Use of allulose to improve intestinal diseases

Abstract

The present specification relates to a composition comprising allulose as an active ingredient, and the use of said composition for the prevention, amelioration or treatment of intestinal diseases, such as leaky gut and / or symptoms of leaky gut.

Description

[Technical Field]

[0001] The present invention relates to a composition for preventing, improving or treating intestinal diseases, which contains allulose as an active ingredient, and the composition for preventing, improving or treating intestinal diseases, such as intestinal leakage or intestinal leakage symptoms, using the composition. [Background technology]

[0002] The functional intestinal barrier is essential for maintaining intestinal and systemic homeostasis, separating the external environment from the tightly regulated internal environment, absorbing nutrients and fluids, and preventing the invasion of pathogens / pathogens and other harmful molecules (e.g., pro-inflammatory bacterial products).

[0003] The intestinal epithelial layer is a major determinant of mucosal gut wall function, and tight junctions (TJs) seal the pericellular space between adjacent epithelial cells, playing a key role in periclinal pathways. Impaired TJ function is known to induce local and systemic inflammation and immune responses, ultimately inducing and / or sustaining systemic inflammatory disorders. Therefore, maintaining a homeostatic gut epithelial barrier is crucial, and a reduction or disruption of gut barrier function is directly linked to increased paracellular and transcellular intestinal permeability, which is characterized by increased invasion of pathogens and the penetration of proinflammatory luminal factors into the systemic circulation, leading to subsequent systemic inflammation and the initiation of numerous diseases. The so-called "leaky gut syndrome" is associated with multiple chronic inflammatory bowel diseases and conditions, including inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), and celiac disease, as well as numerous extraintestinal and systemic diseases and disorders. Increased leaky gut has been linked to intestinal and systemic disease states such as inflammatory bowel disease and neurodegenerative diseases such as Parkinson's disease.

[0004] In recent years, there has been increasing interest in factors that can strengthen the intestinal epithelium through their gut barrier-protecting or restorative properties. A widely applied therapeutic approach is the restoration of imbalanced gut microbial communities. Modulation of symbiotic microbiota through bacteria-based therapy resolves imbalances in the gut microbial community, alleviating inflammation and restoring a healthy microbiota. These bacteria-based therapies currently utilize traditional probiotics, prebiotics, or both. Prebiotics indirectly function to strengthen gut barrier function by promoting the growth of health-beneficial symbionts.

[0005] Therefore, there is an emerging need for substances that can perform a direct function for preventing, improving, or treating functional intestinal intestinal wall homeostatic imbalance and resulting in intestinal leakage or leaky gut syndrome, with little concern about side effects or safety. Summary of the Invention [Problem to be solved by the invention]

[0006] One example of the present invention relates to a composition for preventing, ameliorating, or treating intestinal diseases, such as inflammatory bowel disease, leaky gut, and / or leaky gut syndrome, which comprises allulose as an active ingredient.

[0007] Another example of the present invention relates to a composition containing allulose as an active ingredient for preventing, ameliorating, or treating intestinal diseases caused by a decrease in the expression level of factors related to the Toll-like receptor 4 (TLR4) signaling pathway, such as inflammatory bowel disease, leaky gut, and / or leaky gut syndrome. [Means for solving the problem]

[0008] One example of the present invention relates to a composition for preventing, ameliorating, or treating intestinal diseases, such as inflammatory bowel disease, leaky gut, and / or leaky gut syndrome, which comprises allulose as an active ingredient.

