KR102188416B1 - Complex Medicines for Improving Respiratory Inflammation Caused Fine Dust - Google Patents

Abstract

The present invention relates to a complex formulation for alleviating respiratory inflammation caused by fine dust, which contains a complex extract of Platycodon grandiflorus, Chinese Liquorice, Lonicera japonica, and Mori cortex as an active component. By administering the complex formulation for alleviating respiratory inflammation caused by fine dust according to the present invention in mice, there is a synergistic effect between formulations, so that it is possible to exhibit excellent efficacy of phlegm excretion, reduction of neutrophils, decreased production of IL-17, MIP2, CXCL-1, and TNF-α, decreased immune cells in BAL and lungs, and reduction of lung cell walls and collagen fibers, thereby being able to be very usefully used in alleviating respiratory inflammation induced by fine dust.

Description

Complex Medicines for Improving Respiratory Inflammation Caused Fine Dust {Complex Medicines for Improving Respiratory Inflammation Caused Fine Dust}

The present invention relates to a combination preparation for improving respiratory tract inflammation induced by fine dust, and more particularly, by administering a complex extract composed of Gilgyeong, Licorice, Geumeunhwa, and Sangbaekpi to mice induced with fine dust, thereby improving respiratory tract inflammation. It is an invention that can improve the quality of life.

Fine dust is a substance suspended in the atmosphere with a variety of complex components, and most of it is generated from the exhaust gas of automobiles and dust generated during road driving. The size and chemical composition of the particles determine health effects, and exposure to fine dust is associated with the incidence of respiratory and cardiovascular diseases, and has been reported to increase mortality. In particular, small dust particles with a size of 10 μm or less pose a great threat because they can enter the lungs and blood.

Particles with a diameter smaller than 10 μm and larger than 2.5 μm are called fine dust, and are mainly generated at roadsides and industrial complexes. Particles with a diameter of 2.5㎛ or less are called ultrafine dust, and are produced when cigarette smoke or fuel is burned. The components of the particles play an important role in the toxicity of the human body, and they are mainly composed of combustion particles such as carbon, organic hydrocarbons, nitrates, sulfates, and harmful metal components. They are very small in size and can reach the alveoli deep in the airways via the nose and airways, and the smaller they can pass directly through the alveoli and allow systemic circulation through the blood.

When looking at the symptoms of the body due to fine dust, during acute exposure, cough and breathing difficulties occur due to irritation of the airways, asthma worsens, and arrhythmias occur. Chronic exposure can reduce lung function, increase chronic bronchitis, and increase mortality. In particular, people with heart or lung disease, children, the elderly, and pregnant women are more affected by exposure to fine dust, and even healthy adults experience these symptoms temporarily when exposed to high concentrations.

These symptoms are mainly caused by inflammatory reactions in the bronchioles caused by fine dust. It can also lead to respiratory infections by interfering with the body's defenses that inactivate or remove bacteria from the airways and lungs. The occurrence of cardiovascular disease due to fine dust has been reported to be related to oxidative stress and inflammatory reactions, disorders of the autonomic nervous system and changes in blood coagulation capacity.

First, prevention is the best way to prevent fine dust, but it is better to visit a hospital immediately and receive initial management if symptoms occur due to unavoidable exposure. Second, academic, policy, and diplomatic efforts to reduce air pollution are required.However, when the concentration of fine dust is high, people with respiratory or cardiovascular disease, children, the elderly, and pregnant women should refrain from going out. Since the inhaled fine dust is proportional to the intensity and duration of the activity, it is recommended that healthy adults minimize excessive outdoor activities. Since the concentration of fine dust on the roadside is usually higher, it is better not to exercise on the roadside, wear a yellow dust mask when outdoors, and wash your nose and hands well after going out. Also, if the window is left open, the concentration of fine dust in the room increases due to fine dust introduced from the outside, so the window must be closed. An air filter or air purifier can help. Smoking indoors or lighting candles increases the concentration of fine dust and should be avoided.

