Composition comprising germinated oat extract for preventing or treating inflammatory bowel diseases

Abstract

The present invention relates to a composition comprising a germinated oat (Avena sativa L.) extract for preventing, alleviating or treating inflammatory bowel diseases. The composition improves intestinal permeability and improves tight junction integrity in an inflammatory intestinal environment, and thus can be effectively used in the preparation of medicines, health functional foods, foods or prebiotics for preventing, alleviating or treating inflammatory bowel diseases.

Description

Composition for the prevention or treatment of inflammatory bowel disease containing sprouted oat extract

[0001] The present invention relates to a composition for the prevention or treatment of inflammatory bowel disease comprising a sprouted oat extract with an increased avenanthramide content.

[0002] Inflammatory bowel disease refers to chronic inflammatory diseases in which inflammation or ulcers develop in the intestines due to unknown causes, such as ulcerative colitis (UC), Crohn's disease (CD), and Behcet's disease.

[0003] Inflammatory bowel disease is common worldwide and has recently shown a significantly increasing trend in Korea; however, the causes and mechanisms of the disease are relatively complex and are not yet precisely known.

[0004] Drugs used to treat existing inflammatory bowel disease include steroidal immunosuppressants, 5-aminosalicylic acid (5-ASA) class drugs that block the production of prostaglandins (e.g., sulfasalazine, etc.), and mesalazine. Although anti-inflammatory drugs and immunosuppressants are prescribed as exemplified above, they have side effects such as nausea, vomiting, headache, hemolytic anemia, and male infertility. Furthermore, because there is a significant number of patients who do not respond to treatment or whose efficacy is reduced, there is a continuous demand for the development of safer and more effective treatments.

[0005] Inflammatory bowel disease (IBD) is characterized by damage to the integrity of the epithelial barrier caused by inflammation. Damaged barrier integrity and increased permeability of epithelial tight junctions lead to barrier weakening, a condition known as the "leaky gut." Recent studies have revealed that the barrier is critical not only for IBD but also for various other diseases, including autoimmune diseases associated with barrier integrity. Maintaining and strengthening barrier integrity is essential for the prevention and treatment of IBD and various other diseases. Recently, natural bioactive substances known for their anti-inflammatory effects have garnered attention as important potential therapeutic agents for IBD.

[0006] Oats (Avena sativa L.) contain abundant biologically active components, exhibiting various health-promoting effects including antioxidant, anti-diabetic, antibacterial, anticancer, antihypertensive, immunomodulatory, antihyperlipidemic, anti-obesity, cardioprotective, and anti-inflammatory effects. According to prior research, sprouted oats are known to be effective for atopic dermatitis by regulating the expression of skin binding proteins, improving skin barrier function, and treating skin barrier disruption.

[0007] Oats contain various phenolic compounds, among which avenanthramides (AVNs) are a type of phenolic alkaloid represented by the following chemical formula I and are specifically contained in oats, and the avenanthramide and phenolic compound content of germinated oats is much higher than that of ungerminated oat seeds.

[0008] [Chemical Formula I]

[0009]

[0010] Avenanthramide is known as a powerful antioxidant with established effects including blood pressure regulation, inhibition of skin irritation, and anti-atherosclerosis. Recently, it has been revealed that it can also be used to treat hearing loss and neurodegenerative diseases. As such, research on the applications of avenanthramide is actively underway, and it is gaining attention as a useful component that can be used to treat various diseases.

[0011] However, in the context of inflammatory bowel disease (IBD), the specific effects of the active ingredients of sprouted oats on intestinal permeability and tight junction (TJ) integrity have not been clearly established.

[0012] Accordingly, the inventors adopted an integrated approach for initial in silico evaluation using the Combination-Oriented Natural Product Database with Unified Terminology (COCONUT), which combines bioinformatics analysis and chemical profiling, with inflammatory bowel disease (IBD) as a phenotype associated with the interaction between the bioactive compounds of germinated oats and the genetic and proteomic environment determining tight junction integrity. Furthermore, by focusing on efficacy evaluation through in silico prediction validated by an in vitro model using a Caco-2 cell model, the inventors elucidated the fundamental mechanism of the effect of germinated oats on intestinal permeability and confirmed that germinated oat extract (GOE) has an effect on inflammatory bowel disease by improving tight junction integrity within an inflamed intestinal environment, thereby completing the present invention.

[0013] Prior art literature

[0014] Patent documents

[0015] (Patent Document 0001) Republic of Korea Registered Patent No. 10-1745734

[0016] (Patent Document 0002) Republic of Korea Registered Patent No. 10-1935500

[0017] The objective of the present invention is to solve the above-mentioned problems and to provide a pharmaceutical composition for the prevention or treatment of inflammatory bowel disease comprising a sprouted oat (Avena sativa L.) extract.

[0018] Another objective of the present invention is to provide a health functional food composition for improving inflammatory bowel disease comprising sprouted oat extract.

[0019] Another objective of the present invention is to provide a food composition for preventing or improving inflammatory bowel disease comprising sprouted oat extract.

[0020] Another objective of the present invention is to provide a prebiotic composition for improving intestinal flora comprising germinated oat extract as an active ingredient.

[0021] Another objective of the present invention is to provide a method for preventing, improving, or treating inflammatory bowel disease comprising administering an effective amount of a composition containing sprouted oat extract to a subject.

[0022] Another objective of the present invention is to provide a use for sprouted oat (Avena sativa L.) extract for the production of pharmaceuticals, health functional foods or foods, or prebiotics for improving gut microbiota for the prevention, improvement, or treatment of inflammatory bowel disease (IBD).

[0023] The present invention relates to a composition for the prevention, improvement, or treatment of inflammatory bowel disease (IBD) comprising an extract of sprouted oats (Avena sativa L.). The composition according to the present invention exhibits an effect of preventing, improving, or treating inflammatory bowel disease by improving intestinal permeability and tight junction integrity within an inflamed intestinal environment.

[0024] In one embodiment of the present invention, a pharmaceutical composition for the prevention or treatment of inflammatory bowel disease comprising sprouted oat extract is provided.

[0025] In another embodiment of the present invention, a health functional food composition for preventing or improving inflammatory bowel disease is provided, comprising sprouted oat extract.

[0026] In another embodiment of the present invention, a food composition for preventing or improving inflammatory bowel disease is provided, comprising sprouted oat extract.

[0027] In another embodiment of the present invention, a prebiotic composition for improving intestinal flora is provided, comprising germinated oat extract as an active ingredient.

[0028] In another embodiment of the present invention, a method for preventing, improving, or treating inflammatory bowel disease is provided, comprising administering an effective amount of a composition containing sprouted oat extract to a subject.

[0029] In another embodiment of the present invention, a use of sprouted oat (Avena sativa L.) extract is provided for the preparation of a drug, health functional food or food, or a prebiotic for improving the gut microbiome for the prevention, improvement, or treatment of inflammatory bowel disease (IBD).

[0030] The present invention relates to a composition for the prevention, improvement, or treatment of inflammatory bowel disease comprising a sprouted oat (Avena sativa L.) extract, wherein the composition improves intestinal permeability and improves tight junction integrity within an inflamed intestinal environment, and thus can be effectively used in the manufacture of pharmaceuticals, health functional foods, foods, or prebiotics for the prevention, improvement, or treatment of inflammatory bowel disease.

[0031] Figure 1 shows the qualitative chromatographic analysis of phenols and alkaloids in germinated oat extract, (A) positive mode; (B) negative mode.

[0032] Figure 2 shows the results of a predictive analysis of potential target genes and biological pathways, comprising: (a) a network of complex gene phenotypes (inflammatory bowel disease) in germinated oats; (b) genes related to tight junctions (GO:CC); (c) protein-protein interaction (PPI) analysis of two combined gene sets (complex genes and tight junction genes) of the red network of the top 15 genes in the PPI analysis; and (d) biological processes associated with the 15 genes analyzed through the PPI analysis.

[0033] Figure 3 shows the results of the intestinal integrity assessment, with (a) changes in epithelial electrical resistance (TEER) in a Caco-2 cell monolayer 24 hours after germinated oat treatment; (b) comparative TEER values ​​after 24 hours; and (c) FITC-dextran permeability measurements. Error bars represent standard error (SE) values, and values ​​indicated by different letters in the graph represent significant differences according to Duncan's multiple range test. p < 0.05.

