KR102247364B1 - Composition for improving respiratory diseases using the extract of Euonymus alatus - Google Patents

Abstract

The present invention discloses a composition for improving lung diseases such as chronic obstructive pulmonary disease using an arrow tree extract. Arrow tree extract inhibits the activation of netosis of neutrophil extracellular traps induced by PMA (phorbol myristate acetate), and is induced by CSE of NCI-H292 cells, a bronchial epithelial cell line that produces mucus. It inhibits the expression of IL-8, an inflammatory cytokine, and has the activity of inhibiting the expression of MIP-2, an inflammatory cytokine, in MH-S cells, alveolar macrophages activated by CES and LPS (lipopolysaccaride). In addition, it inhibits lung tissue damage in a mouse model of respiratory disease induced by CSE and PPE, and includes total cells, macrophages, lymphocytes, eosinophils, and neutrophils in BALF. It inhibits the invasion of immune cells and has the activity of inhibiting the secretion of inflammatory cytokines and chemokines in BALF and lung tissue.

Description

Composition for improving respiratory diseases using the extract of Euonymus alatus}

The present invention relates to a composition for improving chronic obstructive pulmonary disease using an arrow tree extract.

Chronic obstructive pulmonary disease (COPD) is a chronic airway disease showing cough, sputum, shortness of breath, decreased expiratory flow, and impaired gas exchange. It is predicted to be the third cause of death in humans in 2012 (Am J Respir Crit Care Med, 2013, 187:347-365; Am J Respir Crit Care Med, 2009, 180:396-406).

COPD is caused by a variety of causes, such as smoking, air pollution, chemicals, occupational factors, and genetic predisposition, and smoking is the main cause, and more than 80% of COPD patients have been found to be smokers (Biol Pharm Bull, 2012, 35:1752-1760). The pathogenesis of COPD involves inflammation, activation of proteolytic enzymes in the lungs, and oxidative stress, and the cells involved in inflammation are mainly neutrophils, macrophages, and T lymphocytes. These inflammatory cells produce reactive oxygen species, various inflammatory cytokines, and various proteolytic enzymes that cause tissue damage (Korean Tuberculosis and Respiratory Society, Respiratory Science Seoul: Gunja Publishing Co.; 2007, p 301-5;Am J Respir Crit Care Med, 1997 155, 1441-1447; Am J Physiol Lung CellMol Physiol, 2010, 298:L262-L269). In particular, neutrophils secrete substances such as elastase, collagenase, proteases such as MPO (myeloperoxidase), arachidonic acid metabolites, and reactive oxygen free radicals to the lungs. It is known to play an important role in the onset of COPD by causing damage, and therefore, it has become an important target in the development of COPD therapeutics (Am J Respir Cell Mol Biol, 2013, 48:531-539; Eur Respir J, 1998, 12:1200-1208).

Recently, it has been reported that the activation of netosis of neutrophil extracellular traps is involved in the pathogenesis of chronic lung/respiratory diseases including COPD (PLoS One. 2014 May 15;9(5):e97784) .; Respir Res. 2015 May 22;16:59.; J Immunol Res. 2017;2017:6710278; Respirology. 2016 Apr; 21(3):467-75).

So far, no drugs have been reported to directly improve COPD, and the current treatment for COPD is to reduce symptoms and complications, such as bronchodilators (β2-agonists, anticholinergics, methylxanthines) and steroids (inhalation, oral). Is mainly used.

It is an object of the present invention to provide a food composition for enhancing respiratory function and improving respiratory diseases using an arrow tree extract, a health functional food comprising the food composition, and a pharmaceutical composition for preventing or treating respiratory disease using an arrow tree extract . Other or specific objects of the present invention will be presented below.

The present inventors, as confirmed in the Examples and Experimental Examples below, the arrow tree extract inhibits the activation of netosis of neutrophil extracellular traps induced by PMA (phorbol myristate acetate), Inhibits the expression of IL-8, an inflammatory cytokine, by CSE in NCI-H292 cells, a bronchial epithelial cell line, and MIP-2, an inflammatory cytokine, also in MH-S cells, alveolar macrophages activated by CES and LPS (lipopolysaccharide). It was confirmed that it inhibits the expression of.

