KR20130107532A - Pharmaceutical composition for prevention and treatment of chronic obstructive pulmonary disease comprising extract of rhus javanica linne as an active ingredient - Google Patents

Abstract

PURPOSE: A pharmaceutical composition for preventing and treating chronic obstructive pulmonary disease (COPD) is provided to reduce the number of macrophages, eosinophils, neutrophils, and lymphocytes in bronchoalveloar lavage fluid (BALF) using a Rhus javanica Linne extract, thereby being used as an active ingredient of pharmaceutical products and health foods for preventing and treating COPD. CONSTITUTION: A pharmaceutical composition for preventing and treating COPD contains a Rhus javanica Linne extract as an active ingredient. COPD includes chronic bronchitis or pulmonary emphysema. A composition for health foods for preventing and treating COPD contains the extract as an active ingredient. A pharmaceutical composition for preventing and treating lung diseases contains the extract as an active ingredient. [Reference numerals] (AA) Total inflammatory cell in waste washing solution (×10^6); (BB) Gallnut

Description

Pharmaceutical composition for prevention and treatment of chronic obstructive pulmonary disease comprising extract of Rhus javanica Linne as an active ingredient

The present invention is a gallant ( Rhus The present invention relates to a pharmaceutical composition for preventing and treating chronic obstructive pulmonary disease (COPD) containing javanica Linne) extract as an active ingredient, and a composition for preventing and improving chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is a chronic bronchitis or emphysema with impaired pulmonary function, or a combination of two diseases, with airway obstruction from the bronchus to the alveoli . Symptoms include persistent cough with long-term sputum, airway obstruction, decreased air velocity, difficulty in breathing, and frequent respiratory infections such as colds. The disease is the sixth leading cause of death in the world and the fourth leading cause of death in the United States. South Korea is also growing rapidly due to smoking and air pollution. The cause of chronic obstructive pulmonary disease is an abnormal chronic inflammatory reaction of the lung to toxic molecules or gas, and various factors such as smoking, urbanization, pollution and respiratory infectious diseases are involved in a complex manner. Smoking among these causes chronic obstructive pulmonary disease (National Heart, Lung, and Blood Institute, Morbidity & Mortality: Chartbook on Cardiovascular, Lung, and Blood Diseases, Global Strategy for the Diagnosis, Management, and Prevention of Chronic Obstructive Pulmonary Disease, 2003).

Smoking causes inflammation of the airways and lung parenchyma. Many types of oxidant, protease, and cytokine, caused by the inflammation of the lungs, are involved in the destruction of pulmonary emphysema, pulmonary vascular remodeling , Chest Med. 21: 67-86 (2000), D'Armiento J, et al., Cell 71: Et al., Chest 117: 684-694, 2000; Finlay GA, et al., ≪ RTI ID = 0.0 > 1997, Wright JL, et al., Inhalation toxicology 10: 969-9, 1997. < RTI ID = 0.0 > 994, 1998).

In particular, emphysema is thought to be caused by the destruction of surrounding tissues by proteolytic enzymes secreted from inflammatory cells. Recent studies have shown that MMPs secreted from alveolar macrophages and neutrophils are important proteolytic enzymes involved in lung destruction (Ohnishi K, et al., Lab Invest. 78: 1077-1087, 1998; Frankenberger M, et al., Mol. Med. 7: 263-270, 2001).

Some studies have shown that smoking and emphysema are not observed in MMP-12 knock-out mice (Hautamaki RD, et al., Science 277: 2002-2004, 1997) MMP-12 secretion was increased in alveolar macrophages of patients with chronic obstructive pulmonary disease (Finlay GA, et al., Am J Respir Crit Care Med 156: 240-247, 1997), chronic obstructive pulmonary disease In the BAL fluid of patients, MMP-2 and MMP-9 were increased (Segura-Valdez L, et al. Chest 117: 684-694, 2000). In addition, recently, administration of MMP inhibitors in smoking-induced guinea-pig emphysema models has been shown to weaken emphysema development (Selman M, et al., Chest 123: 1633-1641, 2003). However, pulmonary parenchymal destruction and pulmonary hypertension in chronic obstructive pulmonary disease are irreversible and progressive, and no treatment has yet been able to prevent progression (National Heart, Lung, and Blood Institute. & Mortality: Chartbook on Cardiovascular, Lung, and Blood Diseases.