[0034] Figure 4 illustrates the effects of sprouted oats on potential inflammatory bowel disease (IBD) biomarkers, including (a) tight junction proteins; (b) inflammatory proteins; (c) cell signaling genes; (d) cell cycle-related genes; (e) cell structure and function; and (f) apoptosis molecules. Error bars represent standard error (SE) values, and values ​​represented by different letters in the graph indicate significant differences according to Duncan's multiple range test. p < 0.05.

[0035] Figure 5 shows the correlation between metabolites and IBD-related biomarkers, where each square represents the Pearson correlation coefficient value (r). Red indicates a positive correlation (0 < r < 1), blue indicates a negative correlation (-1 < r < 0), and asterisks indicate a significant difference (p < 0.05). (a) Correlation analysis for the negative mode of the 25 μg / mL sprouted oat treatment group; (b) Correlation analysis for the positive mode of the 25 μg / mL sprouted oat treatment group.

[0036] Figure 6 is a graph showing experimental results confirming that germinated oat extract promotes the growth of lactic acid bacteria according to one embodiment of the present invention.

[0037] Unless otherwise defined, all terms used in this specification (including technical and scientific terms) may be used in a meaning commonly understood by those skilled in the art to which the present invention pertains. Additionally, terms defined in commonly used dictionaries are not to be interpreted ideally or excessively unless explicitly and specifically defined otherwise.

[0038] As used in this specification, the singular form may include the plural form unless the context clearly indicates otherwise.

[0039] In this specification, when a part is described as “comprising” a certain component, it means that, unless specifically stated otherwise, it does not exclude other components but may include additional components.

[0040] In addition, all numbers and expressions indicating the amounts of components, reaction conditions, etc. described in this specification should be understood as being modified by the term “about” in all cases unless otherwise specified.

[0041] In this specification, “pharmaceuticalally acceptable” means that which is approved by a government or equivalent regulatory body, listed in a pharmacopoeia, or recognized by other general pharmacopoeias for use in animals, more specifically in humans, by avoiding significant toxic effects when used in normal medicinal doses.

[0042] In the present invention, the term "prevention" means suppressing the occurrence of a disease or illness in an individual who has not been diagnosed with having such a disease or illness but has a tendency to contract such a disease or illness. In this specification, the term "treatment" means suppression of the progression of a disease or illness; alleviation of a disease or illness; and / or elimination of a disease or illness. In this specification, the term "improvement" includes the alleviation of symptoms, suppression of the manifestation of such symptoms, delay of manifestation, and elimination of manifestation.

[0043] In addition, the experimental process specified in this specification is identical to the experimental process ordinarily performed in the art unless specifically described otherwise.

[0044] The present invention will be described in detail below.

[0045] One aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of inflammatory bowel disease (IBD) comprising a sprouted oat (Avena sativa L.) extract.

[0046] In this specification, the term “germinated oats” may have a sprout length of an average of 3 to 6 mm, specifically 4 to 5 mm, more specifically 4.2 to 4.7 mm, but is not limited thereto.

[0047] In one embodiment of the present invention, the germinated oats may be oat seeds germinated at room temperature for 2 days with a 2:1 light-dark cycle and then treated with an inducer for 3 days under the same light conditions, and the inducer may be abscisic acid, methyl jasmonate, or a combination thereof, but is not limited thereto.

[0048] The germinated oat extract, which is the active ingredient of the present invention, may be prepared by a method comprising the following steps, but is not limited thereto:

[0049] 1) a step of extracting by adding an extraction solvent to germinated oats; and

[0050] 2) A step of filtering the extract from step 1).

[0051] In the present invention, the germinated oats of step 1) can be cultivated or commercially available without limitation.

[0052] In the present invention, the extraction solvent of step 1) is preferably one or more solvents selected from the group consisting of water and organic solvents, and the organic solvent is more preferably one or more selected from the group consisting of alcohols having 1 to 5 carbon atoms, ethyl acetate, acetone, ether, chloroform, benzene, hexane, and dichloromethane. The alcohol may be selected from the group consisting of methanol, ethanol, propanol, butanol, and isopropanol, and preferably may be ethanol. As an extraction method, hot water extraction, ultrasonic extraction, shaking extraction, Soxhelt extraction, or reflux extraction may be used, and specifically, hot water extraction may be used, but is not limited thereto. In addition, the extraction time may be 1 to 24 hours, specifically 2 to 10 hours, 2 to 9 hours, or 2 to 8 hours, but is not limited thereto. Furthermore, the number of extractions may be 1 to 5 times, but is not limited thereto.

[0053] In addition, the germinated oat extract of the present invention may include a fraction obtained by further fractionating a primary extract obtained using the extraction solvent with a different polarity using an extraction solvent with a different polarity. For example, the germinated oat extract may be a fraction obtained by extracting an active ingredient contained in germinated oats with an alcohol having 1 to 5 carbon atoms, and then further fractionating it with a solvent with a different polarity such as ether, benzene, or hexane.

[0054] Two or more types of solvents may be used for the above fractionation, and each solvent extract may be prepared by using them sequentially or in combination according to the polarity of the solvents, but is not limited thereto.

[0055] The extract prepared through the above process or the fraction obtained by performing the above fractionation process may subsequently be filtered, concentrated, or dried to remove the solvent, and may undergo filtration, concentration, and drying all at once. Specifically, the filtration may be performed using filter paper or a vacuum filter, the concentration may be performed using a vacuum concentrator, for example, a rotary evaporator, and the drying may be performed using a spray drying method, for example.

[0056] In the present invention, the germinated oat extract may be included at a concentration of 5 to 500 μg / ml, specifically 10 to 400 μg / ml, more specifically 15 to 300 μg / ml, even more specifically 20 to 200 μg / ml, and even more specifically 25 to 100 μg / ml, but is not limited thereto.

[0057] In this specification, the term "avenanthramides (AVNs)" refers to alkaloid compounds known to be produced primarily from oats. The avenanthramides may be one or more selected from the group consisting of avenanthramide A, avenanthramide B, avenanthramide C, avenanthramide O, and avenanthramide P. Specifically, they may refer to one or more components selected from the group consisting of avenanthramide A, avenanthramide B, and avenanthramide C, but are not limited thereto.

[0058] The above avenanthramide may also be named anthranilic acid amides and can be represented by the following [Chemical Formula I]:

[0059] [Chemical Formula I]

[0060] .

[0061] In the above chemical formula, n=1 and R 1 is H, and R 2is OH, and R 3 The compound where is H is "avenanthramide A", n=1, and R 1 is OCH3, and R 2 is OH, and R 3 The compound where is H is "avenanthramide B", n=1, and R 1 is OH, and R 2 is OH, and R 3 A compound that is H can be referred to as "avenanthramide C".

[0062] In this specification, the term “inflammatory bowel disease” may be one or more selected from the group consisting of ulcerative colitis (UC), Crohn’s disease (CD), irritable bowel syndrome, intestinal Behcet’s disease, indeterminate colitis, infectious enteritis, bacterial enteritis, viral enteritis, amoebic enteritis, hemorrhagic rectal ulcer, ischemic bowel disease, radiation enteritis, tuberculous enteritis, and leaky gut syndrome.

[0063] In one embodiment of the present invention, the inventors have confirmed that the germinated oat extract improves intestinal permeability and tight junction (TJ) integrity resulting from inflammatory bowel disease, so the extract can be very usefully used as an active ingredient in a pharmaceutical composition for the prevention or treatment of inflammatory bowel disease.

[0064] In addition, according to one embodiment of the present invention, the germinated oat extract can promote the proliferation of beneficial bacteria in the intestines, and specifically, it has been confirmed that it improves the intestinal environment by promoting the proliferation of one or more lactic acid bacteria selected from the group consisting of Lactobacillus plantarum and Lactobacillus delbrueckii. Therefore, the germinated oat extract can be usefully used as an active ingredient in a pharmaceutical composition for the prevention or treatment of inflammatory bowel disease.

[0065] The pharmaceutical composition according to the present invention may additionally include a pharmaceutically acceptable carrier in addition to an active ingredient such as germinated oat extract.

