KR102153338B1 - Pharmaceutical composition for preventing or treating an inflammatory disease, comprising an extract of Canarium subulatum as an active ingredient - Google Patents

Abstract

The present invention relates to a composition for preventing, improving or treating inflammatory diseases, comprising an extract of Canarium subulatum having anti-inflammatory activity as an active ingredient, wherein the methanol extract of Canarium subulatum is produced by an inflammatory reaction. It can effectively reduce the generation of cytokines and their reactants, and can inhibit the expression of signaling proteins involved in immune activity. Furthermore, the anti-inflammatory effect of the extract was also confirmed in animal experiments that caused gastritis. It is expected to be useful in the development of pharmaceuticals and quasi-drug materials and related industries.

Description

Pharmaceutical composition for preventing or treating an inflammatory disease, comprising an extract of Canarium subulatum as an active ingredient {Pharmaceutical composition for preventing or treating an inflammatory disease, comprising an extract of Canarium subulatum as an active ingredient}

The present invention relates to a composition for preventing, improving or treating inflammatory diseases, comprising an extract of Canarium subulatum having anti-inflammatory activity as an active ingredient.

Inflammation is when tissue (cells) is damaged or infected by external infectious agents (bacteria, fungi, viruses, various types of allergens), various inflammatory mediators and immune cells are involved, resulting in the activation of enzymes and mediating inflammation. It exhibits complex physiological reactions such as substance secretion, body fluid infiltration, cell migration, and tissue destruction, and external symptoms such as erythema, swelling, fever, and pain. In normal cases, the inflammatory reaction removes external infectious agents and regenerates damaged tissues to restore the function of living organisms. However, if the inflammatory reaction is excessive or persistent due to the absence of antigens or internal substances, it promotes mucosal damage. , In some cases, it induces rheumatoid arthritis, osteoporosis, sepsis, vascular disease, and cancer. In other words, the inflammatory reaction is the most important reaction among pathological mechanisms according to the host's defense mechanism against wounds and microbial infections, but persistent and excessive inflammatory reactions damage tissues.

Macrophages are the most representative immune cells that regulate this inflammatory response, play an important role in the host's defense mechanism in a harmful environment, and are involved in the process of various diseases such as autoimmune diseases. Inflammation stimulating factors such as lipopolysaccharides can activate macrophages, and activated macrophages are tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) And IL-1β, and by synthesizing inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), nitric oxide , NO) and PGE 2 (prostaglandin E 2 ).

On the other hand, Lipopolysaccharide (LPS) is a component of the outer membrane of Gram-negative bacteria and is known as an inflammatory factor, and macrophages produced in the bone marrow are activated by a trace amount of LPS, and various cytokines, hydrogen peroxide, and H ₂ O ₂ ) secretes various cytotoxic substances such as nitric oxide and functions as an immune system.

Recently, it has been reported that foods and plants have functions that can prevent or treat diseases, and research to search for substances with various physiological functions from various resources around the world has been conducted according to the needs of humans to live healthier and longer. It is actively progressing, and among them, a lot of attention is being focused on compounds contained in plant resources.

Republic of Korea Registration Tok Heo Bulletin 10-1049041

The present inventors analyzed changes in the activity of cytokines and signaling proteins involved in the inflammatory response using a macrophage and gastritis mouse model to reveal the anti-inflammatory efficacy of the extract of Canarium subulatum. Was confirmed experimentally, the present invention was completed based on this.

Accordingly, an object of the present invention is to provide a composition for preventing, improving or treating inflammatory diseases, comprising the extract of Canarium subulatum as an active ingredient.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems that are not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention as described above, the present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases, comprising an extract of Canarium subulatum as an active ingredient.

In addition, the present invention provides a food composition for preventing or improving inflammatory diseases, comprising an extract of Canarium subulatum as an active ingredient.

In one embodiment of the present invention, the inflammatory disease may be gastritis.

In another embodiment of the present invention, the extract may be obtained by extracting methanol with a solvent.

In another embodiment of the present invention, the canarium western latum extract may include one or more components selected from the group consisting of quercetin, luteolin, and campferol.

In another embodiment of the present invention, the canarium western ratom extract may inhibit the production of nitric oxide (NO).