Nut gall (Rhus javanica Linne is an insect beetle caused by the stagnation of the Meliaphis chinensis Bell on the leaves of the Rhus javanica L. or other similar plant, Anacardiaceae, with 50-70% of all gallotannins. Gallic acid and pentagalloyl glucose have been identified as main ingredients.

On the other hand, as a technique for prevention and treatment of chronic obstructive pulmonary disease, Korean Patent No. 10-101866 discloses that a mixed composition including Sukjunghwang, Chunmunmdong, Omiza, Mokdepi, Golden, Passengers and Baekbun is effective for chronic obstructive pulmonary disease Korean Patent Laid-Open No. 10-2009-0103239 discloses that a composition containing an extract of a grapefruit extract as an active ingredient is effective for asthma or chronic obstructive pulmonary disease. In Korean Patent No. 10-0785969, Discloses that a composition comprising fibroblast growth factor 2 (FGF2 or basic fibroblast growth factor, bFGF) as an active ingredient is effective for the prevention or treatment of asthma and chronic obstructive pulmonary disease. However, the therapeutic and prophylactic effects of gall extracts on chronic obstructive pulmonary disease are not known.

Therefore, the present inventors are trying to develop a natural substance having a therapeutic and prophylactic effect for chronic obstructive pulmonary disease. , IL-6 and TNF-α cytokines sharply increased by chronic obstructive pulmonary disease, and have the effect of significantly lowering the amount of MCP-1 and MMP-12, reducing inflammatory cells around the alveoli in tissues and expanding The present invention has been completed by revealing that the alveolar size can be significantly used to prevent and treat chronic obstructive pulmonary disease.

The object of the present invention is a fivefold ( Rhus) It provides a pharmaceutical composition for the prevention and treatment of chronic obstructive pulmonary disease (COPD) containing javanica Linne) extract as an active ingredient.

Another object of the present invention is to provide a composition for the prevention and improvement of chronic obstructive pulmonary disease containing the gall extract as an active ingredient.

In order to achieve the above object, the present invention is a gallant ( Rhus Provided is a pharmaceutical composition for preventing and treating chronic obstructive pulmonary disease (COPD) containing javanica Linne) extract as an active ingredient.

In addition, the present invention provides a composition for preventing and improving chronic obstructive pulmonary disease containing the gall extract as an active ingredient.

Rover of the present invention ( Rhus javanica Linne) extract reduces the number of bronchoalveloar lavage fluid (BALF) macrophages, eosinophils, neutrophils and lymphocytes, and rapidly increases IL-6 and TNF-α cytokines caused by chronic obstructive pulmonary disease, and MCP- Drugs and health for the prevention and treatment of chronic obstructive pulmonary disease as it has a significant effect of lowering the amount of 1 and MMP-12, significantly reducing inflammatory cells around the alveoli in tissues and significantly reducing the size of the expanded alveoli. It can be usefully used as an active ingredient of food.

1 is a diagram confirming the effect of the gall bladder extract of the present invention on the weight change of the cigarette smoke exposure group induced COPD (chronic obstructive pulmonary disease):
CON: control group;
Misery: experimental group; And
CS: Tobacco smoke exposure.
Figure 2 is a diagram confirming the total number of inflammatory cells in the lung washing solution of the COPD-induced tobacco smoke exposure group, the experimental group and the control group administered the gall extract of the present invention;
CON: control group;
Misery: experimental group; And
CS: Tobacco smoke exposure.
Figure 3 is a diagram confirming the distribution of inflammatory cells in the lung washing solution of COPD-induced tobacco smoke exposure group, the experimental group and the control group administered the gall extract of the present invention:
CON: control group;
Misery: experimental group; And
CS: Tobacco smoke exposure.
Figure 4 is a diagram confirming the expression level of inflammatory cytokines in the lung washing solution of the COPD-induced tobacco smoke exposure group, the experimental group and the control group administered the five gall extract of the present invention:
CON: control group;
Misery: experimental group; And
CS: Tobacco smoke exposure.
5 is a diagram confirming the expression level of inflammatory cytokines of the COPD-induced tobacco smoke exposure group, the experimental group and the control group administered the gall bladder extract of the present invention:
CON: control group;
Misery: experimental group; And
CS: Tobacco smoke exposure.
Figure 6 is a diagram confirming the overall morphology of the lung tissue of the COPD-induced tobacco smoke exposure group, the experimental group and the control group administered the five gall extract of the present invention:
CON: control group;
Misery: experimental group; And
CS: Tobacco smoke exposure.

