KR20220169024A - Composition for enhancing pulmonary function having extract of Rehmannia glutinosa Liboschitz var - Google Patents

Abstract

Disclosed is a composition containing a natural complex extract, an effect of which has been verified to alleviate lung damage caused by fine dust through in vitro and animal experiments. The present invention provides the composition for alleviating lung damage caused by fine dust, containing a complex extract of Rehmannia glutinosa, Morus alba, and Liriope muscari as an active ingredient.

Description

Composition for enhancing pulmonary function having extract of Rehmannia glutinosa Liboschitz var}

The present invention relates to a functional natural composite against fine dust, and more particularly, to a composition for improving lung damage caused by fine dust.

Due to air pollution and environmental changes due to industrialization, respiratory diseases caused by fine dust are increasing, and the demand for their prevention or treatment is increasing.

Fine dust is dust (PM10) with a diameter of 10 ㎛ or less among total floating dust in the air. When fine dust is inhaled, it is deposited in the lower bronchus and lung parenchyma, causing damage to the respiratory system, worsening symptoms of existing diseases, and increasing morbidity and mortality. .

In addition, the increase in fine dust shows a significant correlation with the decrease in lung function. After exposure to fine dust, an increase in pulmonary macrophages, neutrophils, and lymphocytes was observed in bronchoalveolar lavage fluid, and an increase in neutrophils in lung tissue and in tracheal tissues. 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, and research on the prevention and treatment of acute exacerbations is required. , Furthermore, the development of reliable natural product-derived materials is required.

[Prior patent literature]

- Korean Registered Patent No. 10-1629706 (registered on June 7, 2016)

- Korean Registered Patent No. 10-1791120 (registered on October 23, 2017)

The present invention is intended to provide a composition containing a natural complex extract, the effect of which is verified to improve lung damage caused by fine dust through in vitro and animal experiments.

In order to solve the above problems, the present invention provides a composition for improving lung damage caused by fine dust, containing a complex extract of Rehmannia glutinosa, Sangyeop, and Gummundong as an active ingredient.

In addition, the composition of the composite extract provides a composition characterized in that 45 to 55% by weight of Rehmannia Root, 15 to 25% by weight of upper leaf, and 25 to 35% by weight of Maulundong based on the extraction target.

In addition, the composite extract provides a composition characterized in that it reduces reactive oxygen species (ROS), inflammatory cytokines IL-6 and TNF-α in alveolar macrophages stimulated with fine dust.

In addition, the composite extract reduces inflammatory cytokines CXCL-1, IL-17, MIP2 and TNF-α in bronchoalveolar lavage fluid (BALF) of a lung injury animal model induced by fine dust, and inflammation-related genes in lung tissue Provides a composition characterized by reducing the expression of phosphorus CXCL-1, NOS-II, COX-2, TRAC, MIP2 and TNF-α, and inhibiting the expression of bronchial inflammation and cough-related factors SDMA, MUC5AC, TRPV1 and TRPA1 do.

In addition, the composition provides a composition characterized in that the pharmaceutical composition.

In addition, the composition provides a composition characterized in that the food composition.

It was confirmed that the composition according to the present invention contains complex extracts of Rehmannia glutinosa, Sangyeol, and Gummundong, and has a significant effect on improving lung damage in an in vitro experiment and an animal model with reduced lung function induced by fine dust.

1 is a graph showing the results of ROS analysis after treating 50 μg/ml of fine dust PM10-like (SRM®CZ120) to MH-S cell lines, which are macrophages of mouse lungs, in an experimental example of the present invention.
Figure 2 is an inflammatory cytokine, IL-6 and TNF-α production analyzed when fine dust PM10-like (50 μg / ml) was treated with MH-S cell line, which is a macrophage of mouse lungs, in an experimental example of the present invention This is the graph showing the result.
Figure 3 is a graph showing the results of analyzing the total number of BAL cells and the total number of lung cells after the end of the fine dust (PM10 + DEP) animal model experiment in the experimental example of the present invention.
Figure 4 shows inflammatory cytokines (CXCL-1 (A), IL-17 (B), MIP2 (C) and It is a graph showing the results of measuring and analyzing TNF- α (D)) by ELISA.
Figure 5 is a graph showing the results of measuring and analyzing airway resistance factors (SDMA, symmetric dimethylarginine) in serum after the end of the fine dust (PM10 + DEP) animal model experiment in the experimental example of the present invention by ELISA.
6 is an inflammatory cytokine gene (TNF-α (F), TARC (C), COX-2 (A), NOS- II (E), MIP-2 (B) and CXCL-1 (D)) expression is measured and analyzed by QRT-PCR.
Figure 7 is a photograph showing the results of measurement and analysis of signal transduction IRAK1 protein expression in lung tissue after the end of the fine dust (PM10D) animal model experiment in the experimental example of the present invention by immunofluorescence tissue staining (Immune histology fluorescent, IHF).
Figure 8 is the result of measuring and analyzing the expression of signaling TNF-α and MCP-1 protein in lung tissue after the end of the fine dust (PM10D) animal model experiment in the experimental example of the present invention by immunofluorescence tissue staining (Immune histology fluorescent, IHF) is a picture showing
9 to 13 are the frequency of white blood cells (lymphocytes, granulocyte) cells through FACS analysis for peripheral blood mononuclear cells (PBMC) after the end of the fine dust (PM10 + DEP) animal model experiment in the experimental example of the present invention. It is a graph showing the result of analyzing (%).
14 to 19 are graphs showing the results of FACS analysis of inflammatory cells in lung tissue (Lung) after the end of the fine dust (PM10 + DEP) animal model experiment in the experimental example of the present invention.
20 to 24 are graphs showing the results of FACS analysis of inflammatory cells in lung lavage fluid (BALF) after the end of the fine dust (PM10 + DEP) animal model experiment in the experimental example of the present invention.
25 is a photograph showing the results of H&E staining of lung tissue (Lung) after the end of the fine dust (PM10 + DEP) animal model experiment in the experimental example of the present invention.

