KR102189109B1 - A composition for improving, preventing and treating of asthmatic containing oriental medicine herbs oil extract as an active ingredient - Google Patents

Abstract

The present invention relates to a composition for the improvement, prevention or treatment of asthma by containing a mixed fat-soluble fraction mixed extract including a fir fat-soluble fraction extract and a peppermint fat-soluble fraction extract as an active ingredient, thereby reducing inflammatory cytokines IL-4 and IL-13 And, since it reduces mucus and inflammatory cells in the bronchi even in histopathology, it is effective in improving, preventing or treating asthma.

Description

A composition for improving, preventing and treating of asthmatic containing oriental medicine herbs oil extract as an active ingredient, containing oil-soluble fraction extract of herbal medicine as an active ingredient}

The present invention relates to a composition capable of improving, preventing, or treating asthma by containing a fat-soluble fraction extract of herbal medicine as an active ingredient.

Asthma is a disease characterized by irritability of the airways to various stimuli. Clinical symptoms such as wheezing, shortness of breath, and cough caused by widespread narrowing of the airways are naturally or reversibly improved by treatment. Can be. Most asthma are allergic, characterized by chronic airway inflammation and bronchial hyperresponsiveness (Minoguchi K and Adachi M. Pathophysiology of asthma. In: Cherniack NS, Altose MD, Homma I, editors) Rehabilitation of the patient with respiratory disease.New York: McGraw-Hill, pp97-104, 1999).

In most countries around the world, including Korea, the number of asthma patients is increasing by about 20-50% every 10 years. According to the results of studies to date, the number of asthma patients worldwide reaches 150 million, and it is reported that about 180,000 deaths per year from asthma. In particular, in Korea, the proportion of children aged 6-7 years old with asthma is 13.3%, the second highest level after Singapore among eight Asian countries.

These asthma can be divided into exogenous asthma and endogenous asthma depending on the cause. In the case of exogenous asthma, it refers to asthma with symptoms when exposed to the causative antigen. A skin test or a bronchial challenge test for the causative antigen is positive, and the onset age is usually young. Usually, house dust and mites are the most causative antigens, and pollen, animal epithelium, and fungi act as causative antigens.

In terms of pathophysiology, asthma is recognized as a chronic inflammatory disease caused by proliferation, differentiation, and activation of inflammatory cells by cytokines produced by Th2 immune cells, migrating to and infiltrating the airways and surrounding tissues (Elias JA, et al. ., New insights into the pathogenesis of Asthma., J. Clin. Invest., 111, pp291-297, 2003). In this case, inflammatory cells such as activated eosinophils, mast cells, alveolar macrophages, etc. secrete various inflammatory mediators (Barnes PJ, Chung KF, Page CP. Inflammatory mediators of asthma: An update., Pharmacol. Rev., 50, pp515-596, 1998).

Cysteine leukotriene, secreted from inflammatory cells by various stimuli such as allergens, cold air, exercise and chemical stimulation, is directly related to the influx and accumulation of eosinophils (Busse WW. Leukotrienes and inflammation. Am. J. Respir. Crit. Care Med., 157, ppS210-S213, 1998). Eosinophils also produce large amounts of cysteine leukotriene, so their increased airway tissue and bronchoalveolar lavage fluid (BALF) is an important factor in exacerbating asthma (Underwood DC, Osborn RR, Newsholme SJ, Torphy TJ, Hay DWP. Persistent airway eosinophilia after leukotriene (LT) D4 administration in the guinea pig.Am. J. Respir. Crit. Care Med., 154, pp850-857, 1996).

Therefore, the production of cytokines such as IL-4 and IL-13, which are involved in the activation of inflammatory cells, and the biosynthesis of cysteine leukotriene secreted from inflammatory cells such as eosinophils through their actions are the main causes of inflammation and allergic reactions and resulting asthma. Many studies are being conducted to develop drugs to inhibit their production.

Korean Patent Registration No. 0822760 Korean Patent Registration No. 0685285

It is an object of the present invention to provide a pharmaceutical composition capable of preventing or treating asthma by containing a fat-soluble fraction extract of herbal medicine as an active ingredient.

In addition, another object of the present invention is to provide a health functional food capable of preventing or improving asthma by containing a fat-soluble fraction extract of herbal medicine as an active ingredient.

The pharmaceutical composition capable of preventing or treating asthma of the present invention for achieving the above object may contain a mixed fat-soluble fraction mixed extract comprising a fir fat-soluble fraction extract and a peppermint fat-soluble fraction extract as an active ingredient.

Each of the fat-soluble fraction extract may be a fat-soluble fraction extract extracted by a supercritical extraction method or a steam distillation extraction method.

