KR102072907B1 - Composition comprising an extract or a fraction of Codonopsis lanceolata for prevention and treatment of anti-inflammatory or asthma - Google Patents

Abstract

The present invention relates to an anti-inflammatory composition or a composition for preventing or treating asthma or a food composition comprising a deodeok extract or fractions thereof, the deodeok extract or fractions thereof of the present invention is a non-toxic, but also associated with nitrogen oxides (inflammatory factors) ( NO) inhibits the production, inhibits the production of IL-1β, IL-6, TNF-α, can be useful in the prevention or treatment of anti-inflammatory or asthma.

Description

Composition comprising an extract or a fraction of Codonopsis lanceolata for prevention and treatment of anti-inflammatory or asthma}

The present invention relates to an anti-inflammatory composition or a composition for preventing or treating asthma or a food composition comprising a deodeok extract or a fraction thereof.

Inflammation refers to the tissue response to damage when foreign antigens, such as bacteria or viruses, enter the body from outside or when cells or tissues are damaged by some cause. Inflammatory cells release various preinflammatory mediators such as free radicals and oxygen species that cause DNA damage in the initiation and promotion of tumors. Inflammation is largely divided into acute and chronic inflammatory. In the case of chronic inflammatory reactions, excessive inflammatory cytokines and chemokines are released, causing damage to surrounding tissues, invasion, metastasis, and transfer of tumor cells. And it promotes angiogenesis and helps tumor growth.

Recently, with the development of molecular biology, attempts have been made to understand inflammatory diseases at the molecular level such as cytokines, and factors affecting these diseases have been identified one by one. Among these factors, nitric oxide (NO) acts as a inflammatory mediator and maintains homeostasis by protecting against pathogenic DNA (Kou and Schroder, Ann Sur 221, 220-235, 1995). NO is produced from L-arginine by three major NOSs: isomers of nitrogen oxide synthase (NOS), neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). NO, which is overproduced by iNOS, reacts with superoxide to form peroxynitrite, which acts as a powerful oxidant and damages cells, activating macrophages by inflammatory stimuli. Let's do it. Macrophages activated by various stimuli and inflammation mediators produce other inflammation-related factors, including tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β), and prostaglandin E2 (PGE2). These are known to be involved in chronic diseases such as inflammatory reactions, cancer, and atherosclerosis by inducing inflammatory sites and activating NF-κB (Lawrence et al., Nat Med., 7, 1291-1297, 2001).

There is an active research to treat inflammatory diseases by controlling the intracellular levels of the factors involved in the above-described inflammatory response. Initially, chemical substances were used to control the intracellular levels of the factors, but side effects are severe. In recent years, studies are being actively conducted to control intracellular levels of these factors by using substances derived from natural products which have no side effects or appear at very low levels.

Accordingly, the present inventors have tried to find a natural material useful for treating inflammatory diseases without causing side effects on the human body, and as a result, the deodeok extract or fractions thereof may be useful for the prevention or treatment of inflammatory diseases without showing toxicity. This invention was completed.

Korea Patent Registration 10-1487065

An object of the present invention to provide an anti-inflammatory composition comprising a deodeok ( Codonopsis lanceolata ) extract or a fraction thereof.

It is another object of the present invention to provide a pharmaceutical composition for preventing or treating asthma, including the extract of Codonopsis lanceolata or a fraction thereof.

Still another object of the present invention is to provide an anti-inflammatory health food composition comprising an extract of Codonopsis lanceolata or a fraction thereof.

Another object of the present invention to provide an anti-inflammatory health food composition comprising a deodeok (Codonopsis lanceolata) extract or a fraction thereof.

Still another object of the present invention is to provide a health food composition for preventing or improving asthma including Codonopsis lanceolata extract or a fraction thereof.

Another object of the present invention is to provide a dietary supplement for preventing or improving asthma, including Deodeok (Codonopsis lanceolata) extract or a fraction thereof.

