KR20230142934A - Composition for improving intestinal function comprising an extract of Corni fructus as an active ingredient - Google Patents

Abstract

The present invention confirms the protective effect of Cornus officinalis water extract against lipopolysaccharide (LPS)-induced intestinal epithelial cell toxicity and intestinal permeability and intestinal inflammation in mice with dextran sulfate sodium (DSS)-induced colitis. Regarding the improving effect of the combination (synbiotics) of Cornus officinalis water extract and Lactobacillus reuteri ( Limosilactobacillus reuteri ), the Cornus officinalis water extract and Lactobacillus reuteri of the present invention and their combination are believed to alleviate colitis symptoms by improving intestinal barrier damage and intestinal inflammation caused by DSS-induced colitis, and controlling short-chain fatty acid content in feces and imbalance of intestinal flora. Therefore, Cornus officinalis water extract, Limosilactobacillus reuteri , and their combination have the potential to be used as health functional food ingredients for intestinal damage caused by colitis.

Description

Composition for improving intestinal function comprising an extract of Corni fructus as an active ingredient}

The present invention relates to a composition for improving intestinal function containing Corni fructus extract as an active ingredient, which can be used in health functional foods or pharmaceutical compositions.

Inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn's disease (CD), is a chronic disease that causes inflammation in the intestines, causing abdominal pain, bleeding in stool, loose stools, and weight loss. It is characterized by the following, and the incidence is increasing worldwide. The exact pathogenesis of IBD is not yet known, but related studies have reported that it is related to abnormal mucosal immune response, destruction of the intestinal barrier, and dysbiosis of the intestinal flora.

The intestinal barrier serves to protect the intestines through physical and chemical defenses. However, when the intestinal mucosal barrier is damaged due to various causes, intestinal damage occurs due to the invasion of pathogens and toxins. Tight junctions affect the permeability of substances by regulating the gap between intestinal cells. When tight junctions become loose due to stimulation or damage in the intestine, intestinal inflammation intensifies due to overproduction of harmful pro-oxidation markers and pro-inflammatory mediators. As a result, when a large amount of toxins and antigens penetrate the intestinal epithelium and enter the bloodstream, the immune system overreacts and the secretion of pro-inflammatory cytokines increases.

The NF-κB activation pathway, a representative pathway of the inflammatory response, plays an important role in the inflammatory response by regulating genes that regulate pro-inflammatory cytokines, chemokines, growth factors, or inducible enzymes such as COX-2 and iNOS. Therefore, by inhibiting NF-κB activation, intestinal damage can be improved by suppressing the expression of inflammatory response mediators.

Excessive inflammatory response appears accompanied by an imbalance of intestinal microorganisms. According to previous studies, compared to healthy people, patients with inflammatory bowel disease are known to have a decreased proportion of lactic acid bacteria such as Lactobacillus , Lactococcus , and Bifidobacterium , and an increase in harmful bacteria. . In addition, short-chain fatty acids produced by intestinal microorganisms fermenting and decomposing fiber supplied to the intestines play a role in forming an intestinal flora environment beneficial to health by reducing the intestinal pH, suppressing the growth of harmful bacteria and inhibiting the activity of harmful enzymes. Due to this effect, short-chain fatty acids are known to lower the incidence of irritable bowel syndrome and inflammatory bowel disease, and reduce the risk of degenerative diseases, cardiovascular diseases, and obesity.

Currently, the most commonly used treatment methods for IBD include pharmacological treatment and surgery to remove the affected part of the gastrointestinal tract, but instead of medical treatment with potential side effects, dietary therapy for prevention and correction is emerging. As research results have reported that functional ingredients present in food can help proliferate beneficial intestinal bacteria and prevent IBD, prebiotics, probiotics, and synbiotics are being used in academia, industry, and consumers. is receiving attention. However, research on this is limited to fiber components, and research on foods that improve intestinal health from natural products containing bioactive substances is lacking.

Beneficial intestinal bacteria such as Lactobacillus are known to suppress inflammatory responses by reducing the NF-κB pathway and the production of inflammatory cytokines that occur through various signaling pathways. In addition, it is reported to protect the intestines by increasing the content of short-chain fatty acids and strengthening the intestinal epithelial barrier function, controlling the penetration of harmful bacteria and toxins. Among them, Lactobacillus reuteri ( Limosilactobacillus reuteri; L. reuteri ) is a type of anaerobic lactic acid bacteria present in the gastrointestinal tract and is known to inhibit the growth of intestinal pathogens by producing organic acids and reuterin, known as an antibacterial compound. It has also been reported to attenuate diseases such as obesity, neurodevelopmental disorders, stress, and intestinal inflammation. Levilactobacillus brevis ( L. brevis ) is a Gram-positive lactic acid bacterium known to produce GABA and is found in the human intestine, vagina, and feces. L. brevis is known to alleviate colitis by controlling inflammation in mice and to have anti-obesity and anti-diabetic effects.

Meanwhile, Cornus officinalis fruit ( Corni fructus ) is the dried mature fruit of Cornus officinalis, a deciduous broad-leaved tree belonging to the Cornaceae family, and is used as a medicinal material. Cornus officinalis contains a large amount of dietary fiber, vitamins, and organic acids, and is reported to be effective in improving kidney health, skin care, obesity, and diabetes. However, despite reports of various physiological activities of Cornus officinalis, there is insufficient research on the activities of Cornus officinalis as a prebiotic and synbiotic in ulcerative colitis. Therefore, in this study, we investigated the potential prebiotics and synbiotics of Cornus officinalis water extract in an animal model of in vitro lipopolysaccharide-induced intestinal epithelial cell (HT-29 cell) damage and dextran sulfate sodium (DSS)-induced ulcerative colitis. Activity was evaluated.

10-2021-0081895

The purpose of the present invention is to provide a health functional food composition for improving intestinal function containing Cornus officinalis extract as an active ingredient.

In addition, another object of the present invention is to provide a pharmaceutical composition for preventing or treating inflammatory bowel disease containing Cornus officinalis extract as an active ingredient.

In order to achieve the above object, the present invention provides a health functional food composition for improving intestinal function containing Cornus officinalis extract as an active ingredient.

