Application of 6-methylcoumarin in the preparation of products for treating intestinal diseases and improving the intestinal barrier

Abstract

This invention, entitled "Application of 6-Methylcoumarin in the Preparation of Products for Treating Intestinal Diseases and Improving the Intestinal Barrier," belongs to the fields of biomedical technology and food. The technical problem to be solved is the poor safety, side effects, and drug resistance of existing IBD clinical drugs. The key technical solutions are the application of 6-methylcoumarin in the preparation of products that improve intestinal barrier function, its application as a treatment for intestinal barrier repair drugs, and its application as a treatment for intestinal barrier-related intestinal diseases.

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine and food, specifically relating to the application of 6-methylcoumarin in the preparation of drugs, health products and functional foods for treating intestinal diseases and improving the intestinal barrier. Background Technology

[0002] Inflammatory bowel disease (IBD) is a chronic, nonspecific inflammatory disease characterized by alternating periods of remission and relapse, primarily involving intestinal immune dysfunction. It mainly includes ulcerative colitis (UC) and Crohn's disease (CD) and is the most common digestive tract disease.

[0003] While current clinical drugs for IBD include aminosalicylic acid derivatives, glucocorticoids, immunosuppressants, and biologics, it is undeniable that these drugs have side effects and drug resistance issues, and there is still an urgent need to develop safer treatments.

[0004] The intestinal barrier is a complex functional structure that maintains the homeostasis of the gut microbiota and serves as the body's "first line of defense" against pathogen invasion; its integrity is crucial for gut health. This barrier system consists of four synergistic components: the mechanical barrier (centered on intestinal epithelial cells and tight junctions), the chemical barrier (such as digestive enzymes, the mucus layer, and antimicrobial substances), the biological barrier (intestinal symbiotic flora and their metabolites), and the immune barrier (intestinal mucosal immune cells and secretory antibodies). These four components work together to maintain the stability of the intestinal environment. However, the intestinal barrier is susceptible to damage from various internal and external factors, including imbalances in inflammation and oxidative stress, pathogen infection, chemical drug stimulation, gut microbiota dysbiosis, and malnutrition. Studies have shown that damage to the intestinal barrier structure has been confirmed as a prominent feature of IBD. When the intestinal barrier is impaired, microorganisms and endotoxins translocate across the intestinal mucosal barrier, thereby promoting enterogenic infections, systemic inflammatory response syndrome, and multiple organ failure. Therefore, maintaining the integrity of the intestinal barrier is of paramount importance.

[0005] Coumarin compounds possess anti-inflammatory, antioxidant, and antibacterial properties. 6-Methylcoumarin (6-MC) is an edible flavoring permitted for use in my country's national food safety standards for food additives, and it has attracted attention due to its high safety, wide availability, and rich aroma.

[0006] Relevant patent documents retrieved:

[0007] This document, published in China (CN112293417A) on November 4, 2020, discloses the application of 6-methylcoumarin in aphidicides and the resulting aphidicide. The aphidicide containing 6-methylcoumarin shows comparable aphid-killing efficacy to other commercial aphidicides, demonstrating broad development prospects and application value. It is an effective and feasible new aphidicide. Furthermore, 6-methylcoumarin is widely available and its preparation process is simple, which helps reduce the cost of aphidicides and facilitates its widespread adoption.

[0008] Relevant non-patent literature retrieved: The journal *Industrial Microbiology*, with the article titled "Study on the Antibacterial Activity and Stability of 6-Methylcoumarin," Volume 55, Issue 3, published in June 2025, discloses the antibacterial activity and stability of 6-methylcoumarin against *Botrytis cinerea*. Using *Botrytis cinerea* as the research object, the in vitro antibacterial activity of 6-methylcoumarin against *Botrytis cinerea* hyphae was determined using the growth rate method. The antibacterial activity of 6-methylcoumarin against *Botrytis cinerea* under different conditions was also measured. The results show that 6-methylcoumarin at different concentrations exhibits certain antibacterial activity against *Botrytis cinerea*, and the dose-inhibition rate relationship is directly proportional, providing a theoretical basis for the use of 6-methylcoumarin in the control of gray mold.

