KR102586723B1 - A composition for preventing, improving or treating inflammatory bowel disease comprising extracts of Trachysalambria curvirostris as an active ingredient - Google Patents

Abstract

The composition of the present invention contains cauliflower extract as an active ingredient, has no cytotoxicity, and can effectively prevent, improve, or treat inflammatory bowel disease by suppressing the expression of inflammatory cytokines in colonic tissue.

Description

A composition for preventing, improving or treating inflammatory bowel disease comprising extracts of Trachysalambria curvirostris as an active ingredient}

The present invention relates to a composition for preventing, improving or treating inflammatory bowel disease containing a cauliflower extract.

Inflammatory bowel disease (IBD) is a disease that causes chronic inflammation of the intestines of unknown cause. Inflammatory bowel disease is typically classified into two types: ulcerative colitis (UC) and Crohn's disease (CD). Ulcerative colitis mainly invades the mucous membrane, frequently forming rubs or ulcers. It is a type of diffuse nonspecific inflammation of the large intestine of unknown cause and is accompanied by various systemic symptoms, including bloody diarrhea. Crohn's disease causes discontinuity of the entire digestive tract from the mouth to the anus, and causes the intestinal tract to spread from the mucosa to the intestines. It is a granulomatous inflammatory lesion of unknown cause in which ulcers, fibrosis, stenosis, and lesions develop in the layers, and is accompanied by systemic symptoms such as abdominal pain, chronic diarrhea, fever, and nutritional disorders.

The above-mentioned inflammatory bowel disease is relatively rare in Asians, but the incidence of inflammatory bowel disease is rapidly increasing in the East as well due to the influence of westernized eating habits.

The cause of the inflammatory bowel disease has not yet been specifically identified, but it is known to be involved in abnormalities in immune function. Immunological factors involved include innate immunity, production of cytokines, activation of CD4, etc. This is known. In particular, cytokines are known to play an important role, and the production of tumor necrosis factor (TNF-α), interleukin (IL)-1, IL-6, and IL-8 at the site of inflammation promotes ulcerative development. It was confirmed that it was significantly increased in patients with inflammatory bowel diseases such as colitis and Crohn's disease.

Accordingly, treatments for inflammatory bowel disease are being sought and applied from various angles, but since a fundamental treatment has not yet been established, the effectiveness of treatment is unsatisfactory and the recurrence rate is known to be high. Currently, 5-aminosalicinic acid drugs that block the production of prostaglandins, such as sulfasalazine, are used as treatments for inflammatory bowel disease. However, it is clear that sulfasalazine is not yet a reliable treatment as side effects such as headache, rash, liver disease, and leukopenia have been reported. Accordingly, the development of an effective inflammatory bowel disease treatment is also urgent and important.

If a treatment for inflammatory bowel disease that does not cause side effects can be developed, it is expected that patients with inflammatory bowel disease can be treated more safely and effectively. However, a treatment without side effects has not yet been developed.

Under this background, the present inventors made intensive research efforts to develop a treatment for inflammatory bowel disease derived from natural products that did not cause side effects. As a result, the cauliflower extract was found to be effective in producing inflammatory cytokines in a mouse colitis model induced by DSS (dextran sulfate sodium). It was confirmed that it could be used for the treatment of inflammatory bowel disease by significantly suppressing and the present invention was completed.

KR 10-2014-0006750 A KR 10-1996487 B

The problem to be solved by the present invention is to provide a composition with excellent efficacy in preventing, improving or treating inflammatory bowel disease, including the extract of Trachysalambria curvirostris as an active ingredient.

The present invention for achieving the above object relates to a food composition for preventing or improving inflammatory bowel disease containing an extract of Trachysalambria curvirostris as an active ingredient.

According to one embodiment of the present invention, the solvent for the flower shrimp extract may be water, alcohol having 1 to 4 carbon atoms, or a mixed solution thereof.

According to one embodiment of the present invention, the inflammatory bowel disease may be a disease selected from Crohn's disease, ulcerative colitis, intestinal Behcet's disease, infectious enteritis, ischemic enteritis, and radiation enteritis.

According to one embodiment of the present invention, the composition can inhibit the expression of inflammatory cytokines in colon tissue.

The inflammatory cytokine may be one or more selected from COS-2, IL-6, IL-1β, and TNF-α.

According to one embodiment of the present invention, the content of the flower shrimp extract contained in the composition may be 100 to 1,000 mg/kg.

Additionally, the present invention relates to a pharmaceutical composition for the prevention or treatment of inflammatory bowel disease containing an extract of Trachysalambria curvirostris as an active ingredient.

According to one embodiment of the present invention, the composition may be for oral administration.

Additionally, the present invention relates to a feed composition for preventing or improving inflammatory bowel disease containing an extract of Trachysalambria curvirostris as an active ingredient.

The composition of the present invention contains cauliflower extract as an active ingredient, has no cytotoxicity, and can effectively prevent, improve, or treat inflammatory bowel disease by suppressing the expression of inflammatory cytokines in colonic tissue.

