KR20230039145A - Composition for Preventing or Treating Gastrointestinal Mucosal Injury - Google Patents

Abstract

The present invention relates to a composition for preventing or treating gastrointestinal mucosal damage comprising 7-hydroxy-6-methoxy-2H-1-benzopyran-2-one as an active ingredient. When using the pharmaceutical composition and/or functional food composition of the present invention, it is possible to prevent, alleviate or treat esophageal and gastrointestinal mucosal damage diseases by effectively increasing the production of mucus and other mucosal protective factors and stabilizing the esophageal and gastrointestinal mucosa.

Description

Composition for preventing or treating gastric mucosal damage {Composition for Preventing or Treating Gastrointestinal Mucosal Injury}

The present invention relates to a composition for preventing or treating gastrointestinal mucosal damage comprising 7-hydroxy-6-methoxy- 2H -1-benzopyran-2-one as an active ingredient.

The gastric mucous membrane is the most superficial layer that protects the stomach wall against gastric acid and various digestive enzymes secreted from the stomach. This can lead to gastrointestinal diseases such as gastritis and gastric ulcer. On the other hand, since the intestinal mucosa plays a role in preventing various bacteria or harmful substances from being absorbed into the blood, when the intestine is weakened or the intestinal mucosa is damaged, problems occur in the body's first defense system. In particular, problems such as leaky gut syndrome (leaky gut syndrome) can occur in which indigestible food, toxins, bacteria, etc. can flow into the blood due to microscopic holes in the intestine. treats it as a foreign substance and causes an excessive immune response, which can cause allergic reactions, atopy due to liver detoxification overload, irritable bowel syndrome, Crohn's disease, and chronic fatigue syndrome.

Meanwhile, an HCl/ethanol-induced gastric ulceration rat model was established by Mizui and Doteuchi in 1983 (Mizui T and Doteuchi M, Effect of polyamines on acidified ethanol-induced gastric lesions in rats. Jpn J Pharmacol 33:939- 945 (1983)). The acidified ethanol induced gastritis model has long been widely used to screen for new gastroprotective agents and study the underlying protective molecular mechanism. In this model, ethanol induces necrotic damage to the gastric mucosa through various mechanisms. For example, ethanol affects hydrogen ion leakage, mucosal blood flow, and gastric acid secretion. Additionally, HCl enhances the action of ethanol in this protocol. Thus, this model represents the most common cause of gastric ulcer in humans. The mucosal layer of the stomach is a viscoelastic gel barrier that serves as the first line of defense against various biological, physical and chemical damages. The major components of the gastric mucus barrier are mucins 5AC (MUC5AC) and MUC6, which are secreted gel-forming mucins that increase body fluid viscosity. The trefoil factor (TFF) family includes three related spasmolytic polypeptides (TFF1-3) each containing two TFF domains. TFF1 and TFF2 are expressed in both duodenal and gastric epithelium. TFF3 is expressed primarily by intestinal goblet cells. TFF provides a scaffold for mucins to protect mucosal integrity, restoring it to its previous state or better after gastric injury. For example, TFF2 maintains mucosal integrity and prevents tumor formation in the stomach. Moreover, TFF is known to interact with mucins. For example, MUC5AC interacts with TFF1 at the gastric mucosal barrier and MUC6 interacts with TFF2. Substances that induce the expression of mucins (MUC5AC or MUC6) or TFF can be promising candidates for the treatment of gastritis. Nam et al. reported that geranylgeranylacetone, an acyclic isoprenoid, protects against ethanol-induced gastric damage by increasing MUC5AC and MUC6 levels (Nam SY, Kim N, Lee CS, Choi KD, Lee HS, Jung HC and Song IS, Gastric mucosal protection via enhancement of MUC5AC and MUC6 by geranylgeranylacetone.Dig Dis Sci 50:2110-2120 (2005)).

The present inventors intensively researched to identify new active ingredients capable of preventing or treating damage to the esophageal and gastric mucosa. As a result, when using a composition containing the 7-hydroxy-6-methoxy- 2H -1-benzopyran-2-one compound, the production of mucus and other mucosal protective factors is increased, and the esophageal and gastric mucosa are stabilized. By identifying that it can be, the present invention was completed.

