KR20180055038A - Composition for treating asthma comprising Platycodon grandiflorum extract - Google Patents

Abstract

A Platycodon grandiflorum extract of the present invention is a natural extract derived from plants and having little side effects on a human body, wherein a composition for treating asthma comprising a Platycodon grandiflorum extract as an active ingredient according to the present invention can effectively treat asthma while minimizing side effects caused by the use of existing chemotherapeutic agents.

Description

 TECHNICAL FIELD The present invention relates to a composition for treating asthma comprising Platycodon grandiflorum extract,

The present invention relates to a composition for treating asthma comprising as an active ingredient a bellflower extract, and more particularly, to a composition for treating asthma that is effective for preventing or ameliorating asthma without adverse side effects by using bellflower, a natural extract.

Asthma is a chronic allergic respiratory disease. It is a condition in which the bronchus in the lung is very sensitive. Sometimes the bronchus narrows, causing the breathing, cramp and breathing sounds to be heard. It is an allergic disease caused by an inflammatory reaction. These symptoms appear repeatedly, seizures, and the bronchial mucosa is swollen by the airway's pathway, which causes the airways to swell, causing the bronchial muscles to spasm, resulting in blockage of the bronchial tree and breathing.

Allergic diseases, such as asthma, are caused by the interaction of genetic and environmental factors, and are usually difficult to cure. Factors causing asthma include causative agents and worsening factors. The most common allergens are house dust mites, pollen, animal hair, dandruff, cockroaches, food, and drugs. Typical deterioration factors include cold, tobacco smoke and indoor pollution, air pollution, food Additives, physical activity such as exercise, climate change, dustiness, and stress.

Asthma is a chronic inflammatory disease of the airways. Airway inflammation and airway hyperalgesia cause symptoms such as coughing, wheezing, difficulty in breathing, chest tightness, and characteristic inflammation of allergic diseases. When allergens are detected, Cells secrete airway constriction mediators such as histamine (bronchoconstriction and inflammatory responses), leukotrienes, and prostaglandin D2 (bronchodilator). Activated eosinophils and TCR invariant natural killer T cells are increased and secrete inflammatory substances such as cytokines (which regulate inflammatory responses in general) and chemokines (inflammatory cell mobilization) that damage airway epithelial cells And symptoms are induced. In addition to the hypertrophy and hyperplasia of smooth muscle cells, growth factors such as VEGF (vascular endothelial growth factor) increase the blood vessels in the airways and increase the airway wall thickness.

Asthma is a chronic, relapsing disease. Therapeutic methods include medication that uses medications to restore symptoms and prevent recurrence, to find causes of asthma, to minimize allergen exposure and to avoid aggravating factors. . In addition, there are therapeutic methods such as immunotherapy which improves the allergic constitution by injecting a small amount of the causative substance.

Among them, asthma medication used for medication is divided into symptom relief agent that alleviates narrowed bronchi in a short time, and disease control agent which suppresses bronchial allergy inflammation and prevents asthma attack. Xanthine drugs, such as theophylline, and anticholinergics (anticholinergics) are widely used as typical symptom relievers. Beta 2 antagonists (agonists), theophylline drugs and anticholinergics And so on.

However, symptoms such as beta-2 agonists can not be treated fundamentally, so they should be used regularly and cause side effects. Steroids can also cause serious side effects such as increased blood sugar, elevated blood pressure, weight gain, mood swings, osteoporosis, and gastric ulcers when used for long periods.

Accordingly, the inventors of the present invention have found that a bellflower extract is effective for the treatment of asthma, while studying natural materials effective for treatment of asthma without causing adverse effects on the body even for a long period of time, thereby completing the present invention .

The present invention provides a composition for treating asthma, which comprises a bellflower extract as an active ingredient.

In order to solve the above-described problems, the present invention provides a composition for treating asthma, which comprises a bellflower extract as an active ingredient.