Figure 1 is a diagram confirming the effect of reducing inflammatory cytokines due to cauliflower extract treatment on the inflammatory response induced by LPS in the mouse macrophage cell line RAW264.7 cells by performing RT-PCR. Figure 1a shows iNOS in RAW264.7 cells. , showing the mRNA expression pattern of IL-6 and TNF-α, which is the result of electrophoresis of RT-PCR products of iNOS, IL-6, and TNF-α, Figure 1b shows the change in iNOS mRNA expression level, Figure 1c is a graph showing the change in the level of TNF-α mRNA expression, and Figure 1d is a graph showing the change in the level of IL-6 mRNA expression.
Figure 2 shows the effect of reducing intestinal inflammatory cytokines by administering cauliflower extract before or after DSS administration in a mouse model in which inflammatory bowel disease was induced by administering DSS, by RT-PCR. Figure 2a shows the mouse model. This shows the mRNA expression pattern of IL-1β, COX-2, IL-6, and TNF-α in colon tissue. RT-PCR products of IL-1β, COX-2, IL-6, and TNF-α were electrophoresed. These are the results, Figure 2b is a graph showing the effect of reducing IL-1β mRNA expression, Figure 2c is a graph showing the effect of reducing COX-2 mRNA expression, Figure 2d is a graph showing the effect of reducing IL-6 mRNA expression, and Figure 2e is a graph showing the effect of reducing TNF-α mRNA expression. . In Figure 2, each experimental group is a normal group (Normal), a control group (DSS), a low-concentration diet group fed with 250 mg/kg of the flower shrimp extract of the example (DSS + flower shrimp L), and a diet of 500 mg/kg of the flower shrimp extract of the example. Represents one high-concentration diet group (DSS+flower shrimp H).
Figure 3 shows the effect of reducing intestinal inflammatory cytokines by administering cauliflower extract before or after DSS administration in a mouse model in which inflammatory bowel disease was induced by administering DSS using ELISA technique. Figure 3a shows the effect of TNF- a reduction effect, Figure 3b shows the IL-6 reduction effect. In Figure 3, each experimental group is a normal group (Normal), a control group (DSS), a low-concentration diet group fed with 250 mg/kg of the flower shrimp extract of the example (DSS + flower shrimp L), and a diet of 500 mg/kg of the flower shrimp extract of the example. Represents one high-concentration diet group (DSS+flower shrimp H).

Hereinafter, the present invention will be described in detail.

The composition for preventing, improving, or treating inflammatory bowel disease of the present invention contains an extract of Trachysalambria curvirostris as an active ingredient.

The term “prevention” of the present invention means anything that suppresses or delays the inflammatory bowel disease.

The term "improvement" of the present invention means at least reducing the degree of symptoms, for example, parameters related to the condition being treated by administration of a composition comprising the cauliflower extract of the present invention.

The term "treatment" of the present invention, unless otherwise specified, means reversing, alleviating, inhibiting the progression of, or preventing the symptoms of inflammatory bowel disease.

Meanwhile, about 2,900 species of shrimp are known worldwide, and about 90 species are known in Korea.

Among these, the flower shrimp ( Trachysalambria curvirostris ) is a crustacean of the genus Trachysalambria of the Penaeidae family of the order Decapoda. It has a gray or brown body and is found in the West and South Seas of Korea. It can be seen mainly in the Gunsan area, which has a mild oceanic climate with severe tidal differences and an average annual temperature of 12.5℃.

The main carotenoids contained in raw flower shrimp are astaxanthin and β-carotene, which account for 75.18-88.86% and 2.86-16.83% of the total carotenoid content, respectively. In addition, the flower shrimp contains trace amounts of echinenone, canthaxanthin, and astacene (Fang Su et al ., Journal of Oceanology and Limnology volume 37, pages706-712 (2019) )). The flower shrimp is rich in nutrients such as protein and calcium, so it is eaten raw, boiled or grilled, and is used in a variety of ways, such as being used as an ingredient for frying and sushi, as well as being dried and processed.

The flower shrimp extract of the present invention is more effective in alleviating, suppressing, or improving inflammatory diseases, especially inflammatory bowel disease, compared to extracts of shrimp of other genera or families of the same content or components such as chitosan or chitin.

The inflammatory bowel disease may be a disease selected from Crohn's disease, ulcerative colitis, intestinal Behcet's disease, infectious enteritis, ischemic bowel disease, and radiation enteritis, and is preferably Crohn's disease or ulcerative colitis, more preferably ulcerative colitis. It can be.

The term “extract” used herein is a broad concept that includes substances obtained by extracting components of natural products, regardless of the extraction method, extraction solvent, or form of the extract, and can be obtained by common extraction methods in the art.

In one embodiment, the solvent for obtaining the flower shrimp extract of the present invention may be a hydrophilic solvent. The hydrophilic solvent may be water, a lower alcohol having 1 to 4 carbon atoms, or a mixed solution thereof. The lower alcohol may include 20 to 80% (v/v) methanol, ethanol, butanol, or propanol. The extraction solvent is not particularly limited, but hot water extract extracted with water works more preferably in preventing, improving, or treating inflammatory bowel disease.

In one embodiment, the flower shrimp extract can be extracted by mixing it with an extraction solvent at a weight ratio of 1:1 to 10, preferably 1:2 to 6, more preferably 1:3 to 5. If the weight ratio of the flower shrimp and the extraction solvent is outside the above range, the active ingredient of the flower shrimp may be extracted in a small amount in the extract.

In one embodiment, the cauliflower hot water extract is prepared by heating cauliflower shrimp at 60 to 120°C, preferably at 80 to 100°C, more preferably at 80 to 90°C for 5 to 60 minutes, preferably at 10 to 30 minutes, more preferably at 80 to 100°C. It can be prepared by extracting for 10 to 20 minutes and then concentrating under reduced pressure.

The flower shrimp extract may be a fraction obtained by extracting the flower shrimp with water, a lower alcohol having 1 to 4 carbon atoms, or a mixed solution thereof and refractionating the extract with a second organic solvent. Preferably, the flower shrimp extract may be a fraction obtained by refractionating the hot water extract of flower shrimp with hexane, ethyl acetate, ethanol, or butanol.