During the supercritical extraction, the solvent may be carbon dioxide.

The fir tree fat-soluble fraction extract and peppermint fat-soluble fraction extract may be mixed in a weight ratio of 1: 0.3-1.

The mixed fat-soluble fraction extract can inhibit the production of interleukin-4 (IL-4) and interleukin-13 (IL-13) in bronchoalveolar lavage fluid.

In addition, the health functional food capable of preventing or improving asthma of the present invention for achieving the above-described other objects may contain a mixed fat-soluble fraction mixed extract including a fir fat-soluble fraction extract and a peppermint fat-soluble fraction extract as an active ingredient. .

The composition capable of improving, preventing or treating asthma of the present invention reduces inflammatory cytokines IL-4 and IL-13 by using a mixture of a fir fat-soluble fraction extract and a peppermint fat-soluble fraction extract, and even in histopathology, mucus in the bronchi And since it reduces the inflammatory cells around the bronchi, it is effective in improving, preventing or treating asthma.

1 is a graph measuring the number of inflammation-related cells in BALF according to the concentration of the mixed fat-soluble extract prepared according to Example 1 of the present invention.
2 is a graph showing the concentration of IL-4 according to the concentration of the mixed fat-soluble extract prepared according to Example 1 of the present invention.
3 is a graph showing the concentration of IL-13 according to the concentration of the mixed fat-soluble extract prepared according to Example 1 of the present invention.
4 is a diagram illustrating the proliferation of goblet cells secreting mucin in the bronchi according to the concentration of the mixed fat-soluble extract prepared according to Example 1 of the present invention.
5 is a diagram illustrating proliferation of inflammatory cells around the bronchi according to the concentration of the mixed fat-soluble extract prepared according to Example 1 of the present invention.
6 is a graph measuring OVA-specific IgE in blood according to the concentration of the mixed fat-soluble extract prepared according to Example 1 of the present invention.

The present invention relates to a composition capable of improving, preventing, or treating asthma by containing a fat-soluble fraction extract of herbal medicine as an active ingredient.

The composition of the present invention may be inhaled with a fragrance or applied to the skin, and may be administered orally or parenterally, and may be used in various ways such as ingestion as food.

Hereinafter, the present invention will be described in detail.

The composition capable of improving, preventing or treating asthma of the present invention contains a mixed fat-soluble fraction mixed extract comprising a fir fat-soluble fraction extract and a peppermint fat-soluble fraction extract as an active ingredient.

The fir tree is an evergreen needle-leaf tall tree of the pine family and grows about 30-40 m tall, and a fir branch is used in the present invention. The fir branches are used to treat uterine bleeding, cold body, diarrhea, gastrointestinal disease, and gum disease.

The main component of peppermint is menthol, and this menthol can be used for medicinal purposes, such as coating agents, pain relievers, stimulants, stomach drugs, and anthelmintics.

The fir tree fat-soluble fraction extract and peppermint fat-soluble fraction extract are mixed in a weight ratio of 1:0.3-1, preferably 1:0.5-0.8. If the content of the peppermint fat-soluble fraction extract based on the fir tree fat-soluble fraction extract is less than the lower limit, the efficacy against asthma may be significantly lowered, and if it exceeds the upper limit, the preference of fragrance decreases, and trouble may occur when applied to the skin. .

Each oil-soluble fraction extract of the present invention may be extracted by a supercritical extraction method or a steam distillation extraction method.

The supercritical extraction method obtains a fat-soluble fraction extract of each of the fir and mint in a supercritical state at a pressure of 300 to 400 bar and a temperature of 40 to 60 °C. At this time, the solvent is not particularly limited as long as it is possible to obtain a fir tree fat-soluble fraction extract and a peppermint fat-soluble fraction extract, but for high extraction efficiency and excellent efficacy against pain, carbon dioxide is used in a critical state (critical temperature of carbon dioxide: 31 ℃, It is preferable to extract at critical pressure: 74 bar).

In addition, the supercritical extraction conditions are different for each substance for high extraction efficiency and excellent efficacy against asthma. Specifically, the firs are extracted in a supercritical state at a pressure of 340 to 360 bar and a temperature of 52 to 54 °C; It is preferable to extract the mint in a supercritical state at a pressure of 350 to 370 bar and a temperature of 46 to 51 °C.

When the pressure during supercritical extraction is less than the lower limit, the fat-soluble fraction may hardly be extracted from the herbal medicine, and when the pressure exceeds the upper limit, the odor may disappear. In addition, when the temperature during the supercritical extraction is less than the lower limit, the extraction efficiency may be low, and when the temperature exceeds the upper limit, the efficacy against asthma may be lowered.