Hereinafter, terms used in the present invention will be described.

The term "prophylactic " as used herein refers to all actions that inhibit or delay the progression of chronic obstructive pulmonary disease (COPD) by administration of the composition of the present invention.

As used herein, the terms " treatment "and" improvement "refer to all actions by which administration of the composition of the present invention improves or alleviates symptoms of COPD.

The term "administering" as used herein is meant to provide any desired composition of the invention to an individual by any suitable method.

The term "individual" as used herein refers to all animals such as humans, monkeys, dogs, goats, pigs or rats having a disease in which symptoms of chronic obstructive pulmonary disease can be improved by administering the composition of the present invention.

The term " pharmaceutically effective amount " as used herein means an amount sufficient to treat a disease at a reasonable benefit or risk rate applicable to medical treatment, including the type of disease, severity, activity of the drug, The time of administration, the route and rate of excretion of the drug, the duration of the treatment, factors including drugs used simultaneously and other factors well known in the medical arts.

Hereinafter, the present invention will be described in detail.

The present invention is a gallant ( Rhus Provided is a pharmaceutical composition for preventing and treating chronic obstructive pulmonary disease (COPD) containing javanica Linne) extract as an active ingredient.

The gallast may be used without limitation, such as grown or commercially available.

The chronic obstructive pulmonary disease is preferably chronic bronchitis or emphysema, but is not limited thereto.

The gall bladder extract is preferably prepared by a manufacturing method comprising the following steps, but not always limited thereto:

1) extracting by adding an extraction solvent to the gallnut;

2) filtering the extract of step 1); And

3) Concentrating the filtered extract of step 2) under reduced pressure and drying.

The extraction solvent is preferably water, alcohol or a mixture thereof. As the alcohol, C ₁ to C ₄ lower alcohol is preferably used, and as the lower alcohol, ethanol or methanol is preferably used. As the extraction method, it is preferable to use shaking extraction, Soxhlet extraction or reflux extraction, but it is not limited thereto. The extraction solvent is preferably extracted by adding 1 to 10 times the amount of dried gallengja. The extraction temperature is preferably 30 to 100 DEG C, but is not limited thereto. In addition, the extraction time is preferably 10 to 48 hours, more preferably 15 to 30 hours, but is not limited thereto. In addition, the extraction number is preferably 3 to 5 times, more preferably 3 times, but not limited thereto.

In the above method, it is preferable to use a vacuum decompression concentrator or a vacuum rotary evaporator for the decompression concentration in step 3), but it is not limited thereto. In addition, the drying is preferably reduced pressure drying, vacuum drying, boiling drying, spray drying or freeze drying, but is not limited thereto.

In a specific embodiment of the present invention, in order to confirm the effect of the gall bladder extract of the present invention on chronic obstructive pulmonary disease (COPD), COPD-induced cigarette smoke exposure group, the experimental group and control group administered the gall bladder extract of the present invention After fabrication, as a result of measuring the weight change, the cigarette smoke exposure group was confirmed that the weight is significantly reduced compared to the control group, the experimental group administered the gall extract of the present invention significantly compared to the COPD-induced cigarette smoke exposure group As a result, it was confirmed that the weight change was reduced (see FIG. 1).

In addition, the inventors of the present invention, in order to determine the effect of the gall extract extract on chronic obstructive pulmonary disease (COPD), the tobacco smoke exposure group classified in the present invention, the experimental group and the control group administered the gall extract of the present invention and immune cells in the lung washing solution of the control group As a result of the analysis, the number of total inflammatory cells was significantly increased in the tobacco smoke-exposed group compared to the control group, and the experimental group to which the gall bladder extract of the present invention was significantly increased compared to the tobacco smoke-exposed group. It was confirmed to decrease (see FIG. 2). In addition, the experimental group administered with the gall of the present invention was confirmed that significantly reduced macrophages, eosinophils, neutrophils and lymphocytes compared to the tobacco smoke exposure group (see Figure 3).

In addition, the present inventors confirmed the expression level of inflammatory cytokines in the pulmonary lavage fluid using ELISA, and the expression levels of IL-6 and TNF-α in the tobacco smoke-exposed group exposed to tobacco smoke for 3 weeks were compared with the control group. Significantly increased, the experimental group administered with the gall bladder extract of the present invention was found to significantly reduce the level of IL-6 and TNF-α compared to the tobacco smoke exposure group (see Figure 4).