In another embodiment of the present invention, the canarium western ratum extract may inhibit the expression of an inducible nitric oxide synthase (iNOS).

In another embodiment of the present invention, the canarium western ratum extract may inhibit phosphorylation of IκBα, IKKα/β, Syk, p85 or Src.

In another embodiment of the present invention, the canarium western ratum extract inhibits the phosphorylation of IκBα, IKKα/β, Syk, p85 or Src, thereby inhibiting the activity of Nuclear Factor κB (NF-κB) can do.

In another embodiment of the present invention, the canarium western ratum extract may reduce nuclear translocation of p50 and p65.

In addition, the present invention provides a method for preventing or treating inflammatory diseases, comprising administering to an individual a pharmaceutical composition comprising an extract of Canarium subulatum as an active ingredient.

In addition, the present invention provides a pharmaceutical composition comprising an extract of Canarium subulatum as an active ingredient, for preventing or treating inflammatory diseases.

The extract of canarium western ratum can effectively reduce the generation of cytokines and reactants produced by the inflammatory reaction and can inhibit the expression of signaling proteins involved in immune activity. The anti-inflammatory effect of is expected to be useful in the development of pharmaceuticals and quasi-drug materials and related industries as it was confirmed in animal experiments that caused gastritis.

1 shows the effect of suppressing the production of nitric oxide caused by lipopolysaccharide (LPS), which is an inflammation-inducing substance in cells, of a methanol extract (hereinafter Cs-ME) of canarium western ratum.
Figure 2 shows the effect of inhibiting the production of nitric oxide by LPS in the peritoneal macrophages of Cs-ME.
Figure 3 shows that Cs-ME has no cytotoxicity to RAW 264.7 cells and macrophages of the peritoneum.
4 shows the results of analysis of major components of Cs-ME through liquid chromatography.
Figure 5 shows that Cs-ME has the effect of reducing the expression of inducible nitric oxide synthase (iNOS) mRNA by LPS.
6 shows the effect of reducing the intranuclear migration of proteins p50 and p65 by LPS when Cs-ME is treated on RAW 264.7 cells.
7 shows the effect of reducing phosphorylation of IKKα/β and IκBα, which are intracellular NF-κB signaling proteins by Cs-ME.
8 shows the effect of reducing phosphorylation of Src and Syk, an intracellular NF-κB signaling protein, by Cs-ME at an initial time.
9 shows the effect of reducing phosphorylation of Src and p85 signaling proteins by Cs-ME treatment when inflammation occurs through Src overexpression.
FIG. 10 shows the effect of reducing phosphorylation of Syk, a signaling protein, by Cs-ME treatment when inflammation occurs through Syk overexpression.
11 is a result of evaluating the effect of Cs-ME on reducing gastric inflammation in an acute gastritis animal model.
12 is a result of comparison of the inhibitory effect of Cs-ME, quercetin, and campferol on the ability to produce nitric oxide (NO) in RAW264.7 cells.
13 illustrates the efficacy of suppressing inflammation through Src/Syk, a target protein of Cs-ME, in the inflammatory signaling system.

The present inventors analyzed changes in the activity of cytokines and signaling proteins involved in the inflammatory response using a macrophage and gastritis mouse model to reveal the anti-inflammatory efficacy of the extract of Canarium subulatum. Was confirmed experimentally, the present invention was completed based on this.

In one embodiment of the present invention, it was confirmed that when Cs-ME was treated with LPS-induced cells, nitric oxide production was inhibited in a dose-dependent manner (see Example 2).

In another example of the present invention, it was confirmed that the RAW 264.7 cells and macrophages were treated with Cs-ME and no cytotoxicity appeared (see Example 3).

In another embodiment of the present invention, it was confirmed that quercetin, luteolin, and campferol were present as main components of Cs-ME using liquid chromatography (see Example 4). .

In another embodiment of the present invention, it was confirmed that Cs-ME has the effect of reducing the mRNA expression of the inducible nitric oxide synthase (iNOS), an inflammation-related cytokine caused by LPS (see Example 5). ).

In another example of the present invention, when Cs-ME was treated with RAW 264.7 cells, it was confirmed that the effect of reducing the intranuclear migration of proteins p50 and p65 by LPS was exhibited (see Example 6).

