KR20220094678A - A composition comprising ginseng for preventing, improving or treating chronic obstructive pulmonary disease by fine dust - Google Patents

Abstract

The present invention relates to a composition for the prevention, alleviation, or treatment of chronic obstructive pulmonary disease caused by fine dust containing red ginseng as an active component. The present invention is the research result of the research project of the Ministry of Agriculture, Food and Rural Affairs, 2015 rural resource complex industrialization support project, local health food masterpiece project.

Description

A composition for the prevention, improvement or treatment of chronic obstructive pulmonary disease caused by fine dust containing red ginseng as an active ingredient

The present invention relates to a composition for preventing, improving or treating chronic obstructive pulmonary disease caused by fine dust containing red ginseng as an active ingredient. The present invention is the research result of the research project of the Ministry of Agriculture, Food and Rural Affairs in 2015 for the rural resource complex industrialization support project for the local health food luxury project.

Chronic Obstructive Pulmonary Disease (COPD) is a chronic lung disease in which continuous respiratory symptoms and airflow limitation appear due to abnormalities in the airways or lung parenchyma, which are generally caused by exposure to harmful particles or gases. In particular, irreversible airflow limitation caused by the reduction of elastic seminal pressure due to stenosis of the small airways due to chronic inflammation and damage to the alveoli is a key feature of COPD. Airflow restriction is confirmed through a lung function test and confirmed when the ratio of forced expiratory volume (FEV1) to forced vital capacity (FVC) is less than 70% in 1 second.

The most important factor in chronic obstructive pulmonary disease is smoking, and external factors such as indoor and outdoor air pollution, socioeconomic status, and respiratory infection interact with host factors such as genes, age, sex, airway hyperresponsiveness, and lung growth. The incidence tends to increase with age, and the prevalence is expected to increase sharply in the future due to an aging population. About 59% of the male population over 70 years of age suffer from chronic obstructive pulmonary disease, and in Korea, the elderly population over 65 years old accounted for 11% of the total population (2010), an increase of 3.8% compared to 10 years ago. It is expected to reach an aged society (15%) and a super-aged society (20%) in 2026, requiring management of chronic obstructive pulmonary disease.

According to the National Institutes of Health, it is currently the fourth most common cause of death in the United States and is expected to become the third leading cause of death worldwide by 2020. Although the negative effects of smoking on health have been of public interest for a very long time, the mechanism of the disease's pathogenesis is still unclear. In particular, it is known that some components contained in tobacco remain deep in the alveoli for a long time after quitting smoking to induce various lung diseases. Furthermore, as the smoking age is gradually decreasing and the smoking rate of women is increasing, the government is experiencing great difficulties in establishing policies related to public health.

In addition, as the concentration of fine dust in the air in Korea has recently increased to a serious level, concerns about the public's lung health are heightening.

EMT (Epithelial-mesenchymal transition) refers to a process in which epithelial cells lose the characteristics of epithelial cells and have the characteristics of mesenchymal cells. EMT is a process associated with airway remodeling in COPD, resulting in progressive lung injury. The mechanism of airway remodeling in COPD is very complex, and recent studies have shown that EMT, the process by which epithelial cells undergo cytoskeletal reorganization with loss of cell-cell contact and acquire a mesenchymal phenotype due to excessive extracellular matrix deposition, is implicated in respiratory restructuring in COPD. known to be involved

Metastatic epithelial cells release extracellular matrix proteins that enhance mobility and promote fiber across the basement membrane and enhance tissue remodeling. In addition, the TGF-β1/Smad pathway is important for inducing EMT. Research on COPD treatment candidates that control the EMT mechanism by exploring the mechanism of the TGF-β1-related pathway in COPD is also being actively conducted.

The present applicant is a basic research on chronic obstructive pulmonary disease, which increases social and economic costs, through EMT research, the leading role of lung epithelial cells, which has been passively recognized in the animal model of chronic obstructive pulmonary disease. By proving the related mechanism and proving the molecular marker network that can regulate it, we want to prove that red ginseng is excellent as a candidate material related to EMT control, thereby identifying the effect of improving lung function.