[0009] The prior art represented by the aforementioned documents has at least the following unresolved technical problems or defects: No application of 6-methylcoumarin in intestinal barrier repair or treatment of intestinal diseases has been found. The relevant evidence is that none of the above-mentioned literature records any research on the use of 6-methylcoumarin in intestinal barrier repair or treatment of intestinal diseases. Summary of the Invention

[0010] The purpose of this invention is to provide: The application of 6-methylcoumarin in the preparation of drugs, health products and functional foods for treating intestinal diseases and improving intestinal barrier function aims to provide more effective means for regulating intestinal diseases and the intestinal barrier.

[0011] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by those skilled in the art. Unless otherwise stated, all patents, patent inventions, and publications cited throughout this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions provided in this chapter shall prevail.

[0012] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0013] Definitions of standard chemical terms can be found in references such as "Molecular Cloning: A Laboratory Manual (4th Edition)," Science Press, October 2013, and "National-level Planning Textbook for Higher Education: Cell Culture Engineering," Higher Education Press, 2012.

[0014] Unless otherwise stated, conventional methods within the scope of this art, such as absorbance detection, weight measurement, DAI scoring, o-toluidine method, length measurement, etc., shall be used. Unless specifically defined, the use of various commercially available products herein shall employ standard techniques. For example, they may be implemented using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein are generally implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0015] Unless otherwise specified, the term "percentage, %" used in this article refers to mass percentage for solid-liquid mixtures and solid-phase-solid mixtures, and volume percentage for liquid-phase-liquid mixtures.

[0016] The term "overnight" used in this article is a common operational term in the fields of biology, microbiology and cell experiments. It refers to the standardized operation of placing a culture system inoculated with microorganisms or cells under suitable conditions and continuously culturing it for one night. The core purpose is to allow the cultured organisms to complete the logarithmic growth phase, reach sufficient biomass, or complete a specific physiological metabolic process. It is usually 12-24 hours, preferably 16-18 hours.

[0017] The terms used in this article are: "DAPI" for 4',6-diamidinyl-2-phenylindole, a nucleus-specific counterstain that emits blue fluorescence for locating and counting cells; "Occludin" for closure protein, a member of the tight junction protein family, and a core detection target for intestinal barrier function; "ZO-1" for tight junction protein 1, a key protein regulating tight junction structure and permeability, which works with Occludin to maintain epithelial barrier integrity; "iF 488" for wheat germ lectin (WGA) probe, a fluorescent marker that emits green fluorescence after being labeled with secondary antibody, facilitating observation under a fluorescence microscope; and "Cy3" for cyanine dye 3, a red fluorescent marker that, when combined with iF 488, enables dual-label fluorescence detection to distinguish the localization of two target proteins.

[0018] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides the use of 6-methylcoumarin in the preparation of products that improve intestinal barrier function.

[0019] The 6-methylcoumarin is an edible flavoring permitted for use in the National Food Safety Standard for the Use of Food Additives (GB2760-2024), with the code S1182.

[0020] The structural formula of the 6-methylcoumarin is as follows: .

[0021] The products mentioned include health foods, functional foods, or drugs.

[0022] The improvement in intestinal barrier function is manifested in at least one of the following ways: (1) Upregulate the relative expression levels of Occludin and / or ZO-1 tight junction protein genes; (2) Inhibits apoptosis of intestinal epithelial cells; (3) Reduce intestinal oxidative stress response.

[0023] Secondly, the present invention provides the application of 6-methylcoumarin in the preparation of drugs for treating intestinal barrier repair.

[0024] The intestinal barrier is selected from at least one of the following: mechanical barrier, chemical barrier, biological barrier and immune barrier.