[0066] The above carrier is one that is commonly used in the formulation of pharmaceutically acceptable ingredients and includes, but is not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above ingredients, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0067] In addition, suitable dosages of the pharmaceutical composition according to the present invention can be prescribed in various ways depending on factors such as the formulation method, mode of administration, patient's age, body weight, gender, pathological condition, food, time of administration, route of administration, excretion rate, and response sensitivity.

[0068] In addition, the pharmaceutical composition of the present invention may be administered orally or parenterally. When administered parenterally, it may be administered via intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, etc., and it is preferable that the route of administration be determined according to the type of disease to which it is applied.

[0069] In addition, the pharmaceutical composition according to the present invention may be prepared in a unit volume form or contained in a multi-volume container by formulation using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily carried out by a person skilled in the art to which the invention belongs. In this case, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or in the form of an extract, powder, granule, tablet, or capsule, and may additionally include a dispersant or a stabilizer.

[0070] In addition, the pharmaceutical composition according to the present invention may further include carriers and vehicles commonly used in the pharmaceutical field. Specifically, it may include, but is not limited to, ion exchange resins, alumina, aluminum stearate, lecithin, serum proteins (e.g., human serum albumin), buffering materials (e.g., various phosphates, glycine, sorbic acid, potassium sorbate, mixtures of partial glycerides of saturated vegetable fatty acids), water, salts or electrolytes (e.g., protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride and zinc salts), colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substrates, polyethylene glycol, sodium carboxymethylcellulose, polyarylates, waxes or wool paper.

[0071] In addition, the pharmaceutical composition according to the present invention may be in the form of granules, powders, coated tablets, tablets, capsules, suppositories, syrups, juices, suspensions, emulsions, drops, injections, or sustained-release formulations of active compounds, and may be administered in various oral or parenteral forms. When formulating, it may be prepared using diluents or excipients such as fillers, extenders, binders, humectants, disintegrants, and surfactants that are commonly used in the pharmaceutical field.

[0072] Another aspect of the present invention provides a health functional food composition for preventing or improving inflammatory bowel disease (IBD), comprising a sprouted oat (Avena sativa L.) extract.

[0073] In the present invention, the health functional food composition may contain 0.0001 to 100 weight% of germinated oat extract, but is not limited thereto.

[0074] Furthermore, since the extraction method of the sprouted oat extract and the types of inflammatory bowel disease described above are identical to those described in the pharmaceutical composition for the prevention or treatment of inflammatory bowel disease containing the sprouted oat extract as an active ingredient, specific descriptions are provided by reference to the above content, and only the specific composition of the health functional food will be described below.

[0075] In one embodiment of the present invention, the inventors have confirmed that the germinated oat extract improves intestinal permeability and tight junction (TJ) integrity caused by inflammatory bowel disease, so the extract can be very usefully used as an active ingredient in a health functional food composition for the prevention or improvement of inflammatory bowel disease.

[0076] In addition, according to one embodiment of the present invention, the germinated oat extract can promote the proliferation of beneficial bacteria in the intestines, and specifically, it has been confirmed that it improves the balance of intestinal microorganisms by promoting the proliferation of one or more lactic acid bacteria selected from the group consisting of Lactobacillus plantarum and Lactobacillus delbrueckii. Therefore, the germinated oat extract can be usefully used as an active ingredient in a health functional food composition for the prevention or improvement of inflammatory bowel disease.

[0077] The health functional food composition of the present invention can be manufactured by a method commonly used in the field of technology, and during such manufacturing, raw materials and ingredients commonly added in the field of technology can be added.

[0078] In addition, the health functional food of the present invention may be used by adding the extract as is or together with other foods or food ingredients, and may be used appropriately according to conventional methods.

[0079] The above-mentioned health functional food may be one or more formulations selected from the group consisting of health functional food preparations such as tablets, capsules, pills, granules, liquids, powders, flakes, pastes, syrups, gels, jellies, and bars, as well as beverages, gums, and candies, but is not limited thereto.

[0080] Another aspect of the present invention provides a food composition for preventing or improving inflammatory bowel disease (IBD) comprising a sprouted oat (Avena sativa L.) extract.

[0081] There are no specific restrictions on the types of the above foods. Examples of the above foods include drinks, meat, sausages, bread, biscuits, rice cakes, chocolates, candies, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, alcoholic beverages, and vitamin complexes, and include all health foods in the conventional sense.

[0082] The above composition may include food-grade acceptable food additives in addition to the active ingredient, and the amount of the active ingredient can be appropriately determined according to the purpose of use (prevention, health, or therapeutic treatment).

[0083] The content of the extract according to the present invention can be appropriately determined according to its purpose of use (for prevention or improvement). Generally, the amount of the extract in the health food can be added in an amount of 0.01 to 15% by weight of the total food weight. However, in the case of long-term consumption for the purpose of health and hygiene or for the purpose of health control, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount greater than the above range.

[0084] The health functional beverage composition of the present invention has no particular restrictions on other ingredients other than containing the extract as an essential ingredient in the indicated proportion, and may contain various flavoring agents or natural carbohydrates as additional ingredients, as in conventional beverages. Examples of the natural carbohydrates mentioned above include monosaccharides, e.g., glucose, fructose, etc.; disaccharides, e.g., maltose, sucrose, etc.; polysaccharides, e.g., dextrin, cyclodextrin, etc., and conventional sugars, and sugar alcohols such as xylitol, sorbitol, erythritol, etc. As flavoring agents other than those mentioned above, natural flavoring agents (taumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) may be advantageously used.

[0085] In addition to the above, the food of the present invention may contain various nutritional supplements, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. Furthermore, the extract of the present invention may contain fruit pulp for the production of natural fruit juices, fruit juice beverages, and vegetable beverages. These ingredients may be used independently or in combination. Although the proportion of these additives is not that critical, it is generally selected in the range of more than 0 to about 20 parts by weight per 100 parts by weight of the extract of the present invention.

[0086] Another aspect of the present invention provides a prebiotic composition for improving intestinal flora, comprising a sprouted oat (Avena sativa L.) extract as an active ingredient.

[0087] The term "improvement of gut microbiota" above means promoting the proliferation or growth of beneficial bacteria in the intestines and inhibiting the proliferation or growth of harmful bacteria in the intestines, while maintaining a balance between beneficial and harmful bacteria in the intestines.

[0088] The term "beneficial intestinal bacteria" above may collectively refer to microorganisms that inhabit the intestines and exert beneficial effects on the human body. For example, beneficial intestinal bacteria may include probiotics.

[0089] The term "intestinal harmful bacteria" above may collectively refer to microorganisms that inhabit the intestines and cause harmful effects to the human body, such as enteritis, and may include Escherichia coli, Clostridium sp., and Staphylococcus sp.

[0090] In the present invention, the term "probiotics" may refer to microorganisms that have a beneficial effect on health within the body, and may include species of the genera Lactobacillus sp., Lactococcus sp., Streptococcus sp., Enterococcus sp., Pediococcus sp., Bifidobacterium sp., etc., specifically Lactobacillus plantarum and Lactobacillus delbrueckii, and more specifically Lactobacillus plantarum subsp. Plantarum and Lactobacillus delbrueckii subsp. It may include one or more selected from the group consisting of Bulagaricus.

[0091] In the present invention, the term "prebiotics" may refer to a component that is utilized by microorganisms including beneficial bacteria to promote the growth or activity of microorganisms, thereby producing a beneficial effect on the health of the host.

[0092] Another aspect of the present invention provides a method for preventing, improving, or treating inflammatory bowel disease (IBD), comprising administering an effective amount of a composition containing sprouted oat extract to a subject.

[0093] Another aspect of the present invention provides a use of sprouted oat (Avena sativa L.) extract for the production of a drug, health functional food or food, or a prebiotic for improving the gut microbiome for the prevention, improvement, or treatment of inflammatory bowel disease (IBD).

[0094] To confirm the therapeutic effect of sprouted oat extract on inflammatory bowel disease, the present invention predicted target biomarkers and molecular mechanisms using network biology and chemoinformatics approaches, and verified the effects of the predicted biomarkers using a cellular model of intestinal inflammation.

