KR20190133637A - Composition for improving chronic obstructive pulmonary disease using an extract of Bark of Poria cocos - Google Patents

Abstract

Disclosed is a composition for alleviating chronic obstructive pulmonary disease (COPD) using an extract of Poria cocos, which has the activities of: inhibiting the expression of an inflammatory cytokine, IL-8, in H292 cells, which are airway epithelial cell lines stimulated by a cigarette smoke extract (CSE), etc.; inhibiting the expression of an inflammatory cytokine, MIP2(IL-8), even in MH-S cells, which are mouse alveolar macrophages activated by CSE; improving respiratory functions in an animal model experiment of COPD induced by CSE; and reducing an infiltrating neutrophil count along with an infiltrating cell count of BALF in an animal model experiment in which COPD is induced by CSE and porcine pancrease elastase (PPE).

Description

Composition for improving chronic obstructive pulmonary disease using an extract of Bark of Poria cocos}

The present invention relates to a composition for improving chronic obstructive pulmonary disease using Poria cocos extract.

Chronic obstructive pulmonary disease (COPD) is a chronic airway disease with irreversible airway obstruction, including coughing, sputum, shortness of breath, decreased exhalation flow rate, and impaired gas exchange. It is predicted to be the third leading cause of death for humanity in 2020 (Am J Respir Crit Care Med, 2013, 187: 347-365; Am J Respir Crit Care Med, 2009, 180: 396-). 406). In the past, COPD was divided into chronic bronchitis and emphysema, but chronic bronchitis is defined based on clinical symptoms and emphysema is classified by anatomical criteria. It is diagnosed collectively with COPD (Eur Respir J, 2007, 30: 993-1013; Respirology 1997, 2 Suppl 1: S1-4). In the case of bronchial asthma, if the airway obstruction is irreversible due to long duration of asthma, it is included in COPD and treated accordingly (Eur Respir J, 2007, 30: 993-1013; Respirology 1997, 2 Suppl 1: S1-4).

COPD is caused by various causes such as smoking, air pollution, chemicals, occupational factors, and genetic predisposition. Among them, smoking is the main cause, and more than 80% of patients with COPD have been identified as smokers. Pharm Bull, 2012, 35: 1752-1760). The pathogenesis of COPD involves chronic inflammation in the bronchus and lung tissue, protease activation in the lung, and oxidative stress. The cells involved in inflammation are neutrophils, macrophages, and T lymphocytes. . These inflammatory cells produce a variety of inflammatory cytokines such as TNF-α, IFN-γ, IL-1β, IL-6, IL-8, IL-18, and various proteases that cause tissue damage. Respiratory Society Seoul: Gunja Publishing Co., 2007, p 301-5; Am J Respir Crit Care Med, 1997 155, 1441-1447; Am J Physiol Lung Cell Mol Physiol, 2010, 298: L262-L269). In COPD animal models induced by tobacco smoke, neutrophil influx, KC (keratinocyte chemoattractant), TNF-α (tumour necrosis factorα), MIP-2 (macropage inflammatory protein 2), MIP-1α and MCP-1 (monocyte chemoattractant protein) In addition, an increase in matrix metalloproteinase 12 (MMP12) and granulocyte macrophage colony-stimulating factor (GM-CSF) were observed (Clin. Sci. 2014, 126 (3): 207).

Neutrophils secrete extracellular substances such as elastase, collagenase, proteases such as myeloperoxidase (MPO), arachidonic acid metabolites (arachidonate), and reactive oxygen free radicals. It is known to play an important role in the development of COPD by causing injury and causing chronic airway inflammation through airway infiltration (Am J Physiol Lung Cell Mol Physiol. 2017, 312 (1): L122-L130; Am J Respir Cell Mol Biol, 2013, 48: 531-539; Eur Respir J, 1998, 12: 1200-1208).

