KR101780900B1 - The composition containing oxyresveratrol for preventing or treating intestinal diseases - Google Patents

Abstract

The present invention relates to a composition for preventing or treating inflammatory intestinal disease containing oxyresveratrol as an active ingredient. According to the present invention, it is possible to prevent or treat various types of inflammatory intestinal diseases by reducing intestinal permeability through formation of tight junctions among intestinal epithelial cells.

Description

[0001] The present invention relates to a composition for preventing or treating intestinal diseases, including oxys resveratrol,

The present invention relates to a composition for preventing or treating intestinal diseases including oxyreservatol.

The junctions between the cells formed by the epithelial cells are classified into adherens junction, desmosome, gap junction, and tight junction. Attachment junctions provide a strong mechanical adhesion between adjacent cells and serve to firmly attach epithelial tissue. Desmosomes are more tightly adhered to the two cells than adhesion junctions, and are connected to intermediate fibers in the cytoplasm. The cell junction consisting of gap junctions has a channel of about 1.5 to 2 nm in diameter, made up of a protein called connexin. Through this channel, intercellular ions and small molecules, or signaling, are made. It is the place where the membrane potential changes between the cells because the ions can move. Adhesive closure is one of the intercellular junctions observed in human epidermis and has the function of sealing adjacent epithelial cells. Unlike gap junctions, it also performs a variety of biological functions including barrier function to regulate the transport of electrolytes and moisture through the intercellular space. Therefore, the material is not free to move in the place where the tight junction is present.

The function of tight junctions is very important in limiting the migration of substances during intercellular junctions by intestinal cells. A tight junction is involved in the transport of water, ions or food nutrients. It is known that it has the function of blocking the inflammatory reaction by microorganisms because it blocks the contact between the microorganisms in the intestine and the outside. In the normal state, the movement of large molecules is restricted, and the immune response is maintained by regulating the migration of food-derived antigens and small amounts of microorganisms. . On the other hand, in the state where the adhesion joint is broken, the intestinal permeability increases as the cell gap spreads. Due to the increased intestinal permeability, a large amount of microorganisms and microorganisms toxins and other pathogenic factors present in the intestines are leaked out. This results in the development of intestinal immune diseases such as inflammatory bowel diseases (IBD) or chronic inflammatory diseases including Crohn's disease and ulcerative colitis. In the case of such a chronic immune disease, it is a chronic disease of unknown origin. In this way, it is very important to maintain close contact in the intestine, since the tight junctions are closely related to the occurrence of metabolic diseases such as diabetes and autoimmune diseases, particularly, intestinal immune diseases.

Adherent synapses are formed by the interaction of proteins such as Claudin, Occludin and Zonual Occludens (ZO). Their expression is also known to be highly correlated with the biological function of the tight junction. Claudin is present in 24 kinds in human body. In the case of Claudin-1, the intestinal permeability decreases as the expression level increases. As an exception, Claudin-2 is a protein that forms intercellular channels, and it has been reported that the intestinal permeability increases with increasing expression amount. Occludin is also induced by the formation of tight junctions as the amount increases, reducing the intestinal permeability. ZO binds to Claudin and Occludin and acts as a medium to make tight junctions harder. Recent studies have shown that the amounts of adhesion proteins such as Claudin-1, Occludin and ZO-1 in IBD patients are significantly lower than those in healthy individuals. Therefore, prevention of intestinal immune diseases caused by increased intestinal permeability , It is suggested that formation of tight junction-inducing proteins such as Claudin, Occludin, and ZO-1 is important.

So far, studies on the effect of tight junction-inducible protein formation have been carried out on flavonoids such as genistein, quercetin and myricetin, and short chain fatty acids (SCFA) including glutamine, galato-oligosaccharides, fucoidan and butyrate.

In order to separate the substances that can reduce intestinal permeability and to prevent the immunological diseases from intestinal tract and to develop them as functional foods and medicinal materials, researches on the separation of substances having the effect of inducing the formation of intimate synapses continue It is.

Non-Patent Document 1. Artis D, Nature Reviews Immunology. 8: 411-420, 2008 Non-Patent Document 2. Tsukita S, Furuse M, Ltoh M., Nature review Molecular cell biology, 2: 285-293, 2001 Non-Patent Document 3. Schneeberger EE, Lynch RD., American Journal of Physiology-Cell Physiology, 286: C1213-C1228, 2004 Non-Patent Document 4: Li N, Lewis P, Samuelson D, Liboni K, Neu J., American Journal of Physiology-Gastrointestinal and Liver Physiology, 287: G726-G733, 2004 Non-Patent Document 5. Berkes J, Viswanathan V, Savkovic S, Hecht G., Gut, 52: 439-451, 2003 Non-Patent Document 6. Groschwitz KR, Hogan SP., Journal of Allergy and Clinical Immunology, 124: 3-20, 2009 Non-Patent Document 7. Menard, Cerf-Bensussan N, Heyman M., Mucosal immunology, 3: 247-259, 2010 Non-Patent Document 8. Camilleri M, Madsen K, Spiller R, Van Meerveld B, Verne G., Neurogastroenterology & Motility, 24: 503-512, 2012 Non-Patent Document 9: Iraha A, Chinen H, Hokama A, Yonashiro T, Kinjo T, et al., World J Gastroenterol 19: 5500-5507, 2013 Non-Patent Document 10. Bruewer M, Samarin S, Nusrat A., Annals of the New York Academy of Sciences, 1072: 242-252, 2006 Non-Patent Document 11. Suzuki T, Hara H., Journal of Nutritional Biochemistry, 22: 401-408, 2011 Non-Patent Document 12. Li N, Neu J., The Journal of Nutrition and Disease, 139: 710-714, 2009 Non-Patent Document 13. Akbari P et al., The Journal of Nutrition, 145: 1064-13, 2015 Non-Patent Document 14. Iraha A et al., World J Gastroenterol, 19: 5500-5507, 2013 Non-Patent Document 15. Peng L et al., The Journal of Nutrition, 139: 1619-1625, 2009

It is an object of the present invention to provide a composition for preventing or treating inflammatory bowel disease including oxir reservatore, which induces formation of tight junction of intestinal epithelial cells.