In order to achieve the above object,

The present invention provides an anti-inflammatory composition comprising a deodeok ( Codonopsis lanceolata ) extract or a fraction thereof.

In addition, the present invention provides a pharmaceutical composition for preventing or treating asthma, including the extract of Codonopsis lanceolata or fractions thereof.

Furthermore, the present invention provides an anti-inflammatory health food composition comprising a deodeok (Codonopsis lanceolata) extract or a fraction thereof.

Furthermore, the present invention provides an anti-inflammatory health food composition comprising Codonopsis lanceolata extract or a fraction thereof.

In addition, the present invention provides a health food composition for preventing or improving asthma including Codonopsis lanceolata extract or a fraction thereof.

Furthermore, the present invention provides a dietary supplement for preventing or improving asthma, which includes Codonopsis lanceolata extract or a fraction thereof.

Deodeok extracts or fractions thereof of the present invention, while not toxic, inhibits the production of inflammation-related factors, such as nitrogen oxides (NO), inhibits the production of IL-1β, IL-6, and TNF-α, thus preventing or inhibiting inflammation It can be useful for treatment.

Figure 1 shows the deodeok extract and fractions thereof.
Figure 2 is the result of measuring the cell viability of the deodeok extract in RAW264.7 cells.
3 is a result showing the inhibition of NO production according to the concentration of ethanol extract in RAW264.7 cells.
Figure 4 is a result showing the inhibition of production of IL-1β according to the concentration of ethanol extract in RAW264.7 cells.
5 is a result showing the inhibition of the production of IL-6 according to the deodeokethanol extraction concentration in RAW264.7 cells.
6 is a result showing the inhibition of production of TNF-α according to the concentration of ethanol extract in RAW264.7 cells.
Figure 7 shows the NO production inhibition of the deodeok fraction in RAW264.7 cells.
Figure 8 shows the inhibition of production of IL-1β of the deodeok fraction in RAW264.7 cells.
Figure 9 shows the inhibition of production of IL-6 of the deodeok fraction in RAW264.7 cells.
10 is a result showing the inhibition of TNF-α production of the deodeok fraction in RAW264.7 cells.
Figure 11 shows the cell viability of the deodeok extract in lung tissue of asthma model mice (a, vehicle control; b, asthma induction; c, dexamethasone; d, 50 mg / kg / day C. lanceolata e, 250 mg / kg / day C. lanceolata f, 500 mg / kg / day C. lanceolata .)
12 is a result showing the inhibition of T cell (CD3) related factors of the deodeok extract in immunostained lung tissue of asthma model mice (a, vehicle control; b, asthma induction; c, dexamethasone; d, 50 mg / kg) / day C. lanceolata e, 250 mg / kg / day C. lanceolata f, 500 mg / kg / day C. lanceolata .)
FIG. 13 shows T cell (CD4) related factors of Deodeok extract in immunostained lung tissues of asthma model mice (a, vehicle control; b, asthma induction; c, dexamethasone; d, 50 mg / kg / day C. lanceolata) e, 250 mg / kg / day C. lanceolata f, 500 mg / kg / day C. lanceolata .)
Figure 14 shows the T-cell (CD8) related factors of Deodeok extract in immunostained lung tissue of asthma model mice (a, vehicle control; b, asthma induction; c, dexamethasone; d, 50 mg / kg / day C. lanceolata) e, 250 mg / kg / day C. lanceolata f, 500 mg / kg / day C. lanceolata .)
Figure 15 shows the T-cell (CD19) related factors of Deodeok extract in immunostained lung tissue of asthma model mice (a, vehicle control; b, asthma induction; c, dexamethasone; d, 50 mg / kg / day C. lanceolata e, 250 mg / kg / day C. lanceolata f, 500 mg / kg / day C. lanceolata .)

Hereinafter, the present invention will be described in detail.