Recently, excessive formation of neutrophil extracellular traps (NET) has been reported to be associated with lung 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), NET is a network of cytoplasmic and granular proteins bound to entangled chromatin. It is a structure in the form. NET captures and neutralizes bacteria, fungi, and viruses and prevents their transmission and infection (Nat Immunol 15, 1017-1025, 2014), but excessive NET secretion is associated with a variety of infectious and non-infectious diseases. . In particular, the association with lung disease has been noted because NET easily expands in the alveoli and causes lung damage (Nat Rev Microbiol. 2007, 5: 577-82; Front Immunol. 2013, 4: 1). Excessive NETosis can cause COPD as well as asthma, cystic fibrosis, RSV respiratory syncytial virus bronchiolitis, influenza virus infection, bacterial pneumonia, tuberculosis (tuberculosis), transfusion-related acute lung injury, and other lung diseases have been reported, so netosis (NETosis) inhibition is recognized as an important target for the development of these pulmonary disease treatment (Front Immunol. 2016, 7: 311)

Currently, bronchoalveolar lavage (BAL) is used as a means of diagnosing and progressing diseases such as COPD and asthma, and inflammatory cytokines and active oxygen in bronchoalveolar lavage fluid (BALF) of these patients. Inflammatory mediators such as species, leukotriene, and activated complement increase, and neutrophils, which account for less than 5% in normal lungs, account for 80% of all cells (Am J Respir Crit Care Med 154 (1): 76-81, 1996).

Until now, no direct improvement of COPD or asthma has been reported. Current COPD or asthma medications are used to reduce symptoms and complications. Bronchodilators (β2-agonists, anticholiners, methylxanthines) and steroids (inhalation, Oral) is mainly used.

The present invention discloses that the effects of improving the chronic obstructive pulmonary disease of the extract of Bokryeongpi.

It is an object of the present invention to provide a composition for improving chronic obstructive pulmonary disease using the extract of Bokryeongpi.

Other and specific objects of the present invention will be presented below.

The inventors of the present invention inhibit the expression of inflammatory cytokines IL-8 by H292 cells, a bronchial epithelial cell line stimulated by CSE (cigarette smoke extract), as confirmed in the following Examples and Experimental Examples, MH-S cells, mouse alveolar macrophages activated by CES, not only inhibit the expression of the inflammatory cytokine MIP2 (IL-8), but also breathe in animal dodel experiments inducing COPD with CSE (cigarette smoke extract). In animal model experiments in which COPD was induced with CSE and porcine pancreas elastase (PPE), we found that BALF infiltrated cell number and invasive neutrophil count were lowered.

The present invention is provided based on the above-described experimental results, the present invention, in one aspect can be understood as a composition for improving COPD comprising the extract of Bokyeongpi as an active ingredient, in another aspect Bokyeongpi extract as an active ingredient It can be grasped as a composition for improving inflammatory lung disease comprising. In another aspect, the present invention can be understood as a composition for improving lung function or respiratory function comprising the extract of Bokryeongpi as an active ingredient, and in another aspect, the respiratory tract by smoking or fine dust containing the extract of Bokryeongpi as an active ingredient It can be grasped as a composition for improving disease, and in another aspect, it can be grasped as a composition for improving lung damage, including Bokyeongpi extract as an active ingredient.

In the present specification, "extract" refers to the stem, leaves, fruits, flowers, roots, outposts, mixtures thereof, and the like of the plant to be extracted, water, lower alcohols having 1 to 4 carbon atoms (methanol, ethanol, butanol, etc.), methylene chloride, Ethylene, 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 Extract obtained by leaching using the supercritical extraction solvent, such as carbon dioxide, pentane, or the fraction obtained by fractionating the extract, the extraction method is cold-precipitation, taking into account the polarity, degree of extraction, the degree of preservation of the active material, Any method, such as reflux, heating, ultrasonic radiation, supercritical extraction, can be applied. In the case of the fractionated extract, the fraction obtained by suspending the extract in a specific solvent and mixing and standing with a solvent having a different polarity, the crude extract is adsorbed on a column filled with silica gel and the like, and then a hydrophobic solvent, a hydrophilic solvent, or a mixed solvent thereof is added. It is meant to include fractions obtained by mobile phase. In addition, the meaning of the extract includes a concentrated liquid extract or solid extract in which the extraction solvent is removed in a manner such as freeze drying, vacuum drying, hot air drying, spray drying, and the like. Preferably it refers to an extract obtained by using water, ethanol or a mixed solvent thereof as the extraction solvent, more preferably an extract obtained by using a mixed solvent of water and ethanol as the extraction solvent.