[0025] In some embodiments, the intestinal barrier is preferably a mechanical barrier or a combination of a mechanical barrier and at least one of a chemical barrier, a biological barrier, or an immune barrier.

[0026] In some embodiments, the combination is further preferably mechanical barrier and chemical barrier, mechanical barrier and biological barrier, mechanical barrier and immune barrier, mechanical barrier, chemical barrier and biological barrier, mechanical barrier, chemical barrier and immune barrier, mechanical barrier, biological barrier and immune barrier, or mechanical barrier, chemical barrier, biological barrier and immune barrier.

[0027] In some implementations, the intestinal barrier is further preferably a mechanical barrier.

[0028] Thirdly, the present invention provides the use of 6-methylcoumarin in the preparation of drugs for treating intestinal barrier-related intestinal diseases.

[0029] The clinical manifestations of the intestinal disease include impaired intestinal barrier and intestinal oxidative stress.

[0030] In some embodiments, the intestinal barrier is a mechanical barrier or a combination of a mechanical barrier and at least one of a chemical barrier, a biological barrier, or an immune barrier.

[0031] In some implementations, the intestinal barrier is preferably a mechanical barrier.

[0032] In some embodiments, the oxidative stress indicator is selected from at least one of malondialdehyde (MDA), catalase (CAT), reduced glutathione (GSH), and total antioxidant capacity (T-AOC).

[0033] In some implementations, the intestinal disease is preferably selected from inflammatory bowel disease.

[0034] In some embodiments, the inflammatory bowel disease is further selected from ulcerative colitis and / or Crohn's disease.

[0035] In some implementations, the inflammatory bowel disease is further selected from ulcerative colitis.

[0036] The drug described in this invention can be administered via one of the following routes: oral, injection, mucosal, transdermal or local, nasal or inhalation.

[0037] In some embodiments, the route of administration is preferably oral, injection, nasal, or inhalation.

[0038] In some embodiments, the route of administration is further preferably oral or injectable.

[0039] In some embodiments, the route of administration is further preferably oral.

[0040] The drug described in this invention includes 6-methylcoumarin and pharmaceutically acceptable excipients. The specific pharmaceutically acceptable excipients can be selected by those skilled in the art based on the route of administration of the drug; therefore, their specific details are not elaborated here.

[0041] The dosage forms of the health foods and functional foods described in this invention are tablets, hard capsules, soft capsules, oral solutions, granules, gel candies, or powders.

[0042] The health foods and functional foods described in this invention include 6-methylcoumarin and excipients acceptable for use in health foods or functional foods. The specific excipients acceptable for use in health foods or functional foods can be selected by those skilled in the art based on the specific dosage form of the health food or functional food; therefore, specific excipients will not be described in detail here.

[0043] In this invention, Example 1 at least supports the protection scope of "improving intestinal barrier function".

[0044] The effects are summarized from the foregoing explanation and / or the corresponding detection indicators in Example 1, including serum FITC-glucan concentration, expression of tight junction protein ZO-1 (red), and Occludin (green), colonic cell apoptosis, and inhibition of intestinal oxidative stress. Therefore, those skilled in the art can reasonably infer that the improvement of intestinal barrier function, its subordinate concepts, its essentially equivalent technical means, and technical means that can replace it within the scope of conventional and common knowledge based on the existing level of technology should all fall within the protection scope of this invention.

[0045] In this invention, Example 1 at least supports the protection scope of "treatment of inflammatory bowel disease", "intestinal barrier repair" and "treatment of intestinal barrier-related intestinal diseases".