However, the problems to be solved by the present invention are not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

According to one embodiment, there is provided a composition for preventing or treating inflammatory bowel disease comprising oxyresveratrol as an active ingredient.

According to one side, the above-mentioned oxys resveratrol can induce the formation of tight junctions in intestinal epithelial cells.

According to one side, the oxy-resveratrol are intestinal epithelial cells Claudin -1, Occludin, ZO -1, ERK1 / 2, JNK -a1, p-38, PKC -ε, PKC -θ , and Lt ; / RTI > and < RTI ID = 0.0 > PKC- 5 . ≪ / RTI >

According to one side, the ox resveratrol can induce the expression of the intestinal epithelial cell transcription factor cdx-2.

According to one aspect, the inflammatory bowel disease is selected from the group consisting of Chron's disease, ulcerative colitis, intestinal Behcet's disease, intestinal tuberculosis and enteritis. Or more.

According to one aspect, the composition may be a pharmaceutical composition.

According to one aspect, the composition may further comprise a pharmaceutically acceptable carrier.

According to one embodiment, there is provided a food composition for preventing or treating inflammatory bowel disease comprising oxyresveratrol as an active ingredient.

The composition for preventing or treating inflammatory bowel diseases containing the oxir reseratrol of the present invention as an active ingredient induces the formation of tight junctions of intestinal epithelial cells to reduce intestinal permeability and thus can be used as a food allergy source or intestinal toxins It is possible to prevent permeation of harmful substances and prevent or treat intestinal diseases such as Crohn's disease and ulcerative colitis.

FIG. 1 is a graph showing the cell viability of intestinal epithelial cells treated with oxyreservatrix according to an embodiment.
FIG. 2 is a graph comparing the ability to maintain tight junctions in intestinal epithelial cells treated with oxiresteral inhibition according to one embodiment.
FIG. 3 is a graph comparing the transmittance of fluorescent materials in the intestinal epithelial cells treated with oxirestera virus according to one embodiment.
FIG. 4 is a graph comparing the expression of Claudin-1 , Occludin, and ZO- 1 genes in intestinal epithelial cells treated with Oxirreservat according to one embodiment.
FIG. 5 is a photograph showing expression of Claudin-1, Occludin and ZO-1 protein in the intestinal epithelial cells treated with oxis resveratrol according to one embodiment.
FIG. 6 is a photograph showing formation of tight junctions in the intestinal epithelial cells treated with oxis resveratrol according to an embodiment.
7 is a graph comparing the PKC -ε, -θ PKC, PKC -δ gene expression in the field oxy resveratrol treatment in epithelial cells in accordance with one embodiment.
FIG. 8 is a graph comparing ERK1 / 2, JNK- a1, and p-38 gene expression in oxir reseratrol- treated intestinal epithelial cells according to an embodiment.
9 is a graph comparing the PKC inhibitors (GF109203X) oxy and resveratrol-treated sheet epithelial cells -ε PKC, PKC -θ, PKC -δ gene expression, according to one embodiment.
10 is a graph comparing ERK1 / 2, JNK- a1, and p-38 gene expression in intestinal epithelial cells treated with PKC inhibitor (GF109203X) according to one embodiment and oxyreservatrix.
11 is a graph comparing the expression of Claudin- 1 , Occludin and ZO- 1 genes in the intestinal epithelial cells treated with the PKC inhibitor (GF109203X) according to one embodiment and oxyreservatrix .
FIG. 12 is a graph illustrating the effect of the oxir reseratrol-treated intestinal epithelial cell transcription factor (cdx-2) Gene expression.
FIG. 13 is a graph comparing the expression of the intestinal epithelial transcription factor (cdx-2) gene treated with the PKC inhibitor (GF109203X) according to one embodiment and oxyreservatrix.

In the following, embodiments will be described in detail with reference to the accompanying drawings. Like reference symbols in the drawings denote like elements.

Various modifications may be made to the embodiments described below. It is to be understood that the embodiments described below are not intended to limit the embodiments, but include all modifications, equivalents, and alternatives to them.

The terms used in the examples are used only to illustrate specific embodiments and are not intended to limit the embodiments. The singular expressions include plural expressions unless the context clearly dictates otherwise. In this specification, the terms "comprises" or "having" and the like refer to the presence of stated features, integers, steps, operations, elements, components, or combinations thereof, But do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, or combinations thereof.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this embodiment belongs. Terms such as those defined in commonly used dictionaries are to be interpreted as having a meaning consistent with the contextual meaning of the related art and are to be interpreted as either ideal or overly formal in the sense of the present application Do not.

In the following description of the present invention with reference to the accompanying drawings, the same components are denoted by the same reference numerals regardless of the reference numerals, and redundant explanations thereof will be omitted. In the following description of the embodiments, a detailed description of related arts will be omitted if it is determined that the gist of the embodiments may be unnecessarily blurred.

According to one embodiment, there is provided a composition for preventing or treating inflammatory bowel disease comprising oxyresveratrol as an active ingredient. In particular, the above-mentioned oxyreservatrix can induce the formation of tight junctions in intestinal epithelial cells.

In addition, the above-mentioned oxyreservatrix can reduce the permeability of intestinal epithelial cells. Referring to Example 3, since the permeability of the fluorescent substance in the intestinal epithelial cells treated with oxyreservatase is remarkably lowered, the composition containing the oxirebazelecrol of the present invention can reduce permeability of intestinal cells.