In consideration of the above, the present invention, in one aspect, can be understood as a composition for improving COPD comprising an extract of Euonymus alatus as an active ingredient, and in another aspect, an extract of an arrow tree is used as an active ingredient. It can also be understood as a composition for enhancing respiratory function and improving respiratory diseases, and the present invention includes cough, sputum, shortness of breath, airway irritability, airway obstruction, mucus hypersecretion, and expiratory flow rate, which contains an arrow tree extract as an active ingredient. It can also be recognized as a composition for improving lung disease accompanying symptoms of reduction and/or impaired gas exchange. These lung diseases include not only the aforementioned COPD, but also diffuse interstitial lung disease, acute respiratory distress syndrome (ARDS), acute lung injury, etc. In addition to pulmonary diseases due to genetic predisposition, in particular, it includes pulmonary diseases due to fine dust, which is a problem recently. Fine dust containing various components such as carbon components such as soot and organic carbon in living organisms, ionic components such as chlorine, nitric acid, ammonium, sodium, calcium, metal components such as lead, arsenic, and mercury, and polycyclic aromatic hydrocarbons such as benzopyrene, are described above. It is known to cause various lung diseases such as asthma and COPD by depositing in the islands, bronchi, small airways, and alveoli (J Korean Med Assoc, 2014; 57: 763-768).

In the present specification, the term "extract" refers to the stem, leaves, fruits, flowers, roots, and mixtures thereof of the plant to be extracted, water, lower alcohols having 1 to 4 carbon atoms (methanol, ethanol, butanol, etc.), methylene chloride, ethylene, Using acetone, hexane, ether, chloroform, ethyl acetate, butyl acetate, N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1,3-butylene glycol, propylene glycol, or a mixed solvent thereof It refers to an extract obtained by leaching, an extract obtained using a supercritical extraction solvent such as carbon dioxide, pentane, or a fraction obtained by fractionating the extract, and the extraction method includes cold sedimentation, reflux, and Any method, such as heating, ultrasonic radiation, and supercritical extraction, can be applied. In the case of a fractionated extract, a fraction obtained by suspending the extract in a specific solvent and then mixing and policing it with a solvent having a different polarity, the crude extract is adsorbed on a column filled with silica gel, etc., and then a hydrophobic solvent, a hydrophilic solvent, or a mixed solvent thereof is added. It means including the fraction obtained as a mobile phase. In addition, the meaning of the extract includes a concentrated liquid extract or a solid extract from which the extraction solvent has been removed by a method such as freeze drying, vacuum drying, hot air drying, spray drying, or the like. Preferably, it means an extract obtained by using water, ethanol or a mixed solvent thereof as an extraction solvent, more preferably an extract obtained by using a mixed solvent of water and ethanol as an extraction solvent.

In addition, in the present specification, the term "active ingredient" refers to an ingredient that can exhibit a desired activity alone or exhibit activity with a carrier that is not itself active.

In the present invention, the term "respiratory function enhancement" refers to maintaining the original function of the respiratory organs such as the nasal cavity, pharynx, larynx, trachea, bronchi, and lungs in a healthy state, or according to smoking, fine dust, activation of neutrophil netosis, or other respiratory diseases. It refers to any action that improves the function of the respiratory tract, which has been deteriorated due to symptoms, to its original healthy state.

In addition, in the present invention, the "respiratory disease" refers to a disease occurring in the respiratory tract of an individual such as the nasal cavity, pharynx, larynx, trachea, bronchi and lungs, and specifically, the respiratory disease is a respiratory disease caused by smoking or fine dust ; Or, it may mean a lung disease accompanying Netosis, but is not particularly limited thereto. More specifically, the respiratory disease may mean a pulmonary disease accompanied by symptoms of sputum, shortness of breath, airway irritability, airway obstruction, mucus hypersecretion, decrease in expiratory flow rate and/or impaired gas exchange, and more specifically asthma , Chronic obstructive pulmonary disease (COPD), tracheitis, bronchitis, diffuse interstitial lung disease, acute respiratory distress syndrome (ARDS), acute lung injury, cystic fibrosis, capillary It may mean one or more selected from the group consisting of bronchitis, influenza virus infection, pneumonia, tuberculosis, and transfusion-related acute lung injury. , Most specifically, may mean COPD. Meanwhile, it has been recently reported that the activation of netosis of neutrophil extracellular traps is involved in the pathogenesis of chronic respiratory diseases including COPD.