In addition, the present inventors confirmed the expression levels of inflammatory cytokines using real-time RT-PCR, and as a result, IL-6, TNF-α, MCP- It was confirmed that the relative expression levels of 1 and MMP-12 was significantly increased compared to the control group, the experimental group administered the five gall extract of the present invention compared to the cigarette smoke exposure group IL-6, TNF-α, MCP-1 and MMP It was confirmed that the relative expression level of -12 significantly decreased (see FIG. 5).

In addition, the present inventors stained the lung tissue of the tobacco smoke-exposed group, the experimental group and the control group with hematoxylin and eosin (hematoxylin & eosin) to confirm the overall morphology of the tissue. And the expansion of alveolar progress was confirmed that there is serious damage to the lung, the experimental group administered the gall extract of the present invention significantly reduced the inflammatory cells around the alveoli compared to the tobacco smoke exposure group, expanded alveolar size Was significantly reduced (see FIG. 6).

Thus, gall bladder extract of the present invention reduces the number of macrophages, eosinophils, neutrophils and lymphocytes in pulmonary lavage fluid, and rapidly increases IL-6 and TNF-α cytokines, and MCP-1 and MMP- in chronic obstructive pulmonary disease. It has an effect of significantly lowering the amount of 12, significantly reduces the inflammatory cells around the alveoli in the tissue, and significantly reduces the size of the expanded alveoli, which is useful as an active ingredient in the pharmaceutical composition for preventing and treating chronic obstructive pulmonary disease. It can be seen that it can be used.

The composition containing the gall bladder extract of the present invention may further contain one or more active ingredients exhibiting the same or similar functions in addition to the above components.

The composition of the present invention may further comprise a pharmaceutically acceptable additive, wherein pharmaceutically acceptable additives include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose Starch glycolate, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, calcium stearate, , White sugar, dextrose, sorbitol and talc. The pharmaceutically acceptable additives according to the present invention are preferably included in the composition in an amount of 0.1 to 90 parts by weight, but are not limited thereto.

That is, the composition of the present invention can be administered in various formulations of oral and parenteral administration at the time of actual clinical administration. In the case of formulation, a diluent such as a filler, an extender, a binder, a wetting agent, a disintegrant, . ≪ / RTI > Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate and sucrose in the gall bladder extract. ), Lactose (Lactose) or gelatin can be prepared by mixing. In addition to simple excipients, lubricants such as magnesium stearate talc may also be used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions and syrups, and various excipients such as wetting agents, sweetening agents, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used . Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used as the non-aqueous solvent and suspension agent. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The composition of the present invention may be administered orally or parenterally in accordance with the intended method, and may be administered orally, parenterally or intraperitoneally, rectally, subcutaneously, intravenously, intramuscularly, . The dosage varies depending on the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and disease severity.

The dosage of the composition of the present invention varies depending on the weight, age, sex, health condition, diet, time of administration, method of administration, excretion rate and severity of the disease of the patient, the daily dosage is the amount of gall bladder extract 0.0001 to 100 mg / kg, preferably 0.001 to 10 mg / kg, and may be administered 1 to 6 times per day.

The composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers for the prophylaxis and treatment of chronic obstructive pulmonary disease.

In addition, the present invention provides a composition for preventing and improving chronic obstructive pulmonary disease containing the gall extract as an active ingredient.

The gall bladder extract is preferably extracted with water, ethanol or a mixture thereof, but is not limited thereto.

The chronic obstructive pulmonary disease is preferably chronic bronchitis or emphysema, but is not limited thereto.

The gall bladder extract of the present invention reduces the number of macrophages, eosinophils, neutrophils and lymphocytes in pulmonary lavage fluid, and rapidly increases IL-6 and TNF-α cytokines, and MCP-1 and MMP-12 by chronic obstructive pulmonary disease. It has an effect of significantly lowering the amount, significantly reducing inflammatory cells around the alveoli in the tissues, and significantly reducing the size of the expanded alveoli, which is useful as an active ingredient of the health food composition for preventing and improving chronic obstructive pulmonary disease. It can be seen that it can be used.

The health food of the present invention may be added as it is, or used in combination with other food or food ingredients, and may be appropriately used in accordance with conventional methods.

There is no particular limitation on the kind of the health food. Examples of foods to which the Gallja extract may be added include meat, sausages, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, teas, and drinks. , Alcoholic beverages and vitamin complexes, and includes all of the health food in the usual sense.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. Such natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. The ratio of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 ml of the composition of the present invention.

