WO2013187575A1

WO2013187575A1 - Composition comprising hypothemycin as active ingredient for preventing and treating macrophage activation syndrome, hepatocirrhosis, or obesity

Info

Publication number: WO2013187575A1
Application number: PCT/KR2013/001376
Authority: WO
Inventors: 강종순; 박기환; 이창우; 김형진; 오수진; 윤지은
Original assignee: 한국생명공학연구원
Priority date: 2012-06-12
Filing date: 2013-02-21
Publication date: 2013-12-19
Also published as: KR20130138944A; KR101440488B1

Abstract

The present invention relates to a pharmaceutical composition or health food comprising hypothemycin as an active ingredient for preventing and treating macrophage activation syndrome, hepatocirrhosis, or obesity. More particularly, since hypothemycin according to the present invention inhibits the production of TNF-α and the expression of TNF-α mRNA caused by activated macrophages, hypothemycin can be effectively used as an active ingredient for a pharmaceutical composition or health food for preventing and treating macrophage activation syndrome, hepatocirrhosis, or obesity, which are diseases induced by activated macrophages.

Description

【Specification】

[Name of invention]

Macrophage activation syndrome, cirrhosis or obesity prevention composition containing hypomycin as an active ingredient

Technical Field

The present invention relates to a composition for the prevention and treatment of macrophage activation syndrome, liver cirrhosis or obesity using hypothemycin. Background Art

Diseases associated with macrophage activation include macrophage activation syndrome, cirrhosis and obesity. Specifically, macrophage activation syndrome (MAS) is a rheumatic disease that presents symptoms such as acute fever, hepatomegaly, lymphadenopathy, skin and mucous membrane bleeding, and pancytopenia. Due to the excessive activation of macrophages, improper interactions between macrophages and lymphocytes result in increased levels of various cytokines, such as TNF-α, IL-1 a, IL-Ιβ, and IL-6 produced in macrophages and T cells. Occurs. In addition, cirrhosis (liver cirrhosis) is a disease in which normal hepatocytes are replaced with scar tissue and interfere with the function of the liver, and in severe cases, liver damage is very severe and requires liver transplantation. Chronic liver damage caused by various causes commonly lead to liver fibrosis and the mechanism is as follows. Hepatic cells are damaged by various causes, and macrophage cells (Kupffer cells) are activated to secrete various cytokines, thereby activating hepatic stellate cells to generate connective tissue. If hepatic damage persists, stromal repair is performed, in which damaged hepatocytes are replaced by connective tissue, resulting in scars due to overaccumulation of connective tissue in liver tissue, and liver function deteriorated due to hepatocellular damage. Fibrosis and cirrhosis are caused (Rojkind et al., Clinical Hepatology., 1983; Rubin et al., Essential pathology., second, 339-41, 1995). Inhibiting macrophages, one of the cells that make up the immune system, has been shown to block the liver damage process caused by cirrhosis. Such studies are expected to help develop new therapies for cirrhosis that have not yet been treated.

In addition, obesity is a disease associated with macrophages. Specifically, marker expression of M2 macrophages was decreased in adipose tissues of the obese model through a high fat diet, whereas expression of Ml macrophage markers such as TNF ᅳ α was significantly increased. Thus, as obesity progresses, most cell types change from adipose tissue macrphage (M2) phenotype to ATM (Ml phenotype) when it changes from lean adipose tissue to obese adipose tissue. It was confirmed that. In addition, TNF-α secreted by Ml macrophages has an effect of inducing insulin resistance. Therefore, in addition to the increase in the number of macrophages in the course of obesity, the hypothesis that phenotype exchange between M1-M2 is important for macrophage tissue inflammation and insulin resistance has been suggested (Lumeng, CN, J. Bodzin). , and AR Saliel, J Clin Invest, 117 (1), 175-84, 2007).

As described above, studies have shown that diseases such as macrophage activation syndrome, cirrhosis of the liver, and obesity are associated with macrophage activation, but hypomycin inhibits the production of TNF-α secreted from macrophages, thereby The therapeutic effect has not been revealed to date. Therefore, the present inventors confirmed that hypothemycin extracted from a fungal species, Phoma sp., Showed an inhibitory effect on TNF-α production by activated macrophages, and was associated with TNF-a by macrophages. The present invention was completed by elucidating that it can be usefully used as a composition for treating or preventing macrophage activation syndrome, cirrhosis or obesity, which is a disease.