Accordingly, an object of the present invention is to provide a novel pharmaceutical composition for preventing or treating esophageal or gastrointestinal mucosal damage diseases.

Another object of the present invention is to provide a novel functional food composition for preventing or improving esophageal or gastrointestinal mucosal damage diseases.

Another object of the present invention is to provide a functional food composition for increasing the expression of one or more proteins selected from the group consisting of TFF1, TFF2, MUC6, MUC5AC and rPigr in the esophageal or gastric mucosa.

According to one aspect of the present invention, the esophageal or gastrointestinal mucosa comprising 7-hydroxy-6-methoxy- 2H -1-benzopyran-2-one or a pharmaceutically acceptable salt thereof as an active ingredient It provides a pharmaceutical composition for preventing or treating damaged diseases.

The present inventors intensively researched to identify new active ingredients capable of preventing or treating damage to the esophageal and gastric mucosa. As a result, when using a composition containing the 7-hydroxy-6-methoxy- 2H -1-benzopyran-2-one compound, the production of mucus and other mucosal protective factors is increased, and the esophageal and gastric mucosa are stabilized. It was established that it can.

7-Hydroxy-6-methoxy- 2H -1-benzopyran-2-one of the present invention is a compound having the following chemical structure and is a coumarin-based compound:

7-Hydroxy-6-methoxy- 2H -1-benzopyran-2-one compound is a commercially available compound from commercial suppliers of the compound, and natural plants previously known to contain the compound. Compounds extracted from can be used.

Esophageal or gastrointestinal mucosal damage disease of the present invention refers to esophageal or gastrointestinal mucosal damage and various esophageal or gastrointestinal symptoms and diseases caused thereby.

In one embodiment of the present invention, the esophageal or gastrointestinal mucosal damage disease is selected from esophageal mucosal damage, gastric mucosal damage, intestinal mucosal damage, gastritis, gastric ulcer, reflux esophagitis or leaky gut syndrome (Leaky Gut Syndrome) can

Gastritis refers to a case where only the gastric mucosa, which is the first layer of the four-layered gastric wall, is damaged, and is pathologically defined as a state in which inflammatory cells infiltrate the gastric mucosa. Gastritis is divided into acute and chronic according to the temporal concept, and can be classified according to histological findings, anatomical distribution, and pathological mechanisms, and can be variously classified according to endoscopic findings.

Gastritis of the present invention can be divided into acute and chronic as described above, and acute gastritis includes erosive gastritis and acute hemorrhagic gastritis. Acute hemorrhagic gastritis is when the lining of the stomach peels off slightly. Chronic gastritis includes superficial gastritis, atrophic gastritis and metaplastic gastritis. The classification of chronic gastritis is based on endoscopic findings, and superficial gastritis refers to a case in which there is irregular redness on the surface of the stomach on gastric endoscopy or a comb-like red line scratched with a fingernail. Atrophic gastritis refers to a case where inflammation of the stomach lasts for a long time and the gastric mucosa becomes thin enough to show blood vessels. Metabolic gastritis refers to a case in which the gastric mucosa is irritated for a long time and loses its original appearance and changes to the mucous membrane of the small intestine or colon.

In one embodiment of the present invention, the gastritis of the present invention is selected from the group consisting of erosive gastritis, acute hemorrhagic gastritis, superficial gastritis, atrophic gastritis, and metaplastic gastritis.

The gastric ulcer of the present invention refers to a case where more than the second layer (submucosa) of the four-layered gastric wall is damaged and even the gastric muscles are exposed. If gastric ulcers are left untreated, complications of gastric perforation may occur, in which gastric muscle layer is melted and a hole is formed in the gastric wall. In this case, gastric acid or stomach contents spread throughout the stomach, causing inflammation. Gastric bleeding can also occur if blood vessels are exposed at the bottom of the ulcer and rupture.

In the case of reflux esophagitis, it means a disease that causes damage to the esophageal mucosa by refluxing stomach contents into the esophagus.

