KR102273066B1 - Composition for improving chronic obstructive pulmonary disease using an extract of Podocarpus macrophyllus - Google Patents

Abstract

The present invention inhibits NETosis induced by phorbol myristate acetate (PMA), and inhibits the expression of IL-8, an inflammatory cytokine, by H292 cells, a bronchial epithelial cell line stimulated by CSE (cigarette smoke extract). Inhibits the expression of the inflammatory cytokine MIP2 (IL-8) in MH-S cells, which are mouse alveolar macrophages activated by CES, as well as inhibits the activity of the proteolytic enzyme elastase, CES and PPE (porcine pancreas elastase)-induced COPD, lowered the number of neutrophils in BALF and the concentration of inflammatory cytokines such as MIP-2, TNF-α, IL-1β, and IL-6 in BALF or lung tissue Disclosed is a composition for improving chronic obstructive pulmonary disease using a Nahansong extract having lowering activity.

Description

Composition for improving chronic obstructive pulmonary disease using an extract of Podocarpus macrophyllus

The present invention relates to a composition for improving chronic obstructive pulmonary disease using an extract of Nahansong ( Podocarpus macrophyllus ).

Chronic obstructive pulmonary disease (COPD) is a chronic airway disease characterized by irreversible airway obstruction such as cough, sputum, dyspnea, decreased expiratory flow rate, and impaired gas exchange. It is predicted to become the third cause of human death by 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 classified into chronic bronchitis and emphysema, but chronic bronchitis is defined based on clinical symptoms and emphysema is classified based on anatomical criteria. COPD is collectively diagnosed (Eur Respir J, 2007, 30:993-1013; Respirology 1997, 2 Suppl 1:S1-4). In the case of bronchial asthma, if the asthma persists for a long time and the airway obstruction shows an irreversible change, it is included in the 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. Pharm Bull, 2012, 35:1752-1760). The pathogenesis of COPD involves chronic inflammation in the bronchi and lung tissue, protease activation in the lung, and oxidative stress. Cells involved in inflammation are mainly neutrophils, macrophages, and T lymphocytes. . These inflammatory cells produce various inflammatory cytokines such as TNF-α, IFN-γ, IL-1β, IL-6, IL-8, IL-18, and various proteolytic enzymes that cause tissue damage (Korean tuberculosis and The Society of Respiratory Medicine, Seoul: Gunja Publishing House; 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 the cigarette smoke-induced COPD animal model, neutrophil influx, KC (keratinocyte chemoattractant), TNF-α (tumour necrosis factorα), MIP-2 (macropage inflammatory protein 2), MIP-1α and MCP-1 (monocyte chemoattractant protein) , an increase in matrix metalloproteinase 12 (MMP12) and granulocyte macrophage colony-stimulating factor (GM-CSF) was observed (Clin. Sci. 2014, 126(3):207).

In COPD patients, as in asthma patients, increased concentrations of IL-17 in sputum and serum were observed, suggesting that IL-17 may play a certain role in COPD. 17 has been proposed as an important target for inhibition of airway fibrosis in COPD by playing a critical role in airway fibrosis (Int J Chron Obstruct Pulmon Dis. 2017; 12:1247-1254; Chest. 2010 Nov;138(5):1140- 7).

On the other hand, neutrophils secrete substances such as elastase, collagenase, proteases such as MPO (myeloperoxidase), arachidonic acid metabolites (arachidonate), and reactive oxygen free radicals to the lungs. It is known to play a very important role in the pathogenesis of COPD by causing damage 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, it has been reported that excessive formation of neutrophil extracellular traps (NET) is 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 in which cytoplasmic and granular proteins are bound to intertwined chromatin form of a structure. NET captures and neutralizes bacteria, fungi, and viruses, and prevents their spread and infection (Nat Immunol 15, 1017-1025, 2014), but excessive secretion of NET is associated with various infectious and non-infectious diseases. . In particular, the relationship 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 is not only caused by COPD, but also asthma, cystic fibrosis, RSV respiratory syncytial virus bronchiolitis, influenza virus infection, bacterial pneumonia, tuberculosis It has been reported in lung diseases such as (tuberculosis) and transfusion-related acute lung injury, and thus inhibition of NETosis is recognized as an important target for the development of therapeutic agents for these lung diseases (Front Immunol. 2016, 7:311)

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

There have been no reports of drugs that directly improve COPD or asthma so far, and the current treatment for COPD or asthma is to reduce symptoms and complications, and bronchodilators (β2-agonists, anticholinergics, methylxanthines) and steroids (inhaled, Oral) is mainly used.

