KR20200135640A - Composition for improving asthma or chronic obstructive pulmonary disease using an extract of Curcuma longa, Scutellariae Radix, etc - Google Patents

Abstract

The present invention discloses a composition for alleviating asthma or chronic obstructive pulmonary diseases using a Curcuma longa extract, a Scutellaria baicalensis extract which inhibits NETosis induced by phorbol myristate acetate (PMA), inhibits the expression of inflammatory cytokineS IL-6 and/or IL-8 in H292 cells, which are bronchial epithelial cell lines stimulated by a cigarette smoke extract (SEC), etc., inhibits the expression of inflammatory cytokine MIP2 (IL-8) in MH-S cells, which are mouse alveolar macrophages activated by CES, and thus lowers the number of infiltrating cells of BALF in an animal model experiment inducing COPD with CES or CES/PPE.

Description

Composition for improving asthma or chronic obstructive pulmonary disease using an extract of Curcuma longa, Scutellariae Radix, etc. {Composition for improving asthma or chronic obstructive pulmonary disease using an extract of Curcuma longa, Scutellariae Radix, etc}

The present invention relates to a composition for improving asthma or chronic obstructive pulmonary disease using turmeric extract, golden extract, and the like.

Chronic obstructive pulmonary disease (COPD) is a chronic airway disease that shows irreversible airway obstruction, such as coughing, sputum, shortness of breath, decreased expiratory flow, and impaired gas exchange. It is predicted to be the third cause of human death 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 classified into chronic bronchitis and emphysema, but chronic bronchitis is defined on the basis of clinical symptoms and emphysema is classified by anatomical criteria, so many cases of having both in the same patient at the same time are difficult to distinguish clinically. It is comprehensively diagnosed as COPD (Eur Respir J, 2007, 30:993-1013; Respirology 1997, 2 Suppl 1:S1-4). In the case of bronchial asthma, if the duration of asthma is long and the airway obstruction shows irreversible changes, 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, 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 chronic inflammation in the bronchi and lung tissue, activation of proteases in the lungs, and oxidative stress, and the 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, and IL-18, and various proteolytic enzymes that cause tissue damage (Korean tuberculosis and Respiratory Society Respiratory Science 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 an animal model of COPD induced by tobacco smoke, influx of neutrophils, keratinocyte chemoattractant (KC), tumor necrosis factorα (TNF-α), macropage inflammatory protein 2 (MIP-2), MIP-1α and monocyte chemoattractant protein (MCP-1) , MMP12 (matrix metalloproteinase 12) and GM-CSF (granulocyte macrophage colony-stimulating factor) were observed to increase (Clin. Sci. 2014, 126(3):207).

COPD is not considered to be a TH2-type disease such as atopy or asthma, but it has been reported that TH1 cells and TH2 cells are activated in COPD patients. The chemokine TARC/CCL17 and MDC/CCL22 are ligands of the chemokine receptor CCR4 expressed in Th2 cells (Curr. Opin. Immunol. 2002. 14: 129-135), and the TH2 cytokine MDC is the same as in asthma patients. It has been observed that the expression is increased (J Immunol 2008, 181:2790-8; Clin Exp Allergy 2012, 42:994-1005). In addition, TARC/CCL17 and MDC/CCL22 were increased along with TSLP (thymic stromal lymphopoietin) in asthma and COPD, which was confirmed by a significant increase in TARC/CCL17 and MDC/CCL22 in the smoking group compared to the non-smoking group in clinical trials. Became (Ijmmunol. 2008. 181(4):2790-8). As a result, it can be seen that reduction of TARC/CCL17 and MDC/CCL22 can be an important factor in improving COPD.

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

Meanwhile, 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 onset 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 of entangled chromatin with cytoplasmic proteins and granular proteins. It is a form of structure. NET captures and neutralizes bacteria, fungi, and viruses, and plays a role in preventing their spread and infection (Nat Immunol 15, 1017-1025, 2014), but excessive NET secretion 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 NET formation (NETosis) is not only COPD, but also asthma, cystic fibrosis, respiratory syncytial virus bronchiolitis, influenza virus infection, bacterial pneumonia, tuberculosis. (tuberculosis), blood transfusion-related acute lung injury (transfusion-related acute lung injury) has been reported in lung diseases, and therefore netosis (NETosis) suppression is recognized as an important target of the development of these pulmonary disease treatments (Front Immunol. 2016, 7:311)

Currently, bronchoalveolar lavage (BAL) is used as a means to view the diagnosis and progress of diseases such as COPD and asthma, and inflammatory cytokines and free radicals in the 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 lung, increase to an extent that accounts for 80% of all cells (Am J Respir Crit Care Med 154(1):76-81, 1996).