In one embodiment of the present invention, the composition may further include Lactobacillus reuteri ( Limosilactobacillus reuteri ), but is not limited thereto.

In one embodiment of the present invention, the composition may have a protective effect on enterocytes and reduce oxidative stress, but is not limited thereto.

In one embodiment of the present invention, the composition may reduce the levels of TLR4 and NF-κB, but is not limited thereto.

In one embodiment of the present invention, the composition may increase occludin and claudin-1 levels, but is not limited thereto.

In one embodiment of the present invention, the composition may restore intestinal permeability, but is not limited thereto.

In one embodiment of the present invention, the composition may reduce myel peroxidase (MPO) activity, but is not limited thereto.

In one embodiment of the present invention, the composition may reduce the levels of p-IκB, p-NF-κB, and iNOS, but is not limited thereto.

Next, the present invention provides a pharmaceutical composition for preventing or treating inflammatory bowel disease containing Cornus officinalis extract as an active ingredient.

In one embodiment of the present invention, the composition may further include Lactobacillus reuteri ( Limosilactobacillus reuteri ), but is not limited thereto.

The present invention confirmed the protective effect of Cornus officinalis extract against intestinal epithelial cell toxicity induced by lipopolysaccharide (LPS), and its effect on intestinal permeability and intestinal inflammation in mice with colitis induced by dextran sulfate sodium (DSS). Regarding the improvement effect of the combination (synbiotics) of Korean Cornus officinalis water extract and Lactobacillus reuteri ( Limosilactobacillus reuteri ), the Cornus officinalis extract and Lactobacillus reuteri of the present invention and their combination improves intestinal barrier damage and intestinal inflammation caused by DSS-induced colitis, and alleviates colitis symptoms by controlling the content of short-chain fatty acids in feces and the imbalance of intestinal flora.

Figure 1 shows the effect of Cornus officinalis water extract (WCF) on the number of viable cells (a) and (b) and titratable acidity measurements (c) and (d) of Limosilactobacillus reuteri and Levilactobacillus brevis strains in an embodiment of the present invention. This is a diagram showing .
Figure 2 is a diagram showing the effect of Cornus officinalis water extract (WCF) on cell viability (a) and intracellular reactive oxygen species production (b) in HT-29 cells induced by LPS, in an embodiment of the present invention. .
Figure 3 shows the effect of Cornus officinalis water extract (WCF) on the NF-κB pathway in HT-29 cells induced with LPS, according to an embodiment of the present invention. Band image (a) is a diagram showing the expression levels of TLR4 (b) and NF-кB (c).
Figure 4 is a diagram showing the effect of Cornus officinalis water extract (WCF) on TJ (tight junction) protein in HT-29 cells induced by LPS, according to an embodiment of the present invention.
Figure 5 is a diagram showing the effects of PRE, PRO, and SYN on pathological symptoms in mice with DSS-induced colitis, according to an embodiment of the present invention.
Figure 6 is a diagram showing the effects of PRE, PRO, and SYN on intestinal permeability in mice with DSS-induced colitis, according to an embodiment of the present invention.
Figure 7 is a diagram showing the effects of PRE, PRO, and SYN on tight junction proteins in colonic tissue of mice with DSS-induced colitis, according to an embodiment of the present invention.
Figure 8 is a diagram showing the effect of PRE, PRO, and SYN on myeloperoxidase (MPO) activity in colon tissue of mice with DSS-induced colitis, according to an embodiment of the present invention.
Figure 9 is a diagram showing the effects of PRE, PRO, and SYN on the inflammatory response in colonic tissue of mice with DSS-induced colitis, according to an embodiment of the present invention.
Figure 10 is a diagram showing the effects of PRE, PRO, and SYN on the concentration of short-chain fatty acids (SCFA) in the feces of mice with DSS-induced colitis, according to an embodiment of the present invention.
Figure 11 is a diagram showing the effects of PRE, PRO, and SYN on the intestinal microbial community in DSS-induced E. coli, according to an embodiment of the present invention.
Figure 12 is a diagram showing the UPLC-Q-TOF/MS2 chromatogram for the ethyl acetate fraction of Corni fructus in one embodiment of the present invention.

Hereinafter, the present invention will be described in detail through embodiments of the present invention with reference to the attached drawings. However, the following implementation examples are provided as examples of the present invention, and if it is judged that a detailed description of a technology or configuration well known to those skilled in the art may unnecessarily obscure the gist of the present invention, the detailed description may be omitted. , the present invention is not limited thereby. The present invention is capable of various modifications and applications within the description of the patent claims described below and the scope of equivalents interpreted therefrom.

In addition, terminology used in this specification is a term used to appropriately express preferred embodiments of the present invention, and may vary depending on the intention of the user or operator or the customs of the field to which the present invention belongs. Therefore, definitions of these terms should be made based on the content throughout this specification. Throughout the specification, when it is said that a part “includes” a certain element, this means that it may further include other elements rather than excluding other elements, unless specifically stated to the contrary.

Throughout this specification, '%' used to indicate the concentration of a specific substance means (w/w) % for solid/solid, (w/v) % for solid/liquid, and Liquid/liquid is (v/v) %.

In one aspect, the present invention provides a health functional food composition for improving cognitive function in patients with inflammatory bowel disease, comprising Cornus officinalis extract as an active ingredient.

The extract according to the present invention can be obtained by extraction and separation from nature using extraction and separation methods known in the art, and the “extract” defined in the present invention is extracted from Cornus officinalis using an appropriate solvent. For example, it includes crude extract, polar solvent soluble extract, or non-polar solvent soluble extract. As a suitable solvent for extracting the extract from Cornus officinalis, any pharmaceutically acceptable organic solvent may be used. Water or an organic solvent may be used, but is not limited thereto, for example, purified water, methanol ( Alcohols with 1 to 4 carbon atoms including methanol, ethanol, propanol, isopropanol, butanol, acetone, ether, benzene, chloroform ( Various solvents such as chloroform, ethyl acetate, methylene chloride, hexane, and cyclohexane can be used individually or in combination. As an extraction method, any one of the following methods can be selected and used: hot water extraction, cold needle extraction, reflux cooling extraction, solvent extraction, steam distillation, ultrasonic extraction, elution, and compression. In addition, the desired extract may be further subjected to a conventional fractionation process or may be purified using a conventional purification method.

