KR102507548B1 - Composition comprising Panax ginseng extract and Perilla frutescens leaves extract for preventing, ameliorating or treating of respiratory diseases caused by fine dust - Google Patents

Abstract

The present invention relates to a composition for preventing, improving or treating respiratory diseases caused by fine dust comprising a composite extract, which is a mixture of ginseng extract and sesame leaf extract, as an active ingredient, including ginsenoside of ginseng extract and rosemary acid of sesame leaf extract. Reduction of reactive oxygen species (ROS) production, reduction of inflammatory cytokines, reduction of inflammatory active cells, and reduction of airway inflammation and endothelial dysfunction-related biomarkers and cough-related gene expression in models caused by exposure to fine dust due to active ingredients, etc. As the effect was confirmed, it can be usefully used to prevent and improve respiratory diseases caused by fine dust.

Description

Composition comprising Panax ginseng extract and Perilla frutescens leaves 　extract for preventing, ameliorating or treating respiratory diseases caused by fine dust}

The present invention relates to a composition for preventing, improving or treating respiratory diseases caused by fine dust containing ginseng extract and sesame leaf extract as active ingredients.

According to the “2017 Health Statistics” of the Organization for Economic Cooperation and Development (OECD), the death rate of respiratory diseases per 100,000 people in Korea was 70 in 2013, up 2.5 from 67.5 in 2010, and deterioration of the atmospheric environment such as fine dust and ozone is expected to have an effect. being analyzed

Fine dust is particulate matter less than 10 μm in diameter (PM10) among total suspended particles in the air. When fine dust is inhaled, it is deposited in the lower bronchial tubes and lung parenchyma, causing damage to the respiratory system and causing damage to existing diseases. worsens symptoms and increases morbidity and mortality.

According to a report on the effect of fine dust concentration in Seoul on outpatient visits, hospitalization and medical expenses for respiratory and cardiovascular systems (Journal of the Korean Society for Environmental Health 2016), whenever the concentration of fine dust increases, bronchitis, asthma, chronic obstructive pulmonary disease (COPD), It has been shown that the number of patients with angina pectoris is increasing. In addition, asthma is a chronic inflammatory airway disease, and morbidity and mortality caused by it not only cause direct suffering to patients, but also emerge as a serious social and economic problem. % prevalence is a problem in all ages, from infants to the elderly.

In addition, fine dust causes or exacerbates asthma by various mechanisms, such as directly depositing in the airways and alveoli of asthmatic patients, exacerbating airway inflammation and inducing bronchoconstriction. COPD is the 4th leading cause of death worldwide and the 7th leading cause of death in Korea.

As such, an increase in fine dust shows a significant relationship with a decrease in lung function, and an increase in pulmonary macrophages, neutrophils, and lymphocytes is observed in bronchoalveolar lavage fluid after exposure to fine dust, and an increase in neutrophils in lung tissue and tracheal tissue It is known to increase the expression of lymphocytes, mast cells, and IL-8 mRNA, but research on drug treatment for acute exacerbations caused by fine dust is very lacking. It is becoming. However, there is no clear countermeasure against the occurrence of respiratory diseases caused by fine dust.

Accordingly, the inventors of the present invention, while studying natural materials in relation to respiratory diseases caused by fine dust, discovered that a composite extract, which is a mixture of ginseng extract and sesame leaf extract, exhibits a respiratory protection effect due to fine dust, and completed the present invention I did.

An object of the present invention is to provide a composition for preventing, improving or treating respiratory diseases caused by fine dust, comprising a composite extract of a mixture of ginseng extract and sesame leaf extract as an active ingredient.

A specific object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of respiratory diseases caused by fine dust comprising a composite extract of a mixture of ginseng extract and sesame leaf extract as an active ingredient.

Another object of the present invention is to provide a health functional food for preventing or improving respiratory diseases caused by fine dust containing a composite extract of a mixture of ginseng extract and sesame leaf extract as an active ingredient.

In order to achieve the above object, the present invention

In the pharmaceutical composition for preventing or treating respiratory diseases caused by fine dust comprising a composite extract as an active ingredient,

The composite extract provides a pharmaceutical composition for preventing or treating respiratory diseases caused by fine dust, which is a mixture of ginseng extract and sesame leaf extract.

In one example of the present invention, the composite extract is 10 to 90% by weight of ginseng extract based on the total weight of the composite extract; And it may be one containing 10 to 90% by weight of sesame leaf extract.

In one example of the present invention, the ginseng extract may contain a mixture of ginsenosides Rg1 and Rb1.

In one example of the present invention, the mixture of the ginsenosides Rg1 and Rb1 may be included in an amount of 1 to 3% by weight based on the total weight of the ginseng extract.

In one example of the present invention, the sesame leaf extract may contain rosmary acid.

In one example of the present invention, the rosmary acid may contain 1 to 10% by weight of the total weight of the sesame leaf extract.

In one example of the present invention, the complex extract

1) extracting ginseng and sesame leaves with alcohol to prepare ginseng extract and sesame leaf extract, respectively;

2) vacuum concentrating the ginseng extract and sesame leaf extract, respectively; and

3) drying each step after step 2); may be one obtained by including.

In one example of the present invention, the sesame leaf extract may further include a sesame seed extract.

In one embodiment of the present invention, the respiratory disease caused by fine dust is respiratory inflammatory lung disease, chronic obstructive pulmonary disease (COPD), sinusitis, allergic rhinitis, lower respiratory tract infection, acute and chronic bronchitis, pneumonia, bronchial asthma , bronchiectasis, emphysema, pulmonary tuberculosis sequelae, acute respiratory distress syndrome, cough, otitis media, sore throat, tonsillitis, laryngitis, and pulmonary fibrosis.

The present invention is a health functional food for preventing or improving respiratory diseases caused by fine dust containing a complex extract as an active ingredient,

The composite extract provides a health functional food for preventing or improving respiratory diseases caused by fine dust, which is a mixture of ginseng extract and sesame leaf extract.

The composition for preventing, improving, or treating respiratory diseases caused by fine dust containing the composite extract, which is a mixture of ginseng extract and sesame leaf extract according to the present invention as an active ingredient, is a natural material, and is safe for respiratory diseases caused by fine dust without exhibiting toxicity. The disease can be prevented, ameliorated or treated.

More specifically, the composition for preventing, improving or treating respiratory diseases caused by fine dust containing the composite extract, which is a mixture of ginseng extract and sesame leaf extract according to the present invention as an active ingredient, in in vitro assay and in vivo assay, fine dust The effect of reducing the expression of physiologically active substances such as reactive oxygen species (ROS), inflammatory cytokines, and inflammatory cells, biomarkers related to airway inflammation and endothelial dysfunction, and cough-related gene expression was confirmed. It can be usefully used to prevent, improve or treat respiratory diseases caused by dust.

1 shows the results of confirming the ginseng indicator substance of the ginseng extract according to Preparation Example 1-2.
Figure 2 shows the results of confirming the indicator substance of the sesame leaf extract according to Preparation Example 2-1.
Figure 3 shows the results of analyzing the amount of reactive oxygen species (ROS) produced by flow cytometry (FACS) after treating the macrophage cell line of the respiratory tract with the complex extracts according to Examples 1 to 9 of the present invention.
Figure 4 shows the results of confirming the amount of ROS production after treating the macrophage cell line of the respiratory tract with the composite extract according to Examples 2 and 3 of the present invention.
Figure 5A is an analysis of the production of inflammatory cytokines (IL-6 and TNF-a) in the culture medium of the cells after treatment with the macrophage cell line of the respiratory tract stimulated by fine dust with the composite extract according to Examples 1 to 9 of the present invention. which shows the results for Figure 5B is the results of the analysis of the production of inflammatory cytokines (IL-6 and TNF-a) in the cells after treating the macrophage cell line of the respiratory tract stimulated with fine dust with the composite extract according to Examples 2 and 3 of the present invention. is shown.
Figure 6 is lung lavage fluid (BALF), lung (lung), spleen (spleen) and neutrophil cell counts in animal model efficacy evaluation for exposure to fine dust for the composite extract according to Example 3 of the present invention analyze that showed the result. Figure 6A shows the result of analyzing the number of BALF cells, Figure 6B lung cells, Figure 6C spleen, and Figure 6D neutrophil cells.
Figure 7 shows the results of analyzing inflammatory cytokines in bronchial cell lavage fluid through ELISA assay in animal model efficacy evaluation for exposure to fine dust for the composite extract according to Example 3 of the present invention. FIG. 7A shows the analysis results for CXCL1, FIG. 7B for IL-17, FIG. 7C for MIP-2, and FIG. 7D for TNF-a.
Figure 8 is an indicator of airway inflammation and endothelial dysfunction in chronic obstructive pulmonary disease in the animal model efficacy evaluation for exposure to fine dust for the composite extract according to Example 3 of the present invention, SDMA (Symmetric-dimethylarginine) and cough (cough) ) shows the results of analyzing the expression of related genes. Figure 8A shows the mRNA expression analysis results of SDMA, Figure 8B, MUC5AC, Figure 8C, TRPV1, Figure 8D, TRPA1.
Figure 9 shows the results of analyzing the inflammatory cytokine gene expression by qRT-PCR in the animal model efficacy evaluation for exposure to fine dust for the composite extract according to Example 3 of the present invention. FIG. 9A is TNF-a, FIG. 9B is TARC, FIG. 9C is COX-2, FIG. 9D is NOS-II, FIG. 9E is MIP-2, and FIG. 9F is CXCL-1 mRNA expression analysis results.
Figure 10 analyzes the expression of IRAK1 and CD11b proteins, which are allergic asthma signaling pathways, by immunofluorescence in the animal model efficacy evaluation for exposure to fine dust for the composite extract according to Example 3 of the present invention. that showed a result. Figure 10A shows the results of immunofluorescence image analysis, Figure 10B shows the results of image analysis for IRAK1 protein as a graph, and Figure 10C shows the results of image analysis for CD11b protein as a graph.
Figure 11 shows the protein expression of TNF-α and MCP-1, which are mediators related to the pathogenesis of lung diseases caused by fine dust, in the animal model efficacy evaluation for exposure to fine dust for the composite extract according to Example 3 of the present invention. It shows the result of analysis by fluorescence method. FIG. 11A shows the results of immunofluorescence image analysis for TNF-α protein expression, and FIG. 11B shows a graph of the image analysis results for TNF-α protein. 11C shows the results of immunofluorescence image analysis for MCP-1 protein expression, and FIG. 11D shows a graph of the image analysis results for MCP-1 protein.
Figure 12 shows the results of lung tissue analysis through staining (H&E, MT, PAS staining assay) method in animal model efficacy evaluation for exposure to fine dust for the composite extract according to Example 3 of the present invention will be.
Figure 13 shows the results of trachea tissue test analysis through the abPAS staining assay method in the animal model efficacy evaluation for exposure to fine dust for the composite extract according to Example 3 of the present invention.