The present invention discloses the effect of improving asthma or chronic obstructive pulmonary disease of Nahansong extract.

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

Other objects or specific objects of the present invention will be set forth below.

The present inventors, as confirmed in the following Examples and Experimental Examples, the Nahansong extract inhibits NETosis induced by PMA (phorbol myristate acetate), the bronchi stimulated by CSE (cigarette smoke extract) Inhibits the expression of IL-8, an inflammatory cytokine, by H292 cells, an epithelial cell line, and inhibits the expression of MIP2 (IL-8), an inflammatory cytokine, in MH-S cells, which are mouse alveolar macrophages activated by CES. In addition, in an animal model experiment in which COPD was induced with CES and porcine pancreas elastase (CES) and porcine pancreas elastase (CES) by inhibiting the activity of elastase, a proteolytic enzyme, the number of neutrophils in BALF was lowered and MIP-2 in BALF or lung tissue, It was confirmed that the concentration of inflammatory cytokines such as TNF-α, IL-1β, and IL-6 was lowered.

The present invention is provided based on the experimental results as described above, and the present invention can be identified as a composition for improving COPD comprising a Nahansong extract as an active ingredient in one aspect, and in another aspect, a Nahansong extract It can be identified as a composition for improving inflammatory lung disease, including as an active ingredient. In addition, in another aspect, the present invention can be identified as a composition for improving lung function or respiratory function comprising the Nahansong extract as an active ingredient, and in another aspect, it is effective against smoking or fine dust containing the Nahansong extract as an active ingredient. It can be identified as a composition for improving respiratory diseases by, and in another aspect, it can be identified as a composition for improving lung damage including Nahansong extract as an active ingredient. In addition, in another aspect, the present invention can be identified as a composition for improving lung disease accompanied by excessive NET formation, that is, netosis, including Nahansong extract as an active ingredient.

As used herein, the term "extract" refers to the stem, leaf, fruit, flower, root, outpost, mixture thereof, etc. 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 It refers to an extract obtained by leaching using a supercritical extraction solvent such as carbon dioxide, pentane, or a fraction obtained by fractionating the extract, and the extraction method is cooling, taking into account the polarity of the active material, the degree of extraction, and the degree of preservation. Any method such as reflux, heating, ultrasonic radiation, or supercritical extraction may be applied. In the case of the fractionated extract, a fraction obtained by suspending the extract in a specific solvent and mixing and standing still with a solvent having a different polarity, and adsorbing the crude extract to a column filled with silica gel, etc., hydrophobic solvent, hydrophilic solvent, or a mixed solvent thereof It is meant to include the fraction obtained as a mobile phase. In addition, the meaning of the extract includes a concentrated liquid extract or solid extract from which the extraction solvent has been removed by 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, and 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" means a component capable of exhibiting the desired activity alone or in combination with a carrier having no activity by itself.

In addition, as used herein, the term "inflammatory lung disease" is a lung disease accompanied by an inflammatory response, and includes asthma, chronic obstructive pulmonary disease (COPD), tracheitis, and bronchitis.

In addition, in the present specification, "improving lung function or respiratory function" refers to improvement in respiratory function of a normal person who is not a disease, recovery of respiratory failure in a patient due to chronic obstructive pulmonary disease, asthma, bronchitis, tracheitis, etc., or respiratory function in a patient means the improvement of

Also, in this 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), and acute lung injury to be. Fine dust containing various components such as carbon components such as soot and 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 It is known to cause various lung diseases such as asthma and COPD by depositing in the airways, bronchial tubes, small airways, and alveoli (J Korean Med Assoc, 2014;57:763-768).

Also, in the present specification, "improving lung damage" means recovery of lung cells or lung tissue damage caused by fine dust or the like, or recovery of lung function deterioration due to lung cell or lung tissue damage.

In addition, in the present specification, "pulmonary disease accompanying netosis" is not only COPD exemplified above, but also asthma, cystic fibrosis, RSV-type bronchiolitis, influenza virus infection (influenza virus) infection, bacterial pneumonia, tuberculosis, and transfusion-related acute lung injury.

Also in this specification, "improvement" is meant to include alleviation, treatment or prevention of a disease or symptom.