There is no limitation to the method for producing the extract of the present invention, and any known method can be used. For example, the extract included in the composition of the present invention can be prepared in powder form from the primary extract extracted by the above-described hot water extraction or solvent extraction method by additional processes such as reduced pressure distillation and freeze-drying or spray drying. In addition, the primary extract was further purified into fractions using various chromatographies such as silica gel column chromatography, thin layer chromatography, and high performance liquid chromatography. You can also get it. Therefore, in the present invention, extract is a concept that includes all extracts, fractions, and purifications obtained at each stage of extraction, fractionation, or purification, as well as their dilutions, concentrates, or dried products.

In addition to containing Cornus officinalis as an active ingredient, the food composition of the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients like a typical food composition.

Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose, etc.; Disaccharides such as maltose, sucrose, etc.; and polysaccharides, such as common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. The above-described flavoring agents include natural flavoring agents (thaumatin), stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.), and synthetic flavoring agents (saccharin, aspartame, etc.). The food composition of the present invention can be formulated in the same way as the pharmaceutical composition and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gum, candy, ice cream, alcoholic beverages, vitamin complexes, health supplements, etc. There is.

In addition, the food composition contains, in addition to the extract as an active ingredient, various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavors, colorants and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, It may contain alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the food composition of the present invention may contain pulp for the production of natural fruit juice, fruit juice beverages, and vegetable beverages.

The functional food composition of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. In the present invention, “health functional food composition” refers to a food manufactured and processed using raw materials or ingredients with functionality useful to the human body pursuant to Act No. 6727 on Health Functional Foods, and refers to a food product that is related to the structure and function of the human body. It means taking it for the purpose of controlling nutrients or obtaining useful health effects such as physiological effects. The health functional food of the present invention may contain common food additives, and its suitability as a food additive is determined in accordance with the general provisions and general test methods of the food additive code approved by the Food and Drug Administration, unless otherwise specified. Judgment is made according to specifications and standards. Items listed in the 'Food Additive Code' include, for example, chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; Natural additives such as dark pigment, licorice extract, crystalline cellulose, kaoliang pigment, and guar gum; Examples include mixed preparations such as sodium L-glutamate preparations, noodle additive alkaline preparations, preservative preparations, and tar coloring preparations. For example, the health functional food in the form of a tablet is made by granulating a mixture of the active ingredient of the present invention with excipients, binders, disintegrants and other additives in a conventional manner, and then adding a lubricant and compression molding, or The mixture can be directly compression molded. In addition, the health functional food in the form of tablets may contain flavoring agents, etc., if necessary. Among capsule-type health functional foods, hard capsules can be manufactured by filling a regular hard capsule with a mixture of the active ingredient of the present invention mixed with additives such as excipients, and soft capsules can be prepared by mixing the active ingredient of the present invention with additives such as excipients. It can be manufactured by filling the mixture with a capsule base such as gelatin. The soft capsule may contain plasticizers such as glycerin or sorbitol, colorants, preservatives, etc., if necessary. The health functional food in the form of a pill can be prepared by molding a mixture of the active ingredient of the present invention and excipients, binders, disintegrants, etc., using a known method. If necessary, it can be coated with white sugar or other coating agent. Alternatively, the surface can be coated with substances such as starch or talc. Health functional food in the form of granules can be manufactured into granules by mixing a mixture of excipients, binders, disintegrants, etc. of the active ingredients of the present invention by a known method, and may contain flavoring agents, flavoring agents, etc., if necessary. You can.

In one aspect, the present invention provides a pharmaceutical composition for preventing or treating degenerative cranial nerve disease in patients with inflammatory bowel disease, comprising Cornus officinalis extract as an active ingredient.

The pharmaceutical composition of the present invention may further include adjuvants in addition to the active ingredients. Any adjuvant known in the art may be used without limitation, but, for example, Freund's complete adjuvant or incomplete adjuvant may be further included to increase immunity.

The pharmaceutical composition according to the present invention can be prepared by incorporating the active ingredient into a pharmaceutically acceptable carrier. Here, pharmaceutically acceptable carriers include carriers, excipients, and diluents commonly used in the pharmaceutical field. Pharmaceutically acceptable carriers that can be used in the pharmaceutical composition of the present invention include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, Examples include calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The pharmaceutical composition of the present invention can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, or sterile injection solutions according to conventional methods. .

When formulated, it can be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations contain the active ingredient plus at least one excipient, such as starch, calcium carbonate, sucrose, lactose, and gelatin. It can be prepared by mixing etc. Additionally, in addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used diluents such as water and liquid paraffin, they contain various excipients such as wetting agents, sweeteners, fragrances, and preservatives. You can. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, tween 61, cacao, laurel, glycerogelatin, etc. can be used.

The pharmaceutical composition according to the present invention can be administered to an individual through various routes. All modes of administration are contemplated, for example, by oral, intravenous, intramuscular, subcutaneous, or intraperitoneal injection.

The dosage of the pharmaceutical composition according to the present invention is selected taking into account the age, weight, gender, physical condition, etc. of the individual. It is obvious that the concentration of the active ingredient included in the pharmaceutical composition can be selected in various ways depending on the target, and is preferably included in the pharmaceutical composition at a concentration of 0.01 to 5,000 μg/ml. If the concentration is less than 0.01 ㎍/ml, pharmaceutical activity may not appear, and if it exceeds 5,000 ㎍/ml, it may be toxic to the human body.

The pharmaceutical composition may be formulated into various oral or parenteral dosage forms.

Dosage forms for oral administration include, for example, tablets, pills, hard and soft capsules, solutions, suspensions, emulsifiers, syrups, granules, etc. These dosage forms contain diluents (e.g. lactose, dextrose, water) in addition to the active ingredient. crose, mannitol, sorbitol, cellulose and/or glycine), lubricants (e.g. silica, talc, stearic acid and its magnesium or calcium salts and/or polyethylene glycol). Additionally, the tablets may contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidine, and in some cases starch, agar, alginic acid. or disintegrants such as sodium salts thereof or effervescent mixtures and/or absorbents, colorants, flavoring and sweetening agents. The formulation can be prepared by conventional mixing, granulating or coating methods.