[0009] One specific example relates to a composition for improving intestinal wall cell permeability, which contains allulose as an active ingredient, and in particular, can prevent damage to intestinal wall cell permeability or improve damaged intestinal wall cell permeability, thereby preventing, improving, or treating intestinal leakage or intestinal leakage syndrome caused by increased intestinal wall cell permeability. [Effects of the Invention]

[0010] The present invention can improve intestinal immunity by simply and effectively improving intestinal leakage in humans and animals with almost no concern about side effects or safety. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 is a diagram showing, in Example 1, that allulose reduced the mRNA and protein expression levels of factors involved in TLR4 / NF-κB signal transduction in Caco-2 cells in which inflammation was induced by LPS. [Figure 2a] FIG. 2a is a diagram showing, in Example 1, that allulose reduced the mRNA and protein expression levels of factors involved in TLR4 / NF-κB signal transduction in Caco-2 cells in which inflammation was induced by LPS. [Figure 2b] FIG. 2b is a diagram showing, in Example 1, that allulose reduced the mRNA and protein expression levels of factors involved in TLR4 / NF-κB signal transduction in Caco-2 cells in which inflammation was induced by LPS. [Figure 3] FIG. 3 shows, in Example 2, that allulose reduced TLR4 / NF-κB signal transduction and the mRNA and protein expression levels of related factors in HT-29 cells in which inflammation was induced by LPS. [Figure 4a] FIG. 4a is a diagram showing, from Example 2, that allulose reduced TLR4 / NF-κB signaling and the mRNA and protein expression levels of related factors in HT-29 cells in which inflammation was induced by LPS. [Figure 4b]FIG. 4b is a diagram showing, from Example 2, that allulose reduced TLR4 / NF-κB signaling and the mRNA and protein expression levels of related factors in HT-29 cells in which inflammation was induced by LPS. [Figure 5] FIG. 5 is a diagram showing, from Example 2, that allulose reduced TLR4 / NF-κB signaling and the mRNA and protein expression levels of related factors in HT-29 cells in which inflammation was induced by LPS. [Figure 6] FIG. 6 is a diagram showing, in Example 3, that allulose reduced the mRNA and protein expression levels of prostaglandin E2 and COX-2 factor in Caco-2 cells in which inflammation was induced by LPS. [Figure 7] Figure 7 shows the results of RT-PCR in Example 3 showing the expression level of mucin, which is involved in intestinal wall protection, in HT-29 cells in which inflammation was induced by LPS, and a graph showing that the expression levels of MUC2, MUC5AC, and MUC5B were reduced. [Figure 8] FIG. 8 is a graph showing the electrical resistance (TEER) values ​​decreased by IFN-γ / TNF-α and increased by allulose in polarized Caco-2 cells according to Example 4. [Figure 9] FIG. 9 is a graph showing the fluorescence intensity increased by IFN-γ / TNF-α through FITC-dextran (4 kD) in polarized Caco-2 cells in Example 5, and the increase in fluorescence intensity by allulose. [Figure 10] FIG. 10 is a graph showing that allulose reduced ROS accumulated through the DCFH-DA fluorescent probe in Caco-2 cells in which inflammation was induced by LPS, resulting in a decrease in intestinal permeability, as shown in Example 7. [Figure 11]FIG. 11 is a graph showing that, in Example 8, the expression levels of mRNA and protein of ZO-1 and occludin, which are tight junction factors that are decreased by inflammation, increased in Caco-2 cells in which inflammation was induced by LPS. [Figure 12] FIG. 12 shows the results of MTT analysis of human intestinal epithelial cells (HT-29 and Caco-2) treated with allulose at various concentrations and cultured for 24 hours, according to Example 9. DETAILED DESCRIPTION OF THE INVENTION

[0012] As used herein, "leaky gut" or "leaky gut syndrome (LGS)" refers to a phenomenon in which intestinal mucosal permeability increases, allowing macromolecular substances to move back and forth between the cells, due to some kind of stimulation or damage to intestinal mucosal cells that maintain a certain intercellular space. This phenomenon results in the intestinal wall not functioning properly, causing macromolecular substances in the blood to leak into the intestinal lumen, or macromolecular substances in the lumen to directly enter the blood (leaky gut), and is a general term for symptoms induced by this phenomenon.

