KR20200027448A - Composition for improving respiratory diseases using the extract of Paliurus ramosissimus - Google Patents

Abstract

The present invention discloses a composition for ameliorating asthma or chronic obstructive pulmonary disease (COPD) by using an extract of Paliurus ramosissimus. A food composition for reinforcing a respiratory function and ameliorating a respiratory disease comprises an extract of Paliurus ramosissimus as an active ingredient. The extract of Paliurus ramosissimus has activities of inhibiting Netosis activation of Neutrophil extracellular traps induced by phorbol myristate acetate (PMA), inhibiting expression of IL-8, i.e., inflammatory cytokines caused by CSE of NCI-H292 cells, i.e., a bronchial epithelial cell line, and inhibiting expression of MIP-2, i.e., inflammatory cytokines even in MH-S cells that are alveolar macrophages activated by CES and lipopolysaccharide (LPS). Further, a respiratory disease mouse model having the extract of Paliurus ramosissimus injected thereinto exhibits an activity of reducing the total cell number of BALF to a level similar to that of a group having Roflumilast (ROF), i.e., a positive control group injected thereinto.

Description

Composition for improving respiratory diseases using the extract of Paliurus ramosissimus}

The present invention relates to a composition for the improvement of asthma or chronic obstructive pulmonary disease (COPD) using the extract of Jujube jujube.

Bronchial asthma is an allergic disease characterized by reversible airway obstruction, airway hypersensitivity (increased airway resistance), hypersecretion of mucus, and high levels of IgE in serum. When TH2 (T helper 2) type immune cells stimulated by various antigens inhaled into the airways produce IL-4, 5, 13, etc., inflammatory cells such as T cells, eosinophils, and mast cells proliferate, differentiate and activate. It is also recognized as a chronic inflammatory disease because it migrates to and infiltrates the tissues around the airways (J Clin Invest, 111: 291-297, 2003; N Engl J. Med. 2001; 44 (5): 350-62; Toshio Hirano Cytokine molecular biology.World Science. 2002). Therefore, in the development of a therapeutic agent for asthma, suppression of infiltration of inflammatory cells such as eosinophils is one of the important targets.

Chronic obstructive pulmonary disease (COPD) is a chronic airway disease that coughs, sputum, shortness of breath, decreases exhalation flow rate, impaired gas exchange, etc., and the incidence of the population increases worldwide each year, 2020 It is predicted to be the third leading cause of human death in years (Am J Respir Crit Care Med, 2013, 187: 347-365; Am J Respir Crit Care Med, 2009, 180: 396-406).

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

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

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

No drugs that directly improve COPD or asthma have been reported, and current COPD or asthma treatments are intended to reduce symptoms and complications. Bronchodilators (β2-agonists, anticholinergics, methylxanthines) and steroids (inhalation, Oral) is mainly used.

It is an object of the present invention to provide a pharmaceutical composition for preventing or treating respiratory disease using a jujube jujube extract, a food composition for enhancing respiratory function and a respiratory disease improvement using a jujube jujube extract, Having Other and specific objects of the present invention will be presented below.

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

Considering the above, the present invention, in one aspect, can be identified as a composition for improving COPD comprising an extract of Paliurus ramosissimus (Paliurus ramosissimus) as an active ingredient, and in another aspect, an extract of Magnolia jujube It can be grasped with a composition for improving asthma, and furthermore, the present invention can also be identified as a composition for enhancing respiratory function and improving respiratory diseases, which includes the extract of jujube jujube as an active ingredient, and the present invention is also a jujube jujube extract. As an active ingredient, cough, sputum, difficulty breathing, airway irritability, airway obstruction, mucus hypersecretion, decrease in exhalation flow rate, and / or can also be identified as a composition for improving lung disease with symptoms of gas exchange disorder. These lung diseases include the aforementioned asthma or COPD, as well as diffuse interstitial lung disease, acute respiratory distress syndrome (ARDS), acute lung injury, etc. In addition to pulmonary diseases caused by contamination, genetic predisposition, etc., it includes pulmonary diseases caused by fine dust, which has recently become a problem. Fine dust containing various components, such as carbon components such as soot and biological 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, etc. It is known to cause various lung diseases such as asthma and COPD by depositing in the trachea, bronchus, small airways, alveoli (J Korean Med Assoc, 2014; 57: 763-768).