So far, no drugs have been reported to directly improve COPD or asthma, and current COPD or asthma treatments are intended to reduce symptoms and complications, including bronchodilators (β2-agonists, anticholinergics, methylxanthines) and steroids (inhalation, Oral), etc. are mainly used.

The present invention discloses that there is an effect of improving asthma or chronic obstructive pulmonary disease such as turmeric extract and golden extract.

An object of the present invention is to provide a composition for improving asthma or chronic obstructive pulmonary disease using turmeric extract, golden extract, and the like.

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

The present inventors, as confirmed in the following Examples and Experimental Examples, turmeric extract, golden extract, etc. inhibit netosis induced by PMA (phorbol myristate acetate), CSE (cigarette smoke extract), etc. Inhibits the expression of inflammatory cytokines IL-6 and/or IL-8 in H292 cells, which are stimulated bronchial epithelial cell lines, and MIP2, an inflammatory cytokine, in MH-S cells, mouse alveolar macrophages activated by CES ( In addition to inhibiting the expression of IL-8), it was confirmed that the number of infiltrating cells of BALF was lowered in an animal model experiment in which COPD was induced with CES or CES/PPE.

The present invention is provided on the basis of the experimental results described above, in one aspect, the present invention, in one aspect, turmeric extract, golden extract, dryweed extract, lobule extract, walnut extract, youngsil extract, fenugreek extract, pepper extract or It can be understood as a composition for improving asthma comprising a mixture of two or more of them as an active ingredient, and in other aspects, turmeric extract, golden extract, dryweed extract, lobule extract, walnut extract, youngsil extract, fenugreek extract, pepper extract, or these In another aspect, it can be understood as a composition for improving COPD comprising a mixture of two or more of them as an active ingredient.In another aspect, turmeric extract, golden extract, dryweed extract, lobule extract, walnut extract, youngsil extract, fenugreek extract, pepper extract, or these It can be recognized as a composition for improving inflammatory lung disease comprising a mixture of two or more of the as an active ingredient. In another aspect, the present invention is a lung function or respiratory function comprising a turmeric extract, a golden extract, a dry leaf extract, a lobule extract, a lobule extract, a youngsil extract, a fenugreek extract, a pepper extract, or a mixture of two or more thereof as an active ingredient Smoking containing turmeric extract, golden extract, dry leaf extract, lobule extract, knuckle extract, youngsil extract, fenugreek extract, pepper extract, or a mixture of two or more thereof as an active ingredient in another aspect Alternatively, it can be identified as a composition for improving respiratory diseases due to fine dust, and in another aspect, turmeric extract, golden extract, dryweed extract, lobule extract, node grass extract, youngsil extract, fenugreek extract, pepper extract, or a mixture of two or more thereof It can be recognized as a composition for improving lung damage comprising as an active ingredient. In another aspect of the present invention, in another aspect, excessive formation of NET containing turmeric extract, golden extract, dry leaf extract, lobule extract, walnut extract, Youngsil extract, fenugreek extract, pepper extract, or a mixture of two or more thereof as an active ingredient, that is, neto It can be recognized as a composition for improving lung diseases accompanying cis.

In the present specification, the term "extract" refers to the stem, leaves, fruits, flowers, roots, outposts, 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, 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 by using, an extract obtained by using a supercritical extraction solvent such as carbon dioxide, pentane, or a fraction obtained by fractionating the extract, and the extraction method is cold precipitation, Arbitrary methods, such as reflux, warming, 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, and adsorbing the crude extract on a column filled with silica gel, etc., and then adding a hydrophobic solvent, a hydrophilic solvent, or a mixture 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 a solid extract from which the extraction solvent has been removed by a method such as freeze drying, vacuum drying, hot air drying, or spray drying. 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" refers to a component capable of exhibiting a desired activity alone, or capable of exhibiting activity together with a carrier that is not itself active.

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

In addition, in the present specification, "improvement of lung function or respiratory function" refers to improvement of respiratory function of a non-disease person, recovery of respiratory failure of a diseased person due to chronic obstructive pulmonary disease or asthma, bronchitis, tracheitis, etc. Means the improvement of.