The present invention will be described in detail below.

Hereinafter, the present invention will be described in more detail through manufacturing examples, examples or test examples including the accompanying drawings. However, the following preparation examples, examples, or test examples are only one reference for explaining the present invention in detail, but the present invention is not limited thereto, and may be implemented in various forms.

Also, unless defined otherwise, all technical and scientific terms have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Terms used in the description in the present invention are merely to effectively describe specific embodiments and are not intended to limit the present invention.

Also, the singular forms used in the specification and appended claims may be intended to include the plural forms as well, unless the context dictates otherwise.

In addition, singular forms of terms used in the present invention may be interpreted as including plural forms unless otherwise indicated.

In addition, in the present invention, the unit of % used unambiguously without particular notice means weight %.

In the present invention, physiologically active substances generated by fine dust mean inflammatory cytokines, inflammatory cells, reactive oxygen species, etc., and mean physiological or pharmacological forms stimulated by fine dust particles or atmospheric particles. do it too

Also, in the present invention, ### means that the physiologically active substances generated by fine dust are significantly increased by fine dust or atmospheric particulates with p<0.01, reliability 99% compared to the normal group.

In addition, * means that the physiologically active substances generated by the fine dust were significantly reduced by the test group, compared to the control group, p <0.05, reliability 95%, with a significance level of 5%. In addition, ** means that the physiologically active substances produced by fine dust were significantly reduced by the test group, compared to the control group, p <0.01, with a reliability of 99%. In addition, *** means that the physiologically active substances generated by fine dust were significantly reduced by the test group compared to the control group with p<0.001 and a reliability of 99.9%.

The present invention is a pharmaceutical composition for preventing or treating respiratory diseases caused by fine dust comprising a composite extract as an active ingredient, wherein the composite extract is a mixture of ginseng extract and sesame leaf extract, a pharmaceutical composition for preventing or treating respiratory diseases caused by fine dust provides

In the present invention, 'composite extract' means a mixture of ginseng extract and sesame leaf extract, which are natural products, and includes all forms formulated using the complex extract. Specifically, it is in powder form. When the composite extract is in powder form or powder form, effects such as reduction of reactive oxygen species (ROS) generation, reduction of inflammatory cytokines, reduction of inflammatory active cells, etc. may be excellent in relation to respiratory diseases caused by fine dust.

As used herein, the term 'prevention' means any action that suppresses or delays the onset of respiratory diseases caused by fine dust by administering the composition of the present invention.

As used herein, the term 'treatment' refers to all activities that improve or beneficially change the symptoms of respiratory diseases caused by fine dust by administration of the composition of the present invention.

The composite extract is 10 to 90% by weight of ginseng extract; And sesame leaf extract may be included in 10 to 90% by weight. Preferably 20 to 50% by weight of ginseng extract; And it may contain 50 to 80% by weight of the sesame leaf extract. In the above content range, the effect of reducing the inflammatory activity of respiratory diseases caused by fine dust may be more excellent.

The ginseng is a perennial plant belonging to the genus Panax, and includes, but is not limited to, Korean ginseng (Panax ginseng), Hwagi ginseng (Panax quinquefolia), Jeonchisam (Samchil, Panax notoginseng), Panax japonicus, and Himalayan ginseng (Panaxa pseudoginseng). ). Panax vietnamensis, Panax elegatior, Panax wangianus, Panax bipinratifidus, Panax angustifolium, etc. are preferred. It may be Korean ginseng.

The Korean ginseng can be divided into fresh ginseng, red ginseng, and white ginseng according to processing, and fresh ginseng means a state in which it is dug up from the ground and not dried. Red ginseng refers to light brown ginseng obtained by selecting 6-year-old fresh ginseng, steaming it in an unpeeled state, and drying it so that the moisture content is less than 14%. White ginseng refers to fresh ginseng with its skin intact, or fresh ginseng peeled and heated to remove moisture.

According to the processing type and method, white ginseng can be divided into main ginseng and non-ginseng. This ginseng refers to the parts of the head, body and legs except for the semi-finished parts or the original form of ginseng is maintained as it is in the manufacturing standards of ginseng.

In addition, misam means the leg part and semi-separated from the head and body in this ginseng, or means a product made with the legs or fine roots separated from the body.

The ginseng extract may contain a mixture of ginsenoside Rg1, which is an indicator substance for promoting DNA and RNA synthesis and immune activity, and ginsenoside Rb1, which is an indicator substance that promotes RNA synthesis and serum protein synthesis. The mixture of ginsenosides Rg1 and Rb1 may be included in an amount of 1 to 3% by weight based on the total weight of the ginseng extract. When the content conditions of the mixture of ginsenosides Rg1 and Rb1 are satisfied, the effect of improving respiratory diseases caused by fine dust may be more excellent.

The perilla leaves refer to the leaves of perilla, and the perilla ( Perilla frutescens var japonica) is an annual plant of the dicotyledonous plant, Lamiaceae, belonging to the clown family, and an annual herbaceous plant in Southeast Asia such as Korea, China, and India. It is widely cultivated. The perilla is a crop that is usually cultivated to squeeze oil.

More specifically, the perilla leaves and perilla leaves can be divided into sesame seeds and sesame leaves. Sesame sprouts mean young shoots, young leaves or young leaves, and sesame leaves mean large leaves or old leaves. Usually, sesame seeds are used as herbs or salads, and sesame leaves are used for wraps and jjangajji.

Perillaaldehyde, rosmary acid, anthocyanin, caffeic acid, etc. have been reported as active ingredients of perilla seeds, and these ingredients have high water solubility, so a high utilization rate can be expected when ingested.

The sesame leaf extract contains rosmary acid, and the rosmary acid may contain 1 to 10% by weight of the total weight of the sesame leaf extract. When the content conditions of rosmary acid are satisfied, in relation to respiratory diseases caused by fine dust, effects such as reduction of inflammatory cytokines, reduction of white blood cell count, reduction of airway inflammation and inflammatory cells may be excellent.

The composite extract, which is a mixture of the ginseng extract and the sesame leaf extract, according to the content of Rg1, Rb1 and rosmary acid in the composite extract, reduces inflammation caused by fine dust, reduces alveolar cell destruction rate, etc. Physiological and pharmacological effects caused by fine dust Enemy effects can be excellent.

The composite extract is prepared by 1) extracting ginseng and sesame leaves with alcohol to prepare ginseng extract and sesame leaf extract, respectively; 2) vacuum concentrating the ginseng extract and sesame leaf extract, respectively; and 3) drying each step after step 2).

More specifically, the ginseng extract among the composite extracts includes the steps of: i) extracting ginseng with alcohol to prepare a ginseng extract; ii) vacuum-concentrating the ginseng extract; and iii) drying after step ii).

In the step i), the ginseng may be white ginseng, and the white ginseng may be white ginseng, which is a mixture of main ginseng and fine ginseng.

In the step ii), vacuum concentration may be performed at 40 to 80 ° C to 3 to 30 brix, but is not limited thereto.

Drying in step iii) may be performed by spray drying or vacuum drying.

In the case of the spray drying, it may be carried out at 70 to 120 ° C, preferably at 90 to 120 ° C, and at 800 to 1800 rpm.

In addition, the vacuum drying may be performed using a vacuum dryer, and may be performed at a pressure of about 200 to 500 mmHg and 40 to 80 ° C, but is not limited thereto.

In addition, the sesame leaf extract of the complex extract is prepared by a) extracting sesame leaf with alcohol to prepare a sesame leaf extract; b) vacuum concentrating the sesame leaf extract; and c) drying after step b).

In step a), washing and drying the sesame leaves at 30 to 60 ° C. for 8 to 48 hours may be further included. In step a), a process of extracting 1 to 5 times at 40 to 80 ° C. for 4 to 10 hours may be further included.

In step b), vacuum concentration may be performed at 40 to 80° C. to 3 to 30 brix, but is not limited thereto.

Between steps b) and c), polysaccharide may be additionally added as a food additive. The polysaccharide may be dextrin, maltodextrin, cyclodextrin, or indigestible dextrin, but is not particularly limited, but maltodextrin is preferred, 10 to 90% (w / w), preferably 10 to 50% (w / w) /w) can be further added, and in the case of the above conditions, the storability of the sesame leaf extract can be increased regardless of the storage temperature such as refrigeration or room temperature. In addition, the content of rosmary acid can be kept constant regardless of temperature.

Drying in step c) may be performed by spray drying, vacuum drying or freeze drying.

The spray drying may be performed at 70 to 120 ° C. at 800 to 1800 rpm, but is not limited thereto.

In addition, the vacuum drying may be performed using a vacuum dryer, and may be performed at a pressure of about 200 to 500 mmHg and 40 to 80 ° C, but is not limited thereto.

In addition, the lyophilization may be performed at -40 to -20 ° C, but is not limited thereto.

Preferably, the sesame leaf extract may further include a sesame seed extract. As the sesame seed extract is further included, the disease improvement effect may be excellent when the sesame leaf extract is used in combination with the ginseng extract. The sesame seed extract may be obtained by: a') preparing a sesame seed extract by extracting the sesame seeds in hot water or a water-alcohol mixed solvent; b') vacuum-concentrating the sesame seed extract; and c') spray drying after step b').

In step a'), the weight ratio of the water-alcohol mixed solvent may be 5:5 to 8:2, and may be extracted at a temperature range of 60 to 80 °C. Both the hot water extraction and the water-alcohol mixed solvent extraction method can achieve the pharmacological effect improvement effect, but the hot water extraction method may be more preferable for improving the pharmacological effect.

The vacuum concentration in step b') and the spray drying in step c') are the same as steps b) and c) of the sesame leaf extract.

The sesame seed extract may be included in 5 to 10% by weight of the total weight of the composition, specifically, the sesame leaf extract and the sesame seed extract may be mixed and included in a weight ratio of 100:5 to 100:25, more specifically 100:10 to 100: It may be mixed and included in a weight ratio of 20.