Looking at the prior art literature, Korean Patent Laid-Open Publication No. 10-2018-0037693 describes a food composition for preventing and improving lung disease. , it is described that the antitussive activity test was performed on the mixed extract fermented product of onion, cornflower oil and licorice. In addition, the Republic of Korea Patent Publication No. 10-2019-0133637 describes a composition for improving chronic obstructive pulmonary disease using a bokryeongpi extract, an experiment using a COPD induction model using CSE and PPE is described. In the first document, red ginseng is described as one of several materials, but experiments or functionality using the COPD model are not described, and in the second document, red ginseng is not used but experiments using the COPD model are described. can be checked

Korean Patent Publication No. 10-2018-0037693 (published on April 13, 2018) Republic of Korea Patent Publication No. 10-2019-0133637 (published on December 3, 2019)

An object to be solved is to provide a composition for preventing, improving or treating chronic obstructive pulmonary disease caused by fine dust.

The solution to the problem is a composition for preventing, improving or treating chronic obstructive pulmonary disease caused by fine dust containing red ginseng extract as an active ingredient.

The red ginseng extract is characterized in that it contains 100 mg/kg based on oral administration of a mouse.

The composition is characterized in that it further comprises an extract of Angelica asiatica.

The extract is characterized in that it contains 20 mg/kg based on oral administration of a mouse.

A solution to the problem is a food composition for preventing or improving chronic obstructive pulmonary disease caused by fine dust containing red ginseng extract as an active ingredient.

The composition according to the present invention has an effect of improving COPD and lung injury in the PM-induced lung injury COPD model of mouse fibroblast NIH3T3 cell line and human fibroblast MRC5 cell line.

It has the effect of improving COPD in the PM-induced lung injury COPD model of the mouse according to the present invention.

1 is a primer used in the experiment, and FIGS. 2 to 17 are graphs or photographs showing the experimental results of Experimental Examples 1 and 2.

The application of the present invention is not limited by the configuration and arrangement of components described in the experimental examples and embodiments of the present invention or shown in the drawings. The present invention is capable of being embodied in other embodiments and of being carried out in various ways. Also, expressions and predicates used in the examples with respect to terms such as the orientation of devices or elements are used only to simplify the description of the present invention, and do not indicate or imply that the related devices or elements simply have to have a specific orientation. . For example, where terms such as “first” and “second” are used in the embodiments and claims describing the present invention, such terms are not intended to indicate or imply relative importance or spirit.

In addition, the terms or words used in the present specification and claims are not to be construed as being limited to conventional or dictionary meanings, and the inventors are in the technical spirit of the present invention in order to explain the terms and concepts of the invention in the best way. It should be interpreted as written based on the corresponding meaning and concept.

Therefore, the present invention is not limited to the experimental examples and examples presented, and those of ordinary skill in the art to which the present invention pertains can use the technical spirit of the present invention and the technical ideas described in the claims to be described below. Various modifications and changes are possible within an equal range.

Although the present invention has been mainly described with reference to the accompanying drawings, it will be apparent to those skilled in the art that various modifications may be made without departing from the technical scope thereof. Accordingly, the scope of the present invention may include many such modifications.

In the present invention, it was carried out using Jinan red ginseng and Cham Angelica, and both red ginseng and Cham Angelica extract were used. Angelica gigas NAKAI ( Angelica gigas NAKAI) is collected in autumn and dried in the sun for use. It is a representative medicine for bohyeol and is known to be effective for anemia, blood circulation disorders due to eohyeol, menstrual irregularities, physical weakness, headache, abdominal pain, and constipation. Main ingredients include decursin, decursinol, and nodakenin. These ingredients are known to help blood circulation, suppress free radicals, and help prevent cancer and dementia.