In another embodiment of the present invention, it was confirmed that phosphorylation of NF-κB signaling proteins IKKα/β, IκBα, Src, Syk or p58 decreased in cells treated with Cs-ME (Examples 7 to 10 Reference).

In another embodiment of the present invention, the gastritis alleviation effect of Cs-ME increases in a concentration-dependent manner, and in particular, the 100mg/kg Cs-ME treatment group shows a higher gastritis alleviation effect than the ranitidine treatment group. Confirmed (see Example 11).

In another embodiment of the present invention, it was confirmed that when the cells were treated with Cs-ME, the inhibitory effect of nitric oxide production was greater than that of each treatment with camphorol and quercetin (see Example 12).

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases, comprising an extract of Canarium subulatum as an active ingredient.

The term "prevention" used in the present invention means any action that suppresses or delays the onset of inflammatory diseases by administration of the pharmaceutical composition according to the present invention.

The term "treatment" used in the present invention means any action in which symptoms caused by an inflammatory disease are improved or advantageously changed by administration of the pharmaceutical composition according to the present invention.

The term "inflammatory disease" used in the present invention is a disease whose main lesion is inflammation, and non-limiting examples of inflammatory diseases include allergic disease, inflammatory bowel disease, atherosclerosis, inflammatory collagen vascular disease, glomerulonephritis, inflammatory skin Disease, sarcoidosis, retinitis, gastritis, hepatitis, enteritis, peritonitis, arthritis, tonsillitis, sore throat, bronchitis, pneumonia, pancreatitis, sepsis, and nephritis, and the like, preferably gastritis, but is not limited thereto.

In the present invention, the kind of the extraction solvent used to obtain the canarium western ratum extract is not particularly limited, and any solvent known in the art may be used. Non-limiting examples of the extraction solvent include water; C ₁ to C ₄ lower alcohols such as methanol, ethanol, propyl alcohol, and butyl alcohol; Polyhydric alcohols such as glycerin, butylene glycol, and propylene glycol; And hydrocarbon solvents such as methyl acetate, ethyl acetate, acetone, benzene, hexane, diethyl ether, and dichloromethane; Alternatively, a mixture thereof may be used, and methanol may be used alone or in combination of two or more, and more preferably 99.9% methanol may be used, but the present invention is not limited thereto.

In addition, after performing the extraction and / or fractionation process of the canarium western ratum extract of the present invention, it can be concentrated or removed by performing a vacuum filtration process or further concentration and/or freeze-drying, and the obtained One canarium western ratum extract can be stored in a quick freezer until use.

Macrophages are the most representative immune cells that regulate the inflammatory response, play an important role in the host's defense mechanism in a harmful environment, and are involved in the progression of various diseases such as autoimmune diseases. Inflammatory stimulating factors such as lipopolysaccharide (LPS) can activate macrophages, and activated macrophages are tumor necrosis factor-α (TNF-α), interleukin-6 (IL). -6) and IL-1β, and by synthesizing inducible nitric oxide synthase (iNOS) and cyclic oxygenase 2 (cyclooxygenase-2, COX-2) to produce nitrogen monoxide ( nitric oxide, NO) and PGE 2 (prostaglandin E 2) are produced.

On the other hand, nuclear factor κB (NF-κB) is a protein that exists everywhere and is specialized in controlling the complex phenomenon. In certain conditions, NF-κB plays a central role in controlling the cellular signals of the body, and among other functions, NF-κB is pro-inflammatory cytokines such as TNF-α and chemokines, which play an important role in controlling the progression of inflammation. (chemokines), and inducible enzymes such as COX-2 are known to control. Therefore, it can be said that NFκB represents an important and very prominent therapeutic target in drugs for treating many inflammatory diseases.

The canarium western ratum extract of the present invention may contain one or more components selected from the group consisting of quercetin, luteolin, and campferol.

The canarium western ratum extract of the present invention can inhibit the production of nitrogen monoxide (NO).

The canarium western ratum extract of the present invention can inhibit the expression of inducible nitric oxide synthase (iNOS).

The canarium western ratum extract of the present invention can inhibit the phosphorylation of IκBα, IKKα/β, Syk, p85 or Src.

By inhibiting the phosphorylation of IκBα, IKKα/β, Syk, p85 or Src, the canarium western ratum extract of the present invention can inhibit the activity of nuclear factor κB (NF-κB).