In addition, the steam distillation extraction method is an extraction method based on the principle of volatilizing essential oil components on the surface or tissue of a medicinal herb with steam and then rapidly cooling and concentrating, a pressure of 10 to 30 bar, a temperature of 100 to 120 °C, and 7 to 9 hours In the state of time, the fat-soluble fraction extract of each of the fir and mint is obtained.

At this time, the steam distillation extraction conditions are different for each substance for high extraction efficiency and excellent efficacy against pain. Specifically, the fir tree is extracted at a pressure of 13 to 14 bar, a temperature of 113 to 115° C., and 7.5 to 8.5 hours; The mint is preferably extracted at a pressure of 15 to 17 bar, a temperature of 115 to 117 °C, and 7 to 8 hours.

When the pressure during steam distillation extraction is less than the lower limit, the oil-soluble fraction may hardly be extracted from the herbal medicine, and when the pressure is greater than the upper limit, the odor may disappear. In addition, when the temperature during the steam distillation extraction is less than the lower limit, the extraction efficiency may be low, and when the temperature exceeds the upper limit, the efficacy against asthma may be lowered. In addition, when the time during the steam distillation extraction is less than the lower limit, the oil-soluble fraction may hardly be extracted from the herbal medicine, and when the time exceeds the upper limit, the odor may be altered.

The supercritical extraction method (yield: 0.4 to 5%) or steam distillation extraction method (yield: 0.2 to 5%) of the present invention has excellent odor and extraction efficiency compared to the case of extraction by other extraction methods such as compression method and solvent dialysis method. It is high and may have better efficacy against asthma. The yield of the compression method and the solvent dialysis method is very low as 0.001 to 0.01%, respectively.

The'extraction efficiency' refers to the amount of the fir tree fat-soluble fraction extract and the peppermint fat-soluble fraction extract.

The composition of the present invention may be adsorbed on a support to slowly release the fragrance and composition for a long time. The support is preferably corrugated porous silica particles, and in order to further adsorb volatile organic compounds contained in the composition of the present invention It is preferable to use porous silica particles whose surface is modified with silanol groups, amine groups or epoxy groups.

The fat-soluble fraction extract prepared as described above may be used as an extract itself or may be powdered for convenience. The drying method performed to powder the extract may include freeze drying, cold air drying, or natural drying, but it is preferable to use a freeze-drying method in order to obtain powder quickly without altering the components.

Meanwhile, in the present specification, the term'containing as an active ingredient' means to include an amount sufficient to achieve the efficacy or activity of the fat-soluble fraction extract. As an example, the fat-soluble fraction extract is used in a concentration of 10 to 1500 mg/kg, preferably 100 to 1000 mg/kg. Since the fat-soluble fraction extract is a natural product and does not have side effects on the human body even if it is administered in an excessive amount, the upper limit of the quantity of the fat-soluble fraction extract included in the composition of the present invention can be selected and carried out by a person skilled in the art within an appropriate range.

The pharmaceutical composition of the present invention may be prepared using a pharmaceutically suitable and physiologically acceptable adjuvant in addition to the active ingredient, and the adjuvant includes excipients, disintegrants, sweeteners, binders, coating agents, expanding agents, lubricants, It is possible to use a lubricant or flavoring agent.

For administration, the pharmaceutical composition may contain one or more pharmaceutically acceptable carriers in addition to the above-described active ingredients, and may be preferably formulated into a pharmaceutical composition.

The formulation form of the pharmaceutical composition may be granules, powders, tablets, coated tablets, capsules, suppositories, solutions, syrups, juices, suspensions, emulsions, drops, or injectable solutions. For example, for formulation in the form of tablets or capsules, the active ingredient may be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. In addition, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included in the mixture. Suitable binders are, but are not limited to, natural sugars such as starch, gelatin, glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, lacquercanth or sodium oleate, sodium stearate, magnesium stearate, sodium Benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.

As acceptable pharmaceutical carriers for compositions formulated as liquid solutions, sterilization and biocompatible, saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added as necessary. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to prepare injectable formulations such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules, or tablets.

Furthermore, it can be preferably formulated according to each disease or ingredient using a method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA as an appropriate method in the field.

The pharmaceutical composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, it may be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, etc., preferably oral administration. .

A suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, mode of administration, age, weight, sex, pathological condition, food, administration time, route of administration, excretion rate and response sensitivity of the patient, Usually, the skilled practitioner can readily determine and prescribe the dosage effective for the desired treatment or prophylaxis. According to a preferred embodiment of the present invention, the daily dosage of the pharmaceutical composition of the present invention is 0.001-10 g/kg.