Experimental Example 1. Experimental model and experimental method

One) in vitro experiment

[Experimental model] The effect of red ginseng was analyzed in the EMT model by PM using the mouse fibroblast NIH3T3 cell line and human fibroblast MRC-5 cell line, respectively . (PM: Particulate matter)

[Experimental group] Untreated group (CTL), PM treated group, NIH3T3 cells PM+Red ginseng (2000ug/ml), NIH3T3 cells PM+Red ginseng (2000ug/ml)+Seaweed (500ug/ml), MRC-5 cells PM+Red ginseng (500ug) /ml), MRC-5 cell PM+red ginseng (500ug/ml)+cham angelfish (150ug/ml) (red ginseng: red ginseng extract, chamomile: chamomile extract)

[Evaluation items] Western blot/real-time PCR (confirm expression of protein markers) / Collagen analysis / Immunofluorescent staining (confirm expression of specific proteins in cells) / Scratch wound healing assay (confirm cell migration) / Matrigel transwell assay (confirm cell invasion)

[Test method]

- Western blot

NIH3T3, MRC5 cells are seeded in a 6-well plate at 5X10 ⁵ cells/well. After 24 hours, PM (National Institute of Standards and Technology) and red ginseng are treated. After 48 hours, extract the protein using RIPA lysis buffer (ATTO), add SDS sample buffer (ELPIS), and boil for 5 minutes. Electrophoresis using SDS-PAGE gel and transfer to membrane. After blocking membrane in 5% sikm milk (BD) for 1 hour, anti-TGF-β1 (Abcam), anti-collagen I (Boster), anti-α-SMA (sigma), anti-E-cadherin (Cell) Signaling), the anti-vimentin (Cell signaling) antibody was reacted overnight at 4°C. After washing 3 times for 10 minutes with 0.1% TBS-T, react with anti-Rabbit-HRP (Jackson Immuno Research) antibody for 1 hour. After washing 3 times for 10 minutes with 0.1% TBS-T, detection is performed using the ECL system (GenDEPOT).

- Real-time PCR

NIH3T3, MRC5 cells are seeded in a 6-well plate at 5X10 ⁵ cells/well. After 24 hours, PM (National Institute of Standards and Technology) and red ginseng are treated. After 48 hours, mRNA was extracted using easy-BLUE (Intron), cDNA was synthesized using the Super-Script First-Strand Synthesis System (Invitrogen), and then real-time PCR was performed using the SYBR Green System (Bioneer). carry out Denaturation is performed once at 95°C for 10 minutes, annealing is performed at 95°C for 5 seconds, and elongation is performed at 60°C for 30 seconds, which is repeated 40 times. Melting is performed by raising the temperature from 55℃ to 95℃. The primer used is shown in FIG. 1 .

- Collagen analysis

NIH3T3, MRC5 cells are seeded in 96-well plate at 1X10 ⁴ cells/well. After 24 hours, PM (National Institute of Standards and Technology) and red ginseng are treated. After 48 hours, 100ul of media was transferred to a tube, 1ml of Sircol Dye Reagent (Biocolor) was added, shaken for 30 minutes, and centrifuged at 12000rpm for 10 minutes. Remove the supernatant, dissolve the pellet with 750ul of cold Acid-Salt Wash Reagent (Biocolor), and centrifuge at 12000rpm for 10 minutes. Remove the supernatant, dissolve the pellet with 250ul Alkali Reagent (Biocolor), and measure the absorbance at 555nm.

- Immunofluorescent staining

NIH3T3, MRC5 cells are seeded on a chamber slide and 24 hours later, PM (National Institute of Standards and Technology) and red ginseng are treated. After 48 hours, fixation was performed with 4% formaldehyde (Intron) for 2 minutes and washed 3 times with PBS for 5 minutes each. Blocking with 5% normal serum (Life Technology) for 1 hour, and anti-TGF-β1 (Abcam), anti-collagen I (Boster), anti-α-SMA (sigma), anti-vimentin (Cell signaling) antibody 4 Incubate overnight at °C. Wash 3 times with PBS for 5 minutes each and react with anti-Rabbit-488 (Jackson Immuno Research) antibody for 1 hour. After washing 3 times with PBS for 5 minutes, and staining the nucleus with DAPI (Vector), observe under a microscope.

- Scratch wound healing assay

NIH3T3, MRC5 cells are seeded in a 6-well plate at 5X10 ⁵ cells/well. After 24 hours, scratch with a yellow tip and treat with PM (National Institute of Standards and Technology) red ginseng. Observe under a microscope at 0, 24, and 48 hours.

