WO2013081425A1 - Composition for preventing or treating asthma, comprising perillae semen extracts as active ingredients - Google Patents

Abstract

The present invention relates to a composition for preventing or treating asthma, comprising perillae semen extracts as active ingredients. According to the present invention, perillae semen extracts obtained from perillae semen are administered to a mouse animal model resulting in superior inhibitory effects of airway hyperreactivity and inhibiting pulmonary infiltrates with eosinophilia to thus not only inhibit the generation of IL-4 and IL-5 which are inflammatory cytokines, but also to promote the generation of interferon gamma and IL-10 to induce immunological tolerance and prevent and treat symptoms of asthma. Thus, the perillae semen extracts may be used as a pharmaceutical composition for treating asthma and as an ingredient for functional health food.

Description

Composition for the prevention or treatment of asthma, containing the device extract as an active ingredient

The present invention relates to a composition for the prevention or treatment of asthma and health functional food containing the device extract as an active ingredient.

Bronchial asthma is an allergic inflammatory disease in the bronchus, which causes inflammation of the bronchial air that enters and exits the body, thereby increasing the sensitivity of the bronchus. Inflammation of the bronchus ultimately thickens the airway muscles and leads to remodeling.

The causes of this asthma include genetic predisposition and environmental factors. Environmental factors include air pollutants, various allergens and industrial dusts, which produce allergen-specific IgE antibodies by immune responses, and the generated IgE binds to receptors on mast cell surfaces in various organs and causes allergic inflammation. Causes Internal factors include genetic factors, especially the fifth chromosome of humans, the β2-adrenoceptor genes, and the 11th, 12th, 14th and 16th chromosomes of humans, which are associated with increased IgE and bronchial hypersensitivity. It is known to cause a reaction.

Asthma, when exposed to the causative allergen, increases the Th2 immune response, suppresses the Th1 immune response, and causes airway inflammation characterized by infiltration of eosinophils. Therefore, various inflammatory cells such as eosinophils, lymphocytes, monocytes, etc. are infiltrated into the bronchus, and various kinds of cytokines, chemotactic factors, and chemical mediators are secreted from them, leading to allergic inflammation.

On the other hand, there are treatment methods for modulating the immune response of bronchial asthma, but there are avoidance therapy and desensitization therapy for the causative allergen, but it is practically impossible to avoid causative allergens.

In addition, conventional drug treatment using steroids, β2-agonists, and chromones (Cromone) is difficult to treat and refractory asthma patients who do not respond to this trend are increasing. Some limited desensitization therapy is performed only for exogenous bronchial asthma caused by allergens, but there are serious side effects such as anaphylaxis.

The most commonly used and most effective treatment is steroid (Steroid), but there is a problem that long-term use may cause serious side effects such as diabetes, hypertension, osteoporosis, glaucoma head. Therefore, there is a need for a therapeutic agent that induces a new concept of area involvement. Immune involvement is achieved by interleukin-10 (IL-10), which is released from regulatory lymphocytes. Asthma treatments that release IL-10 have not yet been invented.

In recent years, researches are being attempted to develop such asthma treatments from natural products. In the prior art, Korean Patent Application Publication No. 2011-0106242 discloses a composition for treating inflammation, allergy or asthma disease, containing paprika extract, and Korea. Korean Patent Application Publication No. 2011-00717 discloses a composition containing a white paper extract having anti-asthmatic activity.

However, the device has not yet been studied for its potential as a treatment for asthma.

In order to solve the problems of the prior art, the present inventors completed the present invention by confirming that the device extract can be used as a new therapeutic agent having excellent asthma treatment effect from natural products.

Therefore, it is an object of the present invention to provide a composition for the prevention or treatment of asthma comprising the extract (Perillae semen) as an active ingredient.

In addition, another object of the present invention is to provide an immune tolerance inducer comprising an extract of Perillae semen having the activity of promoting the production of IL-10 as an active ingredient.

In addition, another object of the present invention to provide a health functional food for the prevention or improvement of asthma comprising the extract (Perillae semen) as an active ingredient.

In order to achieve the object of the present invention as described above, the present invention provides a composition for the prevention or treatment of asthma comprising the extract (Perillae semen) as an active ingredient.

In one embodiment of the present invention, the device extract may be an extract extracted using water.

In one embodiment of the present invention, the composition may be administered to an individual in need thereof in an amount of 0.5 ~ 60mg / g bwt.

In one embodiment of the present invention, the device extract inhibits the production of inflammatory cytokines IL-4 and IL-5, and promotes the production of IFN-γ and IL-10 to prevent asthma through immune tolerance or Has therapeutic activity

In one embodiment of the present invention, the device extract inhibits allergic reactions by Th2 lymphocytes through promoting the production of IL-10, and induces a response by Th1 lymphocytes, which produces IL-10 produced by regulatory lymphocytes. This is because the immune is achieved by inducing.