[Detailed description of the name] [Technical Challenges]

An object of the present invention is to provide a pharmaceutical composition for the prevention and treatment of macrophage activation syndrome, liver cirrhosis or obesity containing hypothemycin as an effective phase.

It is an object of the present invention to provide a food composition for preventing and improving macrophage activation syndrome, liver cirrhosis or obesity, including hypothecin as an active ingredient.

Technical Solution

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing and treating macrophage activation syndrome comprising a hypothemycin or a pharmaceutically acceptable salt thereof as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for preventing and treating cirrhosis of the liver, including hypomycin or a pharmaceutically acceptable salt thereof as an active ingredient. In addition, the present invention provides a pharmaceutical composition for the prevention and treatment of obesity, including hypomycin or a pharmaceutically acceptable salt thereof as an active ingredient.

The present invention also provides a food composition for preventing and ameliorating macrophage activation syndrome, including hypomycin or a pharmaceutically acceptable salt thereof as an active ingredient.

In another aspect, the present invention provides a food composition for preventing and improving liver cirrhosis comprising hypothemycin or a pharmaceutically acceptable salt thereof as an active ingredient.

In addition, the present invention provides a food composition for preventing and improving obesity, including hypomycin or a pharmaceutically acceptable salt thereof as an active ingredient.

The present invention also provides a method of treating macrophage activation syndrome comprising administering to a subject suffering from macrophage activation syndrome an effective amount of hypothecin or a pharmaceutically acceptable salt thereof.

The present invention also provides a method of treating macrophage activation syndrome comprising administering to a subject an effective amount of hypothemycin or a pharmaceutically acceptable salt thereof. Provide preventive measures.

The present invention also provides a method of treating cirrhosis, comprising administering to a subject suffering from cirrhosis an effective amount of hypothecin or a pharmaceutically acceptable salt thereof.

The present invention also provides a method for preventing cirrhosis, comprising administering to a subject an effective amount of hypomycin or a pharmaceutically acceptable salt thereof. _,

The present invention also provides a method of treating obesity comprising administering to a subject suffering from obesity an effective amount of hypothemycin or a pharmaceutically acceptable salt thereof.

The present invention also provides a method of preventing obesity, comprising administering to a subject an effective amount of hypothecin or a pharmaceutically acceptable salt thereof.

The present invention also provides a composition comprising hypothecin or a pharmaceutically acceptable salt thereof for use in the prevention, amelioration or treatment of macrophage activation syndrome.

The present invention also provides compositions comprising hypothemycin or a pharmaceutically acceptable salt thereof for use in the prevention, amelioration or treatment of cirrhosis.

The present invention also provides compositions comprising hypothemycin or a pharmaceutically acceptable salt thereof for use in the prevention, amelioration or treatment of obesity.

The present invention also provides the use of an effective amount of hypothemycin or a pharmaceutically acceptable salt thereof for the preparation of a composition for the prevention, amelioration or treatment of macrophage activation syndrome.

The present invention also provides the use of an effective amount of hypothemycin or a pharmaceutically acceptable salt thereof for the preparation of a composition for the prevention, amelioration or treatment of cirrhosis. In addition, the present invention provides a use for the use of an effective amount of hypothecin or a pharmaceutically acceptable salt thereof in the preparation of a composition for the prevention, amelioration or treatment of obesity. [Effective Effect].

Since pretreatment of hypothemycin of the present invention inhibits TNF ᅳ α production and TNF-α mRNA expression by activated macrophages, macrophages are diseases induced by the activated macrophages. It can be usefully used as an active ingredient of a prophylactic or therapeutic pharmaceutical composition or a prophylactic and improving food composition for any one disease selected from the group consisting of activation syndrome, cirrhosis and obesity.

[Brief Description of Drawings]

1 is a graph showing the cytotoxicity evaluation of hypothemycin.

2 is a graph showing the inhibitory effect of hypothemycin on lipopolysaccharide induced TNF-a production in RAW 264.7 cells.

Figure 3 is a graph showing the inhibitory effect of hypothemycin on lipopolysaccharide induced TNF-a mRNA expression in RAW 264.7 cells.

4 is a graph showing the effect of hypothemycin on lipopolysaccharide induced nitric oxide (NO) production in RAW 264.7 cells.

