Composition for treating chronic obstructive pulmonary disease, comprising novel compound as active ingredient

Abstract

The present invention relates to a composition for preventing, alleviating or treating chronic obstructive pulmonary disease, comprising a novel compound as an active ingredient.

Description

Composition for treating chronic obstructive pulmonary disease comprising a novel compound as an active ingredient

[0001] The present invention relates to a composition for treating chronic obstructive pulmonary disease comprising a novel compound as an active ingredient.

[0002]

[0003] Chronic obstructive pulmonary disease (COPD) is a chronic airway disease characterized by irreversible airway obstruction, including coughing, sputum production, dyspnea, reduced expiratory flow rate, and impaired gas exchange. The number of people with COPD is increasing worldwide every year, and it is predicted that it will become the third leading cause of death worldwide by 2020. In the past, COPD was divided into chronic bronchitis and emphysema, but chronic bronchitis was defined based on clinical symptoms, and emphysema was classified based on anatomical criteria. Since both conditions often coexist in the same patient and clinical differentiation is difficult, they are diagnosed collectively as COPD. In the case of bronchial asthma, if asthma has persisted for a long time and airway obstruction has shown irreversible changes, it is included in COPD and treated accordingly.

[0004] COPD is caused by various factors such as smoking, air pollution, chemicals, occupational factors, and genetic predisposition. Among them, smoking is identified as the main cause, and it has been revealed that more than 80% of COPD patients are smokers. The pathogenesis involves chronic inflammation in the bronchial tubes and lung tissue, activation of proteases in the lungs, and oxidative stress. Cells involved in inflammation include 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, as well as various proteases that cause tissue damage. In an animal model of COPD induced by cigarette smoke, an increase in neutrophil influx, keratinocyte chemoattractant (KC), tumor necrosis factor α (TNF-α), macrophage inflammatory protein 2 (MIP-2), MIP-1α and monocyte chemoattractant protein (MCP-1), matrix metalloproteinase 12 (MMP12), and granulocyte macrophage colony-stimulating factor (GM-CSF) has been observed. In addition, as in asthma patients, the concentration of IL-17 in the sputum and serum has been observed to increase in COPD patients, suggesting that IL-17 may play a certain role in COPD. In an animal model of COPD, IL-17 has been suggested as an important target for inhibiting airway fibrosis in COPD because it plays a crucial role in airway fibrosis.

[0005] Meanwhile, neutrophils are known to play a very important role in the development of COPD by secreting substances such as proteases such as elastase, collagenase, and myeloperoxidase (MPO), arachidonic acid metabolites (arachidonate), and reactive oxygen free radicals outside the cells, causing lung damage and chronic airway inflammation through airway infiltration.

[0006] Recently, excessive formation of neutrophil extracellular traps (NETs) has been reported to be associated with lung diseases including COPD. NETs are mesh-like structures composed of interwoven chromatin, cytoplasmic proteins, and granular proteins. NETs capture and neutralize bacteria, fungi, and viruses, preventing their spread and infection. However, excessive NET secretion has been associated with various infectious and non-infectious diseases. In particular, their association with lung diseases has been noted because NETs easily expand in the alveoli, causing lung damage. Excessive NET formation (NETosis) has been reported in lung diseases such as COPD, asthma, cystic fibrosis, respiratory syncytial virus bronchiolitis, influenza virus infection, bacterial pneumonia, tuberculosis, and transfusion-related acute lung injury, and therefore, inhibition of NETosis is recognized as an important target for the development of therapeutics for these lung diseases.

[0007] Currently, bronchoalveolar lavage (BAL) is used as a means to diagnose and monitor the progress of diseases such as COPD and asthma. In the bronchoalveolar lavage fluid (BALF) of these patients, inflammatory mediators such as inflammatory cytokines, reactive oxygen species, leukotrienes, and activated complement increase, and neutrophils, which account for less than 5% of normal lung cells, increase to account for 80% of all cells.

[0008] To date, no drugs have been reported to directly improve COPD or asthma, and current COPD or asthma treatments mainly use bronchodilators (β2-agonists, anticholinergics, methylxanthines) and steroids (inhaled, oral) to reduce symptoms and complications.

[0009] Accordingly, the inventors of the present invention were able to complete the present invention by studying various physiological activities using a novel compound and confirming that the novel compound has the effect of inhibiting lung tissue damage and inflammatory response, and thus revealing that it can be easily used as a treatment for chronic obstructive pulmonary disease.