In one embodiment of the present invention, the inhibition of allergic reaction by Th2 lymphocytes may occur through the inhibitory activity of IgE antibody production of the device extract.

In one embodiment of the present invention, the response induced by Th1 lymphocytes may be caused by the IgG, IgG2a antibody production promoting activity of the device extract.

In addition, the present invention provides an immunotolerant inducer comprising a perillae semen extract having an activity of promoting the production of IL-10 as an active ingredient.

In addition, the present invention provides a health functional food for the prevention or improvement of asthma comprising the extract (Perillae semen) as an active ingredient.

In one embodiment of the invention, the food is selected from the group consisting of beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gums, candy, ice cream, alcoholic beverages, vitamin complexes and health supplements It may be.

According to the present invention, as a result of administering the device extract obtained from the device to the mouse animal model induced asthma, it is excellent in suppressing the airway hyperresponsiveness, inhibits the infiltration of eosinophils into the bronchoalveolar alveoli and the inflammatory cytokine IL- The pharmaceutical composition for treating asthma by inhibiting the production of 4 and IL-5, as well as promoting the production of interferon gamma and IL-10 to prevent and treat the symptoms of asthma by induction of immune tolerance. And it can be used as a functional food material.

Figure 1 shows the results of analyzing the bronchial airway resistance of the asthma-induced mouse model treated group and the untreated control group of the present invention.

Figure 2 shows an optical microscope photograph of eosinophil cells infiltrated from the group treated with the device extract of the present invention and the untreated control group in the mouse model induced asthma.

3 is a bar graph showing the change in cell numbers of eosinophils, macrophages, lymphocytes and epithelial cells infiltrated into the bronchus in the group treated with the device extract of the present invention and the control group in the asthma-induced mouse model. .

4 is isolated from lungs from the group treated with the device extract of the present invention and the untreated control in the mouse model induced asthma, followed by RT-PCR to perform IL-4, IL-10 and interferon-gamma The photograph shows a comparative analysis of the amount of expression.

Figure 5 is a collection of bronchial alveolar lavage fluid from the group treated with the device extract of the present invention and an untreated control group in the mouse model induced asthma, and the amount of cytokines present in the bronchoalveolar lavage fluid measured spectrophotometer The results are shown.

Figure 6 shows the analysis results of bronchial airway resistance after varying the intake of the device extract of the present invention in the mouse model induced asthma.

FIG. 7 shows serum obtained from a group treated with the device extract of the present invention and an untreated control group in an asthma-induced mouse model, and the amount of IgE antibody and IgG antibody in the serum was measured by immunoenzyme method. It is shown.

The present invention is the first time that the device can be used as a new therapeutic agent for preventing and treating asthma, as well as the fact that the device extract can treat and prevent asthma through the immune tolerance mechanism through promoting the production of IL-10 According to the present invention, the present invention is characterized in that it provides a composition for preventing or treating asthma comprising the extract of Perillae semen as an active ingredient.

The device (Perillae semen) used as a material of the pharmacologically active ingredient in the present invention is not yet well known for its pharmacological efficacy, and has only antioxidant activity, and contains general ingredients, fats and oils, fatty acids and amino acid composition. As for the general ingredients, the water content is 7.5%, crude fat is 33.2%, crude protein is 16.3%, crude ash is 2.8%, crude fiber is 6.5%, and soluble nitrogen is 33.7%. Nitrogen is the main component and contains a significant amount of protein. In addition, the amino acid composition is the most essential amino acid in histidine 21.6mg%, phenylalanine 20.1mg% most, non-essential amino acid glutamic acid 66.9mg%, aspartic acid 32.5mg% high content, The ratio of essential amino acid to amino acid is known as 41.2%, but little research has been conducted on other pharmaceutical efficacy.

Therefore, the present inventors performed the following experiments to determine whether such a device can be used as a therapeutic agent for the treatment of asthma, that is, according to one embodiment of the present invention, After the administration of the extract, the degree of improvement in asthma was analyzed by analyzing the inhibitory effect of bronchial airway resistance and eosinophils and inflammatory cells into the bronchoalveolar algae. Airway resistance was reduced by about three times.

Airway resistance is caused by airway obstruction by airway hypersensitivity, a hallmark of bronchial asthma. The device extract according to the present invention was able to effectively reduce airway resistance. In addition, the number of eosinophils infiltrated with bronchial alveoli was significantly reduced in the group treated with the device extract compared to the control. Asthma is an eosinophilic chronic bronchitis, which shows anti-inflammatory effects through eosinophil suppression.

