KR101990199B1 - Pharmaceutical composition for the prevention or treatment of tuberculosis comprising 3,6-dihydroxyflavone or pharmaceutically acceptable salts thereof as an active ingredient - Google Patents

Abstract

The present invention relates to a composition for antibacterial treatment against Mycobacterium tuberculosis comprising 3,6-dihydroxyflavone (3,6-DHF) or a pharmaceutically acceptable salt thereof as an active ingredient; And a composition for preventing or treating tuberculosis. The 3,6-dihydroxyflavone of the present invention binds to the active site of mtKASIII with high affinity to inhibit the activity of the enzyme, thereby inhibiting the growth of M. tuberculosis, M. tuberculosis, and XDR tuberculosis, and inhibiting LPS stimulated human lung fibroblast Inhibits the expression of inflammatory cytokines and MAPK family. Therefore, the 3,6-dihydroxyflavone can be effectively used as an active ingredient of a composition for preventing or treating tuberculosis.

Description

[0001] The present invention relates to a pharmaceutical composition for preventing or treating tuberculosis comprising 3,6-dihydroxyflavone or a pharmaceutically acceptable salt thereof as an active ingredient, and a pharmaceutical composition for preventing or treating tuberculosis comprising 3,6-dihydroxyflavone or other acceptable salts thereof as an active ingredient}

The present invention relates to a composition for antibacterial treatment against Mycobacterium tuberculosis comprising 3,6-dihydroxyflavone (3,6-DHF) or a pharmaceutically acceptable salt thereof as an active ingredient; And a composition for preventing or treating tuberculosis.

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis ( Mycobacterium tuberculosis ). Mycobacterium tuberculosis floats in the air. When people around you breathe in, When inhaled, the tubercle bacillus enters the lungs and causes infection. When infected with Mycobacterium tuberculosis, macrophages, fibroblasts and dendritic cells are activated, leading to local inflammation and granuloma. Infection with M. tuberculosis can be a chronic inflammatory disease caused by mutual or staggered regulatory processes and proinflammatory processes, which can affect the development and progression of other diseases. On the other hand, cells such as interferons, interleukins, cytokines such as tumor necrosis factor, and cells such as macrophages, fibroblasts, regulatory T cells and type 1 assisted lymphocytes, Are the main factors involved. These factors have a dual function of promoting or inhibiting a local inflammatory response and can be modulated by an immune response by TNF- [alpha], IL-6, IL-12 and IL-1 [beta] cytokines.

In early patients with tuberculosis infection, the proliferation of Mycobacterium tuberculosis can be suppressed without completely eradicating tuberculosis, which leads to the stage of chronic tuberculosis infection. When progressing to chronic tuberculosis, tuberculosis bacillus forms granuloma caused by phagocyte. These granulomas are surrounded by peripheral mantle of fibroblasts. The main function of fibroblasts appears to involve or separate mycobacteria by secretion of collagen at the periphery of granuloma (J. Immunol. 2007, 178, 3767-3776). Fibroblasts are the most abundant cell types within the connective tissue that can be infected by Mycobacterium tuberculosis (Hum. Immunol. 2013, 74, 722-729). The interaction of other immune cells with fibroblasts is an integral component of the granulomatous structure that occurs in response to M. tuberculosis infection, leading to immunomodulatory functions that control Mycobacterium tuberculosis infection. Mycobacterium tuberculosis has been reported to upregulate STAT3 (signal transducer and activator of transcription 3), p38 MAPK (p38 mitogen-activated protein kinase) and NF-kB dependent MMP-1 in human lung fibroblast MRC-5 cells (Am J. Respir. Cell Mol Biol., 2010, 43, 465-474).

With the development of various anti-tuberculosis agents, worldwide tuberculosis infection rates between 2000 and 2014 have been reported to decrease by about 1.5% per year. In addition, between 1990 and 2015, mortality and morbidity rates for tuberculosis have been reported to have dropped to about 47% and 40%, respectively. Recently, however, drug-resistant Mycobacterium tuberculosis has developed in the stage of treatment of tuberculosis, and therefore, the treatment is hampered. Drug resistant Mycobacterium tuberculosis can be classified into two broad categories. First, tuberculosis strains classified as multi-drug resistant (MDR) are resistant to the two most potent anti-tuberculosis drugs, isoniazid and rifampicin. In addition, extensively drug resistant (XDR) strains may also be used in combination with isoniazid or rifampicin, fluoroquinolone, and at least one secondary anti-tuberculosis drug, such as capreomycin, kanamycin ) Or amikacin (Emerg. Infect. Dis. 2008, 14, 1700-1706). Patients infected with the strain require special treatment, including expensive, potentially toxic secondary drugs to manage toxic reactions or complications. Therefore, there is a need to develop new drugs in a new way to combat MDR and XDR tuberculosis.

Flavonoids refer to polyphenolic secondary metabolites that are present in abundant amounts in plants. Flavonoids consist of two phenyl groups linked together in the form of a heterocyclo ring (C6-C3-C6). The flavonoid compounds reported to date are the largest phytonutrient group, of which about 5,000 are known. Antimicrobial, anti-inflammatory, anti-tuberculosis and anticancer activities of various flavonoids are known.

As a result of efforts to develop a therapeutic agent for tuberculosis against MDR and XDR mycobacteria without cytotoxicity, the present inventors have found that the activity of β-ketoacyl acyl carrier protein synthase III (mtKASIII) (3,6-dihydroxyflavone, 3,6-DHF) inhibited the growth of M. tuberculosis and M. tuberculosis by inhibiting the growth of M. tuberculosis, Since the fibroblasts exhibit significant anti-inflammatory activity by inhibiting the phosphorylation of inflammatory cytokines and MAPK family, it has been confirmed that 3,6-DHF can be effectively used as an active ingredient of a composition for preventing or treating tuberculosis, .

An object of the present invention is to provide an antibacterial composition for tubercle bacillus which has an antibacterial activity against multidrug-resistant tuberculosis and broad-spectrum tuberculosis strains; A pharmaceutical composition for preventing or treating tuberculosis; And a health food for preventing or improving tuberculosis.

In order to achieve the above object, the present invention provides a pharmaceutical composition comprising 3,6-dihydroxyflavone (3,6-DHF) or a pharmaceutically acceptable salt thereof, As an active ingredient, a pharmaceutical composition for preventing or treating tuberculosis, comprising:

[Chemical Formula 1]

.

