KR20150083327A - Composition comprising an extract or a fraction of Euonymus alatus for preventing or treating asthma - Google Patents

Abstract

The present invention relates to a pharmaceutical composition and a food composition comprising a Euonymus alatus extract and a fraction thereof as active ingredients for preventing, treating, and alleviating asthma. A Euonymus alatus extract and a fraction thereof of the present invention can be effectively used as a composition for preventing, treating, and alleviating asthma and as an ingredient with excellent effects of treatment and no toxicity overcoming side effects of a current medicine for asthma, which is capable of reducing airway hyperresponsiveness, inhibiting infiltration of inflammatory cells inside bronchial tubes, inhibiting IgE generation inside bronchoalveolar lavage fluid and serum, and inhibiting expression of Th2 cytokines (IL-4, IL-5, and IL-13) in lung.

Description

The present invention relates to a composition for preventing or treating asthma,

The present invention relates to an arrow- The present invention relates to a pharmaceutical composition and a food composition for prevention, treatment and improvement of asthma comprising an extract or fraction thereof as an active ingredient.

Asthma is a lung disease characterized by chronic airway inflammation and airway hyperresponsiveness. It is caused by air pollutants, dust, allergens, and the like. In particular, asthma is an inflammatory respiratory disease causing dyspnea, and about 3 million people in Korea have asthma. Asthma is a disease characterized by colorless asthma, dyspnea, sneezing, severe cyanosis due to lack of oxygen, and chest pain. Recently, it has been rapidly increasing due to exposure to air pollution and various chemical substances and westernization of diet. Especially, (Lemanske RF Jr., et al., JAMA 1997; 278: 1855-1873; Global Initiative for Asthma, NIH Publication No. 02-3659, 2002). However, in asthmatic patients, the cause and pathogenesis of the disease is not clear (Global Initiative for Asthma, NIH Publication No. 02-3659, 2002, p50-66). In this pathogenesis, Th2 (T helper type 2) type of immune response is exaggerated, resulting in increased secretion of interleukin-4, 5, and 13 (Liu et al., 2006; Williams et al. 2012), many inflammatory cells including eosinophils migrate and infiltrate into lung tissue in association with this reaction (Zhou et al., 2011). In addition, inflammatory cells release a variety of proinflammatory and chemoattractant factors, further exacerbating the inflammatory response, increasing mucus secretion in intratracheal goblet cells, and causing airway hyperresponsiveness (Chibana et al., 2008). Because of this series of reactions, patients with asthma exhibit clinical symptoms such as dyspnea, cyanosis, and chest pain.

Currently, steroids, bronchodilators and antibiotics are mainly used as medicines that are being used to treat asthma. Steroids and antibiotics are used in the treatment of asthma through immune responses and inhibition of inflammatory responses, and bronchodilators are used to counteract clinical manifestations such as dyspnea. The most widely used inhaled corticosteroids have excellent therapeutic effect, but when used for a long period of time, adrenal suppression, bone density reduction, growth disorder, eye and skin complications, collagen synthesis And the like. In addition, persistent beta-2 agonists, such as salmeterol and formeterol, have been shown to be protective against asthma attacks, . And long-term use has been found to have side effects, limited use as a treatment for asthma. The development of natural products or new compounds that overcome these side effects, are less toxic, and have excellent therapeutic effects are constantly under way.

Arrow wood ( Euonymus alatus ) is a woody plant distributed in Northeast Asia centered on the Korean peninsula. It is a woody plant which has been born since ancient times and has suffered from mental anxiety, mental anxiety, uterine bleeding, epilepsy, Has been used to repair the back. (Park et al., J. Ethnopharmacol., 102, 326-335, 2005; Cha et al., J. Ethnopharmacol., 85, 163- 167, 2003), there is no known prophylactic or therapeutic activity of astringents of the horseshoe extract, fractions thereof or compounds isolated therefrom.

Accordingly, the present inventors have made intensive efforts to find a natural substance useful for prevention or treatment of asthma without causing adverse effects on the human body. As a result, it has been found that an arrow egg extract or its fractions can be useful for prevention or treatment of asthma without showing toxicity And completed the present invention.

An object of the present invention is an arrow tree (Euonymus The present invention also provides a pharmaceutical composition for preventing or treating asthma comprising the extract or fraction thereof as an active ingredient.

Another object of the present invention is to provide a method and apparatus for detecting the presence of an arrowhead The present invention also provides a food composition for preventing or ameliorating asthma comprising as an active ingredient an alatus extract or a fraction thereof.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating asthma comprising an extract of Euonymus alatus or a fraction thereof as an active ingredient.

Specifically, the prophylactic or therapeutic pharmaceutical composition for asthma of the invention arrow tree (Euonymus alatus extract or fractions thereof.