In addition, according to another embodiment of the present invention, in the case of the group treated with the device extract in the mouse animal model induced asthma, the number of macrophages infiltrated into the bronchial alveolar compared with the control group compared to the control group It was significantly increased, and was significantly suppressed compared to the eosinophil control. These results show that the present inventors have the effect of inhibiting the inflammatory response in the airway causing the asthma extract asthma.

On the other hand, asthma is a chronic inflammatory disease caused by the proliferation, differentiation and activation of inflammatory cells by cytokines produced by Th2 immune cells, migration and invasion into airway and airway tissues (Elias JA, et al. , J. Clin. Invest., 111, p291-297, 2003), wherein key mediators for inducing inflammatory and allergic diseases are leukotriens, prostaglandines and platelet activating factors. factor, PAF) and the like, which are produced from precursor arachidonic acid by phospholipase A2, cyclooxygenase and lipoxygenase.

Leukotriene constitutes a group of locally acting hormones produced in vivo from arachidonic acid, and important leukotrienes include leukotriene B4 (LTB4), leukotriene C4 (LTC4), leukotriene D4 (LTD4) and leukotriene E4 (LTE4). have. Biosynthesis of these leukotrienes begins by the enzyme 5-lipoxygenase acting on arachidonic acid to produce an epoxide known as leukotriene A4, which is produced by successive enzymatic reaction steps (LTB4, LTC4, LTD4, LTE4). Is converted to). Among them, cysteinyl leukotrienes (LCC4, LTD4, LTE4) is the substance that induces the strongest bronchial contraction action and eosinophil increase (Barnes PJ, et al., Pharmacol. Rev., 50, p515-596, 1998).

Eosinophils also produce large amounts of cysteine leukotriene, so their increase in airway tissue and bronchoalveolar lavage fluid (BALF) is an important factor exacerbating asthma (Underwood DC, et al., Am. J. Respir Crit Care Med., 154, p850-857, 1996). Therefore, inhibiting eosinophils is an important part of treating one inflammation of asthma. Asthma's inflammatory response is mainly caused by cytokines such as IL-4, IL-5 and IL-13 produced from inflammatory cells. (Gani, F. et al., Tecenti. Prog. Med., 89, p520-528, 1998)

Asthma is a complex disease involving several chemical media, cytokines, inflammatory cells, etc. Therefore, treatment for one cytokine, chemical media or a single cell is undesirable.

In particular, asthma, an airway allergy, is an airway hypersensitivity disease caused by allergic inflammation and is not a general inflammatory response by Th1 lymphocytes and neutrophils, but an allergic inflammatory response by Th2 lymphocytes and eosinophils.

Therefore, in the case of asthma treatments currently under development, methods using cytokine antibodies, that is, IL-2 antibodies, IL-5 antibodies or antagonistic cytokines of IL-8, anti-IgE antibodies, etc. have been tried. These substances are only substances that cause or worsen, and most of these substances are low molecular weight proteins, causing side effects and continuous unavailability due to their use.

Therefore, the development of a new concept as a treatment for asthma, that is, the need for the development of treatments through immune tolerance, has started to emerge, and researches to treat the treatment of asthma by the mechanism of immune tolerance are continuing. Is made through IL-10 of cytokines, and research has been attempted to develop interleukin-10 as an asthma therapeutic agent, but interleukin is a protein that causes a decrease in drug efficacy due to an immune response to heterologous proteins after prolonged administration. There is also a problem that antigen-antibody reactions occur and adversely affect other organs. Therefore, researches on therapeutic methods through substances capable of promoting their production are continuing, rather than using IL-10 itself.

In this regard, the inventors of the present invention, in order to determine whether the asthma treatment effect of the device extract of the present invention is a mechanism through immunotolerance in the embodiment of the present invention asthma-induced mice treated with the device extract group and untreated control group After pulmonary separation from the lung, the expression level of mRNA of IL-10 was analyzed by RT-PCR, and further, by measuring the amount of cytokines in the bronchial lavage lavage fluid of mice.

As a result, it was shown that the mRNA expression level of IL-10 was increased in the group treated with the device extract compared to the control group not treated (see FIG. 4). On the other hand, the expression level of the inflammatory cytokine IL-4 was shown to be decreased, and the expression level of interleukin-gamma was increased.

In addition, as a result of analyzing the cytokine amount at the protein level rather than the mRNA level, as shown in FIG. 5, the amount of IL-4 and IL-5 is asthma-induced mouse group in which the urea extract treatment group did not process urea extract. In contrast, the amount of IL-10 and interferon-gamma was significantly increased in the urea extract treatment group.

Through these results, the present inventors have found that the device extract can prevent and treat asthma through the mechanism of immune tolerance through promoting the production of IL-10 in asthma disease.