[0095] The efficacy of sprouted oat extract was verified through in vitro studies, showing a significant improvement in epithelial electrical resistance (TEER) and a decrease in fluorescent isothiocyanate (FITC) permeability. Analysis of mRNA expression of IBD-related biomarkers in Caco-2 cells revealed a significant increase in mRNA levels of TJ proteins, including TJP1, TJP2, occludin, and claudin-1, compared to the inflammatory group. Furthermore, sprouted oat extract significantly reduced mRNA expression levels of inflammatory cytokines such as TNF-α, IL-6, and CXCL8. The underlying molecular mechanism of the sprouted oat extract was identified by combining COCONUT and chemical profiling analysis. Through these results, the efficacy of the food component was confirmed using big data-based network biology, and the sprouted oat extract of the present invention can be used for the prevention or treatment of IBD.

[0096] The present invention will be explained in more detail below through the following examples and experimental examples. However, the following examples and experimental examples are merely illustrative of the present invention, and the scope of the present invention is not limited thereto.

[0097] <Example 1> Preparation of Sprouted Oat Extract

[0098] Sprouted oat extract powder was obtained from Imagine the Next Green Revolution, Inc. (INGR, Yongin, Korea), and avenanthramide-rich sprouted oats were cultivated in smart farms using molecular switch technology.

[0099] Specifically, oat seeds (Avena sativa cv Daeyang) were germinated at 25°C for 2 days under a light-dark cycle of 16 hours:8 hours, and then treated with the inducers abscisic acid (ABA) and methyl jasmonate (MJ) at 22°C under the same light conditions for 3 days. Afterward, the germinated oats were harvested and washed, then hot-air dried at 40°C for 1 day, and the dried germinated oats were subjected to a first hot-water extraction at 120°C for 5 hours. After the first extraction, the residue was rehydrated with distilled water and subjected to a second extraction at 105°C for 3 hours. Both the first and second extracts were treated with 0.2% (v / v) 1,4-alpha-D-glucan glucanohydrolase (BAN 480 LS, Novozymes) at 75°C for 1 hour, then 0.2% (v / v) amylase (Termamyl 2X, Novozymes) was added and the reaction was carried out for an additional hour at 95°C. The enzymes were inactivated by heating at 105°C for 1 hour, then the mixture was filtered through a 1 μm filter and concentrated to a Brix level of 20 or higher at 65°C, and finally spray-dried using a spray dryer (SPRAY DRYER FS-2.0D, Fine ST Co.). The spray-dried sprouted oat flour was dissolved in a 30% ethanol solution, and the following in vitro experiments were conducted.

[0100] <Experimental Example 1> Profiling of Phenolic Compounds and Qualitative Analysis of Germinated Oats and Quantitative Evaluation of Avenanthramide

[0101] Analysis method

[0102] Combination of High Performance Liquid Chromatography and Q Exactive Orbitrap Mass Spectrometry (HPLC / Q Exactive Orbitrap MS)

[0103] Qualitative analysis of the germinated oat extract obtained in Example 1 above was performed using a Thermo Vanquish (Thermo Fisher, Waltham, Massachusetts, USA) instrument. A Waters Cortects T3 (2.1 mm × 150 mm, 1.6 μm particle size; water) column was used for chromatographic analysis.

[0104] Chromatographic analysis was performed using mobile phase A, which contained 0.1% formic acid in water, and mobile phase B, which contained 0.1% formic acid in acetonitrile, through gradient elution at a flow rate of 0.25 mL / min.

[0105] The mass spectrometry conditions are as follows:

[0106] Ion source type, heated electrospray ionization (H-ESI); cation mode voltage, 3.5 kV; anion mode voltage, 3 kV; cover gas flow rate, 50 (Arb); auxiliary gas flow rate, 10 (Arb); sweep gas flow rate, 1 (Arb); ion transfer tube temperature 320℃.

[0107] The mass spectrometer was operated in a scan range of 100–1500 m / z, with a full width at half maximum (FWHM) of 70,000 for MS1 and 17,500 FWHM for MS2. Data-dependent acquisition was performed in topN mode by selecting the top 10 precursor ions for fragmentation using stepwise normalized collision energy (NCE) settings of 10, 30, and 50. The above settings facilitate detailed analysis and identification of compounds based on isotope distribution patterns, ion fragmentation patterns, and accurate mass measurements by utilizing an advanced mass spectrum library and analysis software.

[0108] Analysis results

[0109] As a result of analyzing phenols, alkaloid chemicals, and avenanthramides in germinated oats using HPLC-MS / MS, the components of germinated oats were identified through qualitative analysis, and as a result, a total of 23 compounds were identified in spray-dried germinated oats (Fig. 1).

[0110] Table 1 shows details such as retention time, ionization mode, precursor, and generated ions, as well as chromatograms of the results of the quantitative analysis of major components of germinated oat compounds via HPLC-MS / MS. Additionally, Table 2 shows the results of the quantitative analysis of avenanthramide contained in spray-dried germinated oat extract, where each value represents the mean ± standard deviation.

[0111] No.R.T (Min) Analyte Name Molecular Formula Addduct Theoretical Mass Measured Mass 14.14Dianthoside C 12 H 16 O8[M+Na]+311.074288.125.21Gentisic acidC7H6O4[MH]-153.0193322199094715437.63Protocatechuic aldehydeC7H6O3[MH]-137.02441759990944138413.77Romucosine D.C. 21 H 23 NO5[M+Na]+392.1468435360905369.2514.03VanillinC8H8O3[MH]-151.04006766390944152615.03LyxoflavinC 17 H 20 N4O6[M+H]+377.1455608120905376.1715.11Avenanthramide 1cC 16 H 13 NO5[M+H]+300.08664896809057299.1815.174-COUMARATEC9H8O3[M+HCO2]-366.1194401799095164915.864-Feruloylquinic acidC17 H 20 O9[M-H]-367.10345576790945368.11017.516-Hydroxy-7-methoxycoumarinC 10 H8O4[M-H]-191.034982283909471921117.72FERULATEC 10 H 10 O4[M-H]-193.05063234790947194.11217.97Avenanthramide EC 17 H 15 NO5[M+H]+314.1022990320905313.11319.73Procyanidin B2C 30 H 26 O 12 [M+H]+579.1497027240905578.11421.92Avenanthramide CC 16 H 13 NO6[2M-H]-629.1412978199094629.1401523.36(S)-Annocherine AC 17 H 15 NO4[2M-H]-593.1929394679095593.1871623.51Avenanthramide AC 16 H 13 NO5[2M-H]-597.1514685799095597.1501723.98ZanthodiolineC 16 H 19 NO5[M+Na]+328.1155434080905328.1171824.18Avenanthramide BC 17 H 15 NO6[2M-H]-657.1725979479095657.1711924.8Avenanthramide 2C 18 H 17 NO7[M+H]+360.1077783360905359.12025.12NedocromilC 19 H 17 NO7[M-H]-370.09322543190945371.12125.94Avenanthramide 1sC 18H 17 NO6[M+H-H2O]+326.1022990320905343.12226.2Avenanthramide GC 16 H 13 NO5[M+H]+300.08664896809057299.12326.35Avenanthramide LC 18 H 15 NO5[M+H]+326.10229903209057325.12426.44Avenanthramide 2fd-1C 19 H 17 NO6[M+Na]+378.09480796409053355.12526.81Avenanthramide 2fd-2C 19 H 17 NO6[M+H]+356.11286371609054355.1

[0112] 화합물함량 (mg / kg)Avenanthramide A2648.21 ± 117.14Avenanthramide B4643.577 ± 81.53Avenanthramide C2752.36 ± 50.68

[0113] In negative mode, gentisic acid, protocatechuic aldehyde, vanillin, 4-coumarate, 4-feruloylquinic acid, 6-hydroxy-7-methoxycoumarin, ferulate, procyanidin B2, and nedocromil were detected, and in positive mode, dianthoside, romucosine D, lyxoflavin, avenanthramide 1c, and zanthodioline were identified. In addition, AVN A, AVN B, AVN C, AVN E, AVN G, AVN 2, AVN 1s, AVN L, AVN 2fd-1, AVN 2fd-2, and (S)-Annocherine A were detected in both negative and positive modes.

[0114] <Experimental Example 2> In silico: Gene-Phenotype Network Approach

[0115] Silico experiments were used to predict potentially important biomarkers that can be controlled by germinated oats.