Canarium western ratum extract of the present invention can reduce the nuclear translocation of p50 and p65 (nuclear translocation).

Pharmaceutical compositions for the prevention or treatment of inflammatory diseases comprising the extract of Canarium western ratum according to the present invention as an active ingredient, respectively, are powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc. It can be formulated and used in the form of external preparations and sterile injectable solutions, and preferably has a formulation of cream, gel, patch, spray, ointment, warning agent, lotion, linenment, pasta or cataplasma. have. Carriers, excipients, and diluents that may be included in the composition containing the canarium western ratum extract include lactose, dextrose, sucrose, oligosaccharide, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, Gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oils. In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and these solid preparations include at least one excipient in the extract such as starch, calcium carbonate, sucrose. ) Or lactose (lactose), gelatin, etc. are mixed to prepare. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, liquid solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweetening agents, fragrances, and preservatives may be included. . Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like can be used.

The preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and weight of the patient, the severity of the disease, the drug form, the route and duration of administration, but may be appropriately selected by those skilled in the art. Administration may be administered once a day, or may be divided several times. The above dosage does not in any way limit the scope of the present invention.

As another aspect of the present invention, the present invention provides a food composition for preventing or improving inflammatory diseases, comprising an extract of Canarium subulatum as an active ingredient.

The food composition includes a health functional food composition.

As used herein, the term "improvement" refers to any action that at least reduces the severity of a parameter related to the condition being treated, for example, symptoms.

In the health functional food composition of the present invention, the active ingredient may be added to food as it is or may be used with other foods or food ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of use (for prevention or improvement). In general, in the manufacture of food or beverage, the composition of the present invention is added in an amount of 15% by weight or less, preferably 10% by weight or less based on the raw material. However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of health control, the amount may be less than the above range.

The health functional food composition of the present invention is not particularly limited to other ingredients other than containing the active ingredient as an essential ingredient in the indicated ratio, and may contain various flavoring agents or natural carbohydrates as additional ingredients, such as a normal beverage. have. Examples of the natural carbohydrates described above include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose, and the like; And polysaccharides, for example, common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (taumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of the natural carbohydrate can be appropriately determined by the choice of a person skilled in the art.

In addition to the above, the health functional food composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and heavy weight agents (cheese, chocolate, etc.), pectic acid and salts thereof, Alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonates used in carbonated beverages, and the like may be contained. These components may be used independently or in combination. The proportion of these additives can also be appropriately selected by a person skilled in the art.

Hereinafter, a preferred embodiment is presented to aid the understanding of the present invention. However, the following examples are provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

[ Example ]

Example 1. Experiment method

Example 1-1 Canarium Western Latum ( Canarium subulatum ) Preparation of extract

To prepare a Canarium subulatum methanol extract (Cs-ME), the aerial parts of Canarium subulatum were dried and pulverized to make a fine powder sample, and then the sample ( 125g) was extracted using 99.9% methanol (1,000 ml) with an ultrasonic grinder (sonicator). Thereafter, it was evaporated to dryness under reduced pressure to obtain a final methanol extract of Canarium subulatum.

Example 1-2. Cell culture method

Murine macrophage cell line RAW264.7 cells in RPMI 1640 medium containing 100 IU/ml of penicillin and 100 μg/ml of streptomycin and 10% Fetal Bovine Serum (FBS) Using a 100 mm cell culture dish (cell culture dish) was cultured at a density of 70-80%. In addition, the human cell line HEK293 cells were cultured in a 100 mm cell culture dish at a density of 70-80% using DMEM medium containing 100 IU/ml of penicillin and 100 μg/ml of streptomycin and 10% fetal bovine serum. .

Example 1-3. Western Blotting (Western blotting)

60mm of the murine macrophage cell line, RAW264.7 cells the penicillin 100 IU / ml streptomycin and 100 μg / ml and the cells cultured using RPMI 1640 medium containing fetal bovine serum with 10% to 7X10 ⁶ cell / ml concentration Pre-incubated in a dish. Then, each fraction was treated and stimulated with a stimulant (Lipopolysaccharide) after a certain period of time, and after a certain period of time according to the drug, the cells were collected and awakened using a lysis buffer to obtain a Western sample. And the protein concentration of each sample was measured by holding bovine serum albumin (BSA) as a standard.

