KR20250031503A - Composition for improvement, prevention or treatment of chronic respiratory disease with Potentilla chinensis bioconversion - Google Patents

Abstract

The present invention relates to a composition for improving, preventing or treating chronic respiratory diseases, comprising a bioconversion product of Potentilla chinensis. The bioconversion product of Potentilla chinensis according to the present invention effectively suppresses inflammation and mucoprotein expression increased by LPS in mucosal epithelial cells, thereby having excellent efficacy in improving, preventing and treating chronic respiratory diseases.

Description

Composition for improvement, prevention or treatment of chronic respiratory disease with Potentilla chinensis bioconversion

The present invention relates to a composition for improving, preventing or treating chronic respiratory diseases comprising a bioconversion product of Potentilla chinensis.

Respiratory epithelial cells maintain appropriate humidity in the respiratory tract through mucus secretion, and protect the respiratory tract from external substances and infectious agents. Mucus is a substance covering the respiratory epithelial surface, and it has physical protection and lubrication functions, and it discharges attached external substances through ciliary movement and maintains appropriate humidity. However, excessive secretion of mucus worsens lung function and causes inflammatory respiratory diseases. In respiratory inflammatory diseases such as chronic obstructive pulmonary disease or asthma, the properties of mucus change and excessive secretion occur.

This hypersecretion of mucus can worsen the course of inflammatory respiratory diseases, so the control of hypersecretion of mucus is very important. The main component that determines the physical and biochemical properties of this mucus is mucin, which is composed of high molecular weight glycoproteins. Mucin is a glycoprotein whose production is regulated by mucin genes, and it plays the biggest role in determining the rheological, physicochemical, or biochemical properties of mucus. About 20 mucin genes have been discovered so far, and the protein structure of mucus expressed by these genes is largely divided into two types: secretory mucin and membrane-attached mucin that acts as a cell receptor. Secretory mucins are known as MUC2, MUC5AC, MUC5B, and MUC8, and membrane-attached mucins are known as MUC1, MUC4, MUC11, MUC13, MUC15, MUC16, and MUC20. Among these, secretory mucins, MUC5AC and MUC5B, are known to play a biologically important role, and much research is currently being conducted on them.

There are several substances that increase mucus gene expression and mucus secretion in mucosal epithelial cells. For example, cytokines and inflammatory substances such as interleukin-1β (IL-1β), lipopolysaccharide (LPS), and phorbol myristate acetate (PMA) increase mucus secretion in the respiratory tract. Among these, LPS is a substance located in the outer membrane of Gram-negative bacteria that stimulates macrophages to induce reactive oxygen species (ROS), cytokines, and inflammatory mediators. In chronic respiratory inflammatory diseases such as chronic obstructive pulmonary disease and asthma, reactive oxygen, cytokines, and inflammatory mediators increase the expression of MUC5AC and MUC5B mucus genes, and the properties of mucus may change or excessive secretion of mucus may occur, which may worsen the course of the disease.

Accordingly, the inventors of the present invention completed the present invention by confirming the inhibitory effect of the bioconversion product of Wei Ling Chae on inflammation and mucoprotein expression increased by LPS in mucosal epithelial cells after much effort.

Korean Patent Registration No. 10-1569876 (November 11, 2015) discloses a pharmaceutical composition containing a mixed herbal extract for preventing or treating respiratory diseases. Korean Patent Registration No. 10-1103393 (December 30, 2011) discloses a composition containing a herbal extract for preventing and treating respiratory diseases.

One object of the present invention is to provide a use for improving, preventing or treating chronic respiratory diseases, which comprises a culture solution obtained by inoculating an Aspergillus Kawachii strain into a medium containing an extract of Wei Ling Chae and culturing it, or a filtrate obtained by filtering the culture solution, or a dry powder obtained by drying the filtrate.

The present invention provides a pharmaceutical composition for preventing or treating chronic respiratory disease, which contains a culture solution obtained by inoculating an Aspergillus Kawachii strain into a medium containing an extract of Wei Ling Chae and culturing it, or a filtrate obtained by filtering the culture solution, or a dry powder obtained by drying the filtrate.

At this time, the extract of the above-mentioned Weolreungchae is preferably extracted by adding ethanol as an extraction solvent to the Weolreungchae.

At this time, the chronic respiratory disease may be, for example, any one selected from asthma, chronic obstructive pulmonary disease, and acute respiratory distress syndrome.

Meanwhile, the present invention provides a food composition for improving chronic respiratory disease, which contains a culture solution obtained by inoculating an Aspergillus Kawachii strain into a medium containing an extract of Wiryungchae and culturing it, or a filtrate obtained by filtering the culture solution, or a dry powder obtained by drying the filtrate.