As long as the active ingredient in the composition of the present invention can exhibit COPD improvement activity, other lung disease improvement activity, respiratory function enhancement, and respiratory disease improvement activity, the active ingredient may be in an arbitrary amount (effective amount) according to the specific use, formulation, purpose of combination, etc. Wherein, a typical effective amount will be determined within the range of 0.001% to 20.0% by weight based on the total weight of the composition. Here, "effective amount" refers to the intended functional and pharmacological effect, such as COPD improvement effect, when the composition of the present invention is administered to a mammal, preferably a human, to which the composition of the present invention is administered during the administration period according to the advice of a medical expert, etc. It refers to the amount of the active ingredient contained in the composition of the present invention. Such effective amounts can be determined empirically within the range of ordinary skill in the art.

Subjects to which the composition of the present invention can be applied (prescribed) are mammals and humans, particularly preferably humans.

In order to enhance the convenience of taking or ingestion through addition of similar activities such as anti-inflammatory activity, anti-allergic activity, etc., in addition to the active ingredient, the composition of the present invention has already been verified for safety in the art and is known to have the corresponding activity. It may further contain a compound or natural extract of.

These compounds or extracts include compounds or extracts, pharmaceuticals, etc. listed in the pharmacopoeia of each country (“Korean Pharmacopoeia” in Korea), health functional food code in each country (“health functional food standards and standards” notified by the Ministry of Food and Drug Safety in Korea), etc. In accordance with the laws of each country governing the manufacture and sale of products (“Pharmaceutical Affairs Act” in Korea), laws governing the manufacture and sale of compounds, extracts, and health functional foods that have been licensed as items (in Korea, “The Act on Health Functional Foods”) ”), a compound or extract whose functionality is recognized may be included.

For example, according to the Korean 「Health Functional Food Act」, anti-inflammatory ("arthritis improvement") functions are recognized, such as MSM (dimethylsulfonylmethane) and N-acetylglucosamine, and anti-allergic ("relieving hypersensitivity immune reactions") functions are recognized. Enterococcus faecalis heat-treated dry powder, complexes such as guava leaf extract, complexes such as vinegar extract, lobule extract, picaopreto powder, and PLAG (1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol). It will correspond to the compound or extract, and is not particularly limited thereto.

One or more of these compounds or natural extracts may be included in the composition of the present invention together with the active ingredient.

In a specific aspect, the composition of this invention can be grasped as a food composition. In addition, the food of the present invention may include health functional (sex) food. In the present invention, the term "health functional food" refers to a food manufactured and processed in the form of tablets, capsules, powders, granules, liquids and pills using raw materials or ingredients having useful functions for the human body. Here, the term "functionality" refers to obtaining useful effects for health purposes such as controlling nutrients or physiological effects on the structure and function of the human body. The health functional food of the present invention can be prepared by a method commonly used in the art, and at the time of production, it can be prepared by adding raw materials and ingredients commonly added in the art. In addition, the formulation of the health functional food may be prepared without limitation as long as it is a formulation recognized as a health functional food. The food composition of the present invention can be prepared in various forms of formulation, and unlike general drugs, it has the advantage of not having side effects that may occur when taking the drug for a long time using food as a raw material, and is excellent in portability. Health functional foods can be consumed as an adjuvant to improve COPD and further enhance respiratory function and improve respiratory disease.

The food composition of the present invention may be prepared in any form, such as beverages such as tea, juice, carbonated beverages, ionized beverages, processed oils such as milk, yogurt, gums, rice cakes, Korean confectionery, bread, Foods such as confectionery and noodles, tablets, capsules, pills, granules, liquids, powders, flakes, pastes, syrups, gels, jelly, and health functional food preparations such as bars can be prepared.

In addition, the food composition of the present invention can be classified as a product as long as it conforms to the enforcement regulations at the time of manufacture and distribution in terms of legal and functional classification. For example, it is a health functional food according to the Korean 「Health Functional Food Act」, or confectionery, bean, tea, and beverages according to each food type according to the food code of the Korean 「Food Sanitation Act」 (``Food Standards and Standards'' notified by the Ministry of Food and Drug Safety). , Special use food, etc.