[0010]

[0011] The purpose of the present invention is to solve the above-described problems and to provide a composition for preventing, improving or treating chronic obstructive pulmonary disease by applying a novel compound.

[0012]

[0013] To achieve the above purpose, a pharmaceutical composition for preventing or treating chronic obstructive pulmonary disease according to one embodiment of the present invention comprises a novel compound as an active ingredient.

[0014] A health functional food composition for preventing or improving chronic obstructive pulmonary disease according to another embodiment of the present invention comprises a novel compound as an active ingredient.

[0015]

[0016] According to one embodiment of the present invention, a composition for preventing, improving or treating chronic obstructive pulmonary disease, which comprises a novel compound as an active ingredient, effectively suppresses damage to lung tissue and inflammatory response by reducing the expression levels of inflammatory cytokines IL-6 and TNF-α in lung tissue with chronic obstructive pulmonary disease to the ranges of 20 to 40 pg / ml and 2 to 5 pg / ml, respectively, thereby being usefully used in a pharmaceutical or health functional food for preventing, improving or treating chronic obstructive pulmonary disease.

[0017]

[0018] Figure 1 shows the HPLC analysis results of OIL and BKC.

[0019] Figure 2 shows the results of analyzing the substance types of each PEAK in the HPLC analysis of BKC.

[0020] Figure 3 shows the results of cytotoxicity measurement.

[0021] Figures 4 and 5 show the results of measuring the anti-inflammatory effect in the lungs.

[0022] Figure 6 shows the results of measuring the thickness of the airway wall, and Figure 7 shows the results of measuring the diameter of the alveoli.

[0023] Figure 8 shows the results of measuring the efficacy of inhibiting the infiltration of inflammatory cells.

[0024] Figure 9 shows the results of measuring the expression level of inflammatory cytokines.

[0025] Figure 10 shows the results of measuring the anti-inflammatory effect through dyeing.

[0026] Figures 11 and 12 show the results of measuring the expression level of inflammatory enzymes.

[0027]

[0028] Hereinafter, the present invention will be described in detail with reference to the attached drawings, using exemplary embodiments. However, the following exemplary embodiments are provided as illustrative examples of the present invention. If a detailed description of a technology or configuration well known to those skilled in the art is judged to unnecessarily obscure the gist of the present invention, such detailed description may be omitted, and the present invention is not limited thereby. The present invention is capable of various modifications and applications within the scope of the following claims and equivalents interpreted therefrom.

[0029] In addition, the terms used in this specification are terms used to appropriately express preferred embodiments of the present invention, and may vary depending on the intention of the user or operator, or the customs of the field to which the present invention belongs. Therefore, the definitions of these terms should be determined based on the contents throughout this specification. Throughout the specification, when a part is said to "include" a certain component, this does not mean that other components are excluded, but rather that other components may be included, unless specifically stated otherwise.

[0030] Throughout this specification, '%' used to indicate the concentration of a particular substance is %(w / w) for solid / solid, %(w / v) for solid / liquid, and %(v / v) for liquid / liquid, unless otherwise stated.

[0031]

[0032] Hereinafter, the present invention will be described in more detail.

[0033] The present invention relates to a pharmaceutical composition for preventing or treating chronic obstructive pulmonary disease, comprising a novel compound as an active ingredient. Here, the novel compound may include a walnut extract.

[0034] In the present invention, walnut (Juglans regia L.) is a natural material widely distributed throughout the world, and it has been reported that the fruit of walnut is rich in alpha-linoleic acid (ALA), an omega-3 unsaturated fatty acid, and is known to be rich in vitamins B1 and B2. Various physiologically active substances exist in the pericarp, shell, kernel, bark, and leaves of walnuts, respectively. The method of using walnuts in the present invention is not particularly limited, but may be used as a powder pulverized after drying, or more preferably, may be used in the form of an extract. It includes an extract obtained by extraction treatment of walnuts, a diluted or concentrated solution of the extract, a dried product obtained by drying the extract, a conditioned or purified product of the extract, or a mixture thereof, the extract itself, an extract of all formulations that can be formed using the extract, and a solid remaining after extracting oil.