According to one side, the oxy-resveratrol are intestinal epithelial cells Claudin -1, Occludin, ZO -1, ERK1 / 2, JNK -a1, p-38, PKC -ε, PKC -θ , and Lt ; / RTI > and < RTI ID = 0.0 > PKC- 5 . ≪ / RTI > Referring to the examples, oxis resveratrol can increase expression of Claudin-1, Occludin and ZO-1 genes required for formation of tight junctions. In addition, Claudin-1, Occludin and -ε of PKC protein kinase (PKC) for generating a ZO-1, PKC -θ, PKC -δ and ERK1 / 2 in the MAPK pathway which is activated by PKC path, JNK -a1 , p-38 gene expression may also be increased.

According to one side, the ox resveratrol can induce the expression of the intestinal epithelial cell transcription factor cdx-2. Referring to Example 12, the expression of Caudal-related homeobox (cdx-2) gene, which is a transcription factor that affects gene expression related to tight junctions, can also be increased by oxyreservatrix.

According to one aspect, the inflammatory bowel disease is selected from the group consisting of Chron's disease, ulcerative colitis, intestinal Behcet's disease, intestinal tuberculosis and enteritis. Or more.

The composition of the present invention may be a pharmaceutical composition, and thus may contain not only the composition of the present invention but also one or more known active ingredients having antiinflammatory activity.

The composition of the present invention may further comprise a pharmaceutically acceptable carrier.

The term "pharmaceutically acceptable carrier " in the present invention means a liquid or solid filler that is involved in carrying or transporting any subject composition or ingredient from one organ or part of the body to another organ, Refers to a pharmaceutically acceptable material, composition or vehicle, such as a diluent, excipient, solvent or encapsulating material, wherein the composition of the present invention further comprises a pharmaceutically acceptable carrier, excipient or diluent in addition to the active ingredient described above for administration . Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

In addition, the composition of the present invention can be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols or the like, oral preparations, suppositories or sterilized injection solutions according to a conventional method have. In detail, when formulating, it can be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants and the like which are usually used. Solid formulations for oral administration include, but are not limited to, tablets, pills, powders, granules, capsules, and the like. Such a solid preparation can be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose, lactose, gelatin and the like in the composition. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Examples of liquid formulations for oral use include suspensions, solutions, emulsions, syrups and the like, but are not limited thereto. In addition to water and liquid paraffin which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, Preservatives, and the like. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like. Examples of the suppository base include withexol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

In addition, the composition of the present invention may be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically), preferably orally, depending on the intended method. The dosage varies depending on the condition and the weight of the patient, the degree of the disease, the drug form, the administration route and the period, but can be appropriately selected by those skilled in the art. May be administered once or several times a day according to need, and may be used alone or in combination with methods using surgery, hormone therapy, drug therapy and biological response modifiers for the prevention or treatment of intestinal diseases .

Meanwhile, the composition for preventing or treating inflammatory bowel disease of the present invention may be a food composition as well as a pharmaceutical composition, and may be added as it is or may be used together with other food or food ingredients.

Examples of the food include dairy products such as meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums and ice cream, various soups, drinks, tea, drinks, alcoholic beverages, And includes all health functional foods in a conventional sense.

The composition of the present invention may contain various flavoring agents or natural carbohydrates as an additional ingredient as well as ordinary health functional foods. The natural carbohydrates may be monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like.

In addition, the composition of the present invention can be used for various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, A carbonating agent used in beverages, and the like. In addition, the composition of the present invention may contain flesh for the production of natural fruit juice, fruit juice beverage and vegetable beverage. These components may be used independently or in combination.

Example  One: Oxy Resveratrol Intestinal epithelial cells (intestinal epithelial cell) Toxicity confirmation

1-1) Intestinal epithelial cells ( intestinal epithelial cell culture Oxy Resveratrol  administration

10% FBS, 100 units / mL penicillin, 100 ㎍ / mL streptomycin in MEM medium, 0.1 mmol / l non-essential amino acids, 1 mmol / l sodium pyruvate by the addition of 5% at 37 ℃ incubator CO ₂ -95% Caco-2 cells were cultured in air. If the cells were grown to 80% confluence, the cells were removed with 0.25% trypsin, and the cells were adjusted to 1.25 × 10 ⁵ cells / mL and seeded in 96-wells at 200 μl. The cells stabilized in a 37 ° C incubator for one day were treated with oxireservatol (10, 20, 30 ㎍ / mL) and cultured for 24 hours.

1-2) Oxy Resveratrol  Toxicity measurement

Then, the supernatant was removed and 5 mg / mL stock MTT (3- (4,5-Dimethylthiazol-2-yl) - 2,5-Diphenyltetrazolium Bromide, Amresco, USA) And incubated for 1 hour in a 37 ° C incubator. When purple crystals were formed, 100 μl of DMSO was added to remove the supernatant, and the OD value was measured at a wavelength of 540 nm using a microplate reader.

FIG. 1 is a graph showing the cell viability of intestinal epithelial cells treated with oxyreservatrix according to an embodiment.

Referring to FIG. 1, it was confirmed that although resveratrol was treated at various concentrations, there was no effect on the viability of cells, and thus, it was confirmed that oxyreservatol did not have cytotoxicity.

Example  2: Confirmation of tight junction recovery ability

2-1) Intestinal epithelial cells ( intestinal epithelial cell culture

MEM medium supplemented with 10% fetal bovine serum (FBS), 100 units / mL penicillin, 100 ㎍ / mL streptomycin, 0.1 mmol / l non-essential amino acids and 1 mmol / l sodium pyruvate, Caco-2 cells were cultured with CO ₂ -95% air composition. If the cultured cells grew to 80% confluence, cells were removed using 0.25% trypsin. Cells were adjusted to 7 × 10 ⁴ cells / mL, and 300 μl of cells were added to the Trans 24 well insert. 900ol of media was dispensed into the basolateral. After 2 to 3 days of washing with media and incubation for 14 days, all wells were used for the experiment when the value reached 600 ~ 700 Ω.