.

The present invention also relates to a pharmaceutical composition comprising, as an active ingredient, 3,6-dihydroxyflavone (3,6-DHF) or a pharmaceutically acceptable salt thereof, , And a health food for preventing or improving tuberculosis.

In a preferred embodiment of the present invention, the 3,6-DHF or a pharmaceutically acceptable salt thereof may have an antibacterial activity against Mycobacterium tuberculosis which is Mycobacterium tuberculosis .

In one preferred embodiment of the present invention, the Mycobacterium tuberculosis may be a multi-drug resistant Mycobacterium tuberculosis or an extensively drug resistant Mycobacterium tuberculosis.

In a preferred embodiment of the present invention, the 3,6-DHF or a pharmaceutically acceptable salt thereof is administered to a patient in need of such treatment, wherein IL-1β, IL-6, IL-12, TNF- Lt; RTI ID = 0.0 > MMP-1. ≪ / RTI >

In a preferred embodiment of the present invention, the 3,6-DHF or a pharmaceutically acceptable salt thereof may inhibit phosphorylation of ERK1 or p38 MAPK upon inducing inflammation in the lung.

The present invention also relates to a pharmaceutical composition comprising, as an active ingredient, 3,6-dihydroxyflavone (3,6-DHF) or a pharmaceutically acceptable salt thereof, , And an antibacterial composition against M. tuberculosis.

In another preferred embodiment of the present invention, the antimicrobial composition may be any one selected from the group consisting of a pharmaceutical composition, a food additive, a feed additive, a nasal composition, and a quasi-drug composition.

The 3,6-dihydroxyflavone of the present invention binds to the active site of the β-ketoacyl acyl carrier protein synthase III (mtKASIII) of Mycobacterium tuberculosis with high affinity to inhibit the enzyme activity, Dihydroxyflavone can be usefully used as an effective ingredient of a composition for preventing or treating tuberculosis, since it inhibits the growth of LPS and suppresses the expression of inflammatory cytokines and MAPK family in LPS-stimulated human lung fibroblasts .

FIG. 1 is a graph showing the effect of 3,6-dihydroxyflavone (3,6-DHF) on Mycobacterium tuberculosis and 3,6,3 ', 4'-tetrahydroxyflavone (3,6,3', 4'- ): ≪ tb >< tb >
FIG. 1A is a graph showing the inhibitory effect of 3,6-DHF on the growth of Mycobacterium tuberculosis strain P887; FIG.
1B is a view for confirming the effect of inhibiting the growth of 3,6-DHF on the M22 strain, which is a multi-drug resistant bacterium of Mycobacterium tuberculosis;
FIG. 1C shows the effect of inhibiting the growth of 3,6-DHF on the X24 strain, which is a broad-spectrum resistant bacterium of Mycobacterium tuberculosis;
FIG. 1D shows the effect of inhibiting the growth of 3,6,3 ', 4'-THF on Mycobacterium tuberculosis strain P887;
FIG. 1E shows the effect of inhibiting the growth of 3,6,3 ', 4'-THF on the M22 strain, which is a multi-drug resistant bacterium of Mycobacterium tuberculosis; And
FIG. 1F shows the effect of inhibiting the growth of 3,6,3 ', 4'-THF on the X24 strain which is a broad-spectrum resistant bacterium of Mycobacterium tuberculosis.
Figure 2 shows the results of confirming cytotoxicity of 3,6-DHF to mammalian cells in vitro.
FIG. 3 shows the effect of inhibiting the expression level of inflammatory cytokines in MRC-5 cells stimulated by LPS:
FIG. 3A shows the results of confirming the inflammatory response induced by the LPS treatment and the mRNA level of the inflammatory cytokine with or without 3,6-DHF treatment; FIG.
FIGS. 3B to 3F show the results of confirming the level of protein expression of inflammatory cytokines according to the presence or absence of the inflammatory reaction induced by LPS treatment and the treatment with 3,6-DHF.
Figure 4 shows the effect of inhibiting the expression level of inflammatory cytokines according to the concentration of 3,6-DHF in MRC-5 cells stimulated by LPS:
Figure 4a is the result of confirming TNF-a expression level; Figure 4b is the result of confirming IL-1 [beta] expression levels; And Figure 4C are the results of confirming IL-12 expression levels.
FIG. 5 is a graph showing activation levels of MAPK-related proteins according to 3,6-DHF treatment in MRC-5 cells stimulated by LPS:
FIG. 5A is a Western blot result showing the induction of inflammation by LPS treatment and the phosphorylation level of ERK, JNK and p38 protein according to the presence or absence of 3,6-DHF treatment;
Figures 5b to 5d show the results of quantitative analysis of phosphorylation levels of ERK, JNK and p38 proteins.
Figure 6 shows the results for confirming the binding affinity with 3,6-DHF and mtKASIII proteins.
Figure 7 shows a docking model in which 3,6-DHF binds to the active site of the mtKASIII protein.

Hereinafter, terms of the present invention will be described.

As used herein, the term "prophylactic " refers to any action that inhibits the onset or delays the onset of the administration of the composition. In the present invention, "improvement" or "treatment" means any action in which administration of the composition improves or alters the symptoms of the disease.

The term "administering" as used herein means providing a given material to a patient in any suitable manner, and the administration route of the composition of the present invention may be administered orally, May be administered parenterally. The composition may also be administered by any device capable of transferring the active agent to the target cell.

Hereinafter, the present invention will be described in detail.

The present invention relates to a pharmaceutical composition comprising 3,6-dihydroxyflavone (3,6-DHF) or a pharmaceutically acceptable salt thereof as an active ingredient and having a structure of the following formula A pharmaceutical composition for preventing or treating:

[Chemical Formula 1]

.

.

In the pharmaceutical composition of the present invention, "3,6-dihydroxyflavone (3,6-DHF") is a flavonoid compound, which is isolated from plants, commercially available, Any of those produced by a chemical synthesis method may be used.

In the pharmaceutical composition of the present invention, "3,6-DHF" or a pharmaceutically acceptable salt thereof is characterized by having an antibacterial activity against Mycobacterium tuberculosis , which is Mycobacterium tuberculosis , Specifically, it is preferable that the M. tuberculosis be at least one of multi drug resistant mycobacteria or extensively drug resistant mycobacteria. However, the present invention is not limited thereto.

