (MT), which affects the lungs and causes serious infections. Recently, drug-resistant tuberculosis strains have presented serious problems because they are resistant to most anti-tuberculosis agents. In particular, multidrug-resistant (MDR) strains are resistant to first-generation anti-tuberculosis agents such as rifampicin and isoniazid, and drug resistant (XDR) strains are resistant to capreomycin, amikacin resistant to second generation anti-tuberculosis agents such as kanamycin. Therefore, it is necessary to develop new anti-tuberculosis drugs against multi-drug-resistant (MDR) strains and drug-resistant (XDR) strains.

Flavonoids are compounds present in foods, including vegetables, fruits and tea, and are rich in polyphenols. Flavonoids are also known to have excellent anti-allergic, anti-inflammatory, antioxidant, anti-bacterial and anti-cancer activities. Due to the active activity of flavonoid, it is a trend to be utilized for the treatment of various diseases.

Raminetin is present in fruits such as cloves, coriander, and apples, Western cherries, and berry. Recently, the present inventors have reported on the anti-inflammatory activity of rhamethine, and it has been shown that rhamethine inhibits mouse mTNF (tumor necrosis factor) -α, mouse mMIP (macrophage inflammatory protein) -1 and mMIP -2 cytokine production. In addition, it has been reported that isosamnetin has anti-tuberculosis and anti-inflammatory activity as an abundant compound in apples, cherries, blackberries and pears.

Fibroblasts are a major cell type within connective tissue and can be easily infected by M. tuberculosis. M. tuberculosis activates MMP (matrix metalloproteinase) -1 through the signaling pathway of NF (nuclear factor) -KB and p38 mitogen-activated protein kinase (MAPK) in human fibroblast MRC-5.

Ramanetin has an anti-inflammatory activity through MAPK signaling pathway in RAW264.7 cells stimulated by LPS, and binds to ERK1 (extracellular signal-regulated kinase 1) and JNK (c-Jun N-terminal kinase) It has been reported.

The present invention is intended to provide a pharmaceutical composition for preventing or treating tuberculosis comprising rhametin, a rhamethine derivative or a pharmaceutically acceptable salt thereof as an active ingredient.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

The present invention provides a pharmaceutical composition for preventing or treating tuberculosis comprising Rhamnetin, Rhamnetin derivative or a pharmaceutically acceptable salt thereof as an active ingredient.

The ramethine may be represented by the following formula 1:

 [Chemical Formula 1]

.

.

The ramethine derivative may be represented by the following formula (2): < EMI ID =

(2)

.

.

The composition may contain the above-mentioned rhamethine at a concentration of 0.1 μg / ml to 1000 μg / ml.

The composition may be one that reduces the secretion of cytokines selected from the group consisting of TNF-a, IL-1 beta, IL-6, IL-12 and MMP-1.

The composition may be one which reduces phosphorylation of p38 MAPK or ERK.

The above-mentioned tuberculosis is selected from the group consisting of multidrug-resistant (MDR) tuberculosis, drug-resistant (XDR) tuberculosis, pulmonary tuberculosis, pleural tuberculosis, tuberculosis lymphoma, cerebral tuberculosis, spinal tuberculosis, .

The present invention also provides a health functional food composition for preventing or improving tuberculosis comprising Rhamnetin, Rhamnetin derivatives or a pharmaceutically acceptable salt thereof as an active ingredient.

The ramethine may be represented by the following formula 1:

[Chemical Formula 1]

.

.

The ramethine derivative may be represented by the following formula (2): < EMI ID =

(2)

.

.

The composition may contain the above-mentioned rhamethine at a concentration of 0.1 μg / ml to 1000 μg / ml.

The composition may be one that reduces the secretion of cytokines selected from the group consisting of TNF-a, IL-1 beta, IL-6, IL-12 and MMP-1.

The composition may be one which reduces phosphorylation of p38 MAPK or ERK.

The above-mentioned tuberculosis is selected from the group consisting of multidrug-resistant (MDR) tuberculosis, drug-resistant (XDR) tuberculosis, pulmonary tuberculosis, pleural tuberculosis, tuberculosis lymphoma, cerebral tuberculosis, spinal tuberculosis, .