Best Mode for Implementation of the Invention

Hereinafter, the present invention will be described in detail. . The present invention provides a pharmaceutical composition for preventing and treating macrophage activation syndrome comprising hypothemycin or a pharmaceutically acceptable salt thereof as an active ingredient. In addition, the present invention provides a pharmaceutical composition for preventing and treating liver cirrhosis, comprising an active ingredient of hypothemycin or a pharmaceutically acceptable salt thereof. In addition, the present invention provides a pharmaceutical composition for the prevention and treatment of obesity, including hypothecin or a pharmaceutically acceptable salt thereof as an active ingredient. The hypomycin is a compound represented by the following Formula 1.

[Formula 1]

The general chemical names of the compounds are ((laR, 3S, 4S, 9S, 15bR, VII) -3, 4, 12—tri hydr oxy-14-me t hoxy-9-me t hy 1 -3, 4, 8, 9-tetrahydro-laH-benzo [c] oxireno [2,3-e] [l] oxacyclotetradecine-5, 11 (2H, 1 5bH) -dione).

The hypothemycin compounds according to the present invention can be used in the form of pharmaceutically acceptable salts, and acid addition salts formed by pharmaceutically acceptable free acid are useful as salts. Acid addition salts include inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, aliphatic mono and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and alkanes. Obtained from non-toxic organic acids such as dioates, aromatic acids, aliphatic and aromatic sulfonic acids. These pharmaceutically toxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate phosphate, monohydrogen phosphate, dihydrogen phosphate metaphosphate, pyrophosphate chloride, bromide and iodide fluoride. Ryde, Acetate, Propionate, Decanoate, Caprylate Acrylate, Formate, Isobutyrate, Caprate, Heptanoate Propiolate, oxalate, malonate, succinate, suverate, sebacate, fumarate, maleate, butyne-1,4-dioate, nucleic acid-1,6-dioate benzoate, chlorobenzoate, Methylbenzoate, dinitro benzoate, hydroxybenzoate, hydroxybenzoate, phthalate, terephthalate benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenyl Butyrate, citrate, lactate, β-hydroxybutyrate, glycolate, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate or mandelate .

The acid addition salts according to the invention can be dissolved in conventional methods, for example, by dissolving hypothemycin compounds in an excess of aqueous acid solution, and using these salts with a miscible organic solvent, such as methane, using ethanol, acetone or acetonitrile. It can be prepared by precipitation. The mixture may also be prepared by evaporating a solvent or excess acid, evaporating and drying, or by suction filtration of the precipitated salt.

Bases can also be used to make pharmaceutically acceptable metal salts. Alkali metal or alkaline earth metal salts are obtained, for example, by dissolving the compound in an excess of 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 pharmaceutically suitable to prepare sodium, potassium or calcium salt as the metal salt. Corresponding silver salts are also obtained by reacting an alkali or alkaline earth metal salt with a suitable silver salt (eg silver nitrate).

The hypothemycin compounds according to the invention include not only their pharmaceutically acceptable salts, but also their isomers or possible solvates or hydrates which can be prepared therefrom.

In addition, the hypomycin compound according to the present invention may be commercially available or synthesized using conventional synthetic methods known in the art of organic synthesis.

The hypomycin may inhibit TNF-α production by activated macrophages. Can be.

The hypomycin may inhibit TNF-α mRNA expression by activated macrophages.

Wherein by the activated macrophage TNF-a produced, and hypo theme who is capable of inhibiting the TNF-a mRNA expression was 1 μΜ to 100 μΜ concentration is preferably, 1 _μ Μ to 50 μΜ more preferably 3 μΜ to 50 y Μ is most preferred but not limited thereto.

The activated macrophages may be activated by lipopolysaccharide (LPS), IFN-γ, IL-4 or GM-CSF, and specifically may be activated by lipopolysaccharide, It is not limited to this.

In a specific embodiment of the present invention, the pretreated macrophage RAW 264.7 (TIB-71, ATCC) cells with hypothemycin at concentrations ranging from 3, 10, 30, 100 and 300 nM for 1 hour. After culturing with lipopolysaccharide and measuring the cytotoxicity using cell proliferation kit II (Roche Applied Science), it was confirmed that hypothemycin did not cause special cytotoxicity until 100 nM (FIG. 1). .