The term "extract" used in this specification while referring to flower shrimp includes not only the crude extract obtained by treating the shrimp with an extraction solvent, but also the processed product of the flower shrimp extract. For example, cauliflower extract can be prepared in powder form by additional processes such as reduced pressure distillation, freeze drying, or spray drying.

In one embodiment, the flower shrimp extract includes the steps of washing and drying the flower shrimp; Grinding dried flower shrimp and then extracting with solvent; and concentrating and filtering the flower shrimp extract.

In one embodiment of the present invention, lipopolysaccharides (LPS: lipopolysaccharides), which are inflammatory response inducing factors, and the shrimp extract of the present invention are treated with mouse macrophages to increase the expression of inflammatory cytokines such as iNOS, IL-6, and TNF-α. After induction, it was compared with the negative control group treated only with LPS. As a result, it was specifically confirmed that inflammatory cytokines were reduced in a concentration-dependent manner when treated with the flower shrimp extract of the present invention.

In addition, when the cauliflower extract of the present invention was administered to a mouse model in which inflammatory bowel disease was induced by administering DSS, the cauliflower extract produced IL-1β, COX-2, IL-6 and TNF- It was specifically confirmed that it is effective in preventing, improving, or treating inflammatory bowel disease by suppressing or reducing the expression of inflammatory cytokines such as α. That is, the composition of the present invention inhibits or reduces the expression of inflammatory cytokines such as IL-1β, COX-2, IL-6, and TNF-α in inflamed intestinal tissue, thereby preventing or improving inflammatory bowel disease. desirable for treatment.

The present invention relates to a food composition for preventing or improving inflammatory bowel disease containing cauliflower extract as an active ingredient.

The 'food composition' includes, in addition to the flower shrimp extract as an active ingredient, food raw materials that can be used as foods as described in the standards and specifications for foods commonly used in food production ('food code'), and food additives listed in the food additive code. .

There is no need to specifically limit it, but examples include proteins, carbohydrates, fats, nutrients, seasonings, and flavoring agents. The carbohydrates include monosaccharides, such as glucose, fructose, etc.; Disaccharides such as maltose, sugar, lactose, etc.; Oligosaccharides or polysaccharides such as dextrin, starch syrup, cyclodextrin, etc.; Sugar alcohols such as xylitol, sorbitol, erythritol, etc. can be used. The flavoring agent may be a natural flavoring agent (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) or a synthetic flavoring agent (saccharin, aspartame, etc.).

In an exemplary embodiment, the cauliflower extract may be present in the composition in any amount desired to provide effective inflammatory bowel disease prevention, improvement, or treatment efficacy. According to one aspect, the cauliflower extract may be 0.01 to 99% by weight, 0.05 to 95% by weight, preferably 0.1 to 90% by weight, more preferably 0.5 to 80% by weight, more preferably 1 to 70% by weight. It may be included in weight percent, 2 to 60 weight percent, or 5 to 50 weight percent.

The cauliflower extract, which is an active ingredient in the food composition, varies depending on the condition, body weight, and presence, degree, and duration of the disease of the consumer, but may be appropriately selected by a person skilled in the art. For example, based on the daily dosage, 1 to 5,000 mg/kg, preferably 5 to 2,000 mg/kg, more preferably 10 to 1,000 mg/kg, more preferably 100 to 1,000 mg/kg, even more preferably. Typically, it may be 200 to 800 mg/kg, more preferably 300 to 700 mg/kg, and even more preferably 400 to 600 mg/kg. The number of administrations does not need to be particularly limited, but can be adjusted by a skilled artisan within the range of three times a day to once a week. In the case of long-term intake for health and hygiene purposes or health control, it may be below the above range.

The food composition does not need to be particularly limited, but may be, for example, powder, granule, tablet, capsule, pill, extract, jelly formulation, tea bag formulation, or beverage formulation.

In addition, the above-mentioned shrimp extract can be added to general foods to provide functionality for preventing or improving inflammatory bowel disease. There is no need to specifically limit the foods that can be added, but for example, the food standards in accordance with Article 7 of the Food Sanitation Act and confectionery, bread or rice cake, processed cocoa products or chocolate, processed meat or egg products, processed fish products, tofu or jelly, noodles, tea, coffee, beverages, special-purpose foods, sauces, etc. as exemplified in the standards ('Food Code of Conduct'). It can be added to seasoned foods, dressings, kimchi, salted seafood, pickled foods, stewed foods, alcoholic beverages, dried raisins, and other foods. In addition, it can be added to dairy products, processed meat products, packaged meat, and egg products as exemplified in the livestock product processing standards and ingredient specifications ('Livestock Product Code') pursuant to Article 4 of the Livestock Products Sanitation Management Act.

Meanwhile, the food composition containing the flower shrimp extract as an active ingredient can be used alone as a “health functional food for preventing or improving inflammatory bowel disease.”

The above ‘health functional food’ refers to food manufactured (including processing) in accordance with legal standards using raw materials or ingredients with functional properties useful to the human body (Article 3, Paragraph 1 of the Health Functional Food Act). The terminology or scope of the above 'health functional food' may vary depending on the country, but it is also called 'Dietary Supplement' in the United States, 'Food Supplement' in Europe, 'Health Functional Food' or 'Health Functional Food' in Japan. It may be classified as ‘Food for Special Health Use (FoSHU)’ or ‘Health Food’ in China.

The above food composition or health functional food may additionally contain food additives, and its suitability as a food additive is determined by the specifications and standards for the relevant item in accordance with the general provisions of the ‘Food Additive Code’ and general test methods, etc., unless otherwise specified. Follow.