[0035] The term “extract” used in the present invention means an extract obtained by leaching stems, leaves, fruits, flowers, roots, whole plants, mixtures thereof, etc., using 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, an extract obtained by using a supercritical extraction solvent such as carbon dioxide or pentane, or a fraction obtained by fractionating the extract, and any method such as cold maceration, reflux, heating, ultrasonic radiation, or supercritical extraction may be applied as the extraction method, taking into consideration the polarity of the active substance, the degree of extraction, and the degree of preservation. In the case of a fractionated extract, it may mean a fraction obtained by suspending the extract in a specific solvent and then mixing and allowing it to settle with a solvent of different polarity, or a fraction obtained by adsorbing the crude extract on a column filled with silica gel or the like and then using a hydrophobic solvent, a hydrophilic solvent, or a mixed solvent thereof as a mobile phase. In addition, the meaning of the extract may include 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. For example, it may mean an extract obtained by using water, ethanol, or a mixed solvent thereof as an extraction solvent. As a specific example, it may mean an extract obtained by using a mixed solvent of water and ethanol as an extraction solvent.

[0036] In this embodiment, the walnut extract may be a solid fraction from which oil has been removed from walnuts.

[0037] In this embodiment, the novel compound can suppress the expression of one or more inflammatory response factors selected from IL-6, TNF-α, iNOS, and COX-2, and specifically, can reduce the expression levels of inflammatory cytokines IL-6 and TNF-α in lung tissue with chronic obstructive pulmonary disease to the ranges of 20 to 40 pg / ml and 2 to 5 pg / ml, respectively.

[0038] Additionally, the novel compound in this embodiment can reduce the expression of one or more inflammatory response factors selected from iNOS and COX-2 by 20% or more.

[0039] Additionally, the novel compound in this example can reduce alveolar wall thickness and alveolar diameter.

[0040] In this embodiment, the novel compound may further include one or more selected from among a turmeric (Curcuma longa) extract, a Scutellaria baicalensis extract, a water chestnut (Trapa japonica) extract, a Perilla frutescens extract, a Polygonum avivulare extract, a Rosae multiflorea extract, a Trigonella foenum extract, a Piper nigrum extract, a red ginseng extract, a barley tree extract, a fragrant oil extract, a Poria cocos bark extract, a needle flower extract, a dandelion extract, a Lupin pine extract, a pepper plant extract, a rhubarb extract, and a Bupleurum rhizome extract, which are already known to have effects of improving asthma, COPD, lung function, or respiratory function.

[0041] Additionally, the novel compound in this embodiment may further include allysol b acetate.

[0042] In addition to chronic obstructive pulmonary disease (COPD), the present invention has the effect of improving inflammatory lung disease, lung function or respiratory function improvement, respiratory disease caused by smoking or fine dust, lung damage improvement, and lung disease accompanied by necrosis.

[0043] For example, in the present example, the novel compound inhibits NETosis induced by PMA (phorbol myristate acetate), inhibits the expression of IL-8, an inflammatory cytokine, in H292 cells, a bronchial epithelial cell line, stimulated by CSE (cigarette smoke extract), and inhibits the expression of MIP2 (IL-8), an inflammatory cytokine, in MH-S cells, a mouse alveolar macrophage cell line, activated by CES. In addition, in an animal model experiment in which COPD was induced by CES and PPE, the novel compound can reduce the number of infiltrating cells and neutrophils in BALF, restore airway obstruction and alveolar destruction, and reduce the concentration of inflammatory cytokines such as IL-1β, MIP-2, IL-6, and TNF-α in BALF.

[0044] The term “chronic obstructive pulmonary disease (COPD)” used in the present invention refers to a disease in which the airways become narrow due to persistent inflammation in the airways caused by cigarette smoke, fine dust, air pollution, or toxic inhaled substances, gradually leading to airway obstruction.

[0045] The term "inflammatory lung disease" used in the present invention means a lung disease accompanied by an inflammatory response, including asthma, chronic obstructive pulmonary disease (COPD), tracheitis, and bronchitis.

[0046] The term "improvement of pulmonary function or respiratory function" used in the present invention means improvement of respiratory function in a normal person without a disease, recovery of respiratory function failure in a patient with a disease such as chronic obstructive pulmonary disease, asthma, bronchitis, or tracheitis, or improvement of respiratory function in a patient with a disease.