In addition, in the present specification, the "active ingredient" means a component that can exhibit the desired activity alone or in combination with a carrier which is itself inactive.

In addition, in the present specification, "inflammatory lung disease" is a lung disease accompanied by an inflammatory response is meant to include asthma, chronic obstructive pulmonary disease (COPD), tracheitis, bronchitis (bronchitis).

In addition, in the present specification, "improvement of pulmonary function or respiratory function" refers to improvement of respiratory function of a normal person who is a non-disease, recovery of respiratory insufficiency of the disease due to chronic obstructive pulmonary disease or asthma, bronchitis, bronchitis, or respiratory function of the sick Means improvement.

In addition, in the present specification, "respiratory disease caused by smoking or fine dust" means, in addition to including asthma and COPD, diffuse interstitial lung disease, acute respiratory distress syndrome (ARDS), acute lung injury to be. Fine dust containing various components such as carbon components such as soot and biological organic carbon, ionic components such as chlorine, nitric acid, ammonium, sodium and calcium, metal components such as lead, arsenic and mercury, and polycyclic aromatic hydrocarbons such as benzopyrene Also, it is known to deposit various bronchial diseases such as asthma and COPD by depositing in bronchial tubes, small airways, alveoli (J Korean Med Assoc, 2014; 57: 763-768).

In addition, in the present specification, "lung damage improvement" means the recovery of pulmonary cell or lung tissue damage caused by fine dust or the like or the recovery of pulmonary function deterioration due to lung cell or lung tissue damage.

In addition, in this specification, "improvement" is meant to include alleviation, treatment or prevention of a disease or condition.

The active ingredient in the composition of the present invention may be included in any amount (effective amount) depending on the use, formulation, etc., as long as it can exhibit an effect of improving COPD, improving lung function or respiratory function, and the like. It will be determined in the range of 0.001% to 15% by weight based on the basis. Here, the term "effective amount" refers to the intended medical, such as COPD improvement, pulmonary function or respiratory function improvement effect when the composition of the present invention is administered to a mammal, preferably a human, during the administration period according to the suggestion of a medical professional or the like. It refers to the amount of the active ingredient included in the composition of the present invention, which can exhibit a pharmacological effect. Such effective amounts can be determined experimentally within the range of ordinary skill in the art.

The composition of the present invention, in addition to the extract of Bokyeongpi as an active ingredient, in order to increase the effect of improving asthma, COPD, lung function or respiratory function, turmeric known to have an effect of improving asthma, COPD, lung function or respiratory function ( Curcuma longa extract, Scutellaria baicalensis extract, Trapa japonica extract, Perilla frutescens extract, Polygonum avivulare extract, Rosae multiflorea extract, Fenugreek extract, Trigonella foenum extract Piper nigrum ) extract or a mixture of two or more thereof may be additionally included as an active ingredient.

The composition of this invention can be grasped | ascertained as a food composition in a specific aspect.

The food composition of the present invention may be prepared in any form, for example, beverages such as tea, juice, carbonated beverages, ionic beverages, processed oils such as milk, yogurt, gums, rice cakes, sweets, breads, Foodstuffs such as confectionery, cotton, tablets, capsules, pills, granules, liquids, powders, flakes, pastes, syrups, gels, jelly, bars, and health functional food preparations such as bars. In addition, the food composition of the present invention can distinguish any product as long as it conforms to the enforcement regulations at the time of manufacture and distribution in the legal and functional divisions. For example, confectionery, soybeans, teas, etc. according to each food type, or health functional foods in accordance with Korea's "Health Functional Foods Act" or Food Code of Korea "Food Sanitation Law" Beverages, special purpose foods, and the like.

The food composition of the present invention may include food additives in addition to the active ingredient. Food additives can generally be understood as substances which are added to the food, mixed or infiltrated in the manufacture, processing or preservation of the food, which must be ensured because of its daily and long-term intake with the food. The Food Additives Code of Korea, which regulates the manufacture and distribution of foods ("Food Sanitation Law" in Korea), provides limited food additives in terms of ingredients or functions. In the Korean Food Additives Code (KFDA notice and standards), food additives are classified into chemical synthetics, natural additives, and mixed preparations in terms of ingredients. These food additives are sweeteners and flavoring agents in terms of function. , Preservatives, emulsifiers, acidulants, thickeners and the like.