Leaky Gut Syndrome, also called leaky gut syndrome, is when the cells of the intestinal mucosa maintain a gap and become loose or damaged due to any stimulation or damage, and foreign substances such as various bacteria or harmful substances pass through the intestine and enter the bloodstream. As a result, various immune reactions occur, which means all the symptoms that are problematic.

In the case of using a pharmaceutical composition for preventing or treating esophageal or gastrointestinal mucosal damage disease comprising 7-hydroxy-6-methoxy- 2H -1-benzopyran-2-one as an active ingredient of the present invention, Various diseases can be effectively prevented or treated.

In one embodiment of the present invention, the composition of the present invention has an activity to increase the expression of one or more selected from the group consisting of TFF1, TFF2, MUC1, MUC4, MUC6, MUC5AC and rPigr. TFF1 and TFF2 are spasmolytic polypeptides each containing two trefoil factor (TFF) domains, and TFF provides a scaffold for mucins to protect mucosal integrity to restore the previous state after gastric injury. It is known to be restored to or better condition. Mucins 5AC (MUC5AC) and MUC6 are major components of the gastric mucus barrier, secreted gel-forming mucins that increase body fluid viscosity. The aforementioned TFF and mucins are known to interact, for example, MUC5AC interacts with TFF1 in the gastric mucosal barrier and MUC6 interacts with TFF2, and the present invention induces the expression of mucins (MUC5AC or MUC6) and TFF. The composition of the present invention can be effectively used for the treatment of gastritis.

The 7-hydroxy-6-methoxy- 2H -1-benzopyran-2-one compound of the present invention can be used in the form of a pharmaceutically acceptable salt, and the salt is a pharmaceutically acceptable free acid (free acid). acid) can be used. Acid addition salts are formed with inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, and aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and alkanes. It is obtained from non-toxic organic acids such as dioate, aromatic acids, aliphatic and aromatic sulfonic acids, and organic acids such as acetic acid, benzoic acid, citric acid, lactic acid, maleic acid, gluconic acid, methanesulfonic acid, 4-toluenesulfonic acid, tartaric acid and fumaric acid. These pharmaceutically non-toxic salts include sulfate, pyrosulfate, bisulphate, sulphite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, ioda Id, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate , sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitro benzoate, hydroxybenzoate, Toxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycol but is not limited to late, maleate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate or mandelate.

The acid addition salt according to the present invention is produced by dissolving the flavanone derivative of formula 1 in an organic solvent such as methanol, ethanol, acetone, methylene chloride, acetonitrile, etc. by a conventional method, for example, and adding an organic or inorganic acid. It may be prepared by filtering and drying the precipitate, or by distilling the solvent and excess acid under reduced pressure and then drying or crystallizing in an organic solvent.

In addition, a pharmaceutically acceptable metal salt may be prepared using a base. An alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and evaporating and drying the filtrate. At this time, it is pharmaceutically suitable to prepare sodium, potassium or calcium salts as metal salts. In addition, the corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable negative salt (eg, silver nitrate). In addition, the present invention includes not only the thienopyrimidine derivative represented by Formula 1 and pharmaceutically acceptable salts thereof, but also possible solvates, hydrates, stereoisomers and the like that can be prepared therefrom.

The pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier in addition to the active ingredient. The pharmaceutically acceptable carrier included in the pharmaceutical composition of the present invention is one commonly used in formulation, and includes lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, including, but not limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil; it is not going to be The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like in addition to the above components. Suitable pharmaceutically acceptable carriers and agents are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

A suitable dosage of the pharmaceutical composition of the present invention is variously prescribed by factors such as formulation method, administration method, patient's age, weight, sex, medical condition, food, administration time, administration route, excretion rate and response sensitivity. It can be. On the other hand, the dosage of the pharmaceutical composition of the present invention is preferably 0.001-1000 mg/kg (body weight) per day.

The pharmaceutical composition of the present invention can be administered orally or parenterally, and in the case of parenteral administration, it can be administered by topical application to the skin, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, etc. . More specifically, considering that the pharmaceutical composition of the present invention is applied for the treatment of esophageal and gastrointestinal mucosal damage, it is preferably administered orally.

