KR20160022190A - A composition for the treatment or prevention of asthma comprising erythronium japonicum - Google Patents

Abstract

The present invention relates to a composition comprising an Erythronium japonicum extract as an active ingredient for preventing, alleviating, or treating asthma. Specifically, the composition comprising an Erythronium japonicum extract as an active ingredient for preventing or treating asthma of the present invention can be a pharmaceutical composition or a composition comprising an Erythronium japonicum extract as an active ingredient for preventing or alleviating asthma. The Erythronium japonicum extract of the present invention has excellent effects in preventing, alleviating, or treating asthma. In addition, Erythronium japonicum as a raw material of the Erythronium japonicum extract, which has been used as a food for a long period of time, is harmless to the human body, and has no side effects, thereby being efficiently used for preventing, alleviating, or treating asthma or asthma diseases.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for prevention or treatment of asthma comprising an extract from Aspergillus oryzae as an active ingredient,

The present invention relates to a composition for preventing or treating asthma comprising an effective ingredient derived from a natural product, specifically, a plant extract as an effective ingredient.

About 235 million people worldwide are suffering from asthma in 2011, and the incidence is estimated to range from 1% to 18% of the total population, although the incidence varies widely depending on the environment. In Korea, the prevalence of asthma in children has increased from 5.6% in 1981 to 10.1% in 1990, and the number of asthmatic patients is increasing in adults. In particular, the prevalence rate is 12.7% .

The asthma is a disease that repeatedly shows symptoms of paroxysmal dyspnea, and includes bronchial asthma and cardiac asthma, and narrow asthma mainly means bronchial asthma. Bronchial asthma is one of the most common diseases caused by allergies. It reacts with allergens and reagins immediately upon binding to the Fc receptor of IgE on the surface of mast cells, thereby releasing chemical substances, .

The asthma is known to be caused by various causes such as dust mites, infections caused by fungi or cockroaches, allergenic substances such as pet hair or pollutants, or smoking.

Specifically, the asthma is an immune-related disease closely related to helper T cells. The helper T cells can be divided into Th1 cell and Th2 cell depending on the type of cytokine that induces secretion. The Th1 cell mainly secretes interleukin-2 (IL-2) or interferon-gamma (IFN-γ), and the Th2 cell secretes IL-4, IL-5 and IL-13. In addition, the Th2 cell promotes the differentiation of B cells (B-cells) and the change of IgG to IgE. It has been reported that asthma is caused by destruction of the balance between Th1 cell and Th2 cell.

The asthma is an immune-related disease and can be induced or suppressed by various cytokines. More specifically, IL-6, which is known to modulate the function of CD4 T cells (CD4 T-cells), is known to modulate asthma induction. On the other hand, IL-12 regulates the balance of Th1 and Th2 cells and is known to promote the production of IFN-y, which is associated with the inhibition of asthma. In addition, IL-4 and IL-13 are known to be involved in inflammatory processes, airway hyperresponsiveness, abnormal growth of mucus-secreting goblet-cells, eosinophil infiltration, mucus hypersecretion, or B-cell activation , And is reported to be a major factor in the development of asthma. IL-5 is also involved in the differentiation, activation and migration of eosinophils and is known to be associated with asthma.

Regarding the above-mentioned asthma, an organ dilator, a corticosteroid, a leukotriene modifier, a theophylline or an anti-IgE agent has been proposed as a conventional therapeutic method. However, despite the administration of such a therapeutic agent, And some problems have been raised. For example, cortico steroid air intoxication agents widely used as therapeutic agents for asthma have been reported to have various adverse effects and their use is decreasing.

As described above, the asthma is a refractory immune disease related to the respiratory system, and various therapies and therapies for treatment are being studied, but the use thereof is limited due to problems of each therapeutic agent.

Accordingly, there is an increasing tendency to search for a substance effective for the treatment of asthma from natural materials derived from natural materials which do not cause side effects or drug resistance.

KR10-1358405B KR10-2014-0018589A KR10-1050003B KR10-2013-0048108A

It is another object of the present invention to provide a composition for treating or preventing asthma comprising a natural substance harmless to human body as an effective ingredient.

In order to accomplish the above object, the present invention provides a composition for treating or preventing asthma comprising an extract as an active ingredient. The composition for treating or preventing asthma may be a pharmaceutical composition.

The extract may be one selected from the group consisting of alcohols having 1 to 5 carbon atoms, water, and mixtures thereof. Specifically, the extract can be extracted with water.

In addition, the present invention provides a food composition for improving or preventing asthma, which comprises an extract of an insect as an active ingredient. The food composition may be a food, a food additive or a beverage, and may preferably be a functional food composition.

The extract may be one selected from the group consisting of alcohols having 1 to 5 carbon atoms, water, and mixtures thereof. Specifically, the extract can be extracted with water.

Further, the present invention provides a use of an irritant for the purpose of achieving the object, specifically, an application for treatment, improvement or prevention of asthma in an irritable lotion.

As used herein, unless otherwise specified, an active ingredient refers to a component that provides a therapeutic, ameliorative, or prophylactic effect of asthma in a composition, particularly a pharmaceutical composition or a food composition.