As used herein, the term "arrow tree (Euonymus alatus ) "is a plant belonging to the family Thunb . It is distributed mainly in Korea, Japan, Sakhalin, and China, and it is common in mountain wilderness. It is 3 m in height and 2 ~ 4 wings in twigs. Leaves are opposite to each other and have short petiole, with oval or inverted egg shape, with serrate on margin and no hair. Hemorrhage in one room, has been used in the former hemorrhage.

In the present invention, the term " arrowwood extract " means an extract obtained by extracting an arrow tree. The arrowhead extract has a volume of water ranging from about 2 to 20 times, preferably about 3 to 5 times the dry weight of the arrowhead crushed material, and from ₁ to 4 carbon atoms (C ₁ to 4) such as methanol, ethanol, ₄ ) or a mixed solvent having a mixing ratio of about 1: 0.1 to 1:10 of them is used as an elution solvent, and the extraction temperature is 20 to 100 ° C, preferably room temperature, and the extraction period is about Extraction can be carried out using extraction methods such as hot water extraction, cold extraction, reflux cooling extraction or ultrasonic extraction for 12 hours to 4 days, preferably 3 days. However, if it is a method for extracting substances having a preventive or therapeutic activity for asthma Can be used without limitation. Preferably, the extract is continuously extracted one to five times by cold extraction, filtered under reduced pressure, and the filtrate is concentrated under reduced pressure at 20 to 100 ° C, preferably at room temperature, in a vacuum rotary condenser to be dissolved in water, a lower alcohol or a mixed solvent thereof But the present invention is not limited thereto. Examples thereof include a dry matter obtained by drying an extract, a diluent or concentrate of an extract, an extract, or the like, as long as it can exhibit the preventive or therapeutic activity of the asthma of the present invention, Or both of these controlled release products or tablets. The barberry extract can be extracted from various organs of natural, hybrid, and variant plants and extracted from, for example, roots, shoots, stems, leaves, fruits, or plant tissue cultures.

The term " fraction " in the present invention means a product obtained by a fractionation method for separating a specific component or a specific group from a mixture containing various constituents. The arrowhead fraction of the present invention can be obtained by suspending an arrowhead extract and then fractionating it with a polar solvent such as water, methanol or ethanol or a non-polar solvent such as hexane or ethyl acetate to obtain a polar solvent fraction and a non-polar solvent fraction, respectively have. Specifically, after the arrowhead crude extract is suspended in distilled water or the like, water having a volume of about 1 to 100 times, preferably about 1 to 5 times the volume of the suspension, an alcohol having 1 to 4 carbon atoms (C ₁ ) to 4 (C ₄ ) , A polar or nonpolar solvent such as ethyl acetate, hexane, butanol, or a mixed solvent thereof is added, and the polar or non-polar solvent soluble layer is extracted and separated from 1 to 10 times, preferably 2 to 5 times have. Further, a conventional fractionation process may be performed (Harborne JB Plant Pathology, 1998, 3rd Ed. P6-7). More specifically, the above-mentioned arrowhead extract was suspended in water and then extracted successively with water, methanol or a mixed solvent thereof [10: 90 → 100: 0 (v / v)] as a solvent, Fractions can be obtained, but are not limited thereto (Production Example).

It has been confirmed that the bark extract or its fractions according to the present invention can reduce the secretion of cytokines or IgE selected from the group consisting of IL-4, IL-5 and IL-13, thereby being effective for preventing or treating asthma Can be used.

The term " asthma " in the present invention is a comprehensive disease name collectively referred to as various diseases characterized by inflammation of the airways leading to organs, bronchi, bronchioles, and alveoli, , Clin Exp Allergy 2005; 35: 1254-62). In particular, asthma is a condition in which the bronchus in the lung is very sensitive. Sometimes the bronchus narrows and the breathing, choking, and breathing sounds can be heard, causing severe coughing. Allergy caused by bronchial allergy inflammation Disease. The most common symptoms of asthma include dyspnea, cough, wheezing (wheezy breathing), a symptom reliever (bronchodilator) that alleviates the narrowed bronchus in a short time, or a disease modifier that prevents asthma attacks by inhibiting bronchial allergic inflammation Anti-inflammatory agents, and leukotriene modulators). In the present invention, the asthma may be bronchial asthma, allergic asthma, atopic asthma, nonatopian asthma, exercise induced asthma, aspirin asthma, cardiogenic asthma or alveolar asthma, But is not limited thereto.

In the present invention, the term "prevention" means all actions that inhibit or delay the onset of asthma by the administration of the composition, and "treatment" means any action do.

