KR20210020221A - Composition of an extract of ligularia fischeri - Google Patents

Abstract

The present invention relates to a composition comprising a Ligularia fischeri extract as an active ingredient and to a production method thereof. The composition is effective for preventing or treating inflammatory diseases.

Description

Composition comprising extract of Gomchwi (COMPOSITION OF AN EXTRACT OF LIGULARIA FISCHERI)

The present invention relates to a composition comprising an extract of Gomchwi as an active ingredient and a method of manufacturing the same.

Inflammation is a protective response of a living body against various stimuli such as external infectious agents (bacteria, fungi, viruses, various types of allergens), and generally indicates the pathological condition of an abscess. The inflammatory reaction includes all processes that attempt to remove external infectious agents and restore and regenerate necrotic cells or tissues when invasion is applied to cells or tissues of a living body. Therefore, local blood vessels, various tissue cells in body fluids, and immune cells are involved in this series of reactions. With the recent development of molecular biology, a broad understanding of inflammatory reactions such as gene expression of various cytokines involved in inflammatory diseases and transcription factors that regulate them has become possible.

Among them, NF-kB is an important transcription factor required for the expression of many inflammatory genes. NF-kB plays an important role in the expression of cytokines such as TNF-α and IL-6 that mediate responses to inflammatory stimuli in allergic inflammatory responses. When such NF-κB is activated, it increases the gene expression of iNOS (inducible nitrous oxide systems) and produces an excessive amount of NO. Since NO is closely related to the inflammatory reaction, a substance capable of regulating the generation and expression of enzymes involved in the production is attracting attention as a preventive and therapeutic agent for inflammatory diseases. In mammals, the following NF-kB family has 5 members: RelA/p65, RelB, c-Rel, NF-kB1/p50, and NF-kB2/p52. The p50-RelA/p65 heterodimer is a typical member of the Rel family of transcription factors. In addition, it binds to the promoter of NF-kB dependent inflammatory genes and induces the expression of these genes. p65 is an essential element of NF-kB. NF-kB activity is regulated by RelA/p65 acetylation and deacetylation, which are mediated by histone acetyltransferases (HATs) and histone deacetylases (HDACs).

Histones play an important role in regulating gene transcription activity. Histone acetylation/deacetylation, regulated to act in two directions by HDACs and HATs, is a major epigenetic event that plays an important role in inflammation. Histone acetylation is associated with increased binding of the transcription factor NF-kB. HDAC and HAT play a major role in regulating the pro-inflammatory response. Modified HAT and HDAC activity can lead to a number of diseases including cancer, diabetes, cardiac hypertrophy and asthma. High glucose induces NF-kB activity by aggregation of the transcriptional coactivator molecule p300 having endogenous HAT activity, resulting in histone acetylation and proinflammatory cytokine secretion by DNA unwinding. Histone acetylation is associated with increased activity of NF-kB, which results in increased acetylation of the RelA/p65 subunique of NF-kB.

HDAC is also an important target for anti-inflammatory drug development as it plays an important role in the production of various inflammatory cytokines and immune regulation. HDAC has been reported to participate in the inflammatory response by binding to p65 (RelA), one of the subunits of NF-κB.

Genes Dev, 2000, 14, 55; Expert Rev. Anticancer Ther. 2010, 10, 935; Am. J. Trans. Res, 2011, 3, 166

It is an object of the present invention to provide a pharmaceutical composition for the prevention or treatment of inflammatory diseases comprising as an active ingredient an extract of Gomchwi alcohol having the ability to inhibit the activity of histone acetyltransferase and the ability to inhibit the production of inflammatory cytokines.

In addition, it is an object of the present invention to provide a food composition for preventing or improving inflammatory diseases, comprising as an active ingredient an extract of Gomchwi alcohol having the ability to inhibit the activity of histone acetyltransferase and inhibit the production of inflammatory cytokines.

Finally, an object of the present invention is to provide a method for preparing a Gomchwi alcohol extract having the ability to inhibit the activity of histone acetyltransferase and the production of inflammatory cytokines.

An exemplary embodiment of the present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases comprising the alcohol extract of Gomchwi as an active ingredient.

Another exemplary embodiment of the present invention provides a food composition for preventing or improving inflammatory diseases comprising an alcohol extract of Gomchwi as an active ingredient.

