KR101367214B1 - A pharmaceutical for treating or preventing asthma - Google Patents

Abstract

The invention sukjihwang (熟地黃, Rehmannia glutinosa ), Pinellia ternata ), Citrus reticulata ), licorice ( Glycyrrhiza) glabra ) , Gil-kyung (桔梗, Platycodon grandiflorum ), Schizandra chinensis ) and compositions for the prevention and treatment of bronchial asthma containing a mixed extract as an active ingredient and to a dietary supplement.
The composition according to the present invention is not cytotoxic and inhibits NO production, treatment of bronchial tissue, inhibition of airway resistance, inhibition of inflammatory substances, increase of anti-inflammatory substances, inhibition of T cell activation, inhibition of lung tissue proteins STAT4 and STAT6 And hepatotoxicity.

Description

A composition for the prevention and treatment of bronchial asthma

The invention sukjihwang (熟地黃, Rehmannia glutinosa ), Pinellia ternata ), Citrus reticulata ), licorice ( Glycyrrhiza) glabra ) , Gil-Gyeong (桔梗, Platycodon grandiflorum ), Schisandra (五味子, Schizandra) chinensis ) and compositions for the prevention and treatment of bronchial asthma containing a mixed extract as an active ingredient and to a dietary supplement.

The composition according to the present invention is not cytotoxic and inhibits NO production, treatment of bronchial tissue, inhibition of airway resistance, inhibition of inflammatory substances, increase of anti-inflammatory substances, inhibition of T cell activation, inhibition of lung tissue proteins STAT4 and STAT6 And hepatotoxicity.

In general, asthma is classified as cardiac asthma and bronchial asthma, the former is a seizure dyspnea that can be seen in heart disease or high blood pressure, it can be seen as a hypersensitive state of the respiratory center due to pulmonary congestion or tissue congestion.

Bronchial asthma is a chronic lung disease in which the bronchus is narrowed and inflamed, making it difficult to breathe and wheezing when breathing.

During an asthma attack, the muscles surrounding the bronchus contract, causing the airway to become very narrow, which blocks part of the airway. If the bronchial inflammation creates mucus sputum and fills the narrowed airway, the airway may be completely blocked.

A substance that can cause asthma is an allergen (allergen), which can cause hypersensitivity of the immune system.

Here, in the inflammatory process of the body, inflammatory factors such as excess NO (nitric oxide) and PGE ₂ (prostaglandin E ₂ ) are formed by inducible NO synthase (iNOS, NOS-II) and COX2 (cyclooxygenase2). do.

Here, NOS-II is expressed when exposed to lipopolysaccharide (LPS) and various inflammatory-induced cytokines, and NOS-II is the most important and pathologically important function of NOS-II. Known.

In bronchial asthma, activated inflammatory cells, such as eosinophils, vitreous cells, and T lymphocytes, are deposited in the airways. In particular, eosinophils are made in the bone marrow and activated by moving into the airways. Cytokines secreted by T helper (Th) cells play an important role in this process.

In particular, Th1 cells secrete IFN-γ and Th2 cells secrete IL-4, IL-5, IL-10, and IL-3. Th2 cytokine expression is expressed in bronchoalveolar lavage fluid or lung tissue of asthma patients. It was found to increase.

In the past, bronchial dilators and anti-inflammatory agents have been mainly used as treatments for bronchial asthma, and emergency or severe asthma patients have used oral steroids or Omalizumab (Xolair).

However, they have a rapid response to treatment, but the symptoms are worse when they are stopped and there is a risk of shock. Therefore, there is an urgent need for complementary products to replace them.

On the other hand, the inventors of the present invention to develop a composition for the prevention and treatment of bronchial asthma using natural products, using the ripening sulfur, hwangja, dermis, licorice, Gilyeong, Schizandra, briefly look at this.

Sukji-hwang ( Rehmannia glutinosa ) is a steamed, dried, sun-dried stem after processing the stem of the worms, nourishing the liver, nourishing, moisturizing, and raising blood. (補) is known as a medicine that has the effect of filling the bone marrow (骨髓).