Interleukin-4 (IL-4), interleukin-5 (IL-5) and interleukin-13 (IL-13), which are cytokines produced by Th2 (helper T cell Type 2) cells, are important mediators of bronchial asthma (Coyle AJ. Et al. 1995. Am J Respir Cell Mol Biol 13 (1): 54-9; Nakajima H. et al 1992. Am Rev. Respir Dis 146 (2 Wills-Karp, M. et al 1998. Sci. 282 (5397): 2258-61; Cohn et al., 2004; Medoff et al., 2008).

Specifically, when the antigen causing bronchial asthma is firstly removed by macrophages and the macrophages of the alveoli activated in this process stimulate B cells to produce IgE, IgE is a method of activating mast cells, Responsible for the reaction. In addition, B cells exposed to asthma-inducing antigens are differentiated into Th2 cells by stimulating CD4 T cells, and the differentiated Th2 cells secrete IL-4, IL-5, and IL-5 in lung tissue and bronchoalveolar lavage (BALF) And IL-13, and these promoted cytokines are known to induce bronchial hyperresponsiveness and airway obstruction resulting in asthma reactions.

Therefore, the suppression of the secretion of cytokine selected from the group consisting of IL-4, IL-5 and IL-13 or suppression of the secretion of IgE can be suppressed.

In the examples of the present invention, IL-4, IL-5 and IL-13 were produced from the bronchoalveolar lavage fluid, and IL-4, IL-5 and IL- (Example 5, Table 2), and ii) the amount of secreted specific IgE in serum and egg albumin was measured. As a result, it was found that the amount of serum IgE in serum IgE and albumin-specific IgE were significantly increased in the asthma-induced group compared to the normal control group, but significantly decreased in the administration group of arrowhead extract or its fraction (Example 6, Table 3). This demonstrates that the barnyardgrass extract or its fractions are effective for the treatment of asthma.

It has also been confirmed by the present invention that the arrowhead extract or its fractions can reduce the number of inflammatory cells around the bronchi or blood vessels or reduce the mucus secretion of the bronchi epithelial cells.

Bronchoalveolar lavage was performed in patients with asthma, and airway inflammation was confirmed. As a result, lymphocytes, mast cells, eosinophils and activated macrophages were increased in bronchoalveolar lavage fluid. Therefore, it is known that asthma is generalized as airway inflammation disease, and most inflammatory cells are activated and secrete various mediators to induce asthma, and 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). On the other hand, asthma is a kind of asthmatic symptom, because airway stenosis and infiltration of inflammatory cells into the bronchi, goblet cells are formed, secretion of mucus is prominent, and collagen deposition is prominent. It is known.

As an example of the present invention, inflammation in lung tissues was observed through H & E (Hematoxylin & Eosin) staining, and mucous secretion amount was observed through PAS (periodic acid Schiff) staining. As a result, And infiltration of a wide range of inflammatory cells around the blood vessels was observed. On the other hand, infiltration of inflammatory cells around the bronchi and blood vessels was reduced in both the arrowhead extract and the fraction-administered group thereof (Example 7, Fig. 4) In the induction group, mucin secretion was increased in the bronchial epithelial goblet cells, whereas in both the arrowhead extract group and the fraction-treated group thereof, mucin secretion of the bronchial epithelial goblet cells was remarkably reduced (Example 7, Fig. 5) . These results also show that the barnyardgrass extract or its fractions are effective for the treatment of asthma.

The pharmaceutical compositions comprising the barnyard extract or fractions thereof of the present invention may further comprise suitable carriers, adducts or diluents conventionally used in the manufacture of pharmaceutical compositions. At this time, the content of the arrow egg extract or its fraction contained in the composition is not particularly limited, but it may include 0.001% by weight to 99.9% by weight, preferably 0.01% by weight to 50% by weight based on the total weight of the composition have.

The pharmaceutical composition may be any one selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterilized aqueous solutions, nonaqueous solvents, suspensions, emulsions, And may be oral or parenteral formulations of various forms. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules, and the like, which may contain one or more excipients such as starch, calcium carbonate, sucrose or lactose lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like may also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the non-aqueous solvent and the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

The composition of the present invention may be administered in a pharmaceutically effective amount.

The term "pharmaceutically effective amount" as used herein means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level will vary depending on the species and severity, age, sex, The type of drug, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts.

The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. And can be administered singly or multiply. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without adverse effect, and can be easily determined by those skilled in the art.

The composition of the present invention can generally be administered in an amount of 0.001 to 1000 mg / kg, preferably 0.05 to 200 mg / kg, more preferably 0.1 to 100 mg / kg, However, the desired dosage can be suitably selected by those skilled in the art depending on the condition and the weight of the individual, the degree of disease, the drug form, the administration route and the period.

The composition is not particularly limited as long as it is an object for asthma, and any composition can be applied. For example, any non-human animal such as a monkey, a dog, a cat, a rabbit, a guinea pig, a rat, a mouse, a cattle, a sheep, a pig or a chlorine can be used. Subcutaneous, intraperitoneal, intrapulmonary, and intranasal, and may be administered by a suitable method, including localized administration, if necessary, for localized treatment. The preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and body weight of the individual, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art.