At this time, the extract of the above-mentioned Weolreungchae is preferably extracted by adding ethanol as an extraction solvent to the Weolreungchae.

At this time, the chronic respiratory disease may be, for example, any one selected from asthma, chronic obstructive pulmonary disease, and acute respiratory distress syndrome.

The bioconversion product of Wei Ling Chae according to the present invention effectively suppresses inflammation and mucoprotein expression increased by LPS in mucosal epithelial cells, thereby showing excellent efficacy in improving, preventing and treating chronic respiratory diseases.

Figure 1 shows the results of confirming the antioxidant efficacy of the extract of Wei Ling Chae (PCE) and its biotransformation product (PCB) according to concentration (a) DPPH free radical scavenging activity confirmation result, b) ABTS cation radical activity confirmation result). The indicated values represent the mean ± SEM of the values of three repeated analyses, and * indicates a significant difference (p<0.05) from the group treated with AA (ascorbic acid) as the control group.
Figure 2 shows the results of confirming the cytotoxicity of the extract of Wei Ling Chae (PCE) and its biotransformation product (PCB) according to concentration (a) Cytotoxicity results for NCI-H292 cells, b) Cytotoxicity results for Raw264.7 cells). The values shown represent the mean ± SEM of the values of three repeated analyses, and * indicates a significant difference (p<0.05) from the control group.
Figure 3 shows the results of examining the effects of PCE and its biotransformation product (PCB) on the inflammatory response induced by LPS in Raw264.7 cells at different concentrations. (a) Results of examining the effect on NO production, b) Results of examining the effect on TNF-α expression, c) Results of examining the effect on IL-1β expression, d) Results of examining the effect on PGE2 secretion, e) Results of western blot examining the effect on the level of COX-2 protein production, and f) A graph quantifying the effect on the level of COX-2 protein production. The values shown represent the mean ± SEM of three repeated analyses, and * indicates a significant difference (p<0.05) from the LPS treatment group.
Figure 4 shows the results of examining the effects of PCE and its biotransformation product (PCB) on the expression of inflammatory genes (MUC5AC and MUC5B) induced by LPS in NCI-H292 cells according to their concentrations (a) MUC5AC protein production level, b) MUC5B protein production level, c) MUC5AC mRNA expression level, and d) MUC5B mRNA expression level). The indicated values represent the mean ± SEM of three repeated analyses, and * indicates a significant difference (p<0.05) from the LPS treatment group.

In one embodiment of the present invention, it was confirmed that the bioconversion product of Wei Ling Chae effectively suppresses inflammation and mucoprotein expression increased by LPS in mucosal epithelial cells, thereby having excellent effects on the improvement, prevention and treatment of chronic respiratory diseases. Specifically, it was confirmed that the bioconversion product of Wei Ling Chae according to the present invention effectively suppresses inflammation and mucoprotein secretion induced by LPS, and particularly excellent antioxidant activity in human respiratory mucosal cells.

Accordingly, the present invention provides a pharmaceutical composition for preventing or treating chronic respiratory disease, which contains a culture solution obtained by inoculating an Aspergillus Kawachii strain into a medium containing an extract of Wiryungchae and culturing it, or a filtrate obtained by filtering the culture solution, or a dry powder obtained by drying the filtrate.

Potentillae Herba (萎陵菜) refers to the whole plant of Potentilla chinensis Ser., a perennial plant in the Rosaceae family. Potentillae Herba is named so because it grows flat on the ground like a scab. It is native to Korea, China, and Japan, and is said to have been eaten as an famine relief plant, while in Japan it has only been used as an antipyretic by the private sector. It is known that medicine using Potentillae has almost no odor, tastes bitter, and is cold in nature. In addition, Potentillae is used to reduce large intestine fever, and in oriental medicine, it is used for dysentery, uterine bleeding, intestinal bleeding, hemorrhoidal bleeding, nosebleed, hemoptysis, blood in the urine, and fever due to cancer.

At this time, the extract of the above-mentioned Weolreungchae is preferably extracted by adding ethanol as an extraction solvent to the Weolreungchae.

In one embodiment of the present invention, it was confirmed that the bioconversion product of the present invention has better antioxidant efficacy, anti-inflammatory efficacy, and mucus protein expression inhibition efficacy compared to the extract of the present invention at the same treatment concentration.

The term “prevention” as used in the present invention means any act of suppressing or delaying the onset of a chronic respiratory disease by administering a pharmaceutical composition according to the present invention.

The term "treatment" as used in the present invention means any act by which symptoms of a chronic respiratory disease are improved or beneficially changed by administration of a pharmaceutical composition according to the present invention.