Deodeok Codonopsis lanceolata ) Anti-inflammatory or asthma preventive or therapeutic pharmaceutical composition comprising an extract or a fraction thereof

The present invention provides a pharmaceutical composition for anti-inflammatory or asthma prevention or treatment comprising Codonopsis lanceolata extract or a fraction thereof.

As used herein, the term "extract" refers to a formulation prepared by squeezing the herbal medicine with an appropriate leachate and evaporating the leachate, but not limited thereto, but is not limited thereto, and an extract obtained by extraction, a dilution or concentrate of the extract, and an extract. It may be a dried product obtained by drying the above, these adjusted products, or a refined product. The deodeok ( Codonopsis lanceolata ) extract can be prepared using common extraction methods, separation and purification methods known in the art. The extraction method is not limited thereto, but may be preferably a method such as hot water extraction, hot water extraction, cold needle extraction, reflux cooling extraction, or ultrasonic extraction.

In the present invention, the extraction solvent may be water, alcohols having 1 to 4 carbon atoms or mixed solvents thereof, and preferably ethanol.

In one embodiment of the present invention, the deodeok extract was prepared using 30%, 50%, 70% and 100% ethanol as a solvent.

Codonopsis lanceolata extract or fractions thereof of the present invention may be administered in various oral and parenteral formulations during clinical administration, and when formulated, commonly used fillers, extenders, binders, wetting agents, disintegrants, surfactants. It is prepared using diluents or excipients.

Solid formulations for oral administration include tablets, patients, powders, granules, capsules, troches, and the like, which may comprise at least one excipient, eg, in one or more Codonopsis lanceolata extracts or fractions thereof of the present invention. For example, it is prepared by mixing starch, calcium carbonate, sucrose, lactose or gelatin. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Liquid preparations for oral administration include suspensions, solvents, emulsions, or syrups, and include various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. Can be.

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, and the like. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerol, gelatin and the like can be used.

In addition, the effective dosage of Codonopsis lanceolata extract or fractions thereof of the present invention to the human body may vary depending on the age, weight, sex, dosage form, health condition and degree of disease of the patient, and generally about 0.001-100 mg / kg / day, preferably 0.01-35 mg / kg / day. Based on an adult patient weighing 70 kg, it is generally 0.07-7000 mg / day, preferably 0.7-2500 mg / day, once a day at regular intervals according to the judgment of the doctor or pharmacist. Multiple doses may be administered.

Deodeok Codonopsis lanceolata Anti-inflammatory or asthma preventive or improved health functional food and health food composition comprising an extract or a fraction thereof

When the codonopsis lanceolata extract of the present invention or a fraction thereof is used as a nutraceutical and health food, there is no particular limitation on the type of food. Examples of the food to which the extract of Codonopsis lanceolata of the present invention or a fraction thereof may be added include drink, meat, sausage, bread, biscuit, rice cake, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, Dairy products, including ice cream, various soups, beverages, alcoholic beverages and vitamin complexes, dairy products and dairy products, and includes all the health functional foods and health foods in the conventional sense.

Nutraceuticals and health food compositions containing Codonopsis lanceolata extract or fractions thereof according to the present invention may be added as is to foods or used with other foods or food ingredients, and may be appropriately used according to conventional methods. . The mixed amount of the Codonopsis lanceolata extract or a fraction thereof may be suitably determined depending on the purpose of use (prevention or improvement). In general, the amount of the composition in the nutraceutical and health food can be added to 0.1 to 90 parts by weight of the total food weight. However, in the case of long-term intake for the purpose of health maintenance or health control, the amount may be below the above range, and since there is no problem in safety, the Codonopsis lanceolata extract or a fraction thereof may be at least above the above range. Can also be used as.

The health functional food and the health food composition of the present invention are not particularly limited to other ingredients except for containing the present invention Codonopsis lanceolata extract or fractions thereof as essential ingredients in the indicated ratios, and various flavors or Natural carbohydrates and the like may be included as additional components. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 nutraceutical and health food composition of the present invention.