2-2) Tight junction maintenance ability Measurement by time

270 ㎕ of cold HBSS (hank's balanced salt solution) was added to a Trans 24 well insert, transferred to a new Trans 24 well containing 900 의 of cold HBSS, and stabilized for 30 minutes. Subsequently, 30 μl of oxyreservatrix (10, 20, 30 ㎍ / mL) diluted in HBSS was added and measured every 4 hours.

FIG. 2 is a graph comparing the ability to maintain tight junctions in intestinal epithelial cells treated with oxiresteral inhibition according to one embodiment.

Referring to FIG. 2, when oxygen resveratrol was treated according to one embodiment of the present invention, it was confirmed that tight junctions were restored over time. In particular, as the concentration of oxyreservatrix increased (30 / / mL) And the synaptic recovery ability was improved.

Example  3: Confirmation of long-term transmission

10% in MEM medium FBS, 100 units / mL penicillin, 100 ㎍ / mL streptomycin, 0.1 mmol / l non-essential amino acids, 1 mmol / l sodium by the addition of 5% CO ₂ -95% of pyruvate 37 ℃ incubator Caco-2 cells were cultured in air. If the cells were grown to 80% confluence, the cells were removed with 0.25% trypsin, and the cells were adjusted to 7 × 10 ⁴ cells / mL. 300 μl of cells were added to the trans 24 well inserts. 900ol of media was dispensed into the basolateral. After 2 to 3 days of washing with media for 10 to 14 days, all of the wells were used for the experiment when the values reached 600 to 700 Ω.

The cultured cells were treated with oxireservatol (10, 20, 30 / / mL) and cultured for 12 hours. Then, 1 mg / mL of FITC-dextran 4 kDa (SIGMA 46944) was treated and further cultured for 6 hours. The FITC-dextran 4 kDa concentration of the basolateral medium was measured using a fluorescence spectrometer at excitation 490 nm and emission wavelength 520 nm.

FIG. 3 is a graph comparing the transmittance of fluorescent materials in the intestinal epithelial cells treated with oxirestera virus according to one embodiment.

3, it was confirmed that the permeability of FITC-dextran 4 kDa was significantly lowered in the group treated with oxyreservatol compared with the negative control group (NC) not treated with oxyreservatrol. As a result, Is improved.

Example  4: On ox resveratrol  by Claudin -One , Occludin  And ZO -One  Expression

In order to identify at the RNA level the protein necessary for the formation of tight junctions, Claudin- 1, Occludin and ZO- 1 .

4-1) Intestinal epithelial cells ( intestinal epithelial cell culture Oxy Resveratrol  administration

10% FBS, 100 units / mL penicillin, and 100 ㎍ / mL streptomycin, 0.1 mmol / L non-essential amino acids, 1 mmol / L sodium pyruvate was added to 5% at 37 ℃ incubator CO ₂ -95 in MEM medium Caco-2 cells were cultured in the% air composition. If the cultured cells reached 80% confluence, the cells were detached using 0.25% trypsin, and the number of cells was adjusted to 2 × 10 ⁵ cells / mL. Cells were seeded in 6-well plates and stabilized in a 37 ° C incubator for one day. Oxy-resveratrol (10, 20, 30 / / mL) was treated the next morning and cultured for 24 hours.

4-2) RNA isolation and Expression level  Measure

RNA was isolated using Trizol. The conversion of the separated RNA into cDNA using a Revert Aid First Strand cDNA kit (Fermentas , EU) and then, DyNamo ™ HS SYBR Green qPCR kit (FINNZYMES, Finland) with a reagent StepOnePlus ^TM PCR was performed using a Real-Time PCR System (Applied Biosystems, Foster City, Calif., USA). The sequences of the primers used are as follows.

GAPDH : Sense: 5'- AAG GGT CAT CAT CTC TGC CC-3 ', Antisense: 5'-GTG ATG GCA TGG ACT GTG GT- 3'

Claudin - 1 : Sense: 5'- CGA TGA GGT GCA GAA GAT GA -3 ', Antisense: 5'- CCA GTG AAG AGA GCC TGA CC -3'

Occludin : Sense: 5'- TTT GTG GGA CAA GGA ACA CA 3 ', Antisense: 5'- TCA TTC ACT TTG CCA TTG GA T -3'

ZO - 1 : Sense: 5'- TGA GGC AGC TCA CAT AAT GC -3 ', Antisense: 5'- GGT CTC TGC TGG CTT GTT TC -3'

(2-deltadelta Ct method) of the gene expression amount of the sample (negative control: NC) which was not subjected to any treatment.

FIG. 4 is a graph comparing the expression of Claudin-1 , Occludin, and ZO- 1 genes in intestinal epithelial cells treated with Oxirreservat according to one embodiment.

4, the expression levels of Claudin- 1 , Occludin and ZO-1 genes were increased in a concentration-dependent manner in the group treated with oxis resveratrol (10, 20, 30 ㎍ / mL) .

Example  5: On ox resveratrol  by Claudin -One, Occludin  And ZO -1 protein expression

10% in MEM medium FBS, 100 units / mL penicillin, 100 ㎍ / mL streptomycin, 0.1 mmol / L non-essential amino acids, 5% CO 2 -95% to 37 ℃ incubator by the addition of 1 mmol / L sodium pyruvate Caco-2 cells were cultured in air. If the cultured cells reached 80% confluence, the cells were detached using 0.25% trypsin, and the number of cells was adjusted to 2 × 10 ⁵ cells / mL. Cells were seeded in a 60 mm dish and stabilized in a 37 ° C incubator for one day. The following morning, oxy resveratrol (10, 20, 30 / / mL) was treated and cultured for 48 hours. Subsequently, Pro-prep (intron, Korea) was used to obtain intracellular proteins. The protein loading of each sample was adjusted using the Bradford assay. The primary Ab used was GAPDH (Thermo MA5-15738, 1: 5,000), Claudin-1 (GeneTex GTX15098, 1: 100), Occludin (GeneTex GTX114949, Zo- Ab was Anti-mouse (Thermo NCI 1430KR, 1: 50,000), Anti-rabbit (Thermo NCI 1460KR, 1: 10,000).