The composition for preventing or treating tuberculosis according to the present invention comprises as an active ingredient ramethine, a rhamethine derivative or a pharmaceutically acceptable salt thereof, wherein the ramethine is TNF-α, IL-1β, IL-6, IL-12 and MMP-1, and reduce the phosphorylation of p38 MAPK or ERK.

In addition, as a natural substance-derived compound, rhamethine is low in cytotoxicity and has an anti-tuberculous activity and can be effectively used for effectively preventing or treating tuberculosis.

Figure 1 shows anti-tuberculosis activity of rhamnetin and isosamanthen.
Figure 2 shows the cell survival relationship of NIH3T3 and MRC-5 with rhamethine and isosamanthen.
FIG. 3 shows the expression levels of TNF-α (a) and IL-12 (b) in the MRC-5 cell stimulated with IFN-γ according to the concentration of laminectin treatment.
FIG. 4 shows RT-PCR results of (a) mRNA expression level (bf) of MRC-5 cells stimulated with IFN-y, respectively. Bars represent standard deviations for independent experiments ± 3 mean expression levels. ** P <0.005, *** P <0.001. RTI ID = 0.0 &gt; IFN-y. &Lt; / RTI &gt;
FIG. 5 shows mRNA expression level (bf) protein expression of IFN-y stimulated MRC-5 cells. The bar represents the standard deviation of the mean ± 3 independent experiments. ** P &lt; 0.005, *** P &lt; 0.001 were expressed as cells treated with IFN-y.

The present inventors have found that Rhamretin and its derivative Isorhamnetin, a natural flavonoid rich in clove and blackberry, are effective against M. tuberculosis such as H37Rv, MDR, and XDR clinical isolates Confirming that it inhibits anti-mycobacterial activity and pulmonary inflammation, and demonstrates the specific mechanism of action of the anti-inflammatory action of rhamethine, thus completing the present invention.

As used herein, "Rhamnetin" is a naturally occurring compound and is known to exist mainly in fruits such as clove, coriander, and apple, western cherry, berry and the like and has anti-inflammatory activity.

As used herein, " Isorhamnetin "is one of the rhametin derivatives and is present in fruits such as apples, cherries, blackberries and pears and is known to have anti-tuberculosis and anti-inflammatory activity.

Hereinafter, the present invention will be described in detail.

Pharmaceutical composition for preventing or treating tuberculosis

The present invention provides a pharmaceutical composition for preventing or treating tuberculosis comprising rhametin, a rhamethine derivative or a pharmaceutically acceptable salt thereof as an active ingredient.

Specifically, the rhamethine may be represented by the following formula (1).

[Chemical Formula 1]

In addition, the laminectin derivative may be represented by the following general formula (2), and may be named as isomorphine as the isoform of the general formula (1).

(2)

The isothiocyanates represented by the formula (2) and the rhamethine represented by the formula (1) have higher anti-tuberculosis activity than their derivatives tamarixetin and quercetin. Namely, since ramethine and its derivatives exhibit isoforms, they have the same hydrophobicity but the position of methoxy group (-OCH ₃ ) in the benzene ring is different, so that the difference in the interaction effect with the target protein , Anti-tuberculosis activity may be different.

The pharmaceutical composition for the prevention or treatment of tuberculosis according to an embodiment of the present invention may contain the above-mentioned rhamethine at a concentration of 0.1 μg / ml to 1000 μg / ml, and a concentration of 100 μg / ml to 200 μg / But is not limited thereto. At this time, when the concentration of the rhamethine in the composition is less than the above range, the antibacterial activity against the Mycobacterium tuberculosis is deteriorated. When the concentration of the rhamethine exceeds the above range, there is a problem of cytotoxicity.

In addition, the composition may reduce the secretion amount of cytokine selected from the group consisting of TNF- ?, IL-1 ?, IL-6, IL-12 and MMP-1.

Cytokines and chemokines are secreted in the initial immune response of M. tuberculosis. IFN-γ is a major cytokine that activates T cells and NK cells and activates macrophages, and activates macrophages and activates NO synthase (NOS), cyclooxygenase-2, IL-1β And IL-12 upregulation and activation. Specifically, mRNA expression and protein expression of IL-1β, IL-6, IL-12, TNF-α and MMP-1 are decreased by treatment with IFN-γ-stimulated human lung fibroblast MRC-5 I could confirm.