The pharmaceutical composition of the present invention is prepared in unit dosage form by formulation using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by those skilled in the art. or it may be prepared by incorporating into a multi-dose container. At this time, the dosage form is, for example, oral (tablets, capsules, powders), buccal, sublingual, rectal, vaginal, intranasal, topical or parenteral (intravenous, corpus cavernosum, intramuscular, subcutaneous and intraductal). Including) may be a dosage form. For example, the pharmaceutical composition according to the present invention may be in the form of a tablet containing starch or lactose, either alone or in the form of a capsule containing excipients, or as an elixir or suspension containing flavoring or coloring chemicals. It can be administered orally, buccally or sublingually in all forms. Liquid formulations may contain suspending agents (e.g. methylcellulose, semi-synthetic glycerides such as witepsol or mixtures of apricot kernel oil and PEG-6 esters or PEG-8 and caprylic/capric glycerides). together with pharmaceutically acceptable excipients such as mixtures of glycerides). Solid preparations for oral administration include tablets, pills, powders, granules, capsules, troches, etc., and these solid preparations contain at least one excipient such as starch, calcium carbonate, It may be prepared by mixing sucrose or lactose or gelatin. In addition to simple excipients, lubricants such as magnesium styrate and talc may also be used. Liquid formulations for oral administration include suspensions, internal solutions, emulsions, or syrups. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. can

Formulations for oral administration include sterilized aqueous solutions, non-aqueous solutions, suspension solutions, emulsions, freeze-dried formulations, suppositories, and the like. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending solvents. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerol, gelatin, and the like may be used.

In addition, when injected parenterally, for example, intravenously, intracavernosal, intramuscularly, subcutaneously, and intraluminally, it is most preferable to use it in the form of a sterile aqueous solution, in which case the solution is isotonic with blood. It may also contain other substances (eg salts or monosaccharides such as mannitol or glucose).

According to another aspect of the present invention, the present invention is a functional food for preventing or improving esophageal or gastrointestinal mucosal damage disease comprising 7-hydroxy-6-methoxy-2H-1-benzopyran-2-one as an active ingredient composition is provided.

In one embodiment of the present invention, the esophageal or gastrointestinal mucosal damage disease of the present invention is esophageal mucosal damage, gastric mucosal damage, intestinal mucosal damage, gastritis, gastric ulcer, reflux esophagitis or leaky gut syndrome (Leaky Gut Syndrome). .

In one embodiment of the present invention, the gastritis of the present invention is selected from the group consisting of erosive gastritis, acute hemorrhagic gastritis, superficial gastritis, atrophic gastritis, and metaplastic gastritis.

The functional food composition of the present invention includes 7-hydroxy-6-methoxy-2H-1-benzopyran-2-one, which is the same compound as the pharmaceutical composition according to another aspect of the present invention, and It relates to a functional food composition that exhibits an effect of preventing or improving various diseases that can be prevented or treated by a medical composition and the same diseases. In order to avoid excessive complexity of the description, the contents are used and the description is omitted.

The functional food composition of the present invention includes components commonly added during food preparation, and may include, for example, proteins, carbohydrates, fats, nutrients, and/or seasonings. For example, when prepared as a drink, a flavoring agent or natural carbohydrate may be included as an additional ingredient in addition to the active ingredient. For example, natural carbohydrates include monosaccharides (eg, glucose, fructose, etc.); disaccharides (eg, maltose, sucrose, etc.); oligosaccharide; polysaccharides (eg, dextrins, cyclodextrins, etc.); and sugar alcohols (eg, xylitol, sorbitol, erythritol, etc.).

As the flavoring agent, natural flavoring agents (eg, thaumatin, stevia extract, etc.) and synthetic flavoring agents (eg, saccharin, aspartame, etc.) may be used.

According to another aspect of the present invention, the present invention provides TFF1, TFF2, MUC6, TFF1, TFF2, MUC6, It provides a functional food composition for increasing the expression of one or more proteins selected from the group consisting of MUC5AC and rPigr.