The food composition of the present invention may contain food additives in addition to the active ingredients. Food additives can generally be understood as substances that are added to food and mixed or infiltrated in manufacturing, processing, or preserving food. Since they are consumed daily and for a long time with food, their safety must be ensured. Food additives with guaranteed safety are limited in terms of ingredients or functions in the Food Additives Code according to the laws of each country governing the manufacture and distribution of food (the “Food Sanitation Act” in Korea). In the Korean Food Additives Code (“Food Additive Standards and Standards” notified by the Ministry of Food and Drug Safety), food additives are classified into chemical synthetic products, natural additives, and mixed preparations in terms of ingredients.These food additives are sweeteners and flavors in terms of function. It is categorized into agents, preservatives, emulsifiers, acidulants, and thickeners.

Sweeteners are used to impart a suitable sweetness to food, and natural or synthetic ones may be used. Preferably, a natural sweetener is used, and examples of the natural sweetener include sugar sweeteners such as corn syrup solids, honey, sucrose, fructose, lactose, and maltose.

Flavoring agents can be used to enhance taste or aroma, and both natural and synthetic can be used. Preferably, it is the case of using a natural one. In the case of using natural ones, the purpose of nutrient enhancement can be combined in addition to flavor. As a natural flavoring agent, it may be obtained from apples, lemons, tangerines, grapes, strawberries, peaches, or the like, or from green tea leaves, roundtails, bamboo leaves, cinnamon, chrysanthemum leaves, jasmine, and the like. In addition, you can use those obtained from ginseng (red ginseng), bamboo shoots, aloe vera, and ginkgo. The natural flavoring agent may be a liquid concentrate or a solid extract. In some cases, synthetic flavoring agents may be used. As synthetic flavoring agents, esters, alcohols, aldehydes, terpenes, and the like may be used.

As a preservative, calcium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate, EDTA (ethylenediaminetetraacetic acid), etc. can be used, and as an emulsifier, acacia gum, carboxymethylcellulose, xanthan gum, pectin And the like may be used, and as the acidulant, arithmetic, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, phosphoric acid, and the like can be used. In addition to the purpose of enhancing taste, the acidulant may be added so that the food composition has an appropriate acidity for the purpose of inhibiting the growth of microorganisms.

As the thickening agent, a suspending agent, a settling agent, a gel-forming agent, a swelling agent, and the like may be used.

In addition to the food additives described above, the food composition of the present invention may include a physiologically active substance or minerals known in the art for the purpose of supplementing and reinforcing functionality and nutritional properties and ensuring stability as a food additive.

Examples of such physiologically active substances include catechins contained in green tea, vitamins such as vitamin B1, vitamin C, vitamin E, and vitamin B12, tocopherol, dibenzoyl thiamine, etc., and minerals include calcium preparations such as calcium citrate, magnesium stearate. Magnesium preparations such as, iron preparations such as iron citrate, chromium chloride, potassium iodide, selenium, germanium, vanadium, and zinc.

In the food composition of the present invention, the food additive described above may be included in an appropriate amount to achieve the purpose of addition according to the product type.

Regarding other food additives that may be included in the food composition of the present invention, reference may be made to the food code of each country or the food additive code.

The composition of the present invention may be understood as a pharmaceutical composition in other specific embodiments.

The pharmaceutical composition of the present invention may be prepared in an oral dosage form or a parenteral dosage form according to an administration route by a conventional method known in the art, including a pharmaceutically acceptable carrier in addition to the active ingredient. Here, the route of administration may be any suitable route including a local route, an oral route, an intravenous route, an intramuscular route, and direct absorption through mucosal tissue, and two or more routes may be used in combination. An example of a combination of two or more routes is a case in which two or more formulations of drugs are combined according to the route of administration, for example, when one drug is first administered by an intravenous route and the second drug is administered by a local route.

Pharmaceutically acceptable carriers are well known in the art depending on the route of administration or formulation, and specifically, reference may be made to the pharmacopoeia of each country, including the “Korean Pharmacopoeia”.