[0047] The term "respiratory disease caused by smoking or fine dust" used in the present invention includes, in addition to asthma and COPD, diffuse interstitial lung disease, acute respiratory distress syndrome (ARDS), and acute lung injury. Fine dust, which contains various components such as carbon components such as soot and organic carbon, ionic components such as chlorine, nitrate, ammonium, sodium, and calcium, metallic components such as lead, arsenic, and mercury, and polycyclic aromatic hydrocarbons such as benzopyrene, is known to cause various lung diseases such as asthma and COPD by depositing in the upper respiratory tract, bronchi, small airways, and alveoli (J Korean Med Assoc, 2014; 57:763-768).

[0048] The term "improvement of lung damage" used in the present invention means recovery of damage to lung cells or lung tissue caused by fine dust, etc., or recovery of decreased lung function due to damage to lung cells or lung tissue.

[0049] The term "pulmonary disease accompanied by necrosis" used in the present invention includes not only COPD as exemplified above, but also asthma, cystic fibrosis, respiratory syncytial virus bronchiolitis, influenza virus infection, bacterial pneumonia, tuberculosis, and transfusion-related acute lung injury.

[0050] As used herein, “improvement” means alleviating, treating or preventing a disease or symptom.

[0051] In this specification, “active ingredient” means an ingredient that exhibits the desired activity alone or can exhibit the activity together with a carrier that is inactive in itself.

[0052] The composition of the present invention may contain, in addition to the above-mentioned effective ingredient, a pharmaceutically acceptable carrier, excipient or diluent, and may be in various oral or parenteral dosage forms. When prepared as an oral dosage form, it may be prepared in the form of a powder, granules, tablet, pill, sugar-coated tablet, capsule, liquid, gel, syrup, suspension, wafer, etc., together with a suitable carrier, according to a method known in the art. At this time, 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, and wheat starch; celluloses such as methylcellulose, ethylcellulose, sodium carboxymethylcellulose, and hydroxypropylmethylcellulose; polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, polyols, and vegetable oils. In the case of formulation, the formulation may include diluents and / or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants, as necessary.

[0053] When the pharmaceutical composition of the present invention is prepared as a parenteral dosage form, it can be formulated in the form of eye drops, injections, transdermal administration agents, nasal inhalants, or suppositories using a suitable carrier according to a method known in the art. When formulated as eye drops, suitable carriers include sterile water, saline, isotonic solutions such as 5% dextrose, and, if necessary, benzalkonium chloride, mephylparaben, ethylparaben, etc. can be added for preservative purposes. When formulated as an injection, suitable carriers include sterile water, ethanol, polyols such as glycerol or propylene glycol, or mixtures thereof, and preferably, Ringer's solution, PBS (phosphate buffered saline) containing triethanolamine, sterile water for injection, isotonic solutions such as 5% dextrose, etc. When formulated as a transdermal agent, it can be formulated in the form of ointments, creams, lotions, gels, external solutions, pastes, liniments, aerosols, etc. In the case of nasal inhalation, it can be formulated in the form of an aerosol spray using a suitable propellant such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, etc. When formulated as a suppository, the base can be witepsol, tween 61, polyethylene glycols, cacao butter, laurin butter, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, sorbitan fatty acid esters, etc.

[0054] Specific formulations of pharmaceutical compositions are known in the art and can be found, for example, in Remington's Pharmaceutical Sciences (19th ed., 1995), which is incorporated herein by reference.

[0055] The composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment. The effective dosage level may be determined based on the type and severity of the patient's disease, the activity and sensitivity of the drug to the drug, the time of administration, the route of administration and excretion rate, the duration of treatment, concomitant drugs, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or in multiple doses. It is important to administer an amount that achieves the maximum effect with the minimum amount without causing side effects by taking all of the above factors into consideration, and this can be easily determined by those skilled in the art.

[0056] The preferred dosage of the pharmaceutical composition of the present invention may range from 0.001 mg / kg to 10 g / kg per day, preferably from 0.001 mg / kg to 1 g / kg, depending on the patient's condition, weight, sex, age, severity of the condition, and route of administration. Administration may be administered once a day or divided into several times. Such dosage should not be construed as limiting the scope of the present invention in any way. The composition of the present invention may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers.