Hereinafter, the present invention will be described in detail through preferred embodiments. Prior to this, the terms or words used in this specification and claims should not be construed as being limited to the usual or dictionary meaning, and the inventor appropriately uses the concept of the term in order to explain his/her invention in the best way. Based on the principle that it can be defined, it should be interpreted as meaning and concept consistent with the technical spirit of the present invention. Therefore, since the configurations of the embodiments described in this specification are merely the most preferred embodiments of the present invention and do not represent all of the technical ideas of the present invention, various equivalents and modifications that can replace them at the time of this application It should be understood that there may be Also, throughout the specification, when a part "includes" a certain component, it means that it may further include other components, not excluding other components, unless otherwise stated.

The present invention discloses a composition for improving lung damage caused by fine dust, comprising a complex extract of Rehmannia glutinosa, Sangyeol, and Gummundong as an active ingredient.

The Rehmannia Root is a perennial herb belonging to the ginseng family, and its roots and rhizomes are used as medicine. As a main medicinal ingredient of Samultang, it is used for symptoms such as internal heat, dry throat, and thirst caused by weakness among various chronic diseases.

In addition, the upper leaf has been used as a medicine in the private sector as a leaf of the Moraceae tree, and the upper leaf contains various physiologically active substances as well as proteins, amino acids, vitamins, minerals and a large amount of dietary fiber.

In addition, the rhubarb is a plant of the family Liliaceae, has an antioxidant action, promotes blood flow in the coronary artery and protects against ischemia of the heart muscle, and is reported to exhibit a sedative action, lowering blood sugar, and white staphylococcus aureus and Escherichia coli It is known to exhibit antibacterial activity from

In the present invention, there is no limitation on the form or properties of the composite extract, and it may be a solution, a concentrate, or a solid or powder from which the solvent used in preparing the extract is removed.

The complex extract may be prepared using a solvent extraction method. Water is selected as the extraction solvent used in preparing the extract using the solvent extraction method, and hot water extraction may be preferably used. The extraction temperature may be 80 to 100 °C, preferably 90 to 10 °C, and more preferably 95 to 100 °C. At this time, in consideration of the manufacturing yield, the extraction is performed with a water amount of 5 to 30 times and an extraction time of 1 to 10 hours, preferably a water amount of 5 to 20 times, an extraction time of 1 to 8 hours, and more preferably a water amount Extraction can be performed with 8 to 15 multiples and extraction time of 2 to 5 hours.

In the present invention, the compounding ratio of the composite extract may be 45 to 55% by weight of Rehmannia Root, 15 to 25% by weight of upper leaf, and 25 to 35% by weight of Rehmannia Root, preferably 47 to 53% by weight, based on the amount added during solvent extraction. , 17 to 23% by weight of the upper leaf and 27 to 33% by weight of the upper leaf, and more preferably 49 to 51% by weight of Rehmannia Root, 19 to 21% by weight of the upper leaf and 29 to 31% by weight of the upper leaf.

The complex extract implements the effect of improving lung damage caused by fine dust, for example, as an in vitro experiment, in alveolar macrophages stimulated by fine dust, reactive oxygen species (ROS), inflammatory cytokine IL- 6 and TNF-α, and in bronchoalveolar lavage fluid (BALF) of an animal model of lung injury induced by fine dust as an animal experiment, reducing inflammatory cytokines CXCL-1, IL-17, MIP2 and TNF-α , Reduces the expression of inflammation-related genes CXCL-1, NOS-II, COX-2, TRAC, MIP2 and TNF-α in lung tissue, and suppresses the expression of bronchial inflammation and cough-related factors SDMA, MUC5AC, TRPV1 and TRPA1 confirmed that

The composition according to the present invention is provided in the form of a pharmaceutical composition or a food composition.

