WO2024123075A1 - Composition for improving intestinal function using turnip extract - Google Patents

Abstract

The present invention relates to a composition for improving intestinal function using a turnip extract, the composition having the activity of: promoting the expression of MUC2, which is a major mucin protein of the intestinal (small intestine and/or large intestine) mucosal layer, in LS174T cells, which are colon cancer cell lines, without showing any particular cytotoxicity; promoting the expression of ZO-1, which is a major protein of the intestinal tight junction, in Caco2 cells, which are colon cancer cell lines; increasing the number of lymphocytes in the blood in animal experiments; promoting the production of IgA in the small intestine; increasing the production of Granulocyte-macrophage colony-stimulating factor (GM-CSF) of Peyer's patches; and increasing the size and number of lymphocytic follicle compartments in Peyer's patches.

Description

Composition for improving intestinal function using turnip extract

The present invention relates to a composition for improving intestinal function using turnip ( Brassica Rapa . L. or B. Rapa horenisis . L) extract.

The intestinal tract wall is structurally composed of intestinal epithelial cells, mucus layer, and various substrates.

The epithelial cell layer has selective permeability to nutrients, electrolytes, and moisture and functions as a biochemical barrier, as well as a physical barrier that protects against intestinal toxins, antigens, and intestinal microorganisms.

The intestinal mucosa layer physically separates the intestinal epithelium from harmful compounds, enzymes, and microorganisms introduced into the luminal environment of the intestine, forming a primary barrier against harmful substances and promoting smooth movement of food entering the intestine. . The intestinal mucosal layer is mainly composed of mucin, a glycoprotein, and this mucin glycoprotein forms the gel of the mucosal layer. In particular, MUC2 mucin is an important component of the mucosal layer and plays a critical role in protecting the intestinal epithelium and maintaining intestinal homeostasis (International Journal of Biological Macromolecules 164 (2020) 884-891). When MUC2 mucin is deficient, the composition of the mucous membrane changes. Inflammatory bowel disease occurs, and in ulcerative colitis, it is known that less MUC2 mucin is secreted, resulting in thin and discontinuous mucous membranes (Oncotarget 8 (2017) 71456-71470).

The route by which substances are absorbed in the small intestine includes a selective permeation route through epithelial cells (transcellular pathway) and a paracellular pathway through tight junctions, which are junctions between epithelial cells. Substances with low molecular weight pass through the intestinal wall through tight junctions, which are gaps between epithelial cells.

Tight junctions are a network of proteins between epithelial cells, and these proteins include integral transmembrane proteins such as occludin, claudin, and junctional adhesion molecules, and ZO. It is broadly classified into plaque proteins such as -1 (zonula occludens proteins-1). The intrinsic transmembrane protein uses ZO-1 (zona occludens-1) and cingulin, which are plate proteins present in the cytoplasm, as mediators, to actin, a cytoskeletal muscle, and a number of cytosolic regulatory proteins. ), and performs a biological barrier function that regulates adhesion between cells and the movement of substances through the space around cells. ZO-1, an occludin protein such as the plate protein, is composed of multiple domains and is known to play an essential role in regulating the permeability of the intestinal wall by binding to various junction proteins (Mol Biol Cell. 2006 Apr; 17( 4): 1922-1932, Ann N Y Acad Sci. 2022 Aug;1514(1):21-33;2021 Dec;161(6):1924-1939).

When tight junction proteins are not expressed normally or lose their activity and tight junctions become loose, the intestinal wall does not function as a sufficient defense barrier, which increases the permeability of harmful substances. Ultimately, mucosal immune system disruption and inflammatory response are induced, resulting in endotoxin. It may cause intestinal-related or systemic diseases such as endotoxemia and inflammatory bowel disease (Ministry of Food and Drug Safety Functional Evaluation Guide for Health Functional Foods, Intestinal Health, September 2020). This condition in which tight junctions become loose and the permeability of the intestinal wall increases is called leaky gut syndrome (Ministry of Food and Drug Safety Functional Evaluation Guide for Health Functional Foods, Related to Intestinal Health, September 2020).