In the pharmaceutical composition of the present invention, "3,6-DHF or a pharmaceutically acceptable salt thereof" may exhibit anti-inflammatory activity when the lung is inflamed. In connection with the anti-inflammatory activity, the level of expression of an inflammatory cytokine selected from the group consisting of IL-1β, IL-6, IL-12, TNF-α, IFN-γ and MMP- . In addition, it is preferable to inhibit phosphorylation of ERK1, JNK or p38 MAPK, which is an associated protein whose expression increases upon inflammatory reaction, but it is not limited thereto.

Specifically, "3,6-DHF" of the present invention acts as an inhibitor against β-ketoacyl acyl carrier protein synthase III (mtKASIII) of Mycobacterium tuberculosis, and thus can exhibit antibacterial activity against Mycobacterium tuberculosis. In connection with the mtKASIII, 3,6-DHF penetrates into the membrane protein due to its hydrophobic nature and binds to the active site with high affinity to mtKASIII, thereby inhibiting the enzyme activity, thereby enabling effective anti-tubercular activity.

In a specific example, the inventors of the present invention have found that 3,6-DHF has an anti-tuberculosis effect. As a result, it has been found that 3,7-DHF is effective against mycobacterium tuberculosis (Mycobacterium tuberculosis) H ₃₇ R _v , multidrug-resistant mycobacteria, , 6-DHF was superior to 3,6,3 ', 4'-tetrahydroxyflavone, which is a control group, in antitubercular activity and inhibited the growth of the strain.

In order to confirm the effect of inflammation caused by Mycobacterium tuberculosis, the present inventors examined the level of pro-inflammatory cytokine in LPS-stimulated human lung fibroblast MRC-5 pretreated with 3,6-DHF DHF was found to decrease mRNA expression or protein expression levels of IL-1β, IL-6, IL-12, TNF-α and INF-γ or MMP-1, 6-DHF was found to have an anti-inflammatory effect.

In addition, the present inventors confirmed that 3,6-DHF suppresses the phosphorylation of ERK-1, JNK and p38 in the MAPK family up-regulated by Mycobacterium tuberculosis. As a result, 6-DHF inhibited inflammation in the lungs.

Therefore, the 3,6-DHF of the present invention exhibits an anti-tuberculosis effect and inhibits the level of inflammatory cytokines and MMP-1 expression, ERK1, JNK and p38 MAPK phosphorylation, thereby inhibiting 3,6-DHF against tuberculosis And can be usefully used as an effective ingredient of the composition.

The 3,6-DHF of the present invention can be used in the form of a pharmaceutically acceptable salt, and the acid addition salt formed by a pharmaceutically acceptable free acid is useful as a salt. 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, Dioleate, aromatic acid, aliphatic and aromatic sulfonic acids. 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 invention can be prepared by a conventional method, for example by dissolving the 3,6-DHF in an excess of the acid aqueous solution and treating the salt with a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile ≪ / RTI > By heating an equal amount of 3,6-DHF and an acid or alcohol in water, followed by evaporating and drying the mixture, or by suction filtration of the precipitated salt.

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 3,6-DHF in an excess of an alkali metal hydroxide or 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. In addition, the corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable salt (for example, silver nitrate).

In addition, the 3,6-DHF of the present invention includes not only pharmaceutically acceptable salts, but also all salts, hydrates and solvates which can be prepared by conventional methods.

The addition salt according to the present invention can be prepared by a conventional method, for example, by dissolving the above 3,6-DHF in an organic solvent such as acetone, methanol, ethanol, acetonitrile, etc., And then precipitating or crystallizing the acid solution. Subsequently, in this mixture, a solvent or an excess acid is evaporated and dried to obtain an additional salt, or the precipitated salt may be produced by suction filtration.

When the composition of the present invention is used as a medicine, the pharmaceutical composition containing the 3,6-DHF of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient can be administered orally or parenterally , And the like, but the present invention is not limited thereto.

Examples of the formulations for oral administration include tablets, pills, light / soft capsules, liquids, suspensions, emulsions, syrups, granules and elixirs. These formulations may contain, in addition to the active ingredient, a diluent (e.g., lactose, dextrose, (E.g., silica, talc, stearic acid and magnesium or calcium salts thereof and / or polyethylene glycols), such as, for example, water, rosin, sucrose, mannitol, sorbitol, cellulose and / or glycine. The tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidine and may optionally contain additives such as starch, agar, alginic acid or its sodium salt A disintegrating or boiling mixture and / or an absorbent, a colorant, a flavoring agent, and a sweetening agent.

The pharmaceutical composition containing 3,6-DHF or a pharmaceutically acceptable salt thereof of the present invention as an active ingredient can be administered parenterally, and parenteral administration can be carried out by subcutaneous injection, intravenous injection, intramuscular injection, It depends on the injection method. In this case, in order to formulate the formulation for parenteral administration, the above-mentioned 3,6-DHF or a pharmaceutically acceptable salt thereof is mixed with water or a stabilizer or a buffer to prepare a solution or suspension, which is then administered in an ampoule or vial unit dosage form Can be manufactured. The compositions may contain sterilized and / or preservatives, stabilizers, wettable or emulsifying accelerators, adjuvants such as salts and / or buffers for the control of osmotic pressure, and other therapeutically useful substances, Or may be formulated according to the coating method.

The dose of 3,6-DHF of the present invention to the human body may vary depending on the patient's age, weight, sex, dosage form, health condition, and disease severity, and is based on an adult patient weighing 60 kg , It is generally 0.001 to 1,000 mg / day, preferably 0.01 to 500 mg / day, and may be administered once or several times a day at a predetermined time interval according to the judgment of a doctor or pharmacist.

In addition, the present invention provides a health food for preventing or improving tuberculosis comprising the above-mentioned 3,6-DHF or a pharmaceutically acceptable salt thereof as an active ingredient.

In the pharmaceutical composition of the present invention, "3,6-DHF" or a pharmaceutically acceptable salt thereof is characterized by having an antibacterial activity against Mycobacterium tuberculosis , which is Mycobacterium tuberculosis , Specifically, it is preferable that the M. tuberculosis be at least one of multi drug resistant mycobacteria or extensively drug resistant mycobacteria. However, the present invention is not limited thereto.