Furthermore, the composition may reduce phosphorylation of p38 MAPK or ERK.

Specifically, as shown in FIG. 5, it was confirmed that rhamethine reduced phosphorylation of p38 MAPK and ERK by 48.5% and 46.1%, respectively, and phosphorylation of JNK did not show any significant change. In other words, it was confirmed that rhamethine did not decrease JNK activity.

Furthermore, the present invention relates to a pharmaceutical composition for preventing or treating tuberculosis comprising as an active ingredient a rhamethine, a rhametin derivative and a pharmaceutically acceptable salt thereof, wherein said tuberculosis is multidrug-resistant (MDR) Tuberculosis, tuberculosis, drug-resistant (XDR) tuberculosis, pulmonary tuberculosis, pleural tuberculosis, lymphatic tuberculosis, brain tuberculosis, intestinal tuberculosis, spinal tuberculosis, and kidney tuberculosis.

The pharmaceutical composition of the present invention may comprise a pharmaceutically acceptable salt of rhamethine. The term &quot; pharmaceutically acceptable &quot; of the present invention refers to those which are physiologically acceptable and do not normally cause an allergic reaction or the like when administered to humans, and the salts include pharmaceutically acceptable free acids acid is preferred.

The pharmaceutically acceptable salt of rhamethine may be an acid addition salt formed using an organic acid or an inorganic acid, and the organic acid may be, for example, formic acid, acetic acid, propionic acid, lactic acid, butyric acid, isobutyric acid, trifluoroacetic acid, There may be mentioned acetic acid, maleic acid, malonic acid, fumaric acid, succinic acid, succinic monoamide, glutamic acid, tartaric acid, oxalic acid, citric acid, glycolic acid, glucuronic acid, ascorbic acid, benzoic acid, phthalic acid, salicylic acid, anthranilic acid, dichloroacetic acid, , p-toluenesulfonic acid or methanesulfonic acid. The inorganic acid includes, for example, hydrochloric acid, bromic acid, sulfuric acid, phosphoric acid, nitric acid, carbonic acid or boric acid. The acid addition salt may preferably be in the form of a hydrochloride or an acetate, more preferably in the form of a hydrochloride.

The above-mentioned acid addition salts may be prepared by a) directly mixing the ramethine and the acid, b) dissolving and mixing it in the solvent or the aqueous solvent, or c) placing the ramethine in the solvent or acid in the solvent and mixing them It is prepared by a general salt production method.

Separately, additionally saltable forms include, but are not limited to, the salts of gabapentin, gabapentin, pregabalin, nicotinate, adipate, hemimarate, cysteine, acetylcysteine, methionine, arginine, lysine, Aspartate and the like.

In addition, the pharmaceutical composition for preventing and treating tuberculosis of the present invention may further comprise a pharmaceutically acceptable carrier.

The pharmaceutically acceptable carrier may further include, for example, a carrier for oral administration or a carrier for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. The carrier for parenteral administration may also contain water, suitable oils, saline, aqueous glucose and glycols and the like. In addition, stabilizers and preservatives may be further included. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. Other pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, Pa., 1995).

The pharmaceutical composition of the present invention can be administered to mammals including humans by any method. For example, it can be administered orally or parenterally, and parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, , Intranasal, enteral, topical, sublingual or rectal administration.

The pharmaceutical composition of the invention may be formulated into oral or parenteral dosage forms according to the route of administration as described above. When formulated, one or more buffers (e.g., saline or PBS), antioxidants, bacteriostats, chelating agents (e.g., EDTA or glutathione), fillers, extenders, binders, adjuvants Side), suspending agents, thickening agents, disintegrating agents or surfactants, diluents or excipients.