Meanwhile, the intestinal tract is constantly exposed to potential immune stimuli such as various microorganisms and foreign proteins, so it has the largest number of immune cells among living tissues. Gut-associated lymphatic tissue (GALT) is the largest lymphoid tissue in the body. It is present in the mucosa and submucosa of the intestine and contains lymphocytes that aggregate, so it is responsible for the IgA immune response and biological immunity within the intestinal immune system. It plays a very important role in defense (Adv Exp Med Biol 635:1-14, 2008). Peyer's patch, a characteristic tissue of GALT, is a tissue that is easily observed with the naked eye and is distributed throughout the small intestine and is concentrated in the ileum of the small intestine. Peyer's patch is an inducing site for IgA production and is composed of T cells, B cells, macrophages, dendritic cells, etc. Additionally, M cells (micro enfold cells), which are antigen-sampling cells, are present in the intestinal tract. Acts as a key lymphoid organ (J Immunol 180: 1293-1294, 2008; J Dairy Sci 86: 3321-3329, 2003). M cells present in Peyer's patches activate immune cells by ingesting soluble antigens, bacteria, or viruses from the intestinal lumen through pinocytosis or phagocytosis and moving them to lymphoid cells (Proc Natl Acad Sci USA 101: 6110-6115, 2004; FEMS Immunol Med Microbiol 52: 2-12, 2008). These activated immune cells and the cytokines produced and secreted by these immune cells are called mesenteric lymph nodes. It regulates not only intestinal immunity but also systemic immunity by regulating the growth, migration, and differentiation of circulating immune system or hematopoietic cells such as bone marrow cells and white blood cells through the thoracic duct (J Immunol 176: 7533-7541, 2006)

The present invention discloses the intestinal function improving activity of a turnip extract, particularly the intestinal barrier function improving activity and the intestinal immune function improving activity.

The purpose of the present invention is to provide a composition for improving intestinal function using turnip extract.

Another object of the present invention is to provide a composition for improving intestinal barrier function using turnip extract.

Another object of the present invention is to provide a composition for improving intestinal immune function using turnip extract.

Other or specific purposes of the present invention will be presented below.

As confirmed in the examples below, the present invention promotes the expression of MUC2, a major mucin protein in the intestinal (small intestine and/or large intestine) mucosal layer, in LS174T cells, a colon cancer cell line, while the turnip extract does not show any particular cytotoxicity, , promotes the expression of ZO-1, a major protein of intestinal tight junctions, in Caco2 cells, a colon cancer cell line, and also increases the number of lymphocytes in the blood in animal experiments, promotes the production of IgA in the small intestine, and promotes the production of GM-CSF (Granulocyte) in Peyer's patches. It was completed by confirming that it increased the expression of the -macrophage colony-stimulating factor) gene and increased the size and number of lymphocytic follicular compartments in Peyer's patches.

The present invention is provided based on these experimental results. In one aspect, the present invention can be viewed as a composition for improving intestinal function containing turnip extract as an active ingredient, and in another aspect, the present invention can be viewed as a composition for improving intestinal function containing turnip extract as an active ingredient. It can be viewed as a composition for improving function, and in another aspect, it can be viewed as a composition for improving intestinal immune function containing turnip extract as an active ingredient, and in another aspect, it can be seen as a composition for improving intestinal barrier function containing turnip extract as an active ingredient. It can be identified as a composition for preventing or treating diseases according to.

In this specification, “extract” refers to the stem, leaf, fruit, flower, root, etc. of the plant to be extracted, mixed with water, lower alcohol with 1 to 4 carbon atoms (methanol, ethanol, butanol, etc.), methylene chloride, ethylene, acetone, hexane, Extract obtained by leaching using ether, chloroform, ethyl acetate, butylacetate, N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1,3-butylene glycol, propylene glycol, or mixed solvents thereof. , refers to an extract obtained using a supercritical extraction solvent such as carbon dioxide or pentane, or a fraction obtained by fractionating the extract. The extraction method is cold immersion, reflux, heating, and ultrasonic radiation, taking into account the polarity of the active substance, degree of extraction, and degree of preservation. , any method such as supercritical extraction can be applied. In the case of a fractionated extract, the extract is suspended in a specific solvent and then mixed with a solvent of different polarity to form a fraction obtained by adsorbing the crude extract onto a column filled with silica gel and then mixed with a hydrophobic solvent, a hydrophilic solvent, or a mixture of these. It is meant to include fractions obtained using the mobile phase. In addition, the meaning of the extract includes concentrated liquid extract or solid extract from which the extraction solvent has been removed by methods such as freeze-drying, vacuum drying, hot air drying, and spray drying. Preferably, it refers to an extract obtained using water, ethanol, or a mixed solvent thereof as an extraction solvent.