In addition to the above, the health food of the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acids and salts thereof, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, , A carbonating agent used in carbonated drinks, and the like. It may also contain flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not critical, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

The present invention also provides a pharmaceutical composition for the prevention and treatment of lung disease (lung disease) containing the gall bladder extract as an active ingredient.

In addition, the present invention provides a composition for preventing and improving pulmonary disease, comprising a five-fold extract as an active ingredient.

The gall bladder extract of the present invention reduces the number of macrophages, eosinophils, neutrophils and lymphocytes in pulmonary lavage fluid, and rapidly increases IL-6 and TNF-α cytokines, and MCP-1 and MMP-12 by chronic obstructive pulmonary disease. It has an effect of significantly lowering the amount, significantly reducing inflammatory cells around the alveoli in the tissues, and significantly reducing the size of the expanded alveoli, which is useful as an active ingredient of the health food composition for preventing and improving chronic obstructive pulmonary disease. It can be seen that it can be used.

Hereinafter, the present invention will be described in detail with reference to the following Examples, Experimental Examples and Production Examples.

However, the following Examples, Experimental Examples and Preparation Examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following Examples, Experimental Examples and Preparation Examples.

< Example  1> 5 times ( Rhus javanica Linne ) Preparation of extract

The gall bladder extract of the present invention proceeded to the following experiment by dissolving the gall bladder powder purchased from SUN TEN (Taipei, Taiwan) in water.

< Example  2> Chronic Obstructive Pulmonary Disease chronic obstructive pulmonary disease ; COPD ) Manufacture of animal models

<2-1> Preparation and breeding of experimental animals

Six-week old female C57BL / 6 (Orient Bio, Korea) mice weighing 20-25 g were used as experimental animals, and mice were divided into 6 mice per group. Water and feed were allowed to eat freely. The temperature in the breeding room was maintained at 21 ~ 24 ℃ with 40 ~ 60% humidity, and the day and night periods were 12 hours respectively.

<2-2> COPD  cause

To produce COPD-induced mouse models, mice were exposed to cigarette smoke using a smoking chamber.

Specifically, the mice prepared in Example <2-1> were exposed to smoke for 3 minutes per day using a reference cigarette 3R4F (University of Kentucky, USA) for 30 minutes, . The same procedure as above was repeated four times, and COPD-induced mouse models were prepared by exposing to smoke for five times a week under the same conditions.

<2-3> Experimental group  Classification

In order to confirm the effect of the gall bladder extract on chronic obstructive pulmonary disease, experimental groups were divided.

Specifically, the normal mouse group not exposed to cigarette smoke was a control group (control, CON), and a COPD-induced mouse group produced by exposing cigarette smoke to the method of Example <2-2> was exposed to cigarette smoke group ( Cigarettes smoking, CS) and the mouse group in which oral administration of the gall bladder extract having a concentration of 100 mg / kg to the mice once a day were classified into experimental groups.

< Experimental Example  1> Chronic Obstructive Pulmonary Diseases COPD ) To confirm the effect of inhibiting weight loss

To measure the body weight change of the cigarette smoke exposure group, the experimental group and the control group classified in the above Example <2-3>, the body weights of the mice in each group on the start of the experiment (day 0) were measured using the precision balance E06120. Then, mice of each group were weighed at intervals of 2 days.

As a result, as shown in Figure 1, the cigarette smoke exposure group was confirmed that the weight is significantly reduced compared to the control group, the experimental group administered the five gall extract of the present invention significantly compared to the cigarette smoke exposure group significantly changed body weight It was confirmed that the decrease (Fig. 1).

< Experimental Example  2> chronic obstructive pulmonary disease COPD ) To confirm the reduction effect of immune cell increase

Cannula was inserted into the air of the tobacco smoke exposure group, the experimental group and the control group classified in the above Example <2-3>, then the phosphate buffered saline was slowly injected, A bronchoalveloar lavage fluid (BALF) was obtained. Then, immune cells in the lung lavage fluid were attached to a slide glass using a hemacytometer, and Diff-Quick staining was carried out to sort 500 cells into each type.

As a result, as shown in Figure 2, after 3 weeks of exposure to tobacco smoke, the cigarette smoke exposure group significantly increased the total number of inflammatory cells compared to the control group, the experimental group administered the five-fold extract of the present invention cigarette smoke exposure Compared with the group, it was confirmed that the total number of inflammatory cells significantly decreased (FIG. 2).