[0057] In addition, the present invention relates to a health functional food composition for preventing or improving chronic obstructive pulmonary disease, which comprises a novel compound as an active ingredient.

[0058] Since the health functional food composition of the present invention includes the novel compound described above, descriptions of the overlapping contents with the novel compound of the present invention described above are omitted to avoid excessive complexity of the present specification due to descriptions of overlapping contents.

[0059] The health functional food of the present invention can be used by adding the novel compound as is or in combination with other foods or food ingredients, and can be used appropriately according to a conventional method. There is no particular limitation on the type of the health food. Examples of foods to which the novel compound can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen and other noodles, gum, ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and all health foods in the conventional sense are included. A health beverage containing the composition of the present invention may contain various flavorings or natural carbohydrates as additional ingredients, like a conventional beverage. The natural carbohydrates mentioned above are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. Sweeteners are used to provide appropriate sweetness to foods, and both natural and synthetic sweeteners can be used in the food composition of the present invention. For example, corn syrup solids, honey, sucrose, fructose, lactose, maltose, etc. can be used as natural sweeteners. The proportion of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g, per 100 g of the composition of the present invention.

[0060] In addition to the above-mentioned effective ingredients, the health functional food of the present invention may further contain various nutrients, catechins contained in green tea, etc., vitamins such as vitamin B1, vitamin C, vitamin E, and vitamin B12, tocopherol, dibenzoylthiamine, calcium preparations such as calcium citrate, magnesium preparations such as magnesium stearate, iron preparations such as ferric citrate, chromium chloride, potassium iodide, selenium, germanium, vanadium, zinc electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, fruit pulp for the production of fruit juice or vegetable beverages may be further contained. These ingredients may be used independently or in combination. The proportion of these additives is not particularly important, but is generally selected in the range of 0.01 to 2 parts by weight per 100 parts by weight of the composition of the present invention.

[0061] Flavoring agents are used to enhance taste or aroma, and both natural and synthetic ones can be used. When natural ones are used, they can also serve the purpose of enhancing nutrition in addition to flavor. Natural flavoring agents include those obtained from apples, lemons, tangerines, grapes, strawberries, and peaches, as well as those obtained from green tea leaves, Polygonum multiflorum, bamboo leaves, cinnamon, chrysanthemum leaves, and jasmine. Those obtained from ginseng (red ginseng), bamboo shoots, aloe vera, and ginkgo biloba can also be used. Natural flavoring agents can be liquid concentrates or solid extracts. In some cases, synthetic flavoring agents can be used, and synthetic flavoring agents such as esters, alcohols, aldehydes, and terpenes can be used. Preservatives that can be used include calcium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate, EDTA (ethylenediaminetetraacetic acid), etc.; emulsifiers that can be used include acacia gum, carboxymethylcellulose, xanthan gum, pectin, etc.; and acidulants that can be used include citric acid, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, phosphoric acid, etc. In addition to the purpose of enhancing taste, acidulants can be added to ensure that the food composition has an appropriate acidity for the purpose of inhibiting the growth of microorganisms. Thickeners that can be used include suspending agents, sedimentation agents, gel-forming agents, and puffing agents.

[0062]

[0063] Hereinafter, the present invention will be described in more detail using examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0064]

[0065] Manufacturing Example 1: Manufacturing of an animal model of chronic obstructive pulmonary disease

[0066] Chronic obstructive pulmonary disease (COPD) was induced in rats using cigarette smoke extract (CSE). CSE was obtained by collecting the smoke from two reference cigarettes (IR5F, University of Kentucky, USA) using an impinger. LPS and CSE were administered directly into the lungs of rats (N=5 / group) five times daily for three weeks.

[0067]

[0068] <Example 1>

[0069] Walnut oil (hereinafter referred to as “OIL”) was obtained by pressing and extracting walnuts.

[0070]

[0071] <Example 2>

[0072] In Example 1, oil was extracted and a solid substance (hereinafter referred to as 'BKC') from which the remaining oil was removed was obtained.

[0073]

[0074] Experimental Example 1: Component Analysis of Walnut Extract

[0075] The oil obtained in Example 1 and the BKC obtained in Example 2 were analyzed by HPLC, and the types of substances in each PEAK of the HPLC of BKC were analyzed, and the results are shown in Figures 1 and 2.