[0046] The effects of this invention are summarized from the foregoing explanation and / or the corresponding detection indicators in Example 1, including colon length, pathological damage evaluation of colonic tissue, serum FITC-glucan concentration, expression of tight junction proteins ZO-1 (red) and Occludin (green), colonic cell apoptosis, and inhibition of intestinal oxidative stress. Therefore, those skilled in the art can reasonably infer that the 6-methylcoumarin of this invention can treat inflammatory bowel disease and repair intestinal barrier damage. Therefore, its subordinate concepts, its essentially equivalent technical means, and technical means that can be replaced within the scope of conventional and common knowledge based on existing technology should all fall within the protection scope of this invention.

[0047] Example 1 of this invention at least supports the scope of protection of "application in the preparation of products that improve intestinal barrier function".

[0048] Based on the foregoing explanation and / or the corresponding detection indicators in Example 1, such as serum FITC-glucan concentration, expression of tight junction protein ZO-1 (red), and Occludin (green), colonic cell apoptosis, and inhibition of intestinal oxidative stress, it can be summarized that the 6-methylcoumarin of the present invention has the effect of improving intestinal barrier function. Furthermore, 6-methylcoumarin is a food additive raw material, meeting the requirements for use in health products, functional foods, and pharmaceuticals. Therefore, those skilled in the art can reasonably presume that products that improve intestinal barrier function, their subordinate concepts, their essentially equivalent technical means, and technical means that can replace them within the scope of conventional and common knowledge based on the existing level of technology should all fall within the protection scope of the present invention.

[0049] In this invention, Example 1 at least supports the scope of protection for "use as a preparation of a drug for treating intestinal barrier repair" and "use as a preparation of a drug for treating intestinal barrier-related intestinal diseases".

[0050] Based on the foregoing explanation and / or the corresponding detection indicators in Example 1, including colon length, pathological damage evaluation of colonic tissue, serum FITC-glucan concentration, expression of tight junction proteins ZO-1 (red) and Occludin (green), colonic cell apoptosis, and inhibition of intestinal oxidative stress, it can be summarized that 6-methylcoumarin of the present invention can treat inflammatory bowel disease and repair intestinal barrier damage. Simultaneously, 6-methylcoumarin is a food additive raw material, meeting the requirements for use in health products, functional foods, and pharmaceuticals. Therefore, those skilled in the art can reasonably infer that its application in "preparing a drug for treating intestinal barrier repair," "preparing a drug for treating intestinal barrier-related intestinal diseases," its subordinate concepts, its essentially equivalent technical means, and technical means that can replace it within the scope of conventional and common knowledge based on the existing technical level should all fall within the protection scope of the present invention.

[0051] The present invention has at least the following beneficial effects: 1. This invention is the first to discover that the food additive 6-methylcoumarin has novel effects in improving the intestinal barrier and treating inflammatory bowel disease. Example 1 demonstrates that 6-methylcoumarin, in an in vivo model, achieves the technical effect of repairing intestinal barrier damage and treating inflammatory bowel disease through multiple synergistic mechanisms, including reducing intestinal permeability, upregulating tight junction proteins, inhibiting intestinal epithelial apoptosis, and alleviating intestinal oxidative stress.

[0052] 2. The 6-methylcoumarin of the present invention is a food additive permitted for use in China. The raw material source is clear and the use is highly safe. It meets the raw material requirements of pharmaceuticals, health foods and functional foods. It has important application value and broad prospects in the field of preparing drugs, health foods and functional foods that improve intestinal barrier function.

[0053] 3. 6-Methylcoumarin has high safety and no obvious side effects. It can replace some traditional IBD treatment drugs and effectively solve the problems of poor safety, side effects and drug resistance of existing clinical drugs. Attached Figure Description

[0054] Figure 1 The therapeutic effect of 6-MC on UC mice is shown below; where A is a schematic diagram of the animal experiment and dosing regimen; B is the weight change curve of mice during the dosing period; C is the DAI score of mice during the dosing period; D is a photograph of mouse colon tissue; E is the length of mouse colon; F is the intestinal permeability measured by serum FITC-glucan concentration; G is the H&E staining result of mouse colon tissue; H is the histological score of mouse colon; compared with the DSS group, P < 0.05 P < 0.01, P < 0.001, P < 0.0001.