In addition, representative formulations for parenteral administration are injectable formulations, and solvents for injectable formulations include water, Ringer's solution, isotonic saline solution, or suspension. The sterile fixed oil of the injectable preparation can be used as a solvent or suspending medium, and any non-irritating fixed oil, including mono- and di-glycerides, can be used for this purpose.

Additionally, the injectable preparation may use fatty acids such as oleic acid.

Hereinafter, the present invention will be described in more detail through examples. However, the above examples and experimental examples are presented as examples of the present invention, and if a detailed description of a technique or configuration well known to those skilled in the art is judged to unnecessarily obscure the gist of the present invention, the detailed description is omitted. It can be done, and the present invention is not limited thereby. The present invention is capable of various modifications and applications within the description of the claims described below and the scope of equivalents interpreted therefrom.

<Materials and experimental methods>

<Preparation Example 1> Preparation of materials and extracts

Cornus officinalis fruit ( Corni fructus ) used in this experiment was grown in Gurye-gun, Jeollanam-do, dried with hot air at 50°C, purchased on July 22, 2020, powdered, and used. 20 g of Cornus officinalis and 1 L of distilled water were mixed and extracted using a reflux cooler at 40°C for 2 hours, and this extract was used as No. 2 Filtered through filter paper (Whatman Inc, Kent, UK). The extracted water extract of Corni fructus (WCF) was concentrated in vacuum and then freeze-dried. The yield of WCF was found to be 49.61%, and it was stored at -20°C and used in the experiment.

<Preparation Example 2> Probiotic strain culture conditions

The lactic acid bacteria strains used in the experiment were Lactobacillus reuteri ( Limosilactobacillus reuteri ; L. reuteri ) KCTC 3594 and Lactobacillus brevis ( Levilactobacillus brevis ; L. brevis ) KCTC 3498 derived from adult feces, Korea Research Institute of Bioscience and Biotechnology, Biological Resources Center (KCTC) , Jeongeup, Korea). The strain was plated on MRS-agar, and the resulting single colony was inoculated into MRS liquid medium and cultured at 37°C for 48 hours.

<Preparation Example 3> Measurement of viable cell count

The strain culture was centrifuged at 12,000 × g, and the pellet was resuspended in 0.85% NaCl. The suspended strain was diluted to show an absorbance value of 0.2 at 660 nm using a microplate reader (Epoch 2, BioTek Instruments, Inc., Winooski, VT, USA) and used in the experiment. The growth curves of L. reuteri and L. brevis were adjusted to pH 6.3~6.5 in glucose-free MRS liquid medium, respectively, and then WCF and fructo-oligosaccharide (FOS) were used as positive controls, and 2% (w/w/w) each was used in the medium. v) was added. Then, the strain in the logarithmic growth phase was inoculated (1%, v/v) and cultured at 37°C for 48 hours for measurement. After 0, 4, 8, 12, 24, and 48 hours, the culture medium was serially diluted with 0.85% NaCl solution, and 0.1 mL was dispensed onto plate medium and plated. Afterwards, they were cultured at the optimal temperature under anaerobic conditions and colony forming units were measured and calculated.

<Preparation Example 4> Measurement of titratable acidity

To measure the titratable acidity of L. reuteri and L. brevis , 1% (v/v) phenolphthalein reagent was added to 1 mL of culture medium cultured for 0, 4, 8, 12, 24, and 48 hours using the method mentioned above. As an indicator, the consumption of 0.1 N sodium hydroxide solution consumed in the titration was converted to lactic acid content (%) and calculated using Equation 1 below.

[Equation 1]

<Preparation Example 5> Cell culture

HT-29 cells, which originate from the human body and have characteristics of mature intestinal epithelial cells, were purchased from the Korea Cell Line Bank (Seoul, Korea) and used. 25 mM sodium bicarbonate, 25 mM HEPES, 10% fetal calf serum, 50 units/ Cultured in RPMI 1640 medium containing mL penicillin and 100 μg/mL streptomycin. Cell culture was maintained at 37°C and 5% CO ₂ conditions.

<Preparation Example 6> Measurement of intestinal epithelial cell protection effect and oxidative stress content

To measure the cell survival rate of WCF against LPS-induced HT-29 cell death, MTT reduction assay was performed. Cells were distributed at 1×10 ⁴ /well in a 96 well plate and grown for one day. After treatment with WCF at concentrations of 5, 10, and 20 μg/mL and FOS used as a positive control at a concentration of 50 μg/mL, 30 minutes later, LPS was diluted in distilled water to make a concentration of 1 μg/mL, and the cells were pretreated for 24 hours. . After incubation, 10 μL of MTT stock solution (5 mg/mL) was added, cultured at 37°C for 3 hours, the culture was removed, and DMSO was added to terminate the reaction, resulting in 570 nm (determination wave) and 690 nm (reference wave). was measured using a microplate reader (Epoch 2, BioTek Instruments, Inc.).

The intracellular ROS content was measured using the dichlorofluorescin diacetate (DCF-DA) method (Karlsson et al., 2010). Cells pretreated in a black 96 well plate were mixed with 50 μM DCF-DA solution and cultured at 37°C for 50 minutes. Afterwards, the culture medium was removed and 100 μL of sterile saline solution (PBS) was added. Afterwards, the fluorescence intensity was measured at the excitation wavelength (485 nm) and emission wavelength (emission filter, 535 nm) using a fluorescence microplate reader (Infinite 200, Tecan Co., SanJose, CA, USA).

<Preparation Example 7> Colitis animal model design

C57BL/6 (male, 6 weeks) mice used in this study were purchased from Samtako (Osan, Korea) and were conducted under the approval of the Animal Experiment Ethics Committee of Gyeongsang National University (IACUC approval number: GNU-210216-M0020). They were raised in an environment maintaining a temperature of 22±2°C and a relative humidity of 50±5%. After the mice went through an adaptation period for 1 week after being brought in, the normal control group (CON), DSS treated group (DSS), DSS+WCF treated group (PRE), DSS+ L. reuteri treated group (PRO), DSS+WCF+ L. reuteri treatment group (SYN), the samples were fed for 3 weeks and DSS was dissolved in sterilized drinking water for 6 days.