At this time, the chronic respiratory disease may be, for example, any one selected from asthma, chronic obstructive pulmonary disease, and acute respiratory distress syndrome.

Specifically, chronic respiratory diseases (CRD) are chronic diseases of the airways and other structures of the lungs, and the most representative ones are asthma, chronic obstructive pulmonary disease, and acute respiratory distress syndrome. In particular, chronic respiratory diseases are characterized by the aggravation of the disease through hypersecretion of mucus. There is no known cure for chronic respiratory diseases, but currently, treatments that help to expand the main airways and improve dyspnea are being used to control symptoms and improve quality of life.

Asthma is an inflammatory airway obstructive disease that causes breathing difficulties. Asthma patients complain of symptoms such as wheezing (wheezing sound), shortness of breath, and coughing due to obstruction of the airway. Symptoms tend to be worse at night than during the day. In addition, they may have difficulty exhaling (breathing) due to excessive phlegm (mucus) formation.

Chronic obstructive pulmonary disease (COPD, COLD, COAD) is a type of obstructive lung disease that chronically causes poor airflow. It usually worsens over time. The main symptoms include shortness of breath, coughing, and phlegm. Most patients with chronic bronchitis also have chronic obstructive pulmonary disease. Chronic obstructive pulmonary disease is caused by various irritants causing inflammation in the lungs, narrowing the small airways and destroying lung tissue, which causes emphysema. Worldwide, 329 million people, or nearly 5% of the world's population, suffer from chronic obstructive pulmonary disease. The economic cost of chronic obstructive pulmonary disease in 2010 was estimated at 2.1 billion dollars. Chronic obstructive pulmonary disease was the third leading cause of death in 2012, causing more than 3 million deaths.

Acute respiratory distress syndrome (ARDS) is a respiratory failure caused by acute inflammation in the human lungs. In ARDS, the permeability of the alveolar capillary membrane increases due to various causes, causing fluid to leak into the alveolar space, resulting in hypoxia and inflammatory acute lung injury. Representative symptoms of ARDS include dyspnea, increased respiratory rate, tachycardia, and bluish skin color.

The pharmaceutical composition of the present invention may additionally contain one or more effective ingredients exhibiting the same or similar function as the bioconversion product of the present invention described above.

The pharmaceutical composition of the present invention may additionally contain a pharmaceutically acceptable carrier in addition to the bioconversion product of Wei Ling Chae.

The type of carrier that can be used in the present invention is not particularly limited, and any carrier commonly used in the relevant technical field can be used. Non-limiting examples of the carrier include lactose, dextrose, sucrose, sorbitol, mannitol, saline solution, sterile water, Ringer's solution, buffered saline, albumin injection solution, xylitol, erythritol, maltitol, maltodextrin, glycerol, ethanol, etc. These may be used alone or in combination of two or more.

In addition, the pharmaceutical composition of the present invention may be used by adding other pharmaceutically acceptable additives, such as antioxidants, excipients, diluents, buffers, or bacteriostatic agents, if necessary, and may be used by additionally adding surfactants, binders, fillers, bulking agents, wetting agents, disintegrants, dispersants, or lubricants.

In the pharmaceutical composition of the present invention, the bioconversion product of Wei Ling Chae may be included in an amount of 0.00001 wt% to 99.99 wt% based on the total weight of the pharmaceutical composition, preferably 0.1 wt% to 90 wt%, more preferably 0.1 wt% to 70 wt%, and even more preferably 0.1 wt% to 50 wt%, but is not limited thereto and may be variously changed depending on the condition of the administration subject, the type and degree of specific symptom, etc. If necessary, it may also be included in the total content of the pharmaceutical composition.

That is, the pharmaceutically effective amount, effective dosage of the pharmaceutical composition of the present invention may vary depending on the formulation method of the pharmaceutical composition, the administration method, the administration time, and/or the administration route, and may vary depending on various factors including the type and degree of the response to be achieved by the administration of the pharmaceutical composition, the type, age, weight, general health condition, symptoms or degree of the disease, sex, diet, excretion, drugs used simultaneously or simultaneously in the subject, other components of the composition, and similar factors well known in the medical field, and a person having ordinary knowledge in the relevant technical field can easily determine and prescribe an effective dosage for the intended treatment. For example, the daily dosage of the pharmaceutical composition of the present invention is about 0.01 to 1,000 mg/kg, preferably 0.1 to 100 mg/kg, and may be administered once or several times a day in divided doses.

The pharmaceutical composition of the present invention may be administered once a day or may be administered in several divided doses. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. Taking all of the above factors into consideration, it may be administered in an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art.