In addition, as shown in Figure 3, only macrophage (Macrophage) is distributed in the lung washing solution of the control group, macrophage, eosinophil, neutrophil (Neutrophil) and lymphocyte (Lymphocyte), which are inflammatory cells in the tobacco smoke exposure group It was confirmed that the number of was increased significantly. In addition, the experimental group administered with the gall of the present invention was confirmed that the number of macrophages, eosinophils, neutrophils and lymphocytes significantly reduced compared to the tobacco smoke exposure group (Fig. 3).

< Experimental Example  3> Chronic Obstructive Pulmonary Diseases COPD ) Inflammatory cytokine ( Inflammatory cytokine ) Production inhibitory effect

<3-1> Measurement of protein amount

Quantitative Sandwich Enzyme Immunoassay Kit of ELF and TNF-α in Lung Washing Solution for Immune Signaling Agents of Lung Washing Fluid (Quantitative sandwich enzyme-linked immunoassay kit (BD, USA) was measured using.

Specifically, a capture antibody was diluted with a coating buffer (0.1 M carbonate, pH 9.5) using a protocol of OptEIA (BD Bioscience, USA), and the plate was immersed in a 96-well plate 96-well plate), and then coated at 4 ° C overnight. Then, the coated plate was washed three times with wash buffer (PBS / Tween-20 0.05%), and 200 μl / well of assay solution (BD Bioscience, San Diego, CA, USA) After wells were dispensed, they were blocked at room temperature for 1 hour. Subsequently, the wells were washed three times with wash buffer, and 100 μl of a standard and a sample were dispensed. After reacting at room temperature for 2 hours, the plate was washed five times with wash buffer, and Avidin-HRP 100 [mu] l of bound detection antibody was added to each well. After 1 hour of reaction at room temperature, the plate was washed 10 times with a washing buffer, and 100 μl of a Substrate Solution (TMB Substrate Reagent; Pharmingen, BD Bioscience, USA) was added per well. After reacting in the dark at room temperature for 30 minutes, 50 μl of 2NH ₂ SO ₄ was added and measured at 450 nm / 570 nm wavelength with a Microplate reader (Molecular Devices, Sunnyvale, USA) within 30 minutes.

As a result, as shown in FIG. 4, the expression levels of IL-6 and TNF-α of the tobacco smoke-exposed group exposed to tobacco smoke for 3 weeks were significantly increased compared to the control group, and the experimental group to which the five-fold extract of the present invention was administered. Compared to the cigarette smoke group, the levels of IL-6 and TNF-α were confirmed to be significantly reduced (FIG. 4).

<3-2> mRNA  Measurement of expression level

<3-2-1> Whole ( total ) RNA  detach

Lungs were extracted from the tobacco smoke exposed group, experimental group and control group classified in Example <2-3> to isolate total RNA according to the manufacturer's protocol in an RNeasy mini kit (QIAGEN, USA). 600 μl of RLT buffer was added to the lung tissue, pipetted and lysed, 600 μl of 70% ethanol was added, and the mixture was mixed by pipetting. The 700 μl sample was transferred to an RNeasy mini column placed in a 2 ml collection tube and centrifuged at 8,000 × g for 15 sec. The flow-through was discarded and the remaining sample was transferred to an RNeasy mini column And the same method was used. 700 [mu] l RW1 buffer was added to the RNeasy mini-column and centrifuged at 8,000 x g for 15 seconds. The RNeasy mini-column was transferred to a new 2-ml collection tube and 500 μl RPE buffer was added to the RNeasy mini-column and centrifuged at 8,000 × g for 15 seconds. 500 [mu] l RPE buffer was added to the column again and centrifuged at 8,000 x g for 2 minutes. The RNeasy mini-column was transferred to a new collection tube, centrifuged at full speed for 1 minute, and discarded. The RNeasy mini-column was transferred to a 1.5 ml collection tube, and 50 μl of RNase-free water was added to the RNeasy mini-column and centrifuged at 8,000 × g for 1 minute. The isolated total RNA was quantitated with a spectrophotometer (ND-1000, NanoDrop Technologies Inc. USA), and the separated total RNA was electrophoresed on 1% agarose gel and ethidium-bromide ) (Et-Br, Sigma-Aldrich, USA).