Based on the value obtained as above, perform SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide gel electrophoresis) with each sample amount that becomes the protein concentration, and use a PVDF membrane using a wet blotting method. After blotting the protein, the membrane was blocked with 3% bovine serum albumin, and the target protein antibody and signaling protein antibody (p50, p65, Lamin A/C, p-IκBα, IκBα, p-IKKα/β) , IKKα/β, p-Src, Src, p-Syk, Syk, p-p85, β-actin) solution was used for primary treatment, and the secondary antibody solution was treated and washed again after washing step. In addition, an enhanced chemiluminescence (ECL) solution was evenly dispensed on the membrane in a dark room, and photosensitized with an X-ray film.

Example 2. Macrophage-derived cytotoxic substance Nitric Oxide (NO) Generating ability Measure

The mouse macrophage cell line RAW264.7 was adjusted to a concentration of 1×10 ⁶ cells/ml using RPMI 1640 medium containing 100 IU/ml of penicillin and streptomycin (100 μg/ml) and 10% fetal bovine serum. Then, it was inoculated into a 96-well plate, and pre-incubated for 18 hours at 5% CO ₂ and 37°C.

Then, the medium was removed, and 50 μl of Cs-ME prepared at a 4-fold concentration and a medium containing 50 μl of LPS (final concentration 1 μg/ml) were simultaneously treated and cultured. After 24 hours, 100 μl of the supernatant was transferred to another 96 well plate, and NO quantification was performed using Griess solution (0.5% naphthylethylenediamine dihydrochloride, 5% sulfanilamide, 25% H ₃ PO _{4 ).} ) Was used to measure the absorbance at 540 nm, and a calibration curve was prepared using sodium nitrite (0 to 100 μM) as a standard material.

As a result, as shown in FIGS. 1 and 2, when only LPS was administered, the production of nitric oxide increased rapidly, but when Cs-ME was administered together, it was confirmed that the production of nitric oxide was suppressed in a dose-dependent manner.

Example 3. Cs-ME treated In macrophage line cell Survival rate (cell viability)

MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-difinyltetrazolium bromide (3-[4,5-dimethylthiazol-2-yl]-2,5-diphinyltetrazolium bromide)) Analysis was performed using an assay method. Cells of 1 × 10 ⁶ cells/ml were plated on a 96-well plate and cultured in a CO ₂ incubator at 37° C. for an incubation time corresponding to each immunization test condition.

Then, 10 µl MTT solution (stock concentration: 5 mg/ml) was added and further reaction was induced for 3 hours. To complete the reaction and dissolve the formazan crystal, 100 μl MTT stopping solution (10% sodium dodecyl sulfate in 0.01M HCl) was additionally added to each well. Cell viability was calculated through the OD value obtained by measuring the absorbance at 570 nm of the amount of MTT reduced to formazan.

As a result, as shown in FIG. 3, even if Cs-ME was treated on RAW 264.7 cells and peritoneal macrophages, it was confirmed that the cell viability was high, so that the cytotoxic effect was not significant.

Example 4. Liquid Chromatography Analysis of major components of Cs-ME through

To confirm the active ingredient of Cs-ME, high performance liquid chromatography (HPLC) was performed using an AZURA Analytical HPLC system (KNAUER Corp.).

Analysis conditions (described below)

HPLC System: AZURA Analytical HPLC (KNAUER Corp.)

Column: Phenomenex, Gemini 5μ C18 110A, 250×4.60mm 5μm

menstruum

Solvent A: 0.1% TFA in H ₂ O

Solvent B: 0.08% TFA in 95% MeCN + 5% H ₂ O

Flow rate: 1.0ml/min

Wavelength: 370nm (Quercetin, Luteolin, Kaempferol)

As a result, as shown in FIG. 4, it was confirmed that quercetin, luteolin, and campferol were present as major components of Cs-ME.