The RAW 264.7 cells were pretreated with hypothemycin 3 10 and 30 nM for 1 hour and then treated with lipopolysaccharide (LPS) to supernatant the degree of TNF-α using ELISA kit (R & D systems INC J). As a result, it was confirmed that hypothemycin statistically significantly inhibited the production of TNF-α at 3 μΜ, 10 μΜ and 30 yM (FIG. 2).

After culturing RAW 264.7 cells, pretreatment with hypothemycin and RNA extracted from lipopolysaccharide-treated RAW 264.7 cells confirmed the mRNA expression level using qRT-PCR method, and TNF-α at 10 μΜ and 30 μΜ. It was confirmed that the expression of mRNA was statistically significantly suppressed (FIG. 3).

In addition, in the case of RAW 264.7 cells cultured with liposome 264.7 cells, pretreatment with hypothemycin and lipopolysaccharide, TNF-a It was confirmed that NO production was inhibited up to 30 μΜ (FIG. 4).

Therefore, pretreatment of hypothemycin of the present invention inhibits TNF-α production and TNF-a mRNA expression by activated macrophages, so macrophage activation syndrome, a disease induced by hypothecin-activated macrophages, It can be usefully used as an active ingredient of a prophylactic and therapeutic pharmaceutical composition for preventing or treating a disease selected from the group consisting of cirrhosis and obesity. Hypomycin of the present invention can be administered parenterally during clinical administration and can be used in the form of general pharmaceutical preparations.

Hypomycin of the present invention can be administered in a variety of actual parenteral formulations, and when formulated, it is formulated using a diluent or excipient such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc., which are commonly used. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspending solvent, propylene glycol, polyethylene glycol, injectable ester such as vegetable oil ethyl oleate, such as olive oil, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, uririnji, glycerogelatin and the like can be used.

Hypothecins of the present invention can be used in combination with a variety of carriers accepted as pharmaceuticals, such as saline or organic solvents, and carbohydrates such as glucose, sucrose or dextran to increase stability or absorption. Antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers can be used as medicaments.

The effective dose of hypomycin of the present invention is 0.0001 to 100 mg / kg, preferably 0.01 to 10 mg / kg, and may be administered once to three times a day. In the pharmaceutical composition of the present invention, the total effective amount of the hypomycin of the present invention is A bolus form black may be administered to a patient in a single dose by infusion or the like for a relatively short period of time, and the fractional treatment method in which multiple doses are administered for a long time by a treatment protocol). Since the concentration is determined in consideration of various factors such as the age and health status of the patient as well as the route and frequency of treatment of the drug, in view of this point, the present invention can be used by those skilled in the art. Appropriate effective dosages for the particular use of hypothemycin as pharmaceutical compositions may be determined. In another aspect, the present invention provides a food composition for preventing and improving macrophage activation syndrome comprising hypomycin or a pharmaceutically acceptable salt thereof as an active ingredient.

In addition, the present invention provides a food composition for preventing and improving liver cirrhosis comprising hypothemycin or a pharmaceutically acceptable salt thereof as an active ingredient.

In addition, the present invention provides a food composition for preventing and improving obesity, including hypothecin or a pharmaceutically acceptable salt thereof as an active ingredient. The food composition may be a health food, but is not limited thereto.

The hypomycin is a compound represented by the following Formula 1.

[Formula 1]

The general chemical name of the compound is ((laR, 3S, 4S, 9S, 15bR, Z) -3,4, 12-tr i hydr oxy-14-me t hoxy-9-me t hy 1 -3, 4, 8,9-tetr ahydro-laH-benzo [c] oxi r eno [2, 3-e] [1] oxacyc lotetr adec i ne_5, 11 (2H, 1 5bH) -dione).

There is no particular limitation on the kind of the food composition. Examples of foods to which the substance may be added include dairy products, including soups, meats, sausages, breads, biscuits, rice cakes, chocolates, candy, snacks, sweets, pizza, ramen, other noodles, gums, ice cream, various soups, Beverages include alcoholic beverages and vitamin complexes, and include all dietary supplements in the conventional sense.

Hypomycin of the present invention can be added to a food product as it is, or used with other foods or food ingredients, and can be suitably used according to a conventional method. The mixed amount of the active ingredient can be suitably determined according to the purpose of use (prevention or improvement). In general, the amount of hypomycin in the health functional food may be added to 0.1 to 90 parts by weight of the total food weight. However, in the case of long-term intake for health and hygiene or health control purposes, the amount may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety.