In the composition of the present invention, the active ingredient may be included in any amount (effective amount) depending on the use, formulation, etc., as long as it can exhibit asthma improvement, COPD improvement, lung function or respiratory function improvement effect, etc. It will be determined within the range of 0.001 wt % to 15 wt % based on the total weight. As used herein, the term "effective amount" refers to a mammal, preferably a human, to which the subject is applied, when the composition of the present invention is administered during the administration period according to the suggestion of a medical professional, asthma improvement, COPD improvement, lung function or respiratory function improvement effect, etc. Refers to the amount of the active ingredient included in the composition of the present invention, which can exhibit a medical and pharmacological effect. Such effective amounts can be determined empirically within the ordinary ability of one of ordinary skill in the art.

In addition to Nahansong extract as an active ingredient, the composition of the present invention is turmeric, which is already known to have an effect of improving asthma, improving COPD, improving lung function or respiratory function, in order to increase the effect of improving asthma, improving COPD, improving lung function or respiratory function. ( Curcuma longa ) extract, golden ( Scutellaria baicalensis ) extract, honeysuckle ( Trapa japonica ) extract, leaf leaf ( Perilla frutescens ) extract, node grass ( Polygonum avivulare ) extract, Youngsil ( Rosae multiflorea ) extract, fenugreek ( Trigonella foenum ) extract, black pepper ( 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, and ionic beverages, processed oils such as milk and yogurt, gums, rice cakes, Korean sweets, bread, Foods such as confectionery and noodles, tablets, capsules, pills, granules, liquids, powders, flakes, pastes, syrups, gels, jellies, bars, and the like can be manufactured into health functional food preparations. In addition, the food composition of the present invention may have any product classification as long as it conforms to the enforcement regulations at the time of manufacture and distribution in legal and functional classification. For example, it is a health functional food according to the "Health Functional Food Act" of Korea, or confectionery, bean curd, tea, It may be beverages, special-purpose foods, and the like.

The food composition of the present invention may contain food additives in addition to the active ingredients thereof. Food additives can be generally understood as substances that are added to and mixed with or infiltrated into food in manufacturing, processing or preserving food. Since they are consumed daily and for a long period of 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 Ordinance in accordance with the laws of each country that regulates the manufacture and distribution of food (“Food Sanitation Act” in Korea). In the Korean Food Additives Code (Ministry of Food and Drug Safety Notice "Food Additive Standards and Specifications), food additives are classified into chemically synthetic products, natural additives, and mixed preparations in terms of ingredients. These food additives are sweeteners and flavorants in terms of function. , preservatives, emulsifiers, acidulants, thickeners, etc.

The sweetener is used to impart appropriate sweetness to food, and both natural and synthetic ones may 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 fragrance, and both natural and synthetic ones may be used. Preferably, it is a case where a natural thing is used. In the case of using a natural one, the purpose of nutritional enhancement in addition to flavor may be concurrently used. As a natural flavoring agent, it may be obtained from apple, lemon, tangerine, grape, strawberry, peach, etc., or it may be obtained from green tea leaf, dandelion, bamboo leaf, cinnamon, chrysanthemum leaf, jasmine, etc. In addition, those obtained from ginseng (red ginseng), bamboo shoots, aloe vera, ginkgo biloba, etc. can be used. The natural flavoring agent may be a liquid concentrate or a solid extract. In some cases, a synthetic flavoring agent may be used, and the synthetic flavoring agent may include esters, alcohols, aldehydes, terpenes, and the like.

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, and pectin can be used. acidulant, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, phosphoric acid and the like may be used as acidulants. Acidulant may be added so that the food composition has an appropriate acidity for the purpose of inhibiting the growth of microorganisms in addition to the purpose of enhancing the taste. As the thickening agent, a suspending agent, a settling agent, a gel-forming agent, a bulking agent, and the like can be used.

The food composition of the present invention may contain, in addition to the food additives described above, bioactive substances or minerals known in the art for the purpose of supplementing and reinforcing functionality and nutrition and with guaranteed stability as food additives.

Examples of such physiologically active substances include catechins contained in green tea and the like, vitamins such as vitamin B1, vitamin C, vitamin E, and vitamin B12, tocopherol, dibenzoylthiamine, and the like. Magnesium preparations, such as iron preparations, such as iron citrate, chromium chloride, potassium iodide, selenium, germanium, vanadium, zinc, etc. are mentioned.

