WO2012128496A2

WO2012128496A2 - Pharmaceutical composition for preventing or treating asthma containing maackiain as an active ingredient

Info

Publication number: WO2012128496A2
Application number: PCT/KR2012/001810
Authority: WO
Inventors: 이형규; 안경섭; 장하영; 김세미; 오세량; 김두영; 김정희
Original assignee: 한국생명공학연구원
Priority date: 2011-03-24
Filing date: 2012-03-13
Publication date: 2012-09-27
Also published as: KR101295079B1; KR20120111756A; WO2012128496A3

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating asthma containing maackiain as an active ingredient. The maackiain of the present invention can be used to advantage as a composition for preventing, treating and alleviating asthma because it has an outstanding effect in suppressing the production of ovalbumin and in particular IgE in serum and bronchoalveolar lavage fluid and in suppressing the expression of Th2 cytokines in the lungs, in addition to suppressing the volume of active oxygen created in the respiratory tract and suppressing the infiltration of inflammatory cells into the bronchi and relieving respiratory-tract hypersensitivity which constitute problems that cause asthma in in-vivo studies.

Description

【Specification】

[Name of invention]

Pharmaceutical composition for the prevention or treatment of asthma, containing macchiin as an active ingredient

The present invention relates to a pharmaceutical composition for preventing or treating asthma, which contains macia as an active ingredient.

Background Art

Asthma is a bronchial hypersensitivity reaction to various irritants that come in when you breathe.Inflammation and swelling of the bronchial mucous membranes and the narrowing of the bronchus cause coughing and shortness of breath, accompanied by wheezing. The bronchial asthma treatment is classified into a controller and a releasing agent.

Inhaled glucocorticosteroids, anti-allergic drugs, and long acting bronchodilators have been used to prevent asthma exacerbations in the long term. Symptom relievers use bronchodilators, which are used to urgently relieve symptoms such as acute asthma attacks and bronchial infarction. In either case, there is no major problem in relieving the symptoms of mild asthma disease, but there are significant problems in treating asthma in the middle and late stages, and serious side effects and incompatibility of high-dose inhaled steroids are emerging. It is becoming. Long-term use of drugs is inevitable due to the nature of asthma, but conventional asthma treatments such as steroids and smooth muscle relaxants have many side effects such as decreased immune function, infection, and action on the heart. As there is an urgent need for development of new drugs having a therapeutic effect, there is a demand for an asthma treatment that can solve side effects of conventional drugs. Therefore, long-term or fast-acting β2-agonists or corticosteroids inhalants are currently used for alleviation of symptoms. Therefore, new drugs with fewer side effects based on new action points are needed to treat asthma. Do. Recently, as part of the fundamental allergy treatment around the world, treatment with immunomodulators has been attempted. It is key to activate the Thl immune response, which is suppressed by excessive Th2 immunity commonly seen in allergic patients, and to reduce IgE. For example, Zolare, an asthma treatment product using an anti-IgE antibody, which was commercially available in August 2003, is also known as CpG ODN (oligodeoxy-), a specific DNA of bacteria known to strongly activate Thl immune response. nucleotides are currently in clinical trials. In addition, the development of products using antibodies such as various kinds of cytokines and chemokines that are characteristic of allergic diseases (produced due to imbalance of the immune system) has been attempted. Currently, GSK's Advair / Seretide, Merck's Singulair, Boehringer Ingelheim's Spiriva, and AstraZeneca's Syrabicort are currently taking positions in blockbuster drugs.

However, sales of some drugs will likely inevitably decline due to generic competition due to patent expiration. As the market grows, competition for generic launches and new drug launches will intensify. Asthma, an allergic inflammatory disease that affects 10% of the world's population, is a representative allergic disease that continues to increase with the rate of environmental pollution. According to the National Cardiovascular Research Institute and the European Respiratory Society, annual costs for asthma are While US $ 16.1 billion is estimated at US $ 16.3 billion in the EU and long-term drug use is inevitable due to the nature of asthma, conventional asthma treatments such as steroids and smooth muscle relaxants have many side effects such as decreased immune function, infections, and effects on the heart. Since there is almost no side effect of the drug, the development of a new drug having an underlying pathological improvement or therapeutic effect is urgently required, and thus an asthma treatment agent that can solve the side effect of the conventional drug is required. There are many papers and patents showing the effects of asthma treatment. The situation is. As a conventional composition for the prevention and treatment of asthma, Korean Patent Publication No. 2008-023570 discloses asthma and allergic activity containing crude extract of Sal via miltiorrhiza BUNGE, polar solvent soluble extract or nonpolar solvent soluble extract as an active ingredient. Disease composition It discloses, and discloses a composition for preventing and treating asthma containing the Republic of Korea Patent No. 2004-097820 alpha lipoic acid as an active ingredient. In addition, Korean Patent Publication No. 20.10-062450 discloses a composition for the prevention and treatment of allergic, inflammatory or asthmatic diseases containing Kluyveromyces marxianus, which is a strain isolated from Kefir grain. Various kinds of extracts, compounds or strains have been studied to treat asthma, but there is still a need for the development of new therapeutic agents useful for the treatment of asthma. Therefore, the present inventors are working to develop a new asthma-derived therapeutic agent derived from natural products, and the macchiine compound reduces airway hypersensitivity in vivo, inhibits the production of free radicals in the airways, and invades inflammatory cells in bronchus. It was confirmed that it can be useful in the prevention or treatment of asthma by inhibiting and completed the present invention.