The food composition comprising the hypomycin of the present invention as an active ingredient is not particularly limited to other ingredients except the fermented product as an essential ingredient in the indicated ratio, and various flavors or natural carbohydrates, such as ordinary drinks, may be used. It may contain as an additional component. 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 sugars such as conventional sugars such as polysaccharides such as dextrin, cyclodextrin and the like, and xylyl, sorbitol and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (for example rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 g of the composition of the present invention. 1 Flavoring agents such as minerals (electrolytes), synthetic and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH adjusting agents, Stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like. In addition, the hypomycin of the present invention may contain flesh for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components can be used independently or in combination. The proportion of such additives is not so critical but the hypomycin of the present invention is generally selected in the range of 0.1 to about 20 parts by weight per 100 parts by weight. The present invention also provides a method of treating macrophage activation syndrome comprising administering an effective amount of hypomycin to a subject suffering from macrophage activation syndrome.

The present invention also provides a method of preventing macrophage activation syndrome comprising administering to a subject an effective amount of hypothecin.

The present invention also provides a method of treating cirrhosis comprising administering an effective amount of hypomycin to an individual suffering from cirrhosis.

The present invention also provides a method of preventing cirrhosis, comprising administering to the subject an effective amount of hypothecin.

The present invention also provides a method of treating obesity comprising administering an effective amount of hypomycin to an obese individual.

The present invention also provides a method for preventing obesity comprising administering to a subject an effective amount of hypothecin. The present invention also provides a composition comprising hypomycin for use in the prevention, amelioration or treatment of macrophage activation syndrome.

The present invention also provides a composition comprising hypothecin for use in the prevention, amelioration or treatment of cirrhosis of the liver. The present invention also provides compositions comprising hypothemycin for use in the prevention, amelioration or treatment of obesity.

The present invention also provides the use of an effective amount of hypothemycin for the preparation of a composition for the prevention, amelioration or treatment of macrophage activation syndrome.

The present invention also provides the use of an effective amount of hypomycin for the preparation of a composition for the prevention, improvement or treatment of cirrhosis.

In addition, the present invention provides a use for the use of an effective amount of hypothemycin in the preparation of a composition for the prevention, improvement or treatment of obesity.

Hereinafter, the present invention will be described in detail by Examples and Preparation Examples.

However, the following Examples and Preparation Examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following Examples and Preparation Examples. Example 1 Culture of RAW 264.7 Cells, a Mouse Macrophage Cell Line

In order to culture the RAW 264.7 cells were carried out as follows.

Specifically, the cell line used in the present invention is RAW 264.7 (TIB—71, ATCC) cells, which are mouse macrophage lines, and the culture of these cells is 10¾> fetal bovine serum, 100 U / ml penicillin (penici Hn). And DME medium (Dulbecco's modified Eagle's medi urn) containing 100 yg / ml of streptomycin (Invi torgen Life Tehchnologies) were used. All cells were incubated in 37 ^° C., 95% wet air / 5% CO ₂ incubator and passaged in fresh culture every 2-3 days to maintain cells.

Example 2 Cytotoxicity Evaluation of Hypotemacin

In order to evaluate the cytotoxicity of hypomycin was carried out as follows. Specifically, the RAW 264.7 cell .5 x ^'10 ^5. Dispense 200 wells into 96-well microplates (microplates) at a concentration of eel ls / ml Hypothemycin (Enzo Life Sciences, Catalog #: ALX-380-116-M001) is pretreated for 1 hour at a wide range of concentrations of 3, 10, 30, 100 and 300 nM, followed by 200 ng / ml lipopoly A saccharide (lipopolysaccharide) was treated and incubated for 24 hours. Assessment of cytotoxicity was performed using Cell Proliferation Kit II (Roche Applied Science) and 1 mg / ml sodium

3 '— [1_ (phenyl ami nocarbonyl) -3,4-tetrazol ium] -bis (4-methoxy-6-nitro) benzene sulfonic acid hydrate and 0.0383 mg / ml N ^to me t hy 1 di benzopy r az Dilute i me methyl sulfate to 50: 1 to make an XTT labeling complex. After removing 50 from the culture solution, 50 XTT-labeled complexes were added to each well and left for 1 hour, and then absorbance was measured at 490 nm (reference wavelength, 650 通). As a result of confirming the degree of cytotoxicity induced by hypomycin, it was confirmed that up to 100 nM of hypothemycin did not cause special cytotoxicity (FIG. 1).