In the present specification, the term "extract" refers to the stem, leaves, fruit, flowers, roots, mixtures, etc. of plants to be extracted, water, lower alcohol 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), dimethylsulfoxide (DMSO), 1,3-butylene glycol, propylene glycol or a mixed solvent thereof. An extract obtained by leaching, an extract obtained by using a supercritical extraction solvent such as carbon dioxide or pentane, or a fraction obtained by fractionating the extract, and the extraction method is cold immersion, reflux, considering the polarity, extraction degree, and storage degree of the active substance. Any method such as heating, ultrasonic radiation, and supercritical extraction can be applied. In the case of fractionated extracts, the fractions obtained by suspending the extracts in a specific solvent and mixing and policing with a solvent of different polarity, adsorb the crude extract on a column filled with silica gel, etc., and then use a hydrophobic solvent, a hydrophilic solvent or a mixed solvent thereof. It means that it contains the fraction obtained as a mobile phase. In addition, the meaning of the extract includes a concentrated liquid extract or a solid extract in which the extraction solvent is removed by a method such as freeze drying, vacuum drying, hot air drying, spray drying, and the like. Preferably, it is 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, the term "active ingredient" in the present specification means a component that can exhibit the desired activity alone or in combination with a carrier that is not active itself.

The term "reinforcing respiratory function" in the present invention maintains the original function of the respiratory organs such as the nasal cavity, pharynx, larynx, trachea, bronchi, and lungs in a healthy state, or may cause smoking, fine dust, neutrophil activation, or other respiratory diseases. Refers to any act that improves the functioning of the respiratory organs that have been reduced due to symptoms to a healthy state.

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

The active ingredient in the composition of the present invention, anti-inflammatory activity, anti-allergic activity, asthma improvement activity, COPD improvement activity, other lung disease improvement activity, as long as it can exhibit respiratory function improvement and respiratory disease improvement activity, specific use, formulation It may be included in any amount (effective amount) according to the like, the typical effective amount will be determined within the range of 0.001% by weight to 20.0% by weight based on the total weight of the composition. Herein, the term “effective amount” means intended functional and pharmacological effects, such as anti-inflammatory effect and anti-allergic effect, when the composition of the present invention is administered to a mammal, preferably a person, to which the object is applied, according to the recommendation of a medical expert or the like. It refers to the amount of active ingredients contained in the composition of the present invention. Such effective amounts can be determined experimentally within the range of ordinary skill in the art.

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

The composition of the present invention, in addition to the active ingredient, to increase the anti-inflammatory effect, anti-allergic effect, asthma improvement effect, in addition to any compounds or natural extracts already known to have safety in the art and known to have the corresponding activity It can contain.

These compounds or extracts include compounds or extracts, medicines, etc. that are listed in the Pharmacopoeia of each country ("Korea Republic of Korea Pharmacopoeia"), and health functional food exhibitions of each country ("Korea Food and Drug Administration's" health functional food standards and standards "). Laws governing the manufacture and sale of compounds or extracts and health functional foods that have been granted an item in accordance with the laws of each country governing the manufacture and sale of foods (the "Pharmaceutical Law" in Korea) ("Act on Health Functional Foods in Korea") ”) May contain a compound or extract that is recognized for its functionality.

For example, MSM (dimethylsulfonylmethane), N-acetylglucosamine, etc., whose anti-inflammatory ("arthritis improvement") functionality was recognized, was recognized in accordance with Korea's "Health Functional Food Act." Enterococcus faecalis heat treated dry powder, complexes such as guava leaf extract, sage extract, leaflet extract, complexes such as picopreto powder, PLAG (1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol), etc. It may correspond to a compound or extract, but is not particularly limited thereto.

These compounds or natural extracts may be included in the composition of the present invention together with one or more of its active ingredients.