Respiratory diseases caused by fine dust include respiratory inflammatory lung disease, chronic obstructive pulmonary disease (COPD), sinusitis, allergic rhinitis, lower respiratory tract infections, acute and chronic bronchitis, pneumonia, bronchial asthma, bronchiectasis, emphysema, and pulmonary tuberculosis. It may be a disease selected from the group consisting of aftereffects, acute respiratory distress syndrome, cough, otitis media, sore throat, tonsillitis, laryngitis, and pulmonary fibrosis, but any respiratory disease caused by fine dust is possible.

The pharmaceutical composition of the present invention may be prepared using pharmaceutically suitable and physiologically acceptable adjuvants in addition to the above active ingredients, and the adjuvants include excipients, disintegrants, sweeteners, binders, coating agents, expanding agents, lubricants, and lubricants. agents or flavoring agents may be used.

The pharmaceutical composition may be preferably formulated as a pharmaceutical composition by including one or more pharmaceutically acceptable carriers in addition to the above-described active ingredients for administration.

The pharmaceutical dosage form of the composition according to the present invention may be used in the form of a pharmaceutically acceptable salt thereof, and may also be used alone or in combination with other pharmaceutically active compounds as well as in a suitable set.

Formulations of the pharmaceutical composition may be granules, powders, tablets, coated tablets, capsules, suppositories, solutions, syrups, juices, suspensions, emulsions, drops, or injectable solutions. For example, for formulation in the form of tablets or capsules, the active ingredient may be combined with an oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. In addition, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included in the complex extract 1. Suitable binders include, but are not limited to, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tracacanth or sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.

In compositions formulated as liquid solutions, acceptable pharmaceutical carriers are sterile and biocompatible, and include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added if necessary. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to prepare formulations for injections such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, or tablets.

Furthermore, using a method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA as an appropriate method in the field, it can be preferably formulated according to each disease or component.

The pharmaceutical composition of the present invention can be administered orally or parenterally, and in the case of parenteral administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, etc. can be administered.

The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, administration method, patient's age, weight, sex, medical condition, food, administration time, administration route, excretion rate and reaction sensitivity, and is preferred. The dosage varies depending on the condition and body weight of the patient, the severity of the disease, the drug form, the route and duration of administration, but can be appropriately selected by those skilled in the art.

However, for desirable effects, the composite extract of the present invention is preferably administered at 0.0001 to 100 mg/kg per day, preferably at 0.001 to 100 mg/kg. Most preferably, it is administered at 50 to 100 mg/kg. Administration may be administered once a day, or may be administered in several divided doses. The dosage is not intended to limit the scope of the present invention in any way.

The composition of the present invention can be administered to mammals such as rats, mice, livestock, and humans through various routes. All modes of administration are contemplated, eg oral, rectal or intravenous, intramuscular or subcutaneous injection.

The pharmaceutical composition of the present invention may be prepared in a unit dose form by formulation using a pharmaceutically acceptable carrier or excipient, or prepared by placing it in a multi-dose container. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet or capsule, and may additionally contain a dispersing agent or stabilizer.

The present invention is a health functional food for preventing or improving respiratory diseases caused by fine dust containing a composite extract as an active ingredient, wherein the composite extract is a mixture of ginseng extract and sesame leaf extract, preventing or improving respiratory diseases caused by fine dust Provide health functional food for improvement.

In the present invention, 'health functional food' refers to a food manufactured and processed by using a specific ingredient as a raw material or by extracting, concentrating, refining, mixing, etc. a specific ingredient contained in a food raw material for the purpose of supplementing health. It refers to food designed and processed to sufficiently exert biological control functions such as biological defense, regulation of biological rhythm, prevention and recovery of disease, etc. function can be performed.

The health functional food is a food effective for supplying nutrients to organisms such as humans, non-human animals, and plants or improving biological functions, and can be prepared according to a method commonly used in the field. Health functional foods may include various food additives such as sweeteners, flavoring agents, emulsifiers, seasonings, vitamins, minerals, amino acids, preservatives, antioxidants, and coloring agents. The form of the health functional food is not particularly limited, and preferably, various forms such as beverages, pills, sweets, chewing gum, bread, animal feed, and plant nutrients can be freely selected.

Hereinafter, the present invention will be described in detail through preparation examples, examples and test examples. However, these examples are intended to illustrate the present invention, and the scope of the present invention is not to be construed as being limited by these examples, and various changes or modifications can be made to those skilled in the art within the spirit and scope of the present invention. It is self-evident.

Preparation Example 1: Preparation of ginseng extract (Panax ginseng) by drying method

Preparation Example 1-1: Preparation of ginseng extract by vacuum drying method

2kg of white ginseng, a mixture of 1.4kg of main ginseng and 0.6kg of fine ginseng, was first extracted at 70°C for 6 hours by adding 70% alcohol of 10 times the amount of white ginseng, adding 70% of 5 times the amount of white ginseng, and then heating it to 70°C. The second and third extractions were performed for 6 hours. Then, the extract was concentrated in vacuo to 20brix at 60~70℃. Then, vacuum drying was performed at a pressure of 200 to 500 mmHg and 40 to 80° C. using a vacuum dryer to obtain 7.19 to 8.97 g of ginseng extract in powder form.

Preparation Example 1-2: Preparation of ginseng extract (Panax ginseng) by spray drying method

2kg of white ginseng, a mixture of 1.4kg of main ginseng and 0.6kg of fine ginseng, was first extracted at 70°C for 6 hours by adding 70% alcohol of 10 times the amount of white ginseng, adding 70% of 5 times the amount of white ginseng, and then heating it to 70°C. The second and third extractions were performed for 6 hours. Then, the extract was concentrated in vacuo to 20brix at 60~70℃. Then, spray drying was performed at 110° C. and 1280 rpm to obtain 214±36 g of ginseng extract in powder form.

Among the methods for producing ginseng extract by the vacuum drying method of Preparation Example 1-1 and the spray drying method of Preparation Example 1-2, the yield of the ginseng extract was further confirmed in the manufacturing method by the spray drying method of Preparation Example 1-2. In the present invention, the effect was confirmed by using the ginseng extract by the spray drying method of Preparation Example 1-2 as a composite extract. In addition, in the present invention, the powdery ginseng extract obtained from the ginseng extract by the spray drying method of Preparation Example 1-2 was named PG.

The powdery ginseng indicator substance of the ginseng extract by the spray drying method of Preparation Example 1-2 was confirmed using LC, and the results are shown in FIG. 1. From Figure 1, Rg1 and Rb1 peaks were confirmed as active ingredients.

As a result of measuring the contents of Rg1 and Rb1 in PG obtained from the ginseng extract by the spray drying method of Preparation Example 1-2, PG contained a mixture of Rg1 and Rb1 at 29.6 mg/g.

Preparation Example 2: Sesame leaf extract by drying method (Perilla frutesens) Manufacture

Preparation Example 2-1: Sesame leaf extract by spray drying method (Perilla frutesens) Manufacture

The sesame leaves were washed three times in clear water, and then 30.8 g of sesame leaves dried for 24 hours at 50 ° C in a far-infrared dryer was mixed with 70% alcohol of 10 times the amount of dried sesame leaves, and extracted three times for 6 hours at 70 ° C. Extraction was performed. Then, the extracted extract was filtered through a 300 mesh filter cloth, and then vacuum concentrated to 20 brix at 60 to 70 °C. Then, 20% (w / w) of dextrin was added and spray-dried at 110 ° C. at 1280 rpm to obtain a powdered sesame leaf extract (yield 3.08%).

Preparation Example 2-2: Sesame leaf extract by vacuum drying method (Perilla frutesens) Manufacture

The sesame leaves were washed three times in clear water, and then 30.8 g of sesame leaves dried for 24 hours at 50 ° C in a far-infrared dryer was mixed with 70% alcohol of 10 times the amount of dried sesame leaves, and extracted three times at 70 ° C for 6 hours. Then, the extracted extract was filtered through a 300 mesh filter cloth, and then vacuum concentrated to 20 brix at 60 to 70 °C. Then, it was vacuum dried at a pressure of 200 to 500 mmHg and 40 to 80 ° C. using a vacuum dryer to obtain a powdered sesame leaf extract.

Preparation Example 2-3: Sesame leaf extract by freeze-drying method (Perilla frutesens) Manufacture

The sesame leaves were washed three times in clear water, and then 30.8 g of sesame leaves dried for 24 hours at 50 ° C in a far-infrared dryer was mixed with 70% alcohol of 10 times the amount of dried sesame leaves, and extracted three times for 6 hours at 70 ° C. Extraction was performed. Then, the extracted extract was filtered through a 300 mesh filter cloth, and then vacuum concentrated to 20 brix at 60 to 70 °C. Then, freeze-drying was performed at -40 to -20 ° C using a freeze dryer to obtain a powdered sesame leaf extract.

Among the methods for preparing sesame leaf extract by the spray drying method of Preparation Example 2-1, the vacuum drying method of Preparation Example 2-2 and the freeze-drying method of Preparation Example 2-3, the rosemary acid content according to the storage temperature of the extract was measured at both refrigeration and room temperature. As a result of confirmation, the rosemary acid content of the sesame leaf extract obtained by the spray drying method of 2-1 is higher than that of the sesame leaf extract obtained by the vacuum drying method of Preparation Example 2-2 and the freeze-drying method of 2-3. It was confirmed that it was maintained continuously for more than 5 days within 53.55 (mg / g) at room temperature, and in the present invention, the powdery sesame leaf extract obtained from the sesame leaf extract by the spray drying method of Preparation Example 2-1 was used as a composite extract. confirmed.

The powdery sesame leaf extract obtained from the sesame leaf extract by the spray drying method of Preparation Example 2-1 was named PF. As a result of measuring the rosmary acid content of the obtained PF, the PF contained 53.55 mg/g of rosmary acid. In addition, as a result of confirming the active ingredient of the sesame leaf extract by the spray drying method of Preparation Example 2-1 by LC, it was confirmed that the peak of rosmary acid appeared as shown in FIG.

Preparation Example 3: Preparation of Perilla frutesens Young leaves (PFY)

500g of sesame seeds were washed and dried 3 times at 50°C for 24 hours, then extracted 3 times at 70°C for 6 hours using a water-alcohol mixed solvent with a weight ratio of 6:4, followed by 20brix at 60-70°C. concentrated in vacuo. Then, spray drying was performed at 110° C. and 1280 rpm to obtain an extract powder of sesame seeds.