On the other hand, the immunological mechanism of allergic disease is mainly described as "hygiene hypothesis" (hygiene hypothesis, imbalance of Th1 / Th2 response). Normally, the Th1 and Th2 responses are balanced by antagonizing each other.However, in order to develop allergic diseases including bronchial asthma, the Th1 response is reduced and the imbalance of the immune system where the Th2 response is enhanced is essential. Factor (Mosmann TR, et al., J Immunol. 136: 2348-57.1986). In allergic diseases such as allergic rhinitis and asthma, abnormal Th2 immune responses caused by IL-4, IL-5, and IL-13 cause eosinophilic inflammation, and in eosinophil inflammation, Th1 and Th2 cytokines antagonize each other and are selected. Inhibition of the Th2 response is important in suppressing allergic inflammation (Wilson MS, et al., J Exp Med 202: 1199-212, 2005; Park Y, et al. J Allergy Clin Immunol 108: 570-6 , 2001).

In addition, factors that determine the differentiation between Th1 and Th2 cells include a number of factors, such as interaction between antigen-presenting cells and T cells, the amount of antigen exposed, and cytokine secretion. Plays a key role in determining / Th2 response. A representative cytokine that increases the Th2 response is IL-4, and IL-12 is known as a cytokine that increases the Th1 response.

Therefore, the present inventors confirmed that the extract of the device has an immune tolerant effect through the production of IL-10 and a response conversion (induction) effect of Th2 lymphocytes to Th1 lymphocytes involved in a general inflammatory response. According to an embodiment, blood is collected from a group treated with the device extract of the present invention and an untreated control group in an asthma-induced mouse, and then to IgG antibodies and Th2 lymphocytes, which are antibodies that drive a response by Th1 lymphocytes. The level of IgE antibody, which is the antibody leading to the response, was measured. As a result, the yolk-specific IgE antibody was decreased in the group treated with urea extract, whereas the IgG antibody and IgG1 antibody were decreased. , IgG2a antibody was shown to increase (see Fig. 7).

Therefore, the present inventors have found that the device extract of the present invention suppresses the Th2 response, which is an allergic immune response, and induces immunity to derive a fundamental asthma treatment effect.

Therefore, the present invention provides a composition for the prevention or treatment of asthma comprising the extract Perillae semen as an active ingredient, and further comprises a Perillae semen extract having the activity of promoting the production of IL-10 as an active ingredient. Provide an immune tolerance inducer.

The device that can be used in the present invention can be used as long as it is a commercially available device, the device extract can be obtained through a known extraction method for obtaining an extract from natural products, in particular solvent extraction methods known in the art It can be prepared by.

Extraction solvents include, but are not limited to, solvents selected from the group consisting of methanol, ethanol, propanol, isopropanol, butanol, acetone, ether, benzene, chloroform, ethyl acetate, methylene chloride, hexane and cyclohexane Can be. In addition, the device extract of the present invention can be used in addition to the solvent extraction method, hot water extraction, ultrasonic extraction and reflux extraction method, it can be preferably extracted using water.

In addition, when the extract is prepared using water, the ratio between the device and the water is not particularly limited, but water may be added 1 to 10 times based on the weight of the device.

The temperature during extraction is preferably performed at room temperature under atmospheric pressure and the extraction time depends on the extraction temperature, but is extracted for 1 hour to 6 hours, preferably 1 hour to 3 hours. In addition, the extraction efficiency can be further increased when stirring with a shaker (shaker) during extraction.

The device used for extraction can be harvested, washed and used as is or dried. As a drying method, both dry, shade, hot air drying and natural drying can be used. In addition, in order to increase the extraction efficiency, the device can be used by pulverizing the dry body with a grinder.

In addition, the composition for preventing or treating asthma according to the present invention may include a pharmaceutically effective amount of urea extract alone or may include one or more pharmaceutically acceptable carriers, excipients or diluents. The pharmaceutically effective amount herein refers to an amount sufficient to prevent, ameliorate and treat asthma symptoms.

The pharmaceutically effective amount of the device extract according to the present invention is 0.1 ~ 100 mg / day / kg body weight, preferably 0.5 ~ 60 mg / day / weight kg. However, the pharmaceutically effective amount may be appropriately changed depending on the degree of asthma symptoms, the age, weight, health condition, sex, route of administration and duration of treatment of the patient.

In addition, the inventors of the present invention to determine the optimum amount of treatment of asthma extract for asthma treatment based on the results of asthma improvement and treatment effect on the device extract of the present invention, 0, 0.5 , 10, 25, 50, 100mg dissolved in physiological saline was ingested using potatoes, and asthma treatment was analyzed by measuring the degree of airway resistance.