The pharmaceutical composition may include a pharmaceutically acceptable carrier in addition to the active ingredient, and such a carrier is commonly used in formulation, and includes lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, Calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and minerals. oils and the like, but are not limited thereto. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like in addition to the above components. Suitable pharmaceutically acceptable carriers and agents are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

A suitable dosage of the pharmaceutical composition according to the present invention varies depending on factors such as formulation method, administration method, patient's age, weight, sex, disease condition, food, administration time, administration route, excretion rate and reaction sensitivity. may be prescribed. On the other hand, the dosage of the pharmaceutical composition of the present invention is preferably 0.0001 to 100 mg/kg (body weight) per day. The pharmaceutical composition of the present invention can be administered orally or parenterally, and in the case of parenteral administration, it can be administered by topical application to the skin, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, etc. . Considering that the pharmaceutical composition of the present invention exhibits antioxidant, anti-wrinkle and skin regeneration effects, it is preferably administered orally or topically applied to the skin.

The concentration of the active ingredient included in the composition of the present invention can be determined in consideration of the purpose of treatment, the condition of the patient, the required period, etc., and is not limited to a specific range of concentration.

The pharmaceutical composition of the present invention is prepared in unit dosage form by formulation using a pharmaceutically acceptable carrier or excipient according to a method that can be easily performed by those skilled in the art, or Or it can be prepared by incorporating into a multi-dose container. In this case, the formulation may be prepared in any one formulation selected from injections, creams, patches, sprays, ointments, warnings, lotions, liniments, pastas, and cataplasmas. When the composition is used as an external skin preparation, it may additionally contain a fatty substance, an organic solvent, a solubilizing agent, a thickening agent and a gelling agent, a softening agent, an antioxidant, a suspending agent, a stabilizer, a foaming agent, a fragrance, a surfactant, and water. , ionic or nonionic emulsifiers, fillers, sequestering agents and chelating agents, preservatives, vitamins, blocking agents, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic actives, lipid vesicles, or skin external preparations It may contain adjuvants commonly used in the field of dermatology, such as any other ingredients that are In addition, the components may be introduced in an amount generally used in the field of skin science.

In addition, the food composition includes all types of functional foods, nutritional supplements, health foods, and food additives. Food compositions of this type can be prepared in various forms according to conventional methods known in the art.

For example, as a health food, the composition itself may be prepared and consumed in the form of tea, juice, or drink, or may be granulated, encapsulated, or powdered to be consumed.

In addition, functional foods include beverages (including alcoholic beverages), fruits and their processed foods (e.g., canned fruit, bottled fruit, jam, marmalade, etc.), fish, meat, and their processed foods (e.g., ham, sausage and corned beef). etc.), breads and noodles (e.g. udon, buckwheat noodles, ramen, spaghetti, macaroni, etc.), fruit juice, various drinks, cookies, taffy, dairy products (e.g. butter, cheese, etc.), edible vegetable oil, margarine, vegetable protein, It can be prepared by adding extracts to retort foods, frozen foods, various seasonings (eg, soybean paste, soy sauce, sauce, etc.).

In addition, in order to use the composition of the present invention in the form of a food additive, it can be prepared and used in the form of a powder or concentrate.

The preferred content of the extract in the food composition of the present invention may be 0.001 to 50%, preferably 0.1 to 50%, more preferably 1 to 20% based on the total weight of the food composition.

Hereinafter, the present invention will be described in more detail with specific experimental examples.

Complex extract preparation

With the composition (unit: weight%) shown in Table 1 below, extract Rehmannia (Herbal Medicine Co., Ltd., Korea), Sangyeop (Herbal Medicine Co., Ltd., Korea), and Maekmundong (Herbal Medicine Co., Ltd., Korea) with 10 times the purified water at 100 ° C. for 3 hours. proceeded. After filtering the extract through an 80 mesh net, it was cooled to 60°C, and the cooled extract was concentrated at 65°C for 2 hours to prepare a composite extract.

division Preparation Example 1
(RML_C1) Preparation Example 2
(RML_C2) Preparation Example 3
(RML_C3) Production Example 4
(RML_C4) Rehmannia glutinosa Liboschitz var. 50 50 50 50 Upper lobe (Morus alba L.) 10 20 30 40 Liriope platyphylla 40 30 20 10

Experiment method

(1) Cell culture and fine dust treatment

Alveolar macrophage (MH-S) cells, a macrophage cell line of the respiratory tract, were stimulated with fine dust (PM10) and treated with test substances to perform ROS FACS analysis and IL-6 and TNF-α ELISA measurements.

(2) Animal model efficacy evaluation

1) Application of mice and standard fine dust (PM10D)

As an experimental animal model using 7-week-old male Balb/c mice and applying standard fine dust (PM10), DEP (diesel exhaust particles, diesel combustion dust), standard fine dust (PM10-like, ERM-CZ120) animal model built and fabricated.