Also, as used herein, “improvement of intestinal function” means improvement of intestinal barrier function and/or improvement of intestinal immune function.

Also, as used herein, “improving intestinal barrier function” means strengthening the intestinal mucosa layer and/or improving the barrier function of intestinal tight junctions.

In addition, as used herein, the term "disease caused by impaired intestinal barrier function" refers to a disease caused by impaired intestinal barrier function, that is, destruction of the intestinal mucosa layer and/or impaired barrier function of intestinal tight junctions, such as leaky gut syndrome, endotoxemia, and inflammation. refers to inflammatory bowel disease and/or ulcerative colitis.

In addition, in this specification, “active ingredient” refers to an ingredient that exhibits the desired activity alone or can exhibit activity in combination with a carrier that is not active on its own.

The composition of the present invention may contain the active ingredient in any amount (effective amount) depending on the use, formulation, purpose of mixing, etc., as long as it can exhibit intestinal function improvement activity, etc., and a typical effective amount is based on the total weight of the composition. It will be determined within the range of 0.001% by weight to 20.0% by weight. Here, “effective amount” means that when the composition of the present invention is administered to mammals, preferably humans, to which it is applied during the administration period recommended by medical experts, etc., it can exhibit the intended functional and pharmacological effects, such as improvement of intestinal function. It refers to the amount of active ingredient contained in the composition of the present invention. Such effective amounts can be determined experimentally within the scope of the ordinary ability of those skilled in the art.

In addition to the active ingredients, the composition of the present invention has already been verified for safety in the art in order to enhance and reinforce the effect of improving intestinal function or to improve the convenience of taking or ingesting through the addition of similar activities such as improving gastric function. It may further include any compounds or natural extracts known to have activity. These compounds or extracts include compounds, extracts, and pharmaceuticals listed in compendial documents such as the Pharmacopoeia of each country (in Korea, the “Korean Pharmacopoeia”) and the Code of Health Functional Foods of each country (in Korea, it is the “Standards and Specifications for Health Functional Foods” notified by the Ministry of Food and Drug Safety). The laws of each country that govern the manufacture and sale of compounds or extracts and health functional foods that have received product approval in accordance with the laws of each country (in Korea, this is the Pharmaceutical Affairs Act) (in Korea, it is the “Health Functional Foods Act”) 」) includes compounds or extracts whose functionality has been individually recognized.

For example, according to the Korean Health Functional Food Act, it is listed in the “Health Functional Food Standards and Specifications” or has been individually recognized for its functionality of “helping to proliferate beneficial bacteria in the intestines and suppress harmful bacteria.”

Galactooligosaccharide, guar gum hydrolyzate, soy oligosaccharide, raffinose, lactulose powder, indigestible maltodextrin derived from wheat starch, isomaltooligosaccharide, xylooligosaccharide, coffee manno-oligosaccharide powder, fructo-oligosaccharide, etc., "regulating immunity" Probiotics (VSL#3), which have been recognized for their functionality of “helping intestinal health,” and soy oligosaccharides, raffinose, wood ear mushrooms, fig paste, powdered agar, isomaltooligosaccharide, and xylo, which have been recognized for their functionality of “helping to facilitate bowel movement.” These compounds or extracts include oligosaccharides, coffee manno-oligosaccharides, fructo-oligosaccharides, etc., as well as licorice extract, mastic gum, beeswax alcohol, and artichoke extract, which are recognized for their “helpful stomach health” functionality.

In a specific embodiment, the composition of the present invention can be considered a food composition.

The food composition of the present invention can be manufactured in any form, for example, beverages such as tea, juice, carbonated beverages, and electrolyte drinks, processed oils such as milk and yogurt, gums, rice cakes, Korean snacks, bread, snacks, noodles, etc. It can be manufactured into health functional food preparations such as foods, tablets, capsules, pills, granules, liquids, powders, flakes, pastes, syrups, gels, jellies, bars, etc.

In addition, the food composition of the present invention can have any product classification in terms of legal and functional classification as long as it complies with the enforcement laws and regulations at the time of manufacture and distribution. For example, it is a health functional food according to the Korean 「Act on Health Functional Foods」, or confectionery, beans, tea, and beverages according to each food type according to the food code of the Korean 「Food Sanitation Act」 (Ministry of Food and Drug Safety Notification 「Food Standards and Specifications」) , special purpose food, etc.