The pharmaceutical composition of the present invention is prepared in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person having ordinary knowledge in the technical field to which the present invention belongs. Or it can be made by incorporating it into a multi-dose container. At this time, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet or capsule, and may additionally include a dispersant or a stabilizer.

In addition, the present invention provides a food composition for improving, preventing or treating asthma, containing a fat-soluble fraction extract as an active ingredient.

The food composition according to the present invention may be formulated in the same manner as the pharmaceutical composition and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, alcoholic beverages, confectionery, diet bars, dairy products, meat, chocolate, pizza, ramen, other noodles, gum, ice cream, vitamin complexes, health supplements. Etc.

The food composition of the present invention may include not only a fat-soluble fraction extract as an active ingredient, but also an ingredient commonly added during food production, and includes, for example, protein, carbohydrate, fat, nutrients, flavoring and flavoring agents. . Examples of the aforementioned carbohydrates include monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, oligosaccharides, and the like; And polysaccharides, for example, common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents, natural flavoring agents [taumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.]) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used. For example, when the food composition of the present invention is made of drinks and beverages, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, and various plant extracts, etc. may be further included in addition to the fat-soluble fraction extract of the present invention. .

The present invention provides a health functional food comprising a food composition for improving, preventing or treating asthma comprising the fat-soluble fraction extract as an active ingredient. Health functional foods are foods prepared by adding fat-soluble fraction extract to food ingredients such as beverages, teas, spices, chewing gum, confectionery, or encapsulated, powdered, or suspension. However, unlike general drugs, it has the advantage of not having side effects that may occur when taking drugs for a long time by using food as a raw material. The health functional food of the present invention obtained in this way is very useful because it can be consumed on a daily basis. The amount of the fat-soluble fraction extract added in such health functional foods cannot be uniformly regulated depending on the type of health functional food to be targeted, but it can be added within the range that does not damage the original taste of the food. It is in the range of 0.01 to 50% by weight, preferably 0.1 to 20% by weight. In addition, in the case of health functional foods in the form of pills, granules, tablets or capsules, it is usually added in the range of 0.1 to 100% by weight, preferably 0.5 to 80% by weight. In one embodiment, the health functional food of the present invention may be in the form of a pill, tablet, capsule or beverage.

In addition, the present invention provides a use of a fat-soluble fraction extract for the manufacture of a medicine or food for relieving, ameliorating, preventing or treating pain. As described above, the fat-soluble fraction extract can be used for relieving, ameliorating, preventing or treating pain caused by side effects of chemotherapy.

In addition, the present invention provides a method for alleviating, ameliorating, preventing or treating pain, comprising administering an effective amount of a fat-soluble fraction extract to a mammal.

The term "mammal" as used herein refers to a mammal that is the subject of treatment, observation or experimentation, and preferably refers to a human.

The term "effective amount" as used herein refers to the amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human, which is considered by a researcher, veterinarian, doctor or other clinician, Includes an amount that induces relief of symptoms of the disease or disorder. It is apparent to those skilled in the art that the effective amount and the number of administrations for the active ingredient of the present invention will vary depending on the desired effect. Therefore, the optimal dosage to be administered can be easily determined by those skilled in the art, and the type of disease, the severity of the disease, the amount of active ingredients and other ingredients contained in the composition, the type of formulation, and the age, weight, and general health of the patient. It can be adjusted according to various factors including condition, sex and diet, time of administration, route of administration and secretion rate of the composition, duration of treatment, and drugs used simultaneously. In the prophylaxis, treatment or improvement method of the present invention, in the case of an adult, it is preferable to administer the fat-soluble fraction extract at a dose of 0.001 g/kg to 10 g/kg when administered once to several times a day.

In the treatment method of the present invention, the composition containing the fat-soluble fraction extract as an active ingredient is a conventional method through oral, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, topical, intraocular or intradermal routes. Can be administered.

Hereinafter, a preferred embodiment is presented to aid the understanding of the present invention, but the following examples are only illustrative of the present invention, and it is obvious to those skilled in the art that various changes and modifications are possible within the scope and spirit of the present invention, It is natural that such modifications and modifications fall within the appended claims.

Manufacturing example 1. Using steam distillation extraction method

After drying the fir and mint to a moisture content of 5%, each extract was obtained by steam distillation extraction.

In order to efficiently extract natural essential oils using the steam distillation extraction method, pressure, temperature conditions, and extraction time are very important, so extraction was performed with different conditions for each sample to obtain a high yield.