[0116] 2-1. Comprehensive Analysis of Tightly Ziziped Genes Regulated by Germinated Oat Compounds

[0117] Based on the compound profiling of Experimental Example 1 above, the effect of sprouted oats on gut health function was analyzed.

[0118] Selection of test gene sets

[0119] Network analysis was performed using the COCONUT database and Quick Gene Ontology (GO) to accurately identify genes related to tightly fused and germinated oat plants. Compound-gene association data and gene-phenotype association data were collected using COCONUT data. COCONUT provides a standardized, structured, and integrated database of natural products, which is used to explore components, related efficacies, and target genes, and to investigate diseases potentially affected by genetic alterations caused by medicinal herbs.

[0120] Tight junction-related genes were collected using Gene Ontology annotations, and the genes were collected from 'cellular components' with a particular focus on genes related to tight junctions.

[0121] Of the 25 compounds in sprouted oats, 14 were identified as major compounds associated with IBD.

[0122] The two-layered network consisted of 404 nodes and 1,220 edges (Fig. 2a). The focus was on genes potentially regulated by compounds found in germinated oats, with a particular emphasis on identifying genes related to gut health. A total of 389 genes were mapped to the compound-gene-phenotype network scaffold (Fig. 2a). To identify specific functions related to gut barrier health, genes related to tight junctions in the Homo sapiens species were investigated. Genes related to gut permeability and tight junctions were selected for the Rapid GO Cell Components (CC) category, and among the genes related to tight junction functions, a total of 147 Homo sapiens genes were specifically identified and integrated into a gene set as shown in Fig. 2b.

[0123] 2-2. Confirmation of Interactions Between Target Genes Derived from Germinated Oats and Tightly Connected Genes

[0124] The focus was on identifying genes potentially regulated by compounds contained in sprouted oats, particularly those related to the maintenance and function of tight junctions, which are critical for gut health. To confirm the relationships between tight junction-related genes and compound-driving genes, two gene datasets were combined, and Protein-Protein Interaction (PPI) network analysis was performed on the datasets.

[0125] Analysis method

[0126] Using the above dataset, PPI analysis was performed to identify potential biomarkers associated with both compound-related genes and tight-joint genes. For the PPI analysis, compound-driven phenotype-related genes and tight-joint-related genes were combined, and the PPI analysis was performed using the combined genes. The STRING database (version 12.0) was used for protein-protein network analysis. All genes were mapped to Homo sapiens genes, and a Cystoscope (version 3.10.0) was used for visualization.

[0127] Analysis results

[0128] Fifteen genes were identified in the order of JP1, CTNNB1, OCLN, AKT1, IL6, ACTB, TNF, BCL2, TJP3, TJP2, CXCL8, CCND1, CLDN1, CLDN3, and ALB (Fig. 2c).

[0129] PPI analysis classified the genes shown in Table 3 according to their functions, and as a result, six functions were identified for the classified genes: tight junctions, cell signaling, cell cycle, inflammatory response, apoptosis, and cell structure and function.

[0130] No Gene symbol Annotation Protein node degree DB Source Function 1TJP1Tight junction protein 1, ZO-162Quick GOTight junction2CTNNB1Catenin beta-151Quick GOCell signaling3OCLNOccludin51Quick GOTight junction4AKT1RAC-alpha serine / threonine-protein kinase43COCONUTCell signaling5ACTBActin, cytoplasmic 138Quick GOCell structure and function6TJP3Tight junction protein 333Quick GOTight junction7TJP2Tight junction protein 230Quick GOTight junction8CLDN1Claudin-127Quick GOTight junction9CLDN3Claudin-327Quick GOTight junction10CCND1G1 / S-specific cyclin-D127COCONUTCell cycle11IL6Interleukin-641COCONUTInflammatory12TNFTumor necrosis factor37COCONUTInflammatory13BCL2Apoptosis regulator Bcl-233COCONUTCell apoptosis14CXCL8C-XC motif chemokine ligand 829COCONUTInflammatory15ALBSerum albumin26COCONUTOther

[0131] 2-3. Prediction of Biological Pathways for Inflammatory Response and Barrier Integrity Genes in IBD through Gene Ontology Analysis

[0132] Analysis method

[0133] To identify the mechanism by which sprouted oat extract promotes gut health in Caco-2 cells, biological pathways were analyzed using DAVID (https: / / david.ncifcrf.gov / tools.jsp). The top 15 genes among the PPI analyzed genes were utilized for pathway prediction analysis, which was expressed according to gene ontology biological processes. To identify biological pathways associated with inflammatory bowel disease, only inflammatory bowel disease-related pathways were selected. To select inflammatory bowel disease-related biological pathways, keywords associated with inflammatory bowel disease were collected through the Human Phenotype Ontology (https: / / hpo.jax.org / app / , (Gargano et al., 2023)).

[0134] Biological pathways associated with the aforementioned terms were identified through a phenotypic keyword analysis using Quick GO. Subsequently, using the Human Phenotype Ontology (HPO) keyword as a reference, a sequential analysis was performed focusing on the top 15 genes and biological pathway lists derived from Quick GO. Only biological pathways showing overlap between the analyses were included in the results, and Tableau (version 2022.2.0) was used for visualization. All pathway results show p < 0.05.

[0135] Analysis results

[0136] Through gene ontology enrichment analysis related to IBD, important biological processes including inflammatory response, intracellular signaling, positive regulation of gene expression, negative regulation of the apoptosis process, and positive and negative regulation of cell proliferation were identified. Processes such as "inflammatory response," "cell-cell junction tissue," "positive regulation of gene expression," "positive regulation of wound healing," "maintenance of blood-brain barrier permeability," and "establishment of the endothelial-intestinal barrier" appeared as some of the ranked processes (Fig. 2d).

[0137] The associations between the genes identified through PPI analysis and specific biological processes, along with detailed statistical significance values, are shown in Table 4. All biological processes listed in Table 4 have statistical significance with a p-value of less than 0.05.