In the pharmaceutical composition of the present invention, "3,6-DHF" or a pharmaceutically acceptable salt thereof may exhibit anti-inflammatory activity when inflammation is induced in the lung. In connection with the anti-inflammatory activity, the level of expression of an inflammatory cytokine selected from the group consisting of IL-1β, IL-6, IL-12, TNF-α, IFN-γ and MMP- . In addition, it is preferable to inhibit phosphorylation of ERK1, JNK or p38 MAPK, which is an associated protein whose expression increases upon inflammatory reaction, but it is not limited thereto.

Specifically, "3,6-DHF" of the present invention acts as an inhibitor against β-ketoacyl acyl carrier protein synthase III (mtKASIII) of Mycobacterium tuberculosis, and thus can exhibit antibacterial activity against Mycobacterium tuberculosis. In connection with the mtKASIII, 3,6-DHF penetrates into the membrane protein due to its hydrophobic nature and binds to the active site with high affinity to mtKASIII, thereby inhibiting the enzyme activity, thereby enabling effective anti-tubercular activity.

The 3,6-DHF of the present invention exhibits an anti-tuberculosis effect and inhibits the level of inflammatory cytokines and MMP-1 expression, ERK1, JNK and p38 MAPK phosphorylation. Thus, 3,6- Can be usefully used as an effective ingredient of the present invention.

There is no particular limitation on the kind of the food. Examples of the foods to which the above substances can be added include dairy products including dairy products, meat, sausage, bread, biscuits, rice cakes, chocolate, candies, snacks, confectionery, pizza, ramen and other noodles, gums, ice cream, Beverages, alcoholic beverages and vitamin complexes, dairy products, and dairy products, all of which include health functional foods in a conventional sense.

The 3,6-DHF of the present invention or a pharmaceutically acceptable salt thereof can be added directly to the food or can be used together with other food or food ingredients, and can be suitably used according to a conventional method. 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 3,6-DHF in the health food may be 0.1 to 90 parts by weight of the total food. However, in the case of long-term intake intended for health and hygiene purposes or for the purpose of controlling health, 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 the other ingredients except that it contains the compound of the present invention as an essential ingredient in the indicated ratios and may contain various flavors or natural carbohydrates as an additional ingredient . 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, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavors (tau martin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 of the composition of the present invention.

In addition to the foregoing, the 3,6-DHF or its pharmaceutically acceptable salt of the present invention can be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, , Chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols and carbonating agents used in carbonated drinks. In addition, the 3,6-DHF of the present invention of the present invention or a pharmaceutically acceptable salt thereof may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks. 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 0.1 to about 20 parts by weight per 100 parts by weight of the 3,6-DHF or a pharmaceutically acceptable salt thereof of the present invention.

In addition, the 3,6-DHF of the present invention or a pharmaceutically acceptable salt thereof can be used as an active ingredient of a food additive.

When 3,6-DHF or a pharmaceutically acceptable salt thereof of the present invention is used as a food additive, the antibiotic peptide, or a combination of an antibiotic peptide and an antibiotic, may be directly added or used together with other food ingredients, May be appropriately used depending on the method. The amount of the active ingredient to be mixed can be appropriately determined according to the intended use. Generally, 3,6-DHF or a pharmaceutically acceptable salt thereof of the present invention is added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, based on the raw material. However, in the case of long-term ingestion, the amount may be less than the above range, and since there is no problem in terms of stability, the active ingredient may be used in an amount in the above range.

There is no particular limitation on the kind of the food. Examples of foods to which the above substances can be added include dairy products such as meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, Alcoholic beverages, and vitamin complexes, and includes foods in a conventional sense.

In addition, the present invention provides an antimicrobial composition for M. tuberculosis comprising 3,6-DHF or a pharmaceutically acceptable salt thereof as an active ingredient.

The antimicrobial composition is characterized by having antibacterial activity against Mycobacterium tuberculosis , which is a Mycobacterium tuberculosis , unless specifically limited. Specifically, the Mycobacterium tuberculosis has a multi-drug resistance more preferably at least one of multi drug resistant mycobacteria or extensively drug resistant mycobacteria.

The antimicrobial composition may be any one selected from the group consisting of a pharmaceutical composition, a food additive, a feed additive, a nasal composition, and a quasi-drug composition.

When the 3,6-DHF of the present invention or a pharmaceutically acceptable salt thereof is used as an active ingredient of the composition for nonspecific use, the composition for nonspecifically applicable to the composition of the present invention includes a food preservative, a cosmetic preservative or a pharmaceutical preservative. Preservatives, cosmetic preservatives, and pharmaceutical preservatives of foods mentioned above are additives used to prevent deterioration, decay, discoloration and chemical change of medicines, including antiseptics and antioxidants, which inhibit the growth of microorganisms such as bacteria, fungi and yeast, And functional antibiotics such as inhibiting the growth of microorganisms or sterilizing microorganisms in pharmaceuticals. Ideal conditions for such preservative compositions should be non-toxic and effective in trace amounts.

When 3,6-DHF or a pharmaceutically acceptable salt thereof of the present invention is used as an active ingredient of the composition for nausea, 3,6-DHF or a pharmaceutically acceptable salt thereof is directly added, or another quasi-drug or quasi-drug ingredient And can be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be appropriately determined depending on the purpose of use.

The quasi-drug composition of the present invention may be a disinfectant cleaner, a shower foam, a gagrin, a wet tissue, a detergent soap, a hand wash, a humidifier filler, a mask, an ointment agent, a patch or a filter filler.

Hereinafter, the present invention will be described in more detail with reference to examples and preparation examples. It should be apparent to those skilled in the art that these examples and preparations are only for illustrating the present invention and that the scope of the present invention is not construed as being limited by these examples and preparations .

Identification of anti-tuberculosis activity of 3,6-dihydroxyflavone (3,6-DHF)

In order to confirm the antituberculous effect of 3,6-DHF against Mycobacterium tuberculosis , mycobacterium tuberculosis , multi-drug resistant (MDR) Mycobacterium tuberculosis and extensively drug resistant, The anti-TB activity of 3,6-DHF was measured using Mycobacterium tuberculosis.

Specifically, M. tuberculosis H ₃₇ Rv was used as a laboratory standard strain purchased from ATCC (American Type Culture Collection, strain # 27289), and MDR and XDR tuberculosis strains were cultured in a tuberculosis resource bank Were used. MDR Mycobacterium tuberculosis strains are clinical isolates obtained from patients with multidrug - resistant tuberculosis. XDR tuberculosis strains are clinical isolates obtained from patients with broad - spectrum tuberculosis.