Finally, one embodiment of the present invention, the first step of powdering by drying and pulverizing the gomchwi; A second step of stirring and extracting the gomchwi powder into alcohol, a third step of centrifuging the extracted liquid and filtering and extracting the supernatant obtained by centrifugation; And it provides a method for producing a alcohol extract of Gomchwi comprising a fourth step of concentrating and drying the extracted liquid.

The composition comprising the alcohol extract of Gomchwi according to an exemplary embodiment of the present invention as an active ingredient is effective in preventing or treating inflammatory diseases.

1 is a diagram confirming the activity of inhibiting the activity of acetyltransferase by the alcohol extract of Gomchwi.
2 is a schematic diagram showing the measurement principle of acetyltransferase.
3 is a diagram evaluating the specificity of the ability to inhibit the activity of acetyltransferase by the alcohol extract of Gomchwi.
Figure 4 is a diagram evaluating the ability to inhibit the acetylation of histone / non-histone protein induced during the inflammatory reaction by the inhibition of the activity of acetyltransferase of the alcohol extract of Gomchwi.
5 is a diagram evaluating the inhibitory ability of gene/protein expression of acetyltransferases by alcohol extract of Gomchwi.
6 is a diagram confirming the acetylation inhibitory ability of p65 by the alcohol extract of Gomchwi.
7 is a diagram showing the evaluation of the ability to inhibit expression of a typical inflammatory cytokine, which is a target gene of NF-kB, by alcohol extract of Gomchwi.

Hereinafter, the present specification will be described in more detail.

An exemplary embodiment of the present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases comprising the alcohol extract of Gomchwi as an active ingredient.

In the present specification, when a part "includes" a certain component, it means that other components may be further included rather than excluding other components unless otherwise stated.

In the present specification, the term "active ingredient" refers to an ingredient capable of exhibiting a desired activity by itself or capable of exhibiting activity together with a carrier that is not itself active.

In the present specification, the "prevention" refers to any action that suppresses or delays a disease by administration of the pharmaceutical composition. In addition, the "treatment" refers to any action in which symptoms of a disease are improved or beneficially changed by administration of the pharmaceutical composition.

Gomchwi (Ligularia fischeri) is a dicotyledonous plant, a perennial plant of the Asteraceae family. It grows in highland or deep mountain wetlands, and is distributed in Korea, Japan, China, Sakhalin Island and East Siberia. In oriental medicine, the roots are digged and dried in autumn, called horochil, and prescribed for seawater, whooping cough, asthma, back pain, joint pain, and bruises.

In the exemplary embodiment of the present specification, the concentration of the alcohol is 30% to 80% (v/v).

In another exemplary embodiment, the composition contains the alcohol extract of Gomchwi at a concentration of 5 mg/ml to 100mg/ml.

In the exemplary embodiment of the present specification, the alcohol extract of Gomchwi is an alcohol extract of Gomchwi leaves.

In the exemplary embodiment of the present specification, the composition has the ability to inhibit the activity of histone acetyltransferase. 1 is a diagram confirming the activity of inhibiting the activity of acetyltransferase by the alcohol extract of Gomchwi. 3 is a diagram evaluating the specificity of the ability to inhibit the activity of acetyltransferase by the alcohol extract of Gomchwi. Figure 4 is a diagram evaluating the ability to inhibit the acetylation of histone / non-histone protein induced during the inflammatory reaction by the inhibition of the activity of acetyltransferase of the alcohol extract of Gomchwi. 5 is a diagram evaluating the inhibitory ability of gene/protein expression of acetyltransferases by alcohol extract of Gomchwi. 6 is a diagram confirming the acetylation inhibitory ability of p65 by the alcohol extract of Gomchwi. 1 and 3 to 6, it can be seen that the alcohol extract of Gomchwi according to an exemplary embodiment of the present invention has the ability to inhibit the activity of histone acetyltransferase.

In the exemplary embodiment of the present specification, the composition has an IC ₅₀ value of less than 10 mg/ml with respect to histone acetyltransferase.