[Content of invention]

[Technical problem]

It is an object of the present invention to provide a pharmaceutical composition for preventing or treating asthma, which contains macchiin or a pharmaceutically acceptable salt thereof as an active ingredient.

Another object of the present invention is to provide a health food composition for preventing or improving asthma, which contains macchiin or a pharmaceutically acceptable salt thereof as an active ingredient.

Technical Solution

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention or treatment of asthma, which contains the macaine represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

[Chemistry 1]

In addition, the present invention provides a health food composition for preventing or improving asthma containing macchiine represented by the formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient. Advantageous Effects

The macchiine of the present invention not only reduces airway hypersensitivity that is a problem as a cause of asthma in vivo, inhibits the production of free radicals in the airways, and inhibits inflammatory cell infiltration in bronchus, as well as serum Egg albumin in bronchial alveolar lavage fluid, particularly IgE inhibits the production of, and the effect of inhibiting the expression of Th2 cytokines in the lungs can be useful as a composition for preventing, treating and improving asthma.

[Brief Description of Drawings]

1 is a graph showing the weight change of the mouse according to an embodiment of the present invention.

2 is a graph showing Penh values according to an embodiment of the present invention.

Figure 3 is a graph showing the concentration of IgE in serum according to an embodiment of the present invention.

Figure 4 is a graph showing the concentration of IgE in bronchoalveolar lavage fluid according to an embodiment of the present invention.

Figure 5 is a graph showing the number of inflammatory cells in the bronchoalveolar lavage fluid according to an embodiment of the present invention.

Figure 6 is a fluorescence diagram showing the expression level of Th2 cytokine-in bronchoalveolar lavage fluid according to an embodiment of the present invention.

Figure 7 is a graph showing the expression level of Th2 cytokine in bronchoalveolar lavage fluid according to an embodiment of the present invention.

8 is a graph showing the degree of generation of active oxygen according to an embodiment of the present invention.

9 is a view showing the presence of inflammation around the bronchioles and blood vessels according to an embodiment of the present invention.

Figure 10 is a graph showing the inflammation level around the bronchioles and blood vessels according to an embodiment of the present invention.

[Best form for implementation of the invention]

The present invention is a pharmaceutical composition for preventing or treating asthma containing Maackiain represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient Provide water.

[Formula 1]

Macchiin according to the present invention can be used to extract or used to manufacture, and can be used for sale, preferably can be used to isolate and identify from the roots ^ Sohpora flavescens) ^ \ but is not limited thereto. In order to investigate the effects of the macaine according to the present invention on asthma, it is widely used in the pathogenesis and efficacy of new drugs in bronchial asthma, and excellent egg reaction against egg white albumin antigens to induce egg white albumin as an antigen. One BALB / c mouse airway inflammation model was used (Herz U, et al., Immunol Cell Biol, 74, 209, 15, 1996), and the mouse asthma model was developed in two models: eosinophilic inflammation of the lung and airway hypersensitivity reaction. In some respects, there is a wide range of research on drug reactions similar to bronchial asthma in humans (Dong-Geun Lee, Pediatrics: Vol. 46, No. 10, 952-957, 2003). Among the characteristics of asthma in mice treated with machiin, the airway hyperresponsiveness, the number of inflammatory cells in bronchoalveolar lavage fluid and serum IgE antibodies were significantly decreased compared to the asthma control group. Inhibits the expression of Th2 cytokines including interleukin-4CIL-4), interleukin-6CIL-6) and interleukin (IL-13) secreted in It showed an effect of reducing infiltration. Specifically, inhalation of methacholine to determine the degree of lung capacity decreased by measuring the airway hypersensitivity to determine whether the risk of asthma by Penh level according to the increase in the concentration of methacholine, Penh level in the asthma induction group rapidly increases On the other hand, it was confirmed that the Penh level significantly decreased from 1.6905 to 2.6916 in the macaine-administered group of the present invention (see Table 1 and FIG. 2). In addition, IgE plays an important role in bronchial asthma. When allergens are sensitized Specific IgE antibodies form and attach to the surface of mast cells or basophils. When exposed again to specific allergens, these allergens bind to IgE attached to mast cells, secreting many inflammatory mediators, causing airway inflammation and bronchial contraction. Previous studies have reported that blood IgE levels correlate with the severity of bronchial asthma (Noah TL, et al., Clin I 瞧 unol 2000; 97: 3-49). It is recognized.

Therefore, as a result of measuring the concentration of IgE antibody in serum and bronchoalveolar lavage fluid according to the present invention, the serum IgE antibody concentration in the group administered the macchiine of the present invention compared to the asthma-induced group (23139.51 ± 484.45 ng /) 19102.47 ± 1383.76 ng / i significantly decreased, IgE antibody concentration in the bronchoalveolar lavage fluid was 10.31 sul 1.17 ng / in the group treated with the machiaine of the present invention compared to the asthma-induced group (19.02 ± 2.43 ng /) Significantly decreased (see Tables 2, 3 and 3, 4). In addition, recently, as a result of bronchial alveolar lavage in asthma patients, it was confirmed that lymphocytes, mast cells, eosinophils and activated macrophages were increased in bronchoalveolar lavage. Thus, asthma is commonly referred to as airway inflammatory disease, and various inflammatory cells are activated to secrete various mediators to induce asthma, and the reduction of inflammatory cells is known to be related to the treatment of asthma (Haley KJ, et al., Am J Respir Crit Care Med, 1998; 158: 565-72).