The composition of this invention can be grasped | ascertained as a food composition in a specific aspect. In addition, the food of the present invention may include a health functional (sex) food. In the present invention, the term "health functional food" refers to food produced and processed in the form of tablets, capsules, powders, granules, liquids and pills, etc. using ingredients or ingredients having useful functionality for the human body. Here, the term "functional" means to obtain a useful effect for health use, such as adjusting nutrients or physiological effects on the structure and function of the human body. The health functional food of the present invention can be manufactured by a method conventionally used in the art, and may be prepared by adding raw materials and ingredients commonly added in the art. In addition, the formulation of the health functional food can also be prepared without limitation as long as the formulation is recognized as a health functional food. The food composition of the present invention can be manufactured in various types of formulations, and has the advantage of not having side effects that may occur when taking the drug for a long time by using food as a raw material, unlike general medicines, and is excellent in portability. Health functional foods can be taken as supplements to improve asthma or COPD, and further enhance respiratory function and respiratory disease.

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.

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, beverages according to each food type in the health functional foods in accordance with the Act on Health Functional Foods, or in the Food Code of the Korean Food Sanitation Act , Special purpose foods, and the like.

The food composition of the present invention may contain 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. In the Food Additives Act, which is governed by the laws of each country that regulates the manufacture and distribution of foods ("Food Sanitation Law" in Korea), food additives with guaranteed safety are limited in terms of ingredients or functions. In the Korean Food Additives Code (KFDA Notification and Standards), food additives are classified into chemical compounds, natural additives, and mixed preparations in terms of their components. Agent, preservative, emulsifier, acidulant, thickener and the like.

Sweeteners are used to impart a moderate sweetness to food, and natural or synthetic ones can be used. 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 can be used to enhance the taste or aroma, 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. Also, ginseng (red ginseng), bamboo shoots, aloe vera, ginkgo, etc. can be used. Natural flavors may 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 taste.

As the thickener, suspending implementers, sedimenting agents, gel forming agents, 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 which are known in the art for the purpose of supplementing and reinforcing the functionality and nutritional properties and are guaranteed 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 food or national food additives.

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. The route of administration here can be any suitable route, including the topical route, the oral route, the intravenous route, the intramuscular route, and direct absorption through mucosal tissue, and may be used in combination of two or more routes. An example of a combination of two or more routes is where a drug of two or more formulations according to the route of administration is combined, for example when one drug is administered first by the intravenous route and the other by the local route.

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

When the pharmaceutical composition of the present invention is prepared in an oral dosage form, powders, granules, tablets, pills, dragees, capsules, solutions, gels, syrups, suspensions, wafers are prepared according to methods known in the art with suitable carriers. It may be prepared in a formulation such as. Examples of suitable carriers include lactose, glucose, sucrose, dextrose, sorbitol, sugars such as mannitol, xylitol, starch such as corn starch, potato starch, wheat starch, cellulose, methyl cellulose, ethyl cellulose, sodium carboxymethyl cellulose, Celluloses such as hydroxypropylmethylcellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, polyol, vegetable oil, ethanol, and Cerrol etc. are mentioned. If formulated, appropriate binders, lubricants, disintegrants, colorants, diluents and the like may be included as needed. Suitable binders include starch, magnesium aluminum silicate, starch ferrite, gelatin, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone, glucose, corn sweeteners, sodium alginate, polyethylene glycol, waxes, and the like. Sodium, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, silica, talcum, stearic acid, magnesium salts thereof, calcium salt, polydetylene glycol and the like, and the disintegrants include starch, methyl cellulose , Agar, bentonite, xanthan gum, starch, alginic acid or its sodium salt and the like. Moreover, lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, glycine, etc. are mentioned as a diluent.

When the pharmaceutical compositions of the present invention are prepared in parenteral formulations, they may be formulated in the form of injections, transdermal administrations, nasal inhalants and suppositories with suitable carriers according to methods known in the art. When formulated as an injection, a suitable carrier may be an aqueous isotonic solution or suspension. Specifically, an isotonic solution such as phosphate buffered saline (PBS) containing triethanol amine, sterile water for injection, or 5% dextrose may be used. . 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 inhalers 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 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, weight, sex, age of the patient, 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, it is possible to provide a composition for improving asthma or COPD using the extract of jujube tree. The composition of the present invention is commercialized as a food, especially a health functional food or a drug, and can be usefully used for an asthma improvement effect or a COPD improvement effect, further strengthening respiratory function and improving respiratory disease, coughing, sputum, and difficulty breathing , Airway irritability, airway obstruction, and other lung diseases that accompany symptoms such as mucus hypersecretion, also useful for improvement effects such as diffuse interstitial lung disease, acute respiratory distress syndrome (ARDS), and acute lung injury Can be used.