The functional food composition according to one aspect of the present invention includes 7-hydroxy-6-methoxy-2H-1-benzopyran-2-one, which is the same compound as the pharmaceutical composition according to another aspect of the present invention described above And, it relates to a functional food composition showing the effect of preventing or improving the same diseases as various diseases that can be prevented or treated by the pharmaceutical composition, and the above-mentioned functional food composition and invention according to another aspect of the present invention In order to avoid excessive complexity of the description in this specification, for the overlapping content among the detailed descriptions of the pharmaceutical composition and the functional food composition, the content is used and the description is omitted.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a novel pharmaceutical composition for preventing or treating esophageal or gastrointestinal mucosal damage diseases.

(b) The present invention provides a novel functional food composition for preventing or improving esophageal or gastrointestinal mucosal damage diseases.

(c) The present invention provides a functional food composition for increasing the expression of one or more proteins selected from the group consisting of TFF1, TFF2, MUC6, MUC5AC and rPigr in the esophageal or gastric mucosa.

(d) When the pharmaceutical composition and/or functional food composition of the present invention is used, it effectively increases the production of mucus and other mucosal protective factors and stabilizes the esophageal and gastrointestinal mucosa, thereby preventing, improving or preventing esophageal and gastrointestinal mucosal damage diseases can be cured

Figure 1 shows the results of observing cell viability after administering 7-hydroxy-6-methoxy-2H-1-benzopyran-2-one compound (HMC) by dose to SNU-601 cells.
Figure 2 shows the results of confirming the effect of enhancing the expression of mucosal stabilizing factors by the 7-hydroxy-6-methoxy-2H-1-benzopyran-2-one compound in epithelial cells.
Figure 3 shows the results confirming that the ERK pathway acts as a mucosal stabilization mechanism by 7-hydroxy-6-methoxy-2H-1-benzopyran-2-one compound in epithelial cells.
Figure 4 shows the effect of reducing damage to the gastric mucosa by the 7-hydroxy-6-methoxy-2H-1-benzopyran-2-one compound in the gastric injury model induced by hydrochloric acid-ethanol, which is an injurious mucosal disease model. Indicates the checked result.
5 shows the effect of inducing the expression of mucosal stabilizing factors by 7-hydroxy-6-methoxy-2H-1-benzopyran-2-one compound in the gastric injury model induced by hydrochloric acid-ethanol, which is a model of damaging mucosal disease shows the results of checking.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for explaining the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

Example 1: Cell culture and reagents

SNU-601 (human gastric epithelial) and RGM-1 (rat gastric epithelial) cells were cultured in RPMI 1640 (Gibco BRL) or Dulbecco's modified Eagle's medium (DMEM; Gibco BRL) at 37°C and 5% CO ₂ conditions. Hydrogen peroxide (H ₂ O ₂ ), 7-hydroxy-6-methoxy-2H-1-benzopyran-2-one, dimethyl sulfoxide (DMSO) and Alcian blue 8GX were obtained from Sigma-Aldrich. Antibodies against extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (p38), phosphorylated ERK, and phosphorylated p38 were purchased from Cell Signaling Technology (Danvers, MA, USA).

Example 2: Animals

Six-week-old male Sprague-Dawley rats weighing 180-200 g were purchased from Daehan Biolink (Korea). Prior to experimentation, animals were allowed to acclimate for 7 days. Normal chow and sterile tap water were provided ad libitum . All experimental procedures, including handling and care of adult rats, were approved by the Institutional Animal Care and Use Committee (SKKUIACUC2020-09-25-2) of Sungkyunkwan University, Suwon, Republic of Korea.

Example 3: Hydrochloric acid / ethanol (HCl / EtOH) induced mouse gastric ulcer model

To investigate the mucosal protective effect of 7-hydroxy-6-methoxy-2H-1-benzopyran-2-one, a gastric ulcer model induced by HCl/EtOH was used. Seven-week-old rats were divided into groups of eight rats and treated as described below. For single dose studies, rats were fasted for 24 hours followed by vehicle (negative control) 1.5 mL, 0.5 mg/kg or 1.0 mg/kg of 7-hydroxy-6-methoxy-2H-1-benzopyran-2 -On was administered orally.