Therefore, as a result of measuring the number of inflammatory cells (macrophages, mast cells, eosinophils, lymphocytes) according to the present invention, 524.30 ± 43.22 in the asthma-induced group, 117.60 ± 14.26 in the macaine-administered group of the present invention It was found to decrease significantly.

In particular, it is a cell mainly involved in cellular-mediated immunity and spreads throughout the cells, spleen and respiratory organs, and contains inflammatory proteins, which directly damage airway epithelial cells, increase airway hypersensitivity, and The number of eosinophils known to play an important role in the incidence of asthma by causing degranulation was 71.75 에서 4.63 in the asthma-induced group, whereas it was reduced to 25.40 ± 3.65 in the macaine-administered group of the present invention (Table 4 And FIG. 5). In addition, when antigen is presented to Th2 cells by antigen-presenting cells, Th2-cells are activated, and interleukin, which is a Th2 cytokine factor—4, 5, 6, 9, 13 (IL-4, 5, 6, 9, 13), etc. Minutes In comparison, they cause allergic airway inflammation. In particular, IL-4 and IL-13 stimulate B-cells to increase production of IgE, and IL-6 is known to amplify the inflammatory response (WUls-Karp M, et al., Science 1998; 282: 2258-2261).

Therefore, as a result of analyzing the expression level of cytokines in the lungs according to the present invention, the expression of IL-4, IL-6 and IL-13 is 15.96¾ in the macaine-administered group compared to the asthma-inducing group, respectively. > 10.73% and 16.59% decreases, indicating inhibition of Th2 cytokines (see Tables 5 and 6, 7). Furthermore, as with other inflammatory diseases, oxidative stress is increased in asthma, producing free radicals in macrophages and eosinophils. Increasing oxidative stress is associated with severity and is known to further increase inflammatory reactions and reduce reaction to steroids. As a result of measuring the inhibitory effect on the generation of active oxygen according to the present invention, it was found that the group treated with macaine of the present invention was 55.99% less than the asthma-induced group (see Table 6 and FIG. 8). In addition, invasion of inflammatory cells consisting of eosinophils, neutrophils and macrophages is observed in the bronchus after antigen induction (Oh SW, et al., J, Immunol 2002; 168: 1992-2000). Thus, as a result of evaluating the degree of inflammatory cell invasion in the airway mucosa according to the present invention, the inflammation index around the bronchioles in the asthma-induced group was found to be 3.60 ± 0.22, but the group message to which the macia of the present invention was administered was 1.60 sul 0.22. It was found to decrease significantly, and that around the blood vessels was significantly decreased to 1.808 0.18 in the group treated with macaine of the present invention compared to the asthma-induced group (3.80 ± 0.18) (Table 7 and FIGS. 9 and 10). Reference). Therefore, the macchiine of the present invention can be usefully used as a pharmaceutical composition for the prevention or treatment of asthma. The present invention includes not only the machaine compound represented by Chemical Formula 1, but also pharmaceutically acceptable salts thereof, and possible solvates, hydrates, racemates, or stereoisomers which can be prepared therefrom. The macchiine compound represented by Formula 1 of the present invention may be used in the form of a pharmaceutically acceptable salt, and the salt may be a pharmaceutically acceptable free acid. acid addition salts formed by acid) are useful. 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 als. Obtained from non-toxic organic acids such as canioates, 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, bromine Rhoidide, iodide, fluoride, 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, methoxybenzoate, phthalate terephthalate, benzene Sulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, hydroxybutyrate, glycolate, malate, tartrate, methane Sulfonates, propanesulfonates, naphthalene-1-sulfonates, naphthalene-2-sulfonates or mandelate.

Acid addition salts according to the invention are dissolved in conventional methods, for example by dissolving a compound of formula 1 in an excess of aqueous acid solution. This salt can be prepared by precipitating with a water-soluble organic solvent such as methanol, ethane, acetone or acetonitrile. The same amount of machiine compound represented by the formula (1) and an aqueous acid solution or alcohol may be heated, and then the mixture is evaporated to dryness, or the precipitated salt may be prepared by suction filtration.

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 non-soluble compound salt, and evaporating and drying the filtrate. At this time, as the metal salt, it is pharmaceutically suitable to prepare sodium, potassium or calcium salts. The composition containing the macchiine compound of the present invention, but preferably containing 0.1 to 50% by weight relative to the total weight of the composition is not limited thereto. The pharmaceutical composition of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of a medicament.

The pharmaceutical compositions according to the present invention are formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories, and sterile injectable solutions, respectively, according to a conventional method. Can be used. Carriers, excipients, and diluents that may be included in the compositions of the present invention include lactose, dextrose, sucrose, sorbetle, mannitol, xili, erythritol, malty, starch, acacia rubber, alginate, gelatin Calcium phosphate, calcium silicate, salose, methyl salose, microcrystalline salose, polyvinyl pyridone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. .