Mycobacterium tube suberic particulate tuberculosis H ₃₇ Rv strain (P887), and MDR TB state (M22, M23) and XDR TB state (X24, X59) for including 500 ㎕ the 166 ㎕ stock solution of clinical isolates at 37 ℃ Of 0.5% liquid agarose to prepare a mixture. The mixture was injected into 10 ml injection wells to form microfluid agarose channels surrounding the liquid medium loading wells. After the gel was solidified at room temperature, the strain was fixed. Then, 3,6-DHF (Formula 1) or 3,6,3 ', 4'-tetrahydroxyflavone (3,6,3', 4'-tetrahydroxyflavone) Was added to the medium in a concentration gradient from 0 to 200 [mu] g / ml. The 3,6-DHF and culture medium were dispersed into microfluid agarose channels. The 96 well microfluidic agarose channel chip was incubated at 37 ° C while the boundary between the liquid medium loading wells and the microfluidic agarose channels was incubated at 1, 3, 5, 7, and 7 using an inverted microscope equipped with a 4x magnification lens , And 9 days to confirm the growth of the strain. Dark gray spots of Mycobacterium tuberculosis were converted to white binary digital information by image processing in a time lapse image. Drug susceptibility was assessed by measuring the area surrounded by mycobacteria and MIC ₉₀ values were calculated. The experiment was repeated three times and a statistical test was performed using GraphPad InStat software (version 3.05, GraphPad, USA) (p < 0.05).

[Chemical Formula 1]

(2)

To the results Table 1, and the 3,6-DHF is M. tuberculosis growth as shown in FIG. 1 as the lowest concentration (MIC ₉₀ values) for inhibiting 90%, with respect to the H ₃₇ Rv, MDR and XDR, respectively 25, 100 and 100 [mu] g / ml (Figs. 1A to 1C). In contrast, in the case of 3,6,3 ', 4'-THF used as a control group, it was confirmed that it did not exhibit antibacterial activity against Mycobacterium tuberculosis even at a concentration of 200 μg / ml (FIG. 1d to FIG. 1f).

Since 3,6,3 ', 4'-THF has two more hydroxyl groups (-OH groups) than 3,6-DHF, the level of hydrophilicity is higher than that of 3,6-DHF. Since 3,6,3 ', 4'-THF binds to membranes through the cell membrane of Mycobacterium tuberculosis, 3,6-DHF does not have significant antimicrobial activity, whereas 3,6,3', 4'- It can be expected that it can play an important role in showing antimicrobial activity against the anti-tuberculosis bacteria by moving into the membrane of the Mycobacterium tuberculosis because it can exhibit hydrophobic properties compared to THF. Thus, 3,6-DHF was expected to be able to prevent and / or treat tuberculosis.

Antibacterial activity of 3,6-DHF and 3,6,3 ', 4'-THF on various mycobacteria Strain name Strain type MIC90 ([mu] g / ml) 3,6-DHF 3,6,3 ', 4'-THF P887 H37Rv   25 > 200 M22 MDR 100 > 200 M23 MDR 100 > 200 X24 XDR 100 > 200 X59 XDR 100 > 200

In vitro in virto ) To confirm cytotoxicity of 3,6-DHF on mammalian cells

It was confirmed that 3,6-DHF can exhibit significant antibacterial activity against Mycobacterium tuberculosis, and it was confirmed whether 3,6-DHF is cytotoxic to human cells.

Specifically, MRC-5 cells, which are human lung fibroblasts, were purchased from ATCC and used. MRC-5 cells were cultured in DMEM medium containing 10% FBS (Invitrogen, USA) and antibiotics (100 U / ml penicillin, 100 μg / ml streptomycin; Invitrogen) in a 5% CO ₂ incubator at 37 ° C . During the culture, 3,6-DHF was added to the medium at a concentration of 0 to 400 μM, followed by further incubation for 24 hours. After the completion of the culture, an MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) analysis was performed and the absorbance at 570 nm was measured. The cell viability (%) was calculated by calculating the relative absorbance value of the 3,6-DHF treated group to the untreated control as a percentage. The experiment was repeated three times and the mean value was calculated.

As a result, the survival rate of cells treated with 3,6-DHF at a concentration of 100 占 퐂 / ml was 60% as shown in Fig. The survival rate for MRC-5 cells was 91.8% at the concentration of 25 μg / ml, which is the MIC value of 3,6-DHF, for the Mtb-P887 strain identified in Example 1 above. As a result, it was confirmed that 3,6-DHF can be used as a therapeutic agent having little cytotoxicity and exhibiting anti-TB activity.

Identification of the effect of 3,6-DHF on the expression level of inflammatory cytokines in LPS-stimulated MRC-5 cells

LPS acts as endotoxin on cells and is known to induce the secretion of a variety of inflammatory cytokines. To confirm the specific mechanism by which 3,6-DHF affects the expression of inflammatory cytokines, LPS was treated to induce inflammatory responses to the cells and the level of expression of cytokines was confirmed.

<3-1> Determination of expression levels of inflammatory cytokines at the mRNA level

Specifically, MRC-5 cells were seeded in DMEM medium containing 10% FBS (Invitrogen, USA) and antibiotics (100 U / ml penicillin, 100 ug / ml streptomycin; Invitrogen) in a 5% CO ₂ incubator at 37 ° C And cultured. During the culture, cells were cultured by adding 20 μM 3,6-DHF to the medium, followed by stimulation with 20 ng / ml LPS for 3 hours. Cells were then washed twice with PBS, harvested and centrifuged at 4 ° C for 5 minutes at 1000 rpm to obtain cell pellets. The obtained cell pellet was subjected to total RNA isolation using Trizol Reagent (Invitrogen, Carlsbad, CA), and 1 μg of the separated RNA was reacted with reverse transcriptase to synthesize cDNA. Then, RT-PCR was performed using the primers shown in Table 2 below. PCR was carried out under the following conditions: 5 min at 94 ° C, 30 cycles of 1 min each at 94 ° C, 57 ° C and 72 ° C, and 10 min at 72 ° C for termination step. The PCR products were analyzed by electrophoresis and quantitated by using ImageJ.