In addition, the above-mentioned health functional food includes raw materials notified as 'functional raw materials' or individually recognized raw materials used in "health functional food for preventing or improving inflammatory bowel disease" along with the flower shrimp extract, such as isomalto-oligosaccharide, soybean oligosaccharide, and raffinose. By combining health functional food ingredients related to intestinal health, such as flactooligosaccharide and guar gum hydrolyzate, health functional food for preventing or improving inflammatory bowel disease or leaky gut syndrome can be manufactured.

In addition, the present invention provides a feed composition for preventing or improving inflammatory bowel disease containing flower shrimp extract as an active ingredient. The terms “flower shrimp extract” and “inflammatory bowel disease” in the present invention are as described above.

In addition to the flower shrimp extract as an active ingredient, the 'feed composition' can use food raw materials that can be used as food as described in the Food Standards and Specifications ('Food Code'), and food additives described in the Food Additive Code, and food that can be used as food. Even if they are not raw materials or food additives, raw materials that fall within the scope of single feed in Annex Table 1 of the ‘Standards and Specifications for Feed, etc.’ and raw materials that fall within the scope of supplementary feed in Annex Table 2 can be used.

The 'feed composition' may be an extractant among supplementary feeds according to 'Standards and specifications for feed, etc.', or may be a compound feed containing the supplementary feed.

When preparing a feed composition using the flower shrimp extract as an active ingredient, the content of the flower shrimp extract does not need to be specifically limited as long as it shows prevention or improvement of inflammatory bowel disease, but may be, for example, 0.01 to 99% by weight, and 0.05 to 95 weight%. %, preferably 0.1 to 90% by weight, more preferably 0.5 to 80% by weight, more preferably 1 to 70% by weight, 2 to 60% by weight, or 5 to 50% by weight.

The cauliflower extract, which is an active ingredient in the feed composition, varies depending on the condition, body weight, presence, degree, and period of disease of the ingesting animal, but may be appropriately selected by a person skilled in the art. For example, based on the daily dosage, 1 to 5,000 mg/kg, preferably 5 to 2,000 mg/kg, more preferably 10 to 1,000 mg/kg, more preferably 100 to 1,000 mg/kg, even more preferably. Typically, it may be 200 to 800 mg/kg, more preferably 300 to 700 mg/kg, and even more preferably 400 to 600 mg/kg. The number of administrations does not need to be particularly limited, but can be adjusted by a skilled artisan within the range of three times a day to once a week. In the case of long-term intake for health and hygiene purposes or health control, it may be below the above range.

According to another aspect of the present invention, the present invention relates to a pharmaceutical composition for preventing or treating inflammatory bowel disease containing an extract of Xanthii fructus as an active ingredient.

In addition, the present invention relates to a pharmaceutical composition for animals for the prevention or treatment of inflammatory bowel disease containing cauliflower extract as an active ingredient.

Additionally, the present invention provides a method of treating inflammatory bowel disease by administering the composition to humans or non-human animals.

In addition, the present invention provides a new use of the cauliflower extract for the manufacture of medicine for the prevention or treatment of inflammatory bowel disease, or medicine for animals.

The terms “flower shrimp extract” and “inflammatory bowel disease” in the present invention are as described above.

The 'pharmaceutical composition', 'medicine', 'pharmaceutical composition for animals' or 'medication for animals' may further include appropriate carriers, excipients and diluents commonly used in the production of pharmaceutical compositions, etc., in addition to the calyx extract as an active ingredient. You can.

The 'carrier' is a compound that facilitates the addition of the compound into cells or tissues. The 'diluent' is a compound diluted in water that not only stabilizes the biologically active form of the target compound, but also dissolves the compound.

There is no need to specifically limit the carrier, excipient, and diluent, but for example, lactose, glucose, sugar, 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 oil.

The amount of the pharmaceutical composition, medicine, pharmaceutical composition for animals, or medicine for animals may vary depending on the age, gender, and weight of the patient or animal to be treated, and, above all, the condition of the subject to be treated, the specific category of the disease to be treated, or It will depend on the type, route of administration, and properties of the therapeutic agent used.

The pharmaceutical composition, medicine, pharmaceutical composition for animals, or medicine for animals is appropriately selected depending on the absorption rate of the active ingredient in the body, the excretion rate, the age, weight, sex, and condition of the patient or animal to be treated, and the severity of the disease to be treated. , generally 1 to 5,000 mg/kg per day, preferably 5 to 2,000 mg/kg, more preferably 10 to 1,000 mg/kg, more preferably 100 to 1,000 mg/kg, more preferably 200 to 200 mg/kg. It is preferable to administer 800 mg/kg, more preferably 300 to 700 mg/kg, and even more preferably 400 to 600 mg/kg. The unit dosage form formulated in this way can be administered several times at regular time intervals as needed.

The pharmaceutical composition, medicine, pharmaceutical composition for animals, or medicine for animals may be administered individually as a preventive or therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents.

The pharmaceutical composition, medicine, pharmaceutical composition for animals, or medicine for animals can be formulated and used in oral dosage forms such as powders, granules, tablets, capsules, troches, suspensions, emulsions, syrups, aerosols, etc. according to conventional methods. there is. When formulated, it can be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Furthermore, it can be preferably formulated according to each disease or ingredient using a method disclosed by Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA, as an appropriate method in the field.

The pharmaceutical composition of the present invention can be administered orally or parenterally, and in the case of parenteral administration, it can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, etc., and is preferably administered orally.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, troches, etc. These solid preparations include the compound with at least one excipient, such as starch, calcium carbonate, sugar or lactose, and gelatin. It can be prepared by mixing etc. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral use include suspensions, oral solutions, emulsions, syrups, etc. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. .

The method of treating inflammatory bowel disease involves administering the composition to humans or non-human animals, especially mammals, for example, administering the composition to an individual to be treated with inflammatory bowel disease.