[Equation 1]

division Pressure (bar) Temperature(℃) Time(hr) Recovery rate (%) fir 13-14 113-115 7.5-8.5 4.90 mint 15-17 115-117 7-8 0.54

Manufacturing example 2. Supercritical Use of extraction method

After drying fir and mint to a moisture content of 5%, put it in a supercritical extractor (ILSHIN Auto, Korea) using a supercritical carbon dioxide recirculation method, and then inject carbon dioxide to extract the fat-soluble fraction extract under supercritical conditions. %) was measured. At this time, the flow rate of carbon dioxide is 30 g/min.

division Pressure (bar) Temperature(℃) Time(hr) Recovery rate (%) fir 340-360 52-24 8-8.5 2.43 mint 350-370 46-51 8-9.5 4.51

As shown in Tables 1 and 2 above, it was confirmed that the yield of the fat-soluble fraction extract extracted by supercritical extraction of fir and peppermint, respectively, was higher than that of the fat-soluble fraction extract extracted by steam distillation extraction method.

Thus, hereinafter, an experiment was performed using the fat-soluble fraction extract extracted by the supercritical extraction method.

Example 1. Mixed fat-soluble fraction extract

The fir tree fat-soluble fraction extract and the peppermint fat-soluble fraction extract extracted by the supercritical extraction method were mixed in a weight ratio of 1:0.7.

<Test Example>

Animal experiment

A 6-week-old specific pathogen-free white paper female (Balb/c) (weight: about 13-22 g) was purchased from Orient Co., Ltd. (Seoul, Korea), and then 1% dissolved in aluminum hydroxide in the mouse. Ovalbumin (OVA) solution (OVA dissolved in physiological saline and aluminum hydroxide is mixed in a weight ratio of 1:1, and left at room temperature for 30 minutes to allow OVA to be absorbed in aluminum hydroxide) on days 0, 7, 14 Each intraperitoneal administration was performed once (same site) to induce a systemic allergic immune response. The mice in which the systemic allergic immune response is induced are placed in an acrylic box of a certain standard once a day from the 24th to the 26th (for a total of 3 days), and 1% egg albumin is added to the nebulizer (Nebulizer, NE-U17, OMRON Healthcare Co., Ltd.) Ltd., Japan) inhaled for 20 minutes to prepare a mouse model of egg albumin-induced asthma. The mixed fat-soluble extract of Example 1 or dexamethasone (Sigma Aldrich) was transdermally and aspirated once a day for 6 days during inhalation sensitization. In the normal group and the negative control group, the excipient was administered by transdermal and aspiration at 0.2 mg/kg/day.

-6 groups of mice-

Normal group: 6 mice administered physiological saline for 3 weeks

Negative control: OVA-administered 6 mice_nan albumin-induced asthma mice

Example 1 (25): OVA + extract of Example 1 transdermal & suction administration 6 mice_0.2 mg/kg/day (dosage: 25% by weight; low concentration extract)

Example 1 (50): OVA + extract of Example 1 transdermal & suction administration 6 mice_0.2 mg/kg/day (administration dose: 50 wt%; medium concentration extract)

Example 1 (100): OVA + extract of Example 1 transdermal & suction administration 6 mice_0.2 mg/kg/day (administered dose: 100% by weight; extract of high concentration)

Positive control: OVA + dexamethasone oral administration 6 mice_10 ml/kg/day (dose: 1 mg/kg)

Asthma is an allergic disease caused by an allergic inflammatory reaction of the bronchi, and ovalbumin (OVA), widely known as allergen, was exposed to test animals by intraperitoneal and inhalation methods to induce the allergic disease.

In the case of allergic asthma, an inflammatory reaction is initiated by cytokines such as IL-4 and IL-13, and mucus is produced in endothelial cells of the airways.

Test Example 1. Measurement of the number of inflammation-related cells in bronchoalveolar lavage fluid (BALF)

In order to measure the effect of reducing the number of inflammatory immune cells in the bronchial alveolar lavage solution of oalbumin-induced asthma mice for the mixed fat-soluble extract of the present invention, the following experiment was performed.

The bronchoalveolar labage fluid (BALF) of the mouse was anesthetized the day after the last administration of the test substance, and then 0.5 mL PBS was injected by inserting a tube into the bronchi, and the process of recovering was repeated three times to obtain a bronchoalveolar lavage solution. . After separating the supernatant and the cell pellet by centrifugation at 3,000 rpm for 5 minutes, the blood cells in the bronchoalveolar lavage fluid were stained using a Diff-Quick staining kit (Lot No.: ZS7006, Sysmex, Japan) to measure the number. The cell pellet was suspended in 0.1 mL of PBS, plated on a slide glass, dried, fixed with methanol, immersed in Eosin solution for a total of 5 times at 1 time/second, and immersed in Methylene Blue solution for a total of 5 times at 1 time/second. After washing, drying with DW, sealing with a mounting solution and observing under a microscope, blood cells (eosinophils, neutrophils, macrophages), lymphocytes, total cells, etc. ) Types and numbers were analyzed.