[0013] Symptoms of leaky gut syndrome manifest as various clinical conditions, such as aging, allergies, multiple trauma, rheumatoid arthritis, inflammatory bowel disease, chronic fatigue syndrome, and irritable bowel syndrome. Furthermore, increased intestinal mucosal permeability or damage to the intestinal mucosa allows pathogens, antigens, putrefactive substances, etc. to enter the intestinal mucosa, resulting in inflammatory responses. Endotoxins enter the bloodstream, causing bacterial translocation, intestinal endotoxemia, and various inflammatory and immune responses. The inflammatory bowel disease may be a disease selected from the group consisting of Crohn's disease, ulcerative colitis, intestinal Behçet's disease, infectious enteritis, ischemic enteropathy, and radiation enteritis.

[0014] In a specific example, the leaky gut may be caused by increased intestinal mucosal permeability, increased mucosal permeability due to intestinal mucosal damage or intestinal inflammation, intestinal mucosal damage and / or intestinal inflammation, or decreased expression of TLR4, increased expression of prostaglandin E2 and COX-2, decreased adhesion strength between intestinal epithelial cells, or increased expression of ZO-1 and occludin.

[0015] Therefore, for example, improvement, prevention, or treatment of leaky gut syndrome may include preventing, suppressing, or treating the phenomenon in which the gaps between intestinal mucosal cells loosen, increasing intestinal mucosal permeability, which allows macromolecular substances to pass back and forth.

[0016] In this specification, the use of allulose as an active ingredient for the prevention, amelioration, or treatment of intestinal diseases, such as inflammatory bowel disease, leaky gut, and / or leaky gut syndrome, may be due to one or more effects selected from the group consisting of allulose's ability to enhance intestinal mucosal immunity, its anti-inflammatory effect against intestinal inflammation, its ability to protect the intestinal mucosa, its ability to improve intestinal permeability, and its ability to remove reactive oxygen species (ROS).

[0017] For more details, The intestinal mucosal immunity enhancing ability is achieved by regulating the expression of a factor related to TLR4 / NF-κB signaling and TLR4 / MAPK signaling that regulates inflammatory responses, preferably one or more factors related to TLR4 / NF-κB signaling selected from the group consisting of p50, p65, TNF-α, IL-6, IL-8, COX-2, TLR4, MyD88, IKKα, IKKβ, IKβα, IRAK4, and TRAF6, or one or more factors related to MAPK signaling selected from the group consisting of p38, p-p38, JNK, p-JNK, c-Jun, pc-Jun, c-Fos, and pc-Fos; The anti-inflammatory activity is characterized by decreasing or inhibiting the expression of one or more selected from the group consisting of prostaglandin E2, a pro-inflammatory mediator, and COX-2, a core indicator of inflammatory bowel disease; The intestinal mucosa protecting ability is achieved by decreasing or inhibiting the expression of one or more mucins that protect the intestinal mucosa, selected from the group consisting of MUC2, MUC5AC, and MUC5B; The ability to improve intestinal permeability includes improving the increase in intestinal permeability induced by intestinal wall damage caused by inflammatory factors, improving the adhesion strength between intestinal epithelial cells, or increasing the expression of tight junction factors ZO-1 and occludin, or The reactive oxygen species scavenging ability may be one that is due to increased expression of one or more factors selected from the group consisting of PI3K, AKT, Nrf2, and HO-1, which are factors involved in Nrf2 / HO-1 signaling activated by ROS.

[0018] Also, herein, allulose may be used as a TLR4 inhibitor or antagonist, thereby providing a preventive, ameliorative, or therapeutic use for intestinal diseases caused by TLR4-related signaling pathways, such as inflammatory bowel disease, leaky gut, and / or leaky gut syndrome.

[0019] Specifically, the allulose may be a TLR4 inhibitor that regulates the expression of one or more factors related to TLR4 / NF-κB signaling selected from the group consisting of p50, p65, TNF-α, IL-6, IL-8, COX-2, TLR4, MyD88, IKKα, IKKβ, IKβα, IRAK4, and TRAF6.