Example 3 Measurement of Inhibitory Effect of Hypothemycin on Lipopolysaccharide-induced TNF-α Production

To determine the inhibitory effect of hypothecin on TNF-a production by activated macrophages, it was performed as follows.

Specifically, RAW 264.7 cells cultured from Example 1 were dispensed into 200 wells in 96-well microplates at a concentration of 5 x 10 ⁵ cells / ml, and after stabilizing hypothecin 3, 10 and 30 nM 1 After pretreatment for 6 hours, lipopolysaccharide (LPS) was treated for 6 hours, and the supernatant was used for TNF-α measurement. The measurement of TNF-a was performed according to the manufacturer's recommended method using an ELISA kit (R & D systems INC.). Hypothemycin was purchased from Enzo Life Sciences (Catalogue #: ALX-380-116-M001) and used.

As a result, the production of TNF-α was significantly increased when RAW 264.7 cells were activated. On the other hand, pretreatment with hypothemycin significantly inhibited the production of TNF-α at concentrations of 3 μΜ, 10 μΜ and 30 μΜ that did not induce cytotoxicity (FIG. 2). Example 4 Determination of Hypothemycin Inhibition of Lipopolysaccharide-Induced TNF-α mRNA Expression

To determine the effect of hypomycin on TNF-a mRNA expression by activated macrophages was performed as follows.

Specifically, RAW 264.7 cells were cultured at a concentration of 5 X 10 ⁵ eel ls / ml in 6-well tissue culture plates for RNA acquisition, and the experiments were performed according to the experimental conditions. After incubation, the cells were washed with PBS stored at 4 ^° C, and the cells were collected using a scraper. The supernatant was discarded by centrifugation at # 1,200 rpm and 4 ^° C for 2 minutes to discard the pellet. Got it. Total RNA was extracted using the RNeasy Plus Mini Kit (Qiagen, Valencia, Calif.) According to the manufacturer-provided assay. RNA dissolved in DEPC-DW was measured for absorbance using an A260 / 280 ratio (Versa Max microplate reader, Molecular Devices) to confirm purity and concentration. QRT-PCR was used to confirm the expression level of the transcribed mRNA. After synthesizing the same amount of RNA into cDNA using oligo (dt) i ₅ primer, experiment using the Power SYBR Green PCR Master Mix (Applied Biosystems) reagent and 7500 Fast Real Time PCR System (Applied Biosystems) PCR instrument It was. For gene amplification, the samples were heated ^at 50 ^° C. for 20 seconds at 95 ^° C. for 10 minutes and repeated 45 times at 95 ^° C. for 15 seconds at 56 ^° C. for 30 seconds at 72 ^° C. for 1 minute. The results were expressed as the amplification ratio of the experimental group to naive using the ACt method. The sequence of the primer used for the experiment is as follows. Mouse TNF-α: sense sequence 5'-CCT GTA GCC CAC GTC GTA GC-3 ', antisense sequence 5'-TTG ACC TCA GCG CTG AGT TG-3; Mouse β—act in: sense sequence 5′-TGG AAT CCT GTGGCA TCC ATG AAA C-3 ′, antisense sequence 5′-TAA AAC GCA GCT CAG ™ CAG TCC G-3 ′.

As a result, the expression of TNF-α mRNA was significantly increased when RAW 264.7 cells were activated. On the other hand, pretreatment with hypothemycin significantly inhibited the expression of TNF-a mR A at concentrations of 10 μΜ and 30 iiM that did not cause cytotoxicity (FIG. 3). Example 5 Determination of Effect of Hypothemycin on Lipopolysaccharide-Induced Nitric Oxide (NO) Production

To determine the effect of hypothemycin on lipopolysaccharide-induced nitric oxide (NO) production, it was carried out as follows.

Specifically, RAW 264.7 cells were dispensed at 200 ^ / well in 96-well microplates at a concentration of 5 x 10 ⁵ eel Is / ml, and after stabilizing hypothemycin for 3 hours at 3, 10 and 30 nM for 1 hour Polysaccharides were treated for 24 hours. A new 96-well microplate was dispensed with 50 medium and the same amount of grease reagent (2% (v / v) phosphoric acid containing sulfanilamide and 0.1% naphthylethylene diamide in DW) at room temperature, followed by reaction at 540 nm. Absorbance was measured.