Solid preparations for oral administration include tablets, pills, powders, granules, solutions, gels, syrups, slurries, suspensions or capsules, etc. These solid preparations can be prepared by incorporating into the pharmaceutical composition of the present invention at least one excipient, , Starch (including corn starch, wheat starch, rice starch and potato starch), calcium carbonate, sucrose, lactose, dextrose, sorbitol, mannitol, xylitol, erythritol maltitol, cellulose , Methyl cellulose, sodium carboxymethyl cellulose and hydroxypropylmethyl-cellulose or gelatin. For example, tablets or tablets may be obtained by combining the active ingredient with a solid excipient, then milling it, adding suitable auxiliaries, and processing the mixture into granules.

In addition to simple excipients, lubricants such as magnesium stearate talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions or syrups. In addition to water or liquid paraffin, which is a simple diluent commonly used, various excipients such as wetting agents, sweeteners, fragrances or preservatives may be included .

In addition, crosslinked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may optionally be added as a disintegrant, and may further include an anticoagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent and an antiseptic agent .

For parenteral administration, the pharmaceutical compositions of the present invention may be formulated in accordance with methods known in the art in the form of injectable, transdermal and nasal inhalers, together with suitable non-oral carriers. In the case of such injections, they must be sterilized and protected against contamination of microorganisms such as bacteria and fungi. Examples of suitable carriers for injectables include, but are not limited to, solvents or dispersion media containing water, ethanol, polyols (e.g., glycerol, propylene glycol and liquid polyethylene glycol, etc.), mixtures thereof and / or vegetable oils . More preferably, suitable carriers include isotonic solutions such as Hanks' solution, Ringer's solution, phosphate buffered saline (PBS) containing triethanolamine, or sterile water for injection, 10% ethanol, 40% propylene glycol and 5% dextrose Etc. may be used. In order to protect the injection from microbial contamination, various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like may be further included. In addition, the injections may in most cases additionally include isotonic agents, such as sugars or sodium chloride.

Examples of transdermal dosage forms include ointments, creams, lotions, gels, solutions for external use, pastes, liniments, and air lozenges. In the above, 'transdermal administration' means that the pharmaceutical composition is locally administered to the skin, whereby an effective amount of the active ingredient contained in the pharmaceutical composition is delivered into the skin.

In the case of inhalation dosage forms, the compounds used according to the present invention may be formulated into a pressurized pack or a pressurized pack using a suitable propellant, for example dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gases. It can be conveniently delivered in the form of an aerosol spray from a nebulizer. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve that delivers a metered amount. For example, gelatin capsules and cartridges for use in an inhaler or insufflator may be formulated to contain a compound, and a powder mixture of a suitable powder base such as lactose or starch. Formulations for parenteral administration are described in Remington's Pharmaceutical Science, 15th Edition, 1975. Mack Publishing Company, Easton, Pennsylvania 18042, Chapter 87: Blaug, Seymour, commonly known in all pharmaceutical chemistries.

The pharmaceutical composition for preventing or treating tuberculosis of the present invention can provide a preferable tuberculosis preventive and therapeutic effect when it contains an effective amount of rhamethine. In the present invention, the term "effective amount" refers to an amount exhibiting a further reaction than that of the negative control, and preferably refers to an amount sufficient to improve muscle function. The pharmaceutical composition of the present invention may contain 0.01 to 99.99% of rhamethine, and the remaining amount may be a pharmaceutically acceptable carrier. The effective amount of rhamethine contained in the pharmaceutical composition of the present invention will vary depending on the form and the like of the composition.

The total effective amount of the pharmaceutical composition of the present invention may be administered to a patient in a single dose and may be administered by a fractionated treatment protocol administered over a prolonged period of time in multiple doses. It is important that all of the above elements be administered in an amount that is conducive to maximizing the effect in a minimal amount without side effects, which can be readily determined by one skilled in the art.

The pharmaceutical composition of the present invention may vary in the content of the active ingredient depending on the degree of the disease. In the case of parenteral administration, it is preferably administered in an amount of 0.01 to 50 mg, more preferably 0.1 to 30 mg per kg of body weight per day on the basis of the above-mentioned rhamethine, and in the case of oral administration, May be administered one to several times in an amount of preferably 0.01 to 100 mg, more preferably 0.01 to 10 mg per kg of body weight. However, the dose of the rhamethine is determined based on various factors such as the patient's age, body weight, health condition, sex, severity of disease, diet and excretion rate as well as administration route and frequency of treatment of the pharmaceutical composition, , It will be possible for a person skilled in the art to determine the appropriate effective dose according to the specific use for the prevention and treatment of tuberculosis. The pharmaceutical composition according to the present invention is not particularly limited to the formulation, administration route and administration method as long as the effect of the present invention is exhibited.