[0138] 분류기능 용어전체 목록수해당 항목수유전자폴드 증가율Fold EnrichmentP-값FDRGO_BPmaintenance of permeability of blood-brain barrier158IL6, TJP2, OCLN, CLDN3, TJP3, TJP1, ACTB, CLDN1314.39191926.95198E-175.02E-14GO_BPpositive regulation of gene expression157IL6, CTNNB1, OCLN, TNF, CLDN3, CXCL8, AKT117.491457938.77087E-070.000114GO_BPnegative regulation of apoptotic process157IL6, BLC2, ALB, CTNNB1, TNF, AKT1, TJP116.811234571.10564E-060.000114GO_BPnegative regulation of gene expression156CTNNB1, OCLN, TNF, CLDN3, CXCL8, AKT124.392476492.03778E-060.000168GO_BPcell-cell junction organization155TJP2, OCLN, TJP3, TJP1, CLDN1259.37333332.1046E-097.14E-07GO_BPpositive regulation of cell proliferation155IL6, BLC2, AKT1, TJP1, ACTB11.897859330.0004876290.020553GO_BPpositive regulation of transcription, DNA-templated155IL6, CTNNB1, TNF, AKT1, ACTB8.9070512820.0014421740.04165GO_BPpositive regulation of maintenance of permeability of blood-brain barrier154TJP2, OCLN, TJP3, TJP11037.4933332.96477E-097.14E-07GO_BPestablishment of endothelial intestinal barrier154TJP2, TJP3, TJP1, CLDN1432.28888896.50316E-081.17E-05GO_BPpositive regulation of peptidyl-serine phosphorylation154IL6, BLC2, TNF, AKT155.779211473.7062E-050.00223GO_BPpositive regulation of sequence-specific DNA binding transcription factor activity154IL6, CTNNB1, TNF, AKT143.961581927.54276E-050.004189GO_BPpositive regulation of protein phosphorylation154CCND1, TNF, CLDN3, AKT122.953392330.0005123920.020553GO_BPpositive regulation of apoptotic process154IL6, BLC2, CTNNB1, TNF15.438888890.0016138320.044815GO_BPinflammatory response154IL6, TNF, CXCL8, AKT111.897859330.0033859820.08731GO_BPnegative regulation of cell proliferation154IL6, CTNNB1, CLDN3, CXCL811.228282830.0039856990.098108GO_BPpositive regulation of wound healing153OCLN, CLDN3, CLDN1149.63846150.0001547810.007982GO_BPnegative regulation of apoptotic signaling pathway153BLC2, CTNNB1, TNF114.42941180.0002662980.012818GO_BPnegative regulation of extrinsic apoptotic signaling pathway in absence of ligand153BLC2, TNF, AKT1105.15135140.000315750.014248GO_BPpositive regulation of smooth muscle cell proliferation153IL6, TNF, AKT167.079310340.0007769480.028048GO_BPpositive regulation of MAPK cascade153IL6, CTNNB1, TNF22.619767440.0065959580.12211GO_BPpositive regulation of cell migration153CLDN3, TJP1, CLDN114.571535580.0153188280.22258GO_BPregulation of membrane permeability152TJP2, CLDN3432.28888890.0043108910.098108GO_BPprotein localization to adherens junction152TJP1, ACTB432.28888890.0043108910.098108GO_BPregulation of transmembrane transporter activity152BLC2, ACTB370.53333330.0050276930.098108GO_BPpositive regulation of bicellular tight junction assembly152CLDN3, CLDN1370.53333330.0050276930.098108GO_BPinflammatory response to wounding152IL6, TNF370.53333330.0050276930.098108GO_BPpositive regulation of I-kappaB phosphorylation152TNF, AKT1370.53333330.0050276930.098108GO_BPnegative regulation of lipid storage152IL6, TNF288.19259260.006459860.12211GO_BPpositive regulation of leukocyte adhesion to vascular endothelial cell152IL6, TNF185.26666670.0100319060.172453GO_BPpositive regulation of cyclin-dependent protein serine / threonine kinase activity152CCND1, AKT1129.68666670.0143026190.22258GO_BPpositive regulation of protein localization to cell surface152TNF, AKT1123.51111110.0150127390.22258GO_BPpositive regulation of glial cell proliferation152IL6, TNF117.89696970.0157223840.22258GO_BPregulation of myelination152CTNNB1, AKT1117.89696970.0157223840.22258GO_BPnegative regulation of neurogenesis152IL6, TNF108.07222220.017140250.237986GO_BPpositive regulation of nitric-oxide synthase activity152TNF, AKT1103.74933330.0178484720.238641GO_BPnegative regulation of intrinsic apoptotic signaling pathway152BLC2, AKT192.633333330.0199702940.262156GO_BPpositive regulation of cytokine production involved in inflammatory response152IL6, TNF81.054166670.022792770.293864GO_BPregulation of angiogenesis152IL6, CTNNB170.10090090.026310250.321966GO_BPpositive regulation of glucose import152OCLN, AKT170.10090090.026310250.321966GO_BPextrinsic apoptotic signaling pathway via death domain receptors152BLC2, TNF64.843333330.0284150910.336323GO_BPT cell differentiation in thymus152BLC2, CTNNB164.843333330.0284150910.336323GO_BPbranching involved in ureteric bud morphogenesis152BLC2, CTNNB158.948484850.0312149740.348548GO_BPpositive regulation of chemokine production152IL6, TNF57.638518520.0319137730.348548GO_BPregulation of insulin secretion152IL6, TNF56.385507250.0326121040.348548GO_BPpositive regulation of nitric oxide biosynthetic process152TNF, AKT155.18581560.0333099670.348548GO_BPpositive regulation of JAK-STAT cascade152IL6, TNF55.18581560.0333099670.348548GO_BPpositive regulation of G1 / S transition of mitotic cell cycle152CCND1, AKT150.857516340.0360967480.357012GO_BPintrinsic apoptotic signaling pathway in response to DNA damage152BLC2, TNF50.857516340.0360967480.357012GO_BPregulation of cyclin-dependent protein serine / threonine kinase activity152CCND1, ACTB48.032098770.0381819350.366274GO_BPregulation of G1 / S transition of mitotic cell cycle152CCND1, ACTB48.032098770.0381819350.366274GO_BPpositive regulation of epithelial to mesenchymal transition152IL6, CTNNB147.158787880.0388760670.366274GO_BPpositive regulation of protein localization to plasma membrane152TNF, AKT146.316666670.0395697330.366274GO_BPnegative regulation of cysteine-type endopeptidase activity involved in apoptotic process152TNF, AKT146.316666670.0395697330.366274GO_BPpositive regulation of cell differentiation152CTNNB1, ACTB43.961581920.0416479430.380631GO_BPnegative regulation of fat cell differentiation152IL6, TNF42.520218580.0430310960.388356GO_BPpositive regulation of neuron apoptotic process152CTNNB1, TNF41.83440860.0437219770.389719GO_BPnegative regulation of autophagy152BLC2, AKT141.170370370.0444123940.391046GO_BPpositive regulation of interleukin-8 production152IL6, TNF39.903589740.0457918420.393592GO_BPpositive regulation of interleukin-1 beta production152IL6, TNF38.712437810.047169440.400663GO_BPpositive regulation of tyrosine phosphorylation of STAT protein152IL6, TNF38.143137250.0478575460.401781GO_BPG1 / S transition of mitotic cell cycle152CCND1, BLC237.590338160.0485451910.402869.

[0139] <Experimental Example 3> In vitro experiment

[0140] In vitro experiments were conducted to confirm that the genes predicted by the above silico analysis are regulated by sprouted oat treatment, thereby improving gut health.

[0141] cell culture

[0142] Caco-2 cells (passage 33-37) were purchased from ATCC (Manassas, Virginia, USA). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Biowest, Nuaille, Cholet, France) supplemented with 10% fetal bovine serum (FBS) (Gibco BRL, NY), 1% penicillin-streptomycin (P / S; Corning Inc., NY), and 1% non-essential amino acids (NEAAs; Gibco, Rockville, Maryland, USA) at 37°C in a 5% CO₂ incubator. For the experiment, cells were cultured at 3.75 × 10⁶ in 0.4 μm pore size polyester 6-well Transwell Costar® (Kennebunk, ME, USA). 5 Cells were cultured for differentiation for 21 days at a density of cells / well. The culture medium was replaced every 2 to 3 days, and experimental concentrations with no cytotoxic effect were determined and applied to the study, based on the results obtained from CCK8 analysis.

[0143] Cell viability

[0144] Cell viability was evaluated using the Cell Counting Kit-8 (CCK8) assay (Sigma-Aldrich), a cell proliferation assay based on the cleavage of 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-8).

[0145] Specifically, 1 × 10 cells per well 5 Cells were seeded into 24-well plates at cell densities and cultured for 21 days to induce differentiation; after 21 days, the cultured cells were replaced with serum-free medium. Cells were treated with various concentrations of GOE (1, 2.5, 5, 10, 25, 50, and 100 μg / mL) for 24 hours. After 24 hours of culture, 100 μL of CCK-8 was added to the cells, and they were cultured for 4 hours in a 5% CO2 and 37°C incubator.

[0146] 3-1. Assessment of Intestinal Health Status Using TEER and FITC-Dextran

[0147] The present invention focused primarily on the integrity of tight junctions, which are important in the pathophysiology of inflammatory bowel disease (IBD). An in vitro model reflecting intestinal inflammation observed in IBD was established using an inflammatory cytokine cocktail, and the integrity and permeability of the epithelial barrier were evaluated using epithelial electrical resistance (TEER) and FITC-dextran permeability measurements.

[0148] Trans-epithelial electrical resistance (TEER)

[0149] A combination therapy approach was used to investigate the effects of sprouted oat extract on intestinal barrier integrity under inflammatory conditions. Initially, a cytokine cocktail consisting of 25 ng / mL interleukin (IL)-1β, 10 ng / mL lipopolysaccharide (LPS), 50 ng / mL tumor necrosis factor (TNF)-α, and 50 ng / mL interferon (IFN)-γ was administered to the basolateral side of a transwell to simulate an intestinal inflammatory environment. Simultaneously, sprouted oat extract at concentrations of 2.5 and 25 μg / mL and LPS (10 μg / mL) were introduced into serum-free medium on the apical side. After the simultaneous administration of the sprouted oat extract and cytokine cocktail, TEER values ​​were measured at 0, 3, 6, 12, and 24-hour intervals using the Millicell® ERS (Millipore) system.