Example 1: Preparation of Complex Extract 1 (PG:PF=1:9, referred to as 'CHJ1')

Complex extract 1 was prepared by mixing 10% by weight of the ginseng extract (PG) of Preparation Example 1-2 and 90% by weight of the sesame leaf extract (PF) of Preparation Example 2-1. In vitro assay, it was named CHJ1.

Example 2: Preparation of Complex Extract 2 (PG:PF=2:8, referred to as 'CHJ2')

20% by weight of the ginseng extract according to Preparation Example 1-2; And by mixing with 80% by weight of the sesame leaf extract according to Preparation Example 2-1, composite extract 2 was prepared. It was named CHJ2 in in vitro assay.

Example 3: Preparation of Complex Extract 3 (PG:PF=3:7, referred to as 'CHJ3')

30% by weight of the ginseng extract according to Preparation Example 1-2; And by mixing with 70% by weight of the sesame leaf extract according to Preparation Example 2-1, composite extract 3 was prepared. In vitro assay, it was named CHJ3.

Example 4: Preparation of Complex Extract 4 (PG:PF=4:6, referred to as 'CHJ4')

40% by weight of the ginseng extract according to Preparation Example 1-2; And by mixing with 60% by weight of the sesame leaf extract according to Preparation Example 2-1, composite extract 4 was prepared. In vitro assay, it was named CHJ4.

Example 5: Preparation of Complex Extract 5 (PG:PF=5:5, referred to as 'CHJ5')

50% by weight of the ginseng extract according to Preparation Example 1-2; And by mixing with 50% by weight of the sesame leaf extract according to Preparation Example 2-1, composite extract 5 was prepared. In vitro assay, it was named CHJ5.

Example 6: Preparation of Complex Extract 6 (PG:PF=6:4, referred to as 'CHJ6')

60% by weight of the ginseng extract according to Preparation Example 1-2; And by mixing with 40% by weight of the sesame leaf extract according to Preparation Example 2-1, composite extract 6 was prepared. In vitro assay, it was named CHJ6.

Example 7: Preparation of Complex Extract 7 (PG:PF=7:3, referred to as 'CHJ7')

70% by weight of the ginseng extract according to Preparation Example 1-2; And by mixing with 30% by weight of the sesame leaf extract according to Preparation Example 2-1, a composite extract 7 was prepared. In vitro assay, it was named CHJ7.

Example 8: Preparation of Complex Extract 8 (PG:PF=8:2, referred to as 'CHJ8')

80% by weight of the ginseng extract according to Preparation Example 1-2; And by mixing with 20% by weight of the sesame leaf extract according to Preparation Example 2-1, composite extract 8 was prepared. In vitro assay, it was named CHJ8.

Example 9: Preparation of Complex Extract 9 (PG:PF=9:1, referred to as 'CHJ9')

10% by weight of the ginseng extract according to Preparation Example 1-2; And by mixing with 90% by weight of the sesame leaf extract according to Preparation Example 2-1, composite extract 9 was prepared. In vitro assay, it was named CHJ9.

Example 10: Preparation of Complex Extract 10 (PG: PF+PFY=3:7)

The sesame leaf extract (PF) of Preparation Example 2-1 and the sesame seed extract (PFY) of Preparation Example 3 were mixed at a weight ratio of 100:15 to prepare a mixture of sesame leaf extract and sesame seed extract (PF + PFY). Complex extract 10 was prepared by mixing 30% by weight of the ginseng extract (PG) of Preparation Example 1-2 and 70% by weight of a mixture of the sesame leaf extract and sesame seed extract (PF+PFY).

Test Example 1: Confirmation of the effect of the composite extract of PG + PF ( In vitro assay)

Alveolar macrophage (MH-S) cells, a macrophage cell line of the respiratory tract, were stimulated with fine dust, and the effect was confirmed by treating the composite extracts 1 to 9 of PG + PF of Examples 1 to 9, respectively. At this time, the ginseng extract (PG) of Preparation Example 1-2 was used as Comparative Example 1, and the perilla leaf extract (PF) of Preparation Example 2 was used as Comparative Example 2-1.

PG:PF weight ratio PG:PF weight ratio complex extract
(PG+PF) CHJ1
(Example 1) 1:9 CHJ6
(Example 6) 6:4 CHJ2
(Example 2) 2:8 CHJ7
(Example 7) 7:3 CHJ3 (Example 3) 3:7 CHJ8
(Example 8) 8:2 CHJ4 (Example 4) 4:6 CHJ9
(Example 9) 9:1 CHJ5 (Example 5) 5:5 - - single extract PG
(Comparative Example 1) 1:0 PF
(Comparative Example 2) 0:1

ROS, which is a reactive oxygen species, was analyzed by flow cytometry (FACS), and inflammatory cytokines, IL-6 and TNF-a, were measured by ELISA assay. The fine dust is an atmospheric particulate matter, and PM10 (ERM®-CZ120), a European standard substance, was put in 50% DMSO to a concentration of 50 μg/ml, and then cells were treated and used. The MH-S cells were received from ATCC, and 10% fetal bovine serum (Gibco BRL, Gaithersburg, MD, USA) and 1% antibiotics (Gibco BRL, Gaithersburg, MD, USA) were added to RPMI 1640 medium (Gibco BRL, Gaithersburg, MD, USA), which is a cell culture medium. BRL, Gaithersburg, MD, USA) was added and cultured. In addition, it was cultured in a 5% CO2 incubator maintaining constant temperature (37 ℃) and constant humidity. After subculture 2 to 4 times, it was divided into 2 × 10 ⁴ cells per well in a 12-well plate and cultured for 24 hours. Then, PM10 50ug/ml was treated in each well except for the normal group. Then, the ginseng extract powder of Comparative Example 1, the sesame leaf extract powder of Comparative Example 2, and each composite extract powder of Examples 1 to 9 were all 1 g, and then mixed in 50% DMSO to prepare 1 g / 10 ml did Then, by serial dilution, the final concentrations of PG in Comparative Example 1 and PF in Comparative Example 2 were 400 ug/ml and 200 ug/ml. The PG+PG composite extracts of Examples 1 to 9 were 400ug/ml, 200ug/ml and 100ug/ml. It was then filtered and treated in each well. In the test to confirm the amount of ROS production, the effect was confirmed by first treating the cells with the complex extracts of PG+PF of Examples 1 to 9 at a concentration of 100 ug/ml, and then Example 2 and Example 3, which had good effects, were treated with 400 ug /ml and 200ug/ml, and then treated the cells to confirm the effect on ROS. In addition, in the test for analyzing the production of inflammatory cytokines, the cells were first treated with the complex extracts of PG + PF of Examples 1 to 9 at concentrations of 100 ug and 200 ug / ml, and then confirmed in the culture medium, followed by Example 2 and After making Example 3 400ug/ml and 200ug/ml, the cells were treated to measure the production of inflammatory cytokines in the cells.

Reactive oxygen species ROS of Test Example 1-1 were measured by the DCFH-DA method, which can analyze intracellular ROS, and as described above, the test groups, PG of Comparative Example 1, PF of Comparative Example 2, and Example 1 The composite extracts of PG+PF from 9 to 9 were treated as described above, and then cultured in a 5% CO2 incubator for 24 hours. Then, each test group was treated with DCFH-DA for 30 minutes, and then cells were harvested and measured by flow cytometry.

In the analysis of inflammatory cytokine production in Test Example 1-2 below, the cells of MH-S were subcultured 2 to 4 times, and then divided into 2 × 10 ⁴ cells per well in a 12 well plate and cultured for 24 hours. Then, 50ug/ml of PM10 was treated in each well except for the normal group, and then PG of Comparative Example 1, PF of Comparative Example 2, and PG+PF of Examples 1 to 9 were added to the wells treated with PM10. The composite extracts of were treated as described above. Then, after culturing for 24 hours, MH-S cells and culture medium were recovered, and inflammatory cytokine production was measured by ELISA assay.

The test group treated only with PM10 was named as a control group, PM10-like_CTL.

Test Example 1-1: Confirmation of ROS generation amount

As shown in the results of Figure 3, when the MH-S cell line was treated with the composite extract of PG + PF of Examples 1 to 9 at 100ug / ml, CHJ2 (PG: PF = 2: 8) of Example 2 and It was confirmed that ROS was reduced in CHJ3 (PG: PF = 3: 7) of Example 3 and the two test groups, CHJ5 (PG: PF = 5: 5) of Example 5 and CHJ9 (PG: PF = 5: 5) of Example 9. PF=9 : 1), it was confirmed that ROS was slightly decreased in both test groups.

From the results of the PG + PF complex extracts of Examples 1 to 9, CHJ2 of Example 2 (PG: PF = 2: 8) CHJ3 of Example 3 (PG: PF = 3: 7) Results of the PG + PF complex extract was the best, and as shown in FIG. 4, the ROS reduction effect was reconfirmed for each concentration of 400 ug/ml and 200 ug/ml.

In the results of FIG. 4, except for the normal group, the MH-S cell line, which is a mouse lung macrophage, was treated with 50ug/ml of PM10, and then treated with PG of Comparative Example 1 and PF of Comparative Example 2 at 400ug/ml, respectively. In one test group, it was confirmed that reactive oxygen species (ROS) were reduced by 63% or more statistically significant compared to the control group (PM10-like_CTL).

In addition, from the results of FIG. 4 , it was confirmed that both CHJ2 (PG: PF = 2: 8) of Example 2 and CHJ3 (PG: PF = 3: 7) of Example 3 showed ROS reduction effects. In particular, at a concentration of 200 ug/ml of CHJ3 (PG: PF = 3: 7) of Example 3, ROS is reduced more than 200 ug / ml of CHJ2 (PG: PF = 2: 8) of Example 2 It was confirmed that the ROS reduction effect was more excellent than PG 200ug/ml of Comparative Example 1 and PF 200ug/ml of Comparative Example 2.

Test Example 1-2: Analysis of inflammatory cytokine production

First, as shown in Figure 5A, when the MH-S cell line was treated with 100ug / ml and 200ug / ml of the composite extract of PG + PF of Examples 1 to 9, and the inflammatory cytokines in the culture medium were measured, CHJ2 of Example 2 (PG: PF = 2: 8) and 200ug/ml of CHJ3 (PG: PF = 3: 7) of Example 3 showed excellent inflammatory cytokine inhibitory effects in both test groups. It was confirmed that the CHJ9 (PG : PF = 9 : 1) test group of Example 9 also showed an inhibitory effect on inflammatory cytokines. In the results of the PG + PF composite extracts of Examples 1 to 9, the inhibitory effect of inflammatory cytokines of Examples 2 and 3 was the most excellent, as shown in FIG. 5B, ROS reduction by concentration of 400ug / ml and 200ug / ml The effect was reconfirmed.