The food composition of the present invention may contain food additives in addition to the active ingredients. Food additives can generally be understood as substances that are added to, mixed with, or infiltrated into food when manufacturing, processing, or preserving food. Since they are consumed daily and for a long period of time with food, their safety must be guaranteed. In the food additive code of each country that regulates the manufacturing and distribution of food (in Korea, it is the Food Sanitation Act), food additives with guaranteed safety are limited in terms of ingredients or functions. In the Korea Food Additive Code (Ministry of Food and Drug Safety Notification “Food Additive Standards and Specifications”), food additives are classified into chemical synthetics, natural additives, and mixed preparations in terms of composition. These food additives are classified into sweeteners and flavors in terms of function. It is classified into preservatives, emulsifiers, acidulants, thickeners, etc.

Sweeteners are used to impart an appropriate sweetness to foods, and both natural and synthetic ones can be used in the food composition of the present invention. Preferably, a natural sweetener is used, and natural sweeteners include sugar sweeteners such as corn syrup solids, honey, sucrose, fructose, lactose, and maltose.

Flavoring agents are used to improve taste or aroma, and both natural and synthetic ones can be used. Preferably, natural products are used. When using natural products, it can serve the purpose of enhancing nutrition in addition to flavor. Natural flavoring agents may be obtained from apples, lemons, tangerines, grapes, strawberries, peaches, etc., or may be obtained from green tea leaves, coriander leaves, bamboo leaves, cinnamon, chrysanthemum leaves, jasmine, etc. You can also use things obtained from ginseng (red ginseng), bamboo shoots, aloe vera, and ginkgo nuts. Natural flavors can be liquid concentrates or solid extracts. In some cases, synthetic flavoring agents may be used, and as synthetic flavoring agents, esters, alcohols, aldehydes, terpenes, etc. may be used.

Preservatives include calcium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate, EDTA (ethylenediaminetetraacetic acid), etc., and emulsifiers include acacia gum, carboxymethyl cellulose, xanthan gum, and pectin. etc. may be used, and as acidulants, acidic acid, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, phosphoric acid, etc. may be used. In addition to improving taste, acidulants may be added to ensure that the food composition has an appropriate acidity for the purpose of suppressing the growth of microorganisms. As a thickening agent, a suspending agent, settling agent, gel forming agent, bulking agent, etc. may be used.

In addition to the food additives described above, the food composition of the present invention may contain bioactive substances or minerals known in the art and whose safety is guaranteed as food additives for the purpose of supplementing and reinforcing functionality and nutrition.

Such physiologically active substances include catechins contained in green tea, vitamins such as vitamin B1, vitamin C, vitamin E, and vitamin B12, tocopherol, dibenzoylthiamine, etc., and minerals include calcium preparations such as calcium citrate and magnesium stearate. Magnesium preparations such as iron citrate, iron preparations such as chromium chloride, potassium iodine, selenium, germanium, vanadium, zinc, etc. are included.

The food composition of the present invention may contain the above-described food additives in an appropriate amount to achieve the purpose of addition depending on the product type.

Regarding other food additives that can be included in the food composition of the present invention, each country's food code or food additive code can be referred to.

The composition of the present invention may be considered a pharmaceutical composition in other specific embodiments.

The pharmaceutical composition of the present invention contains a pharmaceutically acceptable carrier in addition to the active ingredient and can be prepared into an oral formulation or a parenteral formulation depending on the route of administration by a conventional method known in the art. Here, the route of administration may be any suitable route, including topical route, oral route, intravenous route, intramuscular route, and direct absorption through mucosal tissue, and two or more routes may be used in combination.

Pharmaceutically acceptable carriers are well known in the art depending on the route of administration or dosage form, and for specifics, reference can be made to the pharmacopoeia of each country, including the “Korean Pharmacopoeia.”