1 is a diagram confirming the activity of inhibiting the activity of acetyltransferase by the alcohol extract of Gomchwi. 3 is a diagram evaluating the specificity of the ability to inhibit the activity of acetyltransferase by the alcohol extract of Gomchwi. 1 and 3, it was confirmed that the activity of inhibiting the activity of histone acetyltransferase of the alcoholic extract of Gomchwi according to an exemplary embodiment of the present invention was confirmed, and the IC of less than 10 mg/ml for the histone acetyltransferase of the alcoholic extract of Gomchi _It can be seen that it has a value of 50.

In addition, the composition according to an exemplary embodiment of the present invention has the ability to inhibit the production of inflammatory cytokines. 7 is a diagram showing the evaluation of the ability to inhibit expression of a typical inflammatory cytokine, which is a target gene of NF-kB, by alcohol extract of Gomchwi. 7, it can be seen that the alcohol extract of Gomchwi according to an exemplary embodiment of the present invention suppresses the production of inflammatory cytokines.

The pharmaceutical composition of the present invention does not increase the medicinal efficacy, but may further include an ingredient that is commonly used in pharmaceutical compositions to improve odor, taste, vision, and the like. In addition, the pharmaceutical composition of the present invention may additionally contain a pharmaceutically acceptable additive. Pharmaceutically acceptable additives include, for example, starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, mannitol, syrup, arabic rubber, pregelatinized starch, corn starch, powder cellulose Hydroxypropyl cellulose, Opadry, sodium starch glycolate, lead carnauba, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, sucrose, dextrose, sorbitol and talc, and the like, but are not limited thereto. Additionally, the pharmaceutical composition may be used alone or may contain a substance having a prophylactic or therapeutic activity against an inflammatory disease that has been used previously.

The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier and may be formulated for oral or parenteral human or veterinary use. When formulating the pharmaceutical composition of the present invention, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants and surfactants may be used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations are at least one excipient, such as starch, calcium carbonate, in the pharmaceutical composition containing the compound of the present invention ( Calcium carbonate), sucrose or lactose, and gelatin can be mixed to prepare. Also, in addition to simple excipients, lubricants such as magnesium, sterate, and talc may be used. Liquid preparations for oral use include suspensions, liquid solutions, emulsions and syrups, but may include various excipients, such as wetting agents, sweeteners, fragrances and preservatives, in addition to water and liquid paraffin, which are commonly used simple diluents. . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, and suppositories. As the non-aqueous solvent and suspension solvent, vegetable oils such as propylene glycol, polyethylene glycol and olive oil, and injectable esters such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like may be used.

The pharmaceutical composition of the present invention may be administered orally or parenterally according to a desired method. The above formulation can be prepared in a form for injection (solution, suspension or emulsion), and it is most preferably prepared for oral use such as tablets, capsules, soft capsules, infusion solutions, granules, and pills.

The composition may be prepared by filling the capsules with the alcohol extract of Gomchwi without an excipient or by uniformly and sufficiently contacting the finely divided solid carrier or liquid carrier, or both. The product can then be shaped into the desired formulation if necessary.

Examples of such carrier excipients include starch, water, saline, Ringer's solution and dextrose solution. Suitable formulations known in the art are preferably those disclosed in Remington's Pharmaceutical Science (latest edition), Mack Publishing Company, Easton PA.

The pharmaceutical composition of the present invention can be administered to an individual to prevent or treat inflammatory diseases. The term "individual" used in the present invention has an inflammatory disease, and includes humans with diseases that can improve symptoms by administering the pharmaceutical composition of the present invention, such as horses, sheep, pigs, goats, dogs, etc. It means a mammal, but preferably a human.

As used herein, the term "administration" means introducing the pharmaceutical composition of the present invention to a subject by any suitable method. The route of administration can be administered orally or parenterally through any general route as long as it can reach the target tissue. In addition, the pharmaceutical composition of the present invention can be administered by any device that allows it to move to target cells.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The term used in the present invention, a pharmaceutically effective amount means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the patient's weight, sex, age, health status, and severity. , Drug activity, sensitivity to the drug, time of administration, route of administration and rate of excretion, duration of treatment, factors including drugs used concurrently, and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or administered in combination with other therapeutic agents, and may be administered sequentially or simultaneously with a conventional therapeutic agent. It can be administered single or multiple. It is important to administer an amount capable of obtaining the maximum effect in a minimum amount without side effects in consideration of all the above factors, and can be easily determined by a person skilled in the art.