In addition to the above, the functional food and health food composition containing the deodeok ( Codonopsis lanceolata ) extract or fractions thereof of the present invention is a variety of nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavoring agents, such as colorants and Neutralizers (cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like. Can be. In addition, the nutraceutical and health food composition of the present invention may contain the flesh for the production of natural fruit juice and fruit juice drinks and vegetable drinks.

These components can be used independently or in combination. The ratio of such additives is not so important but is generally selected in the range of 0.1 to about 20 parts by weight per 100 parts by weight of the nutraceutical and health food compositions containing the Codonopsis lanceolata extract or fractions thereof.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are merely to illustrate the present invention, the contents of the present invention is not limited by the following examples.

Example 1 Preparation of Deodeok Extract

Deodeok roots were washed, lyophilized and sliced into 5 mm size, extracted with hot water, 30%, 50%, 70% and 100% alcohol for 3 times at room temperature, and the extract was concentrated under reduced pressure to obtain a powdered sample. Storage at -20 ℃ was used for the experimental analysis.

Example 2 Preparation of Deodeok Fractions

Deodeok roots were selected and washed, followed by lyophilization, extracted with 30% alcohol and repeated extraction three times at room temperature, and then concentrated under reduced pressure to obtain an extract. The concentrated extract was freeze-dried to n-hexane, Methylene chloride, Ethyl acetate, Butyl alcohol, D.W. Repeated extraction three times for each solvent, such as, and then, the extract was concentrated completely with a vacuum condenser to obtain a fraction (see Figure 1).

Preparation Example 1 RAW 264.7 Macrophage Culture

RAW 264.7 cells were used in DMEM medium containing 10% fetal bovine serum (FBS). RAW 264.7 cells were dispensed with 2 × 10 ⁵ in 24 well plates and incubated at 5% CO _2, 37 ° C. This was observed under a microscope to confirm that the cells were differentiated and used for the test.

Experimental Example 1 Measurement of Cell Viability of Deodeok Extract (MTT assay)

MTT assay was performed to determine the toxicity of the deodeok extract against RAW 264.7 macrophages. That is, RAW264.7 cells in subculture were adjusted to 96 well plates, and then, after adding 1 hour of incubation, the extracts were treated with LPS 1 ㎍ / ml in each well, and 10% SDS (0.1 N after 4 hours). 100 μl of HCL) was treated to react with light blocking for 18 hours. Using an ELISA reader, the absorbance of each well was measured at 570 nm and compared with the absorbance of the control group.

As a result, as shown in Figure 2, the deodeok extract did not show cytotoxicity.

Experimental Example 2 Determination of Anti-inflammatory Activity of Deodeok Extract

Experimental Example 2-1 Measurement of NO Production in Macrophages

1 × 106 cells / ml of RAW264.7 cells were suspended in DMEM containing 5% FBS, and then the cell suspension was attached to 800 µl each in a 12well plate. The samples were processed and the concentration of LPS, a macrophage stimulant, was 1 μg / ml. After 24 hours, 100 μl of the culture supernatant was removed, transferred to a 96 well plate, and 100 μl of Griess Reagent (Sigma) was added thereto. After reacting with light blocking at room temperature for 10 minutes, the absorbance was measured at 540 nm using an ELISA reader. It was. The nitric oxide secreted by macrophages was calculated from the sodium nitrite standard calibration curve.

NO production inhibitory effect was the highest in 100% extract, but did not show a significant difference from 30% ~ 70% extract (see Figure 3).

Experimental Example 2-2 Production of Cytokines (IL-1β, IL-6, TNF-α) of Deodeok Extract

In order to measure the cytokine, the cells were dispensed into 6-well plates, treated with Deodeok extract, and then treated with LPS after 1 hour. After LPS treatment, the media was collected for 6-12 hours and cytokine was measured. Collected media was stored at -70 ° C until measurement. IL-1β, IL-6, TNF-α were measured using an ELISA Kit (Pierce endogen, Rockford, IL, USA).