Major pharmacological actions include hypoglycemia, cardiovascular and diuretic effects of the circulatory system, liver function protection and antibacterial effects.

Half, Pinellia ternata ) is a tuberroot of Chunnamseong and perennial herb, and is known as a useful medicine for treating any phlegm.

Major pharmacological actions include antitussive expectoration, pneumoconiosis prevention and treatment effect, anti-earth action and detoxification.

Citrus reticulata ) is a skin of mature fruit, which is known as an medicinal herb that has the effect of communicating qi, drying and damping phlegm.

Major pharmacological actions include antitussive expectoration, antiulcer and antigastric secretion, cardiovascular cardiovascular, antiallergic, and antibacterial effects.

Licorice (甘草, Glycyrrhiza glabra ) is known as an effective medicine that does not slow down and speeds up the middle, harmonizes, soaks and detoxifies the dryness of the lungs, and harmonizes all medicines.

The main pharmacological actions include anti-digestive ulceration, spasm relief, anti-inflammatory, antiviral and detoxification, hepatoprotective and antitussive expectoration.

Gilkyung ( Platycodon grandiflorum ) removes the phlegm, relaxes the throat, straightens the lungs, loosens the symptom, removes the wet and dry blood, and relieves the blood. It is known as a medicine that warms the inside, digests food well, breaks hard things, discharges pus, and makes gugyu.

Major pharmacological actions include antitussive expectoration, obesity suppression, hypoglycemic action and gastric ulcer effect.

Schisandra (五味子, Schizandra chinensis ) is known as a medicinal herb that uses fruit and has the effect of converging intermittent, nourishing kidney, producing essence, converging sweat and converging tablets.

Major pharmacological actions include respiratory central stimulation, cardiovascular physiology, blood circulation disorders improvement, and the like.

On the other hand, when reviewing the prior patents for the composition for treating asthma using natural products, asthma therapeutic pharmaceuticals containing licorice, urea, Jeonho, Schisandra chinensis, Astronomical dong, Sangbaekpi and lobules as main ingredients in Korean Patent Application Publication No. 10-2004-0023199 Appropriate compositions are described.

An object of the present invention is to provide a composition and health functional food using natural products for the prevention and treatment of bronchial asthma.

The invention sukjihwang (熟地黃, Rehmannia glutinosa ), Pinellia ternata ), Citrus reticulata ), licorice ( Glycyrrhiza) glabra ) , Gil-Gyeong (桔梗, Platycodon grandiflorum ), Schisandra (五味子, Schizandra) chinensis ) to solve the technical problem by providing a composition for the prevention and treatment of bronchial asthma containing a mixed extract as an active ingredient.

In addition, the weight ratio of Sukji sulfur, hwangja, dermis, licorice, gilyeong and Schisandra chinensis to provide a composition for the prevention and treatment of bronchial asthma, characterized by the same, to solve the technical problem.

In addition, the solvent for the mixed extraction is to provide a composition for the prevention and treatment of bronchial asthma, characterized in that 70% methanol, to solve the technical problem.

Extract itself of the present invention is a drug that can be used with confidence even for long-term administration for the purpose of prevention because there is little toxicity and side effects.

The compositions of the present invention can be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral formulations, external preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods. Carriers, excipients and diluents that may be included in the composition comprising the extract include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose , Microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydride, hydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used.

Solid form preparations for oral administration include tablets, pills, powders, granules, capsules and the like, and such solid form preparations contain at least one or more excipients, for example, calcium carbonate, sucrose, etc. Or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium styrate talc are also used.

Oral liquid preparations include suspending agents, liquid solutions, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. .

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

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

Preferred dosages of the compositions of the present invention vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. However, for the desired effect, it is preferable to administer at 0.0001 g / kg to 10 g / kg per day.

The administration may be carried out once a day or divided into several doses. Accordingly, the dosage is not limited in any way to the scope of the present invention.

In addition, the composition of the present invention can be administered to various mammals such as rats, mice, livestock, humans. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

The present invention provides a health functional food for the prevention and improvement of bronchial asthma containing the extract obtained by the above method as an active ingredient.