The pharmaceutical composition for preventing or treating tuberculosis of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy or biological response modifiers.

Health functional food composition for preventing or improving tuberculosis

The present invention provides a health functional food composition for preventing or improving tuberculosis comprising Rhamnetin, rhamnetin derivatives or a pharmaceutically acceptable salt thereof as an active ingredient.

The details of the above-mentioned ramethine are as described above.

The food composition of the present invention includes all forms of functional foods, nutritional supplements, health foods, food additives and feeds, It is targeted for eating. Food compositions of this type may be prepared in a variety of forms according to conventional methods known in the art.

The food composition according to the present invention can be prepared in various forms according to a conventional method known in the art. Common foods include but are not limited to beverages (including alcoholic beverages), fruits and processed foods (eg, canned fruits, jam, maamalade, etc.), fish, meat and processed foods (Eg butter, chewing), edible vegetable oil, margarine (such as corn oil, etc.), breads and noodles (eg udon, buckwheat noodles, ramen noodles, spaghetti, macaroni, etc.), juice, various drinks, cookies, , Vegetable protein, retort food, frozen food, various kinds of seasoning (for example, soybean paste, soy sauce, sauce, etc.). The nutritional supplement is not limited thereto, but it can be prepared by adding the rhamethine of the present invention to capsules, tablets, rings and the like. For example, the health functional food may be prepared by liquefying, granulating, encapsulating and powdering laminectin itself of the present invention in the form of tea, juice, and drink so as to be able to drink (health drink) It can be ingested. In addition, in order to use the rhamethine of the present invention in the form of a food additive, it may be used in the form of a powder or a concentrated liquid. In addition, it can be prepared in the form of a composition by mixing together with known active ingredients known to be effective for preventing and improving tuberculosis and rhamethine of the present invention.

When the rhamethine of the present invention is used as a health drink, the health beverage composition may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. The above-mentioned natural carbohydrates include monosaccharides such as glucose and fructose; Disaccharides such as maltose, sucrose; Polysaccharides such as dextrin, cyclodextrin; Xylitol, sorbitol, erythritol, and the like. Sweeteners include natural sweeteners such as tau Martin and stevia extract; Synthetic sweetening agents such as saccharin and aspartame, and the like can be used. The ratio of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 mL of the composition of the present invention.

The amount of rhamethine of the present invention may be contained as an effective ingredient of a food composition for preventing and improving tuberculosis. The amount of ramethine is not particularly limited as long as it is effective for achieving tuberculosis prevention and remedy action, By weight based on 100% by weight of the total composition. The food composition of the present invention may be prepared by mixing together with other active ingredients known to be effective in compositions for preventing and improving tuberculosis in combination with rhamethine.

In addition to the above, the health food of the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid, salts of pectic acid, alginic acid, salts of alginic acid, organic acid, protective colloid thickener, pH adjusting agent, Glycerin, an alcohol or a carbonating agent, and the like. In addition, the health food of the present invention may contain natural fruit juice, fruit juice drink, or pulp for the production of vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not critical, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

[Preparation example]

Lambentin (purity 99%) used in the following examples was purchased from Sigma-Aldrich (St. Louis, Mo., USA). The ramethine was dissolved in dimethyl sulfoxide (DMSO) to produce 10 mg / ml stock solution.