In addition, in the present specification, "respiratory disease caused by smoking or fine dust" means including asthma, 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 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 trachea, bronchi, small airways and alveoli (J Korean Med Assoc, 2014;57:763-768).

In addition, in the present specification, "improving lung damage" refers to recovery of damage to lung cells or lung tissue due to fine dust or the like or recovery of decreased lung function due to damage to lung cells or lung tissue.

In addition, in the present specification, "pulmonary disease accompanying netosis" refers to asthma (asthma), cystic fibrosis, RSV bronchiolitis (respiratory syncytial virus bronchiolitis), influenza virus infection (influenza virus) as well as COPD exemplified above. infection), bacterial pneumonia, tuberculosis, and transfusion-related acute lung injury.

In addition, in the present 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 an arbitrary amount (effective amount) depending on use, formulation, etc., as long as it can exhibit an effect of improving asthma, COPD, lung function or respiratory function, etc., and a typical effective amount is the composition It will be determined within the range of 0.001% to 15% by weight based on the total weight. Herein, the term "effective amount" refers to the intended mammal, preferably a human, when the composition of the present invention is administered during the administration period according to the advice of a medical professional, etc., asthma improvement, COPD improvement, lung function or respiratory function improvement effect, etc. It refers to the amount of the active ingredient contained in the composition of the present invention that can exhibit medical and pharmacological effects. Such effective amounts can be determined empirically within the range of ordinary skill in the art.

The composition of the present invention can be grasped as a food composition in a specific aspect.

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 and 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 in accordance with the Korean "Health Functional Food Act", or confectionery, bean, tea, etc. 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). It may be beverages, special purpose foods, 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. In the Food Additive Code (“Food Sanitation Act” in Korea) governing the manufacture and distribution of food, food additives with guaranteed safety are limited in terms of ingredients or functions. In the Korean Food Additives Code (KFDA notice "Food Additive Standards and Specifications), food additives are classified into chemical synthetic products, 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, etc.

Sweeteners are used to impart a suitable sweetness to foods, and both natural and synthetic ones can be used in the food composition of the present invention. 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 are used to improve taste or flavor, and both natural and synthetic flavors can be used. Preferably, it is the case of using a natural one. In the case of using natural ingredients, 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, etc., or may be obtained 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 the 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 Etc. 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. The acidulant may be added so that the food composition has an appropriate acidity for the purpose of suppressing the growth of microorganisms in addition to the purpose of enhancing taste. 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 contain 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 magnesium preparations, 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 as described above may be included in an appropriate amount to achieve the purpose of addition according to the product type.

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

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 meaning of "pharmaceutically acceptable" means that the application (prescription) does not have toxicity beyond 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 a method known in the art together with a suitable carrier It can be prepared in such a formulation. Examples of suitable pharmaceutically acceptable carriers include sugars such as lactose, glucose, sucrose, dextrose, sorbitol, mannitol, and xylitol, corn starch, potato starch, wheat starch, and other starches, cellulose, methylcellulose, ethylcellulose, Celluloses such as sodium carboxymethylcellulose and hydroxypropylmethylcellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, polyol, vegetable Yu, etc. are mentioned. In the case of formulation activity, if necessary, it may be formulated including diluents and/or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

When the pharmaceutical composition of the present invention is prepared in a parenteral dosage form, it may be formulated in the form of eye drops, injections, transdermal administration, nasal inhalation, and suppositories according to a method known in the art together with a suitable carrier. When formulated as an eye drop, suitable carriers may include sterile water, saline, and isotonic solutions such as 5% dextrose, and benzalkonium chloride, mefilparaben, ethylparaben, etc. may be added for preservative purposes if necessary. When formulated as an injection, sterile water, ethanol, polyols such as glycerol or propylene glycol, or mixtures thereof may be used as suitable carriers, preferably Ringer's solution, PBS (phosphate buffered saline) containing triethanol amine, or sterilization for injection Water, isotonic solutions such as 5% dextrose can be used. When formulated into a transdermal dosage form, 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 to form an aerosol spray.When formulated as a suppository, the base 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.