WCF was dissolved in PBS at 300 mg/kg of body weight and supplied to mice, and L. reuteri was cultured in MRS medium at 37°C for 24 hours, centrifuged at 13,000 × g, washed twice with PBS, and washed at 660 nm. Diluted to 1 × 109/CFU/mL in absorbance (EEpoch 2, BioTek Instruments, Inc., Winooski, VT, USA). The stock solution was stored at -80°C and used in the experiment, and 200 μL was fed to the experimental animals per day.

<Preparation Example 8> Intestinal permeability experiment

Intestinal permeability was assessed using the FITC-dextran permeability assay. A solution of FITC-dextran (4 kDa) (Sigma Aldrich, St. Louis, MO, USA) was supplied to mice by gavage at 0.4 mg/g body weight, and blood was collected 4 hours later. A standard curve was prepared using a dilution of FITC-dextran in PBS, and the excitation wave at 485 nm and the emission wave at 535 nm were measured using a fluorescence microplate reader (Infinite 200, Tecan Co., San Jose, CA, USA).

<Preparation Example 9> Measurement of serum albumin concentration

Four hours after the intestinal permeability experiment was conducted, the animals were sacrificed by asphyxiation with CO ₂ and blood was collected from the abdominal artery. Afterwards, the blood was centrifuged at 10,000 × g for 10 minutes to separate serum, and serum albumin was analyzed using a serum analyzer (Fujidri-chem 4000i, Fuji film Co., Tokyo, Japan).

<Preparation Example 10> Measurement of myeloperoxidase (MPO) activity in colon tissue

Colon tissue was homogenized in 50 mM potassium phosphate buffer (pH 6.0) containing 0.5% cetrimonium bromide (Alfa Aesar, Haverhill, MA, USA) and centrifuged at 15,000 × g for 15 min at 4°C. The supernatant was reacted by mixing with 16.7 mg o -diacinine dihydrochloride (TCI, Tokyo, Japan) and 50 mM potassium phosphate buffer (pH 6.0) containing 0.0005% hydrogen peroxide, and then processed using a microplate reader (Epoch 2, BioTek). Absorbance was measured at a wavelength of 450 nm using Instruments, Inc.). MPO activity is measured in units (U) of MPO/mg tissue, with 1 unit defined as the amount required to decompose 1 μmol of peroxide/min.

<Preparation Example 11> Western blot assay on intestinal epithelial cells and colon tissue

HT-29 cells are seeded at a concentration of 1×10 ⁴ cells/well, cultured for 24 hours, and then treated with the sample and FOS, a positive control. 24 hours after sample treatment, lipopolysaccharide (LPS) was treated at a final concentration of 1 μM, reacted and cultured for 3 hours, then centrifuged and collected cells were washed twice with PBS and cells containing 1% protease inhibitor cocktails. HT-29 cells were harvested with lysis buffer, and colon tissue was homogenized with a bullet blender and reacted on ice for 30 minutes. After the reaction, it was centrifuged again and the same amount of protein was quantified using the supernatant.

After sampling, the samples were subjected to electrophoresis on SDS-PAGE, transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA), and blocked with 5% skim milk for 1 hour to prevent incorporation of other proteins. The primary antibody is then incubated overnight, washed in Tris-buffered saline (TBST) containing 0.1% tween 20, and incubated with the secondary antibody for 1 hour at room temperature. Western blot images were detected using an iBright imager, and expression density was calculated using image detection software (image J, National Institutes of Health, Bethesda, MD, USA).

<Preparation Example 12> Measurement of short-chain fatty acid content in strain culture medium and mouse feces

The preprocessing process for GC/MS analysis to measure short-chain fatty acids (acetic acid, propionic acid, and butyric acid) in L. reuteri and L. brevis is as follows. A propanol/pyridine mixed solvent (v/v=3:2) and propyl chloroformate were added to the L. reuteri and L. brevis supernatants containing the internal standard (2-ethyl butyric acid). Hexane was added to the reaction mixture, centrifuged at 13,000 × g for 15 minutes, and the hexane layer was used for analysis.

The entire process for measuring short-chain fatty acids in mouse feces is as follows. Weigh 30 mg of mouse feces, add 5 mM NaOH solution and beads, and homogenize. The supernatant was transferred to a new tube and centrifuged at 13,000 × g for 10 minutes, and the obtained supernatant was subjected to the same experiment as the above analysis process.

The instrument used was gas chromatography (GC 2010 plus, Shimadzu, Kyoto, Japan), and the column was DB-5ms (thickness: 0.25 μm, length: 30 m, diameter: 0.25 mm, Agilent, Santa Clara, CA, USA). used. He was used as a carrier gas, and the analysis was performed at a column oven temperature of 40°C, injection temperature of 260°C, total flow of 54.0 mL/min, and total program time of 16.50 min.

<Preparation Example 13> NGS analysis

DNA was extracted from mouse feces to analyze intestinal microorganisms, and high-quality DNA was checked using a Qubit 3.0 Fluorometer (ThermoFisher, Waltham, MA, USA). Amplification was performed through polymerase chain reaction (PCR) using primers targeting the 16S rRNA V3-V4 region, and the amplified and quantified sample was sequenced using MiSeq (Illumina, CA, USA) equipment. was carried out. Paired-end MiSeq Illumina reads (2x300 bp) were processed using QIIME2 (version 2020.08), and the quality of raw data was checked using FastQC (version 0.11.8). After quality inspection, artificial sequences such as adapters and primers were removed and noise removed, and chimeric sequences interfering with the PCR process were removed during the library preparation process to obtain high-quality sequences. The sequence data was then classified to the species level using the Sliva 16S rRNA database.

<Preparation Example 14> Analysis of biologically active substances

To analyze the major phenolic compounds in Cornus officinalis water extract, ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) was used. A UPLC system (Vion, Waters Corp., Milford, MA, USA) was used, and an Acquity UPLC BEH C18 column (100 mm x 2.1 mm x 1.7 μm) (Waters) was used. Samples were analyzed in distilled water containing 0.1% formic acid and ACN containing 0.1% formic acid at a flow rate of 0.6 mL/min for 12 minutes. Compounds separated from the column were analyzed in negative ionization mode, and the measured data were analyzed using UNIFI software. was analyzed using .