FIG. 5 is a photograph showing expression of Claudin-1, Occludin and ZO-1 protein in the intestinal epithelial cells treated with oxis resveratrol according to an embodiment. FIG. 5 (A) is a group of negative control (NC), (B) is treated with 10 μg / ml of ox res reseratol, (C) is treated with 20 μg / ml of ox res reseratol and (D) is treated with 30 μg / ml of ox res reserat.

5, it was confirmed that the expression levels of Claudin-1, Occludin and ZO-1 protein were increased in the group treated with oxis resveratrol, compared with the negative control group (NC) not treated with oxyreservatrix.

Example  6: Oxy Resveratrol  The formation of tight junctions in the treated case was compared by staining.

10% in MEM medium FBS, 100 units / mL penicillin, 100 ㎍ / mL streptomycin, 0.1 mmol / L non-essential amino acids, 5% CO 2 -95% to 37 ℃ incubator by the addition of 1 mmol / L sodium pyruvate Caco-2 cells were cultured in air. If the cultured cells reached 80% confluence, the cells were detached using 0.25% trypsin, and the number of cells was adjusted to 2 × 10 ⁵ cells / mL. Cells were seeded in 24 wells precoated with Coverslip. And incubated with visual observation until a tight junction was formed sufficiently. After 10-14 days of incubation, oxyreservatrix (10, 20, 30 / / mL) was treated and incubated for 5 days. After fixed with 4% formaldehyde, the cells were stained with antibody. The primary Ab used was Claudin-1 (GeneTex GTX15098, 1: 200) and the secondary Ab was Anti-rabbit (Thermo 35553 1: 1,000).

FIG. 6 is a photograph showing formation of tight junctions in the intestinal epithelial cells treated with oxis resveratrol according to an embodiment. FIG. 6 (A) shows the group treated with the negative control (NC), (B) with 10 μg / ml of ox res reseratol, (C) 20 μg / ml of ox res reseratol and (D) with 30 μg / ml of ox res reserat.

6, it was confirmed that the amount of Claudin-1 expression was increased in the group treated with oxis resveratrol compared with the negative control group (NC) not treated with oxyreservatrix, and thus tight junctions were formed thickly.

Example  7: Oxy Resveratrol  For the case of processing PKC - 竜, PKC -θ, PKC -δ Expression

PKC- epsilon, PKC- theta, PKC- delta Claudin-1, Occludin, and protein kinase (PKC), which are necessary for the production of ZO-1, known as proteins that form tight junctions. The expression levels of PKC- ε, PKC- Θ and PKC- δ at the RNA level were compared.

7-1) Intestinal epithelial cells (intestinal epithelial cell) culture and Oxy Resveratrol  administration

10% in MEM medium FBS, 100 units / mL penicillin, 100 ㎍ / mL streptomycin, 0.1 mmol / L non-essential amino acids, 5% CO 2 -95% to 37 ℃ incubator by the addition of 1 mmol / L sodium pyruvate Caco-2 cells were cultured in air. If the cultured cells reached 80% confluence, the cells were detached using 0.25% trypsin, and the number of cells was adjusted to 2 × 10 ⁵ cells / mL. Cells were seeded in 6-well plates and stabilized in a 37 ° C incubator for one day. The following morning, oxy resveratrol (10, 20, 30 [mu] g / mL) was treated and cultured in a 37 DEG C incubator for 24 hours.

7-2) RNA isolation and Expression level  Measure

RNA was isolated using Trizol. The conversion of the separated RNA into cDNA using a Revert Aid First Strand cDNA kit (Fermentas , EU) and then, DyNamo ™ HS SYBR Green qPCR kit (FINNZYMES, Finland) with a reagent StepOnePlus ^TM PCR was performed using a Real-Time PCR System (Applied Biosystems, Foster City, Calif., USA). The sequences of the primers used are as follows.

GAPDH : Sense: 5'-AAG GGT CAT CAT CTC TGC CC-3 ', Antisense: 5'-GTG ATG GCA TGG ACT GTG GT-

PKC - ?: Sense: 5'- GAA CCC GGC GAG GAA ATA CA -3 ', Antisense: 5'-AGG GCA GGA ATG AAG AAC CG -3'

PKC - ?: Sense: 5'- ATG TCG AAT CAG AAC GGGCA -3 ', Antisense: 5'-TAG CAT TCG GCC TTG AGG TT -3'

PKC - δ : Sense: 5'- CCC TTC TGT GCC GTG AAG AT -3 ', Antisense: 5'- GCC CGC ATT AGC ACA ATC TG -3'.

(2-deltadelta Ct method) of the gene expression amount of the sample (negative control: NC) which was not subjected to any treatment.

7 is a graph comparing the PKC -ε, -θ PKC, PKC -δ gene expression in the field oxy resveratrol treatment in epithelial cells in accordance with one embodiment.

7 , PKC - epsilon, PKC - epsilon, and PKC - epsilon were determined depending on the concentration of oxyreservatol (10, 20, 30 g / mL) in the group treated with oxyreservatol compared to the negative control group ?, and PKC- ?, respectively.

Example  8: Oxy Resveratrol  For the case of processing ERK1 /2, JNK -a1, p-38  Expression

ERK1 / 2, JNK- a1, and p-38 are the MAPK pathway genes that are activated by the PKC pathway. Proteins required for the production of Claudin-1, Occludin and ZO-1 are compared at the RNA level.