Another exemplary embodiment of the present invention provides a food composition for preventing or improving inflammatory diseases comprising an alcohol extract of Gomchwi as an active ingredient.

In the present specification, the alcohol extract of Gomchwi may be applied in the same manner as long as it does not contradict each other with the matters mentioned in the aforementioned pharmaceutical composition.

In the present specification, the food composition may be added as it is or may be used together with other foods or food ingredients, and may be appropriately used according to a conventional method.

In the present specification, the "food composition" refers to a food manufactured and processed using raw materials or ingredients having useful functionality for the human body.

There is no particular limitation on the type of food. Examples of foods that can be added to the composition containing the alcohol extract of Gomchwi as an active ingredient include various soups, beverages, teas, drinks, alcoholic beverages, and vitamin complexes, and include all health foods in the usual sense. I can. In addition to the above, the alcohol extract of Gomchwi is a variety of nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin. , Alcohol, carbonated beverages, etc. may contain. In addition, the food composition of the present invention may contain pulp for the production of natural fruit juice, fruit juice beverage and vegetable beverage. These ingredients may be used independently or in combination.

Another exemplary embodiment of the present invention is a first step of powdering by drying and pulverizing gomchwi; A second step of stirring and extracting the gomchwi powder into alcohol, a third step of centrifuging the extracted liquid and filtering and extracting the supernatant obtained by centrifugation; And it provides a method for producing a alcohol extract of Gomchwi comprising a fourth step of concentrating and drying the extracted liquid.

In the exemplary embodiment of the present invention, the first step of drying and grinding the gomchwi may be freeze-dried.

In another exemplary embodiment, the second step of stirring and extracting the gomchwi powder into the alcohol may be mixed into the alcohol having a volume of 2 to 6 times the volume of the gomchwi powder. More specifically, the second step of stirring and extracting the gomchwi powder into the alcohol may be incorporated into the alcohol having a volume of 5 times the volume of the gomchwi powder.

In one exemplary embodiment, the second step of stirring and extracting the gomchwi powder into the alcohol may be stirred at room temperature.

In the exemplary embodiment of the present invention, drying in the fourth step of concentrating and drying the extracted liquid may be freeze-dried.

In the present specification, freeze-drying is a method of freezing the object to be dried at a temperature below the freezing point and removing moisture by sublimation as it is, and may be prepared by a method generally used.

It is possible to provide a pharmaceutical composition comprising as an active ingredient the alcohol extract of Gomchwi prepared by the above production method of the present invention. The pharmaceutical composition may be the same as the description of the pharmaceutical composition described above.

In addition, it is possible to provide a food composition comprising as an active ingredient the alcohol extract of Gomchwi prepared by the above production method of the present invention. The food composition may be the same as the description of the food composition described above.

Hereinafter, examples will be described in detail to aid understanding of the present invention. However, the following examples are for illustrative purposes only, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art.

Example 1. Preparation of alcohol extract of Gomchwi

The washed gomchwi was freeze-dried, dried, and then powdered using a grinder. The powder was mixed with 70% alcohol of 5 times the volume of the powder and extracted while stirring at room temperature. The supernatant obtained by centrifuging the extract was filtered, the extract from which the solid content was removed was concentrated under reduced pressure, and then freeze-dried to prepare the alcoholic extract of Gomchwi.

Example 2. Confirmation of the ability to inhibit the activity of acetyltransferase

In order to confirm the ability to inhibit the activity of acetyltransferase by the alcohol extract of Gomchwi, the alcohol extract of Gomchwi at concentrations of 10mg/ml, 25mg/ml, 50mg/ml, and 100mg/ml prepared in Example 1 was used with acetyltransferase. After reacting for 30 minutes in vitro with the containing nuclear extract, OD (optical density) was measured at 440 nm to observe the activity change.

2 is a schematic diagram showing the measurement principle of acetyltransferase. Specifically, Biovision's HAT activity colorimetric assay kit (Cat. No. K332) was used to measure the activity of acetyltransferase. After the above reagents were mixed and reacted according to the provided protocol, the activity of acetyltransferase was evaluated by measuring OD at 440 nm.