[0020] In one embodiment of the present invention, allulose was shown to have an anti-inflammatory effect by reducing the expression levels of factors involved in TLR4 / NF-κB signaling, an inflammatory response-mediated signaling pathway, in Caco-2 cells induced by LPS through RT-PCR and Western blotting. Another embodiment of the present invention relates to a composition for reducing or improving intestinal permeability and regulating inflammation through intestinal permeability, comprising allulose as an active ingredient. The present invention relates to the function of allulose in protecting or restoring the intestinal epithelial wall. Accordingly, the present invention relates to directly regulating tight junctions in intestinal epithelial cells using allulose, and more specifically, to improving or treating intestinal epithelial cell tight junctions and intestinal permeability in response to TLR4 signaling factors, or preventing or preventing increases in intestinal epithelial cell tight junctions and intestinal permeability in response to TLR4 signaling factors.

[0021] The functionality of allulose according to the present invention is directly related to the improvement, maintenance, and prevention of deterioration of the homeostatic intestinal epithelial intestinal wall, unlike conventional prebiotics which only serve the indirect function of enhancing intestinal wall function by promoting the growth of symbiotic organisms that are beneficial to health.

[0022] The present invention has a beneficial effect on the health of microorganisms independently through direct interaction with intestinal epithelium and immune cells, one of which is directly regulating tight junctions through AMPK-, PKC-, MAPK-, and TLR-related pathways to promote intestinal wall function. The present invention aims to provide a protective function to tight junctions independently of or separately from the microbial community. Therefore, the prevention, improvement, or treatment of leaky gut or leaky gut syndrome may not only directly prevent, improve, or treat leaky gut or leaky gut syndrome, but may also selectively include the prebiotic effect of allulose, which regulates the proliferation of intestinal microorganisms. Of course, the improvement of intestinal wall permeability by allulose according to the present invention may not only directly improve intestinal wall permeability, but may also selectively include a conventionally known prebiotic function.

[0023] The composition of the present invention can be applied to pharmaceutical compositions or food compositions.

[0024] In this study, the efficacy of allulose in preventing, improving, or treating intestinal diseases was demonstrated through specific experiments. In other words, when an inflammatory response occurs due to LPS treatment, the expression of TLR4 and NF-κB p65 genes increases. However, treatment with allulose reduced the expression of TLR4, which is activated by LPS, and also reduced the expression of NF-κB p65 / p50, which is activated by LPS. The anti-inflammatory effect was confirmed by suppressing factors related to TLR4 / NF-κB activation in inflammation-induced intestinal epithelial cells through allulose treatment.

[0025] Furthermore, in HT-29 cells with inflammation induced by LPS, allulose reduced the expression of TLR4, a signal mediating the inflammatory response. This reduced the expression of MyD88, IRAK4, TRAF6, NF-κB signaling (p50, p65, p-p65, Ikkα, Ikkβ, p-Ikkαβ, Ikβα, p-Ikβα), and MAPK signaling (p38, p-p38, JNK, p-JNK, c-Jun, pc-Jun, c-Fos, pc-Fos), thereby reducing COX-2 expression. This anti-inflammatory effect was confirmed by RT-PCR and Western blot analysis, which showed a reduction in the expression of factors related to TLR4 / NF-κB signaling and TLR4 / MAPK signaling.

[0026] Furthermore, in Caco-2 cells in which inflammation was induced by LPS, the expression levels of prostaglandin E2, a pro-inflammatory mediator, and COX-2, a key indicator of inflammatory bowel disease, were confirmed by ELISA and RT-PCR. The results showed that allulose reduced the concentration of prostaglandin E2 and the expression level of COX-2, demonstrating its inflammation-regulating effect.

[0027] In HT-29 cells in which inflammation was induced by LPS, the expression level of mucin, which is involved in protecting the intestinal wall, was confirmed by RT-PCR, and it was found that the expression levels of MUC2, MUC5AC, and MUC5B were reduced, confirming that allulose protects the intestinal periphery.