Platycodon grandiflorum of the present invention is a perennial herbaceous perennial plant and is sometimes referred to as Gakgyeong, Dorat, Gilpyeong, Baekjeong, Gilbong, or Mountain bellflower. Roots are thick, stems grow straight, white juices come out, height is 40-100 cm. Leaves are alternate phyllotaxis, long ovate or broad basal, serrate on margin, petiole free, green on the front, grayish blue on the back, hairless, 4-7cm long and 1.5-4cm wide. In spring and autumn, roots are picked, eaten raw or eaten as herbs, and the main ingredient of the bellflower is saponin. Kikyoku of herbal medicine is peeled off or dried as it is, and it is used for heat treatment, waste heat, tonsillitis, diarrhea in the oriental medicine. It is distributed in Korea, Japan, and China.

The bellflower used for preparing the composition for treating asthma according to the present invention may be diploid or tetraploid, preferably diploid.

The Platycodon grandiflorum extract of the present invention may be any extract, fraction, purified product, diluted solution, concentrated solution, or dried product obtained in each step of extraction, fractionation, or purification (separation, fractionation).

The bellflower extract may be extracted using a conventional solvent known in the art, and the extracted liquid may be used in a liquid form or may be used by concentration and / or drying. The solvent may be water, an anhydrous or lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, propanol, butanol, etc.), a mixed solvent of the lower alcohol and water, acetone, ethyl acetate, chloroform, Butylene glycol may be used. Preferably, extraction is carried out using methanol, ethanol or butane. However, the present invention is not limited thereto. Depending on the organic solvent to be extracted, the extraction degree and the degree of loss of the active ingredient of the extract may differ. Therefore, it is preferable to select an appropriate organic solvent for use. Examples of the extraction method include a cold extraction, an ultrasonic extraction, and a reflux cooling extraction method, but the present invention is not limited thereto.

In one specific example of the present invention, the solvent used for preparing the bellflower extract is preferably a mixture of 40 to 60 wt% of distilled water and 60 to 40 wt% of alcohol alcohol, more preferably 50 wt% of distilled water, 50% by weight.

In one specific example of the present invention, the bellflower extract is prepared by lyophilizing the freeze-dried Bellflower in a solvent in which 50% by weight of distilled water and 50% by weight of alcohol are mixed, extracting at room temperature and then concentrating the extract to a powder by freeze- The dried powder was fractionated.

As one specific example of the present invention, a composition comprising an extract of Bellflower as an active ingredient can be used as a pharmaceutical composition for the treatment of asthma.

When the composition of the present invention is used as a pharmaceutical composition, the effective amount of the bellflower extract is 0.00001 to 15.0% by weight, preferably 0.0001 to 10% by weight, more preferably 0.0001 to 10% by weight based on the total weight of the whole pharmaceutical composition. 5% by weight, and as an active ingredient, a pharmaceutically acceptable carrier other than the bellflower extract may be further contained.

The dosage of the pharmaceutical composition of the present invention is in the range of 0.001 to 300 mg / kg on an adult basis. However, the dose is not limited in any way to the scope of the present invention.

The pharmaceutically acceptable carriers to be contained in the pharmaceutical composition of the present invention are those conventionally used in the present invention and include carbohydrate-type compounds (e.g., lactose, amylose, dextrose, sucrose, sorbitol, mannitol, starch, Corn oil, cottonseed oil, cottonseed oil, cottonseed oil, cottonseed oil, cottonseed oil, cottonseed oil, cottonseed oil, cottonseed oil, cottonseed oil, cottonseed oil, But are not limited to, polyethylene glycol, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like.

The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc., in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition may be formulated in the form of an injectable or external preparation according to a conventional method.

The pharmaceutical composition may prevent or treat cutaneous damage or skin atrophy, which comprises administering the pharmaceutical composition in a pharmaceutically effective amount to mammals such as rats, mice, livestock, humans, and the like in a variety of routes . The above administration method can be carried out in various ways, for example, by intramuscular administration, subcutaneous administration, or local administration by application.

The dosage of the pharmaceutical composition may vary depending on factors such as formulation method, administration method, age, body weight, sex, pathological condition and the like of the patient. The dosage of the pharmaceutical composition of the present invention is in the range of 0.001 to 300 mg / kg on an adult basis. However, the dose is not limited in any way to the scope of the present invention.