As a result, ascertaining the improvement of asthma symptoms according to the intake of the device extract, interestingly, the group ingested in the amount of 25mg showed the best asthma improvement, and then 50mg, 100mg, 10mg and 0.5mg in order (See FIG. 5). Therefore, through these results, the inventors of the present invention, the optimum intake of the device extract that can be used for the asthma treatment effect may be 15 ~ 35mg, preferably 20 ~ 30mg, more preferably 25mg.

In addition, the pharmaceutically acceptable refers to a composition that is physiologically acceptable and does not cause an allergic reaction such as gastrointestinal disorders, dizziness or the like when administered to humans. Examples of such carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In addition, fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers and preservatives may be further included.

In addition, the compositions of the present invention may be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. The formulations may be in the form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, sterile powders.

Compositions for the prevention and treatment of asthma symptoms according to the present invention can be administered via a variety of routes including oral, transdermal, subcutaneous, intravenous or intramuscular, the dosage of the active ingredient being determined by the route of administration, the age, sex, weight and It may be appropriately selected depending on various factors such as the severity of the patient. In addition, the composition for the prevention and treatment of asthma symptoms of the present invention can be administered in parallel with a known compound having the effect of preventing, improving or treating asthma symptoms.

Furthermore, the extract of the device according to the present invention is an extract obtained from the device which is a natural product and has stability which does not cause other side effects in the body, so the device extract of the present invention can be added to food for the purpose of preventing and improving asthma symptoms. , The present invention can be used as a food composition for the prevention or improvement of asthma symptoms comprising the device extract as an active ingredient.

Therefore, the composition for food for the prevention and improvement of asthma symptoms of the present invention can be easily utilized as foods, such as the main ingredients, secondary ingredients, food additives, functional foods or beverages that are effective in preventing and improving asthma symptoms.

As used herein, the food means a natural product or processed product containing one or more nutrients, and preferably means a state in which it can be directly eaten through a certain processing process, and as a general meaning, food It includes all food additives, functional foods and drinks.

Foods to which a composition for preventing and improving asthma symptoms according to the present invention can be added include, for example, various foods, beverages, gums, teas, vitamin complexes, functional foods, and the like. In addition, in the present invention, food includes special nutritional products (e.g., formulated milk, young, infant food, etc.), processed meat products, fish products, tofu, jelly, noodles (e.g. ramen, noodles, etc.), bread, health supplements, seasonings. Foods (e.g. soy sauce, miso, red pepper paste, mixed soy sauce), sauces, confectionery (e.g. snacks), candy, chocolates, gums, ice creams, dairy products (e.g. fermented milk, cheese, etc.), other processed foods, kimchi, Pickled foods (various kimchi, pickles, etc.), beverages (e.g., fruit drinks, vegetable drinks, soy milk, fermented beverages, etc.), natural seasonings (e.g. ramen soup, etc.) are not limited thereto. The food, beverage or food additives may be prepared by a conventional manufacturing method.

In addition, the functional food is a biological defense rhythm control, disease prevention and recovery of a food group or a food composition that has added value to the food by using physical, biochemical, biotechnological techniques, etc. to function and express the function of the food for a specific purpose. It means a food that is designed and processed to fully express the body regulatory function related to the living body, specifically, it may be a health functional food. The functional food may include food acceptable food additives, and may further include appropriate carriers, excipients and diluents commonly used in the manufacture of functional foods.

In addition, in the present invention, the drink refers to a generic term for drinking to quench thirst or enjoy a taste and includes a functional drink. The beverage contains, as essential ingredients, a composition for the prevention and amelioration of the asthma symptom as an essential ingredient, and there are no special limitations on the other ingredients, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. Can be.

Furthermore, in addition to the above, foods containing compositions for the prevention and improvement of asthma symptoms of the present invention include various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavoring agents, such as colorants and fillers (cheese) , Chocolate and the like), pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. The components can be used independently or in combination.

In the food containing the composition for the prevention and improvement of asthma symptoms of the present invention, the amount of the composition according to the present invention may comprise from 0.001% to 90% by weight of the total food weight, preferably from 0.1% by weight to 40% by weight, and in the case of a beverage, may be included in a ratio of 0.001g to 2g, preferably 0.01g to 0.1g based on 100ml, but for health and hygiene purposes or for health control purposes. In the case of long-term ingestion may be below the above range, the active ingredient is not limited to the above range because it can be used in an amount above the above range because there is no problem in terms of safety.

Hereinafter, the present invention will be described in detail with reference to Examples and drawings. However, these examples are intended to illustrate the present invention in more detail, and the scope of the present invention is not limited to these examples.

<Example 1>

Preparation of Device Extract

Perillae semen was purchased on the market, dried and pulverized using a pulverizer. Then, 1 L of water was added to 480 g of the pulverized pulverized element, and concentrated by obtaining a urea extract.