8-1) Intestinal epithelial cells (intestinal epithelial cell) culture and Oxy Resveratrol  administration

10% in MEM medium FBS, 100 units / mL penicillin, 100 ㎍ / mL streptomycin, 0.1 mmol / L non-essential amino acids, 5% CO 2 -95% to 37 ℃ incubator by the addition of 1 mmol / L sodium pyruvate Caco-2 cells were cultured in air. If the cultured cells were grown to 80% confluence, the cells were removed using 0.25% trypsin and the number of cells was adjusted to 2 × 10 ⁵ cells / mL. Cells were seeded in 6-well plates and stabilized in a 37 ° C incubator for one day. Oxy-resveratrol (10, 20, 30 / / mL) was treated the next morning and cultured in a 37 ° C incubator.

8-2) RNA isolation and Expression level  Measure

RNA was isolated using Trizol. The conversion of the separated RNA into cDNA using a Revert Aid First Strand cDNA kit (Fermentas , EU) and then, DyNamo ™ HS SYBR Green qPCR kit (FINNZYMES, Finland) with a reagent StepOnePlus ^TM PCR was performed using a Real-Time PCR System (Applied Biosystems, Foster City, Calif., USA). The sequences of the primers used are as follows.

GAPDH : Sense: 5'- AAG GGT CAT CAT CTC TGC CC -3 ', Antisense: 5'-GTG ATG GCA TGG ACT GTG GT -3'

ERK - 1 : Sense: 5'- TCA GAC TCC AAA GCC CTT GAC -3 ', Antisense: 5'-CGT GCT GTC TCC TGG AAG ATG -3'

ERK - 2 : Sense: 5'- TCC AAC AGG CCC ATC TTT CC -3 ', Antisense: 5'- CCA GAG CTT TGG AGT CAGCA -3'

JNK - a1 : Sense: 5'-GCT TGG AAC ACC ATG TCC TGA -3 ', Antisense: 5'- GTA CGG GTG TTG GAG AGC TT -3'

P- 38 : Sense: 5'- ATG CAT AAT GGC CGA GCT GT -3 ', Antisense: 5'- GGT CAA CTT ACC CAG GGG ATT -3'

(2-deltadelta Ct method) of the gene expression amount of the sample (negative control: NC) which was not subjected to any treatment.

FIG. 8 is a graph comparing ERK1 / 2, JNK- a1, and p-38 gene expression in oxir reseratrol- treated intestinal epithelial cells according to an embodiment.

8, in the group treated with oxyreservatol , ERK1 / 2, JNK - 1, and JNK - 2 were determined depending on the concentration of oxyreservatrix (10, 20, 30 / / a1, and p-38, respectively.

Example  9: PKC  inhibitor Oxy Resveratrol  For the case of processing PKC -ε, PKC-θ, PKC -δ Expression

9-1) Intestinal epithelial cells (intestinal epithelial cell) culture and Oxy Resveratrol  administration

10% in MEM medium FBS, 100 units / mL penicillin, 100 ㎍ / mL streptomycin, 0.1 mmol / L non-essential amino acids, 5% CO 2 -95% to 37 ℃ incubator by the addition of 1 mmol / L sodium pyruvate Caco-2 cells were cultured in air. If the cultured cells reached 80% confluence, the cells were detached using 0.25% trypsin, and the number of cells was adjusted to 2 × 10 ⁵ cells / mL. Cells were seeded in 6-well plates and stabilized in a 37 ° C incubator for one day. The following morning, 8 μM of protein kinase C inhibitor (GF109203X) was treated and cultured for 2 hours. Oxirreservat (30 [mu] g / ml) was then treated and incubated for 24 hours.

9-2) RNA isolation and Expression level  Measure

RNA was isolated using Trizol. The conversion of the separated RNA into cDNA using a Revert Aid First Strand cDNA kit (Fermentas , EU) and then, DyNamo ™ HS SYBR Green qPCR kit (FINNZYMES, Finland) with a reagent StepOnePlus ^TM PCR was performed using a Real-Time PCR System (Applied Biosystems, Foster City, Calif., USA). The sequences of the primers used are as follows.

GAPDH : Sense: 5'-AAG GGT CAT CAT CTC TGC CC-3 ', Antisense: 5'-GTG ATG GCA TGG ACT GTG GT-

PKC - ?: Sense: 5'- GAA CCC GGC GAG GAA ATA CA -3 ', Antisense: 5'-AGG GCA GGA ATG AAG AAC CG -3'

PKC - ?: Sense: 5'- ATG TCG AAT CAG AAC GGGCA -3 ', Antisense: 5'-TAG CAT TCG GCC TTG AGG TT -3'

PKC - ?: Sense: 5'- CCC TTC TGT GCC GTG AAG AT -3 ', Antisense: 5'- GCC CGC ATT AGC ACA ATC TG -3'

(2-deltadelta Ct method) of the gene expression amount of the sample (negative control: NC) which was not subjected to any treatment.

9 is a graph comparing the PKC inhibitors (GF109203X) oxy and resveratrol-treated sheet epithelial cells -ε PKC, PKC -θ, PKC -δ gene expression, according to one embodiment.

9, PKC- epsilon, PKC- theta and PKC- delta expression levels were lowest in the case of treatment with PKC inhibitor alone compared to the negative control group, and PKC inhibitor and oxyreservatrix 30 μg / mL were treated together In one group, the expression levels of PKC- ε, PKC- Θ, and PKC- δ were increased.

Example  10: PKC  With inhibitor Oxy Resveratrol  For the case of processing ERK1 / 2, JNK-a1, p-38 Expression

In order to confirm the activation of the MAPK pathway by the PKC pathway, the PKC inhibitor and oxis resveratrol were treated to compare their expression levels at the RNA level.