As used herein, unless otherwise specified, pharmacologically acceptable means physiologically acceptable and does not cause an allergic reaction such as gastrointestinal disorder, dizziness, or the like, when administered to an animal, preferably a human, do.

The inventors of the present invention have found that an anti-asthmatic effect, specifically, an ovalbumin in a BALB / c mouse (BALB / c mouse) is used as an anti-asthmatic effect in order to find a substance derived from a natural substance, (BALF) of bronchoalveolar lavage fluid (BALF) by administration of hot water extract of the asthma induced by airway hyperresponsiveness (AHR) , WBC counts and neutrophil counts (NE), the degree of IgE production associated with allergen induction, histopathological observation of lung tissue, and immunostaining, And in the search process, it is evaluated that the irritable is excellent in the treatment, improvement and prevention of asthma. From this, .

Hereinafter, the present invention will be described in detail.

The present invention relates to a composition for the treatment or prevention of asthma, which comprises an extract of Aspergillus as an active ingredient. The composition for treating or preventing asthma may be a pharmaceutical composition.

The dog-tooth violet, Erythronium japonicum ) is a perennial plant belonging to the family Liliaceae. The horseshoe larvae grow mainly in fertile soil in high areas, some in mountain valleys, and distributed in Korea and Japan. The leaves are ovate or oval, with purple pattern on green background, plain edge, long oval with lobes, 6 petals, 6 dried, purple back, 6 stamens and 1 pistil . Also, the fruit of the above-mentioned insect is deficient in July to August, and has a wide oval or spherical shape with a capsule. Although the above-mentioned horsetail is a wild flower, it has recently been spotlighted as an ornamental use and has been known to stop vomiting and diarrhea due to its function of improving gastrointestinal function in civilian medicine, and leaves have been edible as herbs.

The insect extract of the present invention can be prepared by extracting with an extraction solvent or extracting with an extraction solvent, followed by fractionation with a fraction solvent.

The insect extract may be one prepared by a conventional method for producing plant extracts. For example, an extracting solvent may be added to the leaves of flowers, leaves, branches, roots, fruits or shells thereof or crushed products thereof, Or extracting with an extraction solvent, and fractionating the crude extract with a fraction solvent.

The extraction solvent may be at least one selected from the group consisting of water and an organic solvent. The organic solvent may be a polar solvent such as an alcohol having 1 to 5 carbon atoms, a diluted alcohol such as ethyl acetate or acetone, a nonpolar solvent such as ether, chloroform, benzene, hexane or dichloromethane, or a mixture thereof. The alcohol having 1 to 5 carbon atoms may be methanol, ethanol, propanol, butanol, isopropanol, etc., but is not limited thereto. In addition, the alcohol dilution water may be an alcohol diluted with water at 50 to 99.9% (v / v). The extraction solvent may be any one selected from the group consisting of alcohols having 1 to 5 carbon atoms, water, and mixtures thereof, more preferably water.

The extract according to the present invention may be a conventional plant extract, specifically, a plant extract prepared according to the method for producing a plant extract. More specifically, the extraction can be performed by adding an extraction solvent to the irrevocably removed impurities. The extraction process may be a cold extraction method, a warm extraction method, a pressure extraction method, or an ultrasonic pulverization extraction method. The extraction process may be performed at, for example, 4 ° C to 120 ° C, or 50 ° C to 90 ° C, or 60 ° C to 80 ° C, but is not limited thereto. The extraction time is not particularly limited, but may be 10 minutes to 12 hours.

Further, the extract extracted with the solvent is then extracted with any one solvent selected from the group consisting of hexane, methylene chloride, acetone, ethyl acetate, ethyl ether, chloroform, water and mixtures thereof, preferably butanol, chloroform and ethyl acetate , More preferably one or more solvents selected from the group consisting of butanol and chloroform, and still more preferably butanol. Two or more kinds of solvents may be used for the fractionation, and the solvent extracts may be sequentially used or mixed according to the polarity of the solvent. In addition, the fractionation process may be performed at, for example, 4 ° C to 120 ° C, or 50 ° C to 90 ° C, or 60 ° C to 80 ° C, but is not limited thereto.

The thus-prepared extract or the fraction obtained by performing the fractionation process may be filtered, concentrated or dried to remove the solvent, and may be subjected to both filtration, concentration and drying. Specifically, the filtration can be performed using a filter paper or a vacuum filter, and the concentration can be reduced by using a vacuum concentrator, for example, a rotary evaporator. The drying can be performed by, for example, freeze drying. The lyophilization method may be performed using a deep freezer.

Specifically, the extract can be obtained by further performing a filtration step, a concentration step, and / or a drying step after the solvent extraction step, selectively or in duplicate. The filtration step may be performed using a filter paper, The process may be carried out through reduced pressure concentration or the like, and the drying process may be performed by a method such as hot air drying or freeze drying.

As a result of the above test, it was confirmed that the extract of the present invention suppresses the increase of leukocyte count, inhibits the production of IgE, which is a cause of allergic reaction, and observes by histopathological observation and immunohistochemistry of the lung, Inhibition of inflammatory cell infiltration and mucus secretion around bronchi and blood vessels, and suppression of tissue changes due to cytokines.