[0055] Figure 2 The therapeutic effect of 6-MC on UC mice is shown in Figure A, where A represents the results of AB-PAS staining; B represents the expression of tight junction proteins ZO-1 (red) and Occludin (green) detected by immunofluorescence staining of mouse intestinal tissue (scale bar = 20 μm); and C represents the detection of colon cell apoptosis by TUNEL staining of colon tissue (green fluorescence).

[0056] Figure 3 The effects of 6-MC on DSS-induced oxidative stress indices in mouse colon tissue were investigated. In this study, A represents MDA content, B represents CAT content, C represents GSH concentration, and D represents T-AOC concentration. All experimental data are expressed as mean ± standard deviation. n=6. One-way ANOVA was used to analyze differences between groups. P<0.05, P<0.01, P<0.001, P<0.0001. Detailed Implementation

[0057] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0058] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0059] Example 1 The efficacy of 6-methylcoumarin (6-MC) in improving intestinal barrier damage and treating inflammatory bowel disease 1. Experimental Methods Fifty male C57BL / 6J mice, all SPF grade, 8 weeks old, weighing 20-23 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The mice were housed under standard laboratory conditions, with free access to autoclaved water and feed. All animals were acclimatized to the environment for one week prior to the experiments. All animal experiments were conducted in accordance with the "Regulations on the Management of Laboratory Animals".

[0060] Mice were randomly divided into 5 groups (n=8 per group). Mice in the model group and experimental group had their drinking water replaced with sodium dextran sulfate (DSS) (MP, catalog number 0216011080) containing 3% for 7 consecutive days to induce ulcerative colitis. During the experiment, mice in the model and experimental groups had free access to the DSS solution daily, which was freshly prepared every two days. Mice in the control group (CON) had free access to distilled water. Simultaneously, the experimental group was divided into a blank solvent group (DSS), a low-dose group (DSS+6-MC, 20 mg / kg), a high-dose group (DSS+6-MC, 40 mg / kg), and a positive control group (DSS+5-ASA, 100 mg / kg), which were administered by gavage for 7 consecutive days (the solvents were 4% DMSO, 40% PEG300, 5% Tween 80, and 51% physiological saline). Figure 1 As shown in Figure A.

[0061] Mice body weight was monitored and recorded at the same time each day. Simultaneously, fecal morphology was observed, and fecal occult blood was detected using a fecal occult blood test kit. The onset and severity of mouse symptoms were recorded, and mice were scored using the DAI (Digital Indication and Analysis) method. At the end of the experiment, mice were sacrificed, and colon length and colonic index were measured. HE staining was used to observe pathological changes in the mouse colon tissue.

[0062] 2. Detection methods and indicators 2.1. DAI Score The Disease Activity Index (DAI) in mice is composed of three indicators: rate of change in body weight, fecal characteristics, and fecal occult blood.

[0063] Body weight change rate: Mouse body weight was monitored daily. Body weight % = body weight on the day of experiment / initial body weight 100%; Percentage weight loss = (Starting weight - Weight on the day of the experiment) / Starting weight 100%; Fecal occult blood test method: o-toluidine method for detecting fecal occult blood in mice.

[0064] Referring to the DAI scoring table in the literature, the severity of clinical symptoms in mice was scored. The DAI score = (weight change + fecal characteristics + fecal bleeding) / 3.

[0065] Table 1 DAI Scoring Table

[0066] 2.2. FITC-glucan Mice were fasted for 12 hours before sampling and were given 400 mg / kg FITC-glucan (Sigma, catalog number MW4000) by gavage. Blood was collected from the orbital cavity 3 hours later. The plasma was separated and centrifuged to collect the supernatant. The serum was diluted with PBS at a ratio of 1:25 and the FITC concentration was measured by a fluorescent microplate reader (standard curve: 0.5 ng / ml - 50 μg / mL).