- Matrigel transwell assay

Mix 10 mg/ml matrigel (corning) and serum free media in a 1:4 ratio and put 100ul each in the upper chamber of the trans-well. After hardening at 37°C for 4 hours, the NIH3T3 and MRC5 cells were dissolved in media containing 1% FBS at 1X10 ⁵ cell/100ul and then treated with PM (National Institute of Standards and Technology) and red ginseng. 600ul of media containing 10% FBS was put into the lower chamber, and after 48 hours, the media and matrigel were removed, and the upper chamber was washed once with PBS. Fixation was performed with 4% formaldehyde (Intron) for 2 minutes, washed twice with PBS, and then permeabilized with 100% methanol (Millipore) for 20 minutes. After washing twice with PBS, stain with Crystal violet (ELPIS) for 15 minutes. After washing twice with PBS, observe under a microscope.

2) in vivo experiment

[Experimental model] Particulate matter(PM)-induced lung injury model

Inject 200 ug of fine dust (PM) per mouse intranasally in a volume of 50 ul 3 times a week. From one week after PM injection, the candidate substance (red ginseng) is administered orally for 2 weeks. After sacrificing a mouse, BALF and lung tissue are isolated, and cytokine ELISA, real-time RT-PCR, Western blot, intracellular cytokine analysis, and immunohistochemical staining (IHC) are performed.

[Experimental group] Untreated group (PBS), PM 200ug, PM+Red ginseng 100mg/kg, PM+Red ginseng 100mg/kg + Korean Angelica 20mg/kg (Red ginseng: Red ginseng extract, Angelica oleifera extract) (See Fig. 11)

[Evaluation items] Body weight/Lung weight / IHC / Western blot / Real-time PCR / Collagen analysis / BALF analysis, Cytokine assay / micro CT analysis

[Test method]

- body weight

Measure the weight of each mouse before the start of the experiment, and observe the change in weight by measuring at intervals of one week after the induction of lung disease until the end of the experiment.

- Lung weight

After completion of the mouse experiment, the lungs are removed and the weight is measured.

- Histopathology skills

After completion of the mouse experiment, the lungs were removed and fixed in 10% Formalin solution 10% (Sigma-Aldrich, Louis, MO, USA) for 24 hours, and then the fixed tissue was washed several times in running water, and 70% alcohol at 4°C. 2 times for 1 hour each, 80% alcohol twice for 1 hour, 95% alcohol for 24 hours, and 100% alcohol (DAEJUNG, Korea) 3 times for 1 hour each. Xylene (DAEJUNG, Korea) was replaced 3 times for 1 hour each, and paraffin (Leica) was replaced twice for 1 hour and 30 minutes each. The tissue was prepared by cutting it into 4 μm and attaching it to a slide. Afterwards, H&E stain, Sirius Red, and IHC were performed.

- H&E stain

The slides were treated in xylene twice for 5 minutes each, followed by 100% alcohol, 95%, 80%, and 70% alcohol for 5 minutes each, and washed with distilled water. Remove unnecessary staining by up and down in 70% alcohol containing 1% HCl for 1 minute in hematoxylin (BBC Biochemical, USA), and stain with eosin for 30 seconds. Alcohol 70%, 80%, 95%, 100% in the order of 1 minute each, after 2 minutes in xylene 2 times, the mountin medium (Vector Laboratories, Inc., USA, CA) was dropped and covered with a slide glass. Afterwards, it was observed under a microscope.

- Sirius Red

It was stained using Sirius Red stain kit (Abcam, Inc., Cambridge, UK). The slides were treated in xylene twice for 5 minutes each, 100% alcohol, 95%, 80%, and 70% in order for 5 minutes each, and washed with distilled water. After staining in sirius red solution for 1 hour, unnecessary staining was removed with Acetic Acid solution, and treated with alcohol 70%, 80%, 95%, 100% for 1 minute each in order, 2 times for 2 minutes in xylene, then mountin medium (vector Laboratories, Inc., USA, CA) was dropped, covered with a slide glass, and observed under a microscope.