Example 5. Inducible nitric oxide synthase from macrophages iNOS )of Secretory ability Measure

To investigate the level of cytokine expression at the transcription level, RAW264.7 cells were cultured in a 6-well plate at 2 x 10 ⁶ for 24 hours, and Cs-ME (50, 100 νg/ml) was pretreated for 30 minutes and then LPS (1 νg/ml). ml) was treated for 6 hours. Then, total RNA was extracted using Trizol reagent. The extracted total RNA was prepared using a First strand cDNA synthesis kit (Thermo scientific), and then the same amount of cDNA was amplified by PCR (polymerase chain reaction). The sense and antisense primer nucleotide sequences of the target protein used at this time were prepared by referring to existing literature, and GAPDH was used as a control gene. PCR amplification was performed using the Hipi PCR kit (Elpis biotech), and the sense and antisense primers of each experimental group cDNA and target proteins, and the control GAPDH primer were dNTP 250 uM, Tris-HCL (pH 8.3) 10 mM, KCl 50 mM. , NgCl _{2 It} was carried out in 20ul of Hipi PCR kit containing 1.5 mM. PCR was performed under conditions of denaturalizing at 95°C for 45 seconds, annealing at 55°C for 45 seconds, and extension at 72°C for 1 minute, and a total of 30 cycles were performed. DNA amplified by PCR was electrophoresed on a 1.5% agarose gel, and the intensity of the fractionated DNA bands was measured.

As a result, as shown in FIG. 5, Cs-ME has the effect of reducing the mRNA expression of the inducible nitric oxide synthase (iNOS), an inflammation-related cytokine caused by LPS, and in particular, Cs-ME is 100 μg/ml), it was confirmed that the effect was clear.

Example 6. Evaluation of transcription factor movement within the nucleus

The murine macrophage RAW264.7 cells were cultured using RPMI 1640 medium containing 100 IU/ml of penicillin and 100 μg/ml of streptomycin and 10% fetal bovine serum in a 100 mm dish at a density of 90% Pre-cultivate. After that, Cs-ME was pretreated for 30 minutes, stimulated with a stimulant (Lipopolysaccharide, LPS), and washed with ice-cold phosphate buffered saline (PBS) after a certain period of time depending on the drug. Homogenization buffer A [20 mM Tris-HCl pH8.0, 10 mM EGTA, 2 mM EDTA, 2 mM DTT, 1 mM PMSF, 25 μg/ml Aprotinin, 10 μg/ml leupeptin ( Leupeptin)] 300 μl were used to collect cells.

Then, the cells were broken at the intensity of Output 4 using a Sonicator, and then centrifuged at 8000 rpm for 15 minutes at 4° C. to separate the supernatant (Cytosol fraction). The pellet (Nuclear fraction) was suspended in 300 μl homogenization buffer B [1% TritonX-100 in homogenization buffer A], and then the cells were broken with a strength of output 4 using a sonicator. The level of intranuclear migration of transcription factors was evaluated using the blotting method.

As a result, as shown in FIG. 6, when Cs-ME was treated on RAW 264.7 cells, the band appeared faintly compared to the untreated group, and it was confirmed that the effect of reducing the intranuclear migration patterns of proteins p50 and p65 by LPS was shown. .

Example 7. Intracellular by Cs-ME NF - κB Signaling protein IKKα /β, IκBα Reduction of phosphorylation

Western blot was performed according to the method of Example 1-3 in order to confirm the effect of reducing phosphorylation of IKKα/β, IκBα, which are intracellular NF-κB signaling proteins by Cs-ME.

As a result, as shown in FIG. 7, it can be seen that phosphorylated IKKα/β and IκBα in the cells treated with Cs-ME are reduced compared to before Cs-ME treatment. The effect of reducing phosphorylation was confirmed.

Example 8. Intracellular by Cs-ME NF - κB Signaling protein Src , Syk Reduction of phosphorylation

Western blot was performed according to the method of Example 1-3 to confirm the decrease in phosphorylation of Src and Syk, which are intracellular NF-κB signaling proteins by Cs-ME.

As a result, as shown in FIG. 8, when Cs-ME was treated, phosphorylated Src and Syk were decreased, resulting in softer p-Src and p-Syk bands. Intracellular NF-κB signaling proteins Src and Syk It was confirmed that phosphorylation was reduced.

Example 9. Src When inflammation occurs through overexpression Src , reduced phosphorylation of p85

Western blot was performed according to the method of Example 1-3 in order to confirm whether the phosphorylation of Src and p85 decreased when Cs-ME was treated when inflammation through Src overexpression occurred.