After 30 minutes, 1.5 mL of 60% ethanol containing 150 mM HCl was administered via oral gavage to induce gastritis. After 1 hour, the rats were sacrificed and the stomach was dissected for analysis. For a multi-dose regimen study, rats were orally administered vehicle or 7-hydroxy-6-methoxy-2H-1-benzopyran-2-one twice a day at 6-hour intervals for 5 days. Animals were fasted for 24 hours prior to final dosing. The experimental procedure after the final dose was the same as in the single dose study. The total length of gastric superficial lesions was measured using ImageJ to calculate the index of each gastric lesion. For normalization, the mucosal damage length of the control group was used. The rate of inhibition of gastric lesions was calculated using the previously known formula shown below:

(%) = (control group gastric lesion length [mm] - drug-administered gastric lesion length [mm]) / control gastric lesion length (mm)

Example 4: Alcian Blue staining

Mucus was quantified relative to the amount of alcian blue bound to the gastric epithelium. The fixed tissues were weighed and incubated in 3% acetic acid for 10 minutes. Then, the tissue was stained with 1% alcian blue (pH 2.5) for 10 minutes and washed with running tap water for 15 minutes. Bound Alcian Blue was eluted by sonication in DMSO for 10 minutes and centrifugation at 6000 rpm for 15 minutes. The supernatant (50 μL) was aliquoted into a 96-well plate and absorbance was measured at 605 nm.

Example 5: MTT assay

SNU-601 cells were seeded in a 96-well plate at 5×10 ³ cells per well and cultured for 24 hours. Cells were then treated with various concentrations of 7-hydroxy-6-methoxy-2H-1-benzopyran-2-one (0.3 to 4.8 μg/mL) for 24 hours. After treatment, all medium was removed and fresh medium containing 10 μL/well of MTT solution (Promega, Madison, WI, USA) was added. Plates were incubated at 37° C. for 2 hours. Purple formazan crystals were dissolved in dimethyl sulfoxide (DMSO) and absorbance was detected at 490 nm. Cytotoxicity was calculated relative to untreated cells.

Example 6: Real-time PCR

SNU-601 cells were seeded in a 6-well plate (1x10 ⁶ cells/mL) and serum-starved for 24 hours. Cells were then treated with different concentrations of 7-hydroxy-6-methoxy-2H-1-benzopyran-2-one compound for 24 hours. RT-PCR was performed as previously described (SJ Hwang, SH Jun, Y. Park, SH Cha, M. Yoon, S. Cho, HJ Lee, Y. Park, Green synthesis of gold nanoparticles using chlorogenic acid and their enhanced performance for inflammation, Nanomed.: Nanotechnol. Biol. Med. 11 (7) (2015) 1677-1688).

Briefly, mRNA was isolated using TRIzol reagent (Invitrogen), and cDNA was synthesized using M-MLV Reverse Transcriptase (Promega). Real-time PCR was performed on a Rotor-Gene Q cycler (Qiagen) using the Rotor Gene SYBR Green RT-PCR kit (Qiagen) according to the manufacturer's instructions. PCR primers were purchased from Bioneer (Korea). Their sequence is as follows:

forward strand human actin 5'-AGAGCTACGAGCTGCCTGAC-3', reverse strand human actin 5'-AGCACTGTGTTGGCGTACAG-3', forward strand human TFF1 5'-CGCCCTCCCAGTGTGTGCAAATAAG-3', reverse strand human TFF1 5'-GAACGGTGTCGTCGAAACAGCAG-3'; forward strand human TFF2 5'-ATGCTGTTTCGACTCCAGTGTCA-3', reverse strand human TFF2 5'-CCACA-GTTTCTTCGGTCTGAGAC-3'; forward strand human TFF3 5'-CCAAGCAAACAATCCAGAGCA-3', reverse strand human TFF3 5'-GCTCAGGACTCGCTTCATGGT-3', forward strand human MUC5AC 5'-CAGCACAACCCCTGTTTCAAA-3', reverse strand human MUC5AC 5'-GCGCACAGAGGATGACAGTCA-3'; Forward strand human MUC6 5′-CTGCCTATACCAGCAATGGA-3′, reverse strand human MUC6 5′-CTGACCCATGTACTTCCGCTC-3′; Forward strand human high molecular immunoglobulin receptor (pIgR) 5′-AGGTGGGTGGGTAGTAGCACG-3′, reverse strand human pIgR 5′-AGTCCCATATTTGGTCCCGAG-3′; forward strand rat GAPDH 5′-GAGCTGAACGGGAAGCTCACTGG-3′, reverse strand rat GAPDH 5′-CCACCTTCTTGATGTCATCAT-3′, forward strand rat TFF1 5′-AGTGCAAGGAAAAGGGTTGC-3′, reverse strand rat TFF1 5′-TCTCAATGACCAGAGGTCGG-3′; forward strand rat TFF2 5′-CCCACTTCCAAACCAAGCGTCG-3′, reverse strand rat TFF2 5′-CAGCAGTGCCCTTCAGTAGT-3′; forward strand rat TFF3 5'-CAAATGTGCCCTGGTGCTTC-3', reverse strand rat TFF3 5'-CCCAGCTCCTATATTGGCCG-3', forward strand rat MUC5AC 5'-ACAACAGACAGACGTGACGG-3', reverse strand rat MUC5AC 5'-GTCATCTG GACAGAAGCAGCCCTC-3'; forward strand rat MUC6 5'-AGGGGGAGAAACAGCCTACA-3', reverse strand rat MUC6 5'-AACACATTGCACCAATCCCG-3'; forward strand rat pIgR 5'-AGTCGCTCTGTTGTGAAGGG-3', reverse strand rat pIgR 5'-ACACCAGTACTTGAGGTGC-3'.

Example 7: Protein extraction and Western blot

SNU-601 cells (50 × 10 ⁶ ) were cultured in 100 mm dishes with or without different concentrations of 7-hydroxy-6-methoxy-2H-1-benzopyran-2-one compound for 24 hours. Cells were then harvested and lysed in lysis buffer (0.5% Triton, 0.1 mM sodium vanadate, 2 mM MgCl ₂ , 1 mM EGTA and 1 mM dithiothreitol). Proteins were resolved by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Membranes were incubated with primary antibodies for 18 hours at 4°C, washed, and then incubated with secondary antibodies for 1 hour at room temperature. Protein bands were visualized by chemiluminescence (FUSION-SL4, Vilber). Polyclonal antibodies against ERK, phosphorylated ERK, p38, phosphorylated p38 and α-tubulin were purchased from Santa Cruz Biotechnology.

Example 8: Statistical Analysis

All data represent the mean (±standard deviation) of assays performed in triplicate, reported as relative values. Statistical analysis performed on the data is as shown in the figure. Differences between treatment and control groups were assessed by Student's t-test or one-way ANOVA followed by post-hoc tests using GraphPad Prism 6 (CA, USA). Statistical significance was set at P<0.05.

Experiment result

1. Confirmation of mucosal stabilizing factor expression enhancement effect of 7-hydroxy-6-methoxy-2H-1-benzopyran-2-one in epithelial cells

Whether 7-hydroxy-6-methoxy-2H-1-benzopyran-2-one increased the expression level of mucosal protective factors in SNU-601 cells was investigated. The optimal 7-hydroxy-6-methoxy-2H-1-benzopyran-2-one concentration was determined by cell viability assays. 1 shows that 7-hydroxy-6-methoxy-2H-1-benzopyran-2-one does not exhibit cytotoxicity in SNU-601 cells at doses ranging from 0.15 to 2.4 μg/mL.

Expression levels of MUC5AC, MUC6, TFF1 and TFF2 were significantly increased in SNU-601 cells when treated with 0.3 or 1.2 μg/mL 7-hydroxy-6-methoxy-2H-1-benzopyran-2-one ( Fig. 2). This suggests that the 7-hydroxy-6-methoxy-2H-1-benzopyran-2-one compound has an effect of restoring mucosal damage by enhancing the expression of mucosal stabilizing factors.