&Lt; 3-2-2 > Real time ) RT - PCR  analysis

In order to confirm the expression level of IL-6, TNF-α, MCP-1 and MMP-12 mRNA in the lung tissue of the tobacco smoke exposure group, the experimental group and the control group classified in Example <2-3>, real-time RT- PCR was performed.

Specifically, cDNA was synthesized with 2 ug of total RNA isolated in Example <3-2-1>. Real time PCR was performed using 45 cycles of 95 ° C (10 seconds), 60 ° C (10 seconds) and 72 ° C (12 seconds) using a TAKARA TP800 Real time PCR system. Then, the relative expression levels of mRNA in the respective cytokines were compared using SYBR Green I Master Mix (Applied Biosystems, Foster City, USA) and the following primers (CosmoGENTECH., Korea).

As a result, as shown in Figure 5, the relative expression of IL-6, TNF-α, MCP-1 and MMP-12 mRNA of the tobacco smoke exposed group exposed to tobacco smoke for 3 weeks significantly increased compared to the control group Experiment with administration of five gall extract of the present invention confirmed that the relative expression of IL-6, TNF-α, MCP-1 and MMP-12 mRNA significantly decreased compared to the tobacco smoke exposure group ( 5).

IL-6 sense primer (SEQ ID NO: 1): 5'-TGC TCC TGA CAA CCA CGG CCT-3 ';

IL-6 antisense primer (SEQ ID NO: 2): 5'-ACA GGT CTG TTG GGA GTG GTA TCC T-3 ';

TNF-α sense primer (SEQ ID NO: 3): 5'-CAA GGG ACA AGG CTG CCC CG-3 ';

TNF-alpha antisense primer (SEQ ID NO: 4): 5'-TAG ACC TGC CCG GAC TCC GC -3 ';

MMP-12 sense primer (SEQ ID NO: 5): 5'-GGC CAT TCC TTG GGG CTG CA -3 ';

MMP-12 antisense primer (SEQ ID NO: 6): 5'-GGG GGT TTC ACT GGG GCT CC -3 ';

CCL-2 sense primer (SEQ ID NO: 7): 5'-TCA CAG TTG CCG GCT GGA GC -3 ';

CCL-2 antisense primer (SEQ ID NO: 8): 5'-CAG CAG GTG AGT GGG GCG TT-3 ';

GAPDH sense primer (SEQ ID NO: 9): 5'-TCT GAC GTG CCG CCT GGA GA-3 '; And

GAPDH antisense primer (SEQ ID NO: 10): 5'-TGG GCC CTC AGA YGC CTG CT -3 '.

< Experimental Example  4> Chronic Obstructive Pulmonary Diseases COPD ) Inhibited the increase of inflammatory cells

The lung tissues of the tobacco smoke exposure group, the experimental group and the control group classified in the above Example <2-3> were removed and immobilized in a fixed solution of 4% paraformaldehyde, followed by dehydration with alcohol to make a paraffin block . The tissue fixed to paraffin was cut to a thickness of 4 ㎛, attached to a slide glass, and then paraffin was removed and stained with hematoxylin and eosin to confirm the overall morphology of the tissue.

As a result, as shown in FIG. 6, the cigarette smoke exposure group was found to have severe damage to the lungs by seeing the infiltration of inflammatory cells and the expansion of alveolar proceeding around the alveoli, and the experimental group to which the gall bladder extract of the present invention was administered. Compared with the tobacco smoke exposure group, it was confirmed that the inflammatory cells around the alveoli were reduced and the alveoli size was significantly decreased (FIG. 6).

Preparation Example 1 Preparation of Pharmaceutical Formulation

<1-1> Sanje  Produce

2 g extract of the present invention

Lactose 1 g

The above components were mixed and packed in airtight bags to prepare powders.

<1-2> Preparation of tablets

100 mg extract of gall of the present invention

Corn starch 100 mg

100 mg of milk

2 mg of magnesium stearate

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

&Lt; 1-3 > Preparation of capsules

100 mg extract of gall of the present invention

Corn starch 100 mg

100 mg of milk

2 mg of magnesium stearate

After mixing the above components, the capsules were filled in gelatin capsules according to the conventional preparation method of capsules.

&Lt; 1-4 >

5 g extract of the present invention

Lactose 1.5 g

Glycerin 1 g

0.5 g of xylitol

After mixing the above components, they were prepared so as to be 4 g per one ring according to a conventional method.

