KR20230116981A - Pharmaceutical composition for improving inflammatory bowel disease comprising Sorbus commixta extracts - Google Patents

Abstract

The present invention relates to a composition for improving inflammatory bowel disease containing a rowan tree extract, and more particularly, to a composition for improving inflammatory bowel disease containing a rowan tree extract exhibiting an anti-inflammatory effect by inhibiting the activity of NLRP3 inflammasome . The composition for improving inflammatory bowel disease according to the present invention can be usefully used for patients who need it by acting usefully on the NLRP3 inflammatory complex control mechanism.

Description

Composition for improving inflammatory bowel disease comprising rowan tree extract {Pharmaceutical composition for improving inflammatory bowel disease comprising Sorbus commixta extracts}

The present invention relates to a composition for improving inflammatory bowel disease containing a rowan tree extract, and more particularly, to a composition for improving inflammatory bowel disease containing a rowan tree extract exhibiting an anti-inflammatory effect by inhibiting the activity of NLRP3 inflammasome .

Rowan ( Sorbus commixta Hedl.) is a deciduous tree belonging to the Rosaceae family and is distributed in East Asia including Korea and Japan. In the West, rowan trees are used as street trees or garden trees, and in the East, including Korea, studies are being conducted using fruits and stems to utilize their functionality. According to a study by Seo et al., four types of rowan trees are naturally distributed in Korea (Reference: Heewon Seo, Ecology of Rowan Trees and Related Relationships by DNA Analysis, Graduate School of Sunchon National University, 2008). As Eunbak et al. said, rowan tree ( S. commixta Hedl.) and horse chestnut tree ( S. amurensis Koehne (= Sorbus pohuashanensis (Hance) Hedlund)) seem to be mixed and distributed (Reference: Folk medicine Rowan herbal medicine Academic study. Park Jong-hee, Journal of Herbal Pharmacy 2009 32-34).

Looking at the use of oriental medicine, there are examples of using rowan wood as a Jeonggongdeung (丁公藤) or as a bone tree (接骨木) with similar properties in Korea, but all of them have different origin plants. According to 『中華本草』, dangma a tree is called Hwachu (花楸), and the fruit is used for cold sores, cold sores, sore throats, and sore throats for its efficacy in treating cold sores and indigestion. poisonous It is recorded that it is used for 慢性气管支炎, 痢疾, etc. with the efficacy of 止痢, so it can be seen that it is a different medicinal material from Dingong藤 or 接骨木 (Reference: Chinese herbal medicine 4th volume p.290-291, Shanghai Science and Technology Publishing House, 1999) .

It is reported that rowan tree contains compounds such as catechin-7-Ο-β-D1apiofuranoside and catechin-7-Ο-β-D-xylopyranoside in branches and lupane-type triterpene and flavonoid in bark ( References: Bhatt et al., 2009; Na et al., 2002).

Components isolated and reported from rowan stems include β-sitosteryl-3-O-β-D-glucopyranoside, cecropiacic acid, lupeol, betulin, betulinic acid, ursolic acid, and β-sitosterol of the triterpenoid family, and catechin-7 of the flavonoid family. -O-β1D-xylopyranoside and catechin-7-O-β-D-apiofuranoside are known, and lignans include (+)-lyoniresinol, lyoniside, tiliamuroside A, nudiposide, ssioriside, prupaside, sorcomiside A and B as phenolic glycosides, And β-pyrufuran, 1, 2, 4-trimethoxydibenzofuran-3, 9-diol and prunasin of the dibenzofuran family are known.

Experimental studies on rowan include antithrombotic activity using rowan stems, anticancer effects using rowan leaves, fruits and bark (References: Lee MK, Lee HY, Lee JH, Oh JS, Chio GP, Kim JH, Kim JD. Anticancer effect of Sorbus commixta Hedl extracts. Korean J. Medicinal Croup Sci. 2002 ; 10: 403-8), Antioxidant effect study of high-pressure solvent extracts (References: Kang MA, Kim MB, Kim JH, Ko YH, Lim SB. Integral antioxidative capacity and antimicrobial activity of pressurized liquid extracts from 40 selected plant species. J Korean Soc Food Sci Nutr. 2010; 39: 1249- 56.), Inhibition of bone loss and cartilage damage by extracts of rowan and local plant Effect (Reference: Moon EJ, Youn YS, Choi BY, Jeing HU, Park JH, Oh MS, Soh YJ, Kim SY. Extracts of Sorbus commixta and Geranium thunbergii inhibit osteoclastogenesis and stimulate chondrogenesis. J. Korea Academia Industrial Cooperation Society. 2010; 11: 3358-65.) and antioxidant and anti-aging effects of rowan branch extracts (Reference: Lim GN, Park MA, Park SN. Antioxidative and Antiaging Effects of Sorbus commixta twig extracts. J. of the Korean Oil Chemists' Soc 2011;28: 482-90.).