<Example 2>

Identification of Asthma Treatment Effect of Delirium Extract from Mouse Animal Model Experiments

<2-1> Analysis of effect on airway resistance of extract

In order to analyze the efficacy of the device extract obtained in Example 1 can cure asthma, the inventors first prepared an asthma mouse model to analyze the effect of the device extract on the resistance in the airways. In other words, 6 weeks-old male C57BL / 6 mice were sensitized yolk to create an asthma-induced mouse model. More specifically, 20 μg of egg yolk was dissolved in 100 μl of physiological saline, and then the same volume of Imject Alum complex was added thereto, and 200 μl of this yolk solution was injected intraperitoneally of the mouse. Thereafter, 2% egg yolk was sensitized daily using a nebulizer (neubilizer, Pari-Inhaler Boy) for 10 minutes every day from day 21 to day 23 of the experiment to prepare a mouse model induced asthma.

Thereafter, from the 23rd to 25th day of the experiment, the device extract prepared in Example 1 was prepared as a freeze-dried powder, and then dissolved in physiological saline to ingest 50 mg of the device extract into mice using potatoes. At this time, as a control group, a group of asthma-induced mice in which physiological saline containing no device extract was ingested with potatoes was used.

Subsequently, the degree of airway resistance induced by airway obstruction was measured to investigate whether the device extracts inhibit airway hypersensitivity, a hallmark of bronchial asthma. After inhaling 2% egg yolk for 10 minutes to sensitize yolk, gradually increase the concentration from 2.5 mg / ml to 25 mg / ml of methacholine for 2 minutes in each mouse group, and then measure the degree of airway resistance. It was calculated as Penh (enhanced pause) using 3000 (Allmedicus, Anyang, Korea).

As a result, as shown in Figure 1, when the device extract of the present invention administered to the asthma mouse model and induced airway hypersensitivity, airway resistance is significantly reduced compared to the control group administered physiological saline instead of the device extract Compared with the control group, the group treated with the device extract reduced airway resistance by about three times.

Therefore, through these results, the inventors found that the device extract has an excellent effect on the asthma treatment effect.

<2-2> Analysis of Inflammatory Inhibitory Effect of Extracts

Experimental mice and control mice used in Example <2-1> (the group treated with the device extract of the present invention to an asthma-induced mouse and the group treated with physiological saline instead of the device extract) after anesthesia chest of each mouse Incision was performed to separate the airways, and then a polyethylene tube was inserted, 1 ml of saline was injected into the airways through the tube, and the lungs were gently massaged to collect bronchoalveolar lavage fliude (BALF). Subsequently, a total of 1.8 ml of the collected bronchoalveolar lavage fluid was centrifuged to separate cells, and a slide was made using cytospin, an automatic cell smearing device. Light-Giemsa staining was performed, and then 100 cells were counted at a × 400 magnification field using an optical microscope to calculate the number of infiltrating eosinophils. In addition, neutrophils and macrophages were also examined by an optical microscope to measure the number of cells.

As a result, as shown in Figure 2, compared to the control group treated with the device extract of the present invention showed that the number of eosinophils as well as the total number of inflammatory cells infiltrated into the bronchoalveolar alveolar due to sensitizing yolk.

In addition, as shown in the results of FIG. 3, in the asthma mouse group treated with the device extract as compared to the asthma mouse group (control) without the device extract, the number of macrophages infiltrated into the bronchoalveolar alveoli was significantly higher than that of the control group. Increased eosinophilic leukocytes were significantly inhibited compared to the control.

These results show that the present inventors have the effect of inhibiting the inflammatory response in the airway causing the asthma extract asthma.

<2-3> Inflammation-related cytokine expression analysis by RT-PCR

Furthermore, the present inventors conducted the following experiment to determine whether the effect of the device extract to prevent or treat asthma is achieved through the control of expression of inflammation-related cytokines. That is, mRNAs were extracted by separating the lungs of the mice used in the experiment of Example <2-2>. Thereafter, 3 μg of mRNA extracted from the lungs of each mouse using Diastar RT-kit was performed using RT-PCR using respective primers capable of amplifying IL-4, IL-10, and IFN-γ. As a result of the PCR, the amplified bands were identified at 399bp for IL-4, 313bp for IL-5, 460bp for IFN-γ, and 300bp for IL-10. Primers used for RT-PCR are as described in Table 1 below. In addition, the PCR reaction conditions were 35 cycles by GAPDH annealing for 1 minute at 58 ℃, 40 ℃ at 72 ℃, IL-4, IFN-γ, IL-10 1 minute at 94 ℃, 1 minute at 56 ℃ Annealing at 72 ° C. for 2 minutes and extension cycle at 35 ° C. for 10 minutes were carried out for 35 cycles. PCR was performed using DNA thermal Cycle 480 (Perkin ElmerNorwalk, USA), and the resulting product was electrophoresed to identify each band at 399 bp for IL-4 and 460 bp for IFN-γ at 1L-10 300 bp.