In the effect of LPS treatment on IL-1β secretion in mouse macrophages, the higher the ethanol concentration as an extractant, the higher the inhibitory activity. In addition, IL-6 secretion inhibitory ability did not show a significant difference between the extraction concentrations were found to inhibit the secretion (see Fig. 4 and 5).

As a result of examining the effect on TNF-α secretion, the higher the ethanol concentration of the extraction solvent, the higher the inhibitory activity, and generally suppressed TNF-α secretion by 20% (see FIG. 6).

Experimental Example 3 Determination of Anti-inflammatory Activity of Extracts from Deodeok Fractions

The anti-inflammatory activity of the deodeok fraction was measured in the same manner as in Experimental Example 2.

As a result, the anti-inflammatory effects of the extracts of Deodeok extract showed that the inhibitory effect was higher in the Ethyl acetate fraction and the n-hexane fraction in all the inflammation-related factors. Inflammatory efficacy was considered (see FIGS. 7, 8, 9, 10).

Experimental Example 4 Determination of Anti-asthma Activity of Deodeok Extract

The roots of Jeju cultivated deodeok were washed, lyophilized and sliced into 5 mm size, and then extracted and concentrated with 30% alcohol and stored in powder at 50 ° C.

To investigate the efficacy of allergic extracts against allergy asthma and immunity, CD3 + T cell, CD4 + T cell, Changes in CD8 + T cells and CD19 + B cells were observed and immunohistochemical analysis was performed.

Paraffin-free tissue sections were treated with methanol containing 3% hydrogen peroxide for 10 minutes to remove peroxidase. Antigen retrieval was also treated with sodium citrate buffer (0.1M) using boiling water. The slides were incubated with normal serum to block nonspecific binding, then CD4 (14-9766, eBioscience, San Diego, CA, USA), CD8 (sc-18913, Santa Cruz Biotechnology, Dallas, TX, USA), CD19 ( 250585, Abbiotec, San Diego, CA, USA) and incubated for 1 hour with a primary antibody (diluted 1: 100 to 1: 200). Then, biotinylated secondary antibody and horseradish peroxidase-conjugated streptavidin were incubated together for 2 hours. Signals were searched using a 3,3-diaminobenzidine tetrahydrochloride substrate chromophore and cells stained with Mayer's hematoxylin. To determine the number of positively stained cells, three separate immunostained lung sections (n = 8 per group) per experimental animal were counted in five randomly selected nonredundant regions (x200-fold).

Investigation of the effects on immune cells in BALF showed that CD3 + T cells, CD4 + T cells, CD8 + T cells, and CD19 + B cells, which increased when allergic reactions were active, showed a concentration-dependent decrease. Allergic and anti-inflammatory effects were thought to be effective for bronchial asthma (see Figures 11, 12, 13, 14, 15).

So far I looked at the center of the preferred embodiment for the present invention. Those skilled in the art will appreciate that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in descriptive sense only and not for purposes of limitation. The scope of the invention is shown in the claims rather than the foregoing description, and all differences within the scope shall be construed as being included in the invention.

Claims (6)

Dedon ( Codonopsis lanceolata ) A pharmaceutical composition for preventing or treating asthma, comprising 30% ethanol extract. delete delete The method of claim 1,
The detoxification 30% ethanol extract is an inflammatory cytokine tumor necrosis factor-alpha (TNF-α; Tumor Necrosis Factor-α), interleukin 6 (IL-6; interleukin 6) or interleukin 1 beta (IL-1β; interleukin 1 beta) ) And nitrogen monoxide (NO). Deodeok (Codonopsis lanceolata) Health food composition for preventing or improving asthma containing 30% ethanol extract. Deodeok (Codonopsis lanceolata) A dietary supplement for asthma prevention or improvement comprising 30% ethanol extract.

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