Inflammatory bowel disease (IBD) is a disease characterized by chronic repeated improvement and exacerbation of inflammation in the entire gastrointestinal tract and intestinal mucosa, increasing the prevalence of colorectal cancer. , UC) and Crohn's Disease (CD) (References: Choi EJ, Lee HJ, Kim WJ, Han KI, Iwasa M, Kobayashi K, et al. Enterococcus faecalis EF-2001 protects DNBS-induced inflammatory bowel disease in mice model. PloS one 2019;14(2):e0210854.)

The cause of IBD has not been clearly identified, and various causes such as environmental, genetic, and psychological factors are assumed. Recently, it has been reported that changes in the intestinal immune system caused by changes in intestinal microbes cause inflammation in the intestinal tissue. (Reference: Korean Association for the Study of Intestinal Diseases (KASID). Easy to understand ulcerative colitis. Seoul: Koonja; 2003, p. 8.) .

Immunomodulatory drugs, steroids, and 5-aminosalicylic acid (5-ASA) are used as treatment methods for IBD, but there are reports that treatment fails in 20-40% of patients, leading to colon resection as a side effect (References). : Jeen YT, Kim J. Advances in ulcerative colitis therapy.The Korean Journal of Medicine 2009;76(6):654-6).

Inflammation is one of the defense mechanisms that protects our body from invasion of pathogens or tissue damage, but when an immune response is strong, cells or tissues are attacked and an inflammatory response in a bad sense that causes chronic immune disease proceeds. Various substances such as pro-inflammatory cytokine and inflammasome are involved (Reference: Lee, J.R., Kim, Y.W., Byun, S.H., Kim, S.C., and Park, S.J. 2015. Anti-inflammatory effects of the fermentation extracts consisting of soybean, red ginseng and Citrus Unshiu Peel. Kor. J. Herbol.30, 59-65.). The inflammasome is a protein complex expressed in immune cells, and proteins normally present in cells form a protein complex by stimulation to activate the inflammasome. Representatively, there are NLRP1 and NLRP3, which are known to bind to caspase-1 to convert pro-IL-1β to an active form and induce the secretion of IL-1β from cells (Reference: Lee, G. 2013. Inflammasomes, multi -cellular protein complex in myeloid cells, induce several metabolic diseases via interleukin-1β maturation. J. Biomed. Res. 14, 195-200.).

NLRP3 is a molecular complex responsible for innate immune defense through maturation of cytokines after inflammatory responses such as interleukin 1β in response to abnormal signals such as infection and metabolic dysregulation.

As an example of using rowan in pharmaceutical form, Korean Registration No. 10-1076482 (October 18, 2011) discloses a pharmaceutical composition for detoxifying smoking toxicity containing rowan extract as an active ingredient, and Registration No. 10-1076482 In No. 2289438 (August 6, 2021), kudzu, barnacle tree, rowan, ginger, jujube, etc., which can treat alcohol and food damage and relieve alcohol poison by removing moisture and heat accumulated in the body. Disclosed is a method for manufacturing a health food composition containing extracts of raisins, rowan tree, duck tree and kudzu for safe and healthy consumption using only 16 natural ingredients of, and a health food composition prepared thereby. Korea Registration No. 10-1508053 (March 26, 2015) discloses a food composition for relieving joint pain comprising 30% to 70% by weight of a hyssop extract and 30% to 70% by weight of a rowan extract.

Korea Publication No. 10-2014-0071738 (June 12, 2014) Korea Publication No. 10-2013-0026376 (March 13, 2013) Korean Registration No. 10-1076482 (October 18, 2011) Korea Registration No. 10-1508053 (March 26, 2015) Korean Registration No. 10-2289438 (08/06/2021)

As a result of intensive research to develop medicinal substances effective for inflammatory bowel disease, the inventors of the present invention found that, as described later, the hot water extract and ethanol extract of rowan tree stems act usefully on the NLRP3 inflammatory complex control mechanism, making them useful for patients who need them. It was found that it can be used and came to complete the present invention.

Therefore, an object of the present invention, in one aspect, is to provide a composition for improving inflammatory diseases that can be usefully used for patients in need thereof by acting usefully on the NLRP3 inflammatory complex control mechanism.

The object of the present invention as described above, in one aspect

It can be achieved by a composition for improving inflammatory bowel disease comprising an organic solvent extract of rowan tree as a main component.

The composition for improving inflammatory bowel disease according to the present invention can be usefully used for patients who need it by acting usefully on the NLRP3 inflammatory complex control mechanism.