In the results of FIG. 5B, in the test group treated with 400 ug/ml of PG of Comparative Example 1 and PF of Comparative Example 2, respectively, the production of IL-6 and TNF-a decreased statistically significantly compared to the control group (PM10-like_CTL). did

In addition, in the results of FIG. 5B, among the test groups treated with the PG+PF composite extracts of Examples 1 to 9 at a concentration of 400 μg/ml, the CHJ2 (PG: PF = 2: 8) of Example 2 and Example 3 It was confirmed that the inflammatory cytokine inhibitory effect was excellent in CHJ3 (PG : PF = 3 : 7).

In particular, in the test group treated with 200ug/ml of CHJ3 (PG: PF = 3: 7) of Example 3, the IL It was confirmed that the production of -6 and TNF-a was further reduced (FIG. 5).

In this way, through the in vitro assay, the animal model efficacy was evaluated with CHJ3 (PG: PF = 3: 7) of Example 3, which had the most excellent effect.

Test Example 2: Evaluation of respiratory protection improvement animal model efficacy against exposure to fine dust (PM10 + DEP) for a composite extract of PG + PF ( In vivo assay)

The effect of improving respiratory protection against exposure to fine dust for CHJ3 (PG : PF = 3 : 7) of Example 3 was confirmed using non-clinical evaluation, animal model evaluation.

By applying standard fine dust (PM10) and DEP (diesel combustion dust) to 7-week-old Balb/c mice, an animal model with respiratory damage was constructed and fabricated, and it was used to evaluate the efficacy of the animal model for improving respiratory protection against fine dust exposure. The animal model constructed above was named an animal model for exposure to fine dust (PM10+DEP).

A 4 mg/ml fine dust mixture (PM10D+DEP), which is a mixture of fine dust (PM10) and diesel exhaust particles (DEP), is diluted in aluminum hydroxide (Alum), and the airway and lung injection method (Intra- Nasal-tracheal (INT) injection) was used to create an animal model that is directly injected into the lungs through the airway by INT injection method three times at 3-day intervals (3 days after drug administration, 6 days after, and 9 days after drug administration), and finally The fine dust mixture (PM10D+DEP) was analyzed 3 days after injection.

In addition, PG of Comparative Example 1 and PF of Comparative Example 2 were prepared at a concentration of 100 mg/kg, respectively, and these were used as test groups.

In addition, CHJ3 (PG : PF = 3 : 7) of Example 3 was prepared at concentrations of 200 mg/kg and 100 mg/kg, and dexamethasone (3 mg/kg) as a positive control group was prepared as a test group.

In addition, in the animal model efficacy evaluation, each test group was prepared in 2 to 3 g, diluted with sterilized phosphate buffered saline (PBS) to have a concentration of 200 mg/kg and 100 mg/kg, and filtered to animal model It was used for efficacy evaluation.

It was administered orally at 11 am every day for 11 days, and the effect of improving respiratory protection against exposure to fine dust was confirmed using an animal model. After the animal model was anesthetized on the last day of 11 days, blood was collected from the heart and collected in a vial containing heparin, and lung lavage fluid, lung tissue, etc. were collected and analyzed.

In the animal model efficacy evaluation, the normal group (Balb/c_Nr), the positive control group (Dexamethasone, 3 mg/kg, PM10D_Dexa 3mg/kg), and the test group treated with only the fine dust mixture as the control group (PM10D_CTL), after fine dust treatment, The test group to which PG of Comparative Example 1 was orally administered was (PM10D_PG), the test group to which PF of Comparative Example 2 was orally administered after fine dust treatment was (PM10D_PF), and the CHJ3 of Example 3 (PG: The test group administered with PF=3 : 7) was named (PM10D_PG+PF). In addition, n = 6 animal model subjects were tested for each test group, and it was confirmed that the animal model did not show any abnormality for 11 days.

Test Example 2-1: Analysis of lung lavage fluid (BALF) and lung cell count in animal model efficacy evaluation for fine dust exposure to PG + PF composite extract

It is known that when exposed to fine dust or chemicals, the number of spleen cells, lung cells, lung lavage fluid cells, and neutrophil cells increase due to splenomegaly. In this regard, the effect of Example 3 on CHJ3 (PG : PF = 3 : 7) was confirmed.

In the results of FIG. 6A, after the end of the animal model experiment for exposure to fine dust (PM10 + DEP), BALF (lung lavage fluid) total cell number was analyzed, and the control group (PM10D_CTL) was significantly higher than the normal group (Balb / c_Nr). It was confirmed that the BAL cell number increased (p<0.001).

Test group (200 mg/kg, p<0.01, 150 mg/kg, p<0.01) orally administered with the positive control group (Dexamethasone, 3 mg/kg) and CHJ3 (PG: PF=3: 7) of Example 3 , significantly decreased compared to the control group (PM10D_CTL).

In addition, CHJ3 (PG of Example 3: PF = 3 : 7) was confirmed that a synergistic effect of decreasing concentration-dependently appeared more in the test group orally administered (Fig. 6A).

From the results of FIG. 6B, it was confirmed that the total number of lung cells in the lung tissue (Lung) increased more than twice as significantly in the control group (PM10D_CTL) compared to the normal group (Balb/c_Nr) (p<0.001). ).

Positive control group (Dexamethasone, 3 mg/kg, p<0.01) and Example 3 CHJ3 (PG: PF=3: 7) orally administered test group (200 mg/kg, 100 mg/kg, p<0.001) was significantly decreased compared to the control group (PM10D_CTL), and the PG of Comparative Example 1 and the PF of Comparative Example 2 (100 mg/kg, p<0.001) were each orally administered. It was confirmed that more appeared (Fig. 6B).

In the results of FIG. 6C, it was confirmed that the control group (PM10D_CTL) showed a significant increase in the number of spleen cells by more than 3 times compared to the total cell number in the spleen tissue (Balb/c_Nr) (p<0.001). .

Test group (200 mg/kg p<0.001, 100 mg/kg, p <0.01), significantly decreased compared to the control group (PM10D_CTL).

In addition, CHJ3 of Example 3 (PG: PF= 3: 7) was confirmed to further exhibit the effect of reducing the number of cells in spleen tissue in the test group orally administered.

The result of FIG. 6D is the result of analyzing the number of neutrophils by cytospin, which can be stained and examined by concentrating cells in the body fluid on a microscope slide using centrifugal force in lung lavage fluid (BALF). . From the results of FIG. 6D, it was confirmed that the control group (PM10D_CTL) showed a significant increase in neutrophil cells compared to the normal group (Balb/c_Nr) (p<0.001).

In addition, the positive control group (Dexamethasone, 3 mg/kg, p<0.001) and the test group orally administered CHJ3 (PG: PF=3: 7) of Example 3 (200 mg/kg, p<0.001, 100 mg/kg , p<0.001), it was significantly decreased in a concentration-dependent manner compared to the control group (PM10D_CTL), and PG of Comparative Example 1 (100 mg/kg, p<0.001) and PF of Comparative Example 2 (100 mg/kg, p< 0.001) was confirmed to show a more concentration-dependent reduction effect than oral administration (FIG. 6D).

Test Example 2-2: Analysis of inflammatory cytokines in bronchopulmonary lavage fluid (BALF) by ELISA assay in animal model efficacy evaluation for fine dust exposure to PG + PF composite extract

Exposure to fine dust or chemicals increases inflammatory cytokines in lung lavage fluid. In this regard, the effect of Example 3 on CHJ3 (PG : PF = 3 : 7) was confirmed.

After the end of the animal model experiment on exposure to fine dust (PM10 + DEP), inflammatory cytokines in lung lavage fluid (BALF) were measured and analyzed by ELISA assay. It was confirmed that the production of inflammatory cytokines increased significantly (p<0.001).

7A shows CXC chemokine (CXCL1, chemokine (C-X-C motif) ligand 1), FIG. 7B shows interleukin 17 (IL-17, Interleukin-17), and FIG. 7C shows macrophage inflammatory protein-2 (MIP-2, macrophage inflammatory protein). , Figure 7D shows the results of analyzing tumor necrosis factor-α (TNF-a).

The positive control group (Dexamethasone, 3 mg/kg) and the test group orally administered with CHJ3 (PG: PF=3: 7) of Example 3 at a concentration of 200 mg/kg showed significant inflammatory cytokines compared to the control group (PM10D_CTL). It was confirmed as shown in FIG. 7 that the concentration-dependent decrease was observed (p<0.001).

In particular, in the test group orally administered CHJ3 (PG: PF = 3: 7) of Example 3 at a concentration of 100 mg / kg, inflammatory cytokines were significantly reduced compared to the control group (PM10D_CTL), as shown by MIP2 in FIG. 7C and It was confirmed in TNF-a in FIG. 7D (p<0.001).

In addition, CHJ3 (PG : PF = 3 : 7) of Example 3 was orally administered than the test group in which PG (100 mg/kg) of Comparative Example 1 and PF (100 mg/kg) of Comparative Example 2 were orally administered, respectively. In the test groups (200 mg/kg, 100 mg/kg), it was confirmed that a synergistic effect of significantly reducing inflammatory cytokines was exhibited.

Experimental Example 2-3: Airway inflammation and endothelial dysfunction biomarker (SDMA) and cough-related gene analysis in animal model efficacy evaluation for fine dust exposure to PG + PF composite extract

In relation to diseases caused by fine dust, the effect of CHJ3 (PG: PF = 3: 7) in Example 3 in relation to airway inflammatory endothelial dysfunction or cough was confirmed by SDMA contained in serum, and the SDMA was analyzed by ELISA assay analyzed. In addition, the effect was confirmed by gene expression analysis of cough-related genes.

In association with fine dust-related cough, chronic intractable cough, or chronic idiopathic cough, MUC5AC, TRPV1, and TRPA1 expression are upregulated. In this regard, the effect of Example 3 on CHJ3 (PG : PF = 3 : 7) was confirmed.

8A is SDMA (Symmetric-dimethylarginine), FIG. 8B is MUC5AC (Mucin 5AC), FIG. 8C is TRPV1 (Transient receptor potential vanilloid 1), and FIG. 8D is TRPA1 (Transient Receptor Potential Cation Channel Subfamily A Member 1) mRNA expression. It shows the result of analysis by real time PCR.