Sweeteners are used to impart a suitable sweetness to foods, both natural and synthetic, which can be used in the food composition of the present invention. Preferably, a natural sweetener is used. Examples of the natural sweetener include sugar sweeteners such as corn syrup solids, honey, sucrose, fructose, lactose and maltose.

Flavoring agents are used for the purpose of improving taste or aroma, and may be used both natural and synthetic. It is the case of using a natural thing preferably. In addition to flavors, the use of natural ones can be combined with nutritional purposes. The natural flavor may be obtained from apples, lemons, citrus fruits, grapes, strawberries, peaches, and the like, or may be obtained from green tea leaves, round leaves, jujube leaves, cinnamon, chrysanthemum leaves, jasmine and the like. In addition, ginseng (red ginseng), bamboo shoots, aloe vera, ginkgo and the like can be used. Natural flavors can be liquid concentrates or solid extracts. In some cases, a synthetic flavor may be used, and as the synthetic flavor, esters, alcohols, aldehydes, terpenes, and the like may be used.

As the preservative, calcium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate, EDTA (ethylenediaminetetraacetic acid) and the like can be used, and as an emulsifier, acacia gum, carboxymethylcellulose, xanthan gum, pectin Etc. may be used, and acidic acid, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, phosphoric acid, and the like may be used. The acidulant may be added so that the food composition is at an appropriate acidity for the purpose of inhibiting the growth of microorganisms in addition to the purpose of enhancing the taste. As the thickener, suspending implementers, sedimenting agents, gel formers, swelling agents and the like can be used.

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

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

In the food composition of the present invention, the food additive as described above may be included in an amount that can 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 or the Food Additive Code according to the laws of each country.

The composition of the present invention may be regarded as a pharmaceutical composition in another specific embodiment.

The pharmaceutical compositions of the present invention may be prepared in oral or parenteral formulations according to the route of administration by conventional methods known in the art, including pharmaceutically acceptable carriers in addition to the active ingredient. "Pharmaceutically acceptable" here means that the subject of application (prescription) is not toxic as far as adaptable without inhibiting the activity of the active ingredient.

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 methods known in the art with suitable carriers It may be prepared in a formulation such as. Examples of suitable pharmaceutically acceptable carriers include sugars such as lactose, glucose, sucrose, dextrose, sorbitol, mannitol and xylitol, starch such as corn starch, potato starch, wheat starch, cellulose, methylcellulose, ethylcellulose, Celluloses such as sodium carboxymethyl cellulose and hydroxypropyl methyl cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, polyol, vegetable Yu etc. can be mentioned. If formulated, it may be formulated to include diluents and / or excipients, such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, if necessary.

When the pharmaceutical compositions of the present invention are prepared in parenteral formulations, they may be formulated in the form of eye drops, injections, transdermal administrations, nasal inhalants, suppositories with suitable carriers according to methods known in the art. When formulated as an eye drop, suitable carriers include sterile water, saline, isotonic solutions such as 5% dextrose, and the like, and benzalkonium chloride, mefilparaben, ethyl paraben, etc. may be added for preservative purposes as necessary. When formulated as an injectable, suitable carriers may be sterile water, ethanol, polyols such as glycerol or propylene glycol, or mixtures thereof. Preferably, PBS (phosphate buffered saline) containing Ringer's solution, triethanol amine, or injectable sterilization is used. Water, isotonic solutions such as 5% dextrose, and the like. When formulated as a transdermal administration, it may be formulated in the form of an ointment, cream, lotion, gel, external solution, pasta, linen, aerosol. Nasal inhalants can be formulated in the form of aerosol sprays using suitable propellants, such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, etc. witepsol), tween 61, polyethylene glycols, cacao butter, laurin paper, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearate, sorbitan fatty acid esters and the like.

Specific formulations of pharmaceutical compositions are known in the art and can be found, for example, in Remington's Pharmaceutical Sciences (19th ed., 1995). The document is considered part of this specification.