[0150] Fluorescein isothiocyanate (FITC)-dextran flux

[0151] High intensity values ​​indicate high transmittance. FITC-dextran solution (100 μg / mL) and germinated oat extract were added to the apical side, and after 24 hours of incubation, the fluorescence was collected towards the basolateral side of the transwell. The collected fluorescence solution was measured at 485 nm and 535 nm (excitation and emission wavelengths, respectively) using a SpectraMax i3x Multi-Mode Microplate Reader.

[0152] Analysis results

[0153] To determine the effects on the integrity and permeability of Caco-2 epithelial cells, epithelial electrical resistance (TEER) and FITC-dextran permeability were measured, and the TEER values ​​in the cocktail treatment group were found to be significantly lower than those in the control group.

[0154] Samples were treated at 25 or 2.5 μl / mL, and a significant effect of the sprouted oat extract treatment group was observed 12 hours after treatment (p < 0.05). In the sprouted oat extract treatment group, TEER values ​​improved in a concentration-dependent manner compared to the cocktail group (Figs. 3a, 3b). In the group administered the cocktail, FITC-dextran flux increased approximately 2.5-fold compared to the control group, and FITC flux was significantly reduced due to sprouted oat treatment (Fig. 3c).

[0155] When treated with sprouted oat extract, barrier function was significantly improved, which was demonstrated by an improvement in TEER values ​​and a decrease in FITC-dextran flux. These results imply that the biologically active components within the sprouted oat extract can exert a protective effect against cytokine-induced barrier disruption, thereby confirming a potential role in regulating intestinal permeability in the context of IBD.

[0156] 3-2. PCR Verification of Gene Regulation Inducing Germination in Oats

[0157] Subsequently, the potential genetic causes of these effects were analyzed, and as a result of predicting genes related to intestinal integrity that may be affected by sprouted oat compounds, 15 candidate genes were identified that are thought to mediate the protective effects of oats on intestinal integrity. To validate the predicted genes, PCR was used in a Caco-2 cell monolayer model focused on various biomarkers.

[0158] RNA isolation and real-time reverse transcription polymerase chain reaction (qRTPCR)

[0159] Total RNA was isolated from Caco-2 cells after treating samples with TRIzol® reagent (Life Technologies, Rockville, Maryland, USA) for 24 hours according to the manufacturer's instructions. After treating the cells with TRIzol and chloroform, the resulting mixture was centrifuged at 12,000 rpm at 4°C for 20 minutes. The supernatant was carefully transferred, and the mixture with an equal amount of isopropanol was centrifuged again under the same conditions to precipitate the mRNA. A NanoDrop™ Lite Spectrophotometer was used to evaluate the amount of total RNA. cDNA was synthesized from total RNA using the Transcriptor First Strand cDNA Synthesis Kit (Roche).

[0160] The top 14 genes identified through protein-protein interaction (PPI) analysis were validated using qRTPCR. mRNA expression levels were relative 2 using normalization with respect to the expression of the housekeeping gene GAPDH. -ΔΔCT It was quantified using a method.

[0161] Analysis results

[0162] Among the 15 genes analyzed by PPI, TJP1, TJP2, TJP3, OCLN, CLDN1, and CLDN3 are associated with tight junction function. TJP1, TJP2, and OCLN, tight junction proteins important for maintaining epithelial barrier integrity, were significantly reduced under cocktail conditions compared to the control group. TJP1, TJP2, and OCLN are important for maintaining epithelial barrier integrity and were significantly upregulated when treated with sprouted oat extract compared to the cocktail group (p = 0.0027, p = 0.0025, p < 0.0001).

[0163] On the other hand, the expression of TJP3 and CLND3, members of the tight junction protein family, did not change significantly in the group treated with sprouted oat extract but showed an increasing trend (Fig. 4a). TNF-α, IL-6, and CXCL8 are inflammatory cytokines upregulated in IBD; the mRNA expression of TNF-α, CXCL-8, and IL-6 increased significantly by about fourfold in the cocktail group, whereas it decreased significantly with sprouted oat extract (p < 0.0001) (Fig. 4b). In addition, CTNNB1 decreased significantly in response to sprouted oat treatment (p < 0.0001), and AKT1 showed a similar trend although it did not show statistical significance; both were regulated by sprouted oat extract treatment (Fig. 4c). In the case of CCND1, different responses were observed depending on the concentration of the extract.

[0164] In the present invention, two concentrations of germinated oat extract, 2.5 μg / mL and 25 μg / mL, were used. The results showed that CCND1 expression decreased at a concentration of 2.5 μg / mL, whereas CCND1 expression increased at a higher concentration of 25 μg / mL (Fig. 4d), indicating a significant increase in the expression of tight junction proteins and a significant decrease in inflammatory cytokine levels. These results confirmed that germinated oat extract has a dose-dependent effect in improving gut health.

[0165] ACTB, a β-actin gene related to cell structure and function, was significantly decreased in the group administered the inflammatory cocktail but significantly increased in the group administered the sprouted oat extract (Fig. 4e). On the other hand, cyclin D (CCND1), which is involved in cell cycle regulation, and BCL-2, which is related to apoptosis, both significantly increased in the cocktail administration group. Bcl-2 expression was significantly decreased in the group administered the sprouted oat extract (Fig. 4f). 14 out of 15 genes were verified (Fig. 4). Through these results, it was confirmed that the sprouted oat extract of the present invention contributes to maintaining barrier integrity by regulating the ACTB gene.

[0166] 3-3. Correlation Analysis of Avenanthramide-Rich Germinated Oat Compounds and IBD-Related Markers at Various Treatment Concentrations

[0167] Figure 5 shows the strength of correlations between the analyzed factors, with red, blue, and white representing positive, negative, and no correlation, respectively. Characteristically, avenanthramide compounds in sprouted oats exhibited a stronger correlation in the negative mode than in the positive mode. While TEER values ​​generally showed a positive correlation with avenanthramide compounds, FITC-dextran permeability test values ​​showed a significant negative correlation with avenanthramide compounds. Among tightly junction genes, TJP1 and CLDN3 showed the strongest positive correlation with avenanthramide compounds, whereas TJP2 and TJP3 showed positive correlations with other phenolic compounds, such as 4-coumarate and nedocromil, rather than avenanthramide. In the context of inflammation-related genes, TNF-α and IL-6 showed significant negative correlations with avenanthramide compounds and phenolic compounds including protocatechusan aldehyde and romucosin D. The effects of CXCL8 showed a negative correlation with non-avenanthramide phenolic compounds such as gentisic acid and 4-coumarate. Cell signaling genes such as akt-1 and CTNNB1 generally showed a negative correlation with avenanthramide compounds, whereas CCND1 showed a positive correlation with non-avenanthramide alkaloids and phenolic compounds in a negative mode. The actin cytoskeleton gene ACTB showed a positive correlation mainly with avenanthramide compounds, while the BCL-2 gene generally showed a significant negative correlation with avenanthramide compounds. These results confirmed specific interactions between sprouted oat metabolites and key biological markers, implying potential therapeutic effects for inflammatory bowel disease.

[0168] <Experimental Example 4> Effect of Germinated Oat Extract on Promoting Lactic Acid Bacteria Growth

[0169] Intestinal lactic acid bacteria are known to improve the balance of intestinal microorganisms by producing organic acids to lower intestinal pH and inhibit the growth of harmful bacteria. Accordingly, in this invention, changes in the proliferation of lactic acid bacteria following treatment with germinated oat extract were measured to confirm the effect of germinated oat extract on the growth of beneficial intestinal bacteria. As a result, it was confirmed that the germinated oat extract of this invention can contribute to the improvement of the intestinal environment by promoting the growth of lactic acid bacteria.

[0170] badge

[0171] MRSA+Sprouted Oat Extract (0%, 1%, 5%)

[0172] MRS broth agar (MRSA)

[0173] : 50 plates of live medium : MB-P1040-P50

[0174] Sprouted oat extract (0%, 1%, 5%)

[0175] Test method - Standard plate method

[0176] ① Dilute 10–25 g (mL) of the sample with the diluent at a ratio of 1:9 (dilute as needed)

[0177] ② Dispense 1 ml of diluted solution onto 2 Petri dishes (KS-P0101).