In this respect, the extract can be used for the treatment, improvement or prevention of asthma, and the extract can be used as an active ingredient or pharmacological ingredient of a composition for treating or preventing asthma, Or prevention composition or a food composition.

In the present invention, asthma refers to a disease in which the bronchus in the lung is very sensitive, sometimes the bronchus narrows, and the breathing, cramp, and breath sounds are heard and the cough is severe. It is an allergic disease caused by the reaction. These symptoms appear repeatedly or seizurefully, with genetic and environmental factors combined. The asthma may be bronchial asthma and cardiac asthma, and in a narrow sense may be bronchial asthma.

Typical symptoms of bronchial asthma include dyspnea, cough, or wheezing. These symptoms appear repeatedly or spontaneously. In actual asthma patients, in addition to the typical asthmatic symptoms described above, there are many cases of atypical symptoms. In other words, the chest tightness, chest tightness, or the throat of the throat may be appealing symptoms such as hanging. Generally, dyspnea deteriorates after taking a cold, or dyspnea and heavy breathing symptoms occur after exercise such as running. Sometimes severe asthma attacks require immediate first aid and inpatient treatment, in which the patient feels fearful of death and, in fact, severe asthma attacks are life-threatening.

The causes of asthma are genetic and environmental factors, and they are mainly caused by their interaction. Specifically, allergic inheritance inherited from parents and surrounding asthma inducing factors interact with each other, resulting in confusion in the immune system, resulting in asthma.

Factors causing asthma include causative agents and worsening factors. Such causative substances are called allergens. Representative allergens include house dust mites, pollen, animal hair, dandruff, or cockroaches. These deteriorating factors include physical activity such as cold, tobacco smoke and indoor pollution, air pollution, food additives, intense exercise, climate change, dustiness, and stress.

The treatment of asthma, especially bronchial asthma, includes the long-term use of medicines that regulate disease in order to quickly recover symptoms and prevent recurrence if symptoms are present, to find the causative agent of asthma, to minimize allergen exposure, There is a avoiding therapy to avoid.

The drug therapy includes a symptom relief agent such as a bronchodilator that alleviates the narrowed bronchus in a short time, and a disease treatment agent that treats or prevents asthma attack by inhibiting bronchial allergy inflammation. In the case of the symptom-relieving agent, the problem of the side effect is small, but the fundamental therapeutic effect can not be exerted. In the case of the disease-treating agent, side effects such as hormone preparations become a problem.

The composition for preventing or treating asthma according to the present invention may contain 0.001 to 99.99% by weight, preferably 0.1 to 99% by weight, of the extract of the present invention, relative to the total weight of the composition. And the content of the active ingredient can be appropriately controlled depending on the purpose of use.

The composition may be used in combination with nutrients, vitamins, electrolytes, flavoring agents, coloring agents, thickening agents, pectic acid and salts thereof, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, A carbonating agent, and the like. The components may be added to the composition independently or in combination. The content of the additional ingredient is preferably in the range of 0.1 to 20,000 parts by weight per 100 parts by weight of the extract.

The composition for treating or preventing asthma comprising the above extract as an active ingredient may be a pharmaceutical composition or a pharmaceutical composition for treating or preventing asthma.

In this respect, the present invention may be a medicinal composition for treating or preventing asthma having the above-mentioned insect extract as an active ingredient and having an effect of treating or preventing asthma or asthma-related diseases.

The medicinal composition for treating or preventing asthma comprising the extract of the present invention as an active ingredient may contain 0.001 to 99.99% by weight, preferably 0.1 to 99% by weight, of the extract of the present invention, based on the total weight of the composition.

The medicinal composition for treating or preventing asthma may further comprise suitable carriers, excipients and diluents conventionally used in the production thereof.

The medicinal composition for treating or preventing asthma comprising the above extract as an active ingredient may be used as an oral dosage form such as powders, granules, tablets, suspensions, emulsions and syrups according to a conventional method.

Examples of carriers, excipients, and diluents that can be included in the pharmaceutical composition for treating or preventing asthma extracts containing the above-mentioned irritant as an active ingredient include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, , Gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

When the medicinal composition for treating or preventing asthma comprising the above extract as an active ingredient is a solid preparation for oral administration, it includes tablets, pills, powders, granules, capsules and the like, The extract is prepared by mixing at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used.

When the medicinal composition for preventing or treating asthma or asthma-related diseases is a liquid preparation for oral administration, the liquid preparations include suspensions, solutions, emulsions, and syrups. Commonly used simple diluents In addition to water and liquid paraffin, various excipients such as wetting agents, sweetening agents, fragrances, preservatives and the like may be included.

The preferred dosage of the medicament for treating or preventing asthma comprising the extract as an active ingredient varies depending on the condition and body weight, degree of disease, drug form, route of administration, and duration, but may be appropriately selected by those skilled in the art. Preferably 0.0001 to 1000 mg / kg per day on the basis of the extract of the present invention, and more preferably 0.01 to 100 mg / kg per day, but the present invention is not limited thereto. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

In yet another aspect, the composition of the present invention may be a food composition containing the above-mentioned insect extract as an active ingredient and having an effect of improving or preventing asthma or asthma-related diseases.