Preferred dosages of the pharmaceutical compositions of the present invention range from 0.001 mg / kg to 10 g / kg per day, preferably 0.001 mg / kg to 1 g, depending on the condition, body weight, sex, age, severity of the patient and route of administration. It can range from / kg. Administration can be done 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 can provide a composition for improving COPD using the extract of Bokryeongpi. The composition of the present invention may be usefully formulated as a food, especially a health functional food or a drug, for the effect of improving COPD, further improving the lung function or respiratory function.

FIG. 1 shows the results of Bokyeongpi extract showing inflammatory cytokine (IL-8) inhibitory activity in H292 cells stimulated by CSE.
Figure 2 shows the results of the MIP-2 inhibitory activity using Bokyeongpi extract MH-S cells stimulated by CSE.
3 is a result showing the improvement of lung function or respiratory function in the animal model experiment that the Bokryeongpi extract COPD-induced by CSE.
Figure 4 shows that Bokyeongpi extract reduced the total cell infiltration in BALF in animal model experiments inducing COPD by CSE and PPE.
Figure 5 shows that Bokryeongpi extract reduced neutrophil infiltration in BALF in animal model experiments inducing COPD by CSE and PPE.

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.

EXAMPLES Improvement Activity of COPD, etc. of Bokyeongpi Extract

1. Sample Preparation

Pulmonary disease improvement activity was confirmed using the extract of Bokryeongpi (extract site: Bokryeong bark). The extract was added to 70% ethanol of 10 times the weight of the dry powder, repeated extraction twice for 6 hours at 50 ℃ filtered and concentrated under reduced pressure and lyophilized to obtain a powder.

2. Experiment Method

2.1 Evaluation of Inflammatory Cytokine (IL-8) Inhibitory Activity Using H292 Cells

Human promyelocytic leukemia cells (Human promyelocytic leukemia cells) obtained from Korea Cell Line Bank were treated with RPMI (10% FBS, 100 U / mL penicillin, 100 mg / mL streptomycin) media treated with 1% DMSO at 2 × 105 cell / mL. The cells were seeded and cultured at 37 ° C., 5% CO 2 for 5 days to differentiate into mature neutrophil phenotype. After 5 days, centrifuged at 1,300 rpm for 5 minutes, HBSS (with Mg +, Ca +, Hanks' balanced salt solution) was treated with 10 mL and washed by centrifugation at 1,300 rpm for 5 minutes. The recovered cells were diluted in HBSS at a concentration of 1 × 10 6 cells / mL, and 100 μL were dispensed into 96 well plates, and 50 μL of each sample was prepared. Thereafter, CSE (cigarette smoke extract, final concentration 2%) was treated with 50 μL of each well and incubated for 3 hours. At this time, CSE was manufactured using the standard tobacco 3R4F for research purposes produced by the University of Kentucky. 5R 3F4F is installed horizontally in a self-made cigarette smoke collector and combusted. The cigarette smoker is collected in 50 mL PBS for 1 minute and 30 seconds with an aspirator, adjusted to 0.9-1.0 at OD320 and used as 100% CSE. It was. After 3 hours, Cytox green was treated with 500 nM and fluorescence was measured at 485/520 nm (excitation / emission maxima) wavelength using a fluorescent plate reader.

2.2 Evaluation of MIP-2 Inhibitory Activity Using MH-S Cells

MH-S cells, a mouse alveolar macrophage line, were purchased from ATCC, and the medium composition used for the culture 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 96 well plates at a concentration of 5 × 10 ⁴ / well and incubated for 24 hours. Subsequently, the stimulant (CSE; final 1% and LPS; final 10 ng / mL) and the sample were diluted with MH-S cell culture medium to reflect the desired final concentration and treated by 250 uL per well. After incubation for 24 hours, the supernatant was collected and used for MIP-2 measurement. The remaining cells were checked for survival by the WST method. MIP-2 was measured by ELISA method, and proceeded according to the manufacturer's protocol. After the supernatant was recovered, the remaining cells were treated with 200 μL / well of WST (Water soluble tetrazolium salt) reagent diluted 1/10 with MH-S medium, and reacted at 37 ° C. in a 5% CO 2 incubator for 20 minutes. The cell survival rate (WST) was confirmed by measurement, and concentrations lower than the control group were excluded from the cytokine analysis.