The subject subject to treatment with inflammatory bowel disease may be a case of having inflammatory bowel disease.

The dosage, administration method, and frequency of administration for the treatment may refer to the dosage, administration method, and frequency of administration of the pharmaceutical composition, medicine, pharmaceutical composition for animals, or medicine for animals.

Hereinafter, the present invention will be described in detail through examples, but the present invention is not limited to the following examples.

<Example>

Example: Flower shrimp extract

2,000 g of distilled water (weight ratio of 1:4) was added to 500 g of dried flower shrimp powder, and then hot water extraction was performed at 60°C for 15 minutes. Afterwards, the extract was centrifuged (8,000 rpm, 4°C, 10 minutes), and the supernatant obtained therefrom was freeze-dried to obtain about 50 g of flower shrimp extract powder (yield: 10.0%).

<Test example>

Experimental equipment and reagents

DMEM medium (Thermo Scientific, USA), 10% inactivated fetal bovine serum (FBS) (GE healtheare, USA), 1% penicillin-streptomycin (GE healtheare, USA), ELISA (enzyme-linked immunosorbent assay) kit (AbFROMTIER, KOR ), chloroform (Daejung, KOR), Hybrid-R (GeneAll, KOR), RT-PCR kit (Bioneer, KOR), Agaro-Power (Bioneer Co, KOR), Shuttle box (Jeungdo, KOR), Maze etc. (Jeungdo, KOR), ethanol (Daejung, KOR), DEPC (diethyl pyrocarbonate) water (Thermo Scientific, USA), agarose (Boo Kyung, KOR), auto clave (JSR, KOR), centrifuge (LABOGENE, KOR), TRIZol (Sigma, USA) enzyme-linked immunosorbent assay (ELISA) reader (Thermo Scientific, USA) and fluoro box (Neo Science, KOR) were used, and RAW 264.7 (KCLB 40071, KOR) cells were purchased from the Korea Cell Line Bank.

Test Example 1: Cytotoxicity test

In order to determine the effect of the sample obtained in the above example on cell viability by the MTS assay, an experiment was performed as follows using the method described in the existing literature (Choi JH et al ., Flowers of Inula japonica attenuate inflammatory responses, Immune Network 2010, 10(5), 145-152).

First, Raw 264.7 cells were distributed at a density of 5×10 ⁴ cell/mL in a 24 well plate and cultured under 5% CO ₂ and 37°C conditions. After 24 hours, the samples according to the examples were treated with each concentration (1.0, 5.0, 10 mg/ml) and cultured for an additional 24 hours. After removing the supernatant, 1 μg/mL MTT solution was added and left for 2 hours. 100 μL of DMSO was added per well to dissolve the formazan crystals for 20 minutes, and the absorbance was measured at 540 nm with an ELISA plate reader. Cell viability was calculated using Equation 1 below, and the results are shown in Table 1 below as a relative ratio to the control group.

[Equation 1]

Cell viability (%) = (absorbance of sample treatment group / absorbance of control group) × 100

sample Cell viability (%) Control group (untreated group) 100
Example 1.0 mg/mL 103.1 5.0 mg/mL 101.6 10.0 mg/mL 96.7

Looking at Table 1 above, it can be seen that the composition according to the example of the present invention does not exhibit significant toxicity up to a concentration of 10.0 mg/mL.

Test Example 2: Confirmation of anti-inflammatory efficacy of flower shrimp extract

2-1. cell culture

RAW 264.7 cells, a mouse macrophage cell line, were distributed to a 48-well plate at 2×10 ⁵ cells/well per well, and then incubated with 10% FBS and 1% antibiotic-antimycotic (10 units/well) under 37°C and 5% CO ₂ conditions. The cells were cultured for 24 hours in DMEM medium supplemented with mL penicillin, 100 μg/mL sterptomycin, and 0.25 μg/mL amphotericin.

The medium was replaced with a new medium, and LPS (1 μg/mL) and samples of the examples were administered to the cells at different concentrations (1, 2.5, 5, and 10 mg/mL), and then cultured for 24 hours.

2-2. Total RNA extraction

Total RNA was extracted from RAW 264.7 cells in which inflammation was induced with LPS using TRIZol, and the extracted RNA was dissolved in diethyl pyrocarbonate water (DEPC) and subjected to reverse transcription polymerase chain reaction.

2-3. Reverse Transcription Polymerase Chain Reaction (RT-PCR)

PCR synthesizes cDNA from mRNA using an RT-PCR kit containing DNA polymerase, buffer, dNTP, and tracking dye, and then interlukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and inducible PCR was performed using primers corresponding to each gene, such as nitric oxide synthase (iNOS), at 94°C for 30 s, 55 to 62°C for 30 s, and 72°C for 1 min, for a total of 35 cycles. The PCR product was electrophoresed on a 1.5% agarose gel and confirmed using a UV transilluminator (Figure 1). The base sequences of each primer used in the analysis are shown in Table 2 below. GAPDH was used as a loading control.

target gene base sequence IL-6 Forward 5’-TTCCATCCAGTTGCCCTTCTTG-3’ Reverse 5’-GGGAGTGGTATCCTCTGTGAAGTC-3’ TNF-α Forward 5’-GGCAGGTCTATTTGGAGTCATTGC-3’ Reverse 5’-ACATTCGAGGTCCAGTGAATTCGG-3’ iNOS Forward 5’-GTTCTCAGCCCAACAATACAAGA-3’ Reverse 5’-GTGGACGGTCGATGTCAC-3’ GAPDH Forward 5’-CATGGCCTTTCGTGTTC-3’ Reverse 5’-CCTGGTCCTCAGTGTAGC-3’

Figure 1 is a diagram confirming the effect of reducing inflammatory cytokines due to cauliflower extract treatment on the inflammatory response induced by LPS in the mouse macrophage cell line RAW264.7 cells by performing RT-PCR. Figure 1a shows iNOS in RAW264.7 cells. , showing the mRNA expression pattern of IL-6 and TNF-α, which is the result of electrophoresis of RT-PCR products of iNOS, IL-6, and TNF-α, Figure 1b shows the change in iNOS mRNA expression level, Figure 1c is a graph showing the change in the level of TNF-α mRNA expression, and Figure 1d is a graph showing the change in the level of IL-6 mRNA expression.