2) Production of animal model with respiratory damage

A 4 mg/ml fine dust mixture (PM10D), which is a mixture of DEP in fine dust (PM10) that damages the respiratory tract, is diluted in Alum (aluminium hydroxide) and injected at 3-day intervals using the INT (Intra-Nazal-Tracheal) injection method. An animal model injecting directly into the lungs through the airway (intra-nasaltracheal injection after 3 days, 6 days, and 9 days after drug administration) was prepared, and sacrificed 3 days after the final fine dust (PM10D) injection.

3) Complex extract concentration and positive control drug administration

The composite extract was orally administered at 200 mg/kg and 100 mg/kg concentration, and dexamethasone (3 mg/kg) as a positive control group was administered orally at 11 am every day for 11 days.

4) Confirmation of causing lung damage

Bronchoalveolar lavage fluid (BALF) was separated and the expression of inflammation-related immune cells was analyzed by FACS, inflammatory cell count, FACS analysis, MUC5AC, IL (Interleukin)-1β, IL-4, IL-6, IL-13, IL -17, IFN(interferon)-γ, eotaxin, KC(keratinocyte chemoattractant), MCP(monocyte chemotactic protein)-1, MIP(macrophage inflammatory protein)-1α and MUC5AC expression were analyzed by ELISA to determine the treatment for each concentration of fine dust. The degree of lung damage induction was evaluated. In addition, neutrophil analysis by Cytospin was performed using Diff-Quik solution, and correlation with lung damage was evaluated through general blood test, serum biochemical test, and blood IgE and SDMA ELISA measurement, and trachea and lung ( The degree of lung damage was evaluated by quantifying alveolar damage, cell infiltration, and collagen deposition through H&E, M-T, PAS, and AB-PAS staining of lung tissue. In addition, changes in the expression of IRAK-1, CD11b, TNF-α, and MCP-1 were explored using IHF in lung tissue.

Experiment result

(1) Fine dust (PM10) induced in vitro composite extract anti-inflammatory effect

1) ROS production analysis

MH-S cell line, which is a macrophage of the mouse lung, was treated with 50 μg/ml of fine dust PM10-like (SRM ^® CZ120), and the results of ROS analysis are shown in FIG. 1 .

Referring to Figure 1, when the composite extract according to Preparation Example 2 (RML_C2) was treated with 100, 200 and 400 μg / ml, ROS was 43.7% (p in a concentration-dependent manner with statistical significance) compared to the control group (PM10-like_CTL) <0.001), 56.1% (p<0.001) and 61.6% (p<0.001) or more. That is, it was confirmed that the ideal mixing ratio (RML_2C-Opt.) was obtained in the case of complex extraction with 50% by weight of Rehmannia glutinosa, 20% by weight of upper leaf, and 30% by weight of rhizomes, according to Preparation Example 2 (RML_C2).

2) Measurement of inflammatory cytokines

When fine dust PM10-like (50 μg / ml) was treated with MH-S cell line, which is a macrophage of mouse lung, the results of analyzing the production of IL-6 and TNF-α, which are inflammatory cytokines, are shown in FIG. 2 .

Referring to Figure 2, when the composite extract according to Preparation Example 2 (RML_C2) was treated with 100, 200 and 400 μg / ml, the production of IL-6 and TNF-α was statistically significant compared to the control group (PM10-like_CTL) Concentration-dependently, IL-6 production decreased by 32.0% (p<0.05), 45.0% (p<0.01) and 56.8% (p<0.001), and TNF-a production decreased by 30.8% (p<0.001) and 38.0% (p<0.001) and 59.4% (p<0.001) decreased. That is, it was confirmed that the ideal mixing ratio (RML_2C-Opt.) was obtained in the case of complex extraction with 50% by weight of Rehmannia glutinosa, 20% by weight of upper leaf, and 30% by weight of rhizomes, according to Preparation Example 2 (RML_C2).

(2) Evaluation of respiratory protection/improvement animal model efficacy against exposure to fine dust (PM10D) of complex extracts

Selective invasion changes of granulocytes (Neutrophil) and eosinophils (Eosinophil), humoral properties, cell-mediated immunity, and mucosal immunity to evaluate the efficacy by setting the dose and oral administration of the complex extract in an animal model with fine dust (PM10+DEP) respiratory damage Functional analysis, inflammation-related gene expression, and histopathological examination were used to evaluate the respiratory protection and improvement efficacy of the candidate substance. Respiratory damage caused by exposure to fine dust (PM10D) The reagents used in the production of animals and the animal model design for respiratory damage caused by exposure to fine dust (PM10D) are summarized in Tables 2 and 3, respectively.

Label Information Cat No. PM10 fine dust (PM10-like) sigma ERM-CZ120 DEP Diesel Particulate Matter sigma #2975 PM10D fine dust (PM10-like) + DEP Dexa dexamethasone sigma #D2915-100mg vehicle Aluminum Hydroxide Gel Adjuvant (1%) + Saline (99%)

No. army Treatment population One Nor BalB/c-Nr 8 2 PM10D_CTL +PM10(Fine dust) + DEP 8 3 PM10D_DEXA (PC) +PM10(Fine dust) + DEP + dexamethasone 3mpk (ip) 8 4 PM10D+2C-Opt-200 +PM10 (Fine dust) + DEP + Preparation Example 2 composite extract: 200 mg/kg 8 5 PM10D-2C-Opt-100 +PM10 (Fine dust) + DEP + Preparation Example 2 Complex Extract: 100 mg/kg 8

Efficacy evaluation items are as follows.