2. Confirmation of mucosal stabilization mechanism of 7-hydroxy-6-methoxy-2H-1-benzopyran-2-one compound in epithelial cells

7-Hydroxy-6-methoxy-2H-1-benzopyran-2-one increased the ERK signaling pathway in a dose-dependent manner in SNU-601 cells (FIG. 3). It was confirmed that 7-hydroxy-6-methoxy-2H-1-benzopyran-2-one has significant anti-ulcerative and anti-secretory properties. These data suggest that 7-hydroxy-6-methoxy-2H-1-benzopyran-2-one has a gastroprotective effect by increasing mucosal protective factors.

3. Confirmation of gastric mucosal damage reduction effect by 7-hydroxy-6-methoxy-2H-1-benzopyran-2-one compound in gastric injury model induced by hydrochloric acid-ethanol, which is a model of damaging mucosal disease

The present inventors found that 7-hydroxy-6-methoxy-2H-1-benzopyran-2-one, a major physiologically active ingredient, showed protective activity against acidified ethanol-induced gastritis and improved mucosal integrity. whether or not it increases was investigated. 0.5 mg/kg and 1.0 mg/kg of 7-hydroxy-6-methoxy-2H-1-benzopyran-2-one were administered. 7-Hydroxy-6-methoxy-2H-1-benzo Pyran-2-one significantly reduced HCl/EtOH-mediated gastric lesions in a concentration-dependent manner ( FIG. 4 ).

4. Confirmation of the expression induction effect of mucosal stabilizing factors by 7-hydroxy-6-methoxy-2H-1-benzopyran-2-one compound in gastric injury model induced by hydrochloric acid-ethanol, which is a model of damaging mucosal disease

Pretreatment with 7-hydroxy-6-methoxy-2H-1-benzopyran-2-one increased MUC5AC, MUC6, rPigr, TFF1 and TFF2 but not TFF3. In particular, 7-hydroxy-6-methoxy-2H-1-benzopyran-2-one greatly enhanced the mRNA expression levels of MUC5AC, TFF1 and TFF2 mRNAs (FIG. 5b).

Claims (8)

7-Hydroxy-6-methoxy-2H-1-benzopyran-2-one or a pharmaceutical composition for the prevention or treatment of esophageal or gastrointestinal mucosal damage, comprising as an active ingredient a pharmaceutically acceptable salt thereof.
The agent according to claim 1, wherein the esophageal or gastrointestinal mucosal damage disease is esophageal mucosal damage, gastric mucosal damage, intestinal mucosal damage, gastritis, gastric ulcer, reflux esophagitis or leaky gut syndrome. academic composition.
The pharmaceutical composition according to claim 2, wherein the gastritis is selected from the group consisting of erosive gastritis, acute hemorrhagic gastritis, superficial gastritis, atrophic gastritis, and metaplastic gastritis.
The pharmaceutical composition according to claim 1, wherein the composition has an activity of increasing the expression of one or more selected from the group consisting of TFF1, TFF2, MUC6, MUC5AC and rPigr. A functional food composition comprising 7-hydroxy-6-methoxy-2H-1-benzopyran-2-one as an active ingredient for preventing or improving esophageal or gastrointestinal mucosal damage diseases.
The method of claim 5, wherein the esophageal or gastrointestinal mucosal damage disease is esophageal mucosal damage, gastric mucosal damage, intestinal mucosal damage, gastritis, gastric ulcer, reflux esophagitis, or leaky gut syndrome. food composition.
The functional food composition according to claim 6, wherein the gastritis is selected from the group consisting of erosive gastritis, acute hemorrhagic gastritis, superficial gastritis, atrophic gastritis, and chemical gastritis.
At least one protein selected from the group consisting of TFF1, TFF2, MUC6, MUC5AC and rPigr in the esophageal or gastric mucosa containing 7-hydroxy-6-methoxy-2H-1-benzopyran-2-one as an active ingredient Functional food composition for increasing expression.

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