In the case of formulation, it is prepared by using a diluent or excipient such as commonly used layering agents, extenders, binders, wetting agents, disintegrating agents and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like. Such solid preparations include at least one excipient such as starch, calcium carbonate, etc. in the composition of the present invention.

It is prepared by mixing (calcium carbonate), sucrose or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspending agents, liquid solutions, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories. Non-aqueous solvents and suspending agents may be used such as propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl acrylate. As a suppository base, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerin gelatin and the like can be used. The composition of the present invention can be administered orally or parenterally, any parenteral administration can be used, systemic or topical administration is possible, systemic administration is more preferred, intravenous administration is most preferred.

Preferred dosages of the compositions of the present invention vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. However, for the desired effect, the composition of the present invention is preferably administered at 0.0001 to 0.03 g / kg, preferably 0.001 to 8 mg / kg per day. Dosing is a day It may be administered once, or may be divided several times. The dosage does not limit the scope of the invention in any aspect. In addition, the present invention provides a health food composition for preventing or improving asthma, containing macchiine represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

Macchiine of the present invention represented by the formula (1) to reduce the airway hypersensitivity that is a problem as a cause of asthma in vivo in vivo, inhibit the amount of free radicals in the airway, and inhibit inflammatory cell infiltration in the bronchus In addition, it has an excellent effect of inhibiting the production of egg albumin, especially IgE, in the serum and bronchial alveolar lavage, and in the expression of Th2 cytokine in the lung, and thus can be useful as a health food composition for preventing and improving asthma ( See Tables 1-7 and Figures 1-10). Macchiin according to the present invention, for the purpose of preventing and improving asthma, the macchiin compound may be added to food supplements, such as food, beverages.

There is no particular limitation on the kind of food. Examples of foods to which the above substances can be added include drinks, meat, sausage bread, biscuits, rice cakes, chocolates, candy, snacks, confectionery, pizza, ramen, other noodles, ¾, dairy products including ice cream, various soups , Beverages, alcoholic beverages and vitamin complexes, dairy products and dairy products, and includes all dietary supplements in the usual sense.

Macchiane compound represented by the formula (1) of the present invention can be added to the food as it is, or used in conjunction with other food or food ingredients, can be suitably used in accordance with conventional methods. The combined amount of the active ingredient can be suitably determined depending on the purpose of use (prevention or improvement). In general, the amount of the compound in the health food can 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 there is no problem in terms of safety, so the active ingredient May be used in an amount above the above range.

The health functional beverage composition of the present invention is not particularly limited to other ingredients except for containing the compound as essential ingredients in the indicated ratios, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. . Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose rounds; Disacarides such as maltose, sucrose and the like; And sugars such as polysaccharides such as conventional sugars such as dextrin, cyclodextrin, and the like, and xylyl, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (tautin, stevia extract (e.g., Rebaudioside A, glycyrzin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of natural carbohydrates is generally about 1 to 20 g, preferably about 5 to 12 g per 100 compositions of the present invention.

In addition to the above, the macchiine compounds of the present invention include various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors such as flavoring agents, coloring and neutralizing agents (such as cheese, chocolate), pectic acid and salts thereof, Alginic acid and its salt organic acid, protective colloid thickener, pH adjuster, stabilizer, preservative, glycerin, alcohol, carbonation agent used for carbonated drinks, and the like. In addition, the macchiine compound of the present invention may contain fruit flesh for the production of natural fruit juices and fruit juice drinks and vegetable drinks.

These components can 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 macchiine compound isolated from the hydrated stack of the present invention. In addition, the present invention provides a method for preventing or treating asthma, which comprises administering the machiaine of Formula 1 or a pharmaceutically acceptable salt thereof to a patient in need thereof.

[Form for implementation of invention]

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

The following examples are merely illustrative of the present invention, but are not limited thereto.

Example 1 Preparation of Machine

Step 1: Preparation of Ginseng Root Extract

After cutting the roots of dried ginseng, 10 L of methyl alcohol is added to 1.0 kg After extraction at room temperature, this was repeated three times to obtain 161 g of methyl alcohol extract. Step 2: Preparation of Ginseng Root Fraction

Dispersing the methyl alcohol extract of step 1 in water, using n-nucleic acid, ethyl acetate and butanol, and sequentially fractionated n ᅳ nucleic acid fraction 34 g, ethyl acetate fraction 53 g, n-butanol fraction 54 g and 20 g of water fractions were obtained. Step 3: Isolation and Identification of Machine Compounds

14.36 g of the ethyl acetate fraction obtained in step 2 was subjected to silica gel column using 15 hexane (v / v) to l / l (v / v) of nucleic acid / chloroform (n-hexane / chloroform) as a solvent elution solvent. Chromatography was carried out, and the fraction obtained was eluted with chloroform / methyl alcohol in 20 chloro (v / v) to l / l (v / v). Compound (160 mg) represented by Formula 1 was obtained, and NMR analysis and mass analysis were performed for structural analysis. As a result, the compound represented by the following Chemical Formula 1 was identified as Maackiain (Journal of natural product-vo 1.58 (12), 1966-1969 (1995)).