The composition containing the platycodon grandiflorum extract of the present invention as an active ingredient has an advantage of achieving the object of improving, improving, or treating asthma in an individual by intra-individual administration, subcutaneous administration, or local administration by application or the like.

On the other hand, the plantain extract of the present invention is a natural extract derived from plants and scarcely adverse to the human body, and can be safely used as an active ingredient in pharmaceuticals and the like.

As described above, the platycodon grandiflorum extract of the present invention is a natural extract derived from plants and having little adverse effects on the human body, and the composition for treating asthma comprising the platycodone extract of the present invention as an active ingredient can be used as a conventional chemical therapeutic agent It is possible to effectively treat asthma while minimizing side effects.

FIG. 1 is a graph showing the effect of the mouse body weight on the 6th day (P6), 3 days, 7 days, 10 days, 14 days, 17 days and 20 days (D3, D7, D10, D14, D17, D20) Respectively. As shown in FIG.
FIG. 2 is a graph showing changes in the amount of the feed used for the 6th day (P6), 3 days, 7 days, 10 days, 14 days, 17 days and 20 days (D3, D7, D10, D14, D17 and D20) And the amount of each of them was measured.
3 is a graph comparing the amounts of WBCs according to the present invention.
Figure 4 is a graph comparing the amount of eosinophil according to the present invention.
5 is a graph comparing the amounts of neutrophils according to the present invention.
6 is a graph comparing the amounts of IgE according to the present invention.
7 is a photograph showing the result of H & E staining according to the present invention.
8 is a photograph showing the results of PAS staining according to the present invention.
Figure 9 is a graphical representation of Western blot results according to the present invention.

Hereinafter, embodiments of the present invention will be described in detail to facilitate understanding of the present invention. However, the embodiments according to the present invention can be modified into various other forms, and the scope of the present invention should not be construed as being limited to the following embodiments. Embodiments of the invention are provided to more fully describe the present invention to those skilled in the art.

In the following examples, asthma was induced in ovalbumin in BALB / c mice and platelet diploid and tetraploid administration was performed to determine the total leukocyte count and cell differentiation coefficient (BALF) in bronchoalveolar lavage fluid (BALF) The anti-asthmatic efficacy was evaluated by analyzing differential cell count (IgE) and H & E staining of the lungs, PAS staining, IHC (Immunohistochemistry) and Western blot analysis.

Manufacturing example  1: Pretreatment of bellflower

The cultivated cultivars were cultivated in 2 - year - old native varieties of diploid and tetraploid bellflower cultivated in vinyl house. The roots of the bellflower were washed to remove the soil from the root of the root completely, and then peeled and washed again with distilled water, and then the water was removed and stored at 80 ° C.

Example 1: diploid  Preparation of bellflower extract

The diploid platelet pretreated in Preparation Example 1 was repeatedly immersed three times in a mixed solvent of 50% by weight of distilled water and 50% by weight of alcohol, and the extract was concentrated under reduced pressure and lyophilized to be powdered. Thereafter, 100 g of the lyophilized powder was fractionated by repeating three times for each solvent using n-hexane, methylene chloride, ethyl acetate, butyl alcohol and distilled water, and the extract was completely concentrated by using a vacuum concentrator and then weighed to 100 mg to obtain 5.6 mL of physiological saline.

Example 2: tetraploid  Preparation of bellflower extract

The tetraploid starch pretreated in Preparation Example 1 was repeatedly immersed in a solvent mixed with 50% by weight of distilled water and 50% by weight of alcohol alcohol, and extracted at room temperature. The extract was concentrated under reduced pressure to give lyophilized powder. Thereafter, 100 g of the lyophilized powder was fractionated by repeating 3 times for each solvent using n -hexane, methylene chloride, ethyl acetate, butyl alcohol and distilled water, and the extract was completely concentrated by using a vacuum concentrator and then weighed to 100 mg to obtain 5.6 mL of physiological saline.

Test Example  1: asthma inducing substance, immunostimulating substance and positive control substance

Ovalbumin (OVA) (Catalog No. A5503, source: Sigma Aldrich, Louis, Mo., USA), which was refrigerated with an asthmatic inducer, was used.