As a result, when RAW 264.7 cells were activated, N0 production was greatly increased as in TNF-α, but pretreatment with hypothecin did not inhibit NO production up to 30 μΜ unlike TNF-α. It was confirmed (FIG. 4). The preparation examples for the compositions of the present invention are illustrated below.

Preparation Example 1 Preparation of Pharmaceutical Formulation

<1-1> Preparation of powder

Hypomycin 20 mg of the present invention

Lactose 20 mg

After mixing the above components, the airtight cloth was filled to prepare a powder.

<1-2> Preparation of the tablet

Hypomycin 10 mg of the invention

Lactose 100 mg

2 mg magnesium stearate

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

<1-3> Preparation of Capsule

Hypomycin 10 rag of the present invention

Crystalline celose 3 rag

Lactose 14.8 mg

Stearic acid magnes 0.2 mg

After mixing the above components, the capsule was prepared in a gelatin capsule according to the conventional method for producing a capsule.

<1-4> Preparation of Liquid

Hypomycin 20 mg of the present invention

10 g of isomerized sugar

5 g of Manni

Purified water

According to the conventional method of preparing a liquid solution, each component is added to the purified water to dissolve it, the lemon flavor is added, the above ingredients are mixed, the purified water is added, the whole is adjusted to 100 with purified water, and then filled in a brown bottle and sterilized. To prepare a liquid solution.

<1-5> Preparation of the injection

Hypomycin of the present invention 10 ug / mt dilute hydrochloric acid BP Up to 1 ml of main sodium chloride BP until pH 7.6 Hypomycin of the present invention is dissolved in a suitable volume of sodium chloride BP, and the pH of the resulting solution is adjusted to pH 7.6 using dilute hydrochloric acid BP, and the volume is adjusted and mixed well using the sodium chloride BP. It was. The solution was filled into a 5 type I ampoule of clear glass, encapsulated under an upper grid of air by dissolving the glass, and sterilized by autoclaving at 120 ^° C. for at least 15 minutes to prepare an injection solution.

<1-6> Preparation of the ring

Hypomycin of the present invention

Lactose 1.5 g

1 g of glycerin

0.5 g of Xili

After mixing the above components, according to the usual method

Prepared.

<1-7> Preparation of granules

Hypomycin 150 rag of the present invention

Soybean Extract 50 mg

Glucose 200 mg

Starch 600 rag

After mixing the components of the group Λ, 30% ethane was added to 100 mg and dried at 60 ^° C to form granules and then laminated to the fabric.

Preparation Example 2 Preparation of Food

Foods containing hypomycin of the present invention are as follows.

Prepared.

<2-1> Preparation of Flour Food 0.5 ~ 5.0 parts by weight of hypothemycin of the present invention is added to wheat flour, and the bread, cake, cookies, crackers and noodles are prepared using this combination to provide a health food for preventing and treating macrophage activation syndrome, cirrhosis and obesity. Prepared.

<2-2> Manufacture of dairy products

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

<2-3> manufacture of wire

Brown rice, barley, rice, and jujube were alphanized by a known method and then dried to be roasted, and then ground to a powder having a particle size of 60 mesh.

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

Hypomycin of the present invention was concentrated under reduced pressure in a vacuum concentrator, dried by spraying and drying with a hot air dryer, and then pulverized with a particle size of 60 mesh to obtain a dry powder.

Cereals, seeds and the dry powder of the hypothemycin of the present invention prepared in the above ratio were prepared.

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

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

Hypomycin of the present invention (3 parts by weight),

Ganoderma lucidum (0.5 parts by weight), and

Foxglove (0.5 part by weight).

<2-4> Preparation of Health Supplements

100 nig of hypomycin of the present invention

Vitamin Complex Proper

Vitamin A Acetate 70 βΕ Vitamin E 1.0 mg

Vitamin Bl 0.13 rag

Vitamin B2 0.15 rag

Vitamin B6 0.5 rag

Vitamin B12 0.2 β

Vitamin C 10 mg

Biotin 10 β

Nicotinamide 1.7 mg

Folic acid 50 ig

Pantothenic Acid Chest 0.5 mg

Mineral Mixtures Proper

Ferrous Sulfate 1.75 rag

Zinc Oxide 0.82 mg

Magnesium Carbonate 25.3 rag

15 mg potassium monophosphate

55 mg of dibasic phosphate

Potassium Citrate 90 mg

Calcium Carbonate 100 rag

Magnesium chloride 24.8 rag

The composition ratio of the vitamin and mineral mixtures described above is a relatively suitable composition for a healthy food in a preferred embodiment, but may be modified arbitrarily, the combination ratio of the above ingredients in accordance with a conventional health food manufacturing method Next, the granules may be prepared and used for preparing a health food composition according to a conventional method.