As a prior art related to fine dust-related diseases, the following Patent Document 001 discloses “a pharmaceutical composition and health functional food for the prevention and treatment of diseases related to heavy metal toxicity and fine dust containing black bellflower extract as an active ingredient” However, this has an effect of inhibiting human lung cell death caused by cadmium and copper, and is different from determining the effect by direct exposure to fine dust.

In addition, the following Patent Document 002 discloses “a composition for protecting cells and tissues against fine dust toxicity including Lactobacillus casei strain”, but a feed composition that suppresses cell damage due to fine dust toxicity and improves resistance to fine dust toxicity There is a difference to be about.

Accordingly, the present inventors completed the present invention by administering a complex extract composed of gilgyeong, licorice, etc. to mice induced with fine dust to improve respiratory tract inflammation in order to solve the problems in the prior art.

Registered Patent Publication No. 10-1791120, 2017. 10. 23.) Registered Patent Publication No. 10-1912258, 2018. 10. 22.)

The present invention has been invented to solve the above-described problems as described above, and the prevention and treatment of respiratory inflammatory diseases by injecting a complex extract composed of gilgyeong, licorice, gold silver flower, and Sangbaek blood into mice induced with fine dust is a task. .

The present invention provides a combination formulation for improving respiratory inflammatory diseases induced by fine dust, characterized in that it comprises a complex extract of Gilgyeong, Licorice, Geumeunhwa, Sangbaekpi as an active ingredient.

On the other hand, solutions to other specific problems according to the present invention are described in the detailed description of the present invention.

By administering the complex formulation for improving respiratory tract inflammation of fine dust according to the present invention to mice, it synergizes between formulations, resulting in excellent efficacy of sputum excretion and reduction of neutrophils, reduction of production of IL-17, MIP2, CXCL-1, and TNF-α, It can be very useful in improving respiratory tract inflammation induced by fine dust because immune cells decrease in BAL and lungs, lung cell walls and collagen fibers are reduced.

1 is a time table of induction and treatment of a model for inducing fine dust of an experimental substance.
2 is a graph showing the results of a toxicity test for a) 24 hours and b) 48 hours after induction into fine dust in MH-S cells according to the present invention.
Figure 3 is a graph measuring the amount of NO production after induction into fine dust in MH-S cells according to the present invention.
Figure 4 is a graph measuring the amount of cytokine production in the supernatant after induction into fine dust in MH-S cells according to the present invention.
5 is a graph calculated by converting sputum discharge after induction into phenol red in experimental animals.
6 is an optical microscope observation photograph of neutrophil cells of each experimental group in the BAL of the induced mouse model of the fine dust-induced model according to the present invention. This is a graph measuring the number of neutrophils in alveolar lavage fluid.
7 is a graph measuring the amount of chemokine produced in the supernatant of the alveolar lavage fluid of the induced mouse model of the fine dust-inducing model according to the present invention.
8 is a photograph and graph of a biopsy analysis through H&E and MT staining of the lungs of the mouse model induced by the microdust-induced model according to the present invention.
9 is a graph showing changes in the absolute cell count of a) BAL and b) lungs of the induced mouse model of the fine dust-induced model according to the present invention.

Hereinafter, a preferred embodiment will be described in detail with respect to the combination preparation for improving respiratory tract inflammation caused by micromerge of the present invention.

The complex formulation for improving respiratory inflammation caused by micromerge according to the present invention is characterized in that it contains a complex extract of Gilgyeong, Licorice, Geumeunhwa and Sangbaekpi as an active ingredient. Each of the components constituting the composite formulation as described above is known to have the following pharmacological effects.

First, Gilkyung (桔梗) dried the roots of Bellflower (Platycodon grandiflorum A. DC.), a perennial herb belonging to the Campanula family. Currently growing or cultivated in Korea, there are white bellflower with white flowers, double bellflower with double flowers, and white double bellflower among them. The main effect of bellflower in oriental medicine is effective for coughing and phlegm. In addition, it is also good for symptoms such as sore throat, pain in the lungs (失音), difficulty urinating (排尿困難), diarrhea, and after-jung (後重), which are closely related to the disorders of the lungs.