Aluminum hydroxide (Alum) (Catalog No. 77161, supplier: Thermo Fisher Scientific lnc, Waltham, Mass., USA), which was refrigerated with the immunosuppressant, was used.

Dexamethasone (DEX) (manufactured by 13099, source: SAMNAM Pharm, Chungnam, Korea), which was stored at room temperature as a positive control, was used.

Sterile physiological saline (N / S) (Lot No .: 12062, source: JW Pharmaceutical, Seoul, Korea) stored at room temperature as the administration material was used.

Test Example  2: Specific pathogen member (SPF) mouse ( BALB / c mouse)

The mouse used in Test Example 1 (source: Samtako Korea) was widely used for asthma test, and the female was used for 6 weeks at the age of 7 weeks in the test. After ingestion, the animals were sanitized in an animal room that was subjected to the test for 6 days and observed at least once a day. During the purification period, red oily magic was identified. During the administration and observation period, identification was made using black oily magic marker, and the animal identification card was attached to the breeding box.

The mice were housed in a breeding area maintained at a temperature of 23 +/- 3 DEG C, a relative humidity of 55 +/- 15%, a ventilation frequency of 10-20 times / hr, an illumination time of 12 hours and an illumination of 150-300 Lux. In a polycarbonate breeding box containing an appropriate amount of rubbing, the animals were raised in a rabbits box with less than 4 rats per feeding period, and the weight of the animals determined to be healthy during the refinement period was measured and ranked, and the average weight of each group was uniformly The distribution was randomly distributed as shown in Table 1 below.

group gender Number of animals Animal number OVA Dose (mg / kg) Dosage amount
(μL / g) Amount of sensitization
(μg / head) Night
(%) DEX sample G1 F 8 1 - 8 20 0 0 0 28 G2 F 8 9 - 16 20 5 0 0 28 G3 F 8 17 - 24 20 5 One 0 28 G4 F 8 25 - 32 20 5 0 Diploid bellflower
500 mg / kg 28 G5 F 8 33 - 40 20 5 0 4-pledged bellflower
500 mg / kg 28

Manufacturing example  2: Preparation of the substance to be administered

2-1. Preparation of peritoneally injectable substance

OVA 20 μg +, Alum 1 mg +, and N / S 500 μL / head. The number of mice to be administered was 45, and was converted into OVA 900 μg + Alum 45 mg + N / S 22.5 mL.

2-2. 5% OVA sol. Preparation of inhalation-administered substance

OVA 1 g + N / S 20 mL (5% OVA Sol.) And administered by vortexing.

2-3. Preparation of Positive Control Substance

Dexamethasone was prepared by dissolving vortexing in a physiological saline solution. However, it was not completely dissolved and was used for vortexing again.

The above-mentioned dosage materials were prepared and used on the day of administration.

Test Example  3: Administration of the drug substance

The dosage form prepared in Preparation Example 2 was administered by OVA intraperitoneal injection and inhalation administration. The intraperitoneal administration was performed once a week for 2 weeks, and the inhalation administration for 5 days from the 15th day, and the platelet extract samples and the positive control substances of Examples 1 and 2 were carried out. However, the administration of the bellflower extract samples of Examples 1 and 2 and the positive control substance proceeded in the morning, and the inhalation administration proceeded in the afternoon. In the OVA mustache group (G1) and OVA induced group (G2) of Table 1, physiological saline was administered.

Experimental Example  1: General symptom observation

The mice in all test groups were observed at least once every day from the date of acquisition to the date of autopsy.

One control group and one control group were killed during the administration period. Other mice had no symptoms during the experiment.

Experimental Example  2: Weight measurement

D3, D7, D10, D14, D17, and D20) at 6 days (P6), 3 days, 7 days, 10 days, 14 days, 17 days and 20 days after administration.

As shown in Fig. 1, no significant difference in body weight between groups was observed.

Experimental Example  3: Measurement of amount of feed

D3, D7, D10, D14, D17, and D20) at 6 days (P6), 3 days, 7 days, 10 days, 14 days, 17 days and 20 days after administration.