1 is a graph measuring the number of inflammation-related cells in BALF according to the concentration of the mixed fat-soluble extract prepared according to Example 1 of the present invention.

As shown in Figure 1, eosinopils, neutropils, lymphocytes, and macrophages in the alveolar lavage fluid of the negative control group in which asthma was induced were on average 6.80 X 10 ⁵ cells/mL, respectively, The total number of cells was increased by about 10 times compared to the normal group without causing asthma in 1.18 X 10 ⁵ cells/mL, 0.77 X 10 ⁵ cells/mL, and 0.96 X 10 ⁵ cells/mL.

On the other hand, it was confirmed that the group to which the mixed fat-soluble extract of Example 1 was administered decreased the number of inflammation-related cells in the alveolar lavage fluid in a concentration-dependent manner, especially compared to the negative control group.

The eosinophils increased in the lungs of asthma patients showed a tendency to decrease in a concentration-dependent manner in all test substance administration groups, and were statistically significant. Neutrophils, lymphocytes, and macrophages showed a tendency to decrease in a concentration-dependent manner in the test substance administration group, and were statistically significant in some administration groups.

The sum of all cells in the counted alveolar lavage fluid showed a tendency to decrease in a concentration dependent manner in the test substance administration group, and was statistically significant.

Test Example 2. Measurement of the inhibitory effect of secretion of inflammatory cytokines (IL-4, IL-13) in BALF

In order to measure the effect of inhibiting the secretion of inflammatory cytokines (IL-4, IL-13) in the serum and bronchoalveolar lavage fluid of oalbumin-induced asthma mice for the mixed fat-soluble extract of the present invention, the following experiment was performed.

Bronchoalveolar lavage supernatant was analyzed according to the manual using an IL-4 ELISA kit (Lot No.; 190443031, eBioscience, CA, U.S.A.) and IL-13 ELISA kit (Lot No.; 192004016, eBioscience).

2 is a graph showing the concentration of IL-4 according to the concentration of the mixed fat-soluble extract prepared according to Example 1 of the present invention, and FIG. 3 is a graph showing the concentration of the mixed fat-soluble extract prepared according to Example 1 of the present invention. It is a graph showing the concentration of IL-13.

2 and 3, IL-4 in the alveolar lavage fluid of the negative control group was 107.3 pg/mg protein, which was increased by about 2.5 times (p<0.05) compared to 39.4 pg/mg protein in the normal group. IL-4 decreased in Example 1 (50) and Example 1 (100) groups, and statistically significant (p<0.05) decreased in Example 1 (100) group.

In addition, IL-13 in the alveolar lavage fluid of the negative control group was 152.2 pg/mg protein, which was about 3 times higher (p<0.01) than the 50.2 pg/mg protein in the normal group. IL-13 decreased in a concentration-dependent manner in all test substance administration groups, and statistically significant (p<0.01) decreased in Example 1(50) and Example 1(100) groups.

Therefore, it was confirmed that the mixed fat-soluble extract of the present invention has an excellent effect of inhibiting the secretion of IL-4 and IL-13, Th2 cytokines secreted from T lymphocytes, which are important for regulating allergic immune responses in asthma, bronchitis or pneumonia.

Test Example 3. Histopathological examination

The lung tissue of the test animal was separated, fixed in 10% neutral formalin, embedding with Paraffin, and sectioned to a thickness of 4 μm. H&E staining was performed to confirm structural changes and the degree of infiltration of inflammatory cells, and PAS staining was performed to confirm the degree of mucus production.

Observation of mucin secretion in the bronchial airways

In order to investigate the effect of the mixed fat-soluble extract according to the present invention on inhibiting the damage to the basement membrane in the bronchi of oalbumin-induced asthma mice, periodic acid-Schiff (PAS) staining was performed on the tissue sample and the results were obtained. It is shown in Figure 4.

Figure 4 is an observation of mucin in the bronchi according to the concentration of the mixed fat-soluble extract prepared according to Example 1 of the present invention.

As shown in FIG. 4, mucus that did not appear in the normal group was secreted excessively in the negative control group that caused asthma, resulting in staining. It was observed that the mucus gradually decreased in the test substance administration group.