Licorice (甘草) is a perennial herb belonging to the legume family, and the roots and stems of European licorice, Manchuria licorice, or other animals and plants have been removed as it is or the pericarp is removed. As a pharmacological action of licorice, adrenal cortical hormone action, anti-inflammatory activity and anti-allergic action, action on the digestive system, detoxification action, action on lipid metabolism, effect on experimental jaundice, antitussive action, analgesic and anticonvulsant action, urinary system And action on the genital system, anti-tumor action, other weight gain action, muscle strength enhancement action, blood pressure increase action, hemolysis action, inhibitory action, antibacterial action, antipyretic action, anthelmintic action against insectworm.

The gold and silver flower buds of honeysuckle are the buds of honeysuckle. The flowers contain about 1% of luteolin and inositol. In addition, saponin, tannin, isochlorogenic acid, chlorogenic acid, etc. Contains. This medicine has a peculiar smell, sweet taste, cold nature, and is used when you have a fever, stuffy chest, and thirst. It is good for inflammation. It is effective in discharging boils, poison caused by skin tears, inflammation of organs, and pus. have. It is also used for dysentery, necrosis of skin tissue due to heat poisoning, and mastitis. It can be applied to colitis, gastric ulcer, cystitis, sore throat, tonsillitis, bronchitis, conjunctivitis and swelling, high fever due to mumps, purulent infections, etc. Pharmacological action has been reported as antibacterial action, anti-inflammatory action, antipyretic action, increased leukocyte phagocytosis action, central nervous system excitation action, serum cholesterol lowering, and ulcer prevention effect.

Sangbaekpi (桑白皮) is a medicinal material made from the root bark of a mulberry tree of the Moraceae family or a plant of the genus. The appearance is semi-tubular or band-shaped, sometimes thinly cut vertically. The outer surface is white or yellowish-brown, and the one with the epidermis attached to it is yellowish-brown, easy to fall off, has many fine vertical wrinkles, and has reddish-brown colored skin. The side cut horizontally is white or light brown, fibrous, and the inner side is dark yellowish brown and flat. Sangbaekpi treats seawater and asthma caused by waste heat and has a diuretic effect. It is used for acute pyelitis and frail edema, has a blood pressure lowering effect, and is also used for nosebleeds and hemoptysis. It is also used for epidemic hepatitis. Pharmacological action has been reported as antitussive, diuretic, lowering blood pressure, sedation, analgesic, antipyretic, antispasmodic, and antibacterial action.

The combination formulation for improving respiratory tract inflammation caused by fine dust was tested by varying the mixing ratio of Gilgyeong, Licorice, Geumeunhwa and Sangbaekpi extract in various ways (see Examples 1 to 5), among which 7:3:7: It is most preferable to set it as a weight ratio of 3.

In addition, the complex extract is preferably mixed after extraction using distilled water at a weight ratio of 10 times as a solvent for each of Gilgyeong, Licorice, Geumeunhwa and Sangbaekpi.

The extraction solvent may be water, alcohol, or a mixture thereof, but it is most preferable to use distilled water. As the extraction method, shaking extraction, Soxhlet extraction, etc. can be listed, but reflux extraction is preferable. The extraction temperature is preferably room temperature, the extraction time is preferably for 2 hours, and the number of extractions is preferably twice.

In the above extraction method, it is preferable to use a vacuum concentrator for concentration, and there may be various methods for drying, but it is preferable to perform lyophilization.

In addition, the extract prepared by the reflux extraction is preferably filtered, concentrated under reduced pressure, and stored frozen at -84°C.

In addition, the combined formulation for improving respiratory inflammation caused by fine dust is most preferably a dosage of 400 mg/kg.

The following examples are intended to explain the present invention in more detail. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to the following examples.

<Preparation Example> Preparation of complex extract

Medicinal herbs such as Gilgyeong, Licorice, Geumeunhwa, and Sangbaekpi were used with support from Omnihub Co., Ltd. For Gilkyung, Licorice, Geumeunhwa, and Sangbaekpi, a reflux extractor (EAMS 9502-06, Seoul, Korea ) For 2 hours, each extract obtained by extracting twice was filtered. This was concentrated with a vacuum distillation apparatus (SB-1000, EYELA, Tokyo, Japan), and the completely dried extract was stored frozen (-84° C.) using a freeze dryer (FD 8508, IlShin, Yangju, Korea). Finally, extracts were prepared with yields of Gilgyeong (47.2%), Licorice (7.8%), Geumeunhwa (7.1%), and Sangbaekpi (16.2%) from 50g of each medicine.