Looking at Figure 1, it can be seen that the expression of iNOS, IL-6, and TNF-α increased by LPS stimulation in RAW264.7 cells is decreased in a concentration-dependent manner by the cauliflower extract.

Test Example 3: Analysis of efficacy of cauliflower extract using inflammatory bowel disease-induced mouse model

3-1. Preparation of experimental animals and construction of inflammatory bowel disease mouse model

There are many ways to establish an animal model for inflammatory bowel disease, but it has been reported that the animal model in which ulcerative colitis is induced with 5% Dextran Sulfate Sodium (DSS) is the best model for studying the therapeutic effect of active ingredients ( Elson CO et al., Gastroenterology. 109:1344, 1995).

This DSS induction model is a relatively simple and highly reproducible method that induces a certain degree of inflammation depending on the administered dose when DSS is mixed with drinking water and is the oldest experiment often used to evaluate the efficacy of new synthetic compounds for controlling colitis. It is one of the models (Ohkusa T. Jpn J Gastroenterol. 82:1372, 1985; Kitajima S. et al., Exp. Animal. 48:137, 1999). Therefore, in the present invention, an ulcerative colitis-inducing mouse model was constructed using DSS to determine whether flower shrimp extract is effective in inflammatory bowel diseases such as ulcerative colitis and Crohn's disease.

The experimental animal used was a 4-week-old male ICR mouse purchased from Orient Co. (KOR).

Mice were preliminarily bred for one week in an animal breeding room maintained at 20 ± 2 ℃, humidity 50 ± 10%, and 12-hour light/dark cycle before being used in the experiment. The experimental animals were separated into four groups of 6 as shown in Table 3 below, The experiment was performed with two animals per cage.

Specifically, in the DSS + Low group and DSS + High group, flower shrimp extract was prepared at concentrations of 250 mg/kg and 500 mg/kg, respectively, using distilled water and orally administered at 100 ㅅl per day for 10 days. Then, 7 days after starting the administration of the flower shrimp extract, colonic inflammation was induced by drinking 5% DSS for 3 days in the DSS group, DSS + Low group, and DSS + High group.

Test group (n=6) 5% DSS Flower Shrimp Extract Normal - - DSS + - DSS+Low + 250 mg/kg/day, dosage 100 ㎕, oral administration for 10 days DSS+High + 500 mg/kg/day, dosage 100 ㎕, oral administration for 10 days

3-2. Effect of suppressing inflammatory cytokine expression in colon tissue - RT-PCR analysis

To confirm whether consumption of flower shrimp extract reduces intestinal inflammatory response, the expression level of inflammatory cytokines (IL-1β, COX-2, IL-6, TNF-α) in colonic tissue of each test group was measured.

For this purpose, after completing the experiment, the experimental animals were sacrificed, each colonic tissue was extracted from them, and total RNA was extracted from the extracted colonic tissues using the Hybrid-R kit. The extracted RNA was used to extract RT- PCR was performed.

Specifically, 1 mL of Ribo Ex was added to each colon tissue, which was then disrupted, and 5 minutes later, 200 ㎕ of chloroform was added and vortexed. Afterwards, the supernatant was obtained by centrifugation at 4°C and 10,000 rpm for 10 minutes. 500 ㎕ of each mixed solution of the supernatant and RB1 was placed in mini column type F, centrifuged at room temperature at 10,000 rpm for 1 minute, and then the solution coming down from the column was removed. Afterwards, 500 ㎕ of SW1 and 500 ㎕ of RNW were added to the column and a centrifugation process was performed. The column was transferred to a new microtube, 50 ㎕ of nuclease free water was allowed to be absorbed into the membrane, and then centrifuged to obtain RNA.

Afterwards, cDNA was synthesized from the extracted RNA using an RT-PCR kit containing DNA polymerase, buffer, dNTP, and tracking dye. The synthesized cDNA was subjected to PCR using primers at 94°C for 30 seconds, 56-66°C for 30 seconds, and 72°C for 1 minute, for a total of 35 cycles. The PCR product was electrophoresed on a 1.2% agarose gel and confirmed using a UV transilluminator (Figure 2). The base sequences of each primer used in the analysis are shown in Table 4 below.

target gene base sequence IL-6 Forward 5’-TTCCATCCAGTTGCCCTTCTTG-3’ Reverse 5’-GGGAGTGGTATCCTCTGTGAAGTC-3’ TNF-α Forward 5’-GGCAGGTCTATTTGGAGTCATTGC-3’ Reverse 5’-ACATTCGAGGTCCAGTGAATTCGG-3’ IL-1β Forward 5’-GCCTTGGGCCTCAAAGGAAAGAATC-3’ Reverse 5’-GGAAGACACAGATTCCATGGTGAAG-3’ COX-2 Forward 5’-GCAAATCCTTGCTGTTCCAATC-3’ Reverse 5’-GGAGAAGGCTTCCCAGTTTTG-3’ GAPDH Forward 5’-CATGGCCTTTCGTGTTC-3’ Reverse 5’-CCTGGTCCTCAGTGTAGC-3’