Therefore, it was confirmed that the mixed fat-soluble extract of the present invention has an excellent effect of improving the damage to the bronchial airways and preventing airway obstruction by inhibiting the secretion and accumulation of mucin in asthma and bronchitis.

Observation of improvement effect on bronchial damage

In order to investigate the effect of the mixed fat-soluble extract according to the present invention to improve the damage in the bronchi of oalbumin-induced asthma mice, the paraffin tissue sample was prepared and stained with H&E (Hematoxylin and Eosin), and the degree of infiltration of inflammatory cells in the bronchi Was observed under a microscope, and the results are shown in FIG. 5.

5 is an observation of proliferation of inflammatory cells around the bronchi according to the concentration of the mixed fat-soluble extract prepared according to Example 1 of the present invention.

As shown in FIG. 5, it was observed that the thickness of the airway was increased in the negative control group compared to the normal group, and inflammatory cells were concentrated around the airway and blood vessels. In the test substance administration group, it was observed that the thickness of the airway gradually decreased and the inflammatory cells decreased.

Therefore, it was confirmed that the mixed fat-soluble extract of the present invention has an excellent effect of protecting the airways in the bronchi by improving and repairing the structural damage of the bronchi in asthma and bronchitis.

Test Example 4. Measurement of OVA-specific IgE in blood

Blood was taken from the abdominal vein of the test animal and centrifuged for 15 minutes at 3,000 rpm. The separated serum was stored at ??70°C until use for the test, and OVA-specific IgE was analyzed according to the manual using an ELISA kit (Lot No.: 0538549, Cayman Chemical, MI, U.S.A.). The obtained results were standardized by quantifying the amount of protein according to the Bradford method.

6 is a graph measuring OVA-specific IgE in blood according to the concentration of the mixed fat-soluble extract prepared according to Example 1 of the present invention.

As shown in FIG. 6, the anti-OVA specific IgE in the blood of the negative control group was 4,134 mg/mL, which was increased by more than 200 times (p<0.01) compared to 197 mg/mL in the normal group. Anti-OVA specific IgE showed a tendency to increase in the test substance administration group, and statistical significance (p<0.05) was shown in Example 1 (100) group.

Asthma is an allergic disease caused by an allergic inflammatory reaction of the bronchi, and symptoms such as shortness of breath, coughing and wheezing appear. These symptoms are mediated by the inflammatory cytokines IL-4, 5, 13, and IL-4 induces the production of antibodies that specifically bind to allergens. When IgE reacts with allergens, it causes an inflammatory reaction and causes asthma or anaphylactic reaction, and eosinophis is a type of white blood cell, which increases the number in the lungs of asthmatic patients. It is the cause of inflammation. IL-5 induces the differentiation and activity of eosinophils. In addition, IL-13 is a major cytokine that causes bronchial hypersensitivity reaction, secretion of mucus and changes in airway structure, and structurally thickens the basement membrane of the airway and the smooth muscles surrounding the airway.

According to the present invention, the number of eosinophils among inflammatory cells in the test group causing asthma was rapidly increased, but decreased in a concentration-dependent manner in the test substance administration group.In addition to eosinophils, neutrophils, lymphocytes, and macrophages ) And other inflammation-related cells were also increased in the asthma-inducing group and then decreased in the test substance-administered group. In addition, as a result of observing lung tissue through H&E staining, eosinophils and inflammatory cells increased in the asthma-inducing group, but decreased in the test substance administration group.

The concentrations of IL-4 and IL-13 increased in the asthma-inducing group and then decreased in a concentration-dependent manner in the test substance-administered group. As a result of PAS staining in lung tissue, excessive secretion of airway mucus in the asthma-inducing group was observed in the test substance-administered group. Decreased. In addition, as a result of H&E staining, the increase in thickness of smooth muscle around the airway observed in the asthma-induced group was decreased in the test substance-administered group.

In conclusion, the composition of the present invention inhibits the secretion of inflammatory cytokines IL-4 and IL-13 in an OVA-induced asthma model to inhibit inflammation-related cell differentiation, inhibit mucus secretion in the airways, and maintain the original airway structure. It is judged to be effective in improving asthma. However, when the composition of the present invention was treated, the anti-OVA specific IgE in the blood was increased in a concentration-dependent manner compared to the asthma-inducing group despite the decrease in IL-4 and IL-13. The composition of the present invention produced anti-OVA specific IgE. This is believed to be due to involvement in other biological mechanisms affecting the disease.