<Example>

Each of the extracts prepared in Preparation Example was prepared by varying the mixing ratio (weight ratio) of Gilkyung: Licorice: Geumeunhwa: Sangbaekpi as shown in the following table.

Gilkyung licorice Gold and silver coins Sangbaek blood Remark Example 1 7 3 7 3 GGX Example 2 7 3 6 4 GGT2 Example 3 7 3 4 6 GGT3 Example 4 7 3 5 5 GGT4 Example 5 7 3 3 7 GGT5

<Comparative Example>

As shown in the following table, extracts of Gamgil-tang (Gamgil-tang, Gilgyeong 7: Licorice 3), and Comparatives 2 to 5 were used as formulations of Gilgyeong, Licorice, Geumeunhwa, and Sangbaekpi, respectively.

Gilkyung licorice Gold and silver coins Sangbaek blood Remark Comparative Example 1 14 6 - - GGT Comparative Example 2 20 - - - PR200 Comparative Example 3 - 20 - - LR200 Comparative Example 4 - - 20 - LF200 Comparative Example 5 - - - 20 MRB200

<Experimental Example 1> Cell culture and fine dust treatment

Induced with standard fine dust and diesel exhaust gas complex (PMD) in the lung macrophage (MH-S) cell system and treated the preparations obtained in Examples 1 to 5 and Comparative Examples 1 to 5, and the effect on cell viability through MTT assay Was evaluated.

In addition, the cells were cultured and induced with standard fine dust and diesel exhaust gas complex (PMD), and the preparations of Examples 1 to 5 and Comparative Examples 1 to 5 were treated to measure the production of inflammatory cytokines TNF-α and INF-γ. In addition, the measurement of the amount of NO generation was measured.

<Experimental Example 2> Exploration of sputum discharge efficacy

As experimental animals, 10 ICR mice (male, body weight 30 to 33 g) were used for each group, and the preparations of Examples 1 to 5 and Comparative Examples 1 to 5 by concentration or a comparative drug, Ambroxol (250 Mg/kg, ambroxol) was orally administered, and 5% phenol red was injected intraperitoneally after 30 minutes. After 30 minutes, cervical vertebrae were dislocated to bleed the abdominal aorta, and then the entire trachea was excised, placed in physiological saline, centrifuged at 3,000 rpm for 10 minutes, and 1N NaOH was added to the supernatant, and the absorbance was measured at 546 nm to measure phenol red. Expectant activity was measured as the concentration of. The sputum discharge effect was converted and calculated based on the phenol red secretion rate of the negative control group.

<Experimental Example 3> Respiratory damage fine dust production

By applying a mixture of standardized fine dust (PM10-like, ERM-CZ120) and diesel combustion dust (DEP) to an animal model, 3 mg/ml of respiratory damage fine dust (PM10-like, ERM-CZ120) and diesel combustion dust It was prepared by diluting 0.6 mg/ml in 1% alum.

<Experimental Example 4> Fine dust-induced lung damage model

In 7-week-old BALB/c male mice, 3 mg/ml of respiratory damage fine dust (PM10-like, ERM-CZ120) and 0.6 mg/ml of diesel-burning dust were diluted in 1% alum to Intra-

Using the Nazal-Tracheal (INT) injection method, we made an animal model that directly injects into the lungs three times (3 days after drug administration, 6 days, and 9 days after administration of the drug) at intervals of 3 days and once a week for 3 weeks. A total of 100 µl each was aspirated through the nose and mouth to induce fine dust. The aspiration method was administered intraperitoneally with 7% chloral hydrate (C8383, Sigma, USA) intraperitoneally to give anesthesia a little and fix the incisors of the mice without movement with a rubber band. It is sacrificed 3 days after the final fine dust (PMD) injection.