[0067] 2.3. Hematoxylin and eosin staining At the end of the experiment, after the mice were euthanized, the colon tissue from the ileocecal region to the anus was taken, stretched naturally, and its length was measured.

[0068] Histopathology: Distal colon tissue from mice was fixed with a general-purpose tissue fixative, dehydrated, embedded in paraffin, and sectioned using a paraffin microtome. After dewaxing, the tissue was stained with hematoxylin and eosin (H&E), dehydrated, mounted, and observed under an optical microscope for histopathological changes. Pathological damage to the colon tissue was evaluated according to methods reported in the literature.

[0069] 2.4. AB-PAS staining Paraffin sections of mouse colon tissue were stained using a combination of Alcian Blue (AB) and Periodic Acid-Schiff (PAS). First, Alcian Blue was used for staining for 5-10 minutes to reveal acidic mucus (blue color), followed by a brief rinse with distilled water. Then, the sections were oxidized with 1% periodic acid solution for 10 minutes. After rinsing with running water, PAS was used for staining with PAS reagent under light-protected conditions for 15-30 minutes to reveal neutral mucus (purple-red color). The sections were then rinsed with running water, and the cell nuclei were counterstained with hematoxylin. Finally, the sections were dehydrated using an ethanol gradient, cleared with xylene, and mounted with neutral resin. Under AB-PAS staining, blue areas indicated the presence of acidic mucus, while purplish-red or red areas indicated the presence of neutral mucus. Goblet cells appeared deep blue, and the remaining tissues were almost colorless.

[0070] 2.5. Immunofluorescence staining Mouse colon tissue sections were dewaxed, rehydrated, and subjected to heat-induced antigen retrieval. They were blocked with 3% H₂O₂ at room temperature in the dark for 25 minutes to block endogenous peroxidase, followed by 5% BSA blocking for 30 minutes. Primary antibody dilution buffer Occludin (1:3000, Servicebio, GB111401) was added, and the sections were incubated overnight at 4°C. HRP-labeled goat anti-rabbit IgG (1:300, Servicebio, GB23303) was incubated at room temperature for 50 minutes, followed by Cy3-Tyramide (Servicebio, G1223) incubation at room temperature in the dark for 10 minutes. The antibody complexes were then eluted by microwave treatment, and primary antibody dilution buffer ZO-1 (1:1000, Servicebio, GB111401) was added, and the sections were incubated overnight at 4°C. HRP-labeled goat anti-rabbit IgG (dilution 1:300, ServiceBio, GB23303) was incubated at room temperature for 50 minutes, followed by incubation with iF488-Tyramide (ServiceBio, G1231) in the dark at room temperature for 10 minutes. Sections were counterstained with DAPI for nuclei for 10 minutes, treated with an autofluorescence quencher, and then mounted. Fluorescence images were observed and captured using a fluorescence microscope.

[0071] 2.6. TUNEL staining After fixation and permeabilization, the samples were incubated with a reaction solution containing TdT enzyme and labeled dUTP at 37°C in the dark for 1-2 hours to allow the enzyme to catalyze the ligation of the label to the 3'-OH end of the DNA break in apoptotic cells. The samples were counterstained with DAPI for 5 minutes, washed, mounted, and observed under a fluorescence microscope.

[0072] 3. Detection of oxidative stress indicators According to the instruction manual, commercially available kits were used to detect the levels of oxidation and antioxidant-related indicators in the tissue homogenate supernatant: Malondialdehyde (MDA) content was determined using the thiobarbituric acid method (Solarbio, BC0020-50T / 48S), with results expressed as nmol / g. Catalase (CAT) activity was determined using a visible light colorimetric method (Solarbio, BC0170-50T / 24S), with results expressed as U / g tissue. Reduced glutathione (GSH) concentration was determined using a microplate method (Solarbio, BC1175-200T / 192S), with results expressed as μg / g tissue. Total antioxidant capacity (T-AOC) was assessed using the ABTS method (Solarbio, BC1310-50T / 48S), with results expressed as μmol / g tissue.