<1-5> Preparation of granules

150 mg extract of gall of the present invention

Soybean extract 50 mg

200 mg of glucose

600 mg of starch

After mixing the above components, 100 mg of 30% ethanol was added and the mixture was dried at 60 캜 to form granules, which were then filled in a capsule.

&Lt; Preparation Example 2 > Production of food

<2-1> Production of flour food

0.5-5.0 parts by weight of the gall bladder extract of the present invention was added to flour, and bread, cake, cookies, crackers and noodles were prepared using the mixture.

<2-2> Production of soups and gravies

0.1-5.0 parts by weight of gall of the extract of the present invention was added to soups and gravy to prepare meat products for health promotion, soups and gravy of noodles.

<2-3> Preparation of ground beef

10 parts by weight of the gall extract of the present invention was added to the ground beef to prepare a ground beef for health promotion.

<2-4> Production of Dairy Products

5 to 10 parts by weight of the gall bladder extract of the present invention was added to milk, and various milk products such as butter and ice cream were prepared using the milk.

&Lt; 2-5 >

Brown rice, barley, glutinous rice, and yulmu were dried by a known method and dried, and the mixture was granulated to a powder having a particle size of 60 mesh.

Black soybeans, black sesame seeds, and perilla seeds were steamed and dried by a conventional method, and then they were prepared into powder having a particle size of 60 mesh by a pulverizer.

The magnolia extract of the present invention was concentrated under reduced pressure in a vacuum concentrator, and the dried product obtained by drying with a sprayer and a hot air dryer was pulverized with a particle size of 60 mesh to obtain a dry powder.

The grains, seeds and the gall extract of the present invention prepared above were formulated in the following ratio.

(30 parts by weight of brown rice, 15 parts by weight of yulmu, 20 parts by weight of barley)

Seeds (7 parts by weight of perilla, 8 parts by weight of black beans, 7 parts by weight of black sesame seeds)

5 g extract of the present invention (3 parts by weight),

(0.5 part by weight),

Rhubarb (0.5 parts by weight).

Preparation Example 3 Preparation of Beverage

<3-1> Production of health drinks

Instant sterilization by homogeneously combining 5 g of gallnut extract of the present invention with subsidiary materials such as liquid fructose (0.5%), oligosaccharide (2%), sugar (2%), salt (0.5%) and water (75%). Then it was prepared by packaging in a small packaging container such as glass bottles, plastic bottles.

<3-2> Preparation of vegetable juice

Vegetable juice was prepared by adding 5 g of gall of the present invention to 1,000 ml of tomato or carrot juice.

<3-3> Production of fruit juice

Fruit juice was prepared by adding 1 g of the gall of the present invention to 1,000 ml of apple or grape juice.

<110> Kyunghee University-Industry Cooperation Foundation <120> Pharmaceutical composition for prevention and treatment of          chronic obstructive pulmonary disease comprising extract of Rhus          javanica Linne as an active ingredient <130> 12p-02-09 <160> 10 <170> Kopatentin 1.71 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> IL-6 sense primer <400> 1 tgctcctgac aaccacggcc t 21 <210> 2 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> IL-6 antisense primer <400> 2 acaggtctgt tgggagtggt atcct 25 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TNF-alpha sense primer <400> 3 caagggacaa ggctgccccg 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TNF-alpha antisense primer <400> 4 tagacctgcc cggactccgc 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MMP-12 sense primer <400> 5 ggccattcct tggggctgca 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MMP-12 antisense primer <400> 6 gggggtttca ctggggctcc 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CCL-2 sense primer <400> 7 tcacagttgc cggctggagc 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CCL-2 antisense primer <400> 8 cagcaggtga gtggggcgtt 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH sense primer <400> 9 tctgacgtgc cgcctggaga 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH antisense primer <400> 10 tgggccctca gaygcctgct 20

Claims (5)

Nut gall (Rhus A pharmaceutical composition for preventing and treating chronic obstructive pulmonary disease (COPD) containing javanica Linne) extract as an active ingredient.
The pharmaceutical composition for the prevention and treatment of chronic obstructive pulmonary disease according to claim 1, wherein the chronic obstructive pulmonary disease is chronic bronchitis or emphysema.
Chronic obstructive pulmonary disease prevention and improvement composition for health food containing the gall extract.
Nut gall (Rhus Pharmaceutical composition for preventing and treating lung disease (lung disease) containing javanica Linne) extract as an active ingredient.
Nut gall (Rhus Health food composition for preventing and improving lung disease containing javanica Linne) extract as an active ingredient.

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