When the pharmaceutical composition of the present invention is prepared as an oral dosage form, it can be prepared as powder, granules, tablets, pills, sugar-coated tablets, capsules, solutions, gels, syrups, suspensions, and wafers along with a suitable carrier according to methods known in the art. It can be manufactured in a dosage form such as: Examples of suitable carriers include sugars such as lactose, glucose, sucrose, dextrose, sorbitol, mannitol, and xylitol, starches such as corn starch, potato starch, and wheat starch, cellulose, methylcellulose, ethylcellulose, sodium carboxymethylcellulose, Cellulose such as hydroxypropylmethylcellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, polyol, vegetable oil, ethanol, grease. Serol, etc. can be mentioned. When formulating, appropriate binders, lubricants, disintegrants, colorants, diluents, etc. can be included as needed. Suitable binders include starch, magnesium aluminum silicate, starch ferrite, gelatin, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone, glucose, corn sweetener, sodium alginate, polyethylene glycol, and wax, and as a lubricant, oleic acid. Examples include sodium, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, silica, talcum, stearic acid, its magnesium and calcium salts, and polydethylene glycol. Disintegrants include starch and methyl cellulose. , agar, bentonite, xanthan gum, starch, alginic acid or its sodium salt, etc. Additionally, diluents include lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, glycine, etc.

When the pharmaceutical composition of the present invention is prepared as a parenteral formulation, it can be formulated in the form of injections, transdermal administration, nasal inhalation, and suppositories along with a suitable carrier according to methods known in the art. When formulated as an injection, an aqueous isotonic solution or suspension can be used as a suitable carrier. Specifically, an isotonic solution such as PBS (phosphate buffered saline) containing triethanolamine, sterile water for injection, or 5% dextrose can be used. . When formulated for transdermal administration, it can be formulated in the form of ointments, creams, lotions, gels, external solutions, paste preparations, linear preparations, and aerol preparations. In the case of nasal inhalation, it can be formulated in the form of an aerosol spray using suitable propellants such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, and carbon dioxide. When formulated as a suppository, the carrier is Wethepsol ( witepsol), Tween 61, polyethylene glycols, cocoa fat, laurel paper, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, sorbitan fatty acid esters, etc. can be used.

Regarding the specific formulation of pharmaceutical compositions, it is known in the art, and references can be made to, for example, Remington's Pharmaceutical Sciences (19th ed., 1995). The above documents are considered a part of this specification.

The preferred dosage of the pharmaceutical composition of the present invention is in the range of 0.001 mg/kg to 10 g/kg per day, preferably 0.001 mg/kg to 1 g, depending on the patient's condition, weight, gender, age, patient's severity, and administration route. It may be in the /kg range. Administration can be done once a day or divided into several times. These dosages should not be construed as limiting the scope of the invention in any respect.

As described above, according to the present invention, a composition for improving intestinal function using turnip extract can be provided.

The composition of the present invention can be commercialized as food or medicine.

Figures 1 and 2 show the results of cytotoxicity evaluation of turnip extract on LS174T cells and Caco-2 cells.

Figure 3 shows the results of evaluating the expression level of the MUC2 mucin gene in turnip extract in LS174T cells.

Figure 4 shows the results of evaluating the expression level of the gene ZO-1, a tight junction protein of turnip extract, in Caco-2 cells.

Figure 5 shows the results of measuring the number of blood lymphocytes in animal experiments.

Figure 6 shows the results of evaluating the degree of IgA production in the small intestine.

Figure 7 shows the results of evaluating the expression level of GM-CSF in small intestine Peyer's patches.

Figure 8 shows the results of evaluating the size and number of lymphocytic follicular compartments in small intestine Peyer's patches.

Hereinafter, the present invention will be described with reference to examples. However, the scope of the present invention is not limited to these examples.

<Example> Evaluation of intestinal barrier improvement and intestinal immunity enhancement activity of turnip extract

1. Preparation of turnip extract

10 times the weight of distilled water was added to the dried turnip root powder, extracted at 70-100°C for 150 minutes, filtered, and the filtrate was concentrated under reduced pressure and freeze-dried to prepare a solid turnip extract ( 73 ). The prepared extract was dissolved in DMSO and used in the experiment.

2. Evaluation of intestinal barrier improvement activity

2.1 Cytotoxicity evaluation

To measure the safety of LS174T and Caco-2 cells, 200 μL of cells at a concentration of 5 × 105 cell/mL were dispensed into 96 well plates. After stabilization in a 5% CO ₂ incubator (37°C) for about 16 to 20 hours, the extract samples of the above examples were treated for 12 hours according to concentration. To measure cytotoxicity, the supernatant was removed and 100 μL of 10% EZ cytox (DoGenBio, Seoul, Korea) dissolved in SFM (Serum Free Media) was added. After 30 minutes, absorbance was measured at 450 nm. Relative cell viability (%) was expressed as a percentage of the negative control. Cell morphology was observed using an inverted microscope (CKX53, OYMPUS, Tokyo, Japan).