[0073] 4. Results Analysis The results are as follows Figure 1 , Figure 2 and Figure 3 As shown.

[0074] like Figure 1 As shown in Figure B, compared with the CON group, the body weight of mice in the DSS group decreased significantly from day 5 of drug administration, and by the end of the experiment, the body weight had dropped to less than 80% of the initial body weight. The trend of body weight loss in mice was significantly alleviated after intervention with 6-MC (20 mg / kg and 40 mg / kg) and 5-ASA, indicating that 6-MC can effectively improve the overall health of colitis mice.

[0075] like Figure 1 As shown in Figure C, the DAI score of mice in the DSS group was significantly increased, while the increase in DAI score was significantly reduced after 6-MC administration, indicating that 6-MC can effectively alleviate the clinical symptoms of colitis.

[0076] like Figure 1 As shown in Figures D and E, DSS modeling resulted in significant shortening, congestion, and edema of the colon in mice. 5-ASA administration showed no significant improvement, while 6-MC (40 mg / kg) administration significantly restored colon length and improved morphology.

[0077] like Figure 1 As shown in Figure F, DSS modeling significantly increased colonic permeability in mice, indicating severe impairment of intestinal barrier function. However, intervention with 6-MC (20 mg / kg and 40 mg / kg) and 5-ASA significantly reduced colonic permeability in mice, demonstrating that 6-MC can effectively reduce intestinal permeability and protect intestinal barrier integrity.

[0078] like Figure 1As shown in Figures G and H, DSS modeling resulted in extensive ulceration of the colonic mucosa in mice, massive infiltration of inflammatory cells, and severe damage to crypt structures. In contrast, intervention with 6-MC (20 mg / kg and 40 mg / kg) and 5-ASA significantly reduced the pathological damage to the colonic tissue, decreased inflammatory infiltration, preserved the crypt structure relatively well, and significantly lowered the histological score.

[0079] like Figure 2 As shown in Figure A, DSS modeling significantly reduced the number of goblet cells in the colonic mucosa of mice, resulting in insufficient mucus secretion and a significant decrease in AB-PAS staining intensity. However, after intervention with 6-MC (20 mg / kg and 40 mg / kg) and 5-ASA, the number of goblet cells and mucus secretion were significantly restored, indicating that 6-MC can effectively protect the colonic mucus barrier function.

[0080] like Figure 2 As shown in Figure B, DSS modeling significantly reduced the expression levels and disordered distribution of tight junction proteins ZO-1 and Occludin in the colonic epithelium of mice, indicating severe impairment of intestinal barrier integrity. However, intervention with 6-MC (20 mg / kg and 40 mg / kg) and 5-ASA significantly restored the expression levels of ZO-1 and Occludin, and their distribution became more continuous and intact, demonstrating that 6-MC can effectively maintain the tight junction structure of the intestinal epithelium and improve intestinal barrier function.

[0081] like Figure 2 As shown in Figure C, DSS modeling significantly increased the number of TUNEL-positive apoptotic cells in the colonic epithelium of mice, indicating that colitis induced a large number of apoptosis. However, intervention with 6-MC (20 mg / kg and 40 mg / kg) and 5-ASA significantly reduced the number of TUNEL-positive cells, suggesting that 6-MC can effectively inhibit colonic epithelial cell apoptosis and alleviate tissue damage.

[0082] like Figure 3 As shown, DSS modeling significantly increased MDA content in mouse colon tissue, indicating severe lipid peroxidation damage; simultaneously, CAT activity, GSH content, and T-AOC levels significantly decreased, indicating severe damage to the intestinal antioxidant system. 6-MC intervention significantly reduced MDA content while significantly increasing CAT activity, GSH content, and T-AOC levels, effectively reversing the intestinal oxidation-antioxidant imbalance, inhibiting oxidative stress, and thus alleviating intestinal epithelial oxidative damage, providing a stable microenvironment for intestinal barrier repair.