Primer sequences for cDNA synthesis and RT-PCR Target sequence direction order SEQ ID NO: IL-1 Forward 5'-GAAGTACCTGAGCTCGCCAGTGAA-3 ' SEQ ID NO: 1 Reverse 5'-AGGTTCTTCTTCAAAGATGAAGGG-3 ' SEQ ID NO: 2 TNF-a Forward 5'-AGCCGCATCGCCGTCTCCTA-3 ' SEQ ID NO: 3 Reverse 5'-CAGCGCTGAGTCGGTCACCC-3 ' SEQ ID NO: 4 IL-12 Forward 5'-CCTGCTGGTGGCTGACGACAAT-3 ' SEQ ID NO: 5 Reverse 5'-CTTCAGCTGCAAGTTGTTGGGT-3 ' SEQ ID NO: 6 MMP-1 Forward 5'-ATTCTACTGATATCGGGGCTTT-3 ' SEQ ID NO: 7 Reverse 5'-ATGTCCTTGGGGTATCCGTGTAG-3 ' SEQ ID NO: 8 IL-6 Forward 5'-GTGTGAAAGCAGCAAAGAG-3 ' SEQ ID NO: 9 Reverse 5'-CTCCAAAAGACCAGTGATG-3 ' SEQ ID NO: 10 beta -actin Forward 5'-ATTGCCGACAGGATGCAGA-3 ' SEQ ID NO: 11 Reverse 5'-GAGTACTTGCGCTCAGGAGGA-3 ' SEQ ID NO: 12

As a result, mRNA expression levels of inflammatory cytokines of TNF-α, IL-6, IL-1β, IL-12 and MMP-1 are shown in FIG. (89%, 85%, 77%, 89% and 70%, respectively) in comparison with one positive control (FIG. 3). The role of TNF-α and IL-1β in the MMP-1 regulatory pathway in tuberculosis patients is well known, and these results suggest that 3,6-DHF of the present invention can be used as an anti-inflammatory agent against pulmonary inflammation caused by tuberculosis Respectively.

<3-2> Confirmation of expression level of inflammatory cytokine at the protein expression level

To determine the level of expression of inflammatory cytokines at the protein level, a sandwich-ELISA assay was performed.

Specifically, MRC-5 cells cultured under the same conditions as in Example <3-1> were treated with 3,6-DHF and stimulated with LPS to induce an inflammatory reaction. Cells were washed twice with PBS, And centrifuged at 1000 rpm at 4 ° C for 5 minutes to obtain cell pellets. The obtained cell pellet was disrupted with a dissolution buffer to obtain an eluate. Then, the eluate obtained above was added to immunoplate wells (SPL, KOREA) coated with an antibody against hTNF-α, hIL-1β and hIL-12 to bind the cytokine to the antibody. After washing, streptavidin-HRP-conjugated hTNF-α, hIL-1β and hIL-12 detection antibody were added and cultured. Finally, 3,3 ', 5,5'-tetramethylbenzidine (3,3', 5,5'-tetramethylbenzidine) was added to develop blue violet, and the absorbance at 450 nm wavelength was measured. Was measured.

As a result, as shown in Fig. 4, TNF-α, IL-1β and TNF-α were significantly reduced in MRC-5 cells stimulated with LPS when 5 and 10 μM of 3,6- And the level of protein expression of IL-12 was decreased (FIG. 4). TNF-α expression level was inhibited in cells treated with 5 μM and 10 μM of 3,6-DHF, indicating 37.6% and 60.8%, respectively, and expression levels of IL-1β were 45.9% and 67.9%, respectively (Figs. 4A and 4B). In addition, it was confirmed that expression levels of IL-12 represent 25.5% and 43.2% (Fig. 4C).

Effect of 3,6-DHF on the activity level of inflammation-related proteins in LPS-stimulated MRC-5 cells

To examine the effect of 3,6-DHF on proteins involved in inflammation in LPS-stimulated human lung fibroblasts, Western blotting of phosphorylation of LPS-induced MAPK family (ERK, JNK and p38 MAPK) Respectively.

Specifically, MRC-5 cells cultured under the same conditions as in Example <3-1> were treated with 3,6-DHF and stimulated with LPS to induce an inflammatory reaction. Cells were washed twice with PBS, And centrifuged at 1000 rpm at 4 ° C for 5 minutes to obtain cell pellets. The obtained cell pellet was disrupted with a dissolution buffer to obtain an eluate. The protein concentration in the supernatant (cytoplasmic extract) was then measured using Bradford assay (Bio-Rad, UK). 20 ㎍ of the same amount of the protein was separated using a 10% SDS-PAGE gel and transferred to a PVDF (polyvinylidene fluoride) microporous membrane (Millipore, USA). Then, the cells were blocked with TBST (25 mM Tris, 3 mM KCl, 140 mM NaCl, 0.1% Tween 20) containing 5% BSA (bovine serum albumin) at room temperature for 1 hour, And reacted at 4 ° C for a day. HRP-conjugated secondary antibody was then added to the blocked primary antibody for 2 hours at room temperature, and this was confirmed using ECL (Pierce chemical co, USA). Cell Signaling Technology, USA), anti-ERK antibody (1: 2000; Cell Signaling Technology, USA), anti-phosphorylated p38 antibody (1: 2000; Cell Signaling Technology, JNK antibody (1: 1000, Cell Signaling Technology, USA), anti-p38 antibody (1: 2000; ) And anti-beta-actin antibody (1: 5000, Sigma-Aldrich) were used. The relative density of each protein band was quantified using Image J software (NIH, Bethesda, USA).

As a result, as shown in Fig. 5, in MRC-5 cells, 3,6-DHF significantly reduced the phosphorylation level of ERK, JNK and p38 MAPK protein and stimulated with LPS and treated with 3,6-DHF The activity of the phosphorylated protein was reduced to about 67%, 56%, and 39% levels compared to the untreated control (FIGS. 5A to 5D). These results indicate that 3,6-DHF can inhibit the activity level of ERK protein most effectively and thus can induce anti-inflammatory effects in patients with tuberculosis.

Determination of the binding ability of 3,6-DHF to mtKASIII protein

β-ketoacyl acyl carrier protein synthase III (KASIII) is a protein produced by Mycobacterium tuberculosis. It is known as an enzyme that plays an important role in the process of initiating the synthesis of type 2 fatty acid, and has recently been presented as a target protein of a new antibiotic. Accordingly, the present inventors have found that 3,6-DHF, which not only exhibits antibacterial activity against Mycobacterium tuberculosis, but also has an anti-inflammatory effect, can exhibit anti-tuberculosis effect through binding to KASIII (mtKASIII) And confirmed this.