[1]

Compound name ： Maackiain

Molecular Formula: C ₁₆ H ₁₂ 0 ₅

Molecular weight ： 284

NMR (300 MHz, DMSO-06) δ 7.25 (IH, d, /=8.4 Hz, Hl), 6.48 (IH, dd, J = 8.4 and 2.4 Hz, H-2), 6.96 (IH, s, H- 7), 6.51 (IH, s, H-10), 6.26 (IH, d, J = 2A Hz H-4), 5.93 (2H, d, 7 = 10.5 Hz, 0CH ₂ 0), 5.49 (IH, d , J = 6.9 Hz), 4.24 (IH, dd, J = 10.8 and 2.1 Hz, H-6 a), 3.64 (IH, t, 7 = 9.3 Hz), 3.5K1H, m, H-6a).

¹³ C NMR (300 MHz, DMS0-G6) δ 132.0 (01), 109.6 (02), 158.7 (C-3), 105.3 (C-4), 156.3 (C ᅳ 4a), 65.7 (C-6 a) , 40.4 (C-6a), 118.4 (C-6b), ^' 105.3 (O 7), 141.0 (C-8), 147.4 (C-9), 93.2 (C-10), 153.7 (C-10a), 77.9 (C-lla), 111.3 (C-llb), 101.0 (C-OCH ₂ 0).

Preparation Example 1 Induction of Experimental Animals and Bronchial Asthma

In order to investigate the effect of the macchian on asthma according to the present invention, 6-week-old Balb / c female mice with an average body weight of about 20 g were used as experimental lamps. Animals not observed were prepared.

200 μΐ, in 2-phosphate solution (pH 7.4) suspended in 2 mg of aluminum hydroxide (A8222, Sigma, St. Louis, M0) and egg white albumin 20 _yg (A5503, Sigma, St. Louis, M0) at 2 week intervals. Sensitization by injection into the abdominal cavity. After the first egg white albumin (0VA) intraperitoneal administration, 1% egg white albumin was inhaled for 30 minutes using an ultrasonic nebulizer from 28 to 30 days.

24 hours after the last antigen administration, airway hypersensitivity was measured, 48 hours after lethal dose of pentobarbital (Entoval, Hallym Pharmaceutical Co., Ltd.), the body weight was measured, and bronchial incision was performed. After washing, the samples were collected. Egg white albumin was administered as a normal control group (NC), not inhaled, egg white albumin was administered as an asthma-induced group (0VA), and bronchial asthma was induced by inhalation. Dexamethasone (dexamethasone; 3 mg / kg, P0: D4902, Sigma, St. Louis, M0) oral administration group, Montelukast (montelukast; 30 mg / kg, P0) 1 hour before egg white albumin inhalation in the control group (Monte) In the orally administered group, the experimental group (γ-mangostin) 1 hour before the egg white albumin inhalation in the group orally administered the macaine (30 mg / mi, P0) of Example 1 was carried out in five groups of each group Rats were used. The weight of the normal control group was 21.60 sul 0.25 g, but the asthma-induced group showed a significant weight loss of 19.72 ± 0.53 g, whereas the dexamethasone group (21.04 ± 0.35 g), the montelukast group (21.08 ± 0.36 g), and the macia of the present invention In the administration group (20.30 土 0.23 g), all drug administration groups were able to confirm weight gain close to the normal control group (see FIG. 1).

Experimental Example 1 Measurement of Airway Hypersensitivity

The airway hypersensitivity of asthma in Macia according to the present invention was determined using a single chamber lethysmography (All Medicus, Seoul). Airway resistance was measured and evaluated by measuring enhanced pause (Penh), a value that reflects the mathematically calculated airway obstruction. Penh was measured for 3 minutes after inhalation of PBS using ultrasonic nebulizer for 3 minutes after measuring the basal value under normal respiration. Afterwards, Penh was measured after inhalation of histamine metacholine (A2251, Sigma, St. Louis, M0), which was used in a general method of diagnosing bronchial asthma, at a concentration of 12, 25, and 50 mg. Equation 1 is shown in the following formula, and the resulting value is expressed as an increase percentage of Penh after inhaling each concentration of methacholine, and the baseline Penh (saline challenge) is expressed as 100%. The results are shown in Table 1 and FIG. 2.

[Equation 1]

expiratory time (sec)

From intake to next intake

RT ^' relaxation time

How long it takes for an exhalation to reach 30% of daily breathing

PEF ： Peak expiration flow

PIF ： Peak inspiration flow

Table 11

As shown in Table 1 and FIG. 2, in the normal control group, the increase in the penh level was increased according to the increase in the concentration of methacholine, but it was significant in the asthma induction group. The sharp increase in penh values was found.

On the other hand, the drug administration group of the Comparative Group 1, 2 and Example 1 (dexamethasone administration group montelukast administration group, macaine administration group) was significantly reduced than asthma group irrespective of the methacholine concentration, this difference was lower than the low concentration of methacholine Inhalation of high concentrations of metacholinol showed more pronounced differences.

Therefore, the macchian according to the present invention can be usefully used for the treatment of asthma because it suppresses airway hypersensitivity which is the cause of asthma.

Experimental Example 2 Measurement of IgE in Serum and Bronchial Alveolar Lavage Solution

In order to determine the effect of macchiine on asthma according to the present invention, the concentration of IgE correlated with the severity of asthma was measured by the immunoenzyme method in the serum and bronchial alveolar lavage fluid.