The composition comprising the extract of the present invention may be used in various ways, such as drugs, foods and drinks for the prevention and improvement of bronchial asthma. Foods to which the extract of the present invention may be added include, for example, various foods, beverages, gums, teas, vitamin complexes, health supplements, and the like, and may be used in the form of powders, granules, tablets, capsules, or beverages. have.

Extracts of the present invention may be added to food or beverages for the purpose of preventing and improving bronchial asthma. At this time, the amount of the extract in the food or beverage is generally the health food composition of the present invention can be added to 1 to 5% by weight of the total food weight and the health beverage composition is 0.02 to 10g, preferably 0.3 to 100ml based It can be added at a rate of 1 g.

The health beverage composition of the present invention, in addition to containing the extract as an essential ingredient in the indicated ratio, has no particular limitation on the liquid component and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks.

Examples of the above-mentioned natural carbohydrates are conventional monosaccharides such as disaccharides such as glucose and fructose, such as maltose, sucrose and the like, and polysaccharides such as dextrin, cyclodextrin and the like. Sugars and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (e.g., Rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of said natural carbohydrates is generally about 1-20 g, preferably about 5-12 g per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring and neutralizing agents (cheese, chocolate, etc.), pectic acid and salts thereof, ilgin acid and salts thereof, organic acids , Protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like. In addition, the compositions of the present invention may contain flesh for the production of natural fruit juices and vegetable beverages. These components may be used independently or in combination. The proportion of such additives is not so critical, but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

The composition according to the present invention is not cytotoxic and inhibits NO production, treatment of bronchial tissue, inhibition of airway resistance, inhibition of inflammatory substances, increase of anti-inflammatory substances, inhibition of T cell activation, inhibition of lung tissue proteins STAT4 and STAT6 And significant effects without hepatotoxicity.

Figure 1a is a view showing the cytotoxicity test results of the hydrothermal extract.
Figure 1b is a diagram showing the cytotoxicity test results of 70% methanol extract.
Figure 2a is a view showing the anti-inflammatory test results of the hydrothermal extract.
Figure 2b is a view showing the anti-inflammatory test results of methanol extract by concentration.
Figure 2c is a diagram showing the anti-inflammatory test results of 70% methanol extract.
Figure 3 is a view showing the experimental process of the extract sample of Example 1 to the bronchial asthma model.
Figure 4 is a view showing the results of bronchial tissue treatment experiment of the extract of Example 1.
5 is a view showing the results of the airway resistance inhibitory effect of 70% MeOH extract.
6 is a view showing the results of the effect of the 70% MeOH extract on the inflammatory and anti-inflammatory substances.
7 is a view showing the results of the T cell activation inhibitory effect of the 70% MeOH extract.
8 is a view showing the results of experiments inhibiting lung tissue proteins STAT4 and STAT6 of 70% MeOH extract.
9a and 9b show the results of hepatotoxicity test of 70% MeOH extract.

The terms and words used in the present specification and claims should not be construed as limited to ordinary or dictionary terms and the inventor may properly define the concept of the term in order to best describe its invention It should be construed as meaning and concept consistent with the technical idea of the present invention.

Therefore, the examples, reference examples, experimental examples, formulation examples, and drawings described in the present specification are merely the most preferred examples of the present invention and do not represent all the technical ideas of the present invention. Therefore, It should be understood that various equivalents and modifications may be substituted for those embodiments.

Example  1. Preparation of Bronchial Asthma Prevention and Treatment Composition

1-1. Heat number  Preparation of extract

First, using a grinder to obtain the ripening sulfur, half, gilyeong, dermis, licorice, Schisandra chinensis powder, and then prepared a total of 600 g each 100 g.

600 g of the powder sample was added to a medicine bath, and 6 L of water was added to extract hot water at 100 ° C. for 3 hours. Next, the hot water extract was filtered through a 0.45 um filter paper and lyophilized to obtain 200 g of a light brown powder sample.

Powder samples were used for the experiments while stored at -80 ℃.

1-2. 70% MeOH  Preparation of extract

First, using a grinder to obtain the ripening sulfur, half, gilyeong, dermis, licorice, Schisandra chinensis powder, and then prepared a total of 600 g each 100 g.