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

In relation to other food additives that may be included in the food composition of the present invention, reference may be made to the Food Standards or Food Additives Code according to the laws of each country.

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

The pharmaceutical composition of the present invention may be prepared as an oral dosage form or a parenteral dosage form according to the route of administration by a conventional method known in the art, including a pharmaceutically acceptable carrier in addition to the active ingredient. Here, "pharmaceutically acceptable" means that it does not inhibit the activity of the active ingredient and does not have toxicity beyond which the application (prescription) target is adaptable.

When the pharmaceutical composition of the present invention is prepared as an oral dosage form, powders, granules, tablets, pills, dragees, capsules, liquids, gels, syrups, suspensions, wafers, together with a suitable carrier according to methods known in the art. It can be prepared in a formulation such as In this case, examples of suitable pharmaceutically acceptable carriers include sugars such as lactose, glucose, sucrose, dextrose, sorbitol, mannitol, and xylitol, starches such as corn starch, potato starch, wheat starch, cellulose, methylcellulose, ethylcellulose, Cellulose such as sodium carboxymethylcellulose and hydroxypropylmethylcellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, polyol, vegetable and oil. In the case of formulation activity, the formulation may be formulated by including a diluent and/or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, and a surfactant, if necessary.

When the pharmaceutical composition of the present invention is prepared for parenteral use, it may be formulated in the form of eye drops, injection, transdermal administration, nasal inhalation, or suppository together with a suitable carrier according to methods known in the art. In the case of formulation with eye drops, sterile water, saline, isotonic solutions such as 5% dextrose, etc. can be used as suitable carriers, and benzalkonium chloride, mefilparaben, ethylparaben, etc. can be added as needed for preservative purposes. When formulated as an injection, a suitable carrier may be sterile water, ethanol, polyol such as glycerol or propylene glycol, or a mixture thereof, preferably Ringer's solution, PBS (phosphate buffered saline) containing triethanolamine, or sterilized for injection. Water, an isotonic solution such as 5% dextrose, etc. may be used. When formulated for transdermal administration, it can be formulated in the form of ointments, creams, lotions, gels, external solutions, pasta agents, liniment agents, air rolls, and the like. In the case of nasal inhalants, it can be formulated in the form of an aerosol spray using a suitable propellant such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, etc., and when formulated as a suppository, the base is Witepsol ( witepsol), tween 61, polyethylene glycols, cacao fat, laurin fat, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearate, sorbitan fatty acid esters, and the like can be used.

Specific formulations of pharmaceutical compositions are known in the art, and reference may be made to, for example, Remington's Pharmaceutical Sciences (19th ed., 1995). This document is considered a part of this specification.

A 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's severity, and administration route. It can be in the range /kg. Administration may be performed once or divided into several times a day. Such dosages should not be construed as limiting the scope of the invention in any respect.

As described above, according to the present invention, it is possible to provide a composition for improving COPD using the Nahansong extract. The composition of the present invention is commercialized as a food, particularly a health functional food or drug, and may be usefully used for the effect of improving COPD and further improving the effect of lung function or respiratory function.

1 is a result showing the NETosis inhibitory activity of Nahansong extract.
2 is a result showing the Elastase inhibitory activity of Nahansong extract.
3 is a result showing the inflammatory cytokine (IL-8) inhibitory activity in H292 cells stimulated by CSE of Nahansong extract.
4 is a result showing the MIP-2 inhibitory activity of Nahansong extract using MH-S cells stimulated by CSE.
5 is a result showing that Nahansong extract reduces the total number of infiltrating cells in BALF in an animal model experiment induced by CSE and PPE.
6 is a result showing that Nahansong extract reduces the number of infiltrating immune cells in BALF in an animal model experiment induced by CSE and PPE.
7 shows inflammatory cytokines such as IL-1β, MIP-2, IL-6, TNF-α, and IL-17 in BALF and lung tissue in an animal model experiment in which Nahansong extract induced COPD by CSE and PPE. The result shows that the concentration of

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> Improvement activity of nahansong extract, such as lung disease

1. Sample Preparation

The Nahansong extract was obtained in powder form by adding 10 times the weight of 70% ethanol to the dry powder of Nahansong (extraction site: leaf, flower), extracting twice at 50°C for 6 hours, filtration, concentration under reduced pressure, and freeze-drying.