The pharmaceutical composition of the present invention can be additionally used in combination with various methods such as hormone therapy and drug therapy to prevent or treat chronic respiratory diseases.

The term "administration" used in the present invention means introducing the pharmaceutical composition of the present invention to a patient by any appropriate method, and the administration route and administration method of the pharmaceutical composition of the present invention may each be independent, and any administration route and administration method may be followed without particular limitation, as long as the pharmaceutical composition can reach the desired site.

The above pharmaceutical composition can be administered orally or parenterally, and can be formulated and used in various suitable dosage forms for oral or parenteral administration.

Non-limiting examples of oral administration preparations using the pharmaceutical composition of the present invention include oily suspensions, troches, lozenges, tablets, aqueous suspensions, prepared powders, granules, emulsions, hard capsules, soft capsules, syrups or elixirs.

In order to formulate the pharmaceutical composition of the present invention for oral administration, a binder such as sorbitol, mannitol, starch, amylopectin, cellulose, lactose, saccharose or gelatin; a lubricant such as magnesium stearate, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax; an excipient such as dicalcium phosphate; a disintegrant such as corn starch or sweet potato starch; and a flavoring agent, syrup, sweetener, etc. can also be used. Furthermore, in the case of capsules, in addition to the above-mentioned substances, a liquid carrier such as fatty oil can be additionally used.

As a parenteral administration method of the pharmaceutical composition of the present invention, intramuscular administration, transdermal administration, intravenous administration, intraperitoneal administration, or subcutaneous administration may be used, and a method of applying, spraying, or inhaling the composition to the diseased area may also be used, but is not limited thereto.

Non-limiting examples of parenteral preparations using the pharmaceutical composition of the present invention include injections, suppositories, ointments, powders for application, oils, powders for respiratory inhalation, aerosols for sprays, creams, etc.

In order to formulate the pharmaceutical composition of the present invention for parenteral administration, a sterile aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a freeze-dried preparation, an external preparation, etc. can be used. As the non-aqueous solvent and the suspension, a vegetable oil such as olive oil, propylene glycol, polyethylene glycol, an injectable ester such as ethyl oleate, etc. can be used.

When the pharmaceutical composition of the present invention is formulated as an injection, the pharmaceutical composition of the present invention may be prepared as a solution or suspension by mixing it in water with a stabilizer or buffer, and this may be formulated for unit dose in an ampoule or vial.

When the pharmaceutical composition of the present invention is formulated as an aerosol, a propellant or the like can be mixed together with additives so that the water-dispersed concentrate or wet powder can be dispersed.

When the pharmaceutical composition of the present invention is formulated into an ointment, oil, cream, powder for application, external skin preparation, etc., the formulation may be formulated using animal oil, vegetable oil, wax, paraffin, polyethylene glycol, silicone, bentonite, silica, talc, starch, tragacanth, cellulose derivatives, zinc oxide, etc. as a carrier.

Meanwhile, the present invention provides a food composition for improving chronic respiratory disease, which contains a culture solution obtained by inoculating an Aspergillus Kawachii strain into a medium containing an extract of Wiryungchae and culturing it, or a filtrate obtained by filtering the culture solution, or a dry powder obtained by drying the filtrate.

At this time, the extract of the above-mentioned Weolreungchae is preferably extracted by adding ethanol as an extraction solvent to Weolreungchae. A specific description thereof has been mentioned above, and a separate description is omitted to avoid unnecessary duplication.

At this time, the chronic respiratory disease may be, for example, one selected from asthma, chronic obstructive pulmonary disease, and acute respiratory distress syndrome. A specific description thereof has been mentioned above, and a separate description is omitted to avoid unnecessary duplication.

The term "improvement" of the present invention means any act by which a chronic respiratory disease is improved or beneficially changed by administration of the composition of the present invention.

In the food composition of the present invention, it is preferable that the bioconversion product of the above-mentioned Weirungchae is included in an amount of 0.00001 to 50 wt% relative to the food composition. If it is less than 0.00001 wt%, the effect is insignificant, and if it exceeds 50 wt%, the increase in effect relative to the amount used is insignificant, making it uneconomical.

The food composition of the present invention may be, for example, any one selected from among noodles, gum, dairy products, ice cream, meat, grains, caffeinated beverages, general beverages, chocolate, bread, snacks, confectionery, candy, pizza, jelly, alcoholic beverages, alcohol, vitamin complexes, health functional foods, and other health supplements, but is not necessarily limited thereto.

The above “health functional food” refers to food manufactured and processed using raw materials or ingredients with functionality useful to the human body according to Act No. 6727 on Health Functional Foods, and “functionality” refers to consumption for the purpose of obtaining useful effects for health purposes, such as regulating nutrients for the structure and function of the human body or physiological effects.