6 g of 70% MeOH was added to 600 g of a powder sample, followed by solvent extraction at room temperature for 24 hours. Next, the extract was filtered through a 0.45 um filter paper, dried, and concentrated to 600 mL of a dark brown solution.

Concentrated solution samples were used for the experiment while storing at -80 ℃.

Reference Example  One. In vitro ( in vitro ) Prepare active experiments

1-1. Laboratory Animal and Reagent Preparation

Females of C57BL / 6 6-week old mice were purchased from Orient Bio Co., Ltd.

Cell culture reagents (Fetal bovine serum (FBS), RPMI-1640, penicillin-streptomycin, etc.) were purchased from Gibco BRL (Grand Island, USA), the culture tank was purchased from Corning (Rochester, USA).

Among the reagents used in the experiment, chloroform, HEPES, sodium dodesyl sulfate (SDS), acrylamide, bisacrylamide, LPS (Serotype: 055: B5), and Tris-HCl were purchased from SIGMA (St. Louis, USA).

1-2. Culture of Inflammatory Macrophages

To obtain inflammatory macrophages, 2.5 ml of Thioglucollate was injected intraperitoneally in C57BL / 6 mice, sacrificed by cervical dislocation 3 days later, and then shaken well by injecting 7 ml of RPMI 1640 with 10% FBS intraperitoneally, and again Leaked with a syringe.

Spilled peritoneal cells were centrifuged for 5 min at 1,200 rpm at 4 ° C, suspended in RPMI 1640 containing 10% FBS and supplemented with 1% penicillin, counting the cells, and counting the cells at 1x10 ⁶ cells / ml for 6 cm dishes or plates. Incubated for 3 hours in a 5% CO2, 37 ℃ incubator so that the macrophages adhere to the bottom and discarded the supernatant, and the cells attached to the dish or plate were examined as inflammatory macrophages.

Experimental Example  One. Example  Cytotoxicity Test of 1 Extract

1-1. Sample Preparation

By diluting each of the hydrothermal extract and 70% MeOH extract according to Example 1, an experimental sample was prepared at a concentration of 25 ug / ml, 50 ug / ml, 100 ug / ml, and 200 ug / ml.

1-2. Cytotoxicity experiment

The survival rate of macrophages was measured by MTT assay based on MTT reduction, which is converted to the purple formazan product by mitochondrial dehydrogenase of dense cells.

The exponential growth cells were suspended at a density of 1 × 10 ⁶ cells / ml in RPMI 1640 medium and treated with AETD at concentrations of 0.1 mg / ml, 0.3 mg / ml, 0.5 mg / ml and 1.0 mg / ml.

After 4 hours of incubation, MTT solution was added to incubate at a concentration of 1 mg / ml, followed by another 2 hours of incubation.

MTT-formazan product was dissolved by adding the same volume of lysis buffer (20% SDS solution containing 50% n, n-dimethylformamide, pH 4.7) and incubated for 20-24 hours.

The amount of formazan measured the amount absorbed at 570 nm.

1-3. Cytotoxicity Test Results

Figure 1a is a cytotoxicity test results of the hydrothermal extract, Figure 1b is a diagram showing the cytotoxicity test results of the 70% methanol extract.

First, the extract according to Example 1 of the present invention was named "OMC-2010", and according to the experimental sample described for each experiment, "OMC-2010" includes all or only one of the hydrothermal extract and the methanol extract. To reveal.

Experimental sample extracts were pretreated with macrophages in different concentrations, and after 24 hours, cell viability was measured.

Referring to Figures 1a and 1b, it can be seen that the hydrothermal extract and 70% methanol extract did not lower the cell viability except for 200 ug / ml.

Therefore, it can be seen that the extract according to Example 1 of the present invention has no toxic effect on macrophages.

Experimental Example  2. Example  Anti-inflammatory test of 1 extract

2-1. Sample Preparation

By diluting the hot water extract according to Example 1 to prepare a test sample at a concentration of 25 ug / ml, 50 ug / ml, 100 ug / ml.

In addition, 30%, 50%, 70%, 100% MeOH extract was prepared for the experimental sample at the same concentration.