Regarding the specific formulation of the pharmaceutical composition is known in the art, for example, Remington's Pharmaceutical Sciences (19th ed., 1995) and the like can be referred to. These documents are 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. These dosages 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 asthma or COPD using a turmeric extract, a golden extract, and the like. The composition of the present invention is commercialized as a food, particularly a health functional food or a drug, and may be usefully used for an asthma improvement effect or COPD improvement effect, and further for an effect of improving lung function or respiratory function.

1 and 2 are results showing the NETosis inhibitory activity such as turmeric extract and golden extract.
3 is a result showing the inhibitory activity of inflammatory cytokines (IL-6 & IL-8) in H292 cells stimulated by CSE, such as turmeric extract and golden extract.
4 and 5 are results showing MIP-2 inhibitory activity using MH-S cells stimulated by CSE such as turmeric extract.
6 and 7 are results showing that the total number of infiltrating cells in BALF is reduced in an animal model experiment in which a node pull extract or the like induces COPD by CSE or CES/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.

<Example> Improvement activity of asthma and COPD such as turmeric and golden extract

1. Sample preparation

Turmeric (root, Curcuma longa ) extract, golden (root, Scutellaria baicalensis ) extract, dried stalk (outpost, Trapa japonica ) extract, leaflet (leaves, Perilla frutescens ) extract, mardi grass ( polygonum avivulare ) extract, youngsil (fruit, Rosae) multiflorea ) extract, fenugreek (seed, Trigonella foenum ) extract, and pepper (fruit, Piper nigrum ) extract were used as samples.

The extract was obtained in the form of powder by adding 10 times the weight of 70% ethanol to the dry powder of the plant part to be extracted, extracting it twice at 50°C for 6 hours each, followed by filtration, concentrated under reduced pressure, and freeze-dried.

2. Experiment method

2.1 In vitro experiment

2.1.1 NETosis inhibition activity test

HL-60 cells (Human promyelocytic leukemia cells) distributed from the Korea Cell Line Bank were converted to 2×105 cells/mL in RPMI (10% FBS, 100 U/mL penicillin, 100 mg/mL streptomycin) medium treated with 1% DMSO. The cells were seeded and cultured at 37° C. and 5% CO2 for 5 days to differentiate into a mature neutrophil phenotype. After 5 days, after 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 to wash. The recovered cells were diluted in HBSS at a concentration of 1×10 6 cells/mL, dispensed into 96 well plates at 100 μL, and 50 μL of samples prepared at each concentration were treated. Then, PMA (phorbol myristate acetate, Sigma) prepared at 400 nM was treated with 50 μL of each well and incubated for 3 hours. After 3 hours, 500 nM of Cytox green was treated and fluorescence was measured at a wavelength of 485/520 nm (excitation/emission maxima) using a fluorescent plate reader.

2.1.2 Evaluation of Inflammatory Cytokine (IL-6 & 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 passaged and cultured 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. Then, using Serum-free RPMI1640 medium, irritants (CSE; final 2% or PPE (porcine pancreas elastase, Sigma); final 0.01 U/mL or PM2.5 (fine particulate matter than 2.5 ㎛, Sigma); final 10 μg/mL) and the sample were diluted to reflect the desired final concentration, and then treated at 500 μL per well. At this time, PPE was dissolved in PBS and stored frozen, and PM2.5 was dissolved in DMSO and stored frozen (2 weeks or less). After overnight incubation, the supernatant was recovered and used for IL-8 and IL-6 measurement, and the remaining cells were tested for survival by WST (water-soluble tetrazolium salt assay). Human IL-8 and IL-6 were ELISA In this case, the supernatant for IL-8 measurement was diluted 5 times and the supernatant for IL-6 was 40 times diluted and analyzed after the supernatant was recovered, and then the remaining cells were analyzed. At 450 nm, water soluble tetrazolium salt (WST) reagent diluted 1/10 with RPMI1640 + 10% FBS + 1% P/S medium was treated with 500 μL/well and reacted for 50 minutes in a 37°C, 5% CO ² incubator. The absorbance was measured to confirm the cell viability (WST), and the concentration lower than the control group was excluded from the cytokine analysis.