Table 1 Primer sequence

		Primer sequence	Annealing temperature	cycle
IL-4	Forward direction	5'-ATGGGTCTCAACCCCCAGCAT-3 '	56 ℃	35
IL-4	Reverse	5'-GCTCTTTAGGCTTTCCAGGAAGTC-3 '
IFN-	Forward direction	5'-TGAACGCTACACACTGCATCTTGG-3 '	56 ℃	35
IFN-	Reverse	5'-CGACTCCTTTTCCGCTTCCTGAG-3 '
IL-10	Forward direction	5'-AGCTGGACAACATACTGCTAACC-3 '	56 ℃	40
IL-10	Reverse	5'-TCATTCATGGCCTTGTAGACAC3-3 '
GAPDH	Forward direction	5'-TCTACCCACGGCAAGTTCAA-3 '	58 ℃	35
GAPDH	Reverse	5'-GGATGACCTTGCCCACAGC-3 '

As shown in FIG. 4, the expression level of the cytokine expression of IL-4 was significantly decreased in the experimental group treated with the device extract compared to the control group, whereas the expression levels of IFN-γ and IL-10 were significantly reduced. Was increased in the experimental group (element extract treatment group) compared to the control group.

From these results, the present inventors have found that the device extract of the present invention can prevent, inhibit and treat asthma with immune tolerance through promoting the production of IL-10 cytokines.

<2-4> Inflammation-related cytokine expression analysis at the protein level

In Example <2-3>, if the inhibitory activity of inflammation-related cytokine production of the device extract according to the present invention was confirmed at the gene level by PCR method, in this Example, the expression level of the actually expressed cytokine was expressed at the protein level. Confirmed. To this end, bronchoalveolar lavage fluid was collected from each mouse used in the experiment of Example <2-2> and stored at −70 ° C., and cytokine IL-4, 5, 10 and INF-γ were quantified by the Quantikine kit. It was measured by the immunoassay using (R & D Systems).

More specifically, 50 μl of analytical dilution RD1 per well was put into a 96-microwell plate for immunoenzyme, the plate was covered with a cover, and then left at room temperature and then removed, and each sample was left at room temperature for 2 hours. After washing 5 times with a washing buffer, 100 μl of each mouse cytokine-conjugate was added to each well and allowed to stand at room temperature for 3 hours. Again washed three times with the washing solution, 100 ㎕ each substrate was placed and allowed to stand at room temperature for 30 minutes. Wash three times and add reaction stop solution and mix well. After 30 minutes, absorbance (optical density, OD) was measured with a spectrophotometer (BEP II plus, Germany). In addition, the bronchoalveolar lavage fluid was anesthetized with ketamine for each of the mice, the incision was made after the thoracic incision, and the airway was incised slightly to intubate the polyethylene tube. Then, after injecting 1 ml of saline solution, the lungs were gently massaged, and bronchial alveolar lavage fluid (Bronchoalveolar lavage fluid, BALF) was collected and used.

As a result, as shown in Figure 5, the group administered with the device extract of the present invention was significantly reduced IL-4 and IL-5 compared to the asthma-induced mouse group (group not treated with the device extract) On the other hand, IL-10 and INF-r were significantly increased in the group receiving the urea extract compared to the group not.

Therefore, through these results, the present inventors can improve the airway hypersensitivity symptoms by the device extracts to promote the activation of tidal lymphocytes to increase the production of IL-10, thereby inhibiting the Th2 allergic reaction and activating the opposite Th1 response. In addition, it was found that the inflammatory response can be suppressed by suppressing the inflammatory cells.

<Example 3>

Analysis of Asthma Treatment Effect by Treatment Concentration

Furthermore, the present inventors looked at the concentration of the device extract which can derive the maximum effect by treating the asthma mouse with the concentration of the device obtained in the present invention for each concentration, and was carried out in Example <2-1> As an asthma mouse model, the dry powder of the device extracts were dissolved in different amounts of physiological saline, and then ingested in the mouse with potatoes. Each intake dose was 0, 10, 25, 50, 100mg. It was dissolved in saline and consumed. Then, the degree of airway resistance was measured in the same manner as in Example <2-1>.

As a result, as shown in Figure 6, by confirming the degree of improvement of asthma symptoms by varying the intake of the device extract, interestingly the group ingested in an amount of 25mg appeared to have the best improvement in asthma, 50mg, In the order of 100mg, 10mg. Therefore, through these results, the inventors found that the optimum of the device extract can be used for the asthma treatment effect is 25mg.