<Preparation example 15> Statistical processing

All experiments were repeated and expressed as mean ± standard deviation, and each mean value was subjected to analysis of variance using SAS software (version 9.4, SAS institute, Cary, NC, USA) and Ducan's multiple range test. The significance between samples was verified within the 5% level using test).

<Example 1> Measurement of viable cell count and titratable acidity

The results of measuring the number of viable bacteria and titratable acidity to determine whether Cornus officinalis water extract (WCF) can help the proliferation of beneficial bacteria ( L. reuteri and L. brevis ) are shown in Figure 1. In the medium supplemented with WCF, L. reutri showed a significantly higher survival number (9.07 × 10 ⁸ CFU/mL) compared to the positive control (FOS) treated strain (8.16 × 10 ⁸ CFU/mL) after 12 hours of culture. , and L. brevis showed a significantly higher survival number (8.89×10 ⁸ CFU/mL) compared to the FOS-treated strain (8.10×10 ⁸ CFU/mL) after 24 hours of culture. Although the values of titratable acidity were slightly different between the two strains, acidity increased after 8 hours, and the increase slowed after 12 hours. The FOS-treated group showed no significant difference from the control group after 24 hours of incubation, while the highest acidity was 1.125 for L. reuteri and 0.477 for L. brevis during WCF treatment at 24 hours of incubation. These results indicate that WCF had a positive effect on the growth and growth conditions of L. reuteri and L. brevis and helped produce organic acids, which are considered to have potential for industrialization as prebiotics.

<Example 2> Short-chain fatty acid content in culture medium

The results of measuring the short-chain fatty acid content after 48 hours of culture are shown in Table 1. Short-chain fatty acids were detected and quantified through comparison of retention time with short-chain fatty acid standard solution and library search. In L. reuteri , the acetic acid content was highest in the culture medium treated with WCF (control (3.79 mM), FOS (7.63 mM), and WCF (12.36 mM)), and propionic acid was detected only in the culture medium treated with WCF (1.01 mM). ) was done. Acetic acid was detected in the L. brevis culture medium, and there was no significant difference between control (6.65mM), FOS (6.24mM), and WCF (6.83mM).

Control 2% FOS 2% WCF L. reuteri Acetic acid 3.79 ± 1.00 ^c 7.63 ± 0.06 ^b 12.36± ^0.62a Propionic acid - - 0.10± 0.09 ^a L. brevis Acetic acid 6.65 ± 0.45 ^a 6.24± 0.38 ^a 6.83± ^0.85a Propionic acid - - -

<Example 3> Measurement of intestinal epithelial cell protection effect and oxidative stress content

To confirm the effect of WCF on the proliferation of HT-29 cells, the results of measuring cell viability using the MTT assay are shown in Figure 2a. Compared to the control group (100.00%), the LPS-treated group showed a relatively low cell survival rate of 82.54% due to cytotoxicity. On the other hand, when WCF was treated at concentrations of 5, 10, and 20 μg/mL, respectively, the cell survival rate was 106.22%, 116.17%, and 130.07%, which was significantly higher than that of the positive control, FOS (93.46%).

In the DCF-DA assay, when the DCF-DA reagent enters the cell, it is oxidized by oxygen radicals, deacetylated to DCF, and converted into a substance that generates fluorescence. By measuring this, the degree of reactive oxygen species (ROS) production can be determined. The results confirming the effect of WCF on LPS-induced oxidative stress are shown in Figure 2b. Compared to the control (100.00%), the LPS-treated group showed an increase in intracellular reactive oxygen species (ROS) to 116.43%, and FOS showed a tendency to slightly decrease to 112.69%, but was statistically significant compared to the LPS-treated group. There was no difference. However, WCF-treated cells showed oxidative stress occurrence levels of 110.19%, 108.80%, and 101.88%, respectively, and it was confirmed that Cornus officinalis water extract significantly reduced intracellular ROS content compared to the LPS-treated group.

<Example 4> Confirmation of NF-κB signaling pathway in intestinal epithelial cells

Toll-like receptors (TLRs) are known to recognize the unique structures of pathogens and induce inflammatory and innate immune responses against these pathogens. Among them, TLR4 is an LPS receptor and stimulates cells to activate NF-κB and induce the production of inflammatory mediators such as TNF-α, IL-1, and monocyte chemotactic protein-1. NF-κB is an important signaling substance in the inflammatory process and is activated by LPS to bind to target genes in the nucleus and secrete inflammatory cytokines. To confirm the inflammatory response mechanism in HT-29 cells induced by LPS, the expression levels of TLR4, known as the LPS receptor, and NF-κB, a protein that regulates the inflammatory response, were measured (Figure 3). As a result of measuring TLR4 and NF-κB, LPS-treated cells (1.58 and 2.76, respectively) showed increased protein expression compared to the control group (1.02 and 0.61, respectively) (Figure 3). On the contrary, in cells treated with WCF at different concentrations, the expression of TLR4 and NF-κB was significantly reduced, and there was no significant difference from the control group (WCF 5; 1.26, 2.14; WCF 10; 0.93, 1.95, respectively) , WCF 20; 0.83, 1.48).

<Example 5> Measurement of tight junction protein changes in intestinal epithelial cells

What affects the permeability of intestinal epithelial cells is the tight junction (TJ) protein that exists between cells. TJs perform various functions such as connecting and joining adjacent epidermal cells to regulate the movement of electrolytes and water, as well as transmitting intracellular signals and regulating cell division. It is connected by claudin and occludin proteins arranged in a chain to seal between the two cells. However, when pathogenic bacteria interact with epithelial cells and damage tight junctions, abnormal electrolyte and water movement, tissue inflammation, etc. occur, causing gastrointestinal diseases, and research results have reported that LPS increases intestinal permeability through an intracellular mechanism. there is.