The results are shown in Figures 1 and 2. As confirmed in Figures 1 and 2, turnip extract did not show any particular cytotoxicity up to 10 μg/mL.

2.2 Evaluation of MUC2 mucin gene expression ability

Evaluation of mucin gene (MUC2) expression was performed using qRT-PCR (Quantitative real-time polymerase chain reaction).

The level of MUC2 mucin gene expression was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR).

LS174T cells (5×10 ⁵ cell/mL) were dispensed at 500 μL each into a 24 well plate and stabilized in a 5% CO ₂ incubator (37°C) for about 16 to 20 hours. And the extract samples of the above examples were treated at each concentration for 12 hours. To confirm the level of gene expression at the mRNA level, RNA was isolated using the Total RNA Extraction Kit (iNtRON BIOTECHNOLOGY, Daejeon, Korea). The isolated RNA was reverse transcribed into cDNA using ReverTra AceTM qPCR RT Master Mix (TOYOBO, Osaka, Japan), and then qPCR was performed using SYBR® Green Realtime PCR Master Mix using Magnetic Induction Cycler PCR Machine (biomolecular systems, Coomera, Australia). The process was carried out using reagents (TOYOBO, Osaka, Japan). The base sequences of the primers used in this experiment are shown in Table 1. Changes in mRNA expression were expressed using the 2 ^-△△Ct method with the GAPDH gene control set to 1.

The results are shown in Figure 3, which shows that turnip extract promotes MUC2 mucin gene expression in a concentration-dependent manner compared to the negative control (NC) in LS174T cells. In Figure 3, the positive control (PC) is propionate.

2.3 Evaluation of gene expression level of ZO-1, a tight junction protein

Evaluation of the gene expression level of the tight junction protein ZO-1 was also performed using qRT-PCR (Quantitative real-time polymerase chain reaction).

Caco2 cells (5×10 ⁵ cell/mL) were dispensed at 500 μL each into a 24 well plate and stabilized in a 5% CO 2 incubator (37°C) for 16 to 20 hours. And the extract samples of the above examples were treated for 12 hours at each concentration. To confirm the level of gene expression at the mRNA level, RNA was isolated using the Total RNA Extraction Kit (iNtRON BIOTECHNOLOGY, Daejeon, Korea). The isolated RNA was reverse transcribed into cDNA using ReverTra AceTM qPCR RT Master Mix (TOYOBO, Osaka, Japan), and then qPCR was performed using the Magnetic Induction Cycler PCR Machine (biomolecular sy stems, Coomera, Australia) using SYBR® Green Realtime PCR Master. This was carried out using Mix reagent (TOYOBO, Osaka, Japan). The base sequences of the primers used in this experiment are shown in Table 2. Changes in mRNA expression were expressed using the 2 ^-△△Ct method, with the GAPDH control set to 1.

The results are shown in Figure 4, which shows that turnip extract promotes ZO-1 gene expression in a concentration-dependent manner. In Figure 3, the positive control (PC) is propionate.

3. Evaluation of intestinal immune-promoting activity

3.1 Lymphocyte number measurement

To measure the immune-boosting ability of the sample, 6-8 week old BALB/c mice were divided into negative control (NC), positive control (propionate administration group, PC), low-dose experimental group (L), and high-dose experimental group (H). Divided (n=6), only regular feed was fed to the negative control group, and regular feed was fed to the positive control group along with 304mg/kg/day?? of propionate. It was dissolved in DW (distilled water) and administered orally, and the experimental group was fed regular feed, and the extract sample of the example was administered at a dose of 1 mg/kg/day (low dose, L) and 5 mg/kg/day (high dose, H). It was dissolved in DW and administered orally. After 3 weeks, the mice were sacrificed and blood (300-500 μL/mouse) was collected from the sacrificed mice. To measure the proportion of immune cells, a drop of blood was taken before it clotted and placed on one end of a glass slide. Prepare an extra slide, place it in front of the blood drop at an angle of 30° to 45°, and gently smear the slide to create an ideal zone. Afterwards, the number of lymphocytes in the smeared blood was measured using Wright-Giemsa solution (15022; MUTO PURE CHEMICALS CO., Japan), and the results are shown in Figure 5.

Referring to FIG. 5, it can be seen that the extract samples of the examples generally increase the number of lymphocytes depending on the administered dose. Here, the positive control group (PC) is the propionate administration group.