As used herein, the term " food " means a natural product or a processed product containing one or more nutrients. Preferably, the term " food " Is intended to include foods, food additives, health functional foods and beverages.

Foods to which the extract of the present invention can be added include, for example, various foods, beverages, gums, tea, vitamin complexes, and functional foods. In addition, the food of the present invention may contain special nutritional foods (such as crude oil, infant formula, etc.), meat products, fish products, tofu, jelly, noodles (such as ramen, (Such as soy sauce, soybean paste, kochujang, and mixed potato), sauces, confectionery (eg, snacks), dairy products (eg fermented milk, cheese, etc.), other processed food, kimchi, (E.g., fruit, vegetable beverages, two oils, fermented beverages, etc.), natural seasoning (e.g., ramen soup, etc.).

The food, beverage or food additive may be prepared by a conventional production method.

In the present invention, the functional food refers to a food group which is imparted with added value to function and express the function of the food by using physical, biochemical, biotechnological techniques and the like, the regulation of the biological defense rhythm of the food composition, Means a food which is processed and designed so that the body control function regarding the body is sufficiently expressed to the living body.

The functional food may include a food-acceptable food-aid additive, and may further comprise suitable carriers, excipients and diluents conventionally used in the production of functional foods.

In the present invention, beverage is a generic term for drinking or enjoying a taste, and is intended to include functional beverages.

There is no particular limitation on the ingredients other than the above-mentioned beer extract as an active ingredient as an essential ingredient in the indicated ratios, and may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages.

Examples of such 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 extract (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 5 to 12 g per 100 ml of the composition of the present invention. In addition, the composition of the present invention may further contain flesh for the production of natural fruit juice, fruit juice drink, and vegetable drink.

In addition to the above, the composition of the present invention can be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and intermediates such as cheese and chocolate, pectic acid and its salts, Colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like.

These components can be used independently or in combination. The proportion of such additives is not so critical, but may be selected in the range of 0 to 20,000 parts by weight per 100 parts by weight of the extract of the present invention.

The functional beverage according to the present invention can be used to control the bio-defense rhythm of the beverage group or beverage composition to which the added value is imparted so that the function of the beverage acts on the specific purpose by physical, biochemical or biotechnological techniques, Means a beverage which has been designed so that the body control function related to restoration and the like is sufficiently expressed in the living body.

The above-mentioned functional beverage is not particularly limited to the other ingredients except that it contains the extract of the present invention as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages.

Examples of such 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 extract (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 may generally be about 1 g to 20 g, preferably 5 g to 12 g per 100 ml of the composition of the present invention.

In addition, in the food composition for improving or preventing asthma, the amount of the irritable extract may be 0.01 to 15% by weight of the total food, and the beverage composition may contain 0.02 g to 5 g , Preferably from 0.3 g to 1 g.

Further, the present invention relates to the use of an irritant, specifically, the use of the irritant for the treatment, improvement or prevention of asthma. The insect extract may be an insect hot water extract.

As a result of examining the asthma animal model by the test animal, the increase in leukocyte count, the histopathological observation of the lung, and the immunohistochemistry showed that the asthma extract, specifically the hot water extract, Inhibits cell infiltration and mucus secretion, and is expected to be effective for the treatment or prevention of asthma or asthma-related diseases. Thus, it is evaluated that the use for treating or preventing asthma or asthma-related diseases is evaluated to be excellent.

The extract of the present invention was found to inhibit the increase of leukocyte, especially neutrophil and eosinophil, to inhibit the production of IgE causing asthma, to inhibit the inflammatory reaction around the bronchus and the blood vessel, It is confirmed that it suppresses infiltration and mucus secretion. Since the insect which is the active ingredient is used as a food, it is expected that the insect extract prepared therefrom will not cause any side effects. Thus, it is very effective as a new preventive or therapeutic agent for asthma and the like It is believed to be highly valued.