- IHC

The slides were treated in xylene twice for 5 minutes each, followed by 100% alcohol, 95%, 80%, and 70% alcohol for 5 minutes each, and washed with distilled water. After blocking in 5% normal goat serum (life technologies, USA) for 20 minutes, the primary antibody, anti-α-SMA antibody (Abcam, Inc., Cambridge, UK) was reacted at 4°C. Wash several times with distilled water, react with a secondary antibody, IgG-conjugated horseradish peroxidase (HRP) (Bethyl, TX, USA) for 30 minutes, and then wash several times with distilled water. 3, 3′-diaminobenzidine as substrate (Sigma, St Louis, MO, USA) was reacted for 10 minutes, washed several times with distilled water, and treated with alcohol 70%, 80%, 95%, 100% in order for 1 minute each , After treatment twice in xylene for 2 minutes each, mountin medium (vector Laboratories, Inc., USA, CA) was dropped, covered with a slide glass, and observed under a microscope.

- Western blot

This is a method for confirming the protein expression level in the tissue. After completion of the experiment, the lungs are removed and the tissue is ground into single cells. Extract the protein using RIPA lysis buffer (ATTO), add SDS sample buffer (ELPIS), and boil for 5 minutes. Electrophoresis using SDS-PAGE gel and transfer to membrane. After blocking membrane in 5% sikm milk (BD) for 1 hour, anti-TGF-β1 (Abcam), anti-collagen I (Boster), anti-α-SMA (sigma), anti-E-cadherin (Cell) Signaling), the anti-vimentin (Cell signaling) antibody was reacted overnight at 4°C. After washing 3 times for 10 minutes with 0.1% TBS-T, react with anti-Rabbit-HRP (Jackson Immuno Research) antibody for 1 hour. After washing 3 times for 10 minutes with 0.1% TBS-T, detection is performed using the ECL system (GenDEPOT).

- Real-time PCR

This is a method for detecting a change in a gene (mRNA) in a tissue. After the end of the experiment, the lung tissue is removed and replaced with single cells. mRNA was extracted using easy-BLUE (Intron), cDNA was synthesized using the Super-Script First-Strand Synthesis System (Invitrogen), and real-time PCR was performed using the SYBR Green System (Bioneer). Denaturation is performed once at 95°C for 10 minutes, annealing is performed at 95°C for 5 seconds, and elongation is performed at 60°C for 30 seconds, which is repeated 40 times. Melting is performed by raising the temperature from 55℃ to 95℃. The primers used are as follows: Total RNA in each sample is detected, cDNA synthesis is performed, and mRNA is detected and quantified by PCR.

- Collagen analysis

It was measured using SirCol collagen assay kit (Biocolor, France). It is measured using bronchoalveolar lavage fluid or lung tissue. To 0.5 mg of lung tissue, add 1 ml of acetic acid, 100 μl of Acid Neutralizing Reagent, and 200 μl of Isolation & Concentration Reagent, mix, and react overnight. A tissue sample is prepared by centrifugation at 12000 rpm for 10 minutes to remove the supernatant, and 50 μl of the tissue sample or bronchoalveolar lavage solution and 1 ml of sircol dye reagent are mixed for 30 minutes. After centrifugation at 4°C and 12,000 rpm for 10 minutes, the supernatant is discarded, 750 μl of Acid-salt wash reagent is added, washed, and centrifuged at 12,000 rpm for 10 minutes. Discard the supernatant, add 1 ml of Alkali Reagent, dissolve, and measure absorbance at 555 nm.

- BALF analysis

After completion of the experiment, open the chest to expose the airway, insert a syringe into the airway, add 800 μl of PBS, circulate twice for washing, and collect 500 μl again to obtain a bronchoalveolar lavage solution. Centrifuge the bronchoalveolar lavage solution at 4°C, 1200rpm, for 5 minutes, and the supernatant is stored separately to measure cytokine and collagen, and the separated cells are dissolved in 600ml of PBS and centrifuged on a cytospin at 1500rpm for 7 minutes. After attaching, using Diff-Quik reagent, immerse in Diff-Quik fixative for 20 seconds, wash, dip in Diff-Quik solution Ⅰ for 50 seconds, wash, immerse in Diff-Quik solution Ⅱ for 20 seconds and wash, and then wash with a mounting medium (vector Laboratories). , Inc., USA, CA) is dropped, covered with a slide glass, and total cells and inflammatory cells are measured.