1 is a graph showing whether rowan extract inhibits the production of IL-1β, an LPS-induced inflammatory cytokine.
Figure 2 is a graph showing whether rowan extract inhibits the production of IL-6, an LPS-induced inflammatory cytokine.
Figure 3 is a graph showing whether rowan extract inhibits the production of TNF-α, an LPS-induced inflammatory cytokine.
Figure 4 is a photograph confirming whether rowan extract inhibits nigericin-induced NLRP3 inflammasome activation.
5 is a graph showing the results of comparison of IL-1β in nigericin-induced activity of NLRP3 inflammasomes in BMDM.
6 is a result confirming whether rowan extract inhibits MSU crystal-induced NLRP3 inflammasome activation.
7 is a graph showing the results of comparison of IL-1β in the MSU-induced activity of NLRP3 inflammasomes in BMDM.
Figure 8 is a graph showing whether rowan extract inhibits intestinal inflammation in an inflammatory bowel disease animal model induced by DSS, and Figure 8A is a result of measuring the intestinal length of a mouse.
9 is a graph showing the comparison results of the pro-inflammatory cytokine IL-1β in the DSS-induced rectum of mice.
10 is a graph showing the comparison results of the pro-inflammatory cytokine IL-6 in the DSS-induced rectum of mice.

In one aspect, the present invention provides a composition for improving inflammatory bowel disease, characterized in that it contains an organic solvent extract of rowan stems as a main component.

The present invention, in a further aspect,

The extract is

a1) obtaining an extract by adding 10 to 25 times the purified water or an organic solvent to the dried herbal medicine and extracting at a temperature of 60 to 110 ° C. for 2 to 72 hours;

a2) filtering the extract of the above step with filter paper to filter out solids;

a3) Filtering the solids and concentrating the obtained filtrates to a brix of 20 or more for 4 to 8 hours under a reduced pressure of -0.08 to -0.09 MPA at a temperature of 50 to 65 ° C.: and

a4) a process of low-temperature or freeze-drying the concentrate obtained in the above step under vacuum at a temperature of 55 to 65 ° C. for 24 to 48 hours; to provide a composition for improving inflammatory bowel disease, characterized in that the extraction.

The present invention, in another further aspect,

The composition is a medicine or health functional food, and the rowan extract provides a composition for improving inflammatory bowel disease, characterized in that it is administered at a dose of 800 mg / kg.

The present invention, in another further aspect,

The composition provides a composition for improving inflammatory bowel disease, characterized in that it exhibits an anti-inflammatory effect by inhibiting the activity of the NLRP3 inflammasome.

Hereinafter, the composition for improving inflammatory bowel disease comprising the rowan tree extract of the present invention as a main component will be described in more detail according to a preferred embodiment.

The terms or words used in this specification and claims are not limited to the usual or dictionary meanings, and the inventor can properly define the concept of the term in order to explain his or her invention in the best way. Based on this, it should be interpreted as a meaning and concept consistent with the technical spirit of the present invention.

Therefore, since the embodiments described in this specification and the configurations shown in the drawings are only the most preferred embodiments of the present invention, it is understood that there may be various equivalents and modifications that can replace them at the time of this application. shall.

In addition, terms used in the present invention are only used to describe specific embodiments, and are not intended to limit the present invention. Singular expressions include plural expressions unless the context clearly dictates otherwise.

Rowan extract according to the present invention

a1) obtaining an extract by adding 10 to 25 times the purified water or organic solvent to the dried herbal medicine (stem) and extracting at a temperature of 60 to 110 ° C. for 2 to 72 hours;

a2) filtering the extract of the above step with filter paper to filter out solids;

a3) Filtering the solids and concentrating the obtained filtrates to a brix of 20 or more for 4 to 8 hours under a reduced pressure of -0.08 to -0.09 MPA at a temperature of 50 to 65 ° C.: and

a4) It may be preferable to extract by a process of low-temperature or freeze-drying the concentrate obtained in the above step at a temperature of 55 to 65 ° C. for 24 to 48 hours under vacuum.

If necessary, the steps a3) and a4) may be omitted.

Here, as the extraction solvent, at least one selected from water, a lower alcohol having 1 to 4 carbon atoms, a polyhydric alcohol, or a mixture thereof may be used. Methanol, ethanol, etc. can be used as a lower alcohol having 1 to 4 carbon atoms, and butylene glycol, propylene glycol, pentylene glycol, etc. can be used as a polyhydric alcohol. As the mixture, a mixture of water and a lower alcohol, a mixture of water and a polyhydric alcohol, a mixture of a lower alcohol and a polyhydric alcohol, or a mixture of water, a lower alcohol and a polyhydric alcohol may be used.

Following the drying process, a packaging and sterilization process may be further included to suit product conditions.