- BALF: inflammatory cell count, FACS analysis, MUC5AC, cytokines

- Blood: blood cell analysis, blood clinical indicators, IgE, IL-6, IL-17, SDMA ELISA measurement

- Biopsy: H&E, PAS staining of trachea and lung

- Lung: Gene expression (CCR3, IL-15, IL-13, MUC5AC, eotaxin, TARC, TNF-α, etc.)

- Lung: Exploring expression changes of IRAK-1, CD11b, TNF-α and MCP-1 using IHF

1) BAL and lung total cell count analysis

The results of analyzing the total number of BAL cells and the total number of lung cells after the end of the fine dust (PM10 + DEP) animal model experiment are shown in FIG. 3 .

Referring to FIG. 3, the control group PM10D_CTL) showed a significant increase in the number of BAL cells compared to the normal group (Balb/c_Nr) (p<0.01). The positive control group (Dexamethasone, 3 mg/kg) and the composite extract according to Preparation Example 2 (RML_2C-Opt.) administration group (200 mg/kg, 100 mg/kg, p<0.01) significantly decreased compared to the control group (PM10D_CTL) did In lung tissue, the control group (PM10D_CTL) showed a significant increase in Lung cell number more than twice as compared to the normal group (Balb/c_Nr) (p<0.01). The positive control group (Dexamethasone, 3 mg/kg) and the composite extract according to Preparation Example 2 (RML_2C-Opt.) administration group (200 mg/kg p<0.01, 100 mg/kg) significantly decreased compared to the control group (PM10D_CTL) .

2) Analysis of inflammatory cytokines in BAL fluid (BALF)

After the end of the fine dust (PM10+DEP) animal model experiment, inflammatory cytokines (CXCL-1 (A), IL-17 (B), MIP2 (C) and TNF-α (D)) were detected in lung lavage fluid (BALF) by ELISA The results of measurement and analysis are shown in Figure 4.

Referring to FIG. 4, it was found that the control group (PM10D_CTL) significantly increased the production of inflammatory cytokines compared to the normal group (Balb/c_Nr) (p<0.001). The positive control group (Dexamethasone, 3 mg/kg) and the composite extract according to Preparation Example 2 (RML_2C-Opt.) administration group (200 mg/kg) were significantly reduced in a concentration-dependent manner compared to the control group (PM10D_CTL) (p<0.01) (See Figures 4, A to D). In addition, IL-17 (B) and TNF-α (D) in the composite extract (RML_2C-Opt.) (100 mg / kg) according to Preparation Example 2 were significantly reduced compared to the control group (PM10D_CTL) (p <0.05) (See Figures 4, B and D).

3) Analysis of bronchopulmonary function index (SDMA) and lung tissue cough-related genes (MUC5AC, TRPV1 and TRPA1) in blood

After the end of the fine dust (PM10 + DEP) animal model experiment, the airway resistance factor (SDMA, symmetric dimethylarginine) in serum was measured and analyzed by ELISA, and the results are shown in FIG. 5A.

Referring to FIG. 5A, compared to the normal group (Balb/c_Nr), the control group (PM10D_CTL) significantly increased the SDMA level by about 30.8% or more (p<0.001). The positive control group (Dexamethasone, 3 mg/kg) and the composite extract according to Preparation Example 2 (RML_2C-Opt.) administration group (200 mg/kg) were significantly reduced compared to the control group (PM10D_CTL) (p<0.01).

Results of measuring and analyzing cough-related gene expression in lung tissue are shown in FIGS. 5B to 5D.

5B to 5D, compared to the normal group (Balb/c_Nr), the control group (PM10D_CTL) significantly increased the mRNA expression of cough-related genes (MUC5AC, TRPV1 and TRPA1) (p<0.05, p<0.01). ). Compared to the control group (PM10D_CTL), the positive control group (Dexamethasone, 3 mg/kg, p<0.05, p<0.01) and the composite extract (RML_2C-Opt.) administration group (200 mg/kg, p<0.01) according to Preparation Example 2 Gene expression was significantly reduced in a concentration-dependent manner.

4) Lung tissue inflammatory cytokine gene expression analysis (QRT-PCR)

Inflammatory cytokine genes (TNF-α(F), TARC(C), COX-2(A), NOS-II(E), MIP-2( B) and CXCL-1 (D)) expression were measured and analyzed by QRT-PCR, and the results are shown in FIG. 6 .

Referring to Figure 6, compared to the normal group (Balb / c_Nr), the control group (PM10D_CTL) increased inflammatory cytokine gene expression more than twice significantly (p <0.001). The positive control group (Dexamethasone, 3 mg/kg) and the composite extract according to Preparation Example 2 (RML_2C-Opt.) administration group (200 mg/kg) were significantly reduced in a concentration-dependent manner compared to the control group (PM10D_CTL).