As shown in Fig. 2, no significant difference in the amount of feed used was observed.

Experimental Example  4: Autopsy and analysis results

All mice were autopsied on the day of the last autopsy (day 20, D20). Bronchoalveolar lavage fluid (BALF), spleen, and lungs were collected from four animals in each group. Serum was collected from all mice.

4-1. Analysis of spleen and lungs

For autopsy, 1/2 of the sample was frozen in liquid nitrogen and stored in a cryocooler for PCR / Western blot analysis. Half of the sample was stored in 10% formalin solution for histopathology and immunohistochemistry And used for immunohistochemistry.

4-2. BALF analysis

The first BALF was taken in tube # 1, the second, and the third BALF taken in tube # 2. The tubes were centrifuged at 3000 rpm for 5 minutes, then transferred to the supernatant 3, and the remaining pellet was mixed with 20 μL of PBS. The second tube was centrifuged at 3000 rpm for 5 minutes. After discarding the supernatant, the pellet was mixed with 60 μL of PBS and mixed with 20 μL of 1 time, and a total of 80 μL of hemolysis was performed.

4-3. Blood drawing

Blood collection was performed by heart sampling.

4-4. Observation of differential cell count

As shown in FIG. 3, WBC was significantly increased in the OVA-induced group compared to the control group, and decreased similarly in the platelet diploid and tetraploid group compared with the positive control group. In diploids, OVA was significantly lower than that of positive control.

As shown in Fig. 4, eosinophil was significantly increased in the OVA-induced group compared with the control group, and decreased in the bellied diploid and tetraploid body similarly to the positive control group.

As shown in FIG. 5, neutrophil was significantly increased in the OVA-induced group compared to the control group, and decreased similarly in the platelet diploid and tetraploid group compared with the positive control group. In diploids, OVA was significantly lower than that of positive control.

As shown in Fig. 6, IgE was significantly increased in the OVA-induced group compared to the control group, and decreased in the bloody diabetes mellitus group than in the OVA-induced group. In the tetraploid group, there was no difference from the OVA induced group.

As shown in FIG. 7, eosinophil, mucus, and epithelial hyperplasia were increased in the OVA-induced group compared to the control group, whereas the positive control group was decreased in the control group and decreased in the platelet-derived diploid and tetraploid group than in the OVA-induced group It was found that in diploids, it decreased more than tetraploid.

As shown in Fig. 8, mucus was increased in the OVA-induced group compared with the control group, and the positive control group was decreased in the similar manner to the control group. As shown in Fig. 8, In the case of the tetraploid.

As shown in FIG. 9, in the Western blot, the amount of TNF-α was increased in the OVA-induced group compared to the control group, and the amount of TNF-α was decreased in the positive control group similarly to that of the control group. The expression level of TNF-α was decreased than that of the positive control.

 Regarding the changes of WBC, eosinophil and neutrophil in BALF, WBC, eosinophil and neutrophil were decreased in the group treated with diploid and tetraploid bloom extract compared to the control group. And decreased further in the group treated with the bellflower extract. IgE changes were decreased in the group treated with Dextrothorrhizae extract, but not in the group treated with Dextranate extract. Histological analysis of the lungs revealed that the H & E staining showed decreased mucin, eosinophil infiltration and hyperplasia, which are typical histopathological features of asthma in the group treated with diploid and tetraploid roots extract. The results of PAS staining for confirming mucus showed that mucus was decreased in the group treated with diploid and tetraploid bellflower extracts. As a result of western blotting, it was confirmed that TNF-α was reduced in the diploid and tetraploid Doradji extract-administered group than in the positive control group. As a result, it was shown that the extracts of diploid and tetraploid bellflower showed anti - asthmatic effect. Especially, it was confirmed that anti - asthmatic effect was exerted in diploid bellflower extract rather than tetraploid.

Claims (2)

A composition for prevention and treatment of asthma, comprising a bellflower extract as an active ingredient. The composition for preventing and treating asthma according to claim 1, wherein the bellflower is diploid or tetraploid.

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