Figure 2 shows the effect of reducing intestinal inflammatory cytokines by administering cauliflower extract before or after DSS administration in a mouse model in which inflammatory bowel disease was induced by administering DSS, by RT-PCR. Figure 2a shows the mouse model. This shows the mRNA expression pattern of IL-1β, COX-2, IL-6, and TNF-α in colon tissue. RT-PCR products of IL-1β, COX-2, IL-6, and TNF-α were electrophoresed. These are the results, Figure 2b is a graph showing the effect of reducing IL-1β mRNA expression, Figure 2c is a graph showing the effect of reducing COX-2 mRNA expression, Figure 2d is a graph showing the effect of reducing IL-6 mRNA expression, and Figure 2e is a graph showing the effect of reducing TNF-α mRNA expression. . In Figure 2, each experimental group is a normal group (Normal), a control group (DSS), a low-concentration diet group fed with 250 mg/kg of the flower shrimp extract of the example (DSS + flower shrimp L), and a diet of 500 mg/kg of the flower shrimp extract of the example. Represents one high-concentration diet group (DSS+flower shrimp H).

Looking at Figure 2, it can be seen that the increased production of inflammatory cytokines in intestinal tissue inflamed by DSS was significantly reduced in the test group to which the cauliflower extract of the present invention was added. In particular, in the case of the flower shrimp high-concentration diet group (DSS + flower shrimp H), the production of inflammatory cytokines IL-1β, IL-6, and TNF-α was confirmed to be reduced close to the normal group.

3-3. Inhibitory effect of inflammatory cytokine expression in colon tissue - ELISA analysis

To confirm whether consumption of flower shrimp extract reduces intestinal inflammatory response, the expression level of inflammatory cytokines (IL-6, TNF-α) in the colonic tissue of each test group was measured.

Specifically, the colon tissue _extracted from each test group was homogenized in 1 The contents of cytokines IL-6 and TNF-α were measured. Specifically, according to the manufacturer's protocol, 100 ㎕ of antigen was added, incubated at 37°C for 2 hours, and then washed three times before adding the substrate, which was repeated at each step. After adding 100 ㎕ of secondary antibody, incubating at 37 ℃ for 1 hour, adding 100 ㎕ of color source, incubating at 37 ℃ for 30 minutes, adding 100 ㎕ of substrate, incubating at room temperature for 10 minutes, and then adding 100 ㎕ of stop solution. Then, the absorbance was measured at a wavelength of 450 nm using a microreader.

Figure 3 shows the effect of reducing intestinal inflammatory cytokines by administering cauliflower extract before or after DSS administration in a mouse model in which inflammatory bowel disease was induced by administering DSS using ELISA technique. Figure 3a shows the effect of TNF- a reduction effect, Figure 3b shows the IL-6 reduction effect. In Figure 3, each experimental group is a normal group (Normal), a control group (DSS), a low-concentration diet group fed with 250 mg/kg of the flower shrimp extract of the example (DSS + flower shrimp L), and a diet of 500 mg/kg of the flower shrimp extract of the example. Represents one high-concentration diet group (DSS+flower shrimp H).

Looking at Figure 3, it can be seen that the inflammatory cytokines IL-6 and TNF-α, which increased in intestinal tissue inflamed by DSS, were significantly reduced in the test group to which the cauliflower extract of the present invention was added. In particular, in the case of the flower shrimp high-concentration diet group (DSS + flower shrimp H), the amount of inflammatory cytokine production was confirmed to be reduced close to the normal group.

3-4. Measurement of intestinal length

Ulcerative colitis is a chronic, idiopathic inflammatory bowel disease of the colon, with clinical characteristics such as rectal bleeding, diarrhea, abdominal pain, weight loss, and fever. Therefore, in the present invention, the colon of the ulcerative colitis-induced mouse model was separated and its length was measured and shown in Table 5 below.

test group Chapter length (cm) percentage(%) Normal 10.22±1.26 100 DSS 6.89±0.34 67.41 DSS+Low 8.26±0.68 80.82 DSS+High 9.77±1.13 95.60

Looking at Table 5 above, it can be seen that the intestine length of the DSS group was significantly shorter by about 2/3 than that of the normal group. Meanwhile, it was confirmed that the intestinal length of all groups that consumed flower shrimp extract significantly increased compared to the DSS group. In particular, in the case of the flower shrimp high-concentration diet group (DSS + flower shrimp H), it was confirmed that the intestine length was almost close to the normal group.

From these results, it can be seen that flower shrimp extract effectively improves inflammation caused by DSS.

<Manufacturing example>

Below is an example of preparation for the flower shrimp extract of the present invention.

<Production Example 1> Production of food

<1-1> Manufacturing of health drinks

A health drink containing the cauliflower extract of the present invention with the ingredients and contents shown in Table 6 below was prepared according to a conventional method.

Mix the ingredients in Table 6 according to a typical health drink manufacturing method, stir and heat at 85°C for about 1 hour, filter the resulting solution, place it in a sterilized 2 L container, seal, sterilize, and store in the refrigerator. Used for manufacturing the functional beverage composition of the present invention.

The composition ratio is a preferred embodiment of mixing ingredients relatively suitable for beverages of preference, but the mixing ratio may be arbitrarily modified according to regional and ethnic preferences such as demand class, country of demand, and intended use.

ingredient content Flower shrimp extract of examples 500 mg citric acid 1,000 mg oligosaccharide 10g plum concentrate 2.0g taurine 1.0 g Purified water remaining amount Sum 1,000 g

<1-2> Manufacturing of flour food

0.5 to 5.0% by weight of the flower shrimp extract of the present invention was added to flour, and bread, cakes, cookies, crackers, and noodles were manufactured using this mixture.