Serum and bronchial alveolar lavage fluid collected from each group was dissolved in 96-well folate (EUSA plate) in egg white albumin (OVA) at a concentration of 20 ug / ^ in 0.1 M NaHC0 _{3 complete} solution at pH 8.3 and at 4 ^° C. After overnight coating, the nonspecific reaction was inhibited with PBS containing 1% bovine serum albumin, and the serum samples were reacted with 1: 400 for 2 hours at room temperature. After washing well, anti-mouse IgE monoclonal antibody was repeated 300 fold for 2 hours, followed by HRP-conjugated goat anti-rat IgG polyclonal A HRP-conjugated goaf ant i -rat IG polyclonal A ) Was circulated 4000 times for 1 hour at room temperature and washed.

Color development was reflected by 3.3 '5.5'-tetramethylbenzaidine substrate, and then the absorbance at 650 nm was measured. IgE results in serum are shown in Table 2 and FIG. 3, and IgE results in bronchoalveolar lavage fluid are shown in Table 3 and FIG. 4.

Table 2

As shown in Table 2 and Figure 3, as a result of measuring the concentration of IgE antibody in the serum according to the present invention, the concentration of IgE antibody in serum compared to the asthma-induced group (0VA) (23139.51 ± 484.45 ng /) In the group administered with macchiin (Example 1), 19102.47 土 Significantly decreased to .76 ng /.

[Table 3]

In addition, as shown in Table 3 and FIG. 4, the concentration of IgE antibody in the bronchoalveolar lavage fluid was 10.31 ± 1.17 ng / m in the group treated with the macchiin of the present invention compared to the asthma-induced group (19.02 ± 2.43 ng / i). Significant decrease in £.

Therefore, the machiain according to the present invention can be usefully used in the treatment of asthma because it inhibits the concentration of IgE associated with asthma.

Experimental Example 3 Analysis of Inflammatory Cells in Bronchoalveolar Lavage Solution

In order to determine the effect of the macchian on asthma according to the present invention was measured the increase and decrease of the number of inflammatory cells associated with asthma.

Immediately after each group of bronchial alveolar lavage fluid was recovered, trypan blue was stained, and the total number of cells except dead cells was counted using a hemocytometer, followed by Cytospin Π. After smearing of the sample, Diff-Quick staining (Sysmex, Switzerland) was performed to differentially calculate eosinophils and other inflammatory cells. The results are shown in Table 4 and FIG. 5.

Table 4

As shown in Table 4 and FIG. 5, the total number of inflammatory cells was 19.24 ± 1.22 in the normal control group, 524.30 ± 43.22 in the asthma-induced group, 89.24 ± 6.98 in the dexamethasone group (Comparative Example 1), and the montelukast group (Comparative Example 2). At 167.68 ± 8.80, Macia of the invention In the group treated with phosphorus (Example 1), 117.60 ± 14.26 was significantly reduced in the drug-treated group compared to the asthma-induced group.

In addition, the number of other inflammatory cells was significantly reduced to 92.20 ± 23.86.

In particular, the number of eosinophils known to play an important role in the development of asthma, which are known to play an important role in the development of asthma, are cells that invade tissue and are involved in cellular mediated immunity, spread throughout the cell, spleen and respiratory tract, and contain inflammatory proteins. 0.00 ± 0.00, 71.75 ± 4.63 in asthma-induced group, 8.24 土 1.31 in texametasone group (Comparative Example 1), monterucaste group (Comparative Example 2), 27.88 土 2.80, Macchiin-treated group of the present invention (Example In 1), 25.40 ± 3.65 was significantly decreased in the drug-treated group compared to the asthma-induced group.

Therefore, the macchiine of the present invention may be useful for treating asthma because it not only reduces inflammatory cells associated with asthma, but also reduces the number of eosinophils, which have a great effect on asthma.

Experimental Example 4 Cytokine mRNA Analysis

In order to determine the effect of the macaine according to the present invention on asthma, the cytokine mRNA associated with asthma was measured and analyzed.

In the supernatant of bronchial alveolar lavage fluid collected from each group, the tree was extracted from the lung to extract the expression level of Th2 cytokines, interleukin-4 (IL-4), interleukin (IL-6), and interleukin (IL-13). RNA was isolated using sol (Invitrogen) and reverse transcriptase polymerase chain react ion (quantitative RT—PCR, Pr omega, WI) was performed. The inhibitory effect was measured by evaluating the relative densitometric value (RDV). The results are shown in Table 5 below and FIGS. 6 and 7.

Table 5

As shown in Table 5, the expression of Th2 cytokines IL-4, IL-6, and IL-13 in the supernatant of bronchoalveolar lavage fluid compared to asthma-induced group 1, 2 and the macaine of the present invention were compared. In the drug-administered group, all decreased, but in particular, the macaine-administered group of Example 1 showed the most excellent inhibitory effect, and 15.96%, 10.73%, and 16.59% of the asthma-induced groups, respectively.

Therefore, the macchiine of the present invention may be useful for treating asthma because it reduces the concentration of the Th2 cytokine factors IL-4, IL-6, and IL-13 associated with asthma.