<5-1> Confirmation of affinity of 3,6-DHF for mtKASIII protein

First, we examined the affinity of 3,6-DHF for binding to mtKASIII. Specifically, expression of recombinant mtKASIII was induced. A fab H gene DNA sequence encoding the mtKASIII protein having an additional 6 × His tag at its C-terminus was inserted into the pET15b expression vector, which was then transformed into E. coli strain DH5-α. The amplified vector was transformed into E. coli BL-21 (DE3) strain and overexpressed for 16 to 25 hours in IPTG induction. Escherichia coli overexpressing mtKASIII was recovered, disrupted by ultrasonic disruption, and loaded on a HiTrap Chelating HP column to isolate and purify mtKASIII.

The binding affinity of 3,6-DHF or 3,6,3 ', 4'-THF was measured by RF5301PC spectrofluorophotometer (Kyoto, Japan) at 25 ° C in the purified mtKASIII. To this end, add 50 mM Tris-Cl (pH 8.0) containing 50 mM NaCl to a 1:10 ratio of protein and inhibitor (3,6-DHF or 3,6,3 ', 4'-THF) The compound was titrated with 3 μM mtKASIII protein solution. Protein samples were added to a 2 ml cuvette and absorbance was measured at 10 ㎚ stimulus wavelength and emission wavelength. The fluorescence quantum yield of mtKASIII was determined by ascertaining the degree of release of tryptophan residues as the concentration of the compound increased. Dissociation constants (K _d ) were measured using the difference in the intensity of the light depending on the addition of 3,6-DHF or 3,6,3 ', 4'-THF.

As a result, as shown in FIG. 6, fluorescence titration curves for confirming the affinity of 3,6-DHF or 3,6,3 ', 4'-THF to mtKASIII bind to each compound The concentration of tryptophan residues was determined according to the degree of quenching. Both 3,6-DHF and 3,6,3 ', 4'-THF strongly bind to mtKASIII and have binding affinity constants of 2.29 x 10 ⁶ M ^-1 (Fig. 6A) and 5.13 x 10 ⁶ M ^-1 (Fig. 6B). 3,6,3 ', 4'-THF has two more -OH groups compared to 3,6-DHF, but has a similar level of binding affinity. However, since 3,6-DHF exhibits a higher level of anti-tuberculosis activity than 3,6,3 ', 4'-THF, the hydrophobicity property of penetrating the TB bacteria membrane is important for anti-tuberculous activity It can be seen as a role.

<5-2> Analysis of docking model of mtKASIII and 3,6-DHF

In order to confirm more specifically the process of binding of 3,6-DHF to the active site of mtKASIII and inhibiting the activity of mtKASIII, a binding site in which 3,6-DHF binds to mtKASIII was confirmed by docking simulation. The active site of mtKASIII was expected to be where the substrate of the reporter was bound. The X-ray crystal structure of mtKASIII (2qo1.pdb) was used to predict binding of 3,6-DHF and 3,6,3 ', 4'-THF. 3,6-DHF and 3,6,3 ', 4'-THF were docked to mtKASIII on a Linux platform using CDOCKER (Biovia, San Diego, CA, USA). The shape of the final binding was reconfirmed with a minimized procedure and determined as the pose with the lowest binding energy and confirmed the desired binding for protein-ligand binding interactions.

As a result, as shown in Fig. 7, the hydroxyl group of the A-ring 6 and the hydroxyl group of the C-ring 3 in the 3,6-DHF structure of the following formula 1 were reacted with the carbonyl group oxygen of Tyr304 and Gly209, respectively, in the active site of mtKASIII Hydrogen bonds (Fig. 7). The A-loop of 3,6-DHF was additionally confirmed to have hydrophobic interactions with mtKASIII, Ile189, Leu207, Val212 and Ala246. Accordingly, the present inventors have made it possible to increase the binding affinity between 3,6-DHF and mtKASIII through the hydrophobic interaction, and it is expected that the hydrophobic interaction acts as a main binding force here. Thus, it was confirmed that the 3,6-DHF of the present invention acts as an activity inhibitor for mtKASIII, blocking the initial process of synthesizing the second type fatty acid, and thus it can be used as an effective anti-tuberculosis agent.

[Chemical Formula 1]

The preparation examples for the composition of the present invention are illustrated below.

&Lt; Preparation Example 1 > Preparation of pharmaceutical preparations

<1-1> Preparation of powder

0.1 g of 3,6-DHF of the present invention

Lactose 1.5 g

Talc 0.5 g

The above ingredients were mixed and filled in an airtight container to prepare powders.

<1-2> Preparation of tablets

0.1 g of 3,6-DHF of the present invention

Lactose 7.9 g

Crystalline cellulose 1.5 g

0.5 g of magnesium stearate

After mixing the above ingredients, tablets were prepared by direct tableting method.

&Lt; 1-3 > Preparation of capsules

0.1 g of 3,6-DHF of the present invention

Corn starch 5 g

4.9 g of carboxycellulose

After mixing the above components to prepare a powder, the powder was filled in a hard capsule according to a conventional preparation method of a capsule to prepare a capsule.

&Lt; 1-4 > Preparation of injection

0.1 g of 3,6-DHF of the present invention

Sterile sterilized water for injection

pH adjuster

(2 ml) per ampoule according to the usual injection preparation method.

<1-5> Preparation of liquid agent

0.1 g of 3,6-DHF of the present invention

10 g per isomer

5 g mannitol

Purified water quantity

Each component was dissolved in purified water in accordance with a conventional method for producing a liquid agent, and the lemon flavor was added in an appropriate amount, followed by mixing the above components. Then, purified water was added to adjust the total volume to 100 ml, and the solution was filled in a brown bottle and sterilized to prepare a liquid preparation.

&Lt; Preparation Example 2 > Preparation of health food

<2-1> Production of flour food

0.5 to 5.0 parts by weight of 3,6-DHF of the present invention was added to wheat flour, and the mixture was used to prepare bread, cake, cookies, crackers and noodles.

<2-2> Production of soups and gravies

0.1 to 5.0 parts by weight of 3,6-DHF of the present invention was added to soups and gravies to prepare health-enhancing meat products, noodles and juices.

<2-3> Preparation of ground beef

10 parts by weight of 3,6-DHF of the present invention was added to ground beef to prepare ground beef for health promotion.