2.1.3 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 culture was RMPI-1640 (WELGENE, cat. LM 011-01), 10% FBS (WELGENE, cat. S 001-07), and It was 1% Penicillin-Streptomycin (WELGENE, cat. LS 202-02), 14.3 mM 2-Mercptoethanol (SIGMA). MH-S cells were seeded in a 96 well plate at a concentration of 5×10 ⁴ /well and cultured for 24 hours. Thereafter, the stimulus (CSE; final 1% and LPS; final 10 ng/mL) and the sample were diluted to reflect the desired final concentration with MH-S cell culture medium and treated with 250 uL per well. After culturing for 24 hours, the supernatant was recovered and used for MIP-2 measurement, and the remaining cells were checked for viability by WST method. MIP-2 was measured by ELISA, and was carried out according to the manufacturer's (BD) protocol. After recovering the supernatant, the remaining cells were treated with 200 μL/well of a water soluble tetrazolium salt (WST) reagent diluted 1/10 with MH-S medium and reacted for 20 minutes in a 5% CO2 incubator at 37°C, and then absorbance at 450 nm. Cell viability (WST) was confirmed by measurement, and concentrations having a lower viability than the control group were excluded from cytokine analysis.

2.2 Measurement of BALF cell count in animal experiments that induced COPD by CSE

BALB/C mice (male, 5 weeks old) were purchased from Orient Bio, and were purified for 1 week, and were grouped with n=7. The sample treatment group was orally administered the sample at a concentration of 200 mg/kg for 28 days from one week before the start of COPD induction to before dissection, and the same amount of PBS was administered to the sample-free group (control) and the COPD group. At this time, the Roflumilast group, a positive control, was administered orally at 10 mg/kg farms once a week after induction. COPD was induced by intraperitoneal administration of 100% Cigarette Smoke Extraction (CSE) once/week, a total of 200 μL three times. After the end of the experiment, 350 μL of 1.2% Avertin was administered intraperitoneally to anesthetize the mouse and then dissection was performed. 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.

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 in the same amount, and 12 uL was injected into the Accuchip channel, and total cells were automatically counted with a cell counter (ADAM-MC, NANOENTEK. INC, Korea).

2.3 BALF cell count measurement in animal experiments inducing COPD by CSE and PPE

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 a 200 mg/kg sample for 24 days from one week before COPD induction to pre-dissection, and the same amount of PBS was administered to the negative control group (Naive) and the COPD group. Roflumilast administration group, an approved COPD treatment group, was used as a positive control, and the Roflumilast administration group was orally administered 10 mg/kg agriculturally 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 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 was proceeded the next day without CSE treatment. I did. The dissection was performed after anesthetizing with isoflurane using an inhalation anesthetic device. 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.

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 was mixed with Accustain T solution in the same amount, and 12 uL was injected into the Accuchip channel, and total cells were automatically counted with a cell counter (ADAM-MC, NANOENTEK. INC, Korea).

3. Experiment results

3.1 In vitro test results

3.1.1 NETosis inhibitory activity evaluation result

The results of evaluating the NETosis inhibitory activity of each sample are shown in FIGS. 1 and 2. These results show that turmeric extract and the like inhibit Netosis in proportion to the treatment concentration.

3.1.2 Results of evaluation of inhibitory activity of inflammatory cytokines (IL-6 & IL-8) using H292 cells

The results are shown in FIG. 3. Referring to these results, it can be seen that turmeric extract or the like generally inhibits inflammatory cytokines IL-6 and/or IL-8 in a concentration-dependent manner in H292 cells.

3.1.3 MIP-2 inhibitory activity evaluation results using MH-S cells

The results are shown in FIGS. 4 and 5. 5 shows that turmeric extract and the like show MIP-2 inhibitory activity in MH-S cells in a concentration-dependent manner.

3.2 Results of BALF cell count measurement in COPD-induced animal experiments by CSE

The results of the total number of infiltrating cells in BALF are shown in FIG. 6. Turmeric extract and the like reduced the total number of infiltrating cells in BALF.

3.2 Results of BALF cell count in animal experiments induced by COPD by CSE and PPE

The results of total cells infiltrating BALF are shown in FIG. 7. The extract, etc., reduced the total number of infiltrating cells in BALF.

Claims (4)

A composition for improving asthma or COPD, including turmeric extract, golden extract, drywall extract, lobule extract, knuckle extract, youngsil extract, fenugreek extract, pepper extract, or both as active ingredients.
The method of claim 1,
The extract is a composition, characterized in that water, ethanol or a mixed solvent extract thereof.
The method according to claim 1 or 2,
The composition is a composition, characterized in that the food composition.
The method according to claim 1 or 2,
The composition is a composition, characterized in that the pharmaceutical composition.

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