10-1) Intestinal epithelial cells (intestinal epithelial cell) culture and Oxy Resveratrol  administration

10% in MEM medium FBS, 100 units / mL penicillin, 100 ㎍ / mL streptomycin, 0.1 mmol / L non-essential amino acids, 5% CO 2 -95% to 37 ℃ incubator by the addition of 1 mmol / L sodium pyruvate Caco-2 cells were cultured in air. If the cultured cells reached 80% confluence, the cells were detached using 0.25% trypsin, and the number of cells was adjusted to 2 × 10 ⁵ cells / mL. Cells were seeded in 6-well plates and stabilized in a 37 ° C incubator for one day. The following morning, 8 μM of PKC inhibitor (GF109203X) was treated and cultured for 2 hours. Oxirreservat (30 [mu] g / ml) was then treated and incubated for 24 hours.

10-2) RNA isolation and Expression level  Measure

RNA was isolated using Trizol. The conversion of the separated RNA into cDNA using a Revert Aid First Strand cDNA kit (Fermentas , EU) and then, DyNamo ™ HS SYBR Green qPCR kit (FINNZYMES, Finland) with a reagent StepOnePlus ^TM PCR was performed using a Real-Time PCR System (Applied Biosystems, Foster City, Calif., USA). The sequences of the primers used are as follows.

GAPDH : Sense: 5'- AAG GGT CAT CAT CTC TGC CC -3 ', Antisense: 5'-GTG ATG GCA TGG ACT GTG GT -3'

ERK - 1 : Sense: 5'- TCA GAC TCC AAA GCC CTT GAC -3 ', Antisense: 5'-CGT GCT GTC TCC TGG AAG ATG -3'

ERK - 2 : Sense: 5'- TCC AAC AGG CCC ATC TTT CC -3 ', Antisense: 5'- CCA GAG CTT TGG AGT CAGCA -3'

JNK - a1 : Sense: 5'-GCT TGG AAC ACC ATG TCC TGA -3 ', Antisense: 5'- GTA CGG GTG TTG GAG AGC TT -3'

P- 38 : Sense: 5'- ATG CAT AAT GGC CGA GCT GT -3 ', Antisense: 5'- GGT CAA CTT ACC CAG GGG ATT -3'

(2-deltadelta Ct method) of the gene expression amount of the sample (negative control: NC) which was not subjected to any treatment.

10 is a graph comparing ERK1 / 2, JNK- a1, and p-38 gene expression in intestinal epithelial cells treated with PKC inhibitor (GF109203X) according to one embodiment and oxyreservatrix.

10, the expression level of ERK1 / 2, JNK- a1, and p-38 was lowest in the case of treatment with PKC inhibitor alone compared to the negative control, and the PKC inhibitor and oxyreservatrix 30 ㎍ / The expression of ERK1 / 2, JNK- a1 and p-38 was increased in one group.

Example  11: PKC  With inhibitor Oxy Resveratrol  For the case of processing Claudin -One , Occludin  And ZO -One  Expression

11-1) Intestinal epithelial cells (intestinal epithelial cell) culture and Oxy Resveratrol  administration

10% in MEM medium FBS, 100 units / mL penicillin, 100 ㎍ / mL streptomycin, 0.1 mmol / L non-essential amino acids, 5% CO 2 -95% to 37 ℃ incubator by the addition of 1 mmol / L sodium pyruvate Caco-2 cells were cultured in air. If the cultured cells reached 80% confluence, the cells were detached using 0.25% trypsin, and the number of cells was adjusted to 2 × 10 ⁵ cells / mL. Cells were seeded in 6-well plates and stabilized in a 37 ° C incubator for one day. The following morning, 8 μM of PKC inhibitor (GF109203X) was treated and cultured for 2 hours. Oxirreservat (30 [mu] g / ml) was then treated and incubated for 24 hours.

11-2) RNA isolation and Expression level  Measure

RNA was isolated using Trizol. The conversion of the separated RNA into cDNA using a Revert Aid First Strand cDNA kit (Fermentas , EU) and then, DyNamo ™ HS SYBR Green qPCR kit (FINNZYMES, Finland) with a reagent StepOnePlus ^TM PCR was performed using a Real-Time PCR System (Applied Biosystems, Foster City, Calif., USA). The sequences of the primers used are as follows.

GAPDH : Sense: 5'- AAG GGT CAT CAT CTC TGC CC -3 ', Antisense: 5'-GTG ATG GCA TGG ACT GTG GT -3'

Claudin - 1 : Sense: 5'- CGA TGA GGT GCA GAA GAT GA -3 ', Antisense: 5'- CCA GTG AAG AGA GCC TGA CC -3'

Occludin : Sense: 5'- TTT GTG GGA CAA GGA ACA CA 3 ', Antisense: 5'- TCA TTC ACT TTG CCA TTG GA T -3'

ZO - 1 : Sense: 5'- TGA GGC AGC TCA CAT AAT GC -3 ', Antisense: 5'- GGT CTC TGC TGG CTT GTT TC -3'

(2-deltadelta Ct method) of the gene expression amount of the sample (negative control: NC) which was not subjected to any treatment.

11 is a graph comparing the expression of Claudin- 1 , Occludin and ZO- 1 genes in the intestinal epithelial cells treated with the PKC inhibitor (GF109203X) according to one embodiment and oxyreservatrix .

Referring to Figure 11, if the process only, PKC inhibitor in comparison to the negative control is to check the Claudin-1, the lowest amount Occludin and ZO -1 expression, as the group treated with the PKC inhibitor oxy resveratrol 30 ㎍ / mL , The amount of expression of Claudin- 1 , Occludin and ZO-1 was increased.

Example  12: Oxy Resveratrol  Transcription factor (cdx-2) expression in the treated case

Caudal-related homeobox (cdx-2) is a transcription factor that is expressed by the MAPK pathway.