[0083] Verification of technical effectiveness and / or analysis of technical problem solving This invention utilizes a DSS-induced ulcerative colitis (UC) mouse model for validation, including dynamic assessment of intestinal barrier function, such as the Disease Activity Index (DAI score), weight change rate, and colon length, as well as assessing changes in intestinal permeability using a FITC-glucan assay to detect serum fluorescence concentration. Phenotypic and inflammatory regulation analysis involves histopathological examination (H&E, AB-PAS staining) to observe pathological changes in colon tissue and goblet cell loss, and immunofluorescence staining to detect tight junction proteins (ZO-1, Occludin) and TUNEL staining to detect colon cell apoptosis. Simultaneously, malondialdehyde (MDA) content, catalase (CAT) activity, reduced glutathione (GSH) content, and total antioxidant capacity (T-AOC) levels are used to detect oxidative stress in colon tissue. This demonstrates that 6-MC, in the in vivo model, achieves its technical efficacy in repairing intestinal barrier damage and treating inflammatory bowel disease through multiple synergistic pathways, including reducing intestinal permeability, upregulating tight junction proteins, inhibiting intestinal epithelial apoptosis, and alleviating intestinal oxidative stress.

[0084] Meanwhile, 6-methylcoumarin is a food additive permitted for use in China. It is highly safe and has no obvious side effects. It can replace some traditional IBD treatment drugs and effectively solve the problems of poor safety, side effects and drug resistance of existing clinical drugs. It has important application value and broad prospects in the fields of pharmaceuticals, health foods and functional foods.

[0085] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. Use of 6-methylcoumarin in the preparation of a product for improving intestinal barrier function.

2. Use according to claim 1, characterized in that, The product includes a health food, a functional food or a medicine.

3. Use according to claim 2, characterized in that, The improvement of intestinal barrier function is at least one of the following: (1) up-regulating the relative expression level of Occludin and / or ZO-1 tight junction protein gene; (2) inhibiting intestinal epithelial cell apoptosis; (3) reducing intestinal oxidative stress.

4. Use of 6-methylcoumarin in the preparation of a medicine for treating intestinal barrier repair.

5. Use according to claim 4, characterized in that, The intestinal barrier is at least one of the following: mechanical barrier, chemical barrier, biological barrier and immune barrier.

6. Use according to claim 5, characterized in that, The intestinal barrier is a mechanical barrier or a combination of a mechanical barrier and at least one of a chemical barrier, a biological barrier or an immune barrier.

7. Use of 6-methylcoumarin in the preparation of a medicine for treating intestinal diseases related to intestinal barrier.

8. Use according to claim 7, characterized in that, The intestinal barrier is a mechanical barrier or a combination of a mechanical barrier and at least one of a chemical barrier, a biological barrier or an immune barrier.

9. Use according to claim 7, characterized in that, The intestinal disease is characterized by intestinal barrier damage and intestinal oxidative stress.

10. Use according to claim 7, characterized in that, The intestinal disease is inflammatory bowel disease.

11. Use according to claim 10, characterized in that, The inflammatory bowel disease includes ulcerative colitis and / or Crohn's disease.

12. The use according to any one of claims 2 to 11, characterized in that, The administration route of the medicine is one of oral, injection, mucosal, transdermal or topical administration, nasal or inhalation administration.

13. Use according to claim 12, characterized in that, The medicine includes 6-methylcoumarin and pharmaceutically acceptable excipients.

14. Use according to any one of claims 2-3, characterized in that, The dosage form of the health food or functional food is tablet, hard capsule, soft capsule, oral solution, granule, gummy candy or powder.

15. Use according to claim 14, characterized in that, The health food or functional food includes 6-methylcoumarin and acceptable excipients for health food or functional food.

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