[0076] Referring to Figures 1 and 2, it was confirmed that OIL and BKC had different components, and in particular, BKC had a high content of 4-Ethylthio-3,20-dioxo-4-pregnen-17alpha-yl acetate, so it was decided to apply BKC to the experiment. In addition, because it has a molecular formula similar to a corticosteroid of the dioxopregnene series, it was expected to exhibit anti-inflammatory function.

[0077]

[0078] Experimental Example 2: Measurement of cytotoxicity of walnut extract (in vitro)

[0079] BKC was treated at different concentrations and the cell viability was evaluated by measuring the MTT assay after 48 hours, and the results are shown in Figure 3.

[0080] Referring to Figure 3, it was confirmed that toxicity was present starting from 10ug / ml, and it was confirmed that it was safe to apply at 10ug / ml or less.

[0081]

[0082] Experimental Example 3: Measurement of anti-inflammatory effects in the lungs (in vitro)

[0083] VEH (DMSO), BKC 10 mg / kg (hereinafter referred to as 'BKC 10'), BKC 20 mg / kg (hereinafter referred to as 'BKC 20'), and Roflumilast (hereinafter referred to as 'ROF') were sprayed into the lungs of white rats three times a week for three weeks and observed, and the results are shown in Fig. 4. Then, the lungs of white rats were perfused, and tissues were extracted, stained with H&E, and observed, and the results are shown in Fig. 5.

[0084] Referring to Figure 4, an inflammation-inducing area (white arrow area) was confirmed in the VEH treatment group, but no inflammation-inducing area was confirmed in the BKC treatment group. BKC 10 showed a degree of inflammation relief similar to that of ROF, and BKC 20 showed a degree of inflammation relief superior to that of ROF.

[0085] Referring to Figure 5, emphysema (enlarged airspace) was observed in the CSE treatment group, but reduced emphysema or normal lung was observed in the BKC 10 treatment group.

[0086]

[0087] Experimental Example 4: Measurement of airway wall thickness and alveolar diameter (in vitro)

[0088] Lung tissues obtained from euthanized rats were fixed in 10% paraformaldehyde and embedded in paraffin blocks. Paraffin sections cut to 4 μm thickness were stained with H&E and observed under a fluorescence microscope (Nikon, Japan) at 200x magnification. Airway wall thickness and alveolar diameter were measured using NIS-element image software (Nikon, Japan), and the results are shown in Figures 6 and 7.

[0089] Referring to Figures 6 and 7, it was confirmed that as the concentration increased in the BKC treatment group, the thickness of the airway wall became thinner and the alveolar diameter decreased. BKC 10 and BKC 20 showed greater reductions in the thickness of the airway wall and the diameter of the alveoli than ROF, indicating that they are more effective in diseases in which the airway narrows and gradually becomes occluded.

[0090]

[0091] Experimental Example 5: Measurement of the efficacy of inhibiting inflammatory cell infiltration (in vitro)

[0092] After instilling 1 mL of PBS through the trachea of ​​a rat, the rat was gently massaged to collect 700 μL of bronchoalveolar lavage fluid (BALF). The collected BALF was separated into a supernatant and a cell pellet by centrifugation at 300 × g for 5 min. The cell pellet was resuspended by adding 700 μL of PBS, and 150 μL of this BALF suspension was centrifuged at 1000 rpm for 10 min at 4°C using a Cytospin device (Centrifuge 5403, Eppendorf, Hamburg, Germany) to attach BALF cells to a slide. Afterwards, cells were stained using Diff-Quick staining reagent according to the manufacturer's protocol (1-5-1 Wakinohamakaigandori, chuo-ku, Kobe, Japan), and the number of macrophages and lymphocytes was counted through microscopic observation, and the results are shown in Fig. 8.

[0093] Referring to Figure 8, it was confirmed that as the concentration increased in the BKC treatment group, the number of total cells, macrophages, and lymphocytes decreased, and 10ug / ml (hereinafter referred to as 'BKC 10') and 20ug / ml (hereinafter referred to as 'BKC 20') showed a greater decrease in the number of total cells and macrophages than ROF, demonstrating a superior effect of inhibiting the infiltration of inflammatory cells.