Symmetric-dimethylarginine (SDMA), a biomarker of airway inflammation and endothelial dysfunction in chronic obstructive pulmonary disease caused by fine dust, in the blood serum of animal models after the end of animal model experiments on exposure to fine dust (PM10 + DEP) ), as a result of measuring and analyzing by ELISA assay, it was confirmed that the control group (PM10D_CTL) showed a significant increase in the level of SDMA by about 32% or more (p <0.001) as shown in FIG. 8A compared to the normal group (Balb / c_Nr). .

The positive control group (Dexamethasone, 3 mg/kg, p<0.05) and the test group orally administered CHJ3 (PG: PF=3: 7) of Example 3 at a concentration of 200 mg/kg were significantly higher than the control group (PM10D_CTL). The decrease was confirmed as shown in FIG. 8A (p<0.05).

In addition, 200 mg/kg of CHJ3 (PG: PF = 3: 7) of Example 3 was higher than the test group in which PG (100 mg/kg) of Comparative Example 1 and PF (100 mg/kg) of Comparative Example 2 were orally administered, respectively. The effect of significantly decreasing in the test group orally administered at a kg concentration was confirmed as shown in FIG. 8A.

In addition, as a result of measuring and analyzing the expression of bronchial inflammation and cough related genes in lung tissue, the control group (PM10D_CTL) showed higher mRNA expression of cough related genes (MUC5AC, TRPV1, TRPA1) compared to the normal group (Balb/c_Nr). Significant increase was confirmed in the results of FIGS. 8B, 8C and 8D (p<0.01).

In addition, the positive control group (Dexamethasone, 3 mg/kg) and the test group orally administered with CHJ3 (PG: PF=3: 7) of Example 3 at a concentration of 200 mg/kg were significantly more concentration-dependent than the control group (PM10D_CTL). It was confirmed that cough-related gene expression was reduced (FIGS. 8B, 8C, and 8D).

In addition, as shown in the results of FIG. 8, CHJ3 (PG: PF = 3 : 7) was orally administered (200 mg/kg, 100 mg/kg), and it was confirmed that cough-related gene expression was further reduced.

Experimental Example 2-4: Inflammatory cytokine gene expression (qRT-PCR) analysis in animal model efficacy evaluation for fine dust exposure for PG + PF composite extract

After the end of the animal model experiment for exposure to fine dust (PM10 + DEP), inflammatory cytokine gene expression in lung tissue was measured and analyzed as shown in FIG. 9 using qRT-PCR (Quantitative real time PCR, qRT-PCR). FIG. 9A is TNF-a, FIG. 9B is TARC, FIG. 9C is COX-2, FIG. 9D is NOS-II, FIG. 9E is MIP-2, and FIG. 9F is CXCL-1 mRNA expression.

Compared to the normal group (Balb/c_Nr), it was confirmed that the control group (PM10D_CTL) showed a significant increase in inflammatory cytokine gene expression more than twice.

In the positive control group (Dexamethasone, 3 mg/kg) and the test group (200 mg/kg, 100 mg/kg) orally administered CHJ3 (PG: PF=3: 7) of Example 3, compared to the control group (PM10D_CTL), inflammatory It was confirmed that the gene expressions of cytokines, TNF-a, TARC, COX-2, NOS-II, MIP-2 and CXCL-1, were significantly decreased in a concentration-dependent manner.

In addition, in the test group orally administered with PF (100 mg/kg) of Comparative Example 2, as shown in the results of NOS-II in FIG. 9D and CXCL-1 in FIG. 9F, a significant decrease was observed compared to the control group (PM10D_CTL). Confirmed.

In addition, in the test group orally administered with PG (100 mg/kg) of Comparative Example 1, it was confirmed that COX-2 gene expression was significantly reduced compared to the control group (PM10D_CTL), as shown in FIG. 9C.

Test Example 2-5: Analysis of IRAK1 and CD11b protein expression (Immune histology fluorescent, IHF), which are allergic asthma signaling pathways caused by fine dust in animal model efficacy evaluation for fine dust exposure to PG + PF composite extract

In order to confirm the effect of CHJ3 (PG: PF = 3: 7) of Example 3 on improving asthma prevention by fine dust, the expression of IRAK1 and CD11b proteins, which are allergic asthma signaling pathways, was confirmed using immunofluorescence.

Figure 10A shows the results of immunofluorescence image analysis, Figure 10B shows the results of image analysis for IRAK1 protein as a graph, and Figure 10C shows the results of image analysis for CD11b protein as a graph.

After the end of the animal model experiment for exposure to fine dust (PM10 + DEP), the signal transmission IRAK1 protein expression in lung tissue was measured and analyzed by immunofluorescence tissue staining (IHF, Immune histology fluorescent). As shown in FIG. 10, the normal group (Balb /c_Nr) compared to the control group (PM10D_CTL), it was confirmed that the expression of IRAK1 protein, which is an allergic asthma signal transducer, was markedly increased. The positive control group (Dexamethasone, 3 mg/kg) and the test groups (200 mg/kg, 100 mg/kg) orally administered with CHJ3 (PG: PF=3: 7) of Example 3 had higher concentrations than the control group (PM10D_CTL). It was confirmed that it was significantly reduced dependently (FIG. 10A, FIG. 10B).

In addition, in the test group orally administered with PF (100 mg/kg, p<0.001) of Comparative Example 2 and PG (100 mg/kg, p<0.05) of Comparative Example 1, respectively, IRAK1 protein expression was higher than that of the control group (PM10D_CTL). A significant decrease was confirmed.

In addition, it was confirmed that the CD11b protein expression significantly increased in the control group (PM10D_CTL) compared to the normal group (Balb/c_Nr). The positive control group (Dexamethasone, 3 mg/kg) and the test group (200 mg/kg p<0.05, 100 mg/kg) orally administered with CHJ3 (PG: PF=3: 7) of Example 3 were the control group (PM10D_CTL) It was confirmed as shown in FIGS. 10A and 10C that the concentration-dependently significantly decreased compared to .

In addition, in the test group to which PF (100 mg/kg, p<0.001) of Comparative Example 2 and PG (100 mg/kg, p<0.05) of Comparative Example 1 were orally administered, respectively, CD11b protein expression was higher than that of the control group (PM10D_CTL). However, it was confirmed that there was no significance.

Test Example 2-6: TNF-α and MCP-1 protein expression (IHF) analysis, which are mediators related to the pathogenesis of lung diseases caused by fine dust in animal model efficacy evaluation for fine dust exposure to PG + PF composite extract

Expression of TNF-α and MCP-1 proteins, which are mediators related to the pathogenesis of lung diseases caused by fine dust, in order to confirm the effect of CHJ3 (PG: PF = 3: 7) of Example 3 in relation to lung diseases caused by fine dust The analysis was confirmed using lung tissue of an animal model for exposure to fine dust.

FIG. 11A shows the results of immunofluorescence image analysis for TNF-α protein expression, and FIG. 11B shows a graph of the image analysis results for TNF-α protein. 11C shows the results of immunofluorescence image analysis for MCP-1 protein expression, and FIG. 11D shows a graph of the image analysis results for MCP-1 protein.

After the end of the animal model experiment on exposure to fine dust (PM10 + DEP), immunofluorescence histological staining (IHF, Immune As a result of measurement and analysis by histology fluorescent), as shown in FIG. 11 , it was confirmed that the TNF-α protein expression significantly increased in the control group (PM10D_CTL) compared to the normal group (Balb/c_Nr).

Control group in the positive control group (Dexamethasone, 3 mg/kg) and the test group (200 mg/kg p<0.01, 100 mg/kg p<0.05) orally administered with CHJ3 (PG: PF=3:7) of Example 3 (PM10D_CTL) was confirmed to be significantly reduced in a concentration-dependent manner (Fig. 11A, Fig. 11B).

In addition, it was confirmed that PF (100 mg/kg, p<0.01) of Comparative Example 1 and PG (100 mg/kg) of Comparative Example 2 significantly decreased TNF-α protein expression compared to the control group (PM10D_CTL), and normal Compared to the group (Balb/c_Nr), it was confirmed that MCP-1 protein expression increased markedly in the control group (PM10D_CTL).

Control group (PM10D_CTL) in the positive control group (Dexamethasone, 3 mg/kg) and the test group (200 mg/kg p<0.01, 100 mg/kg) orally administered with CHJ3 (PG: PF=3: 7) of Example 3 It was confirmed as shown in FIGS. 11C and 11D that the concentration-dependently significantly decreased compared to .

In addition, in each test group orally administered with PF (100 mg/kg) of Comparative Example 2 and PG (100 mg/kg) of Comparative Example 1, MCP-1 protein expression was decreased compared to the control group (PM10D_CTL), but the significance was It was confirmed that there was no (FIG. 11D).

Test Example 2-7: FACS analysis of inflammatory cells of lung tissue of animal models in animal model efficacy evaluation for fine dust exposure to PG + PF composite extract

Respiratory diseases caused by fine dust, such as inflammatory lung disease, chronic obstructive pulmonary disease, bronchial asthma, bronchiectasis, and emphysema, are caused by type 1 helper T (Th1) cells and type 2 helper T (Th2) It is caused by cell imbalance, and interferon-γ (INF-γ) is secreted from Th1 cells, stimulating macrophages and cytotoxic T-cells. Th2 cells secrete interleukin (IL)-4, -5, -9, -13, etc., and they cause allergic airway inflammation caused by fine dust.

In this regard, inflammatory active cells such as CD4+ T cells (CD4 ⁺ ), CD8+ T cells (CD8+), CD4 ⁺ /CD69 ⁺ cells, CD62L ⁺ /CD44 ⁺ cells and Gr-1 ⁺ /CD11b ⁺ cells are the fine dust It is an inflammatory active cell related to T cells related to the induction of allergic airway inflammation by the back. In order to confirm the effect of CHJ3 (PG : PF = 3 : 7) of Example 3 on airway inflammation caused by fine dust, the inflammatory active cells were analyzed.

After the end of the animal model experiment for exposure to fine dust (PM10 + DEP), inflammatory cells in lung tissue (Lung) were confirmed using FACS. The results are shown in Table 2 below.