As a result, as shown in FIG. 9, it was confirmed that when Cs-ME was treated during inflammation through Src overexpression, the effect of reducing phosphorylation of signal transduction proteins Src and p85 appeared. In the case, it was confirmed that the decrease in p-p85 appeared rapidly.

Example 10. Syk When inflammation occurs through overexpression Syk's Reduced phosphorylation

Western blot was performed according to the method of Example 1-3 in order to check whether the degree of phosphorylation of Syk decreased when Cs-ME was treated when inflammation through Syk overexpression occurred.

As a result, as shown in FIG. 10, when Cs-ME is treated when inflammation occurs through Syk overexpression, it can be seen that the p-Syk band is dose-dependently softened compared to the control. I did.

Example 11. Effect of Cs-ME in acute gastritis-induced animal model

Acute gastritis was induced in mice with ethanol (Ethanol) and hydrochloric acid (HCl). First, ICR mice were classified into five groups, and one group did not induce an inflammatory response and the other four groups induced an inflammatory response with ethanol and HCl. Among them, two groups were injected with Cs-ME at 50mg/kg and 100mg/kg, one group was injected with ranitidine, a control compound, and 0.5% CMC, a vehicle of Cs-ME, was injected for two days and then dissected. After measuring the state of the gastric wall, the degree of gastritis induction was quantified.

As a result, as shown in FIG. 12, the gastritis alleviation effect of Cs-ME increased in a concentration-dependent manner when comparing the 50 mg/kg treatment group and the 100 mg/kg treatment group. In particular, it was confirmed that the 100mg/kg Cs-ME treatment group showed a higher gastritis alleviation effect than the ranitidine treatment group.

Example 12. Cs-ME, Quercetin ( Quercetin ), and Camperol ( Kaempferol )of Nitric Oxide (NO) Generating ability compare

The experiment was performed according to the experimental method of Example 2 in order to confirm that the Cs-ME treatment had a greater effect of inhibiting the nitric oxide production ability than the case of the treatment with Quercetin and Campferol alone.

As a result, it was confirmed that, as shown in FIG. 12, when Cs-ME was treated, the nitric oxide production inhibitory effect was greater than that of each treatment with camphorol and quercetin.

The above description of the present invention is for illustrative purposes only, and those of ordinary skill in the art to which the present invention pertains will be able to understand that it is possible to easily transform it into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not limiting.

Claims (11)

A pharmaceutical composition for the prevention or treatment of inflammatory diseases, comprising an extract of Canarium subulatum as an active ingredient,
The inflammatory disease is characterized in that gastritis, a pharmaceutical composition for the prevention or treatment of inflammatory diseases.
delete The method of claim 1,
The extract is a pharmaceutical composition for preventing or treating inflammatory diseases, characterized in that methanol is extracted with a solvent.
The method of claim 1,
The canarium western ratum extract is characterized in that it comprises one or more components selected from the group consisting of quercetin, luteolin, and camperol (Kaempferol), a pharmaceutical for the prevention or treatment of inflammatory diseases Ever composition.
The method of claim 1,
The canarium western ratum extract is characterized in that it inhibits the production of nitrogen monoxide (nitric oxide, NO), a pharmaceutical composition for the prevention or treatment of inflammatory diseases.
The method of claim 1,
The canarium western ratum extract is characterized in that to inhibit the expression of inducible nitric oxide synthase (iNOS), a pharmaceutical composition for the prevention or treatment of inflammatory diseases.
The method of claim 1,
The canarium western ratum extract is characterized in that inhibiting the phosphorylation of IκBα, IKKα / β, Syk, p85 or Src, a pharmaceutical composition for the prevention or treatment of inflammatory diseases.
The method of claim 1,
The canarium western ratum extract inhibits the phosphorylation of IκBα, IKKα/β, Syk, p85 or Src, thereby inhibiting the activity of nuclear factor κB (NF-κB). Pharmaceutical composition for prophylaxis or treatment.
The method of claim 1,
The canarium western ratum extract is characterized in that reducing the nuclear translocation of p50 and p65 (nuclear translocation), a pharmaceutical composition for the prevention or treatment of inflammatory diseases.
As a food composition for preventing or improving inflammatory diseases, comprising an extract of Canarium subulatum as an active ingredient,
The food composition for preventing or improving inflammatory diseases, characterized in that the inflammatory disease is gastritis.

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