1 is a diagram confirming the activity of inhibiting the activity of acetyltransferase by the alcohol extract of Gomchwi. The activity of acetyltransferase was expressed as (100%)% compared to the control group (group containing only NE). As a result, it was confirmed that the activity of acetyltransferase was approximately 35.4% for 10mg/ml of Gomchwi alcohol extract, 13.3% for 25mg/ml, 9.4% for 50mg/ml, and 8.2% for 100mg/ml. I did. _{The IC 50} (half maximal inhibitory concentration) of Gomchwi alcohol extract, which can reduce the activity of acetyltransferase by about 50%, can be expected to be about 5.33mg/ml.

Example 3. Evaluation of specificity of inhibition of acetyltransferase activity

In order to evaluate the specificity of inhibition of the activity of the acetyltransfer enzyme by the alcohol extract of Gomchwi, the extract of Gomchwi prepared in Example 1 at concentrations of 10mg/ml, 25mg/ml, 50mg/ml and 100mg/ml was used as p300, CBP, and pCAF. After reacting with in vitro for 30 minutes, OD (optical density) was measured at 440 nm to observe the change in activity. The measurement principle of acetyltransferase is the same as that mentioned in Example 2.

3 is a diagram evaluating the specificity of the ability to inhibit the activity of acetyltransferase by the alcohol extract of Gomchwi. The activity of acetyltransferase was expressed as (100%)% compared to the control group (group containing only acetyltransferase). Specifically, looking at FIG. 3, for acetyltransferase p300, 36.5% at a concentration of 10mg/ml alcohol extract of Gomchwi, 20.4% at a concentration of 25mg/ml, 14.5% at a concentration of 50mg/ml, and a concentration of 100mg/ml In, 15.3% of the activity of acetyltransferase was confirmed. Therefore, the IC ₅₀ value of the alcohol extract of Gomchwi with respect to p300 can be expected to be about 6.54mg/ml. In addition, for CBP, at a concentration of 10 mg/ml of alcohol extract of Gomchwi, 24.8% at a concentration of 25 mg/ml, 17.3% at a concentration of 50 mg/ml, and 16.0% of acetyltransferase at a concentration of 100 mg/ml. Activity was confirmed, and the IC ₅₀ value of the alcohol extract of Gomchwi against CBP can be expected to be about 7.54mg/ml. Finally, for pCAF, acetyltransferase of 42.3% at a concentration of 10mg/ml of alcohol extract of Gomchwi, 25.0% at a concentration of 25mg/ml, 21.7% at a concentration of 50mg/ml, and 23.5% at a concentration of 100mg/ml. _{Was confirmed, the IC 50} value of the alcohol extract of Gomchwi against pCAF can be expected to be about 9.36mg/ml. As a result of FIG. 3, it was confirmed that the alcohol extract of Gomchwi prepared in Example 1 had an inhibitory ability of acetyltransferase, and in particular, it was confirmed that the sensitivity to p300 is somewhat high.

Example 4. Evaluation of acetylation inhibitory ability of histone and non-histone proteins

In order to observe the effect of the acetyltransferase activity inhibitory ability of the alcohol extract of Gomchwi on the anti-inflammatory activity, 10nM of LPS (lipopolysaccharides) and 25mg/ml and 50mg/ml of Gomchi extract were combined in Raw264.7 cell line for 2 hours. Western blotting was performed by extracting non-histone proteins and histone proteins from the cells.

Figure 4 is a diagram evaluating the ability to inhibit the acetylation of histone / non-histone protein induced during the inflammatory reaction by the inhibition of the activity of acetyltransferase of the alcohol extract of Gomchwi. Looking specifically at Figure 4, it can be seen that the acetylation of non-histone proteins and histones H3K9, H4K8, H4K12, H4K15 and H4K16 is increased when the inflammatory response is induced by LPS treatment. In addition, it was confirmed that the acetylation of non-histone and histone proteins increased by LPS was decreased when the LPS and the alcohol extract of Gomchwi were mixed. As a result of this, it was confirmed that the alcohol extract of Gomchwi had the inhibitory ability of acetyltransferase, and also had the inhibitory ability of acetylation of non-histone and histone proteins induced by the inflammatory reaction.