FIG. 1A is a graph showing the number of leukocyte (WBC) cells per unit volume (x 10 ³ cells / μL), FIG. 1B is a graph showing changes in the number of cells per unit volume FIG. 1C is a graph showing the number of neutrophils (NE, Neutrophil) (x 10 ³ cells / μL). Dexamethasone is a positive control group treated with dexamethasone, and 60 mg / kg is a control group. In the control group, the control group is the control group without any treatment. Induction control means the negative control group causing asthma. The experimental group was treated with 60 mg / kg / day of insecticide extract and 600 mg / kg of insecticidal group treated with 600 mg / kg / day of insecticide.
FIG. 2 is a graph showing the amount of immunoglobulin Ig E and various cytokines produced in association with an allergic reaction according to an embodiment of the present invention. In the graph, Control represents a normal control group without any treatment, and Induc Dexa represents a positive control group treated with dexamethasone, and 60 mg / kg represents an experimental group treated with an extract of Mores (60 mg / kg / day) , And 600 mg / kg means the experimental group treated with the extract of Mores (600 mg / kg / day).
FIG. 3 is a graph showing histopathological changes of lung tissues in an animal experiment using an asthma experimental model according to an embodiment of the present invention, showing H & E (hematoxylin and eosin stain) around blood vessels of bronchi and lung tissue, (Arrow) means infiltration of inflammatory cells, Br means bronchiole, and V means vessel. As shown in FIG. In addition, N / S means a normal control (control) without any treatment, Induc. Means a negative control inducing asthma, and Dexa is a positive control group treated with dexamethasone (Dexamethasone), 60 mg / kg means 60 mg / kg / day, and 600 mg / kg means 60 mg / kg / day, respectively.
FIG. 4 is a photograph showing the pathological histological changes of pulmonary tissues in an animal experiment using an asthma experimental model according to an embodiment of the present invention, after PAS staining of blood vessels around bronchi and lung tissue, Means bronchial bronchial, and V means vessel. In addition, N / S means a normal control (control) without any treatment, Induc. Means a negative control inducing asthma, and Dexa is a positive control group treated with dexamethasone (Dexamethasone), 60 mg / kg means 60 mg / kg / day, and 600 mg / kg means 60 mg / kg / day, respectively.
FIG. 5 is a photograph of lung tissue histopathologically observed after IHC staining (IHC Stain) around blood vessels of bronchi and lung tissues in an animal experiment using an asthma experimental model according to an embodiment of the present invention. FIG. 5B is a photograph showing the result of the assay using an antibody against IL-4, FIG. 5C is a photograph showing the result obtained by using an antibody against TNF-a, FIG. 5D is a photograph showing a result obtained by using an antibody against IL-12p40, FIG. 5E is a photograph showing a result obtained by using an antibody against IL-12p35, FIG. 5F is a photograph showing an antibody against CD8 FIG. 5G is a photograph showing the result of checking using an antibody against CD19, FIG. 5H is a photograph showing the result of checking using an antibody against CD4, FIG. 5I is a photograph showing the result of confirming using IFN-r FIG. 5J is a photograph showing the result of confirming using an antibody against IL-13, FIG. 5K is a photograph showing a result of confirming using an antibody against IL-5, FIG. FIG. 5L is a photograph showing a result obtained by using an antibody against GATA-3, and FIG. 5M is a photograph showing a result obtained by using an antibody against T-bet. In the above photograph, Br denotes a bronchiole, and V denotes a vessel. In addition, N / S means a normal control (control) without any treatment, Induc. Means a negative control inducing asthma, and Dexa is a positive control group treated with dexamethasone (Dexamethasone), 60 mg / kg means 60 mg / kg / day, and 600 mg / kg means 60 mg / kg / day, respectively.

Hereinafter, preferred embodiments of the present invention will be described. The following examples are for illustrative purposes only and are not intended to limit the scope of the invention.

Example  1: Preparation of extract

Erythronium , a native plant of the Suncheon Cho Mountains in 2013, japonicum ) leaves were lyophilized at -20 ℃ and powdered. The powdered extract and fraction are prepared by immersing 300 g of the sample of the irritable powder in 3 L of an aqueous 80% methanol solution, extracting the mixture with warm-up extraction, and sequentially treating the extract with an organic solvent to prepare a fraction Respectively.

More specifically, 3 L of an aqueous 80% methanol solution was added to 300 g of each of the above-mentioned leaves, flowers, stems and roots, and the above-mentioned leaves, flowers, stems and roots were completely immersed in extraction solvents, Lt; / RTI > Extracts were prepared by filtering the lemon extracts of each site produced through the above extraction, collecting the filtrate, concentrating the filtrate under reduced pressure using a vacuum concentrator, and lyophilizing at -20 ° C to obtain a powder.

About 3 L of a mixed solvent prepared by mixing hexane (Hexane), water (H ₂ O) and methanol (methanol) at a volume ratio of 10: 9.5: 0.5 was added to the prepared 80% , Left and right shaking, and left to separate layers. After the layer was separated by the abovementioned leaving, a hexane fraction was prepared by separating the hexane fraction layer in the upper layer and the hexane fraction layer in the aqueous layer in the separating layer. The hexane layer was separated, and the same amount of chloroform (CHCl3) was added to the remaining solution (water layer). The chloroform layer was separated to separate the chloroform layer. The chloroform layer was separated and the remaining solution An ethyl acetate fraction was prepared by adding the same amount of ethyl acetate (EtOAc) and fractionating to separate only the ethyl acetate layer. The ethyl acetate layer was separated and the same amount of butanol (BuOH) was added to the remaining solution And a butanol fraction was separated by separating only the butanol layer, and the remaining solution was used as a water fraction. Each of the fractions of each of the fractions prepared by the above fractions was concentrated under reduced pressure using a vacuum concentrator and then lyophilized at -20 ° C to prepare fractions. The yields of the extracts and fractions prepared are shown in Table 1 below.

Solvent type Extract / fraction content (g) yield(%) 80% MeOH 77.220 26.54 Hexane 2.973 3.85 CHCl3 2.109 2.73 EtOAc 0.233 0.30 BuOH 7.354 9.52 Water 54.75 70.90

The extracts and fractions thus prepared were stored at -20 ° C. until they were used in the experiment, and each extract and fractions were dissolved in DMSO for use in the experiment.