[0028] Figure 8 shows a graph showing the electrical resistance (TEER) values ​​decreased by IFN-γ / TNF-α and the electrical resistance (TEER) values ​​increased by allulose in polarized Caco-2 cells. It was confirmed that the electrical resistance (TEER) values ​​decreased by IFN-γ / TNF-α in polarized Caco-2 cells were the same as the electrical resistance (TEER) values ​​increased by allulose. The electrical resistance (TEER) values ​​increased by allulose indicate that allulose, as an active ingredient, improved the adhesion strength between intestinal epithelial cells.

[0029] In polarized Caco-2 cells, the fluorescence intensity increased by IFN-γ / TNF-α through FITC-dextran (4 kD) was shown to be increased by allulose, indicating the improvement of paracellular permeability using FITC-dextran.

[0030] Furthermore, we show that in Caco-2 cells with LPS-induced inflammation, allulose reduces the accumulated ROS via the DCFH-DA fluorescent probe, thereby reducing intestinal permeability.

[0031] In Caco-2 cells with inflammation induced by LPS, the mRNA and protein expression levels of ZO-1 and occludin, tight junction factors that were decreased by inflammation, were increased by allulose treatment, indicating that allulose improved intestinal permeability, i.e., tight junctions, which were damaged by inflammation.

[0032] In the present specification, allulose is included as an active ingredient, and the allulose may be used alone or in a sugar composition containing allulose, and may be used in liquid or powder form, and the powder may be amorphous or crystalline. The allulose may be purchased as a commercially available product or may be prepared using predetermined raw materials.

[0033] The allulose or sugar composition containing allulose that can be used in the present invention is not particularly limited, and can usually be produced using an enzyme having allulose conversion activity or a microorganism that produces the enzyme, using fructose or sugar as a substrate.

[0034] The enzyme having allulose conversion activity is an enzyme having the activity of converting a substrate containing fructose or sugar into allulose, and may be, for example, an enzyme derived from one or more species selected from the group consisting of Corynebacterium or Microbacterium strains.

[0035] In the present invention, there is a method in which allulose is orally ingested by humans or animals either as it is or in the form of a food, drink, or pharmaceutical.

[0036] The allulose composition may contain allulose as an active ingredient in a total amount of 1 g to 60 g, 5 g to 60 g, 5 g to 50 g, 5 g to 40 g, 5 g to 30 g, 5 g to 25 g, 10 g to 25 g, or 10 g to 15 g per day for a 60 kg adult.

[0037] The allulose usable in the present invention may be in the form of liquid or powder and may be included in the composition of the present invention in various amounts. The allulose may be used as a single component or as a mixed composition containing other sugars.

[0038] The liquid or powder form of allulose may be a composition having a content of 50 (w / w)% or more, 60 (w / w)% or more, 70 (w / w)% or more, 80 (w / w)% or more, preferably 85 (w / w)% or more based on the total solid content. Also, the crystalline form of allulose may be a composition having a content of 98% or more based on the total solid content.

[0039] The dosage form of medicines, quasi-drugs, and supplements containing allulose is not particularly limited, and can be appropriately selected according to the dosage method.For example, in the case of oral administration, it can be made into solid or liquid dosage forms such as powder, tablet, sugar-coated agent, capsule, granule, dry syrup, liquid, syrup, drop, and drink.

[0040] Allulose can be added to various foods, beverages, food additives, and animal feeds during the normal manufacturing process. Allulose has a relative sweetness of 70%, and its quality of taste, physical properties, and processability are similar to those of sucrose, so it can be used in the same way as sugar, such as replacing part or all of sugar in the manufacturing process of various foods, beverages, food additives, medicines, and feeds.

[0041] Specific embodiments of the composition according to the present invention include, for example, drinks, dairy products, edible granules, pastes, seasonings, retort foods, baby foods, fermented foods, preserved foods, processed seafood products, processed meat products, processed grain products and other processed foods, food additives, health foods, animal feeds, and the like.