<1-3> Preparation of soups and gravies

0.1 to 5.0% by weight of the flower shrimp extract of the present invention was added to soup and gravy to prepare health-enhancing meat products and noodle soup and gravy.

<1-4> Manufacturing of dairy products

5 to 10% by weight of the flower shrimp extract of the present invention was added to milk, and various dairy products such as butter and ice cream were manufactured using the milk.

<Preparation Example 2> Preparation of pharmaceutical preparation

<2-1> Production of powder

10 mg of flower shrimp extract of the example

Lactose 20 mg

After mixing the above ingredients, they were filled into an airtight bubble to prepare a powder.

<2-2> Preparation of tablets

10 mg of flower shrimp extract of the example

Corn starch 100 mg

100 mg lactose

Magnesium stearate 2 mg

After mixing the above ingredients, tablets were manufactured by tableting according to a conventional tablet manufacturing method.

<2-3> Manufacturing of capsules

10 mg of flower shrimp extract of the example

3 mg crystalline cellulose

Lactose 14.8 mg

Magnesium stearate 0.2 mg

After mixing the above ingredients, a capsule was prepared by filling a gelatin capsule according to a typical capsule manufacturing method.

<2-4> Preparation of liquid

100 mg of flower shrimp extract of the example

10 g isomerized sugar

5 g mannitol

Proper amount of purified water

According to the usual liquid preparation method, add and dissolve each ingredient in purified water, add an appropriate amount of lemon flavor, mix the above ingredients, add purified water to adjust the total to 100 ml, fill it in a brown bottle, and sterilize to prepare the liquid. Manufactured.

<2-5> Preparation of injections

Flower shrimp extract of the example 100 mg/ml

Dilute hydrochloric acid BP until pH 7.6

Sodium chloride BP for main use up to 1㎖

The mixture of the present invention was dissolved in an appropriate volume of sodium chloride BP for injection, the pH of the resulting solution was adjusted to pH 7.6 using dilute hydrochloric acid BP, and the volume was adjusted using sodium chloride BP for injection and thoroughly mixed. The solution was filled into a 5 ml type-ampoule made of transparent glass, sealed under an upper grid of air by dissolving the glass, and autoclaved sterilized at 120°C for at least 15 minutes to prepare an injectable solution.

<Preparation Example 3> Preparation of pet food composition

In the example, 0.1 kg of flower shrimp extract, 25.5 kg of corn, 15.04 kg of wheat, 8.15 kg of wheat flour, 7.4 kg of rice bran, 18 kg of soybean meal, 1 kg of octopus, 14 kg of chicken by-products, 9 kg of animal fat, 0.3 kg of processed salt, 0.3 tricalcium phosphate. kg, 1 kg of limestone, 0.01 kg of choline chloride, 0.05 kg of vitamins, 0.05 kg of minerals, and 0.1 kg of digestive enzymes were mixed to prepare an animal (dog, pet) feed composition.

Although the present invention has been described in terms of the above-mentioned preferred embodiments, various modifications and variations can be made without departing from the gist and scope of the invention. Additionally, the appended claims cover such modifications or variations as fall within the subject matter of the present invention.

Claims (13)

A food composition for preventing or improving inflammatory bowel disease comprising a hot water extract of Trachysalambria curvirostris as an active ingredient. According to paragraph 1,
The hot water extract of flower shrimp is for preventing or improving inflammatory bowel disease, characterized in that it is prepared by mixing flower shrimp with water at a weight ratio of 1: 1 to 10, extracting at 60 to 120 ° C. for 5 to 60 minutes, and then performing reduced pressure concentration. Food composition. According to paragraph 1,
A food composition for preventing or improving inflammatory bowel disease, wherein the inflammatory bowel disease is a disease selected from Crohn's disease, ulcerative colitis, intestinal Behcet's disease, infectious enteritis, ischemic enteritis, and radiation enteritis. According to paragraph 1,
The composition is a food composition for preventing or improving inflammatory bowel disease, characterized in that it inhibits the expression of inflammatory cytokines in colon tissue. According to paragraph 4,
A food composition for preventing or improving inflammatory bowel disease, wherein the inflammatory cytokine is one or more selected from COS-2, IL-6, IL-1β, and TNF-α. According to paragraph 1,
A food composition for preventing or improving inflammatory bowel disease, characterized in that the content of the flower shrimp hot water extract contained in the composition is 100 to 1,000 mg/kg. A pharmaceutical composition for preventing or treating inflammatory bowel disease comprising a hot water extract of Trachysalambria curvirostris as an active ingredient. In clause 7,
A pharmaceutical composition for preventing or treating inflammatory bowel disease, wherein the inflammatory bowel disease is a disease selected from Crohn's disease, ulcerative colitis, intestinal Behcet's disease, infectious enteritis, ischemic enteritis, and radiation enteritis. In clause 7,
The composition is a pharmaceutical composition for preventing or treating inflammatory bowel disease, characterized in that it inhibits the expression of inflammatory cytokines in colon tissue. According to clause 9,
A pharmaceutical composition for preventing or treating inflammatory bowel disease, wherein the inflammatory cytokine is one or more selected from COS-2, IL-6, IL-1β, and TNF-α. In clause 7,
A pharmaceutical composition for the prevention or treatment of inflammatory bowel disease, characterized in that the content of the flower shrimp hot water extract contained in the composition is 100 to 1,000 mg/kg. In clause 7,
A pharmaceutical composition for preventing or treating inflammatory bowel disease, characterized in that the composition is for oral administration. A feed composition for preventing or improving inflammatory bowel disease comprising a hot water extract of flower shrimp ( Trachysalambria curvirostris ) as an active ingredient.