The food composition of the present invention may include additional ingredients that are commonly used to improve odor, taste, sight, etc. For example, it may include biotin, folate, pantothenic acid, vitamins A, C, D, E, B1, B2, B6, B12, niacin, etc. In addition, it may include minerals such as chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), zinc (Zn), iron (Fe), calcium (Ca), etc. In addition, it may include amino acids such as cysteine, valine, lysine, and tryptophan. In addition, food additives such as preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), coloring agents (tar color, etc.), coloring agents (sodium nitrite, sodium nitrate, etc.), bleaching agents (sodium sulfite), bactericides (bleaching powder and high-purity bleaching powder, sodium hypochlorite, etc.), leavening agents (alum, D-potassium hydrogen tartrate, etc.), reinforcing agents, emulsifiers, thickeners (glucose), film-forming agents, antioxidants (butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), etc.), seasonings (MSG, monosodium glutamate, etc.), sweeteners (dulcin, cyclamate, saccharin, sodium, etc.), flavorings (vanillin, lactones, etc.), gum bases, antifoaming agents, solvents, and improvers can be added. The above additives can be selected depending on the type of food and used in an appropriate amount.

When the food composition of the present invention is used as a food additive, it can be added as is or used together with other foods or food ingredients, and can be used appropriately according to a conventional method.

Hereinafter, the contents of the present invention will be described in more detail through the following examples or experimental examples. However, the scope of the rights of the present invention is not limited to the following examples or experimental examples, but includes modifications of technical ideas equivalent thereto.

Materials and Methods

1. Materials

LPS was purchased from Sigma (St. Louis, MO, USA), RPMI 1640 medium and DMEM medium were purchased from Invitrogen (Carlsbad, CA, USA), and fetal bovine serum (FBS) was purchased from Hyclone Laboratories (Logan, UT, USA). COX-2 was purchased from Cayman, and anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

2. Cell culture and treatment

NCI-H292 cells (Korea Cell Line Bank), a human pulmonary mucoepidermoid carcinoma cell line, were cultured in RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA) containing 100 U/㎖ penicillin, 100 ㎍/㎖ streptomycin, and 10% FBS (fetal bovine serum; GIBCO, Grand Island, NY, USA) at 37℃ under 5% carbon dioxide. Cells were seeded at a density of 1 × 10 ⁶ cells/well in a 6-well plate and cultured. When 70-80% confluent, the cells were replaced with RPMI 1640 medium containing 0.5% FBS and cultured for 24 hours.

3. Antioxidant capacity evaluation

To measure the antioxidant activity, DPPH (1,1-diphenyl-2-picrylhydrazyl) and ABTS (2,2'-azinobis; 3-ethylbenzothiazoline-6-sulfonic acid) were measured. The scavenging activity for DPPH radical was measured according to the method of Blios (1958), and the inhibition rate (%) was calculated by (1-absorbance of the reaction group/absorbance of the control group) × 100. The measurement of ABTS radical cation decolorization was measured according to the method of Fellegrin et al. (1999), and the inhibition rate (%) was calculated by (1-absorbance of the reaction group/absorbance of the control group) × 100.

4. Cytotoxicity evaluation

NCI-H292 cells and Raw264.7 cells were seeded at 5 × 10 ⁴ cells/well in a 96-well culture plate and cultured in RPMI and DMEM containing 10% FBS and 1% streptomycin for 24 hours at 37°C and 5% CO _2. When the cells were 80–90% confluent, the medium was removed and the samples were treated with the concentrations in each well of RPMI and DMEM medium without FBS for 24 hours. After that, cytotoxicity evaluation was performed using MTT (5 mg/mL) reagent. 20 ㎕ of MTT (5 mg/mL) reagent was treated, cultured at 37°C for 3 hours, and the reaction was measured by absorbance at 540 nm.

5. Quantitative real-time PCR analyze

NCI-H292 cells were pretreated with samples at various concentrations, and then treated with LPS at a concentration of 100 ng/㎖ 1 hour later. Raw264.7 cells were pretreated with LPS at a concentration of 1 ㎍/㎖, and then treated with samples at various concentrations 1 hour later. After 8 hours, the cultured cells were washed with cold PBS, and total mRNA was extracted using Quiazol lysis reagent (Qiagen, Greenwich, U.K.).