Example  2: Identification of asthma treatment effect using animal model

Forty BALB / c animals were purchased from central laboratory animals (Korea) and classified into 5 groups. The experimental animals were housed in a polycarbonate breeding box having a constant light-dark cycle of light of 150 Lux to 300 Lux, a temperature of 20 to 26 ° C and a relative humidity of 40% to 60% from 8:00 am to 8:00 pm Four rabbits were kept in each cage, and feed and water were constantly ingested. The animal feeds used were pellet-dried diets (Korea Experimental Animals, Korea) and animal experiments were approved by the animal ethics committee of the Dongshin University.

The experimental animals were purified and stabilized in an animal room for 8 days after the purchase, and were divided into five groups. The experimental animals were divided into a normal control group provided with sterilized water, a negative control group in which asthma was induced with OVA (ovalbumin), a sterilized water treated group, a positive control group administered with 1 mg / kg / day dexamethasone, Day, 60 mg / kg / day, and 60 mg / kg / day, respectively, of the extract prepared in Example 1 and 600 mg / kg / day, respectively .

In particular, 20 μg of OVA (Albumin from chicken egg white, Sigma Chemical Co., USA) and 1 mg Alum (aluminum hydroxide hydrate, Thermo Co., USA) were added to all groups except the normal control at 1 and 7 days, Was added to 0.5 ml PBS buffer (PBS buffer), vortexed and dissolved, and sensitization was performed by intraperitoneally injecting the sensitization solution. After 14 days from the start of the experiment, 5% OVA solution prepared by dissolving OVA in PBS buffer was challenged with a nebulizer system (Aerosol 5210, BUXCO Electronics, Inc., USA). Specifically, the nebulizer head was filled with 20 ml of the 5% OVA solution, and the experimental animals were placed in an experimental animal breeding box connected with the nebulizer distribution chamber and the silicone hose. The duty percentage of the nebulizer was adjusted to 100% , And 5 times for 5 days by continuously spraying 5% OVA solution for 30 minutes. However, in the case of the normal control group, the 5% OVA solution was injected by filling with an excipient, that is, a PBS buffer solution. (Normal control and negative control), dexamethasone (positive control, Samsan Pharmaceutical, Korea), and 60 mg / day of sterile water (normal control and negative control) by direct administration into the stomach using a syringe once a day for 5 days. kg / day and 600 mg / kg / day, respectively. The nebulizer system and the 5% OVA solution were used in the afternoon. All animal experiments were approved by the Dongshin Animal Experimental Ethics Committee and were carried out in accordance with the provisions of the Dongshin Animal Experimental Ethics Committee.

After the experiment, animals were sacrificed in 5 groups and bronchoalveolar lavage fluid (BALF) was collected to determine changes in leukocyte count and immunoglobulin E (IgE) content. (H & E Stain), IHC stain (IHC Stain) and PAS stain (PAS Stain) were performed after isolating the lung tissue and fixing the isolated lung tissue (Carson FL, Hladik C. Histotechnology: A Self-Instructional Text (3 ed.). Hong Kong: American Society for Clinical Pathology Press. Pp. 137-39 (2009)).

First, the changes in the collection of lung bronchial washing solution and the change in leukocyte count were performed in the following manner. After opening the airway of the experimental animal and connecting a catheter containing PBS buffer for lung bronchial washing, 0.4 mL of the PBS buffer was slowly pushed into the lung bronchus, and 0.2 mL to 0.3 mL of the PBS was drained. Then 0.4 mL of PBS buffer solution Slowly pushed in, 0.4 mL was withdrawn, 0.4 mL of PBS buffer was again pushed in and 0.4 mL was withdrawn.

The collected bronchoalveolar lavage fluid (BALF) was centrifuged at 4 ° C and 3,000 rpm for 5 minutes, and the supernatant was separated and stored frozen at -70 ° C. After centrifugation, the supernatant was removed, and the remaining pellet was suspended in 20 uL PBS buffer. After centrifugation at 4 ° C and 3,000 rpm for 5 minutes, the supernatant was discarded, and the supernatant was removed. After adding 60 μL of PBS buffer to the pellet and suspending, the suspended suspension and the frozen suspension were mixed to make a total of 60 μL, followed by hemocyte analysis. The hemocyte analysis was performed using an automatic blood analyzer (ADVIA 2120, SIEMENS, USA). The results are shown in FIG.

As shown in FIG. 1, when the blood cells were observed in the bronchial washing solution, total counts of white blood cells (WBC, FIG. 1A), neutrophil cells (Neutrophil, NE, 1c) and eosinophil (EO, Fig. 1b), the total number of leukocyte counts and neutrophil counts decreased significantly depending on the amount of treatment when the extract was treated after the induction of asthma As a result, it was confirmed that the extracts of Amygdalus can immunologically improve asthma disease. Particularly, in the case of eosinophil (FIG. 1c), which is known to be sensitively related to asthma, the test group treated with the extract of 600 mg / kg / day and the test group treated with the positive control group dexamethasone were almost similar , And it was confirmed immunologically that the above-mentioned extract of the present invention is remarkably superior in improving or treating asthma disease

In addition, the content of IgE increased by iso-switching of IgE, i.e., IgG, which is an immunoglobulin inducing asthma in lung tissues was measured, and the results are shown in Fig. The immunoglobulin measurement was performed using a BD Mouse IgE ELISA set (Cat No: 555248, BD OptElA ^™ , BD Biosciences, USA). The reagents and measurement methods were prepared and performed according to the kit's datasheet and protocol. The measurement results are shown in Fig.