In the composite extract (RML_2C-Opt.) administration group (100 mg/kg) according to Preparation Example 2, NOS-II (E) and TARC (C) mRNA gene expressions were significantly reduced compared to the control group (PM10D_CTL), but COX-2 (A), MIP-2 (B), CXCL-1 (D) and TNF-α (F) mRNA gene expression showed a tendency to decrease compared to the control group (PM10D_CTL), but no significance was shown.

5) Lung tissue signaling IRAK-1 and CD11b protein expression analysis (IHF)

After the end of the fine dust (PM10D) animal model experiment, the signal transmission IRAK1 protein expression in lung tissue was measured and analyzed by immunofluorescence tissue staining (Immune histology fluorescent, IHF), and the results are shown in FIG. 7 .

Referring to FIG. 7 , IRAK-1 protein expression was markedly 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 complex extract (RML_2C-Opt.) administration group (200 mg/kg) according to Preparation Example 2 were significantly reduced in a concentration-dependent manner compared to the control group (PM10D_CTL) (FIG. 7, A and B). In addition, CD11b protein expression was markedly 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 composite extract according to Preparation Example 2 (RML_2C-Opt.) administration group (200 mg/kg p<0.05, 100 mg/kg, p<0.01) were compared to the control group (PM10D_CTL) It was significantly decreased in a concentration-dependent manner (see FIGS. 7, C and D).

6) Lung tissue signaling TNF-α and MCP-1 protein expression analysis (IHF)

After the end of the fine dust (PM10D) animal model experiment, the results of measuring and analyzing TNF-α and MCP-1 protein expression in lung tissue by immunofluorescence tissue staining (Immune histology fluorescent, IHF) are shown in FIG. 8 .

Referring to FIG. 8 , TNF-α protein expression was markedly 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 composite extract according to Preparation Example 2 (RML_2C-Opt.) administration group (200 mg/kg p<0.05, 100 mg/kg, p<0.01) were compared to the control group (PM10D_CTL) It was significantly decreased in a concentration-dependent manner (see FIG. 8, A and B). In addition, MCP-1 protein expression was significantly increased in the control group (PM10D_CTL) compared to the normal group (Balb/c_Nr) (see FIG. 8, C). The positive control group (Dexamethasone, 3 mg/kg) and the composite extract according to Preparation Example 2 (RML_2C-Opt.) administration group (200 mg/kg p<0.001, 100 mg/kg, p<0.05) were compared to the control group (PM10D_CTL) It was significantly decreased in a concentration-dependent manner (see FIGS. 8, C and D).

7) Cell frequency analysis of total leucocytes in PBMC

9 to 9 to analyze the frequency (%) of white blood cells (lymphocytes, granulocyte) cells through FACS analysis for peripheral blood mononuclear cells (　 PBMC) after the end of the fine dust (PM10 + DEP) animal model experiment. 13.

9 to 13, the frequency (%) of lymphocytes, neutrophils, CD3+ T cells, and CD4+ & CD8+ T helper cells decreased in the control group (PM10D_CTL) compared to the normal group (Balb/c_Nr). Compared to the control group (PM10D_CTL), the positive control group (Dexamethasone, 3 mg/kg) and the composite extract (RML_2C-Opt.) administration group (200 mg/kg, 100 mg/kg) were treated with lymphocytes, CD3+ T cells, and CD4+ & CD8+ T helper cell frequency (%) increased in a concentration dependent manner (p<0.01, p<0.001). In addition, the frequency (%) of neutrophils, CD4+CD69+ cells, and CD3+CD49b+ NK cells decreased in the control group (PM10D_CTL) compared to the normal group (Balb/c_Nr).

8) Lung tissue inflammatory cell FACS analysis

The results of FACS analysis of inflammatory cells in lung tissue (Lung) after the end of the fine dust (PM10 + DEP) animal model experiment are shown in Table 4 and Figures 14 to 19.

Referring to Table 4 and FIGS. 14 to 19, the total absolute cell count of inflammatory activity in lung tissue (Lung) (lymphocytes No. FIG. 14B), absolute cell number of neutrophils (FIG. 14B), absolute cell number of eosinophils (FIG. 14D), CD4+ & CD8+ absolute No. (FIG. 15), CD62L-CD44high+ absolute No. (FIG. 17), CD21/CD35+B220+ absolute No. (FIG. 18) and Gr-1+/CD11b+ absolute No. (FIG. 19) calculated As a result, compared to the normal group (Balb/c_Nr), the control group (PM10D_CTL) had a higher total inflammatory activity absolute cell count (CD4+ absolute No., neutrophils absolute cell number, Gr-1+/CD11b+ absolute No., CD3+/CD19+ absolute No., CD21/CD35+B220+ absolute No. and CD62L-CD44high+ absolute No.) were found to increase significantly. The positive control group (Dexamethasone, 3 mg/kg) and the complex extract (RML_2C-Opt.) administration group (200 mg/kg, 100 mg/kg) according to Preparation Example 2 showed a total absolute cell count of inflammatory activity compared to the control group (PM10D_CTL) ( CD4+ absolute No., neutrophils absolute cell count, CD62L-CD44+ absolute No., Gr-1+/CD11b+ absolute No., CD3+/CD19+ absolute No., CD21/CD35+B220+ absolute No.) decreased significantly in a concentration-dependent manner (see FIGS. 14 to 19).