In addition, dilution of 70% MeOH extract, a test sample was prepared at a concentration of 25 ug / ml, 50 ug / ml, 100 ug / ml.

2-2. Anti-inflammatory experiment

Among NOS types, NOS-II is produced by NOS-II absolutely and has a pathologically important function. NOS-II is known to be expressed when exposed to LPS (lipopolysaccharide) and various inflammation-induced cytokines.

Therefore, anti-inflammatory experiments were performed through experiments that inhibited LPS-induced NO production.

In the measurement process, L-arginine, a substrate of NO, is converted to L-citrulline and nitrogen monoxide, which quickly turn into stable nitrogen dioxide, nitrite and nitrate. Grease reagent (0.5% sulfanylamide, 2.5% phosphoric acid and 0.5% naphthylethyleneamine) chemically reacts with nitrite to form a purple azo salt, which is consistent with the concentration of nitrogen monoxide. The absorbance was measured at 540 nm by measuring the nitrite concentration from the salt concentration.

That is, 100 ul of grease reagent was added to each of the control and experimental samples, and the mixture was incubated at 37 ° C. for 10 minutes.

The light absorption of the sample was measured at 540 nm with a spectrophotometer (MD, U.S.A), and the concentration of nitrogen monoxide was calculated from the standard curve of nitrite.

2-3. Anti-inflammatory test results

Figure 2a is an anti-inflammatory test results of the hydrothermal extract, Figure 2b is an anti-inflammatory test results of the methanol extract by concentration, Figure 2c is a view showing the anti-inflammatory test results of the 70% methanol extract, respectively.

In order to examine the anti-inflammatory effect, pre-treatment of hot water extract, methanol extract by concentration, and 70% methanol extract test sample to macrophages of mice, and NO (nitrite oxide) were measured after 24 hours.

In Figure 2a, it can be seen that the hydrothermal extract inhibits NO production in a concentration-dependent manner, and also in Figure 2b, it can be seen that the NO production inhibitory effect of the 70% methanol extract in the methanol extract according to the concentration is remarkable.

In addition, in Figure 2c, 70% methanol extract sample is concentration-dependent, but the effect is more significant than the hydrothermal extract of Figure 2a.

In summary, Experimental Example 1 and Experimental Example 2, both hot water extract and methanol extract have no cytotoxicity and have an effect of inhibiting NO production.

In addition, it can be seen that 70% methanol extract has a significant effect of inhibiting NO production rather than hydrothermal extract.

Therefore, 70% methanol extract may suggest that the inhibitory effect of bronchial asthma may be excellent.

Reference Example  2. living body in vivo ) Prepare active experiments

2-1. Bronchial Asthma Model

Figure 3 is a view showing the experimental process of the extract sample of Example 1 to the bronchial asthma model.

Females of BALB / C 8-week-old mice were purchased from Orient Bio, Inc. (Gyeonggi-do, Seongnam, Korea), and Ovalbumin and alum were purchased from SIGMA (St. Louis, USA).

Female BALB / c mice aged 6 to 8 weeks were OVA sensitized and challenged with OVA spray.

That is, mice were sensitized by intraperitoneal injection of 20 ug / mouse of ovalbumin (OVA, Sigma) adsorbed with aluminum hydroxide (alum) on days 1 and 15, and resensitized three times by OVA spraying mice after 7 days. .

2-2. Biological ( in vivo ) Active Experiment Method

As a control group, saline was administered, anesthetized with diethylether (Wako) at various times, and blood was obtained from retro-orbital sinus plexus and stained with Diff-Quick solution (Merk) for different cell subtype counts. Was

Total leukocyte count in blood was measured after RBC lysis using RBC lysis buffer.

Bronchoalveolar lavage (BAL) was washed twice with HBSS (0.25 ml and 0.20 ml, respectively) through the bronchus to recover approximately 0.4 ml of BAL fluid from each mouse.

Total cell number was counted with a hemocytometer, and smears of BAL cells prepared with Cytospin II for heterologous cell counting were stained with Diff-Quick solution or fixed with PLP solution for immunocytochemistry.