As shown in FIG. 2, the content of IgE inducing asthma in lung tissues was examined. As a result, in the negative control group inducing asthma in the experimental animals, the content of IgE rapidly increased to about 400% In contrast, in the group treated with the extract, the amount of 600 mg / kg / day of the extract was significantly decreased in the case of treatment with the extract of lettuce after the induction of asthma, it was confirmed to be almost similar to the control group treated with dexamethasone and the control group not causing asthma, and it was confirmed that the above-mentioned insect extract had remarkably excellent asthma disease treatment or therapeutic effect

In addition, lung tissues were isolated and the isolated lung tissue was fixed, followed by H & E staining and PAS staining to confirm histopathologic treatment effects on asthma. Specifically, the experimental method is as follows.

First, the lung tissue isolated from the above-mentioned five groups of experimental animals was fixed with a 10% (v / v) formaldehyde solution, and an aqueous 99.9% ethanol solution, a 90% ethanol aqueous solution, an 80% ethanol aqueous solution and a 70% Followed by dehydration in succession, followed by paraffin block. The paraffin-fixed lung tissue was cut to a thickness of 4 μm, and then subjected to H & E staining and PAS staining.

The H & E staining was performed by immersing the filter paper in a solution of heamatoxylin for 1 minute and 30 seconds in a stock solution of filtered heamatoxylin, followed by washing with water for 5 minutes. After staining, the cells were washed with primary distilled water until the tissue appeared blue, and then a clear process and dehydration process were performed. Specifically, after immersion in 95% ethanol for 5 minutes, immersion in Eosin for 5 minutes, immersion in 95% ethanol for 1 minute, immersion in 100% ethanol for 1 minute, immersion in 80% ethanol for 1 minute , And then immersed in 90% ethanol for 1 minute, then immersed in 100% ethanol for 1 minute, and immersed in Xylene (Junsei, Japan) solution for 2 minutes repeatedly for 3 times to make it transparent. Then, Canada Balsam , Kanto, Japan).

The PAS staining was performed by immersing in a xylene (Junsei, Japan) solution for 5 minutes, immersing it for 3 minutes, immersing in 80% ethanol for 1 minute, immersing in 90% ethanol for 1 minute, After immersing for 1 minute, the tissue was then dipped in 0.5% Periodic Acid Solution for 10 minutes to oxidize the tissue. Thereafter, it is washed with running water for 5 minutes, then rinsed with primary distilled water for 3 minutes, immersed in Schiff reagent for 10 minutes, washed with running water for 5 minutes and washed with running tap water. The cells were stained with hematoxylin for 1 minute and 30 seconds after washing. After dyeing, water was removed from the effluent for 10 minutes, followed by clarification and dehydration. Specifically, it was immersed twice in 95% ethanol for 1 minute, and then immersed in the order of 100% ethanol, 80% ethanol, 90% ethanol and 100% ethanol for 1 minute each, and then repeated in a xylene (Junsei, Japan) solution for 2 minutes , And then subjected to tissue sealing with Canadian Balsam (Canada Balsam, Kanto, Japan). The H & E staining and PAS staining results are shown in FIGS. 3 and 4. FIG.

As shown in Fig. 3, it was confirmed that the extract of the irrigation suppresses changes in lung tissue caused by asthma in a concentration-dependent manner. Specifically, as shown in Fig. 3, as compared with the lung tissue of the negative control group (Induc), the negative control group significantly increased inflammatory cell infiltration and mucus secretion, as confirmed by arrows of the negative control group, In the experimental group treated with 60 mg / kg / day of extract, inflammatory cell infiltration and mucin secretion around the bronchi and blood vessels were somewhat inhibited. In the test group treated with 60 mg / kg / day of the extract, (N / S), which is suppressed by inflammatory cell infiltration and mucus secretion around the bronchi and blood vessels rather than dexamethasone, which is a Dexa, It was confirmed histopathologically that it is possible to inhibit the concentration - dependent changes.

As shown in FIG. 4, glycoprotein secretion was confirmed by PAS staining. As a result, inhibition of secretion of glycoprotein was restricted in the experimental group treated with 60 mg / kg / day of the extract, day, the glycoprotein secretion was suppressed to a similar level as that of dexamethasone, which is a positive control (Dexa). Thus, the extract of the present invention can effectively inhibit the pathological change caused by asthma, that is, the secretion of glycoprotein induced by asthma .

Allergen-induced asthma, which is an allergen inducer, is divided into early and late reactions, and cytokine changes are observed at each step. Therefore, the change of cytokine associated with Th1 cell and Th2 cell was observed through immunostaining, and the anti-asthmatic effect of the extract was confirmed. Specifically, the types of antibodies used in the immuno-staining method are as shown in Table 2, including CD4 (helper T cell) and CD8 (cytotoxic T cell) as cell surface markers.