RNA extracted from cells was used to synthesize cDNA according to the manual using the GoscripTM Reverse Transcriptase system (Promega, Madison, WI). Real-time PCR was performed on the synthesized cDNA using Goscrip ^ⓡ qPCR Master Mix (Promega, Madison, WI) and primers. Oligonucleotide primers used in PCR were designed based on previously discovered base sequences, and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as an internal positive control for each reaction. The primers used in the experiment are shown in Table 1 below and were purchased from Bioneer, Korea. The PCR reaction conditions for MUC5AC and MUC5B mRNA were denaturation (95℃, 30 sec), annealing (60℃, 60 sec), and extension (72℃, 60 sec).

Target gene Primer sequence MUC5AC forward 5'-TCA ACG GAG ACT GCG AGT ACA C-3′ MUC5AC reverse 5'-CTT GAT GGC CTT GGA GCA-3' MUC5B forward 5'-CAC ATC CAC CCT TCC AAC-3' MUC5B reverse 5'-GGC TCA TTG TCG TCT CTG-3' GAPDH forward 5'-CCT CCA AGG AGT AAG ACC CC-3' GAPDH reverse 5'-AGG GGT CTA CAT GGC AAC TG-3'

6. ELISA (Enzyme-linked immunosorbent assay) analysis

ELISA was used to measure MUC5AC, MUC5B, PGE2, TNF-α, and IL-1β. First, NCI-H292 cells were pretreated with samples at various concentrations, and then 1 hour later, they were treated with 100 ng/㎖ LPS. Raw264.7 cells were pretreated with 1 ㎍/㎖ LPS, and then 1 hour later, the samples were treated with various concentrations. The supernatants were collected after 24 hours. ELISA analysis was performed using the collected supernatants according to the manufacturer's manual.

7. Western Blot Analysis (Western blot analysis)

Raw264.7 cells were seeded at 5× ¹⁰⁵ cells/well in a 6-well plate and cultured for 24 hours, after which the samples were treated at various concentrations. After 1 hour, LPS (1 μg/ml) was treated and cultured for 24 hours. After 24 hours, the supernatant was removed, washed with cold PBS, and the cells were harvested and protein was extracted by adding RIPA buffer (Thermo, USA) solution. The extracted protein was quantified using a BCA protein kit.

To investigate the protein production of cytokines and COX-2 mucin genes by samples in Raw264.7 cells, Raw264.7 cells were pretreated with 1 μg/㎖ of LPS, and the samples were treated at various concentrations 1 hour later. Then, 20 μg of protein was separated using 10% Mini-PROTEAN® TGX™ Precast Protein Gels (Bio-Rad, Hercules, CA, USA) and transferred to Immobilon®-P PVDF Membrane (Merck KGaA, Darmstadt, Germany). COX-2 and β-actin were used as primary antibodies, and HRP-conjugated mouse antibody and HRP-conjugated rabbit antibody were used as secondary antibodies. After completing the antibody reaction, the reaction was performed using Immobilon Western Chemiluminescent HRP Substrate (Merck KGaA, Darmstadt, Germany) and then measured using LAS-4000 (Fujifilm Life Science, Tokyo, Japan).

To investigate the expression of MUC5AC and MUC5B mucin genes and protein production, NCI-H292 cells were pretreated with samples at various concentrations, and then 1 hour later, LPS (from Pseudomonas aeruginosa 10, L8643, Sigma-Aldrich) was administered at a concentration of 1 μg/mL.

8. Statistics

All experiments were repeated at least three times, and the experimental results were expressed as the mean ± standard deviation. The significance of each experimental result was examined using SPSS version 22.0 (IBM Corp., Armonk, NY, USA). A p value of less than 0.05 compared to the control group was considered significant, and Student's t-test was used.

[ Example 1: Wireungchae Biotransformation products manufacturing]

The wilungchae used in this experiment was purchased directly from (Jecheon Medicinal Herb Living Health) and used after washing.

1. Wireungchae Preparation of extracts

70% ethanol (2,000 ㎖) was added to 500 g of Wireungchae as an extraction solvent, and the extract was stirred at room temperature for 24 hours. Each extract was filtered through Whatman No. 1 filter paper (Whatman Inc., Piscataway, NJ, USA) and then freeze-dried at -85℃ using a freeze-dryer (FD8518, Ilshinbiobase Co., Dongducheon, Korea). The freeze-dried product was dissolved in distilled water and used in the following experiments.

2. Wireungchae Extract of Biotransformation products manufacturing

The above-mentioned extract of Wiryungchae was added to a medium using wheat bran as a carbon source, and Aspergillus Kawachii ( Aspergillus Kawachii ) was inoculated and cultured at 30℃ for 3 days. Aspergillus Kawachii was provided by the Korea Center for Microorganism Conservation (KCCM 32819). After culture was completed, the culture solution was obtained by filtration, and this was freeze-dried to prepare a bioconversion product of Wiryungchae extract. The sample prepared in this way was used in the following experiments at various concentrations.