Preparation Example 3 Preparation of Health Beverage

Hypomycin 100 mg of the present invention

Citric acid 100 mg Oligosaccharide 100 nig Plum concentrate 2 nig

Taurine 100 nig

Purified water is added to the total 500 1

After mixing all the above ingredients according to the conventional healthy beverage production method, and then stirred and heated at 85 ^° C for about 1 hour, the resulting solution is filtered and obtained in one sterilized container, sealed sterilization and refrigerated storage Used to prepare the healthy beverage composition of the invention.

Although the composition ratio is a combination of relatively suitable components for a preferred beverage in a preferred embodiment, the composition ratio may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, demand country, use purpose. The present invention is not limited to the embodiments and manufacturing examples described above, and can be variously modified and changed by those skilled in the art, and can be applied to cosmetics of various uses, including other color cosmetics. It can be used in the manufacture of a medicament, that is, an ointment that can be applied thinly to the human body depending on its efficacy, which is included in the spirit and scope of the invention as defined in the appended claims. Industrial Applicability

As described above, the hypothemycin of the present invention can be used for the prophylactic treatment or improvement of any one or more diseases selected from the group consisting of macrophage activation syndrome, cirrhosis and obesity, which are diseases induced by activated macrophages. It can be usefully used as an active ingredient of a pharmaceutical composition or a health food composition for.

Claims

[Range of request]

[Claim 1]

Pharmaceutical composition for the prevention and treatment of macrophage activation syndrome comprising hypothemycin (hypothemycin) or a pharmaceutically acceptable salt thereof as an active ingredient.

[Claim 2]

The pharmaceutical composition for preventing and treating macrophage activation syndrome according to claim 1, wherein the hypomycin is represented by the following Formula 1.

[Formula 1]

[Claim 3]

The pharmaceutical composition for preventing and treating macrophage activation syndrome, according to claim 1, wherein the hypomycin inhibits TNF-α production by activated macrophages.

[Claim 4]

The pharmaceutical composition for preventing and treating macrophage activation syndrome according to claim 1, wherein the hypomycin inhibits TNF-a mRNA expression by activated macrophages.

[Claim 5] Food composition for preventing and ameliorating macrophage activation syndrome comprising hypomycin or a pharmaceutically acceptable salt thereof as an active ingredient.

[Claim 6]

A pharmaceutical composition for preventing and treating liver cirrhosis comprising hypomycin or a pharmaceutically acceptable salt thereof as an active ingredient.

[Claim 7]

A food composition for preventing and improving liver cirrhosis comprising hypomycin or a pharmaceutically acceptable salt thereof as an active ingredient.

[Claim 8]

Hypomycin or its pharmaceutically acceptable

Obesity prevention and treatment pharmaceutical composition comprising.

[Claim 9]

Hypomycin or its pharmacologically

Obesity prevention and improvement food composition comprising.

[Claim 10]

A method of preventing or treating macrophage activation syndrome, comprising administering to a subject a pharmaceutically effective amount of hypothemycin or a pharmaceutically acceptable salt thereof.

[Claim 11]

A method of preventing or treating liver cirrhosis, comprising administering to a subject a pharmaceutically effective amount of hypothemycin or a pharmaceutically acceptable salt thereof.

[Claim 12]

A method of preventing or treating obesity, comprising administering to a subject a pharmaceutically effective amount of hypothecin or a pharmaceutically acceptable salt thereof.

[Claim 13]

A composition comprising hypothecin or a pharmaceutically acceptable salt thereof for use in the prophylactic improvement or treatment of macrophage activation syndrome.

[Claim 14]

A composition comprising hypothecin or a pharmaceutically acceptable salt thereof for use in the prevention, amelioration or treatment of cirrhosis of the liver.

[Claim 15]

A composition comprising hypothecin or a pharmaceutically acceptable salt thereof for use in the prevention improvement or treatment of obesity.