The drying process may be powdered by vacuum concentration, spray drying or freeze drying according to conventional methods.

The sterilization process may be preferably performed at 80 to 100° C. for 5 to 20 minutes according to a conventional method.

As described below, the caspase-1 expression level of the rowan extract of the present invention was measured through immunological blotting, and as a result, caspase-1 expression was suppressed at a concentration of 200 μg/ml of rowan tree, and IL-1β expression through immunological blotting As a result of measuring the amount, IL-1β expression was suppressed at a concentration of 200 μg/ml rowan tree, and as a result of measuring IL-1β secretion using enzyme-linked immunosorbent assay (ELISA), production of IL-1β at a concentration of 200 μg/ml rowan tree In the DSS-induced ulcerative colitis model, the production of inflammatory cytokines IL-1β and IL-6 was reduced in the group administered with rowan 800 mg/kg orally. Since it is confirmed that there is an anti-inflammatory effect by suppressing the activity, it can be effectively used as a medicine or health functional food for preventing and improving inflammatory bowel disease.

The composition for improving inflammatory bowel disease of the present invention can be particularly preferably used for inflammatory diseases such as Crohn's disease, gastric ulcer, chronic ulcer, and ulcerative colitis without toxicity and side effects, and those skilled in the art can treat those suffering from such diseases, conditions and abnormalities. The alleged recipients can be readily identified.

The pharmaceutical composition containing the organic solvent extract of rowan stems of the present invention can be prepared according to conventional methods according to the purpose of use, oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., and sterilization. It can be formulated and used in various forms such as injections of injection solutions, and can be administered through various routes including oral administration or intravenous, intraperitoneal, subcutaneous, rectal, topical administration, and the like.

These pharmaceutical compositions may further include carriers, excipients, or diluents, and examples of suitable carriers, excipients, or diluents that may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, Starch, gum acacia, alginates, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, amorphous cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. etc. can be mentioned. In addition, the pharmaceutical composition of the present invention may further include fillers, anti-agglomerating agents, lubricants, wetting agents, flavoring agents, emulsifiers, preservatives, and the like.

As a preferred embodiment, solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in the pharmaceutical composition, for example, starch, calcium carbonate, It is formulated by mixing sucrose, lactose, gelatin, etc. In addition, lubricants such as magnesium stearate and talc may be used in addition to simple excipients.

As a preferred embodiment, liquid preparations for oral use may include suspensions, internal solutions, emulsions, syrups, and the like, and various excipients such as wetting agents, sweeteners, Fragrance, preservatives, etc. may be included.

As a preferred embodiment, preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, suppositories, and the like. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Conventional additives such as solubilizers, tonicity agents, suspending agents, emulsifiers, stabilizers, preservatives and the like may be included in the injection.

The organic solvent extract of rowan stems of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level is the type, severity, drug activity, It may be determined according to factors including sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, 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 in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple times. Considering all of the above factors, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art.

In a preferred embodiment, the effective amount of the rowan stem extract in the pharmaceutical composition of the present invention may vary depending on the patient's age, sex, and weight, and is generally 1 to 5,000 mg, preferably 100 to 3,000 mg per kg of body weight. It can be administered daily or every other day or divided into 1 to 3 times a day. However, since it may increase or decrease according to the route of administration, severity of disease, sex, weight, age, etc., the dosage is not limited to the scope of the present invention in any way.

The pharmaceutical composition of the present invention may be administered to a subject through various routes. All modes of administration can be envisaged, for example by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

In the present invention, "administration" means providing a predetermined substance to a patient by any suitable method, and the route of administration of the pharmaceutical composition of the present invention is oral or parenteral through all common routes as long as it can reach the target tissue. can be administered orally. In addition, the composition of the present invention may be administered using any device capable of delivering active ingredients to target cells.

The food composition of the present invention may include sweeteners, flavors, physiologically active ingredients, minerals, and the like in addition to the active ingredients.

Sweeteners can be used in amounts that give the food a moderately sweet taste, and can be natural or synthetic. Preferably, a natural sweetener is used, and examples of the natural sweetener include sugar sweeteners such as corn syrup solids, honey, sucrose, fructose, lactose, and maltose.

Flavors may be used to improve taste or aroma, and both natural and synthetic flavors may be used. Preferably, it is the case of using a natural one. In case of using natural ones, in addition to flavor, the purpose of enhancing nutrition can also be combined. As a natural flavoring agent, it may be obtained from apples, lemons, tangerines, grapes, strawberries, peaches, etc., or obtained from green tea leaves, roundworms, bamboo leaves, cinnamon, chrysanthemum leaves, jasmine, and the like. In addition, those obtained from ginseng (red ginseng), bamboo shoots, aloe vera, ginkgo, etc. can be used. Natural flavors can be liquid concentrates or solid extracts. In some cases, synthetic flavors may be used, and synthetic flavors may include esters, alcohols, aldehydes, terpenes, and the like.