2. Experimental method

2.1 NETosis Inhibitory Activity

HL-60 cells (Human promyelocytic leukemia cells) purchased from the Korea Cell Line Bank were treated with 1% DMSO in RPMI (10% FBS, 100 U/mL penicillin, 100 mg/mL streptomycin) medium at 2×10 ⁵ cell/mL. was seeded and differentiated into a mature neutrophil phenotype by culturing for 5 days at 37°C in a cell incubator, 5% CO _{2 .} After 5 days of centrifugation at 1,300 rpm for 5 minutes ^{, 10 mL of HBSS (with Mg +,} Ca ^+, Hanks' balanced salt solution) was treated, followed by centrifugation at 1,300 rpm for 5 minutes, followed by washing. The recovered cells were ^{diluted in HBSS to a concentration of 1×10 6} cell/mL, and 100 μL of each was dispensed in a 96-well plate, and 50 μL of each sample was treated. After that, 50 μL of PMA (phorbol myristate acetate, Sigma) prepared at 400 nM was treated in each well and incubated for 3 hours. After 3 hours, Cytox green was treated with 500 nM and fluorescence was measured at a wavelength of 485/520 nm (excitation/emission maxima) using a fluorescence plate reader.

2.2 Evaluation of Elastase Inhibitory Activity

Elastase inhibitory activity was measured using EnzChekⓡ Elastase assay kit (InvitrogenTM). After dispensing 50 uL of diluted sample or inhibitor reflecting the desired final concentration in a 96 well plate, 50 uL of DQ elastin was added and incubated at 25°C in a dark place for 1 hour. After that, 50 uL of elastase was added and the enzymatic reaction was performed in the dark for 90 minutes, and then the fluorescence intensity was measured under ex/em=505/515 conditions. At this time, based on the fluorescence intensity of the sample or the inhibitor-free group (100%), the inhibition rate according to the addition of the sample or inhibitor was relatively calculated.

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

H292 cells, a bronchial epithelial cell line, were purchased from the Korea Cell Line Bank and used. ^{H292 cells prepared at a concentration of 2×10 5} cells/mL in a 24-well plate were subcultured at 500 μL/well, and when the cells became more than 80% confluent, they were replaced with serum-free RPMI1640 medium and starvated for 24 hours. After that, the stimulant (CSE; final 2%) and the sample were diluted by reflecting the desired final concentration using serum-free RPMI1640 medium, and 500 μL per well was treated. After overnight incubation, the supernatant was recovered and used for IL-8 measurement, and the viability of the remaining cells was confirmed by WST (water-soluble tetrazolium salt assay). Human IL-8 was measured by ELISA, and the manufacturer (BD Inc.) protocol.At this time, the supernatant for IL-8 measurement was diluted 5 times and analyzed.After collecting the supernatant, the remaining cells were diluted 1/10 with RPMI1640 + 10% FBS + 1% P/S medium. One WST (Water soluble tetrazolium salt) reagent was treated with 500 μL/well and ^{reacted for 50 minutes in an incubator at 37°C, 5% CO 2} , and then absorbance was measured at 450 nm to check the cell viability (WST). Concentrations were excluded from cytokine analysis.

2.4 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 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 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 sample (final; 25, 50, 100 μg/mL) with MH-S cell culture medium to reflect the desired final concentration and dilute the wells. 250 uL per each was treated. After culturing for 24 hours, the supernatant was recovered and used for MIP-2 measurement, and the viability of the remaining cells was confirmed by the WST method. MIP-2 was measured by ELISA method, and proceeded according to the protocol of the manufacturer (R&D, DY452). After recovering the supernatant, the remaining cells were treated with 200 μL/well of water soluble tetrazolium salt (WST) reagent diluted 1/10 with MH-S medium, reacted in an incubator at 37°C, 5% CO2 for 20 minutes, and absorbance at 450 nm Cell viability (WST) was confirmed by measuring, and concentrations with lower viability than the control were excluded from cytokine analysis.

2.5 Evaluation of COPD improvement activity in animal experiments

BALB/C mice (male, 5 weeks old) were purchased from Orient Bio, acclimatized for 1 week, and grouped with n=10. The sample treatment group was orally administered with a sample of 200 mg/kg for 24 days from one week before the start of COPD induction until dissection, and the negative control group (Naive) and the COPD group were administered the same amount of PBS. The Roflumilast administration group, an approved COPD treatment, was used as a positive control, and the Roflumilast administration group was orally administered at a concentration of 10 mg/kg every day after COPD induction started. 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 intranasally once every 7 days for a total of 3 times, and 20 μl of CSE was treated through intranasal for 3 days from the day after PPE treatment. After the last PPE treatment, dissection was performed the next day without CSE treatment. did. Dissection was performed after anesthesia with isoflurane using an inhalation anesthetic. During the experiment, feed and drinking water were provided in the form of self-feeding, and they were reared at 24 ℃ and 60% humidity.