<2-4> Manufacture of dairy products (dairy products)

5 to 10 parts by weight of 3,6-DHF of the present invention was added to milk, and various dairy products such as butter and ice cream were prepared using the milk.

&Lt; 2-5 >

Brown rice, barley, glutinous rice, and yulmu were alfalted by a known method, dried and dispersed, and granulated to a powder having a particle size of 60 mesh.

Black soybeans, black sesame seeds, and perilla seeds were steamed and dried by a conventional method, and then they were prepared into powder having a particle size of 60 mesh by a pulverizer.

The 3,6-DHF of the present invention was concentrated under reduced pressure in a vacuum concentrator, sprayed, and dried with a hot-air drier, and the resulting dried product was pulverized to a size of 60 mesh with a pulverizer to obtain a dried powder.

The above-prepared cereals, seeds and 3,6-DHF of the present invention were blended in the following proportions: grain (30 parts by weight of brown rice, 15 parts by weight of yulmu, 20 parts by weight of barley), seeds 7 parts by weight of black beans, 8 parts by weight of black beans, and 7 parts by weight of black sesame), 3,6-DHF (3 parts by weight), Ganoderma lucidum (0.5 parts by weight) and Rhizophoria (0.5 parts by weight).

<2-6> Production of health supplement foods

100 mg of 3,6-DHF of the present invention

Vitamin mixture quantity

70 [mu] g of vitamin A acetate

Vitamin E 1.0 mg

0.13 mg vitamin B1

0.15 mg of vitamin B2

0.5 mg vitamin B6

0.2 [mu] g vitamin B12

10 mg vitamin C

Biotin 10 μg

Nicotinic acid amide 1.7 mg

50 ㎍ of folic acid

Calcium pantothenate 0.5 mg

Mineral mixture quantity

1.75 mg of ferrous sulfate

0.82 mg of zinc oxide

Magnesium carbonate 25.3 mg

15 mg of potassium phosphate monobasic

Secondary calcium phosphate 55 mg

Potassium citrate 90 mg

100 mg of calcium carbonate

24.8 mg of magnesium chloride

The composition ratio of the above-mentioned vitamin and mineral mixture is relatively mixed with a suitable ingredient for health food. However, the compounding ratio may be arbitrarily changed, and the above ingredients may be 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.

&Lt; Preparation Example 3 > Preparation of health drink

100 mg of 3,6-DHF of the present invention

Citric acid 100 mg

100 mg of oligosaccharide

2 mg of plum concentrate

100 mg taurine

Purified water was added to 500 ml

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was stirred and heated at 85 DEG C for about 1 hour. The resulting solution was filtered to obtain a sterilized 1 liter container which was sealed and sterilized, &Lt; / RTI &gt;

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.

<110> KONKUK UNIVERSITY INDUSTRIAL COOPERATION CORP <120> Pharmaceutical composition for the prevention or treatment of          tuberculosis comprising 3,6-dihydroxyflavone or          acceptable salts thereof as an active ingredient <130> 1062828 <160> 12 <170> Kopatentin 2.0 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer_IL-b1_F <400> 1 gaagtacctg agctcgccag tgaa 24 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer_IL-b1_R <400> 2 aggttcttct tcaaagatga aggg 24 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer_TNF-a_F <400> 3 agccgcatcg ccgtctccta 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer_TNF-a_R <400> 4 cagcgctgag tcggtcaccc 20 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer_IL-12_F <400> 5 cctgctggtg gctgacgaca at 22 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer_IL-12_R <400> 6 cttcagctgc aagttgttgg gt 22 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer_MMP-1_F <400> 7 attctactga tatcggggct tt 22 <210> 8 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer_MMP-1_R <400> 8 atgtccttgg ggtatccgtg tag 23 <210> 9 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer_IL-6_F <400> 9 gtgtgaaagc agcaaagag 19 <210> 10 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer_IL-6_R <400> 10 ctccaaaaga ccagtgatg 19 <210> 11 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer_b-actin_F <400> 11 attgccgaca ggatgcaga 19 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer_b-actin_R <400> 12 gagtacttgc gctcaggagg a 21

Claims (9)

A method for producing β-ketoacyl acyl (3,6-dihydroxyflavone) comprising 3,6-dihydroxyflavone (3,6-DHF) or a pharmaceutically acceptable salt thereof as an active ingredient, A pharmaceutical composition for the prevention or treatment of tuberculosis, multidrug resistant (MDR) tuberculosis and extensively drug resistant (XDR) tubercle bacilli by inhibiting the activity of carrier protein synthase III (mtKASIII)
[Chemical Formula 1]

.
2. The method of claim 1, wherein the Mycobacterium tuberculosis strain is Mycobacterium tuberculosis . 2. The method of claim 1, wherein the Mycobacterium tuberculosis is extensively drug resistant (XDR) ) &Lt; / RTI &gt; for the prevention or treatment of tuberculosis.
delete The method of claim 1, wherein the 3,6-DHF or a pharmaceutically acceptable salt thereof is selected from the group consisting of IL-1β, IL-6, IL-12, TNF-α, IFN- (MDR) tuberculosis and extensively drug resistant (XDR) tuberculosis strains, characterized in that the level of expression of a factor selected from the group consisting of tuberculosis, multidrug resistant (MDR), and extensively drug resistant A pharmaceutical composition for therapeutic use.
4. The method of claim 1, wherein the 3,6-DHF or a pharmaceutically acceptable salt thereof inhibits phosphorylation of ERK1 or p38 MAPK upon induction of inflammation in the lung. , MDR) A pharmaceutical composition for the prevention or treatment of tuberculosis of both M. tuberculosis and extensively drug resistant (XDR) M. tuberculosis strains.
A method for producing β-ketoacyl acyl (3,6-dihydroxyflavone) comprising 3,6-dihydroxyflavone (3,6-DHF) or a pharmaceutically acceptable salt thereof as an active ingredient, Drug-resistant, multi-drug resistant (MDR) tuberculosis and extensively drug resistant (XDR) tuberculosis by inhibiting the activity of carrier protein synthase III (mtKASIII)
[Chemical Formula 1]

.
7. The method of claim 6, wherein the Mycobacterium tuberculosis strain is Mycobacterium tuberculosis . 7. The method of claim 6, wherein the Mycobacterium tuberculosis is extensively drug resistant (XDR) ) Health food for the prevention or improvement of tuberculosis of all tuberculosis strains.


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