As the physiologically active substance, catechins such as catechin, epicatechin, gallocatechin, and epigallocatechin, and vitamins such as retinol, ascorbic acid, tocopherol, calciferol, thiamine, and riboflavin may be used.

Examples of minerals include calcium, magnesium, chromium, cobalt, copper, fluoride, germanium, iodine, iron, lithium, magnesium, manganese, molybdenum, phosphorus, potassium, selenium, silicon, sodium, sulfur, vanadium, zinc, and the like.

In addition, the food composition of the present invention may include a preservative, an emulsifier, an acidulant, a thickener, and the like, if necessary, in addition to the sweetener.

These preservatives, emulsifiers, etc. are preferably added and used in very small amounts as long as the purpose to which they are added can be achieved. A trace amount means a range of 0.0005% by weight to about 0.5% by weight based on the total weight of the food composition when expressed numerically.

Preservatives that may be used include sodium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate, EDTA (ethylenediaminetetraacetic acid) and the like.

Examples of emulsifiers that may be used include gum acacia, carboxymethylcellulose, xanthan gum, pectin, and the like.

Acidulants that may be used include acid salt, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, phosphoric acid, and the like. Such an acidulant may be added to the food composition to have an appropriate acidity for the purpose of inhibiting the growth of microorganisms in addition to enhancing the taste.

As thickeners that can be used, suspending agents, sedimentation agents, gel forming agents, bulking agents and the like may be mentioned.

In a preferred embodiment, the food composition according to the present invention is, for example, honey, malt, glycerin, red ginseng, cornus officinalis, bokryeong, sukjihwang, cinnamon, cordyceps sinensis, grapefruit extract, licorice, barley medicinal leaf powder, western lion extract, St. John's wort , selenium yeast, vitamin C, citric acid, nicotinic acid, sodium benzoate, aspartame, saccharin, pectin, malitol, sorbitol, xylitol, guar gum, skim milk powder, and oligosaccharides. can increase They are suitably used in an amount of about 50 to 80% based on the total weight of the composition of the present invention.

(Example)

Hereinafter, the present invention will be described in more detail based on the following examples. However, the following examples are only for exemplifying the present invention, and the scope of the present invention is not limited or limited by the following examples. Therefore, those skilled in the art to which the present invention belongs can make various changes and modifications to the present invention without departing from the spirit of the present invention, and it is obvious that this also falls within the scope of the present invention. .

1. Animal and cell culture

C57BL/6 mice (male) were purchased from Hanil Laboratories (Jeonju, Republic of Korea) and were acclimatized in a specific pathogen free (SPF) animal facility for at least one week prior to the experiment. Mice were bred in an environment in which a 12:12 hour light/dark cycle, temperature (23±3° C.) and humidity (40-60%) were controlled, and were allowed to freely consume food and water. Bone marrow-derived primary macrophages (BMDMs) were prepared after separating bone marrow from C57BL/6 mice according to a known method. The macrophages were cultured in DMEM medium supplemented with 10% (v/v) fetal bovine serum (Gibco, Carlsbad, Canada), 10,000 unit/mL penicillin, and 10,000 μg/mL streptomycin at 37°C, 5% CO ₂ incubator. cultured in.

2. Reagents

tzenstrasse, Austria)에서, IL-1β 및 IL-1β 전구체에 대한 항체는 R & D Systems (Minneapolis, MN)에서 구입하였다. LPS purified from Escherichia coli was purchased from List biological Laboratories (Campbell, CA) and dissolved in endotoxin-free water for use. MCC 950 was purchased from Invivogen (San Diego, CA), and monosodium urate (MSU) and ATP were purchased from Invivogen (Carlsbad, CA). Antibodies against mouse caspase-1 and mouse caspase-1 precursors are adipogen (Sch

tzenstrasse, Austria), antibodies against IL-1β and IL-1β precursors were purchased from R & D Systems (Minneapolis, MN).

3. Animal model of inflammatory bowel disease induced by dextran sulfate sodium (DSS)

C57BL/6 mice (male, 8 weeks old) were randomly divided into 3 groups (n=4~6/group) as follows: DSS was purchased from MPbio (Solon, OH) and mixed in sterile distilled water for 2 weeks. It was dissolved in % concentration and supplied for free drinking, and rowan extract was orally administered every day. Weight loss, diarrhea, and visual bleeding were observed daily. On day 10, the mice were sacrificed, and the large intestine was separated and the length was measured. Thereafter, the rectal region was cut and proteins were extracted using a T-PER solution purchased from Thermo Scientific (Waltham, MA), followed by enzyme-linked immunosorbent assay.