[ Experimental example 1: According to the present invention Wireungchae Extracts and their derivatives Biotransformation products [Antioxidant activity evaluation]

The antioxidant activities of PCE (PCE), its biotransformation product (PCB), and the synthetic antioxidant vitamin C (ascorbic acid; AA) were compared by measuring DPPH and ABTS scavenging activities.

As a result of the experiment, the extract of Wei Ling Chae and its bioconversion product showed high scavenging activity in a concentration-dependent manner, similar to the positive control group treated with vitamin C. In particular, at the highest treatment concentration of 1,000 ㎍/㎖, the extract of Wei Ling Chae and its bioconversion product showed excellent scavenging activity of 60.5% and 68.5%, respectively (Fig. 1a).

In addition, the ABTS scavenging activity showed a similar trend to the DPPH scavenging activity. That is, the extract of Ginkgo biloba and its bioconversion product showed high scavenging activity in a concentration-dependent manner, similar to the positive control group, the vitamin C treatment group, and in particular, at the highest treatment concentration of 1,000 ㎍/㎖, the extract of Ginkgo biloba and its bioconversion product showed excellent scavenging activity of 73.2% and 80.2%, respectively (Fig. 1b).

Figure 1 shows the results of confirming the antioxidant efficacy of the extract of Wei Ling Chae (PCE) and its biotransformation product (PCB) according to concentration (a) DPPH free radical scavenging activity confirmation result, b) ABTS cation radical activity confirmation result). The indicated values represent the mean ± SEM of the values of three repeated analyses, and * indicates a significant difference (p<0.05) from the group treated with AA (ascorbic acid) as the control group.

[ Experimental example 2: According to the present invention Wireungchae Extracts and their derivatives Biotransformation products [Cytotoxicity evaluation]

MTT assay was performed to determine the cytotoxicity of PCE and its biotransformation product (PCB) using NCI-H292 cells and Raw264.7 cells.

As a result of the experiment, when the extract of Wi-reungchae and its bioconversion product were treated at various concentrations (25, 50, 100 ㎍/㎖), the extract of Wi-reungchae and its bioconversion product did not show any toxicity in both NCI-H292 cells and Raw264.7 cells in the 25 and 50 ㎍/㎖ treatment groups, but in the 100 ㎍/㎖ treatment group, the viability of NCI-H292 cells was confirmed to decrease to less than 70%, indicating that the extract of Wi-reungchae and its bioconversion product inhibited cell viability when treated at a concentration of 100 ㎍/㎖ (Fig. 2).

Therefore, in the following experiments, the extract of Wei Ling Chae and its biotransformation product were treated at concentrations of 25 and 50 ㎍/㎖.

Figure 2 shows the results of confirming the cytotoxicity of the extract of Wei Ling Chae (PCE) and its biotransformation product (PCB) according to concentration (a) Cytotoxicity results for NCI-H292 cells, b) Cytotoxicity results for Raw264.7 cells). The values shown represent the mean ± SEM of the values of three repeated analyses, and * indicates a significant difference (p<0.05) from the control group.

[ Experimental example 3: According to the present invention Wireungchae Extracts and their derivatives Biotransformation products [Anti-inflammatory activity evaluation]

It is known that the early inflammatory response is induced by iNOS (inducible nitric oxide synthase) and COX-2 (cyclooxygenase-2) and inflammatory mediators (NO, PGE2) produced by these proteins.

Therefore, in this experiment, we aimed to compare the anti-inflammatory effects of PCE (polychloroprene extract) and its biotransformation product (PCB).

First, the amount of nitric oxide production significantly decreased in a concentration-dependent manner at all treatment concentrations of the extract of Wei Ling Chae and its bioconversion product. In particular, the amount of nitric oxide production decreased by 15% and 25% in the 50 ㎍/㎖ treatment group, respectively (Fig. 3a).

In addition, the expression of PGE _{2 ,} TNF-α, and IL-1β induced by LPS was significantly decreased in a concentration-dependent manner at all treatment concentrations of the extract of Gastrodia elata and its biotransformation product. In particular, in the group treated with 50 ㎍/㎖ of the biotransformation product of Gastrodia elata extract, PGE _{2 ,} TNF-α, and IL-1β were inhibited by 35%, 58%, and 52%, respectively (Fig. 3 b, c, and d).

Meanwhile, Western blot was performed to investigate the effect of the extract of Wei Ling Chae and its biotransformation product on the expression of COX-2.