Experimental Example 5 Measurement of Reactive Oxygen Species

Increased oxidative stress is known to decrease the response to steroids. In order to find out the effects of the macaine according to the present invention on asthma, as with other inflammatory diseases, the amount of active oxygen produced in asthma was measured. Some bronchial alveolar lavage fluid in each group was washed with phosphate saturated saline (PBS), followed by 2, 7-dichlorofluorescein diacetate (2, its Dichlorofluorescein diacetate; DCFH-DA, 35845, Sigma). , 'St. Louis, MO) was added to stand in the dark room for 10 minutes and measured using a spectrofluorometer (Ex = 480 nm Em = 522 nm), the results as a percentage of the normal control Table 6 And FIG. 8.

Table 6

As shown in Table 6, the amount of active oxygen production in the maciaine-administered group of Example 1 according to the present invention was reduced by 55.99% compared to the asthma-induced group, comparison group 1 (dexamethasone) 39.58% and comparison group 2 (montelukast) 45.84% It was confirmed that the inhibitory effect was higher than that of the group treated with.

Therefore, the macchiine of the present invention reduces the amount of free radicals produced in asthma. Because of its excellent effect, it can be useful for treating asthma.

Experimental Example 6 Histopathological Examination

In the bronchus from which the antigen is raised, infiltration of inflammatory cells consisting of eosinophils, neutrophils and macrophages is observed. Therefore, in order to determine the effect on the asthma of the macchia according to the present invention, the lungs which did not undergo bronchoalveolar lavage were removed and histopathological examination was performed. Extracted lungs that did not undergo bronchoalveolar lavage in each group were subjected to normal formalin fixation and paraffin embedding to make permanent tissue sections 4 μπι thick and stained with each other. After staining with Η & Ε, the salt index was measured and averaged at five sites per tissue section of each subject.

Inflammation index 0 is when no inflammatory cells are found around the bronchus. Inflammation index 1 is when inflammatory cells are observed intermittently. Inflammation index 2 is when one or two or three thin layers of inflammatory cells are observed around the bronchus. Inflammation index 3 was evaluated when five or less inflammatory cell layers were observed in two or three layers around most bronchus, and inflammation index 4 was evaluated when five or more thick inflammatory cell layers were observed around most bronchus. The results are shown in Table 7 and FIGS. 9 and 10.

[Table 7]

In the asthma-induced group according to the present invention, many inflammatory cells, including eosinophils, were infiltrated around the bronchioles, and hyperproliferated epithelial cells and thickened bronchial smooth muscle were also identified.

On the other hand, as shown in Table 7, the drug-administered group significantly reduced infiltration of inflammatory cells. As a result, the asthma-induced group had an inflammation index around the bronchioles of 3.60 ± 0.22, but the comparison group KDEXA) was 1 75. ± 0.19, Comparative Group 2 (Monte) is 2.25 ± 0.22 and the Macchiin-administered group of Example 1 of the present invention is 1.60 ± 0.22 all cause asthma It was found to decrease significantly compared to the group.

In addition, the inflammation index around the blood vessels was 3.80 土 0.18 in the asthma-induced group, the comparative group 1 is 1.50 ± 0.22, the comparative group 2 is 2.50 ± 0.25 and the macchiin-treated group of Example 1 of the present invention is 1.80 ± 0.18 All of which were significantly reduced compared to the asthma-induced group.

Therefore, the macchiine of the present invention has an excellent effect of reducing inflammatory cells composed of eosinophils, neutrophils, and macrophages in the bronchus caused by the antigen, and thus may be useful for treating asthma.

Experimental Example 7 Statistical Analysis

All measurements were expressed as mean 土 error, and the mean value and standard error (mean ± S.E.) For various variables were calculated, and the comparison between each group was performed using SPSS 10.0. Mann-whitney U) assay was performed and analyzed. Statistically, it was determined that there was a significant difference when the p value was less than 0.05. Macchian of the present invention reduces airway hypersensitivity, which is a cause of asthma, inhibits the production of free radicals in the airways, inhibits inflammatory cell infiltration in bronchus, and egg albumin in serum and bronchoalveolar lavage fluid, in particular Since the effect of inhibiting IgE production and inhibiting the expression of Th2 cytokines in the lung is excellent, it can be usefully used as a composition for preventing, treating and improving asthma.

Preparation Example 1 Preparation of Pharmaceutical Formulation

1-1. Manufacture of powder

Macchian 500 rag

Lactose 100 rag

Talc 10 rag The above ingredients are mixed and filled in an airtight cloth to prepare a powder.

1-2. Manufacture of tablets

Macchiane 500 mg

Corn starch 100 mg Lactose 100 rag

Magnesium stearate 2 rag After mixing the above components and tableting according to the manufacturing method of the conventional tablet to prepare a tablet.

1-3. Manufacture of axel

Macchiane 500 mg

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 rag According to the conventional method for producing a capsule, the above ingredients are mixed and layered in a gelatin capsule to prepare a capsule.

1-4. Preparation of Injectables

Macchian 500 rag

Appropriate sterile distilled water for injection

pH adjuster The amount of the above-mentioned ingredient is prepared per ampoule (2 mi) according to the conventional method for preparing an injection.

1-5. Preparation of liquid

Macchiin 100 mg

10 g of isomerized sugar

5 g of Manni

Appropriate amount of purified water Dissolve each of the ingredients in purified water according to the usual method of preparing liquid solution, add the lemon flavor appropriately, mix the above ingredients, add purified water, adjust the whole to purified water, and adjust the total amount to 100. Layered and sterilized to produce liquid do.