[0042] The present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not intended to be limited to the following examples. [Example]

[0043] Example 1: Evaluation of the ability of allulose to inhibit the expression of the TLR4 / NF-κB signal pathway and related factors The anti-inflammatory effect of allulose was confirmed in Caco-2 cells in which inflammation was induced with LPS (Lipopolysaccharide).The gene and protein expression levels of factors involved in TLR4 / NF-κB signaling, which mediates the inflammatory response (p50, p65, TLR4, MyD88, IKKα, IKKβ, IKβα, IRAK4, TRAF6) were confirmed by RT-PCR and Western blot.

[0044] Specifically, 2 x 10 cells were cultured in a 24-well plate. 5 Differentiated Caco-2 cells cultured at a cell / well concentration were simultaneously treated with allulose dissolved in MEM medium (Caco-2 cell culture medium) at concentrations of 5, 10, and 20 mM. After 6 hours, they were treated with LPS (1 μg / ml) to induce inflammation for 24 hours. The cells were cultured in a 5% CO2 incubator at 37°C.

[0045] As a negative control, Caco-2 cells that had not been treated with LPS were used. As a positive control, the differentiated Caco-2 cells were treated with LPS to induce inflammation but not with allulose.

[0046] RNA was extracted from the cells using Trizol, cDNA was synthesized using a cDNA synthesis kit, and gene expression was confirmed by qRT-PCR using primers designed for each gene. The PCR conditions used were denaturation at 95°C for 15 seconds, annealing at 62°C for 30 seconds, and elongation at 72°C for 1 minute. For protein expression analysis, proteins were extracted from the cultured cells using RIPA lysis buffer. The extracted proteins were pretreated at 95°C for 5 minutes and then separated by SDS-PAGE. The proteins were then transferred to a PVDF membrane and blocked with 5% (w / v) BSA solution for 1 hour. Then, primary antibodies for each factor were diluted 1:1000 in 5% (w / v) BSA solution and incubated at 4°C for 1 day. The membrane reacted with the primary antibody was then reacted with the secondary antibody at room temperature for 1 hour in Tris-buffered saline containing 0.1% (v / v) Tween 20. Bands were detected using the LAS 4000 system, and expression density was calculated using Image J software to determine the expression level.

[0047] The experimental results showed that allulose reduced the expression of factors involved in TLR4 / NF-κB signaling, an inflammatory response-mediated signaling pathway, in Caco-2 cells in which inflammation was induced by LPS. Specifically, the results of RT-PCR analysis of mRNA expression levels are shown in Figure 1, and the results of Western blot analysis of protein expression levels are shown in Figures 2a and 2b. In Figures 1, 2a, and 2b, the control (-) indicates the expression of factors involved in TLR4 / NF-κB signaling in Caco-2 cells not treated with LPS, while the control (+) indicates an experimental group in which Caco-2 cells were treated with LPS to induce inflammation, as in the experimental group, but not treated with allulose.

[0048] The experimental results showed that when LPS treatment causes an inflammatory response, the expression of TLR4 and NF-κB p65 genes increases, but allulose treatment reduced the expression of TLR4, which is activated by LPS, and also reduced the expression of NF-κB p65 / p50, which is activated by LPS. Allulose treatment in inflammation-induced intestinal epithelial cells suppressed factors related to TLR4 / NF-κB activation, thereby demonstrating its anti-inflammatory and preventive effects.

[0049] Example 2: Evaluation of the ability of allulose to inhibit the expression of the TLR4 / NF-κB signal pathway and related factors using HT-29 cells HT-29 cells were simultaneously treated with LPS (1 μg / ml) and allulose and allowed to react for 8 hours. Specifically, 2 × 10 HT-29 cells were plated on a 24-well plate. 5 The cells were seeded at a concentration of 1000 cells / well and cultured in an incubator at 5% CO2 and 37°C for 6-7 days. Allulose was dissolved in RPMI 1640 medium, a culture medium for HT-29 cells, at concentrations of 5, 10, and 20 mM. After 8 hours, the cells were treated with LPS (1 μg / ml) to induce inflammation for 24 hours.