In another aspect, the present invention provides a food composition for prevention or amelioration of asthma comprising an arrowhead extract or a fraction thereof as an active ingredient.

The above-mentioned arrow tree, its extract, fraction and asthma are as described above.

The term "improvement" in the present invention refers to all kinds of diseases that are suspected or suspected of diseases such as asthma that are prevented or treated by using a composition comprising an arrowhead extract or a fraction thereof as an active ingredient, It says.

As a remedy, the extract of the present invention or a fraction thereof may be added to the food composition for the purpose of preventing or improving asthma.

The food composition of the present invention may be in the form of pills, powders, granules, infusions, tablets, capsules or liquid preparations. There is no particular limitation on the kinds of foodstuffs to which the extract of the present invention or its fractions can be added , For example, various drinks, gum, tea, vitamin complex, and health supplement foods.

The food composition may be supplemented with other ingredients in addition to the barnyardgrass extract or the fraction thereof, and the kind thereof is not particularly limited. For example, it may contain various herbal medicine extracts, food-acceptable food-aid additives or natural carbohydrates such as ordinary food, but is not limited thereto.

The term "food supplementary additive " in the present invention means a component which can be added to foods in a supplementary manner, and is appropriately selected and used by those skilled in the art as added to produce health functional foods of each formulation. Examples of food-aid additives include flavors such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, colorants and fillers, pectic acid and its salts, alginic acid and its salts, organic acids, , a pH adjusting agent, a stabilizer, a preservative, a glycerin, an alcohol, and a carbonating agent used in a carbonated beverage. However, the types of the food auxiliary additives of the present invention are not limited by the above examples.

Examples of the natural carbohydrate include monosaccharides such as glucose and fructose; Disaccharides such as maltose, sucrose and the like; And polysaccharides such as dextrin and cyclodextrin and sugar alcohols such as xylitol, sorbitol and erythritol. In addition to the above, natural flavorings (such as tau mart), stevia extracts (rebaudioside A, Etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used.

The food composition of the present invention may include a health functional food. The term " health functional food " as used in the present invention refers to foods prepared and processed in the form of tablets, capsules, powders, granules, liquids, and rings using raw materials and ingredients having useful functions in the human body. Here, the term "functionality" means that the structure and function of the human body are controlled to obtain nutritional effects or effects useful for health use such as physiological actions. The health functional food of the present invention can be manufactured by a method commonly used in the art and can be prepared by adding raw materials and ingredients which are conventionally added in the art. Also, unlike general medicine, there is an advantage that there is no side effect that can occur when a medicine is used for a long time by using food as a raw material, and it is excellent in portability.

The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment). Generally, the arrowhead extract or the fraction thereof of the present invention may be added in an amount of 1 to 50% by weight, preferably 5 to 10% by weight, of the raw material composition, but not limited thereto. However, in the case of long-term ingestion intended for health and hygiene purposes or for the purpose of controlling health, the amount can also be used in the above-mentioned range.

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

The bark extract or its fractions of the present invention alleviate airway hyper-responsiveness, inhibit inflammatory cell infiltration in bronchi, inhibit IgE production in serum and bronchoalveolar lavage fluid, inhibit Th2 cytokines (IL-4, IL -5 and IL-13), it is possible to overcome the side effects of current therapeutic agents for asthma, and as a component having no toxicity and excellent therapeutic effect, it is useful as a composition for prevention, treatment and improvement of asthma .

FIG. 1 is a graph showing the results of analysis of arrowhead leaves, stem methanol extract and fractions thereof by means of MPLC (medium pressure liquid chromatography). FIG.
FIG. 2A is a diagram showing the result of analyzing arrowhead leaves, stem methanol extract and fractions thereof by PDA (Photodiode-Array detection) and MS (Mass Spectrometry) using UPLC (Ultra Performance Liquid Chromatography).
FIG. 2B is a graph showing the results of analysis of arrowhead leaves, stem methanol extract and active fractions thereof by UPLC-CAD (Charged Aerosol Detector). FIG.
FIG. 3 is a graph showing the effect of methanol extract (Total) and active fractions of arrowheads on airway hyperresponsiveness in asthmatic animal model induced by egg white.
FIG. 4 is a graph showing effects of methanol extracts and active fractions of arrowhead stem on the infiltration of extensive inflammatory cells around airway and blood vessels in a model of asthma induction animal.
FIG. 5 is a graph showing the effects of methanol extracts and active fractions of the arrows on the mucus secretion in bronchi in an asthma-inducing animal model.

Hereinafter, the present invention will be described more specifically in the following examples. However, these examples are provided only to aid understanding of the present invention, and the present invention is not limited thereto.