[0094]

[0095] Experimental Example 6: Measurement of inflammatory cytokine expression (in vitro)

[0096] PBS equivalent to 10 times the weight of rat lung tissue was added, homogenized, and centrifuged at 12,000 rpm for 20 min. The collected supernatant was used for cytokine analysis (IL-6, TNF-α). For cytokine analysis, 30 μL of the supernatant was taken and the experiment was performed according to the manufacturer's (Quansis biosciences) protocol of the Q-plex ELISA array kit, and the results are shown in Figure 9.

[0097] Referring to Figure 9, it was confirmed that as the concentration of BKC increased, the expression of inflammatory cytokines significantly decreased. In particular, BKC was confirmed to have a superior effect in suppressing IL-6 expression compared to ROF. Therefore, it was found that the BKC treatment group exhibited an anti-inflammatory effect, which could be useful in the prevention or treatment of chronic obstructive pulmonary disease.

[0098]

[0099] Experimental Example 7: Measurement of the anti-inflammatory effect through dyeing (in vitro)

[0100] VEH (DMSO), BKC, and BAY 11-7082 were orally administered to rats three times a week for three weeks, and the lungs of the rats were perfused. The tissues were extracted and stained with H&E for observation. The results are shown in Fig. 10.

[0101] Referring to Figure 10, in the BKC treatment group, reduced emphysema or normal lungs were observed.

[0102]

[0103] Experimental Example 8: Measurement of the expression level of inflammatory enzymes (in vitro)

[0104] RAW264.7 cells were seeded at 1×10 in a 96-well plate. 5After dispensing at a concentration of 10 cells / well and culturing for 12 hours, the cells were pretreated with VEH (DMSO), 2.5 mg / kg of BKC (hereinafter referred to as 'BKC 2.5'), 5 mg / kg of BKC (hereinafter referred to as 'BKC 5'), 10 mg / kg of BKC (hereinafter referred to as 'BKC 10'), 20 mg / kg of BKC (hereinafter referred to as 'BKC 20'), and ROF for 1 hour, treated with CSE and LPS (L6529, Sigma), and then cultured for 24 hours. The expression levels of iNOS and COX-2 in the supernatant obtained by centrifugation were measured by Western blotting analysis, and the results are shown in Fig. 11. The concentrations of iNOS and COX-2 were measured using an enzyme immunoassay (EIA; Amersham Pharmacia) system, and the results are shown in Fig. 12.

[0105] Referring to Figures 11 and 12, it was confirmed that the expression levels of iNOS and COX-2 decreased in the BKC treatment group, making the color of the band appear lighter.

[0106]

[0107] Although exemplary embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and improvements made by those skilled in the art using the basic concept of the present invention defined in the following claims also fall within the scope of the present invention.

[0108] All technical terms used in this invention, unless otherwise defined, have the same meaning as commonly understood by those skilled in the art in the relevant field of the present invention. The contents of all publications cited as references herein are incorporated herein by reference.

Claims

1. Contains a new compound as an active ingredient, A pharmaceutical composition for preventing or treating chronic obstructive pulmonary disease, wherein the novel compound reduces the expression levels of inflammatory cytokines IL-6 and TNF-α in lung tissue with chronic obstructive pulmonary disease to the range of 20 to 40 pg / ml and 2 to 5 pg / ml, respectively.

2. In claim 1, A pharmaceutical composition for preventing or treating chronic obstructive pulmonary disease, wherein the novel compound reduces the expression of at least one inflammatory response factor selected from iNOS and COX-2 by 20% or more.

3. In claim 1, A pharmaceutical composition for preventing or treating chronic obstructive pulmonary disease, wherein the novel compound further comprises at least one selected from among turmeric extract, golden extract, water chestnut extract, leaf extract, knotweed extract, yeongsil extract, fenugreek extract, pepper extract, red ginseng extract, barley tree extract, fragrant oil extract, poria cocos bark extract, needle flower extract, dewdrop extract, Nahan pine extract, pepper green extract, rhubarb extract, and Bupleurum extract.

4. In claim 1, A pharmaceutical composition for preventing or treating chronic obstructive pulmonary disease, wherein the novel compound further comprises allysol b acetate.

5. Contains a new compound as an active ingredient, A health functional food composition for preventing or improving chronic obstructive pulmonary disease, wherein the novel compound reduces the expression levels of inflammatory cytokines IL-6 and TNF-α in lung tissue with chronic obstructive pulmonary disease to the ranges of 20 to 40 pg / ml and 2 to 5 pg / ml, respectively.

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