* Statistical processing of data

Body weight and all measurement results obtained in the experiment were assayed using SPSS (Version 20.0, IBM Corp., USA). Normal group) and negative control group were tested for significance by performing an independent t-test (significance level: one-sided 0.05). One-way analysis of variance (ANOVA) was performed (significance level: 0.05) to confirm the significance between each test group with respect to the negative control group, and a multiple test of Dunnett's t-test was performed (significance level: one-sided 0.05).

Hereinafter, examples of the formulation of the composition containing the extract of the present invention will be described, but the present invention is not intended to limit it, but is intended to be described in detail.

Formulation Example 1. Preparation of powder

500 mg of fat-soluble fraction extract in Example 1

100 mg lactose

10 mg of talc

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

Formulation Example 2. Preparation of tablet

300 mg of fat-soluble fraction extract in Example 1

100 mg corn starch

100 mg lactose

2 mg of magnesium stearate

After mixing the above ingredients, tablets are prepared by tableting according to a conventional tablet preparation method.

Formulation Example 3. Preparation of Capsule

200 mg of fat-soluble fraction extract in Example 1

3 mg of crystalline cellulose

14.8 mg lactose

Magnesium stearate 0.2 mg

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare a capsule.

Formulation Example 4. Preparation of injection

600 mg of fat-soluble fraction extract in Example 1

Mannitol 180 mg

2974 mg of sterile distilled water for injection

Na ₂ HPO _4ㅇ 12H ₂ O 26 mg

It is prepared with the above ingredients per ampoule according to a conventional injection preparation method.

Formulation Example 5. Preparation of liquid formulation

4 g of fat-soluble fraction extract in Example 1

10 g of isomerized sugar

5 g of mannitol

Purified water appropriate amount

According to the usual preparation method of liquid preparation, add each ingredient to purified water to dissolve it, add lemon zest, mix the above ingredients, add purified water and add purified water to adjust the total to 100g, then fill in a brown bottle for sterilization. To prepare a liquid.

Formulation Example 6. Preparation of granules

1,000 mg of fat-soluble fraction extract in Example 1

Vitamin mixture right amount

Vitamin A acetate 70 mg

1.0 mg of vitamin E

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

0.5 mg of vitamin B6

Vitamin B12 0.2 mg

Vitamin C 10 mg

Biotin 10 mg

1.7 mg of nicotinic acid amide

50 mg folic acid

0.5 mg of calcium pantothenate

Suitable amount of inorganic mixture

1.75 mg ferrous sulfate

Zinc oxide 0.82 mg

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Dicalcium phosphate 55 mg

90 mg of potassium citrate

100 mg of calcium carbonate

Magnesium chloride 24.8 mg

The composition ratio of the vitamin and mineral mixture is relatively suitable for granules, but it is possible to arbitrarily modify the mixing ratio. After mixing the above ingredients according to a conventional granule preparation method, granules And can be used to prepare a health functional food composition according to a conventional method.

Formulation Example 7. Preparation of functional beverage

1,000 mg of fat-soluble fraction extract in Example 1

1,000 mg citric acid

100 g oligosaccharides

2 g of plum concentrate

1 g taurine

Total 900 mL with purified water

After mixing the above ingredients according to a normal health drink manufacturing method, stirring and heating the resulting solution at 85°C for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed and sterilized, and stored in a refrigerator. It is used to prepare the functional beverage composition of the invention.

The composition ratio is composed of ingredients suitable for a relatively preferred beverage in a preferred embodiment, but the mixing ratio may be arbitrarily modified according to regional and ethnic preferences, such as the demand class, the country of demand, and the purpose of use.

Claims (6)

A pharmaceutical composition for the prevention or treatment of asthma, characterized in that the fat-soluble fraction extract of fir and the extract of the peppermint fat-soluble fraction contain as an active ingredient a mixed fat-soluble fraction mixed extract in a weight ratio of 1: 0.3-1. The pharmaceutical composition for preventing or treating asthma according to claim 1, wherein each of the fat-soluble fraction extracts is a fat-soluble fraction extract extracted by a supercritical extraction method or a steam distillation extraction method. The pharmaceutical composition for preventing or treating asthma according to claim 2, wherein the solvent for the supercritical extraction is carbon dioxide. delete The pharmaceutical composition for preventing or treating asthma according to claim 1, wherein the mixed fat-soluble fraction extract inhibits the production of interleukin-4 (IL-4) and interleukin-13 (IL-13) in bronchoalveolar lavage fluid. . A health functional food for the prevention or improvement of asthma, characterized in that the fat-soluble fraction extract of fir tree and the extract of peppermint fat-soluble fraction contain as an active ingredient a mixed fat-soluble fraction mixed extract in a weight ratio of 1: 0.3-1.

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