Manufacturing example : Arrowhead leaves, stem extracts and Fraction  Produce

The present inventors removed moisture through natural drying (shade) using a drier (50-55 ° C) and then pulverized to a size of about 1 cm. Based on the dry weight of the pulverized powder sample, 1 L of methanol was added and extracted at room temperature. Then, after filtration and concentration under reduced pressure, 4.91 g of a methanol extract of an arrowhead leaf and stem was obtained. In the subsequent experiments, the obtained extracts of arrowhead leaves and stem methanol were designated as total extracts.

Further, a column (20 mm × 250 mm; Resin: Zeoprep C18, 10 μm) was attached to a medium pressure liquid chromatography (MPLC) apparatus (sepbox 2D 5000) Was loaded in an amount of 2 g. At this time, water / methanol [10: 90 -> 100: 0 (v / v)] was used as a solvent and the elution rate was 40 ml / min and detected at a wavelength of UV 200-400 nm. The analysis time was 150 minutes, and 9 fractions were obtained as shown in Table 1 (FIG. 1, Table 1).

Arrow wood MeOH (%) Analysis time (min) Weight (mg) Fraction 1 0-8 1-11 501.8 Fraction 2 8-28 11-35 132.5 Fraction 3 28-32 35-40 81.1 Fraction 4 32-39 40-48 220.8 Fraction 5 39-50 48-61 357.6 Fraction 6 50-69 61-83 222.8 Fraction 7 69-80 83-96 64.7 Fraction 8 80-89 96-107 155.1 Fraction 9 89-100 107-134 128.4

Example  1: arrowhead leaves, stem extracts and Fraction  analysis

In order to investigate the active fractions of leaves, stem methanol extracts and active fractions obtained in the above-mentioned production examples, they were analyzed by liquid chromatography (UPLC), ultra performance liquid chromatography.

First, for the UPLC analysis, 4.91 g of the methanol extract of the arrowhead obtained in the Production Example and the active fraction were once filtered with a 0.25 占 퐉 membrane filter for UPLC. A column (Waters BEH C18 column, 2.1 x 100 mm, 1.7 mu m) was loaded on a UPLC instrument (Waters UPLC-QTOF-MS) and each fraction filtered was loaded with 5 uL aliquots. At this time, acetonitrile + 0.1% formic acid / water + 0.1% formic acid [10: 90 → 100: 0 (v / v)] was used as a solvent and the elution rate was 0.4 ml / min. Respectively.

Example  2: Albumin albumin  Induced bronchial asthma animal model

In the present invention, 6-week-old BALB / c female mice with an average weight of about 20 g were used as experimental animals. The subjects who did not have any abnormality on basic physical examination after 1 week of adaptation period were included. 200 μl of phosphate buffered saline suspended in 2 mg aluminum hydroxide (A8222, Sigma-Aldrich, MO, USA) and 20 ug of egg albumin (A5503, Sigma-Aldrich) Sensitized. After the administration of the first egg white albumin, 1% egg white albumin was inhaled for 30 minutes using an ultrasonic atomizer (NE-U12, Omron Corp., Japan) from the 21st to the 23rd day. Twenty-four hours after the last egg white albumin exposure, bronchial hyperresponsiveness was measured. After 48 hours, pentobarbital (50 mg / kg, Entobal, Hanil, Korea) was anesthetized by intraperitoneal injection and blood was collected through the abdominal vein. Bronchial alveoli were washed with 1.4 ml of PBS to collect the specimens. Experimental groups were divided into two groups: normal control group (NC, no albumin administration and inhalation group), asthma induction group (OVA, albumin administration and inhalation group), positive control group (Mon, montelukast 30 mg / kg administration + (Fraction 8) were administered at a dose of 30 mg / kg each, followed by administration of egg white albumin and inhalation, respectively. The drug and the sample were orally administered from the 21st to 23rd day after the administration of the first egg white albumin. Seven mice in each group were used. After the experiment, the blood, lung lavage fluid and lung tissue of each group of mice were separated.

Example  3: airway hyperresponsiveness Airway hyperresponsiveness ) Measure

One-chamber plethysmography (All Medicus, Korea) was used to measure airway hyperresponsiveness due to asthma. The degree of airway resistance was assessed by measuring the enhanced pause (Pehn). Pehn was measured at baseline in normal respiration state, and then PBS was inhaled for 3 minutes using an ultrasonic atomizer and then measured for 3 minutes. Penh values were measured after inhaling methacholine (A2251, Sigma-Aldrich) at increasing concentrations of 12, 25 and 50 mg / ml.