- Cytokine assay

IL-6 (BD, San Diego), IL-1β (BD, San Diego) and TNF-α (eBioscience, USA) were measured using bronchoalveolar lavage fluid. Capture antibody is mixed with coating buffer, put into each well by 100 μl, reacted at 4°C overnight, and washed 4 times with washing buffer. Add 200 μl of assay diluent per well, block at room temperature for 1 hour, and wash 4 times with washing buffer. After diluting the bronchoalveolar lavage solution and standard in each experimental group to a concentration of 3 times in the assay diluent solution, 100 μl of each was added to a 96 well plate coated with each capture antibody and reacted at room temperature for 2 hours. After washing 4 times with washing buffer, 100 μl of biotin-conjugate antibody reagent was treated in each well and reacted at room temperature for 1 hour, washed 4 times, and then treated with 100 μl of streptavidin-HRP solution in each well. After incubation at room temperature for 1 hour, it was washed 4 times with washing buffer again. Here, 100 μl of substrate solution was treated and reacted for 5-30 minutes, and then the reaction was terminated by treatment with 50 μl of stop solution, and absorbance was measured at 450 nm using Enzyme-Linked Immunosorbent Assay (ELISA).

- micro CT analysis

Put the mouse into the anesthesia tube connected to the inhalation anesthesia machine, open the O ₂ gas cylinder, and then open the anesthetic machine so that sevofrain (Ilsung Shinyak, Korea) enters the anesthesia tube. When the mouse is anesthetized, it is placed on the CT bed and micro-CT is taken. The tube voltage and tube current of the X-ray generator are 50 kV and 80 μA, respectively, and the CT scans under the condition that a total of 720 images can be obtained when rotated 360° by rotating 0.5° per projection.

Experimental Example 2. Experimental results

One) in vitro experiment

(1) Real-time PCR (mRNA quantitative analysis)

In a lung injury cell model using PM (fine dust) using mouse fibroblast NIH3T3 cells and human fibroblast MRC5 cells, TGF-β1, Collagen I, α-SMA, E-cadherin, Vimentin Confirmed.

As a result, it was found that the mRNA expression levels of TGF-β1, Collagen I, α-SMA, and Vimentin, which were increased when chronic obstructive pulmonary disease or pulmonary fibrosis was induced, decreased when red ginseng was administered, and chronic obstructive pulmonary disease However, when pulmonary fibrosis was induced, it was found that the expression of E-cadherin, which had been lost, was restored again, and this effect was also confirmed in the group treated with red ginseng and Korean Angelfish complex (see Fig. 2, NIH3T3 above, MRC5 below). )

(2) Western blot (protein expression analysis)

In a lung injury cell model using PM (fine dust) using mouse fibroblast NIH3T3 cells and human fibroblast MRC5 cells, TGF-β1, Collagen I, α-SMA, E-cadherin, Vimentin Confirmed.

As a result, it was found that the expression levels of TGF-β1, Collagen I, α-SMA, and Vimentin, which were increased when chronic obstructive pulmonary disease or pulmonary fibrosis was induced, decreased when red ginseng was administered. When disease or pulmonary fibrosis was induced, it was found that E-cadherin, whose expression was off, was restored again, and this effect was also confirmed in the group treated with red ginseng and Angelica keiskei (see Fig. 3, NIH3T3 above, below). MRC5)

(3) Collagen analysis

As a result of checking the intracellular collagen expression level using the Sircol assay kit, it was confirmed that the collagen expression level was decreased by red ginseng in the PM lung injury model, and this effect was also confirmed in the group treated with red ginseng and Korean Angelica. .(See Figure 4, NIH3T3 above, MRC5 below)

(4) Immunofluorescent staining

In the PM lung injury model of NIH3T3 cells and MRC5 cells, it can be confirmed that TGF-β1, Collagen I, α-SMA, and Vimentin were significantly reduced by red ginseng. This effect was also confirmed in the group treated with red ginseng and cham-Anggui. (See FIGS. 5 to 8, NIH3T3 above, MRC5 below)