[0050] Gene and protein expression levels of factors involved in TLR4 / NF-κB signaling and TLR4 / MAPK signaling were confirmed using RT-PCR and Western blot. RNA was extracted from cells using Trizol, cDNA was synthesized using a cDNA synthesis kit, and gene expression levels were confirmed using qRT-PCR with primers designed for each gene. The PCR conditions used were denaturation at 95°C for 15 seconds, annealing at 62°C for 30 seconds, and extension at 72°C for 1 minute. For protein expression analysis, proteins were extracted from cultured cells using RIPA lysis buffer. The extracted proteins were pretreated at 95°C for 5 minutes and then separated by SDS-PAGE. The proteins were then transferred to a PVDF membrane, blocked with 5% (w / v) BSA solution for 1 hour, and incubated with primary antibodies for each factor at a 1:1000 dilution in 5% (w / v) BSA solution at 4°C for 1 day. The membrane was then incubated with secondary antibodies in Tris-buffered saline containing 0.1% (v / v) Tween 20 at room temperature for 1 hour. Bands were detected using the LAS 4000 system, and expression densities were calculated using image detection software (Image J software) to determine expression levels. The experimental results are shown in Figures 3, 4a, 4b, and 5.

[0051] Based on the experimental results, allulose reduced the expression levels of factors related to TLR4 / NF-κB signaling and TLR4 / MAPK signaling, which are inflammatory response-mediated signaling pathways, in HT-29 cells in which inflammation was induced by LPS, as shown by RT-PCR and Western blot. The graphs shown in Figures 3 to 5 show that in Figure 3, the control group (-) shows the expression levels of factors related to TLR4 / NF-κB signaling in Caco-2 cells that were not treated with LPS, and the control group (+) refers to an experimental group in which Caco-2 cells were treated with LPS to induce inflammation, as in the experimental group, but were not treated with allulose.

[0052] The experimental results showed that allulose reduced the expression of TLR4, an inflammatory response-mediating signal, in HT-29 cells induced by LPS. This reduced the expression of MyD88, IRAK4, TRAF6, NF-κB signaling (p50, p65, p-p65, Ikkα, Ikkβ, p-Ikkαβ, Ikβα, p-Ikβα), and MAPK signaling (p38, p-p38, JNK, p-JNK, c-Jun, pc-Jun, c-Fos, pc-Fos), thereby reducing COX-2 expression. The anti-inflammatory effect was confirmed by RT-PCR and Western blot analysis, which showed a reduction in the expression of factors related to TLR4 / NF-κB signaling and TLR4 / MAPK signaling.

[0053] Example 3: Modulation of intestinal inflammatory response to allulose Caco-2 cells were plated in a 24-well plate at 2 × 10 5 Cells were cultured at a concentration of 1000 cells / well. The medium was changed every two days, and cells were simultaneously treated with allulose at concentrations of 5mM, 10mM, and 20mM, then cultured for 6 hours. After LPS (1μg / ml) treatment, the cells were incubated for 12 hours. The supernatant was used for ELISA measurements. RNA was extracted from the cells using Trizol and then synthesized into cDNA using a cDNA synthesis kit. Gene expression levels were confirmed by qRT-PCR using primers designed for each gene. The PCR conditions used were denaturation at 95°C for 15 seconds, annealing at 62°C for 30 seconds, and extension at 72°C for 1 minute.

[0054] Figure 6 is a graph showing the inflammation-modulating effect of allulose, showing a decrease in the expression levels of prostaglandin E2, a pro-inflammatory mediator, and COX-2, a key indicator of inflammatory bowel disease, as determined by ELISA and RT-PCR in Caco-2 cells in which inflammation was induced by LPS, as shown in Example 3. In Figure 6, the control group (-) shows the expression levels of factors related to TLR4 / NF-κB signaling in HT-29 cells that were not treated with LPS, and the control group (+) refers to an experimental group in which HT-29 cells that had been treated with LPS to induce inflammation in the same way as the experimental group, but were not treated with allulose.