As a result, as shown in FIG. 3, in the asthmatic animal model treated with egg white albumin, airway hyperresponsiveness was significantly increased in the asthma induction group as the methylcholine concentration increased compared with the normal control group. On the other hand, the bronchial hyperresponsiveness of montelukast treated group was significantly decreased as the concentration of methacholine increased. In addition, as the concentration of methylcholine in the total extract and fraction thereof treated group was increased, a significant decrease in airway hyperresponsiveness was observed compared to asthmatic induction group. 8).

Example  4: bronchoalveolar lavage fluid ( Bronchoalveolar lavage fluid , BALF ) Separation and Number of inflammatory cells  Measure

The increase of eosinophils is one of the main characteristics of asthma. The bronchoalveolar lavage fluid obtained in Example 2 was stained with trypan blue immediately after collection, and the total number of cells, excluding dead cells, was calculated using a hemocytometer, and then cytotoxin (Cytospin (Sysmex, Switzerland) was used to attach the cells to the slide using a microscope, and the number of inflammatory cells in each sample was measured using a microscope And the results are shown in Table 2 below.

Experimental group Eosinophils (x10 ⁴ ) Large (x10 ⁴ ) Lymphocyte (x10 ⁴ ) Neutrophil (x10 ⁴ ) Total inflammatory cells (x10 ⁴ ) Normal group 0 ± 0 7 ± 2 2 ± 0 0 ± 0 9 ± 2 Asthmatic induction group 87 ± 16 ^# 90 ± 14 ^# 11 ± 3 ^# 9 ± 4 ^# 196 ± 28 ^# Positive control group 28 ± 16 ^* 54 ± 11 ^* 4 ± 2 ^* 3 ± 2 ^* 88 ± 14 ^* Arrow tree extract (Total) 56 ± 16 ^* 76 ± 9 1 ± 0 ^* 4 ± 2 ^* 136 ± 19 ^* Arrow wood fractions 8
(Fraction 8) 33 ± 10 ^* 71 ± 9 1 ± 1 ^* 3 ± 1 ^* 108 ± 13 ^*

As shown in Table 2, the number of inflammatory cells including eosinophils was significantly increased in asthma induction group compared to the normal group. On the other hand, a significant decrease in inflammatory cells was observed in the total and fraction fraction of arrowhead extract (Fraction 8) (Fraction 8), which was significantly higher in the montelukast group Respectively.

Example  5: bronchoalveolar lavage fluid ( BALF ) My cytokine analysis

The amount of IL-4, IL-5 and IL-13 produced in the bronchoalveolar lavage fluid isolated from each individual of Example 2 was measured using an enzyme-liked immunosorbent assay (ELISA) kit (R & D System, USA ). Each cytokine was analyzed according to the manufacturer's test method, and the absorbance at 450 nm was measured with an ELISA reader (Molecular Devices, USA). The results are shown in Table 3 below.

Experimental group IL-4 (pg / mL) IL-5 (pg / mL) IL-13 (pg / mL) Normal group 10.3 ± 2.11 19.4 ± 2.48 32.5 ± 2.44 Asthmatic induction group 54.8 ± 13.95 ^# 37.7 ± 5.66 ^# 86.0 + - 8.33 ^# Positive control group 13.0 ± 5.99 ^* 26.0 + - 3.48 ^* 47.7 ± 5.53 ^* Arrow tree extract (Total) 19.9 ± 6.20 ^* 28.4 ± 2.98 ^* 57.9 ± 9.38 ^* Arrow wood fractions 8
(Fraction 8) 15.1 ± 3.89 ^* 25.1 ± 2.93 ^* 51.8 ± 5.89 ^*

As shown in Table 3, the Th2 type cytokines IL-4, IL-5 and IL-13 were significantly increased in the asthma-induced group compared to the normal group. On the other hand, the positive control group, Montelukast group, and all the test substance group, Th2 cytokine were significantly inhibited compared to the asthmatic group. Particularly, the inhibitory amount was remarkably observed in the group administered with Fraction 8, and it was confirmed that the amount of inhibition was almost similar to that of the positive control group.

Example  6: Serum IgE  And Albumin albumin  singularity IgE  Measure

The blood obtained through the posterior vena cava in Example 2 was reacted at room temperature for 30 minutes and then centrifuged (3000 rpm, 15 min) to obtain serum. ELISA was used for the measurement of serum IgE and albumin - specific IgE. IgE was measured using a commercially available IgE (Biolegend Ins., USA). Egg albumin-specific IgE was measured in a 96-well flat bottom ELISA plate with 0.1 M NaHCO ₃ buffer (pH 8.3) at a concentration of 20 μg / And reacted at 4 ° C for 16 hours. Afterwards, PBS containing 1% bovine serum albumin (BSA) inhibited the non-specific response. Serum samples were diluted 1: 400, reacted at room temperature for 2 hours, and then washed with PBS containing 0.05% Tween 20. After incubation at room temperature for 1 hour, HRP-conjugated goat anti-rat IgG polyclonal A conjugated with peroxidase was reacted at room temperature for 1 hour, and then the color developed by reacting with 3.3'.5.5'-tetramethylbenzidine substrate and absorbance at 450 nm And the results are shown in Table 4 below.