(5) Scratch wound healing assay

As a result of confirming cell mobility in two lung disease cell models, NIH3T3 cell and MRC5 cell, it was confirmed that red ginseng slowed down the accelerated cell mobility in the lung disease model. could also be confirmed. (See Figure 9, NIH3T3 above, MRC5 below)

(6) Matrigel transwell assay

As a result of confirming the permeability of cells in two lung disease cell models, NIH3T3 cell and MRC5 cell, it was confirmed that red ginseng slowed down the accelerated cell mobility in the lung disease model. could also be confirmed. (See Figure 10, NIH3T3 above, MRC5 below)

2) in vivo experiment

(1) Body weight & Lung weight (mouse weight and lung weight)

As a result of measuring the weight change of mice from the beginning of the experiment to the end of the experiment at intervals of one week, there was no significant difference in mouse weight between groups, so it was judged that there was no toxicity to the experimental animals. As a result, it was confirmed that the lung weight increased in the disease group was decreased by red ginseng. In addition, the same effect was found in the red ginseng and chamomile group. (See Fig. 12)

(2) Real-time PCR (mRNA quantitative analysis) & Western blot (protein quantitative analysis)

In order to confirm the effect of red ginseng and red ginseng complex in the lung damage model caused by fine dust (PM), mRNA and protein expression levels were quantified in the mouse lungs of each group. It was confirmed that all of the complexes containing red ginseng had an improvement effect on lung damage (see FIG. 13).

(3) Collagen analysis & micro CT analysis

In order to confirm the effect of red ginseng and red ginseng complex in the lung damage model caused by fine dust (PM), the expression level of collagen in the mouse lungs of each group was checked, and as a result, it was found that it was decreased by red ginseng. It could be seen that a larger decrease was observed by In addition, as a result of CT image analysis of mouse lungs before and after the experiment, it was shown that there was no significant effect by the extract (see FIG. 14).

(4) Analysis of cells in alveolar lavage fluid

In order to confirm the effect of red ginseng and red ginseng complex in the lung damage model caused by fine dust (PM), the distribution of cells in the alveolar lavage fluid was analyzed to determine the effect on total cells in the mouse lungs of each group. , macrophages, no significant changes in lymphocytes were seen (see Figure 15, Total cess, Macrophages, Lymphocytes order)

(5) Analysis of cells in alveolar lavage fluid

In order to confirm the effect of red ginseng and red ginseng complex in the lung damage model caused by fine dust (PM), the alveolar lavage fluid of mice was separated and centrifuged. Cytokine was measured in the supernatant. As a result, TNF-α, IL increased in the disease group. It was found that the secretion of -6 and IL-1β was decreased, respectively. (See Figure 16, TNF-α, IL-6, IL-1β sequence)

(6) Immunohistochemical staining (H&E stain, Sirius red, IHC)

Cell morphology can be confirmed with H&E stain and Sirius red. As a result of observing the degree of infiltration of inflammatory cells in the peripheral and alveolar regions, it was confirmed that the degree of infiltration of inflammatory cells was decreased by red ginseng and Angelica keiskei. -SMA was confirmed to be reduced by red ginseng, and there was no synergistic effect by the Cham Angelica complex group. (See FIG. 17, H&E Stain, Sirius red stain, α-SMA order)

Example 1. Composition for preventing, improving or treating chronic obstructive pulmonary disease caused by fine dust containing red ginseng extract as an active ingredient

The composition according to the present invention contains a red ginseng extract as an active ingredient. Preferably, a hot water extract is used, and it contains 100 mg/kg based on oral administration to mice. Depending on the design conditions, ethanol or water-ethanol mixed solvent extract is used, and the human standard dose can be appropriately adjusted.

The composition according to the present invention contains a red ginseng extract and a chamomile extract (hereinafter, a red ginseng complex) together as active ingredients. Preferably, each extract uses a hot water extract, and the red ginseng extract contains 100 mg/kg based on oral administration of a mouse, and the Angelica asiatica extract contains 20 mg/kg based on an oral administration of a mouse. Depending on the design conditions, ethanol or water-ethanol mixed solvent extract is used, and the human standard dose can be appropriately adjusted.