3.2 Evaluation of IgA production

In the above 3.1 animal experiment, small intestine tissue was collected from sacrificed mice and stored in a PBS/15mL conical tube. The small intestine tissue was cut into small pieces and vortexed vigorously for 5 minutes to extract fluid from the small intestine tissue. Afterwards, the supernatant was separated by centrifugation (3,000 rpm, 20 min, 4°C), and centrifugation was performed again with some of the supernatant under the same conditions. To quantify the total protein level in the supernatant, it was measured using the BCA Protein Assay kit (23225, Thermo Fisher Scientific). Measurement of the content of IgA, a mucosal immunoglobulin, in small intestinal fluid was performed by ELISA using an ELISA kit (BD bioscience, Vancouver, Canada) according to the manufacturer's instructions.

The results are shown in Figure 6. The results in Figure 6 show that the production of IgA increases depending on the administered dose of the example extract sample. Here too, the positive control group (PC) is the propionate administration group.

3.3 Evaluation of GM-CSF expression

The level of GM-CSF expression was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR).

To evaluate the level of GM-CSF expression, in animal experiment 3.1 above, 4 to 6 Peyer's patches were cut from the small intestine wall of the sacrificed mice. Cell suspension was prepared by disrupting tissue from Peyer's patches using a stainless steel mesh. To confirm the expression level of the gene at the mRNA level, RNA was isolated using the Total RNA Extraction Kit (iNtRON BIOTECHNOLOGY, Daejeon, Korea). The isolated RNA was reverse transcribed into cDNA using ReverTra AceTM qPCR RT Master Mix (TOYOBO, Osaka, Japan), and then qPCR was performed using SYBR®Green Realtime PCR Master Mix using Magnetic Induction Cycler PCR Machine (biomolecular systems, Coomera, Australia). This was carried out using reagents (TOYOBO, Osaka, Japan). The base sequences of the primers used in this experiment are shown in Table 3 below, and changes in mRNA expression were expressed using the 2 ^△△Ct method with the GAPDH control set to 1.

primer sequence

gene	Genebank number	forward primer	reverse primer
GM-CSF	NM_009969.4	5'-AAGGTCCTGAGGAGGATGTG-3'	5'-GAGGTTCAGGGCTTCTTTGA-3'
GAPDH	NM_001411843.1	5'-ATGGTGAAGGTCGGTGTGGAAC-3'	5'-TTGATGTGTAGTGGGGTCTCGC-3'

The results are shown in Figure 7, which shows that GM-CSF expression increases depending on the administered dose of the example extract sample. Here too, the positive control group (PC) is the propionate administration group. 3.4 Histological evaluation of Peyer's patches in the small intestine

In order to measure the ability of the sample to enhance intestinal immunity, in the animal experiment in 3.1 above, Peyer's patch of the small intestine of the sacrificed mouse was cut, fixed in formalin solution, and embedded in paraffin. For histological evaluation, paraffin-embedded tissue sections were stained with hematoxylin and eosin (H&E). Afterwards, histological changes in Peyer's plate, one of the immune organs, were observed.

The results are shown in Figure 8. Referring to Figure 8, in the experimental group administered the example extract sample compared to the negative control (NC), the lymphocytic follicular compartments in Peyer's patches were found to be larger and increased in number.

Claims (7)

1. A food composition for improving intestinal function containing turnip extract as an active ingredient.
2. According to paragraph 1,

   A composition in which the extract is an extract of turnip roots in water, ethanol, or a mixed solvent thereof.
3. A food composition for improving intestinal barrier function containing turnip extract as an active ingredient.
4. According to paragraph 3,

   The improvement of intestinal barrier function is strengthening of the intestinal mucosa layer or improving the barrier function of intestinal tight junctions,

   A composition in which the extract is an extract of turnip roots in water, ethanol, or a mixed solvent thereof.
5. A food composition for improving intestinal immune function containing turnip extract as an active ingredient.
6. According to clause 5,

   A composition in which the extract is an extract of turnip roots in water, ethanol, or a mixed solvent thereof.
7. A composition for preventing or treating diseases caused by impaired intestinal barrier function, comprising water, ethanol, or a mixed solvent extract of turnip roots as an active ingredient,

   The composition, wherein the disease is leaky gut syndrome, endotoxemia, inflammatory bowel disease, or ulcerative colitis.

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