Type of antibody (subject to recognition) Cat.No manufacturer T-bet bs-3599R Bioss GATA-3 TA305795 OriGene TNF-a 3053R-100 BioVision 12p35 sc-9350 SANTA CRUZ BIOTECHNOLOGY, INC. 12p40 sc-57258 SANTA CRUZ BIOTECHNOLOGY, INC. IL-4 sc-73318 SANTA CRUZ BIOTECHNOLOGY, INC. IL-5 504301 BioLegend IL-6 PAB16165 Abnova IL-13 sc-1776 SANTA CRUZ BIOTECHNOLOGY, INC. IFN-r sc-74104 SANTA CRUZ BIOTECHNOLOGY, INC. CD4 MCA55GA A Bio-Rad Company CD8 MBS551004 M BioSource CD19 250585 ABBIOTEC ™

The immunostaining was carried out in the following manner.

After the paraffin was fixed, it was immersed twice in xylene (Junsei, Japan) for 5 minutes for paraffin removal and hydration, immersed in 100% ethanol for 3 minutes, immersed in 95% ethanol for 1 minute, Immersed in 80% ethanol for 1 minute, and then washed with primary distilled water for 3 minutes. Then, 3% hydrogen peroxide (H ₂ O ₂ ) methanol solution was pipetted for 10 min to remove endogenous peroxidase, and the plate was washed twice with TBS-T twice for 5 min. After this, a blocking serum of VECTASTAIN Quick KIT (Catalog No. PK-7800) was prepared for immunoblocking, dropped on tissue with a pipette, covered with parafilm, and reacted at room temperature for 1 hour. After the reaction, the cells were washed twice with TBS-T twice for 5 minutes, reacted with the antibody (Primary Antibody) shown in Table 2 for 30 minutes at room temperature, washed twice with TBS-T twice for 5 minutes, Antibody (Biotinylated Secondary Antibody) was reacted at room temperature for 10 minutes and was washed twice with TBS-T for 5 minutes each time. After washing with water, the Chromogen Reaction was dropped on the tissue according to the Liquid DAB Substrate Kit. After observing with a microscope, the degree of color development of the tissue was observed, and the reaction was stopped by soaking in PBS before it became brown. After stopping the reaction, the cells were washed twice with TBS-T for 5 minutes and then subjected to contrast dyeing for 7 seconds using hematoxylin. After the control dyeing, the reaction was stopped by rinsing with primary distilled water and rinsed twice with TBS-T for 5 minutes each time. The result of staining using each of the above antibodies is shown in Fig.

As shown in Fig. 5, in the case of IL-6 (Fig. 5A), in the test group treated with the extract of 60 mg / kg / day, the amount of IL-6 It was confirmed that overexpression of cytokine was slightly improved, and that the overexpression of cytokine was improved to the extent similar to that of dexamethasone treated with the positive control (Dexa) in the test group treated with the extract of 600 mg / kg / day . In the case of IL-4 (Fig. 5B), the overexpression of cytokine was improved only at a dose of 60 mg / kg / day, but remarkably improved in the experimental group treated with 600 mg / kg / day of the extract . In the case of TNF-a (FIG. 5C), it was confirmed that the test group treated with the extract of 600 mg / kg / day contained the test group similar to the non-asthmatic positive control group (N / S). In addition, as a result of confirming the use of antibodies against IL-12p40 (FIG. 5d) and IL-12p35 (FIG. 5e) In addition, as a result of using antibodies against IFN-y (Fig. 5i), IL-13 (Fig. 5J), IL-5 (Fig. 5k), GATA-3 (Fig. When the extract was treated with 600 mg / kg / day of the extract, the effect of overexpression of the cytokine was confirmed.

As shown in FIGS. 5F to 5H, in the case of CD 8 (FIG. 5F), CD 4 (FIG. 5H) and CD 19 (FIG. 5G) day, the cytokine overexpression was found to be improved even in the case of the cell surface marker substance as compared with the negative control group. In the test group treated with the extract of 600 mg / kg / day, the positive control group Dexa) dexamethasone in a dose-dependent manner.

According to the above results, the extract of the present invention effectively suppresses the increase of leukocyte-specific eosinophils and neutrophil counts associated with asthma, suppresses the production of IgE which causes allergy, , It was confirmed that the infiltration of inflammatory cells around the bronchi and blood vessels and the secretion of mucus were suppressed and the tissue change due to cytokine could be suppressed. Treatment, improvement, or prophylaxis.

Claims (6)

Erythronium japonicum ) as an active ingredient. The method according to claim 1,
Wherein the insect extract is extracted with any one selected from the group consisting of alcohols having 1 to 5 carbon atoms, water, and mixtures thereof. The method according to claim 1,
The composition for treating or preventing asthma is a medicinal composition for treating or preventing asthma. Erythronium japonicum ) as an active ingredient. 5. The method of claim 4,
The above-mentioned insect extract is an insect hot-water extract, which is used for improving or preventing asthma. 6. The method of claim 5,
Wherein the food composition is a functional food composition.

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