Cell phenotypes in the lung Evaluation of airway inflammation animal models by induction of fine dust
(PM10D-induced airway inflammation murine model (Absolute No.)
Balb/c_
Normal
PM10D_
CTL
PM10D_
Dexa
3 mg/kg
PM10D_PF
100 mg/kg
PM10D_PG
100 mg/kg
PM10D_
PG+PF
100 mg/kg
PM10D_
PG+PF
200 mg/kg
CD4+
(x10 ⁵ cells)
12.11
±1.23
21.23
±0.2###
15.58
±2.29**
20.08
±6.45
20.03
±0.69
15.50
±0.52***
13.27
±1.51***
CD8+
(x10 ⁵ cells)
4.81
±0.09
14.60
±0.41###
8.03
±1.52
12.02
±3.55
9.96
±1.46
8.01
±0.18***
5.98
±0.40***
CD4+/CD69+
(x10 ⁵ cells)
0.41
±0.12
5.16
±0.79###
1.11
±0.29**
3.29
±0.24
2.34
±0.11**
1.00
±0.34***
0.75
±0.09***
CD62L+/CD44+
(x10 ⁵ cells)
6.51
±1.53
17.39
±0.08###
8.24
±1.64**
9.08
±2.66
9.05
±0.07
5.48
±0.40***
3.76
±0.95***
Gr-1+/CD11b+
(x10 ⁵ cells)
6.15
±1.53
29.53
±21.97
7.02
±0.69
3.78
±0.70
7.73 ±
1.51
3.24
±0.32***
2.99
±0.35***

As shown in Table 2, after the end of the animal model experiment for exposure to fine dust (PM10 + DEP), inflammatory cells were confirmed in the lung tissue (Lung), CD4 + T cells (CD4 ⁺ ), CD8 + T cells (CD8 +), The ratios of CD4 ⁺ /CD69 ⁺ cells, CD62L ⁺ /CD44 ⁺ cells and Gr-1 ⁺ /CD11b ⁺ cells in the test group to which CHJ3 (PG : PF = 3 : 7) of Example 3 was orally administered (200 mg/kg and 100 mg/kg) was confirmed to decrease in a concentration dependent manner. In addition, compared to the test group orally administered with PG of Comparative Example 1 and the test group orally administered with PF of Comparative Example 2, the CD4+ T cells in the test group orally administered with CHJ3 (PG : PF = 3 : 7) of Example 3 (CD4 ⁺ ), CD8+ T cells (CD8+), CD4 ⁺ /CD69 ⁺ cells, CD62L ⁺ /CD44 ⁺ cells and Gr-1 ⁺ /CD11b ⁺ cells were further reduced. As such, it was confirmed that the pharmacological effect of CHJ3 (PG : PF = 3 : 7) of Example 3 on airway inflammation caused by fine dust was very superior to that of PG of Comparative Example 1 and PF of Comparative Example 2.

Experimental Example 2-8: Analysis of inflammatory cells included in lung lavage fluid (BALF) by flow cytometry (FACS) in animal model efficacy evaluation for fine dust exposure to PG + PF composite extract

After the end of the animal model experiment on exposure to fine dust (PM10 + DEP), inflammatory cells included in the lung lavage fluid (BALF) were confirmed using FACS. The results are shown in Table 3 below.

Cell phenotypes in lung lavage fluid
Evaluation of airway inflammation animal models by induction of fine dust
(PM10D-induced airway inflammation murine model (Absolute No.) Balb/c_
Normal
PM10D_
CTL
PM10D_
Dexa
3 mg/kg
PM10D_PF
100 mg/kg
PM10D_PG
100 mg/kg
PM10D_
PG+PF
100 mg/kg
PM10D_
PG+PF
200 mg/kg CD4+
(x10 ⁴ cells)
0.91±
0.11
26.54
±3.39###
6.82±
0.03
16.78±
6.62
17.74±
0.02
11.11±
1.16***
9.76±
1.64*** CD8+
(x10 ⁴ cells)
0.36±
0.13
21.02±
4.01###
4.04±
0.87
15.53±
1.14
16.94±
1.23
12.49±
1.03***
10.66±
2.24*** CD4+/CD69+
(x10 ⁴ cells)
0.67±
0.38
9.63±
0.02
3.35
±0.8
4.74±
0.54
4.52±
0.65**
2.81±
0.29***
2.22±
0.69*** CD62L+/CD44+
(x10 ⁴ cells)
0.33±
0.03
49.52±
4.43
28.8
±3.80
29.58±
3.39**
28.48±
0.90**
20.17±
0.28***
18.72±
2.36***
CD11b+/CGr-1+
(x10 ⁴ cells)
1.77±
0.32
74.40 ±
5.15#
43.09
±2.74
27.55 ±
7.2**
29.11±
5.18**
16.51 ±
1.63***
10.78±
5.22***

As shown in Table 3, fine dust (PM10 + DEP) in the waste washing liquid (BALF) at the end of the animal model experiment As a result of confirming the inflammatory cells, the reduction effect of CD4+ T cells (CD4 ⁺ ), CD8+ T cells (CD8+), CD4 ⁺ /CD69 ⁺ cells, CD62L ⁺ /CD44 ⁺ cells and Gr-1 ⁺ /CD11b ⁺ cells was found in Example 3 In the test group (200 mg/kg, 100 mg/kg) orally administered CHJ3 (PG : PF = 3 : 7), it was confirmed that it appeared in a concentration-dependent manner. In addition, compared to the test group orally administered with PG of Comparative Example 1 and the test group orally administered with PF of Comparative Example 2, CD4+ T cells in the test group orally administered with CHJ3 (PG : PF = 3 : 7) of Example 3 (CD4 ⁺ ), CD8+ T cells (CD8+), CD4 ⁺ /CD69 ⁺ cells, CD62L ⁺ /CD44 ⁺ cells and Gr-1 ⁺ /CD11b ⁺ cells were further reduced. As such, compared to the test group orally administered with PG of Comparative Example 1 and PF of Comparative Example 2, the pharmacology of airway inflammation caused by fine dust in the test group orally administered CHJ3 (PG: PF = 3: 7) of Example 3 It was reconfirmed that the effect was excellent.

Test Example 2-9: Lung tissue analysis through staining assay method in animal model efficacy evaluation for fine dust exposure to PG + PF composite extract

In order to histologically analyze the effects of prevention, treatment and improvement on diseases caused by fine dust for CHJ3 (PG: PF = 3: 7) of Example 3 of the present invention, lungs of animal models for exposure to fine dust Tissues were analyzed by hematoxylin & eosin staining (H&E staining), Masson's trichrome staining (M-T staining), and PAS staining.

As a result of hematoxylin and eosin staining (H&E, Hematoxylin and Eosin Staining) of lung tissue after the end of the animal model experiment on exposure to fine dust (PM10+DEP), compared to the control group (Balb/c_Nr) ( PM10D_CTL) showed a marked increase in the deposition of fine dust (PM10+DEP) around the brochia, increased airway thickness, and infiltration of inflammatory immune cells around the airway and granulocytes (neutrophils, eosinophils), and activated macrophages, which intensified inflammation, confirming that alveolar cell destruction was in progress.

The degree of alveolar cell destruction was confirmed by Masson Trichrome (M-T staining) staining to observe collagen deposition. The micro-organs (tracheal) and alveolar (alveolar) of the (structure) part and inflammation (inflammatory) of the cell part and the degree of infiltration of blood cells could be observed.

As a result of PAS staining to confirm mucous secretion such as bronchitis, in the control group (PM10D_CTL), it was confirmed that many goblet cells secreting mucus were distributed around the airway.

From the results of hematoxylin & eosin staining (H & E staining), MT staining and PAS staining, the positive control group (Dexa. 3 mg / kg) and CHJ3 of Example 3 (PG: PF = 3: 7) at a concentration of 200 mg/kg, the deposition of fine dust (PM10+DEP) around the brochia was significantly reduced compared to the control group (PM10D_CTL), and the airway thickness was normal. , the infiltration of inflammatory immune cells around the airway, granulocytes (neutrophils, eosinophils), and activated macrophages were greatly reduced.

In addition, it was confirmed that the inflammation of the cell part and the degree of blood cell infiltration were suppressed close to the normal group.

In addition, 200 mg/kg of CHJ3 (PG: PF = 3: 7) of Example 3 was higher than the test group in which PG (100 mg/kg) of Comparative Example 1 and PF (100 mg/kg) of Comparative Example 2 were orally administered, respectively. In the test group orally administered at a concentration of kg, the effect of further reducing inflammation due to the infiltration of inflammatory immune cells around the airway, granulocytes (neutrophils, eosinophils), and activated macrophages was confirmed (FIG. 12) .

Test Example 2-10: Trachea tissue test analysis through staining assay method in animal model efficacy evaluation for fine dust exposure to PG + PF composite extract

As a result of staining trachea tissue with AB-PAS staining after the end of the animal model experiment on exposure to fine dust (PM10+DEP), the control group (PM10D_CTL) compared to the normal group (Balb/c_Nr) It was confirmed as shown in FIG. 13 that inflammation due to fine dust (PM10 + DEP) was increased around the brochia, and the content of mucus (musin) in the epithelial cells of the respiratory tract was clearly increased.

The positive control group (Dexa. 3 mg/kg) and the test groups (200 mg/kg, 100 mg/kg) to which CHJ3 (PG: PF=3: 7) of Example 3 was orally administered were bronchial compared to the control group (PM10D_CTL). It was confirmed that the degree of inflammation around the brochia was reduced.

In addition, 200 mg of CHJ3 (PG: PF = 3: 7) of Example 3 was higher than the test group in which PG (100 mg/kg) of Comparative Example 1 and PF (100 mg/kg) of Comparative Example 2 were orally administered, respectively. In the test group orally administered at a /kg concentration, it was confirmed that the content of mucus in respiratory epithelial cells was suppressed closer to that of the normal group (FIG. 13).

Test Example 3: PG + (PF + PFY) composite extract for respiratory protection improvement animal model efficacy evaluation for exposure to fine dust (PM10 + DEP) (In vivo assay )

For the composite extract (PG: PF + PFY = 3: 7) of Example 10 The effect of improving respiratory protection against exposure to fine dust was confirmed using non-clinical evaluation, animal model evaluation.

By applying standard fine dust (PM10) and DEP (diesel combustion dust) to 7-week-old Balb/c mice, an animal model with respiratory damage was established and manufactured, and it was used to evaluate the efficacy of the animal model for improving respiratory protection against exposure to fine dust. . The animal model constructed above was named an animal model for exposure to fine dust (PM10+DEP).

A 4 mg/ml fine dust mixture (PM10D), which is a mixture of diesel exhaust particles (DEP) in fine dust (PM10) that damages the respiratory tract, is diluted in aluminum hydroxide (Alum) and injected into the airways and lungs (Intra-airway and lung injection method). -Nasal-tracheal (INT) injection) 3 times at 3-day intervals (3 days after drug administration, 6 days after, 9 days after) intra-nasal tracheal injection, making an animal model that is directly injected into the lungs through the airway and analyzed 3 days after the final fine dust (PM10D+DEP) injection.