Experimental group Total IgE (ng / mL) OVA-specific IgE (ng / mL) Normal group 84.6 ± 41.41 - Asthmatic induction group 2624.3 ± 286.90 ^# 306.2 ± 71.18 ^# Positive control group 1892.9 ± 139.23 ^* 168.5 ± 55.87 ^* Arrow tree extract (Total) 1988.8 ± 227.16 ^* 224.6 ± 43.73 ^* Arrow wood fractions 8
(Fraction 8) 1844.2 ± 291.90 ^* 178.0 ± 44.83 ^*

As shown in Table 4, total IgE in serum was significantly increased in the asthma-induced group compared with the normal group, whereas the montelukast-treated group, which was positive control group, was significantly decreased compared to the asthma-induced group. Total IgE and total fraction of fraction 8 (Fraction 8) showed significantly lower total IgE than asthma - induced group.

On the other hand, the results of OVA specific IgE were similar to those of total IgE. In asthma - induced group, OVA - specific IgE (IgE) was significantly increased compared with normal group, but the positive control group and the sample - administered group were significantly decreased compared to the asthma - induced group.

Example  7: Airway inflammation  Influence on invasion and mucus secretion

The lungs were removed from the animal model of Example 2 and immediately fixed in a 10% formaldehyde solution. Then, the lungs were washed with flowing water for 8 hours, embedded in epoxy, and then embedded in a microtome . Hematoxylin and eosin staining was performed to observe inflammation in the lung tissue. (2) Mucin secretion was significantly increased when asthma was induced, and periodic acid Schiff (PAS, IMEB) was used to observe the inflammation. , USA). Pathological changes of pulmonary tissues were examined using an optical microscope.

(1) Observation of inflammatory reaction in lung tissue revealed extensive infiltration of inflammatory cells around airway and blood vessels of lung tissue of asthmatic induction group. On the other hand, it was confirmed that infiltration of inflammatory cells was reduced in the montelukast-treated group, and that infiltration of inflammatory cells around the bronchi and the blood vessels was also reduced in both of the group of arrowhead extract (Total) and the fraction of arrowhead fraction 8 (Fraction 8) . This reduction was similarly observed when compared to the montelukast treated group (Fig. 4).

(2) Observation of mucus secretion results in bronchial epithelium showed an increase in mucin secretion in bronchial epithelial cells. On the other hand, mucin secretion was decreased in the montelukast group, and mucin secretion of the bronchial epithelium was remarkably reduced in all of the group of arrowhead extract (Total) or fraction 8 (Fraction 8) (FIG. 5).

From the above results, it was confirmed that the bark extract or its fractions according to the present invention have an inhibitory activity against asthma.

Examples of formulations for the composition of the present invention are illustrated below, but are not limited thereto.

Formulation example  1: Preparation of pharmaceutical preparations

The pharmaceutical preparations containing the bark extract or fractions of the present invention were prepared according to the conventional method as follows.

Formulation example  1-1: Sanje  Produce

2 g < RTI ID = 0.0 >

Lactose 1 g

The above components were mixed and packed in airtight bags to prepare powders.

Formulation example  1-2: Preparation of tablets

100 < RTI ID = 0.0 > mg < / RTI >

Corn starch 100 mg

Lactose 100 mg

2 mg of magnesium stearate

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

Formulation example  1-3: Preparation of capsules

100 < RTI ID = 0.0 > mg < / RTI >

Corn starch 100 mg

Lactose 100 mg

2 mg of magnesium stearate

After mixing the above components, the capsules were filled in gelatin capsules according to the conventional preparation method of capsules.

Formulation example  1-4: Manufacture of rings

1 g each of the bark extract or fraction prepared in the Production Example

Lactose 1.5 g

Glycerin 1 g

0.5 g of xylitol

After mixing the above components, they were prepared so as to be 4 g per one ring according to a conventional method.

Formulation example  1-5: Preparation of granules

150 < RTI ID = 0.0 > mg < / RTI >

Soybean extract 50 mg

200 mg of glucose

600 mg of starch

After mixing the above components, 100 mg of 30% ethanol was added and the mixture was dried at 60 캜 to form granules, which were then filled in a capsule.

Formulation example  2: Preparation of Health Functional Food

Healthy functional foods containing the bark extract or fraction of the present invention were prepared according to the conventional method as follows.

Formulation example  2-1: Manufacture of Flour Food

0.5 to 5.0 wt.% Of an arrowhead extract or fraction prepared in the Preparation Example of the present invention was added to wheat flour, and a bread, a cake, a cookie, a cracker and a noodle were prepared using this mixture to prepare a food for health promotion.