Example 5. Evaluation of the ability of acetyltransferase to inhibit gene and protein expression

QRT-PCR (quantitative real time polymerase chain reaction) was performed to observe whether the gene expression of acetylase transfer enzymes, which directly affect the acetylation of non-histone/histone proteins, was inhibited by the alcohol extract of Gomchwi. Induce an inflammatory response by treating the raw264.7 cell line with 10 mg/mL, 25 mg/mL, 50 mg/mL, and 100 mg/mL concentrations of alcohol extract of Gomchwi for 18 hours and then adding 1 ng LPS for the last 6 hours. I did. At the final 18 hours, cells were harvested, RNA was extracted, cDNA was synthesized by reverse transcription using reverse transcriptase, and used for qRT-PCR. Primers capable of selectively amplifying P300, CBP, and pCAF were synthesized, amplified by the corresponding qRT-PCR, and then the expression changes were compared with the control group.

5 is a diagram evaluating the inhibitory ability of gene/protein expression of acetyltransferases by alcohol extract of Gomchwi. Specifically, looking at FIG. 5, the gene expression of p300 and CBP acetyltransferase increased by about 2 times in the case of p300, 1.9 times, and 1.7 times in the case of CBP by the inflammatory reaction (LPS treatment) compared to the control group, 10 mg/mL, When 25 mg/mL and 50 mg/mL of Gomchwi alcohol extract were pretreated, the expression of p300 gene was the same as that of the control without LPS, and when 100mg/mL of Gomchwi alcohol extract was pretreated, it was 0.4 times larger than the control. Decreased. In addition, the expression of CBP, which was increased by 1.7 times by LPS, was 0.6 times at the concentration of 10 mg/ml of the alcohol extract of Gomchwi, 0.4 times at the concentration of 25 mg/ml, 0.6 times at the concentration of 50 mg/ml by the pretreatment of the alcohol extract of Gomchwi. It was confirmed that it appeared to be 0.7 times at the concentration of pear and 100 mg/ml. In the case of pCAF, no increase in gene expression was observed by LPS treatment, but it was confirmed that gene expression was decreased compared to the control group by pretreatment of alcohol extract of Gomchwi at 10mg/mL concentration and 25mg/mL concentration. In addition, as a result of observing the protein expression of p300, CBP and pCAF through western blotting after protein extraction through the same preparation process, it was confirmed that the alcohol extract of Gomchwi inhibited the protein expression of p300 and CBP. As a result of the above, it was confirmed that the alcohol extract of Gomchwi inhibited the expression of genes and proteins of acetyltransferase.

Example 6. Evaluation of p65's ability to inhibit acetylation

p65 is a major transcription factor mediating the inflammatory response and plays an important role in the expression of inflammatory cytokines. In order for p65 to mediate the inflammatory response, acetylation of p65K310 by inflammatory signaling and its migration to the nucleus are essential. The following experiments were conducted to evaluate whether the acetylation of p65K310 was inhibited through the inhibition of the activity of the acetyltransferase p300 of the alcohol extract of Gomchwi, and successively inhibited intranuclear influx.

(A) Raw264.7 cell line was treated with 10 mg/mL, 25 mg/mL, 50 mg/mL, and 100 mg/mL concentrations of alcohol extract of Gomchwi, respectively, after 18 hours of pretreatment, followed by 1ng LPS for the last 2 hours. Induce an inflammatory response. At the final 18 hours, the cells were harvested, cytoplasmic proteins and nuclear proteins were separated, and western blotting was performed. 6 is a diagram confirming the acetylation inhibitory ability of p65 by the alcohol extract of Gomchwi. Referring to FIG. 6, when the alcohol extract of Gomchwi was treated, the acetylation of p65K310 was decreased, and as the concentration of the etonol extract of Gomchwi was increased, the intranuclear migration of p65 was decreased.

(B) Through a confocal microscope, p65 protein (Green, FITC) in actual cells was stained and its migration was observed. Figure 6 is a diagram confirming the acetylation inhibitory ability of p65 by the alcohol extract of Gomchwi. Looking closely at Figure 6, while observed in both the nucleus and cytoplasm by LPS treatment, the p65 protein was observed only in the cytoplasm in the 50mg/mL alcohol extract pretreatment group of Gomchwi. Through this, it was confirmed that the extract of Gomchwi inhibited the migration to the nucleus through the inhibition of the acetylation of p65.