[Example]

Example 1. Evaluation of anti-tuberculosis activity

The M. tuberculosis strains, H37Rv, MDR, and XDR, were loaded onto 37 ° C inlet wells with liquid agarose. After the bacteria were fixed by coagulation, rhamnetin and isorhamnetin (Sigma-Aldrich, St. Louis, Mo., USA) were slowly loaded with liquid culture media at a concentration of 0 to 200 μg / . During incubation, the wells were imaged on days 1, 3, 5, 7 and 9 to record bacterial cell growth. The point at which dark spots of M. tuberculosis are converted to white is imaged and calculated by determining the total area covered by M. tuberculosis with a MIC ₉₀ value (the minimum limiting concentration that causes 90% inhibition of M. tuberculosis growth) Respectively. All experiments were performed in triplicate and the minimum growth limiting concentrations were shown in Table 1 below

Strain Type of strain Minimum growth limiting concentration (MIC ₉₀ , μM) Laminectin Isolam Netin P887 H ₃₇ Rv 100 50 M22 MDR 200 100 X23 MDR 200 100 X24 XDR 100 100 X59 XDR 100 100

As a result, as shown in Table 1, it can be confirmed that isosamnetin has a minimum growth inhibitory concentration of 1/2 compared to rhamethine for H ₃₇ Rv (P887) and MDR strains (M22, X23) It was confirmed that the strain has the same anti-tubercular activity as the strain XDR (X24, X59).

Example 2. Toxicity Evaluation of NIH3T3 and MRC-5 Cells

Human lung fibroblast MRC-5 cells and mouse embryonic fibroblast-derived cell line NIH3T3 were purchased from the American Type Culture Collection (Manassas, VA, USA).

Toxicity was assessed using the MTT assay (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) assay and the absorbance at 570 nm was measured.

As a result, as shown in Fig. 2, it was confirmed that the cell viability was 73.1% and 93.1% in NIH3T3 cells treated with 200 / / ml of isosamnet and 200 ㎍ / ml of rhamethine.

In addition, it was confirmed that the cell viability was 74.5% and 82.8% in MRC-5 cells treated with 200 / / ㎖ of isosamantine and 200 / / ml of ramethine.

That is, it can be seen that rhamethine has lower cytotoxicity when compared to isosarnetine, and that rheemetin is a more effective anti-tuberculosis preventive or therapeutic composition despite less anti-tuberculin activity than isosamethine It can be seen that.

Example 3. Enzyme-linked immunosorbent assay (ELISA)

The expression of cytokines was measured in MRC-5 cells stimulated with IFN-γ using ELISA.

The stimulated MRC-5 cells were treated with 0.31, 1.58 and 3.16 ng / ml of ramethine, and cultured in an incubator for 24 hours.

 The antibodies to hTNF-alpha (human tumor necrosis factor-alpha) and hIL-12 (human interleukin 12) were immobilized on immunoplates. Stimulated MRC-5 cells were co-cultured with serially diluted compounds and supernatants were added to the precoated plates with antidoady. After washing the plate, a detection antibody solution and 3,3,5,5'-tetramethylbenzidine (TMB) were added. The range of enzyme reaction was measured at 450 nm.

 As a result, as shown in FIG. 3, when 1.58 μg / ml and 3.16 μg / ml of ramethine were treated with IFN-γ stimulated MRC-5 cells, hTNF- α leve ㅣ decreased by 30% and 41%, respectively. In addition, hIL-12 decreased by 40% and 72%, respectively, compared with the negative control group.

Example 4. Reverse Transcription-Polymerase Chain Reaction (RT-PCR)

To investigate the compound-mediated inflammatory response pathway, RT-PCR was performed.

Human lung fibroblast MRC-5 cells were treated with 6.3 μg / ml of ramethine and cultured in an incubator for 3 hours. In addition, human lung fibroblast MRC-5 cells were treated with 6.3ng / ml of ramethine and 20ng / ml of IFN-γ and cultured in an incubator for 3 hours.

Human lung fibroblast MRC-5 cells were cultured for 3 hrs without treatment with ramethine, treated with 6.3 ng / ml ramethine and 20 ng / ml IFN-γ, and cultured in an incubator for 3 hrs. .

The mRNA expression of human interleukin (IL) -1β, hIL-6, IL-12, tumor necrosis factor (TNF) -α and matrix metalloproteinase (MMP) -1 and β- -PCR was performed and the amplified product was visualized using ethidium bromide-stained gels by UV illumination.

As a result, as shown in FIG. 4, IL-1β, hIL-6 and IL-12 in MRC-5 cells treated with IFN-γ and rhamethine in comparison with MRC- , tumor necrosis factor (TNF) -α and matrix metalloproteinase (MMP) -1 mRNA expression levels were reduced by 48%, 19%, 30%, 84% and 52%, respectively.