The experimental group was (i) the normal group without any treatment (Normal), (ii) the control group treated with the inducer (PMD_CT), (iii) the positive control group administered dexamethasone (3 mg/kg, po ) after treatment with the inducer. (PMD_Dexa_3), (iv) Comparative Example 1 extract (200 mg/kg, po ) administration group (GGT-200) after treatment with the inducer, (v) Example 1 extract (100 mg/kg) after treatment with the inducer po ) Administration group (PMD_GGX_100), (vi) Example 1 extract (200 mg/kg po ) administration group (PMD_GGX_200) after treatment with inducing substances, (vii) Example 1 extract (400 mg/kg) after treatment with inducer po ) Administration group (PMD_GGX_400, (viii) after treatment with the inducer was used as an extract of Example 1 (500 mg/kg po ) administration group (PMD_GGX_500). Each experimental group to which the drug was administered was orally administered daily for 12 days, and the experiment was carried out daily. After completion, bronchoalveolar lavage fluid (BALF) and lung tissues of mice in each group were separated.

<Experimental Example 5> Autopsy and inspection plan

After completion of the experiment, the experimental animals were anesthetized with ethyl ether, and blood and bronchoalveolar lavage fluid (BAL) were obtained. Immune cells were identified in the BAL and lungs using a flow cytometer, and IL-17, TNF-α, MIP2, and CXCL-1 were identified in BALF. In addition, after centrifugation of BAL cells, the increase of neutrophils was confirmed with Diff-Quik Stain. The lungs were separated and lung biopsy (H&E, M-T stain) was confirmed.

1) Separation of bronchoalveolar lavage fluid

After blood was collected on the last day of the experiment, a syringe containing 1 ml of FBS free DMEM medium was inserted into the trachea by exposing the airways by opening the chest. After fixing with a string, circulating the bronchoalveolar lavage three times to ). After centrifuging the bronchoalveolar lavage solution at 4° C., 1,750 rpm for 5 minutes, the supernatant was stored frozen to measure cell centrifugation, and the cells isolated from BALF were treated with ACK solution for 3 minutes to dissolve red blood cells, After washing again with 1% FBS free DMEM culture solution, the total number of cells was measured using a hemocytometer.

2) Bronchoalveolar lavage fluid (BALF) separation and total neutrophil cell count measurement

Blood was collected, dissected, and then centrifuged for cell counting of neutrophils in BALF. The precipitated blood cells were separated, stained with 0.04% trypan blue, and the total number of cells was measured. Diff-Quik staining (Romanowsky stain) was treated three times, then washed twice with PBS, and then 9 slides per group were prepared and counted through an optical microscope (Nikon, Japan) at 400x magnification.

3) Lung cell isolation and total cell count measurement

Lungs were removed from mice that did not separate the alveolar lavage fluid (BALF), and the lung tissue was finely sectioned, and then added to 3 ml DMEM medium containing no fetal calf serum (FBS), and 1 mg/ml collagenase IV (C5138). , Sigma) was added and the tissue was digested at least 4 times for 30 minutes in a shaking incubator at 37°C to separate lung cells. The separated lung cells were washed with a medium and then passed through a cell filter (352350, FALCON) to remove non-degraded tissues or impurities other than the cells. From these cells, ACK solution (10-548E, LONZA) was treated at 37°C for 5 minutes to lyse red blood cells, washed again with culture solution, and stained with 0.04% trypan blue (15250061, Invitrogen), and then total number of lung cells Was measured.

4) Immunofluorescent cell staining in bronchoalveolar lavage solution

The isolated BAL and lung cells were adjusted to ⁵ ×10 ⁵ cells and then subjected to immunofluorescence staining at 4°C. PE-anti-CD4e (553047, BD Pharmingen, San Diego, CA), FITC-anti-CD8 (553031, BD Pharmingen), PE-anti-Gr-1, (553128, BD Pharmingen), FITC-anti- respectively. CD69, (55732, BD Pharmingen), FITC-anti-CD11b (553310, BD Pharmingen) were added and reacted at 4° C. for 30 minutes. After the reaction, washing with phosphate buffered saline at least 3 times, using the Cell Quest program (643274, BD Biosciences, San Diego, CA) of a flow cytometer, CD8+CD4+, CD8+CD69+, CD69+CD4+ and Gr-1+Neutrophil+ , Gr-1 + CD11b + cell frequency was analyzed as a percentage (%) and then analyzed in each tissue by applying the total number of cells.