<Example 4>

Analysis of the Effect of Solanum Extract on the Production of Allergen-Specific Sensitive IgE and IgG Antibodies

As described above, asthma is an airway hypersensitivity disease caused by allergic inflammation. In other words, inflammatory reactions occur due to exposure of allergens, wherein general inflammation is known to be led by IgG antibodies, and allergic inflammation is known to be led by IgE antibodies.

The present inventors performed the following experiment to investigate whether the device extract of the present invention affects the IgG and IgE antibody levels.

Blood was collected by puncturing the heart of the mouse for each mouse used in Example <2-2> and stored at −70 ° C., and the levels of egg yolk specific IgE, IgG1 and IgG2 antibodies were measured by immunoenzyme method. Specifically, 5 μg / ml of egg yolk was added to 20 μl per well in a 96-microwell plate for immunoenzyme and covered with a plate, and then left at 4 ° C. overnight. After removing the yolk solution, 300 μl of 1% BSA dissolved in PBS (phosp-hate buffered solution) was added to each well and left at room temperature for 1 hour. After washing three times with PBST (PBS containing 0.05% Tween 20), 100 μl of mouse serum was added to the wells and left at room temperature for 1 hour, washed three times with PBST again, and diluted with a secondary antibody. 100 μl of IgE monoclonal antibody (2 ug / ml in PBS-1% BSA) was added thereto and allowed to stand at room temperature for 1 hour. After washing three times with PBST, 100 μl of Av-HRP diluted 1: 1000 with PBS-1% BSA was added thereto and allowed to stand at room temperature for 30 minutes. After washing 3 times with PBST again, 100 μl of 30% H ₂ O ₂ was added to the substrate buffer in which ABTS was dissolved in 0.1M citric acid, and the mixture was left at room temperature for 30 minutes. 50 μl of SDS / DMF solution dissolved in 200 ml NN-dimethyl formamide (40% SDS) as a colorant was added, and then the optical density (OD) was measured at 405 nm using a spectrophotometer. The same was measured by the same immunoenzyme method, except that the concentration of the secondary antibody was diluted 1: 1 using PBS solution at 1: 10000.

As a result, as shown in Figure 7, the asthma-induced mouse group treated with the device extract of the present invention showed that the production of egg yolk, especially IgE antibody is reduced compared to the asthma-induced mouse group not treated with the device extract, while IgG The degree of production of IgG1 and IgG2 has been shown to be increased.

Therefore, through these results, the present inventors found that the device extract of the present invention can induce asthma, which is a response of Th2 lymphocytes, to a response to Th1 lymphocytes involved in a general inflammatory response, ultimately caused by Th2 cytokines. It was found that allergic asthma can be effectively prevented and treated.

So far I looked at the center of the preferred embodiment for the present invention. Those skilled in the art will appreciate that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in descriptive sense only and not for purposes of limitation. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope will be construed as being included in the present invention.

Claims (10)

1. Asthma (Perillae semen) composition for the prevention or treatment of asthma comprising the extract as an active ingredient.
2. The method of claim 1,

   The device extract is a composition for the prevention or treatment of asthma, characterized in that the extract extracted using water.
3. The method of claim 1,

   The composition is a composition for the prevention or treatment of asthma, characterized in that administered to the individual in need thereof in an amount of 0.5 ~ 60mg / g bwt.
4. The method of claim 1,

   The device extract has the activity of inhibiting the production of inflammatory cytokines IL-4 and IL-5, and promoting the production of IFN-γ and IL-10 to prevent or treat asthma through immune tolerance Composition for the prevention or treatment of asthma.
5. The method of claim 4, wherein

   The device extract is a composition for preventing or treating asthma, characterized in that by inhibiting the allergic reaction by Th2 lymphocytes through the production of IL-10, inducing a response by Th1 lymphocytes.
6. The method of claim 5,

   Inhibition of allergic reaction by Th2 lymphocytes is a composition for the prevention or treatment of asthma, characterized in that occurs through the inhibitory activity of IgE antibody production of the device extract.
7. The method of claim 5,

   Induction of response by Th1 lymphocytes is a composition for the prevention or treatment of asthma, characterized in that occurs through the activity of promoting the production of IgG, IgG1, IgG2a antibody of the device extract.
8. An immunotolerant inducer comprising an extract of Perillae semen having a production promoting activity of IL-10 as an active ingredient.
9. Health functional food for the prevention or improvement of asthma comprising the extract of Perillae semen as an active ingredient.
10. The method of claim 9,

    The food is a health functional food, characterized in that selected from the group consisting of beverages, meat, chocolate, food, confectionery, pizza, ramen, other noodles, gum, candy, ice cream, alcoholic beverages, vitamin complexes and dietary supplements.

Images