Example 7. Evaluation of inflammatory cytokine expression inhibition ability

The activity of acetyltransferase acts as a direct cause of increased expression of genes. Therefore, the effect of inhibition of acetyltransferase activity by alcohol extract of Gomchwi on the expression of inflammatory cytokines, which is a target gene of NF-kB, was evaluated through qRT-PCR (quantitative real-time polymerase chain reaction). Inflammation by treatment with Etonol extract of Gomchwi at concentrations of 10 mg/mL, 25 mg/mL, 50 mg/mL, and 100 mg/mL, respectively, to Raw264.7 cell line by adding 1 ng of LPS for the last 6 hours after 18 hours pretreatment. The reaction was induced. At the final 18 hours, cells were harvested, RNA was extracted, cDNA was synthesized by reverse transcription using reverse transcriptase, and then used for qRT-PCR. Primers capable of selectively amplifying IL-6, IL-1β, and TNF-α were synthesized, amplified by the corresponding qRT-PCR, and the expression changes were compared with the control group. 7 is a diagram showing the evaluation of the ability to inhibit expression of a typical inflammatory cytokine, which is a target gene of NF-kB, by alcohol extract of Gomchwi. Looking closely at FIG. 7, both IL-6 and IL-1β showed a 3,000-fold increase in expression compared to the control group when inducing an inflammatory response by LPS, whereas in the pretreated group of alcohol extract of Gomchwi, IL-6 was 1238, respectively, by concentration. 1395, 1009, and 499 times increased, and IL-1β increased by 2121, 1528, 1291, and 1057 times for each concentration, respectively. In addition, the expression of TNFα was increased by about 13 times by LPS, and when the alcohol extract of Gomchwi was pretreated, it was confirmed that it was increased by 11.8, 12.4, 10.4, and 10.5 times, respectively, by concentration.

As a result of the experiments of Examples 2 to 7, it can be seen that the alcohol extract of Gomchwi according to an exemplary embodiment of the present invention has the ability to inhibit the activity of acetyltransferase and the ability to inhibit the production of inflammatory cytokines. Therefore, it can be confirmed that the alcohol extract of Gomchwi according to an exemplary embodiment of the present invention is effective in preventing or treating inflammatory diseases.

Claims (11)

A pharmaceutical composition for preventing or treating inflammatory diseases, comprising the extract of Gomchwi alcohol as an active ingredient. The method of claim 1,
The concentration of the alcohol is 30% to 80% (v / v) of the inflammatory disease prevention or treatment pharmaceutical composition. The method of claim 1,
The composition is a pharmaceutical composition for preventing or treating inflammatory diseases containing the alcohol extract of the gomchwi in a concentration of 5 mg/ml to 100mg/ml. The method of claim 1,
The alcohol extract of Gomchwi is a pharmaceutical composition for preventing or treating inflammatory diseases that is an extract of alcoholic extract of Gomchwi leaves. The method of claim 1,
The composition is a pharmaceutical composition for preventing or treating inflammatory diseases having the ability to inhibit the activity of histone acetyltransferase. The method of claim 1,
_{The composition has an IC 50} value of less than 10 mg/ml with respect to histone acetyltransferase, an inflammatory disease or a pharmaceutical composition for treatment. The method of claim 1,
The composition is a pharmaceutical composition for preventing or treating inflammatory diseases having the ability to inhibit the production of inflammatory cytokines. Food composition for the prevention or improvement of inflammatory diseases comprising the alcohol extract of Gomchwi as an active ingredient. A first step of drying and pulverizing the gomchwi to powder it;
A second step of stirring and extracting the gomchwi powder into alcohol;
A third step of centrifuging the extracted liquid and filtering and extracting the supernatant obtained by centrifugation; And
A method for producing a alcohol extract of Gomchwi comprising a fourth step of concentrating and drying the extracted liquid. The method of claim 9,
The first step of drying and pulverizing the gomchwi is freeze-drying. The method of claim 9,
The second step of stirring and extracting the gomchwi powder into the alcohol is a method for producing an alcoholic extract of Gomchwi that is mixed in a volume of 2 to 6 times the volume of the gomchwi powder.

Images