4. Statistical analysis

Statistical analysis was performed using GraphPad Prism7 (GraphPad Software, San Diego, CA, USA). Data were expressed in means ㅁ SEM format, one-way ANOVA was applied, and Turkey's test was performed for post hoc test. Statistical significance level was carried out at P-value<0.05, P<0.01, P<0.001.

Preparation Example 1: Preparation of rowan extract

Rowan tree was purchased from Gwangmyeongdang (Ulsan, Korea), and 500 g was hot water extracted in 5 L of tertiary distilled water for 2 hours. The extract was filtered with a filter paper to filter out solids, and the filtrate was combined and concentrated for 6 hours under a reduced pressure of -0.08 to -0.09 MPA at a temperature of 55 ° C. The concentrate was freeze-dried to prepare a hot water extract of rowan.

Subsequently, in the same manner as above, ethanol was used as the solvent to obtain an ethanol extract, and each extract was mixed at a weight ratio of 1:1 to prepare a sample to be used in a subsequent test.

Preparation Example 2: Preparation of tablets

Rowan extract (Preparation Example 1) 10 mg

lactose 60mg

starch 15 mg

magnesium stearate 5mg

crystalline cellulose 15 mg

One embodiment of the present invention is the use of said tablet as an active ingredient of the tablet exemplified above. Tablets can be prepared by conventional methods, and one preferred embodiment is in the form of tablets or soft capsules having conventional enteric coatings (eg, hydroxypropylmethyl cellulose phthalate), sugar coatings or shell coatings.

Preparation Example 3: Preparation of health drink

Based on 100ml of beverage, 1g of rowan stem extract of Preparation Example 2, 1g of citric acid, 100mg of oligosaccharide, 2g of plum concentrate, 1g of taurine, and the remaining amount of purified water up to 100ml were mixed according to the usual health drink manufacturing method, and then about After stirring and heating at 85 ° C. for 1 hour, the prepared solution was filtered and collected in a sterilized 2 L container, sealed and sterilized, and stored in a refrigerator. In this production example, ingredients suitable for a favorite beverage were mixed and formulated in a preferred ratio, but the blending ratio may be modified arbitrarily.

Test Example 1: Inflammation activity assay

BMDM was dispensed on a 48-well plate at a cell count of 1x10 ⁵ , rowan extract of Preparation Example 1 (50, 100, 200 μg/ml) was treated for 1 hour, and 500 ng/ml of LPS was added to the endotoxin-free It was dissolved in water and treated with cells for 16 hours. Thereafter, levels of inflammatory cytokines IL-1β, IL-6, and TNFα in the supernatant were measured by enzyme-linked immunosorbent assay.

The effect of rowan on LPS-induced inflammatory cytokine IL-1β production is shown in FIG. 1 . Figure 1 is a graph showing whether rowan extract inhibits the production of IL-1β, an LPS-induced inflammatory cytokine. Rowan extract could not confirm the inhibition of LPS-induced IL-1β production in BMDM cells.

The effect of rowan on LPS-induced inflammatory cytokine IL-6 production is shown in FIG. 2 . Figure 2 is a graph showing whether rowan extract inhibits the production of IL-6, an LPS-induced inflammatory cytokine. Rowan extract could not be confirmed to inhibit LPS-induced IL-6 production in BMDM cells.

The effect of rowan on LPS-induced inflammatory cytokine TNF-α production is shown in FIG. 3 . Figure 3 is a graph showing whether rowan extract inhibits the production of TNF-α, an LPS-induced inflammatory cytokine. Rowan extract could not confirm the inhibition of LPS-induced TNFα production in BMDM cells.

Test Example 2: Effect of rowan on nigericin-induced IL-1β production

Enzyme-linked immunosorbent assay is a technique for measuring the amount of a small amount of antigen or antibody using an antigen-antibody reaction using an enzyme as a marker, and an ELISA kit (R&D systems, Minneapolis, MN, USA) was used to measure IL-1β. A Duoset ELISA kit (R&D systems, DY401) was used to measure the level of IL-1β in the supernatant of the culture medium or intestinal homogenate, and the experiment was performed according to the protocol provided by the manufacturer. In addition, an ELISA kit (R&D systems, DY406) was used to measure the level of IL-6 in the supernatant of the intestinal homogenate, and the experiment was performed according to the protocol provided by the manufacturer. An ELISA kit (R&D systems, DY410) was used to measure the level of TNFα in the supernatant of the intestinal homogenate, and the experiment was performed according to the protocol provided by the manufacturer.

Immunological blotting, also called Western blotting, is an analysis method performed to determine the presence or amount of a specific protein and mainly uses an antigen-antibody reaction. It is a technique of transferring trace proteins on a gel to a membrane by electrophoresis and detecting them by combining them with antibodies.