The composition of the PLP solution is 2% paraformaldehyde (Nacalai Tesque, Kyoto, Japan), 75 mM L-lysine monohydrochloride (Wako), 10 mM sodium m-periodate (Sigma) in 37.5 mM PBS.

The percentage of different cell numbers was determined by counting at least 300 cells on an optical microscope.

After centrifugation of BAL fluid, the supernatant was stored at -20 ° C for use, and bronchial tissues were fixed with PLP solution and placed in a liquid nitrogen OCT compound.

5 um thick tissue sections were used for counting eosinophils after staining with Diff-Quick solution and for counting CD3-positive cells after immunohistochemical staining.

Experimental Example  3. Example  1 bronchial tissue treatment experiment of extract

3-1. Preparation for experiment

The bronchial asthma model was treated with 0.1 mg / kg and 1 mg / kg of hydrothermal extract and 70% MeOH extract according to Example 1, respectively.

3-2. Bronchial biopsy

For histology, BAL was performed, and then fixative solution (4% paraformaldehyde in PBS) was injected into the lavage catheter, and lung tissue was removed to further fix for 4 hours. Then, it was placed in a paraffin, stained with 4 fragments hematoxylin-eosin (H & E) and examined.

3-3. Bronchial Tissue Treatment Experiment Results

Figure 4 is a view showing the results of the bronchial tissue treatment experiment of the extract of Example 1, it can be seen the bronchial tissue state.

Allergic bronchial asthma is characterized by the infiltration of inflammatory cells and the formation of goblet cells in the strictures of the respiratory tract and bronchial glands, resulting in the deposition of mucus and marked deposition of collagen.

As shown in FIG. 4, after administration of the hydrothermal extract and the 70% MeOH extract, the bronchus appeared similar to the normal population, and the narrowing of the airways and the infiltration of inflammatory cells were shown to be markedly reduced.

In addition, the bronchial tissue treatment effect of the 70% methanol extract was more significant than the hydrothermal extract.

Experimental Example  4. 70% MeOH  Experimental Inhibition of Airway Resistance of Extracts

4-1. Preparation for experiment

Airway resistance values were measured after administration of 2.5%, 10, 20, 50 mg / ml of the 70% methanol extract according to Example 1 for one week.

4-2. Experimental Results of Airway Resistance Suppression

5 is a view showing the results of the airway resistance inhibitory effect of the 70% MeOH extract, it can be seen that 70% methanol extract significantly inhibits airway resistance.

Therefore, bronchial asthma patients have remarkably increased airway resistance, which can help alleviate this phenomenon.

Experimental Example  5. 70% MeOH  Experimental effect of extracts on inflammatory and anti-inflammatory substances

5-1. Preparation for experiment

After treating 0.1, 1 g / kg of the 70% methanol extract according to Example 1, the effect on inflammatory and anti-inflammatory substances was measured.

5-2. BAL  And cell subtype analysis and measurement of cytokine levels

After anesthetizing the mouse, the probe was collected by dissecting the bronchus, inserting the catheter, and washing twice with 0.5 ml saline. The BAL fluid was immediately centrifuged at 270 x g 4 ° C for 7 minutes and the cell pellet was mixed in 1 ml PBS. Cells were counted with a hemocytometer and stained with Diff-Quick solution to analyze cell subtypes.

The amount of cytokines secreted in the BAL fluid was determined by ELISA kit (PharMingen, USA). First, capture antibodies of IL-4, IL-5, IL-12, and INF-γ were coated on 96 wells and left at 4 ° C. for one day. The next day they were washed with PBS, treated with samples and standards and left for 1 hour. After washing three times with PBS, treated with biotinylated secondary antibody and left for 1 hour, washed three times with PBS. After the substrate was treated and left in the dark for 30 minutes, the reaction was stopped and converted to a value using a plate reader at 450 nm.

5-3. Results of effects on inflammatory and anti-inflammatory substances

6 is a view showing the results of the effect of the 70% MeOH extract on the inflammatory and anti-inflammatory substances.

70% MeOH extract significantly inhibits the production of IL-4 and IL-5, which are closely related to bronchial asthma in bronchoalveolar lavage fluid.