The present invention includes a red ginseng extract or a red ginseng complex as an active ingredient and is formulated in a pharmaceutical unit dosage form by adding a pharmaceutically acceptable carrier, excipient or diluent to provide a preventive and therapeutic agent for immune-enhancing and anti-inflammatory diseases. can Here, the carrier, excipient, and diluent include toz, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, undecided. vaginal cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

In addition, the pharmaceutical dosage form may be used in the form of a pharmaceutically acceptable salt, and may be used alone or in combination with other pharmaceutically active compounds as well as in an appropriate group. In addition, when formulating the active ingredient, it can be prepared using a commonly used diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, and a surfactant. In addition, the pharmaceutical dosage forms are each formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injection solutions according to conventional methods to be used. can

In the solid formulation for oral administration, at least one or more excipients, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc. may be mixed with the extract. It can be prepared by mixing. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used.

Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used.

As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like can be used.

The preferred dosage of the extract of the present invention varies depending on the condition and weight of the patient, the degree of disease, age, sex, drug form, administration route and period, but may be appropriately selected by those skilled in the art. However, for a desirable effect, the extract of the present invention is preferably administered at 0.001 to 300 mg/kg, and the administration may be administered once a day, or divided into several administrations. The above dosage does not limit the scope of the present invention in any way.

The extract of the present invention may be administered to mammals such as rats, mice, livestock, and humans by various routes. Any mode of administration can be envisaged, for example, by oral, rectal or intravenous, intramuscular, subcutaneous injection.

By adding a food supplement additive to the active ingredient of the present invention, it is possible to provide an immune-enhancing and anti-inflammatory health functional food composition.

Foods to which the active ingredient can be added include, for example, various foods, beverages, gums, tea, vitamin complexes, health functional foods, and the like.

The amount of the active ingredient in the food or beverage may be added 0.01 to 20% by weight of the total food or beverage weight, and the health beverage composition may be added in a ratio of 0.02 to 5 g, preferably 0.3 to 1 g, based on 100 ml. have.

The health functional beverage composition of the present invention is not particularly limited in other ingredients other than containing the red ginseng extract or red ginseng complex, and may contain various flavoring agents or natural carbohydrates as additional ingredients like a conventional beverage. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose; conventional sugars such as disaccharides such as maltose, sucrose and the like and polysaccharides such as dextrin, cyclodextrin, and the like, and sugar alcohols such as xylitol, sorbitol, erythritol and the like. In addition to the above, natural flavoring agents (taumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) may be advantageously used as flavoring agents other than those described above. The proportion of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition to the above, the red ginseng extract or red ginseng complex of the present invention contains various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavoring agents, colorants and thickeners (cheese, chocolate, etc.), pectic acid and salts thereof. , organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like.

In addition, the red ginseng extract or red ginseng complex of the present invention may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of these additives is not critical, but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the extract of the present invention.

So far, the present invention has been focused on preferred embodiments.

The embodiments described in this specification and the configurations shown in the drawings relate to one most preferred embodiment of the present invention, and do not represent all of the technical spirit of the present invention, so various equivalents and modifications that can be substituted for them are It should be understood that there may be examples.

Therefore, the present invention is not limited to the presented embodiments, and within the equivalent scope of the technical spirit of the present invention and the technical spirit described in the claims to be described below by those of ordinary skill in the technical field to which the present invention pertains. There may be embodiments in which various modifications and changes are possible.

Claims (5)

A composition for the prevention, improvement or treatment of chronic obstructive pulmonary disease caused by fine dust containing red ginseng extract as an active ingredient.
The method according to claim 1,
The composition characterized in that the red ginseng extract contains 100 mg/kg based on oral administration of a mouse.
The method according to claim 1 or 2,
Composition, characterized in that it further comprises an extract of Angelica keiskei.
4. The method according to claim 3,
The composition, characterized in that the extract is characterized in that it contains 20 mg/kg based on oral administration of the mouse.
A food composition for the prevention or improvement of chronic obstructive pulmonary disease caused by fine dust containing red ginseng extract as an active ingredient.

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