In this study, caspase-1 and IL-1β were analyzed and measured. Pro-caspase-1 and caspase-1 were detected by anti-caspase-1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Pro-IL-1β and IL-1β were detected by anti-IL-1β antibody (R&D Systems, Minneapolis, MN, USA). Caspase-1 and IL-1β thus detected are used as indicators of NLRP3 inflammasome activity. And the supernatant was used to analyze IL-1β by ELISA technique. BMDM was dispensed on a 6-well plate at a cell count of 1x10 ⁶ , 100 ng/ml of LPS was dissolved in non-endotoxic water, and the cells were treated for 4 hours. In order to exclude the effect of LPS on the herbal extract, the herbal extract was added after washing the LPS with PBS. Cells were treated with oriental medicine extracts (50, 100, 200 μg/ml) for 1 hour and stimulated with nigericin (10 μM), an inflammasome activator, or monosodium urate (MSU; 500 μg/ml) crystals in a serum-free medium. made it The cells were eluted with RIPA buffer purchased from Biosegae (Seongnam, Republic of Korea), and immunoblot analysis was performed. The supernatant was centrifuged at 20,000g for 10 minutes by adding 1 volume of methanol and 0.25 volume of chloroform. The supernatant was removed, and the mixture obtained by adding 1 volume of methanol was centrifuged at 20,000 g for 10 minutes to obtain a protein precipitate. After drying at room temperature, Laemmli buffer was purchased from bio-rad (Hercules, CA) and resuspended. These samples were subjected to SDS-PAGE followed by electrophoresis on a nitrocellulose membrane. The membrane was probed with a first antibody and a related HRP-conjugated second antibody. Each band was visualized using the ECL system.

The results of measuring the expression of caspase-1 and IL-1β by immunoblotting are shown in FIG. 4 . Figure 4 is a photograph confirming whether rowan extract inhibits nigericin-induced NLRP3 inflammasome activation. In FIG. 4, it was confirmed that truncated caspase-1, which is an activated form of pro-caspase-1, a component of nigericin-induced inflammasome, decreased in a concentration-dependent manner in rowan extract, and also, an activated form of pro IL-1β. It was confirmed that the truncated IL-1β also decreased in a concentration-dependent manner of rowan extract.

5 is a graph showing the results of comparison of IL-1β in nigericin-induced activity of NLRP3 inflammasomes in BMDM. In FIG. 5, it was confirmed by ELISA that rowan extract inhibited nigericin-induced IL-1β production in a concentration-dependent manner. From these results, it was found that the rowan extract inhibited nigericin-induced activation of NLRP3 inflammasome.

Test Example 3: Effect of rowan in MSU-induced IL-1β fish

The results of measuring the expression of caspase-1 and IL-1β by immunoblotting are shown in FIG. 6 . 6 is a result confirming whether rowan extract inhibits MSU crystal-induced NLRP3 inflammasome activation.

In FIG. 6, it was confirmed that truncated caspase-1, an activated form of pro-caspase-1, a component of the MSU crystal-induced inflammasome, decreased in a concentration-dependent manner in rowan extract, but truncated IL-1, an activated form of pro IL-1β. It could not be confirmed that 1β also decreased in a concentration-dependent manner in the rowan extract.

7 is a graph showing the results of comparison of IL-1β in the MSU-induced activity of NLRP3 inflammasomes in BMDM. 7, it can be seen that rowan extract inhibited MSU crystal-induced IL-1β production in a concentration-dependent manner. From these results, it was found that rowan extract inhibited the activation of MSU-induced NLRP3 inflammasome.

Test Example 4: Inhibition of Intestinal Inflammation by Rowan Tree in DSS-Induced Inflammatory Bowel Disease Animal Model

Mice were tested to determine whether rowan extract inhibited intestinal inflammation in the DSS-induced inflammatory bowel disease animal model.

Figure 8 is a photograph and graph showing whether rowan extract inhibits intestinal inflammation in an inflammatory bowel disease animal model induced by DSS, and Figure 8A is a result of measuring the intestinal length of a mouse. It can be confirmed that the intestine length of the mice freely drinking DSS is shortened, and the intestine length of the mice that are orally administered with rowan extract daily is maintained a little longer. However, in the body weight change of the mice in FIG. 8B, even after oral administration of the rowan extract, no weight recovery occurred.

Figure 9 is a graph showing the comparison results of the pro-inflammatory cytokine IL-1β in the DSS-induced rectum of mice, Figure 10 is a graph showing the comparison results of the pro-inflammatory cytokine IL-6 in the DSS-induced rectum of mice am.

From FIG. 9 to FIG. 10, the results of measuring inflammatory cytokines in the mouse rectum can be confirmed.