9) Lung lavage fluid (BALF) inflammatory cell FACS analysis

The results of FACS analysis of inflammatory cells in lung lavage fluid (BALF) after the end of the fine dust (PM10 + DEP) animal model experiment are shown in Table 5 and Figures 20 to 24.

Referring to Table 5 and FIGS. 20 to 24, total inflammatory activity in lung lavage fluid (BALF) (lymphocytes No., FIG. 20B), neutrophils absolute cell number (FIG. 20B), eosinophils absolute cell number (FIG. 20C), Results of calculating CD4+ & CD8+ absolute No. (FIG. 21), CD4+/CD69+ absolute No. (FIG. 22), CD62L-CD44high+ absolute No. (FIG. 23) and Gr-1+/CD11b+ absolute No. (FIG. 24) , Compared to the normal group (Balb/c_Nr), the control group (PM10D_CTL) showed total inflammatory activity (CD4+ absolute No., neutrophils absolute cell number, Gr-1+/CD11b+ absolute No., CD4+/CD69+ absolute No., and CD62L -CD44+ absolute No.) was found to increase significantly. The positive control group (Dexamethasone, 3 mg/kg) and the complex extract (RML_2C-Opt.) administration group (200 mg/kg, 100 mg/kg) according to Preparation Example 2 showed a total absolute cell count of inflammatory activity compared to the control group (PM10D_CTL) ( CD4+ absolute No., absolute cell number of neutrophils, CD62L-CD44+ absolute No., and Gr-1+/CD11b+ absolute No.) were significantly decreased in a concentration-dependent manner compared to the control group (PM10D_CTL) (see FIGS. 20 to 24).

10) Lung tissue analysis

25 shows the results of H&E staining of lung tissue (Lung) after the end of the fine dust (PM10 + DEP) animal model experiment.

25, compared to the normal group (Balb/c_Nr), the deposition of fine dust (PM10 & DEP) around the brochia in the control group (PM10D_CTL) showed a clear increase, and the thickness of the airway was greatly increased, Infiltration of inflammatory immune cells, granulocytes (neutrophils, eosinophils), and activated macrophages around the airway caused inflammation to intensify, resulting in alveolar cell destruction. To observe the degree of destruction of alveolar cells by collagen deposition, Masson's Trichrome (M-T) staining was used to determine tracheol and alveolar structures and inflammatory and blood cell infiltration in the structure part. degree can be observed. Also, in the control group (PM10D_CTL), many goblet cells (PAS staining) secreting mucus were distributed around the airway. The positive control group (Dexamethasone, 3 mg/kg) and the composite extract (RML_2C-Opt.) administration group (200 mg/kg) according to Preparation Example 2 showed fine dust (PM10 & DEP) around the airway compared to the control group (PM10D_CTL). ) was markedly reduced, and the thickness of the airway was reduced close to the normal group, and the infiltration of inflammatory immune cells around the airway, granulocytes (neutrophils, eosinophils) and activated macrophages were greatly reduced, and collagen deposition (collagen deposition) is Masson's Trichrome (M-T) staining, showing inflammation and inflammation of goblet cells and cells that secrete mucus around the tracheol, alveolar, and airways of the structural part. The degree of infiltration of blood cells was also suppressed close to that of the normal group.

Hereinafter, examples of application of food compositions and pharmaceutical compositions containing the complex extract according to the present invention as an active ingredient will be described, but are not limited thereto.

Application Example 1: Preparation of liquid composition

Table 6 below illustrates the composition of a liquid composition containing the complex extract according to the present invention described above as an active ingredient. The liquid composition is prepared by adding and dissolving each of the following components in purified water according to a conventional liquid composition manufacturing method, adding an appropriate amount of lemon flavor, mixing the following components, adding purified water to adjust the total volume to 100 ml, and then filling and sterilizing a brown bottle. can be manufactured.

ingredient content complex extract 200 mg Lee Seonghwadang 10g mannitol 5g Purified water Appropriate amount

Application Example 2: Manufacture of health functional food

Table 7 below shows the health functional food composition containing the complex extract according to the present invention described above as an active ingredient. In the following composition, the composition ratio of the vitamin and mineral mixture shows that a mixture of ingredients suitable for health functional food is mixed as a preferred application example, but it is okay to modify the mixing ratio arbitrarily, and each component according to the normal health functional food manufacturing method After mixing, granules are prepared and can be used for preparing a health functional food composition according to a conventional method.