When the pharmaceutical composition of the present invention is prepared in an oral dosage form, powders, granules, tablets, pills, dragees, capsules, solutions, gels, syrups, suspensions, wafers according to a method known in the art together with a suitable carrier It can be prepared in a formulation such as. Examples of suitable carriers at this time include sugars such as lactose, glucose, sucrose, dextrose, sorbitol, mannitol, and xylitol, starches such as corn starch, potato starch, wheat starch, cellulose, methylcellulose, ethylcellulose, sodium carboxymethylcellulose, Celluloses such as hydroxypropylmethylcellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, polyol, vegetable oil, ethanol, grease Serol, etc. are mentioned. In the case of formulation, appropriate binders, lubricants, disintegrants, colorants, and diluents may be included as needed. Suitable binders include starch, magnesium aluminum silicate, starch ferryst, gelatin, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone, glucose, corn sweetener, sodium alginate, polyethylene glycol, wax, etc., and lubricants include oleic acid. Sodium, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, silica, talcum, stearic acid, magnesium salts and calcium salts thereof, polydecylene glycol, etc., and as disintegrating agents starch, methyl cellulose , Agar, bentonite, xanthan gum, starch, alginic acid, or a sodium salt thereof. Moreover, lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, glycine, etc. are mentioned as a diluent.

When the pharmaceutical composition of the present invention is prepared in a parenteral formulation, it may be formulated in the form of an injection, a transdermal administration, a nasal inhalation and a suppository according to a method known in the art together with a suitable carrier. When formulated as an injection, an aqueous isotonic solution or suspension may be used as a suitable carrier, and specifically, triethanol amine-containing PBS (phosphate buffered saline), sterile water for injection, an isotonic solution such as 5% dextrose, etc. may be used. . When formulated as a transdermal administration, it can be formulated in the form of an ointment, cream, lotion, gel, external solution, pasta, liniment, air roll, and the like. In the case of nasal inhalants, suitable propellants such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, and carbon dioxide can be used in the form of an aerosol spray.When formulated as a suppository, the carrier is Withepsol ( witepsol), tween 61, polyethylene glycols, cacao butter, laurin paper, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, sorbitan fatty acid esters, and the like.

The specific formulation of pharmaceutical compositions is known in the art, and for example, Remington's Pharmaceutical Sciences (19th ed., 1995) may be referred to. This document is considered as part of this specification.

The preferred dosage of the pharmaceutical composition of the present invention is in the range of 0.001 mg/kg to 10 g/kg per day, preferably 0.001 mg/kg to 1 g, depending on the patient's condition, weight, sex, age, patient severity, and route of administration. May be in the /kg range. Administration can be made once a day or divided into several times. Such dosage should not be construed as limiting the scope of the invention in any aspect.

As described above, according to the present invention, it is possible to provide a composition for improving COPD using an arrow tree extract. The composition of the present invention is commercialized as a food, in particular a health functional food or drug, and can be usefully used for the effect of improving COPD, further enhancing respiratory function and improving respiratory diseases, coughing, sputum, shortness of breath, respiratory irritation, Other lung diseases accompanying symptoms such as airway obstruction and mucus hypersecretion, such as diffuse interstitial pulmonary disease, acute respiratory distress syndrome (ARDS), acute lung injury, etc. can also be usefully used for improvement.

Figure 1 shows the results of evaluating the Netosis level (%) for HL-60 cells at the time of treatment by concentration of the arrow tree extract.
2 and 3 show the results of evaluating the IL-8 inhibitory activity upon treatment of the extracts of arrow tree leaves or branches for H292 cells, respectively.
4 shows the results of evaluating the MIP-2 inhibitory activity upon treatment of MH-S cells according to concentrations of the arrow tree extract.
Figure 5 shows the total number of cells counted from bronchoalveolar lavage fluid (BALF) after administration of the arrow tree extract to the PPE/CSE-induced chronic obstructive pulmonary disease (or respiratory disease) mouse model.
6 and 7 show the results of analyzing immune cells (Macrophage, Lympocyte, Neutrophils, and Eosinophil) in BALF through Diff-Quick staining.
8 shows the results of staining the lung tissue extracted from the mouse model.
9 shows the results of analysis of Cytokine (TNF-alpha, MIP-2, IL-1beta, IL-6) in BALF.
Figure 10 is Cytokine from lung tissue (KC, MCP-1, MDC, TARC, GM-CSF, MIP-2, IL-6, TNF-alpha, IL-1beta, INF-gamma, IL-10 and IL-17) It shows the results of analysis.

Hereinafter, the present invention will be described with reference to Examples and Experimental Examples. However, the scope of the present invention is not limited to these Examples and Experimental Examples.

[Example] Sample preparation

An extract of the arrow tree (extracted area: leaves, branches) was prepared to confirm the activity of improving lung disease. The extract was obtained by adding 10 times the weight of 70% ethanol to the dry powder of each natural product to be extracted, and repeatedly extracting it twice at 50°C for 6 hours, filtered, concentrated under reduced pressure, and freeze-dried to obtain a powder.