In addition, as test groups, PG of Comparative Example 1, PF of Comparative Example 2, CHJ3 of Example 3 (PG: PF = 3: 7) and the composite extract of Example 10 (PG: PF + PFY = 3: 7) were all 100 Test groups were prepared at the same concentration in mg/kg. As a positive control group, dexamethasone (3 mg/kg) was orally administered at 11 am every day for 11 days, and the effect of improving respiratory protection against exposure to fine dust was confirmed through an animal model. As a result, after the end of the fine dust (PM10 + DEP) animal model experiment, inflammatory cells in lung tissue (Lung) and lung lavage fluid (BALF) were confirmed using a flow cytometer.

Experimental Example 3-1: Analysis of inflammatory cells in animal model lung tissue by flow cytometry (FASC) in animal model efficacy evaluation for fine dust exposure to PG + (PF + PFY) composite extract

After the end of the animal model experiment for exposure to fine dust (PM10 + DEP), inflammatory cells in lung tissue (Lung) were confirmed using FACS. The results are shown in Table 4 below.

Cell phenotypes in the lung Evaluation of airway inflammation animal models by induction of fine dust
(PM10D-induced airway inflammation murine model (Absolute No.)) Balb/c_
Normal PM10D_
CTL PM10D_
Dexa
3 mg/kg PM10D_PF
100 mg/kg PM10D_PG
100 mg/kg PM10D_
PG+PF
100 mg/kg PM10D_
PG+(PF+PFY)
100 mg/kg CD4+
(x10 ⁵ cells) 12.82
±0.23 26.94
±1.74### 18.03
±2.21** 19.09
±2.21 19.04
±0.12 12.20
±0.41*** 7.41
±1.11*** CD8+
(x10 ⁵ cells) 4.35
±0.09 15.78
±0.55### 11.22
±1.51 12.94
±1.43 11.65
±1.47 7.07
±0.19*** 4.25
±0.21*** CD4+/CD69+
(x10 ⁵ cells) 0.39
±0.15 6.16
±0.72### 2.22
±0.29** 4.11
±0.25 3.99
±0.21** 1.87
±0.49*** 0.65
±0.05*** CD62L+/CD44+
(x10 ⁵ cells) 1.47
±0.39 18.86
±0.08### 9.25
±0.16** 10.01
±2.44 10.54
±0.09 3.42
±0.03*** 2.89
±0.51*** Gr-1+/CD11b+
(x10 ⁵ cells) 6.85
±1.55 31.23
±17.97 11.13
±0.15 5.59
±0.2 8.71
±1.25 3.01
±0.22*** 1.89
±0.45***

As shown in Table 4 above, as a result of confirming inflammatory cells in lung tissue (Lung) after the end of the animal model experiment for exposure to fine dust (PM10 + DEP), CD4+ T cells (CD4 ⁺ ), CD8+ T cells (CD8+) , CD4 ⁺ /CD69 ⁺ cells, CD62L ⁺ /CD44 ⁺ cells and Gr-1 ⁺ /CD11b ⁺ cells in the test group orally administered with the PG + (PF + PFY) complex extract of Example 10 at a concentration of 100 mg / kg cells It was confirmed that the ratio decreased more than other test groups. More specifically, compared to the test group administered with PG of Comparative Example 1 and the test group orally administered with PF of Comparative Example 2, the composite extract of Example 10 (PG: PF + PFY = 3: 7) was orally administered. In the test group, it was confirmed that CD4+ T cells (CD4 ⁺ ), CD8+ T cells (CD8+), CD4 ⁺ /CD69 ⁺ cells, CD62L ⁺ /CD44 ⁺ cells, and Gr-1 ⁺ /CD11b ⁺ cells were further decreased. In addition, CD4+ T cells (CD4 ⁺ ), CD8+ T cells (CD8+), CD4 ⁺ /CD69 ⁺ cells, CD62L ⁺ / In the test group orally administered the composite extract (PG: PF + PFY = 3: 7) of Example 10, the effect of reducing the inflammatory cells of CD44 ⁺ cells and Gr-1 ⁺ /CD11b ⁺ cells was about 2 times more observed It became.

Experimental Example 3-2: Analysis of inflammatory cells included in lung lavage fluid (BALF) of animal model by flow cytometry (FASC) in animal model efficacy evaluation for fine dust exposure to PG+ (PF+PFY) composite extract

After the end of the animal model experiment for exposure to fine dust (PM10 + DEP), inflammatory cells in lung lavage fluid (BALF) were confirmed using flow cytometry (FACS). The results are shown in Table 5 below.

Cell phenotypes in lung lavage fluid Evaluation of airway inflammation animal models by induction of fine dust
(PM10D-induced airway inflammation murine model (Absolute No.)) Balb/c_
Normal PM10D_
CTL PM10D_
Dexa
3 mg/kg PM10D_PF
100 mg/kg PM10D_PG
100 mg/kg PM10D
_PG+PF
100 mg/kg PM10D_
PG+PF+PFY
100 mg/kg CD4+
(x10 ⁴ cells) 0.81±
0.19 40.47
±3.49### 8.84±
0.01 20.45 ±
0.03 21.44±
0.09 8.02±
2.69*** 3.66±
1.11*** CD8+
(x10 ⁴ cells) 0.39±
0.08 38.99±
0.21### 7.45 ±
0.81 21.25±
1.11 22.11±
1.04 8.88±
1.31*** 2.21±
0.11*** CD4+/CD69+
(x10 ⁴ cells) 0.72±
0.69 10.11±
0.14### 3.51
±0.43 4.75±
0.64 4.88±
0.25** 2.21±
0.38*** 1.11±
0.39*** CD62L+/CD44+
(x10 ⁴ cells) 0.37±
0.1 47.09±
5.13### 23.58
±2.80 24.59 ±
0.18** 22.21±
0.04** 12.02±
0.33*** 6.32±
4.36*** CD11b+/CGr-1+
(x10 ⁴ cells) 1.45 ±
0.31 81.74±
6.59### 47.03
±2.21 25.21±
8.2** 26.22±
4.45** 14.59 ±
2.29*** 4.21±
6.58***

As shown in Table 5, after the end of the animal model experiment for exposure to fine dust (PM10 + DEP), in the lung washing fluid (BALF), as a result of confirming the inflammatory cells, CD4+ T cells (CD4 ⁺ ), CD8+ T cells (CD8+ ), CD4 ⁺ /CD69 ⁺ cells, CD62L ⁺ /CD44 ⁺ cells and Gr-1 ⁺ /CD11b ⁺ cells, the composite extract of Example 10 (PG: PF + PFY = 3: 7) was orally administered at a concentration of 100 mg / kg It was confirmed that the concentration-dependent decrease in the administered test group. In addition, the test group orally administered with the composite extract (PG: PF + PFY = 3: 7) of Example 10 than the test group orally administered with PG of Comparative Example 1 and the test group orally administered with PF of Comparative Example 2 It was confirmed that the ratios of CD4+ T cells (CD4 ⁺ ), CD8+ T cells (CD8+), CD4 ⁺ /CD69 ⁺ cells, CD62L ⁺ /CD44 ⁺ cells, and Gr-1 ⁺ /CD11b ⁺ cells were further decreased. In addition, CD4+ T cells (CD4 ⁺ ), CD8+ T cells (CD8+), CD4 ⁺ /CD69 ⁺ cells, CD62L ⁺ /CD44 ⁺ cells than the test group orally administered with CHJ3 (PG: PF = 3: 7) of Example 3 And it was confirmed that the effect of reducing inflammatory cells of Gr-1 ⁺ /CD11b ⁺ cells was about twice or more significantly in the test group orally administered with the complex extract (PG: PF + PFY = 3: 7) of Example 10. . As a result of confirming the results of Test Example 3, by mixing the sesame seed extract and the sesame leaf extract, it was found that the action effect was remarkable when used in combination with the ginseng extract.

Claims (10)

In the pharmaceutical composition for preventing or treating respiratory diseases caused by fine dust comprising a composite extract as an active ingredient,
The complex extract is a mixture of 20 to 30% by weight of ginseng extract and 80 to 70% by weight of sesame leaf extract,
The ginseng extract is a pharmaceutical composition for preventing or treating respiratory diseases caused by fine dust comprising a mixture of ginsenosides Rg1 and Rb1. delete delete According to claim 1,
The mixture of ginsenosides Rg1 and Rb1 is a pharmaceutical composition for preventing or treating respiratory diseases caused by fine dust, comprising 1 to 3% by weight based on the total weight of the ginseng extract. According to claim 1,
The sesame leaf extract is a pharmaceutical composition for preventing or treating respiratory diseases caused by fine dust, which contains rosemary acid. According to claim 5,
The rosmary acid is a pharmaceutical composition for preventing or treating respiratory diseases caused by fine dust, which contains 1 to 10% by weight of the total weight of the sesame leaf extract. According to claim 1,
The complex extract is
1) extracting ginseng and sesame leaves with alcohol to prepare ginseng extract and sesame leaf extract, respectively;
2) vacuum concentrating the ginseng extract and sesame leaf extract, respectively; and
3) a pharmaceutical composition for preventing or treating respiratory diseases caused by fine dust obtained by including; drying each step after step 2). According to claim 1,
The sesame leaf extract further comprises a sesame seed extract, a pharmaceutical composition for preventing or treating respiratory diseases caused by fine dust. According to claim 1,
Respiratory diseases caused by fine dust include respiratory inflammatory lung disease, chronic obstructive pulmonary disease (COPD), sinusitis, allergic rhinitis, lower respiratory tract infection, acute and chronic bronchitis, pneumonia, bronchial asthma, bronchiectasis, emphysema, and pulmonary tuberculosis sequelae. , Acute respiratory distress syndrome, cough, otitis media, sore throat, tonsillitis, a disease selected from the group consisting of laryngitis and pulmonary fibrosis, a pharmaceutical composition for preventing or treating the respiratory tract caused by fine dust. In the health functional food for preventing or improving respiratory diseases caused by fine dust containing a complex extract as an active ingredient,
The complex extract is a mixture of 20 to 30% by weight of ginseng extract and 80 to 70% by weight of sesame leaf extract,
The ginseng extract is a health functional food for preventing or improving respiratory diseases caused by fine dust, including a mixture of ginsenosides Rg1 and Rb1.

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