Test Example 5: Acute toxicity test

A toxicity test was performed on experimental animals of the rowan extract according to Preparation Example 1, and the presence or absence of toxicity was observed. Experimental animals were 60 SD-based rats, 4 and 5 weeks old, male and female weighing 105±4 g and 95±3 g, and tested using the composition of the present invention and distilled water as a negative control group. First, the rats were placed in a laboratory breeding box with a temperature of 22±2° C., a relative humidity of 53±2%, and a light/dark cycle of fluorescent lighting (09:00 on-18:00 off), and an illumination condition of 150-300 Lux for about 1 After acclimatization over a period of about a week, only healthy animals were selected, divided into groups to match the average weight, and forced orally administered once a day in an amount of 20 ml/kg for 14 days, and then changes in general condition and symptoms of poisoning , motility, appearance, autonomic nerves, weight change, and the presence or absence of dead animals were checked.

According to the experimental results, there was no significant change in body weight during the experimental period, but there was no significance, and no dead animals were observed even though the highest concentration of 20ml per kg was selected for the amount of sample, which is the maximum amount that can be administered to test animals. It was not possible to calculate the lethal dose, so the LD ₅₀ was found to be 20ml/kg BW or more, and no specific general symptoms or specific lesions were observed at autopsy.

Test Example 6: sensory test

In this test example, the beverage of Preparation Example 3 and the comparative product (commercial product) were sensory evaluated by 40 volunteers randomly selected by age, and the results are shown in Table 2 below. As sensory items, color, taste, coolness, and comprehensive evaluation items were evaluated using a 5-point scale, and the results were averaged and the results are shown in Table 1 below. The evaluation criteria were set as 5 for very good, 4 for slightly good, 3 for moderate, 2 for poor, and 1 for very poor.

Preparation Example 3 comparative example color 4.5±0.3 3.3±0.3 taste 4.3±0.5 3.2±0.2 refreshing feeling 4.4±0.1 3.2±0.2 Comprehensive evaluation 4.4±0.2 3.3±0.4

From the results of Table 1, it can be seen that the products of Examples of the present invention were generally better than the products of Comparative Examples, and particularly, the preference was high in taste and coolness.

As reviewed above, the following results were obtained in a study to investigate the anti-inflammatory action of rowan tree on the inflammatory complex NLRP3 inflammasome.

1. As a result of measuring the amount of caspase-1 expression through immunoblotting, caspase-1 expression was suppressed at the concentration of 200 μg/ml of rowan tree.

2. As a result of measuring the IL-1β expression level through immunological blotting, IL-1β expression was suppressed at the concentration of 200 μg/ml of rowan tree.

3. As a result of measuring IL-1β secretion using enzyme-linked immunosorbent assay (ELISA), the concentration of 200 μg/ml of rowan tree showed an inhibitory effect on IL-1β production.

4. Cell test results LPS-induced production of IL-1β, IL-6, and TNF-α in BMDM cells was not inhibited in the rowan tree-treated group.

5. In the DSS-induced ulcerative colitis model, the production of inflammatory cytokines IL-1β and IL-6 was decreased in the group administered with rowan 800 mg/kg orally.

Through the above results, it was confirmed that the administration of rowan extract inhibited the activity of NLRP3 inflammasome in the DSS-induced ulcerative colitis mouse model and had an anti-inflammatory effect.

Although the present invention has been described with reference to preferred embodiments, those skilled in the art can variously modify and change the present invention without departing from the spirit and scope of the present invention described in the claims below. You will understand that you can.

Claims (4)

A composition for improving inflammatory bowel disease, comprising an organic solvent extract of rowan stem as a main component. The method of claim 1,
The extract is
a1) obtaining an extract by adding 10 to 25 times the purified water or an organic solvent to the dried herbal medicine and extracting at a temperature of 60 to 110 ° C. for 2 to 72 hours;
a2) filtering the extract of the above step with filter paper to filter out solids;
a3) Filtering the solids and concentrating the obtained filtrates to a brix of 20 or more for 4 to 8 hours under a reduced pressure of -0.08 to -0.09 MPA at a temperature of 50 to 65 ° C.: and
a4) extracting the concentrate obtained in the step above at a low temperature or freeze-drying at a temperature of 55 to 65 ° C. for 24 to 48 hours under vacuum; A composition for improving inflammatory bowel disease, characterized in that extraction. The method of claim 1,
The composition is a pharmaceutical or health functional food, and the rowan extract is a composition for improving inflammatory bowel disease, characterized in that administered at a dose of 800 mg / kg. The method of claim 1,
The composition for improving inflammatory bowel disease, characterized in that exhibiting an anti-inflammatory effect by inhibiting the activity of the NLRP3 inflammasome.