As a result of the experiment, it was confirmed that the group treated with LPS showed an increase in the production of COX-2 protein compared to the group not treated with LPS. On the other hand, the group treated with the extract of Welch's rhizome and its biotransformation product showed a significant decrease in the expression of COX-2 protein compared to the LPS-treated group, indicating that the COX-2 protein inhibition effect showed a similar tendency to the inhibition effect on the production of NO, PGE _{2 ,} TNF-α, and IL-1β described above (Fig. 3e and f). Therefore, it was confirmed that the extract of Welch's rhizome and its biotransformation product had an inflammation inhibition effect by inhibiting COX-2 expression.

Figure 3 shows the results of examining the effects of PCE and its biotransformation product (PCB) on the inflammatory response induced by LPS in Raw264.7 cells at different concentrations. (a) Results of examining the effect on NO production, b) Results of examining the effect on TNF-α expression, c) Results of examining the effect on IL-1β expression, d) Results of examining the effect on PGE2 secretion, e) Results of western blot examining the effect on the level of COX-2 protein production, and f) A graph quantifying the effect on the level of COX-2 protein production. The values shown represent the mean ± SEM of three repeated analyses, and * indicates a significant difference (p<0.05) from the LPS treatment group.

[ Experimental example 4: According to the present invention Wireungchae Extracts and their derivatives Biotransformation products Mucus protein expression Reverse power evaluation]

MUC5AC and MUC5B are glycoproteins secreted from goblet cells of the respiratory mucosa and play an important role in maintaining the function of mucociliary motor cells. However, excessive secretion of glycoproteins is closely related to chronic obstructive pulmonary disease and inflammatory respiratory diseases. LPS, PMA (phorbol myristate acetate), and various ILs are known to increase the production of MUC5AC and MUC5B mucoproteins, and they are known to be regulated by various signal transduction systems.

In this experiment, we confirmed that LPS treatment of NCI-H292 cells induced the expression of MUC5AC and MUC5B mucin genes and the production of mucin proteins. We also analyzed the effects of PCE and its biotransformation product (PCB) on LPS-induced MUC5AC and MUC5B expression and the production of mucin proteins using RT-PCR and ELISA.

As a result of the experiment, the mRNA expression of MUC5AC and the resulting mucoprotein production induced by LPS in NCI-H292 cells were statistically significantly decreased in the group pretreated with the extract of Ginkgo biloba and its bioconversion product. Specifically, as the treatment concentration of the extract of Ginkgo biloba and its bioconversion product increased, the mRNA expression decreased, and a particularly large decrease was observed in MUC5B. In the case of the resulting mucoprotein production, a large decrease in MUC5AC protein was observed. In the treatment groups of the extract of Ginkgo biloba and its bioconversion product, the activity was observed to be higher in the treatment group of the bioconversion product of the extract of Ginkgo biloba (Fig. 4).

Figure 4 shows the results of examining the effects of PCE and its biotransformation product (PCB) on the expression of inflammatory genes (MUC5AC and MUC5B) induced by LPS in NCI-H292 cells according to their concentrations (a) MUC5AC protein production level, b) MUC5B protein production level, c) MUC5AC mRNA expression level, and d) MUC5B mRNA expression level). The indicated values represent the mean ± SEM of three repeated analyses, and * indicates a significant difference (p<0.05) from the LPS treatment group.

In summary, the results above show that the extract of Wei Ling Chae and its biotransformation products are involved in the regulation of mucus hypersecretion in NCI-H292 cells, and that they have the potential to be utilized as novel regulators that can suppress mucus hypersecretion.

Claims (6)

A pharmaceutical composition for preventing or treating a chronic respiratory disease, comprising a culture solution obtained by inoculating an Aspergillus kawachii strain into a medium containing an extract of Wiryungchae and culturing it, or a filtrate obtained by filtering the culture solution, or a dry powder obtained by drying the filtrate.
In the first paragraph,
The above extract of Weolingchae is,
A pharmaceutical composition obtained by extracting Wiryungchae using ethanol as an extraction solvent.
In the first paragraph,
The above chronic respiratory diseases are,
A pharmaceutical composition selected from the group consisting of asthma, chronic obstructive pulmonary disease and acute respiratory distress syndrome.
A food composition for improving chronic respiratory disease, comprising a culture solution obtained by inoculating an Aspergillus kawachii strain into a medium containing an extract of Wiryungchae and culturing it, or a filtrate obtained by filtering the culture solution, or a dry powder obtained by drying the filtrate.
In paragraph 4,
The above extract of Weolingchae is,
A food composition obtained by extracting Weileungchae using ethanol as an extraction solvent.
In paragraph 4,
The above chronic respiratory diseases are,
A food composition selected from asthma, chronic obstructive pulmonary disease and acute respiratory distress syndrome.