2.3 Evaluation of Improvement Activity Using CSE Induced COPD Model

BALB / C, male, and 5 week old mice were purchased from Orient Bio Co., Ltd. and were purified for 1 week, and grouped as n = 7. The sample treatment group orally administered the sample at a concentration of 200 mg / kg for 28 days from one week before the start of COPD induction to dissection, and the negative control group (Naive) and COPD group was administered the same amount of PBS. Roflumilast group, a licensed COPD treatment, was used as a positive control. Roflumilast group was orally administered 10 mg / kg concentrations three times a week after induction. COPD was induced by intraperitoneal administration of 100% Cigarette Smoke Extraction (CSE) once / week, three times in total. At the end of the experiment, 350% of 1.2% Avertin was intraperitoneally administered to anesthetize the mice, followed by dissection. Feed and drinking water were provided in the form of self-feeding during the experiment, and were raised at 24 ℃ and 60% humidity.

2.3.1 Respiratory Function Improvement Experiment

The degree of respiratory improvement was measured using Flexi Vent (SCIREQ). Catheter was injected into the anesthetized mouse airway and connected to the Flexi Vent device, followed by Tissue Damping (G) associated with tissue resistance and reflecting energy dissipation of the alveoli in the Flexi Vent program and closely related to tissue elasticity and energy conservation of the alveoli Tissue Elastnace (H) and Newtonian resistance (Rn) reflecting were measured.

2.4 Evaluation of Improvement Activity Using PPE / CSE Induced COPD Model

BALB / C mice (male, 5 weeks old) were purchased from Orient Bio Co., Ltd. and were purified for 1 week, and grouped at n = 10. The sample treatment group was orally administered 200 mg / kg of the sample for 24 days from one week before the start of COPD induction to dissection, and the negative control group (Naive) and the COPD group received the same amount of PBS. The Roflumilast group, a licensed COPD drug, was used as a positive control, and the Roflumilast group was administered orally at 10 mg / kg concentration daily after initiation of COPD induction. Induction of COPD was carried out using 1.2 units of Porcine Pancreatic Elastase (PPE) and 100% Cigarette Smoke Extraction (CSE). PPE 1.2 unit was injected 3 times through intra nasal once every 7 days. CSE treated 20 μl through intra nasal for 3 days from the day after PPE treatment, but after the last PPE treatment, dissected the CSE the next day. It was. Anatomy was performed after anesthesia with isoflurane using an inhalation anesthesia. Feed and drinking water were provided in the form of self-feeding during the experiment, and were raised at 24 ℃ and 60% humidity.

2.4.1 Total Cell Infiltration in BALF

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

2.4.2 Analysis of Immune Cells in BALF by Diff-Quick Staining

After injecting 1 mL of PBS through the airway of the mouse, lightly massaged to recover 700 μL of bronchoalveolar lavage fluid (BALF). The recovered BALF was separated into supernatant and cell pellet by centrifugation at 300 × g, 5 min. The cell pellet was resuspended by the addition of 700 μL of PBS. The BALF suspension was centrifuged at 1000 rpm, 10 min, and 4 ° C using a Cytospin device (Centrifuge 5403, Eppendorf, Hamburg, Germany) at 150 rpm. Attached. Subsequently, the cells were stained using a Diff-Quick staining reagent according to the manufacturer's protocol (1-5-1 Wakinohamakaigandori, chuo-ku, Kobe, Japan), and microscopic observation was used to determine the number of Macrophages, Lympocytes, Neutrophils, and Eosinophils. Counted.

3. Experimental Results

3.1 Evaluation of Inflammatory Cytokine (IL-8) Inhibitory Activity Using H292 Cells

The results are shown in FIG. Referring to these results, it can be seen that the Bokyeongpi extract significantly inhibits IL-8, an inflammatory cytokine, in a concentration-dependent manner in H292 cells.

3.2 Results of MIP-2 Inhibitory Activity Using MH-S Cells

The results are shown in FIG. Figure 2 shows the extract of Mip-2 in MH-S cells Show suppression.