Figure 1 shows the results of evaluating the IL-8 inhibitory activity in H292 cells treated by concentration of the jujube tree extract.
Figure 2 shows the results of evaluating the inhibitory activity of MIP-2 in the treatment of each concentration of the jujube jujube extract for MH-S cells.
3 and 4, respectively, the total number of cells counted from bronchoalveolar lavage fluid (BALF) and immune cells in BALF through Diff-Quick staining after administration of the jujube tree extract for the PPE / CSE induced respiratory disease mouse model, respectively. It shows the results of the analysis.

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

[Example] Sample preparation

An extract of the medicinal jujube tree (extraction site: leaf) was prepared to confirm lung disease improvement activity. The extract was obtained by adding 10% by weight of 70% ethanol to the dry powder of the leaves of the jujube tree, repeatedly extracting it at 50 ° C for 6 hours twice, filtering it, and then concentrated under reduced pressure and freeze-dried to obtain a powder.

[Experimental Example] Lung Disease Improvement Activity

Experimental Example 1: Netosis inhibitory activity of neutrophil cells

Neutrophil extracellular traps (Netosis) are known to be involved in the pathogenesis of chronic pulmonary / respiratory disease patients including chronic obstructive pulmonary disease (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.). Accordingly, in the model treated with phorbol myristate acetate (PMA) on neutrophil cells, the Netosis activity of each extract was confirmed.

In order to measure the neutrophil netosis activity, HL-60 (Human promyelocytic leukemia cells) cells from a Korean cell line bank were used in the experiment. HL-60 cells were treated with 1% DMSO-treated RPMI (10% FBS, 100 U / mL penicillin, 100 mg / mL)

streptomycin) treated with 2 × 10 ⁵ cells / mL in a medium and cultured for 5 days in a cell incubator (37 ° C., 5% CO ₂ ) to differentiate to show 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 and washed by centrifugation at 1,300 rpm for 5 minutes. Next, diluted in HBSS with 2 × 10 ⁶ cells / mL, dispensed by 50 μL, and treated with 50 μL of the sample of the example prepared at each concentration, and each 100 μL of PMA (in HBSS) prepared at 200 nM. After treating the wells, the cells were cultured for 3 hours (final PMA concentration 100 nM). After 3 hours, Cytox green was treated with 500 nM and measured at a wavelength of 485/520 nm (excitation / emission maxima) using a fluorescent plate reader. Here, PMA is generally used to elicit Netosis activity of neutrophil cells.

The percentage of Netosis inhibitory activity of each sample was measured as a percentage of the PMA-treated control group (experimental control group), and the average value was shown in% in Table 1 (n> 3).

6.25 μg / ml 12.5 μg / ml 25 μg / ml 50 μg / ml Netosis inhibitory activity (%) 97.9 136.8 143.0 264.3

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

H292 cells prepared at a concentration of 2 × 10 ⁵ cells / mL in a 24 well plate were passaged and cultured at 500 μL / well, and when the cells became more than 80% confluent, they were exchanged with serum-free RPMI1640 medium and starvated for 24 hours. After that, dilute the stimulator (CSE; final 2%) and sample (final; 6.25, 12.5, 25 μg / mL) using Serum-free RPMI1640 medium to reflect the desired final concentration, and then treat 500 uL per well Did. After incubation overnight, the supernatant was recovered and used for IL-8 measurement. Human IL-8 was measured by ELISA, and was performed according to the manufacturer's (BD) protocol. At this time, the IL-8 measurement supernatant was diluted 5 times and analyzed.

As a result, it was confirmed that the concentration of IL-8 was suppressed depending on the concentration of the treated jujube tree extract (6.25 μg / ml, 12.5 μg / ml, 25 μg / ml) (FIG. 1).