The results of measuring the expression levels of tight junction proteins occludin and claudin-1 when HT-29 cells were treated with LPS alone and in parallel with WCF are shown in Figure 4. As shown in Figures 4a and 4b, the expression level of occludin was increased in WCF-treated cells (WCF5; 0.95; WCF10; 1.00, WCF20; 1.21) compared to LPS-treated cells (0.74), and was significantly higher than that in the control (0.96). The results were shown. On the other hand, FOS-treated cells (0.89) had no effect on the expression of occludin compared to the LPS-only treatment group. The results of measuring the expression level of Claudin-1 are shown in Figures 4a and 4c. There was no significant difference in LPS-treated cells (1.50) and FOS, WCF 5, and 10 μg/mL concentrations (1.68, 1.73, and 1.77, respectively), but there was a significant difference in WCF 20 μg/mL concentration (2.38) compared to the control (2.42). It was confirmed that there was a significant increase.

<Example 6> Effect of PRE, PRO, and SYN on body weight change, colon length, and serum albumin content in DSS-induced colitis mice

The DSS ulcerative colitis model shows clinical symptoms characterized by weight loss, decreased colon length, and bloody stool. In order to confirm the effect of the sample on these clinical symptoms, the results for weight change rate, colon length, and serum albumin content were measured and are shown in Figure 5. A graph measuring the rate of change in body weight of mice from the first day to the sixth day of DSS induction is shown in Figure 5a. On the last day of DSS induction, the DSS group showed a rapid weight loss rate (70.00%), and the PRE, PRO, and SYN groups showed a significant decrease in body weight. showed a tendency to improve (83.00%, 83.00%, 87.00%). As a result of measuring the albumin concentration in the serum to check the nutritional status of the mouse, the DSS group (1.62 mg/dL) showed a significantly lower albumin value compared to the CON group (2.86 mg/dL), and PRE, PRO , the SYN group showed significantly higher content than the DSS group at 2.2 mg/dL, 2.14 mg/dL, and 1.96 mg/dL, respectively (Figure 5b). In addition, the length from the bottom of the cecum to the top of the anus, that is, the length of the large intestine, was measured at 6.57 cm in the CON group, but was measured at 4.00 cm in the DSS group (Figure 5c). However, the PRE, PRO, and SYN groups measured 4.68 cm, 4.61 cm, and 4.72 cm, respectively, and the colon length increased by 15.57%, 16.88%, and 22.04%, respectively, compared to the DSS group.

<Example 7> Effect of PRE, PRO and SYN on intestinal permeability in colonic tissue of mice with DSS-induced colitis

Evaluation of mouse intestinal permeability was measured using the FITC-dextran method, and the results are shown in Figure 6. Serum FITC-dextran (509.19 μg/mL) in DSS-treated mice was higher than that in the CON group (47.24 μg/mL), and was detected at 384.48 μg/mL, 200.28 μg/mL, and 204.50 μg/mL in the PRE, PRO, and SYN groups, respectively. showed a tendency to significantly decrease compared to the DSS group. Notably, in the sample groups, the lowest serum FITC-dextran was detected in the SYN group, which appears to have helped restore intestinal permeability in mice.

<Example 8> Effect of PRE, PRO, and SYN on TJ (tight junction) protein expression level in colon tissue of mice with DSS-induced colitis

The integrity of the mouse intestinal barrier was assessed by performing Western blot for TJ protein expression levels, and the results are shown in Figure 7. As a result of measuring the expression levels of occludin and claudin-1, compared to the CON group (1.00), the DSS group (0.43, 0.51, respectively) decreased by 2.34-fold and 1.96-fold, respectively, and in the PRE, PRO, and SYN groups, occludin (0.64, 0.61, respectively) Although the protein expression levels of claudin-1 (0.67) and claudin-1 (0.76, 0.74, and 0.86, respectively) were not restored to the level of the CON group, they tended to significantly increase compared to the DSS group.

<Example 9> Effect of PRE, PRO and SYN on MPO activity in colon tissue of mice with DSS-induced colitis

MPO activity, known as an indicator of inflammatory activity, was measured in mouse colon tissue, and the results are shown in Figure 8. MPO increased in the DSS group (9.79 U/mg) compared to the CON group (0.24 U/mg), and the PRE, PRO, and SYN groups decreased MPO activity in the colon compared to the DSS group (6.01 U/mg each , 5.07 U/mg, 3.66 U/mg).

<Example 10> Effect of PRE, PRO and SYN on inflammatory factors in colonic tissue of mice with DSS-induced colitis

TLR4, p-IκBα, p-NF-κB, TNF-α, caspase 1, IL-1β, iNOS, and COX-2, which are important NF-κB signaling pathway factors involved in inflammatory mechanisms in immune cells in mouse colon tissue. The measurement results are shown in Figure 9. As a result, the DSS group showed a tendency for the expression levels of all inflammatory factors to increase to 1.65, 1.90, 2.93, 2.18, 1.31, 1.37, 2.22, and 1.88, respectively, compared to the CON group (1.00). However, the PRE, PRO, and SYN groups showed a tendency to significantly decrease (TLR4; 1.25, 1.15, 1.14, p-IκBα; 1.63, 1.35, 1.13, p-NF-κB; 2.08, 2.37, 1.73, respectively) TNF-α; 1.82, 1.79, 1.66, Caspase 1; 1.02, 0.99, 0.85, IL-1β; 1.06, 1.05, 0.94, iNOS; 1.63, 1.79, 0.83, COX-2; 1.08, 1.15, 1.08). In particular, the SYN group tended to have significantly greater decreases than the PRE and PRO groups in the factors p-IκB, p-NF-κB, and iNOS (0.59-, 0.59- and 0.37-fold decreases, respectively, compared to DSS).

<Example 11> Effect of PRE, PRO and SYN on short-chain fatty acid content in feces of mice with DSS-induced colitis

The results of measuring the short-chain fatty acid content in the feces of DSS-treated mice are shown in Figure 10. The acetic acid, propionic acid, and butyric acid contents tended to decrease in the DSS group, with 81.76mM, 8.85mM, and 7.28mM, respectively, detected compared to the CON group (82.93mM, 18.44mM, 39.47mM). However, the PRE (216.39mM, 28.04mM, 23.20mM, respectively), PRO (200.44mM, 25.79mM, 24.82mM, respectively), and SYN group (254.22mM, 30.12mM, 38.18mM) showed significant improvement. In particular, in the SYN group, the contents of acetic acid, propionic acid, and butyric acid increased by 3.11, 3.40, and 5.24 times, respectively, compared to the DSS group, showing that the short-chain fatty acid content increased compared to the other sample controls.