[Experimental Example] Lung disease improvement activity

Experimental Example 1: Netosis inhibitory activity of neutrophil cells

^{HL-60 cells were seeded at 2×10 5} cells/mL in RPMI (10% FBS, 100 U/mL penicillin, 100 mg/mL streptomycin) medium treated with 1% DMSO, and a cell incubator (37°C, 5% CO). ₂ ) was cultured for 5 days to differentiate into a mature neutrophil phenotype. After 5 days, after centrifugation at 1,300 rpm for 5 minutes, HBSS (with Mg ^+, Ca ⁺ ) was treated with 10 mL, followed by centrifugation at 1,300 rpm for 5 minutes to wash. The recovered cells were ^{diluted in HBSS at a concentration of 1×10 6} cells/mL, dispensed into 96 well plates by 100 μL, and 50 μL of samples prepared at each concentration were treated. Then, 50 μL of PMA or CSE prepared at 400 nM was treated in each well and incubated for 3 hours. After 3 hours, Cytox green (500 nM) was treated and fluorescence was measured at excitation/emission maxima: 485/520 nm using a fluorescent plate reader.

As a result, it was confirmed that the level of Netosis (%) decreased depending on the concentration (12.5 μg/ml, 25 μg/ml, 50 μg/ml, 100 μg/ml) of the treated arrowhead leaf extract (FIG. 1).

Experimental Example 2: Evaluation of inflammatory cytokine (IL-8) inhibitory activity using H292 cells

^{H292 cells prepared at a concentration of 2×10 5} cells/mL in a 24 well plate were passaged and cultured at 500 μL/well, and when the cells became more than 80% confluent, they were exchanged with serum-free RPMI1640 medium and starvated for 24 hours. Thereafter, using Serum-free RPMI1640 medium, irritants (CSE; final 2% or PPE; final 0.01 U/mL or PM2.5; final 10 μg/mL) and arrowhead extract (final; 6.25, 12.5, 25 μg) /mL) was diluted to reflect the desired final concentration, and then treated at 500 uL per well. After incubation overnight, the supernatant was recovered and used for IL-8 measurement. Human IL-8 was measured by the ELISA method, and proceeded according to the manufacturer's (BD) protocol.

As a result, the concentration of IL-8 was inhibited depending on the concentration (6.25 µg/ml, 12.5 µg/ml, 25 µg/ml) of the treated arrow tree leaf extract, and treated with the arrow tree branch extract (6.25 µg/ml). Even in the case, it was confirmed that IL-8 was inhibited (FIGS. 2 and 3).

Experimental Example 3: Evaluation of MIP-2 inhibitory activity using MH-S cells

The MH-S cell line was purchased from ATCC, and the medium composition used for cultivation was RMPI-1640 (WELGENE, cat. LM 011-01), 10% FBS (WELGENE, cat. S 001-07), 1% Penicillin- Streptomycin (WELGENE, cat. LS 202-02), 14.3 mM 2-Mercptoethanol (SIGMA). MH-S cells were seeded in a cell culture 96 well plate (SPL, cat. 30096) at a concentration of ^{5x10 4 /well and cultured for 24 hours.} After that, dilute the stimulant (CSE; final 1% and LPS; final 10 ng/mL) and arrowhead leaf extract (final; 25 μg/mL) with MH-S cell culture medium to reflect the desired final concentration and dilute per well. Treated with 250 uL each. After incubation for 24 hours, the supernatant was recovered and used for MIP-2 measurement. MIP-2 was measured by the ELISA method, and proceeded according to the manufacturer's (R&D, DY452) protocol.

As a result, it was confirmed that MIP-2 inhibitory activity was exhibited upon treatment with an arrow tree leaf extract (25 μg/ml) (FIG. 4).