12-1) Intestinal epithelial cells (intestinal epithelial cell) culture and Oxy Resveratrol  administration

10% in MEM medium FBS, 100 units / mL penicillin, 100 ㎍ / mL streptomycin, 0.1 mmol / L non-essential amino acids, 5% CO 2 -95% to 37 ℃ incubator by the addition of 1 mmol / L sodium pyruvate Caco-2 cells were cultured in air. If the cultured cells reached 80% confluence, the cells were detached using 0.25% trypsin, and the number of cells was adjusted to 2 × 10 ⁵ cells / mL. Cells were seeded in 6-well plates and stabilized in a 37 ° C incubator for one day. Oxy-resveratrol (10, 20, 30 / / mL) was treated the next morning and cultured in a 37 ° C incubator.

12-2) RNA isolation and measurement of expression level

RNA was isolated using Trizol. The conversion of the separated RNA into cDNA using a Revert Aid First Strand cDNA kit (Fermentas , EU) and then, DyNamo ™ HS SYBR Green qPCR kit (FINNZYMES, Finland) with a reagent StepOnePlus ^TM PCR was performed using a Real-Time PCR System (Applied Biosystems, Foster City, Calif., USA). The sequences of the primers used are as follows.

GAPDH : Sense: 5'-AAG GGT CAT CAT CTC TGC CC-3 ', Antisense: 5'-GTG ATG GCA TGG ACT GTG GT-

cdx - 2 : Sense: 5'- GCA GCC AAG TGA AAA CCA GG -3 ', Antisense: 5'- TTC CTC TCC TTT GCT CTG CG -3'

(2-deltadelta Ct method) of the gene expression amount of the sample (negative control: NC) which was not subjected to any treatment.

FIG. 12 is a graph illustrating the effect of the oxir reseratrol-treated intestinal epithelial cell transcription factor (cdx-2) Gene expression.

12, the expression level of cdx-2 in the group treated with oxis resveratrol (10, 20, 30 / / mL) dependent on the concentration of oxis resveratrol Increase in the number of workers.

Example  13: PKC  inhibitor Oxy Resveratrol  Transcription factor ( cdx -2) expression

13-1) Intestinal epithelial cells (intestinal epithelial cell) culture and Oxy Resveratrol  administration

10% in MEM medium FBS, 100 units / mL penicillin, 100 ㎍ / mL streptomycin, 0.1 mmol / L non-essential amino acids, 5% CO 2 -95% to 37 ℃ incubator by the addition of 1 mmol / L sodium pyruvate Caco-2 cells were cultured in air. If the cultured cells reached 80% confluence, the cells were detached using 0.25% trypsin, and the number of cells was adjusted to 2 × 10 ⁵ cells / mL. Cells were seeded in 6-well plates and stabilized in a 37 ° C incubator for one day. The following morning, 8 μM of PKC inhibitor (GF109203X) was treated and cultured for 2 hours. Oxirreservat (30 [mu] g / ml) was then treated and incubated for 24 hours.

13-2) RNA isolation and measurement of expression level

RNA was isolated using Trizol. The conversion of the separated RNA into cDNA using a Revert Aid First Strand cDNA kit (Fermentas , EU) and then, DyNamo ™ HS SYBR Green qPCR kit (FINNZYMES, Finland) with a reagent StepOnePlus ^TM PCR was performed using a Real-Time PCR System (Applied Biosystems, Foster City, Calif., USA). The sequences of the primers used are as follows.

GAPDH : Sense: 5'-AAG GGT CAT CAT CTC TGC CC-3 ', Antisense: 5'-GTG ATG GCA TGG ACT GTG GT-

cdx - 2 : Sense: 5'- GCA GCC AAG TGA AAA CCA GG -3 ', Antisense: 5'- TTC CTC TCC TTT GCT CTG CG -3'

(2-deltadelta Ct method) of the gene expression amount of the sample (negative control: NC) which was not subjected to any treatment.

FIG. 13 is a graph comparing the expression of the intestinal epithelial transcription factor (cdx-2) gene treated with the PKC inhibitor (GF109203X) according to one embodiment and oxyreservatrix.

13, the expression level of cdx-2 was lowest in the case of treatment with PKC inhibitor alone compared with the negative control. In the group treated with PKC inhibitor and oxyreservatrix at 30 ㎍ / mL, the expression level of cdx-2 .

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. For example, if the techniques described are performed in a different order than the described methods, and / or if the described components are combined or combined in other ways than the described methods, or are replaced or substituted by other components or equivalents Appropriate results can be achieved.

Therefore, other implementations, other embodiments, and equivalents to the claims are also within the scope of the following claims.

Claims (8)

It contains oxyresveratrol as an active ingredient,
The pharmaceutical composition for preventing or treating inflammatory bowel disease, wherein the oxyservatrol induces the expression of Claudin-1 in intestinal epithelial cells.
The method according to claim 1,
The pharmaceutical composition for preventing or treating inflammatory bowel disease, wherein the oxyreservatrix induces formation of tight junctions in intestinal epithelial cells.
The method according to claim 1,
The oxir reseratol induces expression of at least one gene selected from the group consisting of Occludin, ZO-1, ERK1 / 2, JNK-a1, p-38, PKC-epsilon, PKC-theta and PKC- ≪ / RTI > or a pharmaceutically acceptable salt thereof, for the prophylaxis or treatment of inflammatory bowel disease.
The method according to claim 1,
The pharmaceutical composition for preventing or treating inflammatory bowel disease, wherein the above-mentioned oxyservatrol induces the expression of intestinal epithelial cell transcription factor cdx-2.
The method according to claim 1,
Wherein the inflammatory bowel disease is one or more selected from the group consisting of Chron's disease, ulcerative colitis, intestinal Behcet's disease, intestinal tuberculosis and enteritis, inflammatory bowel disease comprising one or more selected from the group consisting of Chron's disease, ulcerative colitis, A pharmaceutical composition for preventing or treating disease.
delete The method according to claim 1,
A pharmaceutical composition for preventing or treating inflammatory bowel disease, which further comprises a pharmaceutically acceptable carrier. delete

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