ingredient content ingredient content complex extract 1000 mg folic acid 50 µg vitamin mixture Appropriate amount Calcium Pantothenate 0.5 mg Vitamin A Acetate 70 µg mineral mixture Appropriate amount vitamin E 1.0 mg ferrous sulfate 1.75mg vitamin B1 0.13 mg zinc oxide 0.82 mg vitamin B2 0.15mg magnesium carbonate 25.3mg vitamin B6 0.5mg Potassium Phosphate Monobasic 15mg vitamin B12 0.2 μg Dibasic Potassium Phosphate 55mg vitamin C 10mg potassium citrate 90 mg biotin 10 µg calcium carbonate 100 mg nicotinic acid amide 1.7 mg magnesium chloride 24.8 mg

Application Example 3: Health Beverage Manufacturing

Table 8 below shows functional health beverage compositions containing the complex extract according to the present invention described above as an active ingredient. For example, the following ingredients are mixed according to the usual health drink manufacturing method, stirred and heated at 85 ° C. for about 1 hour, the resulting solution is filtered, collected in a sterilized 2-liter container, sealed and sterilized, and refrigerated. It can be stored and used. The following composition ratio is a mixture of ingredients suitable for a relatively favorite beverage, but it is okay to arbitrarily modify the mixing ratio according to regional and ethnic preferences such as class of demand, country of demand, and purpose of use.

ingredient content complex extract 1000 mg citric acid 1000mg oligosaccharide 100 grams plum concentrate 2g taurine 1 g Purified water mix Total 900 ml

Application example 4: Powder preparation

Table 9 below shows the powder composition containing the complex extract according to the present invention described above as an active ingredient. Powders can be prepared by mixing the following ingredients and filling them into airtight bags.

ingredient content complex extract 200mg lactose 100 mg talc 10mg

Application Example 5: Tablet Manufacturing

Table 10 below shows the tablet composition containing the complex extract according to the present invention described above as an active ingredient. Tablets may be prepared by mixing the following ingredients and then tableting according to a conventional tablet manufacturing method.

ingredient content complex extract 200mg corn starch 100mg lactose 100 mg magnesium stearate 2 mg

Application Example 6: Preparation of capsules

Table 11 below shows the composition of capsules containing the complex extract according to the present invention described above as an active ingredient. Capsules may be prepared by mixing the following ingredients and filling them into gelatin capsules according to a conventional capsule preparation method.

ingredient content complex extract 200mg crystalline cellulose 3mg lactose 14.8mg magnesium stearate 0.2mg

Application Example 7: Preparation of injection

Table 12 below shows the composition of injections containing the complex extract according to the present invention described above as an active ingredient. The injection may be prepared with the following ingredient content per 1 ampoule (2 ml) according to a conventional injection preparation method.

ingredient content complex extract 200mg mannitol 3mg Sterile Distilled Water for Injection 14.8 mg Na ₂ HPO ₄ 12H ₂ O 0.2mg

Application Example 8: Liquid Preparation

Table 13 below shows liquid compositions containing the complex extract according to the present invention described above as an active ingredient. The liquid preparation is prepared by adding and dissolving each of the following ingredients in purified water according to a conventional liquid preparation method, adding an appropriate amount of lemon flavor, mixing the following ingredients, adding purified water to adjust the total volume to 100 ml, and then filling and sterilizing a brown bottle. can

ingredient content complex extract 200mg Lee Seonghwadang 10g mannitol 5g Purified water Appropriate amount

The preferred embodiments of the present invention described above have been disclosed to solve the technical problems, and those skilled in the art will be able to make various modifications, changes, additions, etc. within the spirit and scope of the present invention. , such modifications and changes should be regarded as belonging to the scope of the following claims.

Claims (6)

A composition for improving lung damage caused by fine dust, containing complex extracts of Rehmannia glutinosa, Sangyeol, and Rhododendron sinensis as an active ingredient. According to claim 1,
The composition of the composite extract is a composition, characterized in that 45 to 55% by weight of Rehmannia Root, 15 to 25% by weight of upper leaves and 25 to 35% by weight of Macundong based on the extraction target. According to claim 1,
The composite extract is a composition characterized in that for reducing reactive oxygen species (ROS), inflammatory cytokines IL-6 and TNF-α in alveolar macrophages stimulated with fine dust. According to claim 1,
The complex extract reduces inflammatory cytokines CXCL-1, IL-17, MIP2 and TNF-α in bronchoalveolar lavage fluid (BALF) of a lung injury animal model induced by fine dust, and in lung tissue, an inflammation-related gene A composition characterized in that it reduces the expression of CXCL-1, NOS-II, COX-2, TRAC, MIP2 and TNF-α, and inhibits the expression of bronchial inflammation and cough-related factors SDMA, MUC5AC, TRPV1 and TRPA1. According to claim 1,
The composition, characterized in that the composition is a pharmaceutical composition. According to claim 1,
The composition is a composition, characterized in that the food composition.

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