5) Enzyme Immunoassay (ELISA)

ELISA was performed to measure the amount of IL-17, TNF-α, MIP2, and CXCL-1 in BALF. The capture antibody was mixed in the coating buffer and added to each well at a rate of 100 μl, overnight at 4° C., and washed 4 times with a washing buffer. 200 µl of assay diluent was added per well, blocking at room temperature for 1 hour, and washed 4 times with a washing buffer. Serum and standard of each experimental group were diluted 10 times in an assay diluent solution, and then 100 μl each was added to a 96 well plate coated with each capture antibody, followed by reaction at room temperature for 2 hours. Wash three times with a washing buffer, treat 100µl of biotin-conjugate antibody reagent to each well, react at room temperature for 1 hour, wash twice, and treat 100µl of streptavidine-HRP solution to each well After reacting at room temperature for 1 hour, it was washed twice with a washing buffer. 100 µl of the substrate solution was treated and reacted for 20 minutes, and the reaction was terminated with 50 µl of stop solution, and the absorbance was measured at 450 nm.

6) Histopathology examination

In order to observe the degree of pathological damage to the lung tissue, the lung tissue was cut, fixed in 10% neutral buffered formalin (NBF) for 24 hours, dehydrated with graded alcohol, embedded in paraffin, and a block was made, and then 4㎛ with a tissue sectioner. Thick tissue sections were prepared and stained with hematoxylin & eosin (H&E). To perform H&E staining, slides were immersed in hematoxylin for 1 minute, washed several times in distilled water, immersed in eosin for 30 seconds, and washed several times with flowing distilled water. Then, after washing several times in 70% → 95% → 100% ethanol to remove the dyeing properly, it was soaked in xylene for 1 minute. Finally, permanently attach the cover-slide using the mounting medium xylene. And Masson-Trichrome staining, which is collagen deposition staining, was performed. These samples were observed through an optical microscope at 400 magnification (Nikon, Japan).

7) Statistics processing

Results obtained from various experiments were recorded as mean±standard error of mean, and significance verification was determined using Student's T-test analysis method. The data were analyzed using a one-way ANOVA to determine the statistically significant variance between each group for the individual final points measured, and the statistical significance between each group was a nonpara metric Mann- Whitney test) and Dunnett's multiple comparison test (IBM SPSS statistics version 19.0 statistic software, Inc, IBM, USA) were used, and results (expressed as results, mean) ± standard error of mean The displayed results were expressed as statistical significance in P values: <0.05 (*), <0.01 (**), or <0.001 (***).

<Experiment result>

1) Cell survival was observed through the MTT assay after processing fine dust through the MH-S cell line, which is a macrophage in the lung, and as a result, it was found that the cell survival rate was not affected by all preparations (Figure 2).

2) As a result of observing the amount of NO production in MH-S cells stimulated with fine dust, Example 1 compared to the control group, the extracts of Gilkyung, Licorice, Geumeunhwa and Sangbaekpi respectively, and Examples 2 to 5 of Comparative Examples 2 to 5 It was found that this further inhibited NO generation (Fig. 3).

3) As a result of observing changes in IFN-r and TNF-α, known as inflammatory cytokines in MH-S cells stimulated with fine dust, extracts of the control group, Gilkyung, Licorice, Geumeunhwa and Sangbaekpi respectively, Compared to the formulations of Examples 2 to 5 and Comparative Example 1, Example 1 was found to significantly inhibit IFN-r and TNF-α production (Fig. 4).

4) As a result of examining the effect in the expectorant model, the secretion rate of phenol red in the positive control group (Ambroxol) and the treatment group by concentration in Examples 1 to 5 based on the control group treated with physiological saline was used as the sputum discharge effect As a result of conversion and calculation, a significant effect of sputum discharge was observed in the 400 mg/kg treatment group of Example 1 (Fig. 5).