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

The MH-S cell line was purchased from ATCC, and the medium composition used for 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, the stimulator (CSE; final 1% and LPS; final 10 ng / mL) and sample (final; 25, 50 μg / mL) were diluted with MH-S cell culture medium to reflect the desired final concentration, and 250 per well. Each uL was treated. After incubation for 24 hours, the supernatant was recovered and used for MIP-2 measurement. MIP-2 was measured by ELISA, and was performed according to the protocol of the manufacturer (R & D, DY452).

As a result, it was confirmed that the concentration of MIP-2 was suppressed depending on the concentration (25 µg / ml, 50 µg / ml) of the treated jujube tree extract (FIG. 2).

Experimental Example 4: BALF cell number measurement in animal model

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

BALB / C mice (male, 5 weeks old) were purchased from Orient Bio, purified for 1 week, and grouped with n = 10. In the sample treatment group, 200 mg / kg of the sample was orally administered for 24 days from a week prior to the start of COPD induction to anatomy, and the same amount of PBS was administered to the Naive and COPD groups. In this case, the Roflumilast group, which is a positive control, was orally administered daily at 10 mg / kg farm after induction. Porcine Pancreatic Elastase (PPE) 1.2 unit and 100% Cigarette Smoke Extraction (CSE) were used for COPD induction. PPE 1.2 unit was injected 3 times through intra nasal once every 7 days, and CSE was treated with 20 μl through intra nasal for 3 days from the day following PPE treatment, but after the last PPE treatment, dissection was performed without CSE treatment the next day. Did. Dissection was performed after anesthesia with isoflurane using an inhalation anesthesia. During the experiment, feed and drinking water were provided in the form of autonomous feeding, and kept in an environment of 24 ° C and 60% humidity.

4-2: Total number of cells infiltrated in BALF

After injecting 1 mL of PBS through the airways of the mouse, lightly massaged to collect 700 μL of bronchoalveolar lavage fluid (BALF). After 10 uL of the collected BALF solution was mixed with the Accustain T solution in the same amount, 12 uL was injected into the Accuchip channel, and total cells were automatically counted using a cell counter (ADAM-MC, NANOENTEK.INC, Korea) (FIG. 3).

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

The recovered BALF was separated into a supernatant and a cell pellet through centrifugation at 300 × g, 5 minutes. The cell pellet was resuspended by adding 700 μL of PBS, and 150 μL of this BALF suspension was centrifuged at 1000 rpm, 10 min, 4 ° C. with a Cytospin device (Centrifuge 5403, Eppendorf, Hamburg, Germany) to transfer BALF cells to the slide. Attached. Thereafter, cells were stained according to the protocol of the manufacturer (1-5-1 Wakinohamakaigandori, chuo-ku, Kobe, Japan) using Diff-Quick staining reagent and observed under a microscope (FIG. 4).

As a result, it was confirmed that the total number of BALF cells was decreased in the mouse group treated with the extract of jujube jujube at a level similar to the group administered with the positive control group Roflumilast (ROF).

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

Claims (10)

Food composition for strengthening respiratory function and improving respiratory disease, comprising the extract of oleander jujube tree as an active ingredient. The composition of claim 1, wherein the extract is water, ethanol, or a mixed solvent extract thereof. The composition of claim 1, wherein the respiratory disease is a lung disease accompanied by coughing, sputum, difficulty breathing, airway irritation, airway obstruction, mucus hypersecretion, a decrease in expiratory flow rate and / or disorder of gas exchange. . The composition of claim 1, wherein the respiratory disease is asthma, COPD, diffuse interstitial lung disease, acute respiratory distress syndrome (ARDS) or acute lung injury. The composition of claim 4, wherein the respiratory disease is asthma or COPD. A health functional food comprising the food composition of any one of claims 1 to 5. A pharmaceutical composition for preventing or treating respiratory disease, comprising the extract of the jujube tree as an active ingredient. The composition of claim 7, wherein the extract is water, ethanol, or a mixed solvent extract thereof. The composition of claim 7, wherein the respiratory disease is asthma, COPD, diffuse interstitial lung disease, acute respiratory distress syndrome (ARDS) or acute lung injury. The composition of claim 9, wherein the respiratory disease is asthma or COPD.

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