Preparation Example 2 Preparation of Health Food

Macchian 1000 rag

Vitamin Complex ^'' Proper

Vitamin A Acetate 70 (ig

Vitamin E 1.0 mg

Vitamin 0.13 rag

Vitamin B2 0.15 rag

Vitamin B6 0.5 mg

Vitamin B12 0.2

Vitamin C 10 rag

Biotin 10

Nicotinamide 1.7 rag

Folic acid 50 mg

Calcium pantothenate 0.5 rag

Mineral Mixtures Proper

Ferrous Sulfate 1.75 rag

Zinc oxide 0.82 rag

Magnesium Carbonate 25.3 mg

Potassium phosphate mono 15 rag

Dicalcium Phosphate 55 mg

Potassium citrate 90 rag

100 mg of carbonate

Magnesium Chloride 24.8 mg The composition ratio of the vitamin and mineral mixtures described above is a relatively suitable composition for the health food in a preferred embodiment, but the composition ratio may be arbitrarily modified, according to the conventional health food production method After mixing all the components of the granules, the granules may be prepared and used for preparing a health food composition according to a conventional method.

Preparation Example 3 Preparation of Healthy Drinks Macchian 1000 rag

Citric acid 1000 rag

100 g of sugar

Plum concentrate

Taurine

After adding purified water, the above ingredients were mixed according to the general method for preparing a healthy drink for 900 mi, and then stirred and heated at 85 for about 1 hour. Storage was then used to prepare a healthy beverage composition.

The composition ratio is a relatively suitable composition for the preferred beverage in a preferred embodiment, but may be arbitrarily modified according to the regional and ethnic preferences such as the demand hierarchy, the demand country, the intended use.

Preparation Example 4 Preparation of Other Health Foods

4-1. Manufacture of beverages

522 mg of honey

Chioctosanamide 5 rag

Nicotinamide 10 rag

Riboflavin Sodium Hydrochloride 3 rag

Pyridoxine hydrochloride 2 mg

30 rag Inoshi

Orthoic acid 50 mg

Macchian 0.48-1.28 rag

200 i Using the above composition and content, a beverage was prepared using a conventional method.

4-2. Preparation of Chewing Gum

Gum base 20%

Sugar 76.36-76.76%

Macchian 0.24-0.64% Fruit flavor 1 ¾

Chewing gum was prepared using a conventional method using the water composition of 2 3/4.

4-3. Candy

50-60% sugar

Starch syrup 39.26-49.66%

Macchian 0.24-0.64%

Orange Flavor 0.1% Candy was prepared using conventional methods using the composition and content.

4-4. Manufacture of Flour Food

0.5 to 5 parts by weight of macchiane was added to 100 parts by weight of flour, and the bread, cake, cookies, crackers, and noodles were prepared using the mixture to prepare foods for health promotion.

4-5. Manufacture of dairy products

5-10 parts by weight of macchiane was added to 100 parts by weight of milk, and the milk was used to prepare various dairy products such as butter and ice cream.

4-6. Manufacture of wire

Brown rice, barley, red rice, and yulmu were alphanated by a known method, and then dried and roasted to prepare a powder having a particle size of 60 mesh. Black beans, black sesame seeds, and perilla were also roasted by the known method, and then rinsed with a powder of 60 mesh in a grinder. Cereals and seeds prepared above and macchiine of the present invention were prepared by combining the following ratios. Brown Rice 30%

15% rate Barley 20% Perilla 7% Black Bean 7% Black Sesame 7% Macchiin 3% Ganoderma 0.5% Foxglove 0.5%

Claims

[Claim] [Claim 11] A pharmaceutical composition for preventing or treating asthma, which contains macchiine represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[ One]

[Claim 2]

The pharmaceutical composition of claim 1, wherein the asthma is bronchial asthma, allergic asthma, atopic asthma, nonatopic asthma, exercise-induced asthma, aspirin asthma, cardiac asthma, or alveolar asthma.

[Claim 3]

The pharmaceutical composition of claim 1, wherein the macchiane has an effect of reducing airway hypersensitivity, inhibiting the amount of reactive oxygen generation in the airways, or inhibiting inflammatory cell infiltration in the bronchus.

[Claim 4]

The pharmaceutical composition of claim 1, wherein the macchiine has an effect of reducing the number of inflammatory cells in the bronchus, reducing the concentration of IgE antibodies in the serum, or inhibiting the expression of Th2 cytokines.

[Claim 5]

A healthy food composition for preventing or improving asthma, which contains a macaine represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

[Claim 6]

The health food composition of claim 5, wherein the asthma is bronchial asthma, allergic asthma, atopic asthma, nonatopic asthma, exercise-induced asthma, aspirin asthma, cardiac asthma or alveolar asthma.

[Claim 7]

The healthy food composition according to claim 5, wherein the macchiin has an effect of alleviating airway hypersensitivity, inhibiting the amount of reactive oxygen generation in the airways, or inhibiting inflammatory cell infiltration in the bronchus.

[Claim 8]

6. The health food composition according to claim 5, wherein the macchiine has an effect of reducing the number of inflammatory cells in the bronchus, reducing the concentration of IgE antibodies in the serum, or inhibiting the expression of Th2 cytokines.

[Claim 9]

A method of preventing or treating asthma comprising administering to a patient in need thereof a macaine of Formula 1 or a pharmaceutically acceptable salt thereof.