<Example 12> Effect of PRE, PRO and SYN on the intestinal flora of mice with DSS-induced colitis

The results of analyzing the intestinal flora in mouse feces using NGS analysis are shown in Figure 11. This is the result of analyzing the intestinal flora in each group by phylum, family, genus, and species (Figure 11a-d). At the phylum level, there are about 90 Firmicutes and Bacteroidota . %, and there was no significant difference in the ratio. Unusually, the Verrucomicrobiota phylum accounted for the highest proportion in the PRO group. At the family level, the families that accounted for the largest proportion were the Lachnospiraceae and Muribaculaceae families, and the Lactobacillaceaes family to which lactic acid bacteria belong. The CON and PRO groups showed the highest distribution. At the genus level, the genus Bacillus and Prevotellaceae UCG-001, known as harmful bacteria, were the most abundant in the DSS group (6.14% and 2.31%, respectively), and showed a significant decreasing trend in the PRE, PRO, and SYN groups ( Bacillus : 3.46%, 4.74%, 3.19%, Prevotellaceae UCG-001: 0.81%, 0.00%, 0.65%) (Figure 11e-f). In particular, the genus Prevotellaceae UCG-001 was not detected in the PRO group. In addition, Lactobacillus genus, known as probiotics, showed a lower distribution in the DSS group (0.70%) compared to the CON group (8.89%), and showed a tendency to recover to the CON level in the PRO group (7.41%) (Figure 11g). The genus Anaerotruncu and Lachnospiraceae NK4A136 group , known as short-chain fatty acid producing bacteria, showed lower levels in the DSS group (0.06% and 1.42%, respectively) compared to the CON group (0.66% and 2.90%, respectively) (Figure 11h-i). The Odoribacter genus is a strain known to strengthen intestinal mucosal function by producing butyrate, a type of short-chain fatty acid, and showed lower levels in the DSS group (0.04%) compared to the CON group (0.32%), especially in the PRE group (0.10%). %) showed a similar level to the CON group (Figure 11j). Akkermansia muciniphila is a microorganism that has been reported to protect intestinal barrier function and reduce inflammatory cytokine levels in animal models of colitis. In this study, the DSS group (0.30%) was compared to the CON group (0.45%). The quantitative level decreased, especially in the SYN group (3.35%), and increased significantly more than the CON group (Figure 11k). Previous studies have reported a decrease in Alistipes onderdonkii in mice with DSS-induced colitis. The Alisipes genus is found in people with a healthy gastrointestinal tract and is known to be effective in alleviating colitis. In this study, Alistipes onderdonkii showed the lowest level in the DSS group (0.02%), but showed a tendency to improve in the PRE, PRO, and SYN groups (0.34%, 0.19%, and 0.28%, respectively) (Figure 11l).

<Example 13> Analysis of biologically active substances

The main bioactive substances of Cornus officinalis water extract, which were judged to have an effect of improving intestinal inflammation, were analyzed by UPLC/MS ² in negative ion mode (Figure 12, Table 2). The major compounds detected were identified by comparing the MS ² major fragments with previously reported literature and the Massbank database. A total of four compounds were tentatively identified by MS fragmentation: qunic acid (RT: 0.81 min, m/z: 191; 85, 93, 109, and 127), morroniside (RT: 4.49 min, m/z: 451; 101, 123, 141, 155 and 243), loganin (RT: 4.69 min, m/z: 435, 101, 127, 209 and 227) and cornuside (RT: 5.30 min, m/z: 541; 125, 169 and 347) was identified as the main compound.

Compound RT(min) Formular [M-H] ^-
(m/z) Product ion(m/z) Quinic acid 0.81 C ₇ H ₁₂ O ₆ 191 85, 93, 109, 127, 191 Moroniside 4.49 C ₁₇ H ₂₆ O ₁₁ 451 101, 123, 141, 155, 243 Loganin 4.69 C ₁₇ H ₂₆ O ₁₀ 435 101, 127, 209, 227 Cornuside 5.3 C ₂₄ H ₃₀ O ₁₄ 541 125, 169, 347

Therefore, through the above examples, it was confirmed that the Cornus officinalis extract of the present invention can be used as a health functional food composition for improving intestinal function or a pharmaceutical composition for preventing or treating inflammatory bowel disease, thereby completing the present invention.

As seen above, the present invention has been described as the preferred embodiment mentioned above, but those skilled in the art will find that other regressive inventions can be found by adding, changing, deleting, etc. other components within the same technical scope. However, other embodiments that are included within the scope of the present invention can be easily proposed. Therefore, the embodiments described above are illustrative in all respects and do not limit the scope of the present invention.

Claims (10)

A health functional food composition for improving intestinal function containing Cornus officinalis extract as an active ingredient. According to paragraph 1,
The composition is a health functional food composition for improving intestinal function, further comprising Lactobacillus reuteri ( Limosilactobacillus reuteri ). According to paragraph 1,
The composition is a health functional food composition for improving intestinal function, which has an intestinal cell protection effect and reduces oxidative stress content. According to paragraph 1,
The composition is a health functional food composition for improving intestinal function, which reduces the levels of TLR4 and NF-κB. According to paragraph 1,
The composition is a health functional food composition for improving intestinal function, which increases occludin and claudin-1 levels. According to paragraph 1,
The composition is a health functional food composition for improving intestinal function, which restores intestinal permeability. According to paragraph 1,
The composition is a health functional food composition for improving intestinal function, which reduces bone marrow peroxidase (MPO) activity. According to paragraph 1,
The composition is a health functional food composition for improving intestinal function, which reduces the levels of p-IκB, p-NF-κB and iNOS. A pharmaceutical composition for preventing or treating inflammatory bowel disease containing Cornus officinalis extract as an active ingredient. According to clause 9,
The composition is a pharmaceutical composition for preventing or treating inflammatory bowel disease, further comprising Lactobacillus reuteri ( Limosilactobacillus reuteri ).