Example 5 Western Blot &lt; RTI ID = 0.0 &gt;

In order to investigate the phosphorylation status of JNK, p38 MAPK and ERK in IFN-γ stimulated MRC5 cells, Westin blotting was performed.

Human lung fibroblast MRC-5 cells were treated with 6.3 μg / ml of ramethine. Human lung fibroblast MRC-5 cells were treated with 6.3 ㎍ / ㎖ of ramethine and 20 ng / ml of IFN-γ.

Human lung fibroblast MRC-5 cells were treated with ramenine, 6.3 μg / ml of ramethine and 20 ng / ml of IFN-γ, respectively.

First, the same amount (20 μg) of protein isolated from MRC-5 cells was separated on 10% sodium dodecyl sulfate polyacrylamide gel and transferred to blocked polyvinylidene difluoride membranes. Subsequently, the cells were incubated at 4 ° C for 1 day with primary antibodies against phosphorylated p38 and β-actin, phosphorylated extracellular signal-regulated kinase (ERK), phosphorylated c-Jun N-terminal kinase (JNK)

Subsequently, the cells were washed three times with Tris-buffered saline containing Tween 20, incubated with Luminol / Enhancer solution, and incubated with horseradish peroxidase conjugated with secondary antibodies for 4 hours at room temperature. The relative density of the protein bands was quantified using ImageJ software.

As a result, as shown in Fig. 5, it was confirmed that rhamethine reduced phosphorylation of p38 MAPK and ERK by 48.5% and 46.1%, respectively, and phosphorylation of JNK did not show any significant change. In other words, it was confirmed that rhamethine did not decrease JNK activity.

Example 6. Statistical analysis

For each analysis, three independent samples were used. The mean and variance of three independent experiments were calculated. Statistical tests were analyzed using InStat (GraphPad software, San Diego, Calif., USA). The analysis was performed using Student-Newman-Keus (SNK) method and statistically significant at P <0.05.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

Claims (14)

A pharmaceutical composition comprising Rhamnetin or a pharmaceutically acceptable salt thereof represented by the following formula (1)
Drug-resistant (XDR) pharmaceutical compositions for the prevention or treatment of tuberculosis:
[Chemical Formula 1]

.
delete delete The method according to claim 1,
Characterized in that said composition comprises said ramethine at a concentration of from 0.1 [mu] g / ml to 1000 [mu] g / ml
Drug-resistant (XDR) A pharmaceutical composition for the prevention or treatment of tuberculosis.
The method according to claim 1,
Wherein said composition reduces the secretion of cytokines selected from the group consisting of TNF-a, IL-1 beta, IL-6, IL-12 and MMP-1
Drug-resistant (XDR) A pharmaceutical composition for the prevention or treatment of tuberculosis.
The method according to claim 1,
Wherein said composition is characterized by reducing phosphorylation of p38 MAPK or ERK
Drug-resistant (XDR) A pharmaceutical composition for the prevention or treatment of tuberculosis.
delete A pharmaceutical composition comprising Rhamnetin or a pharmaceutically acceptable salt thereof represented by the following formula (1)
Drug-resistant (XDR) health functional food composition for preventing or improving tuberculosis:
[Chemical Formula 1]

.
delete delete 9. The method of claim 8,
Characterized in that said composition comprises said ramethine at a concentration of from 0.1 [mu] g / ml to 1000 [mu] g / ml
DRUG-RESISTANT (XDR) A health functional food composition for preventing or ameliorating tuberculosis.
9. The method of claim 8,
Wherein said composition reduces the secretion of cytokines selected from the group consisting of TNF- [alpha], IL-1 [beta], IL-6, IL-12 and MMP-1
DRUG-RESISTANT (XDR) A health functional food composition for preventing or ameliorating tuberculosis.
9. The method of claim 8,
Wherein said composition is characterized by reducing phosphorylation of p38 MAPK or ERK
DRUG-RESISTANT (XDR) A health functional food composition for preventing or ameliorating tuberculosis. delete

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