[0178] ③ Dispense the prepared MRSA medium cooled to 43–45°C, mix well, and allow to solidify (if the medium solidifies, add another 3–5 ml to overlap).

[0179] ④ Incubate at 30±1℃ for 24 hours

[0180] Specifically, the lactic acid bacteria Lactobacillus plantarum subsp. Plantarum and Lactobacillus delbrueckii subsp. Bulagaricus were placed in MRS medium supplemented with the sprouted oat extract prepared in Example 1 at concentrations of 2% and 5%, 10 9CFU / mL 1% of probiotics in 10 7 After inoculation at a concentration of CFU / mL, the samples were cultured under aerobic conditions in a 30℃ incubator for 24 hours, and the change in the total number of cultured lactic acid bacteria was measured. The number of lactic acid bacteria was measured using the standard plate method according to General Test Method 8 - 4.5.1 (Measurement of Total Bacterial Count) of the Food Code. 10–25 g (mL) of the sample was diluted with the diluent at a ratio of 1:9, 1 mL was dispensed onto two Petri dishes (KS-P0101), and the number of lactic acid bacteria (CFU / mL) was calculated. As a control, MRS medium (0% germinated oat extract) was used, and the number of lactic acid bacteria was calculated using the same method. The results are shown in Table 5 and Figure 6 below.

[0181] Sprouted oat extract (w / v %) Strain Lactobacillus plantarum subsp.Plantarum(log 10 CFU / mL)Lactobacillus delbrueckiisubsp.Bulgaricus(log 10 CFU / mL)09.10±0.01 A 9.32±0.11 A 29.25±0.05 B 9.39±0.10 B 59.32±0.01 C 9.42±0.10 C

[0182] Data are expressed as mean ± standard deviation (n = 2). Values ​​with different capitalization within the same column showed significant differences according to Tukey's range test (P < 0.05). CFU, colony-forming unit.

[0183] As a result, it was found that both Lactobacillus plantarum and Lactobacillus delbrueckis lactic acid bacteria were significantly increased in the germinated oat extract treatment group (2%, 5%) of the present invention compared to the control group (0%).

[0184] Statistical analysis

[0185] Data were expressed as mean ± standard error (SE). Statistical evaluation was performed using one-way analysis of variance (one-way ANOVA) with SAS 9.4 (SAS Institute, Cary, North Carolina, USA). For post-hoc analysis, Duncan's multiple range test was applied to both TEER and FITC-dextran measurements and PCR data analysis. Statistical significance for comparisons with a P-value less than 0.05 was indicated by a different letter for each group.

[0186] In conclusion, experimental verification of the sprouted oat extract demonstrated positive regulation of permeability and effective regulation of 9 out of 14 tested genes (TJP1, TJP2, OCLN, CLDN1, TNF, IL-6, CXCL8, CTNNB1, CCND1). In particular, sprouted oats effectively regulated genes related to tight junctions and inflammation. Furthermore, the sprouted oat extract was shown to improve the balance of the gut microbiome by promoting the proliferation of beneficial gut bacteria (especially lactic acid bacteria). Therefore, the sprouted oat extract of the present invention can be effectively used for the prevention, improvement, or treatment of inflammatory bowel disease by maintaining the integrity of tight junctions between intestinal epithelial cells, regulating intestinal permeability and inflammatory responses, and simultaneously promoting the growth of beneficial gut bacteria.

[0187] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical spirit or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

A pharmaceutical composition for the prevention or treatment of inflammatory bowel disease (IBD) comprising sprouted oat (Avena sativa L.) extract and a pharmaceutically acceptable carrier. A pharmaceutical composition according to claim 1, wherein the inflammatory bowel disease is one or more selected from the group consisting of ulcerative colitis (UC), Crohn's disease (CD), irritable bowel syndrome, intestinal Behcet's disease, indeterminate colitis, infectious enteritis, bacterial enteritis, viral enteritis, amoebic enteritis, hemorrhagic rectal ulcer, ischemic bowel disease, radiation enteritis, tuberculous enteritis, and leaky gut syndrome. A pharmaceutical composition according to claim 1, wherein the germinated oats are obtained by germinating oat seeds for 1 to 3 days with a 2:1 light-dark cycle and then treating with an inducer for 2 to 5 days with a 2:1 light-dark cycle. A pharmaceutical composition according to paragraph 3, wherein the inducer is abscisic acid, methyl jasmonate, or a combination thereof. In claim 1, the germinated oat extract is 1) a step of extracting by adding an extraction solvent to germinated oats; and 2) Prepared by a method comprising the step of filtering, concentrating, or drying the extract of step 1), and Here, the extraction solvent of step 1) above is one or more solvents selected from the group consisting of water and organic solvents, and A pharmaceutical composition in which the drying of step 2) above is by spray drying. A pharmaceutical composition according to claim 5, wherein the organic solvent is one or more selected from the group consisting of alcohols having 1 to 5 carbon atoms, ethyl acetate, acetone, ether, chloroform, benzene, hexane, and dichloromethane. A pharmaceutical composition according to claim 1, wherein the germinated oat extract improves intestinal permeability and tight junction (TJ) integrity resulting from inflammatory bowel disease. A pharmaceutical composition according to claim 1, characterized in that the germinated oat extract promotes the proliferation of beneficial bacteria in the intestines. A pharmaceutical composition according to claim 8, characterized in that the beneficial bacteria are one or more lactic acid bacteria selected from the group consisting of Lactobacillus plantarum and Lactobacillus delbrueckii. A health functional food composition for the prevention or improvement of inflammatory bowel disease (IBD) comprising sprouted oat (Avena sativa L.) extract. A health functional food composition according to claim 10, wherein the inflammatory bowel disease is one or more selected from the group consisting of ulcerative colitis (UC), Crohn's disease (CD), irritable bowel syndrome, intestinal Behcet's disease, indeterminate colitis, infectious enteritis, bacterial enteritis, viral enteritis, amoebic enteritis, hemorrhagic rectal ulcer, ischemic bowel disease, radiation enteritis, tuberculous enteritis, and leaky gut syndrome. A health functional food composition according to claim 10, wherein the germinated oat extract improves intestinal permeability and tight junction (TJ) integrity resulting from inflammatory bowel disease. A health functional food composition according to claim 10, wherein the health functional food is one or more formulations selected from the group consisting of health functional food preparations such as tablets, capsules, pills, granules, liquids, powders, flakes, pastes, syrups, gels, jellies, and bars, beverages, gums, and candies. A food composition for preventing or improving inflammatory bowel disease (IBD) comprising sprouted oat (Avena sativa L.) extract. A food composition according to claim 14, wherein the inflammatory bowel disease is one or more selected from the group consisting of ulcerative colitis (UC), Crohn's disease (CD), irritable bowel syndrome, intestinal Behcet's disease, indeterminate colitis, infectious enteritis, bacterial enteritis, viral enteritis, amoebic enteritis, hemorrhagic rectal ulcer, ischemic bowel disease, radiation enteritis, tuberculous enteritis, and leaky gut syndrome. A food composition according to claim 14, wherein the food is one or more selected from the group consisting of various drinks, meat, sausage, bread, biscuits, rice cakes, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, soup, beverages, alcoholic beverages, and vitamin complexes. A prebiotic composition for improving intestinal flora containing sprouted oat (Avena sativa L.) extract as an active ingredient. A method for the prevention, improvement, or treatment of inflammatory bowel disease (IBD), comprising administering an effective amount of a composition according to any one of claims 1 to 17 to a subject. In claim 18, the inflammatory bowel disease is one or more selected from the group consisting of ulcerative colitis (UC), Crohn's disease (CD), irritable bowel syndrome, intestinal Behcet's disease, indeterminate colitis, infectious enteritis, bacterial enteritis, viral enteritis, amoebic enteritis, hemorrhagic rectal ulcer, ischemic bowel disease, radiation enteritis, tuberculous enteritis, and leaky gut syndrome. Use of sprouted oat (Avena sativa L.) extract for the manufacture of pharmaceuticals, health functional foods or foods for the prevention, improvement, or treatment of inflammatory bowel disease (IBD), or prebiotics for improving gut microbiota.

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