5) The difference from the positive control group was examined by checking the number of neutrophils through the BAL cell cytospin and Diff-Qick staining of each experimental group and example in the fine dust induction mouse model. In the PMD_control group in which PMD was injected into mice by INT method, the number of neutrophils was significantly increased, indicating that the level of inflammation in the lungs increased. In addition, the number of neutrophils decreased in the group administered with dexamethasone (3 mg/kg) as a positive control group, and the number of neutrophils in the experimental group of Gamgil-tang, the main component of Geumeunhwa, and the main component of Gilgyeong decreased. Could be expected to decrease. In particular, in the experimental group of Example 1, there was a concentration-dependent decrease trend. At an administration concentration of 400 mg/kg, the Example 1 administration group was found to significantly reduce the number of neutrophils than the Comparative Example 1 administration group (Fig. 6).

6) Production of chemokines IL-17, MIP2, GRO-a(CXCL-1), and TNF-α were measured through ELISA in BALF isolated from normal, control, positive control, and experimental mice. In the BALF of the PMD_control group in which PMD was injected into mice by the INT method, all the production of inflammation-related chemokines increased. In the positive control group administered with dexamethasone, the production of IL-17, TNF-α, MIP2 and CXCL-1 decreased statistically at a statistically significant level. In the comparative example and the example 1 administration group, it could be confirmed that the production amount decreased at a statistically significant level. At an administration concentration of 400 mg/kg, the Example 1 administration group was found to significantly reduce CXCL-1, MIP2 and TNF-α expressions than the Comparative Example 1 administration group (Fig. 7).

7) Part of the lung tissue isolated from the animals injected with PMD in mice by the INT method was fixed in 10% formaldehyde solution, followed by H&E and M-T staining. Compared to the normal group, it was confirmed that the lung cell wall was thickened through the H&E staining method of the PMD_control group, and the collagen fibers were increased through the M-T staining method. In the positive control group, the dexamethasone administration group, the comparative example, and the Example 1 administration group, it was confirmed that thinner lung cell walls and collagen fibers were decreased compared to the PMD_control group. At an administration concentration of 400 mg/kg, the Example 1 administration group showed greater inhibition of lung tissue damage than the Comparative Example 1 administration group (Fig. 8).

8) In order to investigate the inhibition of the increase in inflammation-related cells, the total number of cells in BAL and lung tissue was observed. As a result, it was confirmed that the number of BAL and total lung cells increased in the respiratory disease mouse group induced by fine dust, and the increased BAL and total number of lung cells decreased by concentration in the mouse group administered orally administered Example 1. At an administration concentration of 400 mg/kg, the Example 1 administration group significantly reduced BALF and total lung cell count, and the Comparative Example 1 administration group also slightly decreased, but there was no significance (Fig. 9).

The present invention has been described in more detail by examples and experimental examples. However, the above embodiments are merely exemplary to describe the present invention in more detail, and thereby the nature of the technical idea of the present invention is not changed or the scope thereof is not reduced. It is obvious to those skilled in the art that various embodiments and application examples not presented in the above embodiments are possible, so that the true technical protection scope of the present invention is determined by the following claims.

Claims (6)

A complex formulation for improving respiratory tract inflammation of fine dust, characterized in that it contains a complex extract of Gilgyeong, Licorice, Geumeunhwa and Sangbaekpi as active ingredients.
The method of claim 1,
The complex extract is a combination formulation for improving respiratory tract inflammation of fine dust, characterized in that gilgyeong, licorice, gold silver flower and Sangbaekpi are mixed in a weight ratio of 7:3:7:3.
The method of claim 2,
The complex extract is an extract obtained by using distilled water at a weight ratio of 10 times for each of the gilgyeong, licorice, gold silver flower and sangbaekpi.
The method of claim 3,
The extract is a combination formulation for improving respiratory inflammation of fine dust, characterized in that the reflux extraction twice for 2 hours at room temperature. The method of claim 4,
The extract obtained by the reflux extraction is filtered, concentrated under reduced pressure, and stored frozen at -84°C.
The method of claim 5,
The complex extract is a complex formulation for improving respiratory tract inflammation, characterized in that 400 mg / kg administered.

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