In addition, IFN-γ and IL-12, which are anti-inflammatory active substances, are important factors for inhibiting asthma, and it can be seen that 70% MeOH extract increases IFN-γ and IL-12.

Experimental Example  6. 70% MeOH  Inhibitory Effect of Extracts on T Cell Activation

6-1. Preparation for experiment

After treating 0.1, 1 g / kg of the 70% methanol extract according to Example 1, the effect of inhibiting T cell activation was measured.

6-2. T cell activation inhibition effect analysis method

Isolation of T cells was performed by attaching cells suspended in RPMI medium containing 10% fetal bovine serum to a plastic culture dish at 37 ° C. and 5% CO ₂ for 1.5 hours to remove adherent cells, followed by biotinylated anti-CD3 antibody (PharMingen, San Diego, CA; labeling and washing the cells with T cell separation, attaching with streptavidin-conjugated microbeads (Miltenyl Biotec, Germany), washing, and separating T cells using column and magnetic power (MACS). .

The purity of the purely isolated T cells is analyzed using a flow cytometer, and more than 95% purity cells are used for the experiment. The measurement and analysis of the cytokines IFN-γ produced by Th1 cells and the cytokines IL-4 and IL-10 produced by Th2 cells were performed by intracellular staining using a flow cytometer.

Briefly, T cells were incubated with brefeldin A (Sigma) for 4 hours, washed, and fixed with 2% paraformaldehyde. After staining with FITC conjugated anti-CD4 antibody, permeabilizing with 0.5% saponin (Sigma) and staining with PE-conjugated anti-interleukin antibody, cytokine production was analyzed by flow cytometer.

6-3. T cell activation inhibitory effect

7 is a view showing the results of the T cell activation inhibitory effect of 70% MeOH extract.

CD23 is activated in the bronchial asthma model, but the activity is inhibited in 70% methanol extract.

Experimental Example  7. 70% MeOH  Extract Lung tissue  protein STAT4  And STAT6  Inhibitory effect experiment

7-1. Experimental sample

The inhibitory effect of STAT4 and STAT6 on lung tissue administered with 1 g / kg of 70% MeOH extract was examined.

7-2. Lung tissue  protein STAT4  And STAT6  Measure

Immunoblotting was performed to measure the expression level of iNOS. BAL fluid and purified iNOS concentrated by Microcon 10 (Millipore) were loaded by non-reducing SDS-PAGE and transcribed onto membrane (Immobilon PVDF). The transcribed membrane was then blocked with 5% skim milk, and blots were incubated with fibronectin-absorbed polyclonal anti-iNOS and NF-kB goat IgG for 24 hours. After washing several times, rabbit IgG anti-goat IgG conjugated with horseradish peroxidase, which is blocked and peroxidase-bound, is applied as blots. After several washes, the blots are sufficiently wetted in chemoluminescence (ECL) solution and exposed to x-ray films.

Westernblotting is performed to measure the gelatinolytic activity of MMP, -9 and NF-kB. BAL fluid and MMP enriched by Microcon 10 (Millipore) were loaded with non-reducing SDS-PAGE and membrane (Immobilon PVDF ) Is transferred to. After the membrane was blocked with 2.2% skim milk, blots were incubated with fibronectin-absorbed polyclonal anti-STAT4, 6 rabbit IgG for 24 hours. After washing several times, rabbit IgG anti-sheep IgG conjugated with horseradish peroxidase, which is blocked and peroxidase-bound, is applied as blots. After several washes, the blots are sufficiently wetted in chemoluminescence (ECL) solution and exposed to x-ray films.

Tissues were harvested with RIPA (-) lysis buffer (1% triton, 1% deoxycholate, and 0.1% NaN ₃ in PBS), and the proteins were quantified with BCA solution.

Obtained 20 ug protein electrophoresis on 10% gel, transfer to nitrocellulose paper, blocking with 10% skim milk for 1 hour.

After washing with PBS-T (phosphate buffered saline with 0.1% Tween 20), the antibody was treated with STAT 4 or the like, washed with PBS-T for 1 hour, and then treated with secondary antibody for 30 minutes.