3.3 Results of improvement activity in CSE-induced COPD model

3.3.1 Respiratory Function Test Results

The results are shown in FIG. Figure 3 shows that Bokyeongpi extract improves lung function or respiratory function by activating Tissue Damping (G) and Tissue Elastnace (H) and suppressing Newtonian resistance (Rn) in COPD-induced animal models with CSE. .

3.4 Results of Assessment of Improvement Activity in PPE / CSE-Induced COPD Models

3.4.1 Total Cell Infiltration in BALF

The analysis results of cytokines in BALF are shown in FIG. 4. Bokyeongpi extract reduced the total number of cells infiltrated in BALF.

3.4.2 Analysis of Immune Cells in BALF by Diff-Quick Staining

The microscopic photograph of the infiltrating neutrophil cell number in BALF is shown in FIG. 5. 5 shows a decrease in neutrophil cells.

Claims (26)

COPD improving composition comprising the extract as an active ingredient.
The method of claim 1,
The composition, in addition to the Bokyeongpi extract as an active ingredient, turmeric extract, golden extract, dried extract, leaflet extract, bark grass extract, Youngsil extract, fenugreek extract, pepper extract or a mixture of two or more thereof, characterized in that Composition.
The method of claim 1,
The extract is a composition characterized in that the water, ethanol or a mixed solvent extract thereof.
The method according to any one of claims 1 to 3,
The composition is a composition, characterized in that the food composition.
The method according to any one of claims 1 to 3,
Wherein said composition is a pharmaceutical composition.
Inflammatory lung disease improvement composition comprising the extract as an active ingredient.
The method of claim 6,
The composition, in addition to the Bokyeongpi extract as an active ingredient, turmeric extract, golden extract, dried extract, leaflet extract, bark grass extract, Youngsil extract, fenugreek extract, pepper extract or a mixture of two or more thereof, characterized in that Composition.
The method of claim 6,
The extract is a composition characterized in that the water, ethanol or a mixed solvent extract thereof.
The method according to any one of claims 6 to 8,
The composition is a composition, characterized in that the food composition.
The method according to any one of claims 6 to 8,
Wherein said composition is a pharmaceutical composition.
Composition for improving lung function or respiratory function comprising the extract of Bokryeongpi as an active ingredient.
The method of claim 11,
The composition, in addition to the Bokyeongpi extract as an active ingredient, turmeric extract, golden extract, dried extract, leaflet extract, bark grass extract, Youngsil extract, fenugreek extract, pepper extract or a mixture of two or more thereof, characterized in that Composition.
The method of claim 11,
The extract is a composition characterized in that the water, ethanol or a mixed solvent extract thereof.
The method according to any one of claims 11 to 13,
The composition is a composition, characterized in that the food composition.
The method according to any one of claims 11 to 13,
Wherein said composition is a pharmaceutical composition.
Respiratory disease improvement composition by smoking or fine dust containing the extract as an active ingredient.
The method of claim 16,
The respiratory disease is asthma, COPD, diffuse interstitial lung disease, acute respiratory distress syndrome or acute lung injury characterized in that the composition.
The method of claim 16,
The composition, in addition to the Bokyeongpi extract as an active ingredient, turmeric extract, golden extract, dried extract, leaflet extract, bark grass extract, Youngsil extract, fenugreek extract, pepper extract or a mixture of two or more thereof, characterized in that Composition.
The method of claim 16,
The extract is a composition characterized in that the water, ethanol or a mixed solvent extract thereof.
The method according to any one of claims 16 to 19,
The composition is a composition, characterized in that the food composition.
The method according to any one of claims 16 to 19,
Wherein said composition is a pharmaceutical composition.
Composition for improving lung damage by smoking or fine dust comprising the extract of Bokryeongpi as an active ingredient.
The method of claim 22,
The composition, in addition to the Bokyeongpi extract as an active ingredient, turmeric extract, golden extract, dried extract, leaflet extract, bark grass extract, Youngsil extract, fenugreek extract, pepper extract or a mixture of two or more thereof, characterized in that Composition.
The method of claim 22,
The extract is a composition characterized in that the water, ethanol or a mixed solvent extract thereof.
The method according to any one of claims 22 to 24,
The composition is a composition, characterized in that the food composition.
The method according to any one of claims 22 to 24,
Wherein said composition is a pharmaceutical composition.

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