2.5.1 Total number of infiltrating cells 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). 10 uL of the recovered BALF solution was mixed with Accustain T solution, 12 uL was injected into the Accuchip channel, and total cells were automatically counted with a cell counter (ADAM-MC, NANOENTEK. INC, Korea).

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

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

2.5.3 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 used for cytokines and chemokines analysis (IL-1β, MIP-2, IL-6, TNF-α, IL-17). After taking 30 uL of the supernatant for the analysis of cytokines and chemokines, the experiment was performed according to the protocol of the manufacturer (Quansis biosciences) of the Q-plex ELISA array kit.

3. Experimental results

3.1 NETosis inhibitory activity evaluation result

The results of evaluation of NETosis inhibitory activity are shown in FIG. 1 . Referring to Figure 1, it can be seen that the Nahansong extract inhibits NETosis in proportion to the treatment concentration.

3.2 Elastase inhibitory activity evaluation result

Elastase inhibitory activity evaluation results are shown in FIG. 2 . Nahansong extract inhibited Elastase in a concentration-dependent manner.

3.3 Results of evaluation of inflammatory cytokine (IL-8) inhibitory activity using H292 cells

The results are shown in FIG. 3 . Referring to these results, it can be seen that the Nahansong extract clearly inhibits IL-8, an inflammatory cytokine, in H292 cells.

3.4 Results of evaluation of MIP-2 inhibitory activity using MH-S cells

The results are shown in FIG. 4 . Figure 4 is Nahansong extract MIP-2 in MH-S cells shows restraint.

3.5 Evaluation results of COPD improvement activity in animal experiments

3.5.1 Total number of infiltrating cells in BALF

The analysis results of cytokines in BALF are shown in FIG. 5 . Nahansong extract decreased the total number of infiltrating cells in BALF.

3.5.2 Immune Cell Analysis Results in BALF by Diff-Quick Staining

The results of analysis of immune cells in BALF are shown in FIG. 6 . Nahansong extract significantly reduced all four immune cells (Eosinophil, Neutrophil, Lympocyte, Macrophasge). At the bottom of FIG. 6, there is shown a photograph of the number of infiltrating neutrophil cells in the BALF under a microscope, and it can be seen that the neutrophil cell count was also clearly reduced in this microscopic photograph.

3.5.3 Analysis of Cytokines in BALF

The analysis results of cytokines in BALF are shown in FIG. 7 . Nahansong extract shows that BALF lowers the concentration of inflammatory cytokines such as IL-1β, MIP-2, IL-6, TNF-α, and IL-17.

Claims (32)

As a food composition for improving respiratory diseases comprising Nahansong extract as an active ingredient,
The respiratory disease is cystic fibrosis, chronic obstructive pulmonary disease (COPD), tuberculosis, pneumonia, bronchiolitis, transfusion-related acute pulmonary disease, or diffuse interstitial lung disease, the composition. delete According to claim 1,
The extract is water, ethanol, or a mixture thereof, characterized in that the extract, the composition. delete delete delete delete According to claim 1,
The respiratory disease is characterized in that COPD, the composition. As a pharmaceutical composition for the prevention or treatment of respiratory diseases comprising Nahansong extract as an active ingredient,
The respiratory disease is cystic fibrosis, chronic obstructive pulmonary disease (COPD), tuberculosis, pneumonia, bronchiolitis, transfusion-related acute pulmonary disease, or diffuse interstitial lung disease, the composition. 10. The method of claim 9,
The composition is characterized in that the extract is water, ethanol, or a mixed solvent extract thereof. delete delete delete 10. The method of claim 9,
The respiratory disease is characterized in that COPD, the composition. As a health functional food for improving respiratory diseases comprising the food composition of claim 1, 3 or 8,
The respiratory disease is cystic fibrosis, chronic obstructive pulmonary disease (COPD), tuberculosis, pneumonia, bronchiolitis, transfusion-related acute lung disease, or diffuse interstitial lung disease, health functional food. delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete

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