Then, after sufficiently washing with PBS-T, the band was confirmed by ECL detection solution (Amersham).

7-3. Lung tissue Protein Bill STAT4  And STAT6  Inhibitory effect test results

8 is a view showing the results of experiments inhibiting lung tissue proteins STAT4 and STAT6 of 70% MeOH extract.

As shown in Figure 8, when STAT4, hexavalent bronchial asthma in the lung tissue is induced to produce inflammatory substances through the phosphorylation process it can be seen that 70% methanol extract inhibited the phosphorylation process.

Experimental Example  8. 70% MeOH  Extract Hepatotoxicity  Experiment

8-1. Preparation for experiment

Females of BALB / C 8-week-old mouse were purchased from Orient Bio Co., Ltd. (Sungnam, Gyeonggi-do, S. Korea).

70% methanol extract according to Example 1 was administered 0, 1, 5, 10 mg / kg, each group was composed of six groups of 1 group.

The doses were administered at daily intervals for one week, and serum serum Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured.

8-2. Hepatotoxicity  Experiment result

9a and 9b show the results of hepatotoxicity test of 70% MeOH extract.

No group died during the observation period, and no weight change was observed in comparison with the normal group.

In terms of hepatotoxicity, there was some toxicity at 10, 5 mg / kg, but not at 1 mg / kg.

In summary, Experimental Example 1 to Example 8, 70% methanol extract is not cytotoxic and the effect of inhibiting NO production is more remarkable than the hydrothermal extract.

In addition, after administration of 70% methanol extract, the bronchus appeared similar to the normal population, and its effect was more pronounced than that of the hydrothermal extract.

In addition, 70% methanol extract is remarkably effective in inhibiting airway resistance.

In addition, 70% methanol extract inhibits the production of inflammatory substances IL-4 and IL-5, and has an effect of increasing the anti-inflammatory substances IFN-γ and IL-12.

In addition, 70% methanol extract is remarkably effective in inhibiting T cell activation.

In addition, 70% methanol extract has a significant effect on inhibiting lung tissue proteins STAT4 and STAT6.

It can also be seen that 70% MeOH extract did not show hepatotoxicity.

Claims (6)

Sukjihwang (熟地黃, Rehmannia glutinosa), contrary (半夏, Pinellia ternata), dermis (陳皮, Citrus reticulata), licorice (甘草, Glycyrrhiza glabra), gilgyeong (桔梗, Platycodon grandiflorum), Schisandra chinensis (五味子, Schizandra chinensis) mixed Contains extracts as active ingredients,
The weight ratio of the ripening sulfur, hwangja, dermis, licorice, Giltyeong and Schisandra chinensis are the same,
The solvent for the mixed extraction is 70% by weight methanol,
Efficacy of inhibiting NO production when the mixed extract is administered 0.1-1 g / kg at a concentration of 25-100 ug / ml (1 day, mouse basis), and an effect of increasing the expression of IL-12 in BAL fluid, CD23 A composition for the prevention and treatment of bronchial asthma, characterized by suppressing the expression of and the effect of inhibiting the phosphorylation of STAT4 and STAT6 protein in lung tissue.
delete delete delete Sukjihwang (熟地黃, Rehmannia glutinosa), contrary (半夏, Pinellia ternata), dermis (陳皮, Citrus reticulata), licorice (甘草, Glycyrrhiza glabra), gilgyeong (桔梗, Platycodon grandiflorum), Schisandra chinensis (五味子, Schizandra chinensis) mixed Contains extracts as active ingredients,
The weight ratio of the ripening sulfur, hwangja, dermis, licorice, Giltyeong and Schisandra chinensis are the same,
The solvent for the mixed extraction is 70% by weight methanol,
Efficacy of inhibiting NO production when the mixed extract is administered 0.1-1 g / kg at a concentration of 25-100 ug / ml (1 day, mouse basis), and an effect of increasing the expression of IL-12 in BAL fluid, CD23 Health functional food for the prevention and improvement of bronchial asthma, characterized by suppressing the expression of and the effect of inhibiting the phosphorylation of STAT4 and STAT6 protein in lung tissue.
delete

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