Formulation example  2-2: Dairy products ( dairy products )

5 to 10% by weight of an arrowhead extract or fraction prepared in the preparation example of the present invention was added to milk, and various dairy products such as butter and ice cream were prepared using the milk.

Formulation example  2-3: Solar  Produce

Brown rice, barley, glutinous rice, and yulmu were dried by a known method and dried, and the mixture was granulated to a powder having a particle size of 60 mesh. Black soybeans, black sesame seeds, and perilla seeds were steamed and dried by a conventional method, and then they were prepared into powder having a particle size of 60 mesh by a pulverizer. Each of the bark extracts or fractions prepared in the preparation examples of the present invention was concentrated under reduced pressure in a vacuum concentrator, dried by spraying and dried in a hot air drier, and pulverized to a size of 60 mesh by a pulverizer to obtain a dry powder.

The dried grains, seeds and arrowhead extracts or fractions prepared above were blended in the following proportions.

Cereals (30% by weight of brown rice, 15% by weight of yulmu, 20% by weight of barley)

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

Dried powder (3% by weight) of an eggplant extract or fraction,

(0.5% by weight),

(0.5% by weight)

Formulation example  3: Manufacturing of beverages

Formulation example  3-1: Health drink  Produce

1000 mg < / RTI > of the extracts or fractions of arrowheads, respectively,

Citric acid 1000 mg

100 g of oligosaccharide

Plum concentrate 2 g

Taurine 1 g

Purified water was added to a total of 900 ml

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was stirred and heated at 85 ° C for about 1 hour. The resulting solution was filtered to obtain a sterilized 2-liter container, which was sealed and sterilized, It is used in the production of the health beverage composition of the invention.

Although the composition ratio is a mixture of the components suitable for the preferred beverage as a preferred embodiment, the blending ratio may be arbitrarily varied according to the regional and national preferences such as the demand level, the demanding country, and the intended use.

Formulation example  3-2: Manufacture of vegetable juice

Healthy vegetable juices were prepared by adding 5 g of each of the bark extracts or fractions prepared in the preparation examples of the present invention to 1,000 ml of tomato or carrot juice.

Formulation example  3-3: Manufacture of fruit juice

Health enhancing fruit juice was prepared by adding 1 g of each of the bark extracts or fractions prepared in the preparation examples of the present invention to 1,000 ml of apple or grape juice.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are illustrative in all aspects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention, rather than the above detailed description, and all changes or modifications derived from the equivalents thereof will be included in the scope of the present invention.

Claims (15)

Arrow wood ( Euonymus alatus extract or a fraction thereof as an active ingredient for the prevention or treatment of asthma.
The method of claim 1, wherein the extract is water, carbon number ₁ (C 1) to 4 will be extracted by using alcohol or a mixed solvent of (C ₄₎ composition.
The composition of claim 1, wherein the extract is extracted with methanol.
The method of claim 1, wherein the fraction by fraction using alcohol, chloroform, ethyl acetate, hexane, butanol, or a mixed solvent of a methanol extract of the arrow wood water, carbon number ₁ (C 1) to ₄ (C 4) ≪ / RTI >
The composition of claim 1 wherein said fraction is obtained by fractionating the methanol extract of arrowheads with water, methanol or a mixed solvent thereof.
2. The composition of claim 1, wherein the arrowhead ( Euonymus alatus ) is an arrow tree stem, a leaf or a mixture thereof.
The composition of claim 1, further comprising a pharmaceutically acceptable carrier, excipient, or diluent in the edible oil extract or fraction thereof.
2. The composition of claim 1, wherein the bark extract or fraction thereof reduces the secretion of cytokines selected from the group consisting of IL-4, IL-5 and IL-13.
2. The composition of claim 1, wherein the hornblend extract or fraction thereof reduces the secretion of IgE.
2. The composition of claim 1, wherein the bark extract or fraction thereof reduces the number of inflammatory cells around the bronchi or blood vessels or decreases the mucus secretion of the bronchial epithelial cells.
A food composition for prevention or amelioration of asthma comprising an extract of Eunonymus alatus or a fraction thereof as an active ingredient.
12. The method of claim 11, wherein the extract is water, carbon number ₁ (C 1) to 4 will be extracted by using alcohol or a mixed solvent of (C ₄₎ composition.
12. The composition of claim 11, wherein the extract is extracted with methanol.
12. The method of claim 11, wherein the fraction by fraction using alcohol, chloroform, ethyl acetate, hexane, butanol, or a mixed solvent of a methanol extract of the arrow wood water, carbon number ₁ (C 1) to ₄ (C 4) ≪ / RTI >
12. The composition of claim 11, wherein the fraction is obtained by fractionating an arrowhead methanol extract with water, methanol or a mixed solvent thereof.

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