Experimental Example 4: Measurement of the number of BALF cells in an animal model

4-1: PPE+CSE-induced respiratory disease mouse model

BALB/C mice (male, 5 weeks old) were purchased from Orient Bio, and purified for 1 week, and grouped with n=10. The sample treatment group was orally administered 200 mg/kg of the sample (arrow tree extract) for 24 days from one week before the start of COPD induction to pre-dissection, and the Naive and COPD groups were administered the same amount of PBS. At this time, the positive control, Roflumilast group, was administered orally at 10 mg/kg per day after induction. For COPD induction, 1.2 units of Porcine Pancreatic Elastase (PPE) and 100% Cigarette Smoke Extraction (CSE) were used. 1.2 unit of PPE was injected three times through intra nasal once every 7 days, and 20 µl of CSE was treated through intra nasal for 3 days from the day after PPE treatment, but after the last PPE treatment, the dissection proceeded the next day without CSE treatment. I did. The dissection was performed after anesthetizing with isoflurane using an inhalation anesthesia. During the experiment, feed and drinking water were provided in the form of self-catering, and were raised in an environment of 24 ℃ and 60% humidity.

4-2: Total number of cells infiltrated into BALF

After injecting 1 mL of PBS through the airway of the mouse, it was gently massaged to recover 700 μL of bronchoalveolar lavage fluid (BALF). 10 uL of the recovered BALF solution was mixed with the Accustain T solution in the same amount, and 12 uL was injected into the Accuchip channel, and then total cells were automatically counted with a cell counter (ADAM-MC, NANOENTEK. INC, Korea) (FIG. 5).

4-3: Analysis of immune cells in BALF through Diff-Quick staining

The recovered BALF was separated into a supernatant and a cell pellet through centrifugation under conditions of 300×g and 5 minutes. The cell pellet was resuspended by adding 700 μL of PBS, and 150 μL of this BALF suspension was centrifuged at 1000 rpm, 10 min, 4°C with a Cytospin device (Centrifuge 5403, Eppendorf, Hamburg, Germany), and the BALF cells were placed on the slide. Attached. Thereafter, cells were stained according to the manufacturer's protocol (1-5-1 Wakinohamakaigandori, chuo-ku, Kobe, Japan) using a Diff-Quick staining reagent, and the number of Macrophages, Lympocytes, Neutrophils, and Eosinophils were determined through microscopic observation. Counted (Fig. 6, Fig. 7).

4-4: Histological examination

Lung tissue was removed from the dissected mouse, the mesenchyme of the lung was removed, and then fixed in 10% neutral buffered formalin for 3 days. The fixed tissue was dehydrated with ethyl alcohol and xylene, embedded with paraffin, trimmed, and cut into 5 um thickness. After attaching the prepared section to the slide, it was stained with H&E to confirm the general shape and observed under a microscope (FIG. 8).

4-5: Analysis of Cytokines in BALF

After injecting 1 mL of PBS through the airway of the mouse, it was gently massaged to recover 700 μL of bronchoalveolar lavage fluid (BALF). The recovered BALF was centrifuged at 300×g for 5 minutes to separate the supernatant, and then analyzed for cytokines and chemokines (TARC, KC, MCP-1, MDC, GM-CSF, MIP-2, TNF-α, IL- 6, IL-1β, IL-17, IL-10, IFN-γ). After taking 30 uL of the supernatant for analysis of cytokines and chemokines, the experiment was performed according to the protocol of the manufacturer of the Q-plex ELISA array kit (Quansis biosciences). The results are shown for each cytokine in FIG. 9.

4-6: Analysis of Cytokines in Lung Tissue

PBS corresponding to 10 times the weight of the lung tissue was added, pulverized with a homogenizer, and centrifuged at 12,000 rpm for 20 min. The recovered supernatant was analyzed for cytokines and chemokines (TARC, KC, MCP-1, MDC, GM-CSF, MIP-2, TNF-α, IL-6, IL-1β, IL-17, IL-10, IFN-γ ). After taking 30 uL of the supernatant for analysis of cytokines and chemokines, the experiment was performed according to the protocol of the manufacturer of the Q-plex ELISA array kit (Quansis biosciences). The results are shown in Fig. 10, respectively.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing the technical spirit or essential features thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not limiting. The scope of the present invention should be construed as including the meaning and scope of the claims to be described later rather than the above detailed description, and all changes or modifications derived from the equivalent concepts within the scope of the present invention.

Claims (10)

A food composition for improving COPD, comprising an arrow tree extract as an active ingredient. The composition of claim 1, wherein the extract is water, ethanol, or a mixed solvent extract thereof. delete delete delete Health functional food for improving COPD comprising the food composition of claim 1 or 2. A pharmaceutical composition for preventing or treating COPD, comprising an arrow tree extract as an active ingredient. The composition of claim 7, wherein the extract is water, ethanol, or a mixed solvent extract thereof. delete delete

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