[0055] In the experimental results, allulose was used in Caco-2 cells in which inflammation was induced by LPS to check the expression levels of prostaglandin E2, a pro-inflammatory mediator, and COX-2, a key indicator of inflammatory bowel disease, using ELISA and RT-PCR. The results showed that the concentration of prostaglandin E2 and the expression level of COX-2 were reduced, confirming its inflammation-regulating effect.

[0056] Example 4: Evaluation of the intestinal mucosa protective ability of allulose using HT-29 cells The amount of mucin expression in allulose material was confirmed in HT-29 cells in which inflammation was induced with LPS. Specifically, HT-29 cells were cultured in a 24-well plate at 2 × 10 5 Cells were seeded at a concentration of 1000 cells / well and cultured in a 5% CO2, 37°C incubator for 6–7 days. HT-29 cells were simultaneously treated with 5, 10, and 20 mM allulose dissolved in RPMI 1640 medium, a cell culture medium. After 8 hours, they were treated with LPS (1 μg / ml) to induce inflammation for 24 hours. After washing with PBS, the cells were subjected to RNA extraction using Trizol, followed by cDNA synthesis using a cDNA synthesis kit. Expression levels of mucin genes (MUC2, MUC5AC, and MUC5B), which protect the intestinal mucosa, were confirmed by qRT-PCR using the designed primers. The PCR conditions used were denaturation at 95°C for 15 seconds, annealing at 62°C for 30 seconds, and extension at 72°C for 1 minute.

[0057] Based on the experimental results, the expression level of mucin, which is involved in intestinal wall protection, in HT-29 cells in which inflammation was induced by LPS was confirmed by RT-PCR, and a graph showing that the expression levels of MUC2, MUC5AC, and MUC5B were reduced is shown in Figure 7. In Figure 7, the control group (-) indicates the expression level of factors involved in TLR4 / NF-κB signaling in HT-29 cells not treated with LPS, and the control group (+) indicates an experimental group in which HT-29 cells were treated with LPS to induce inflammation in the same way as the experimental group, but were not treated with allulose.

[0058] According to the results of the experiment, the expression level of mucin, which is involved in intestinal wall protection, was confirmed by RT-PCR in HT-29 cells in which inflammation was induced by LPS, and it was found that the expression levels of MUC2, MUC5AC, and MUC5B were reduced, confirming that allulose protects the intestinal area.

[0059] Example 5: Evaluation of allulose's improvement of intestinal permeability (Testing the electrical resistance of the upper and lower layers of intestinal cells) Using allulose as an active ingredient, the transepithelial electrical resistance (TEER) between epithelial cells was measured as an indicator of the adhesion strength between intestinal epithelial cells, and the electrical resistance values ​​of the upper and lower layers of intestinal cells in response to the allulose material were examined.

[0060] Specifically, 5 × 10 Caco-2 cells were placed on a transwell insert. 4 Cells were seeded in the upper layer at a concentration of 100 cells / well, and 600 μL of medium was added to the lower layer without cells and cultured. The medium was changed every two days and cultured for 3 weeks to allow for polarization. After polarization, the upper layer containing the cells was pre-treated with IFN-γ (10 ng / ml) for 24 hours, treated with various concentrations of allulose for 1 hour, and then treated with TNF-α (10 ng / ml) for 6 hours. After the reaction was completed, electrical resistance (TEER) values ​​were measured using a Millicell ERS-2 Voltohmmeter.

[0061] Based on the experimental results, the electrical resistance (TEER) values ​​decreased by IFN-γ / TNF-α in polarized Caco-2 cells and the electrical resistance (TEER) values ​​increased by allulose are shown in Figure 8. It was confirmed that the electrical resistance (TEER) values ​​decreased by IFN-γ / TNF-α in polarized Caco-2 cells were the same as the electrical resistance (TEER) values ​​increased by allulose.

[0062] Example 6: Evaluation of allulose permeability across the upper and lower layers of enterocytes This means that when cells that have suffered intestinal wall damage due to inflammatory factors are treated with the fluorescently labeled protein FITC-dextran (4kD), permeability increases.To confirm the effect of allulose as an active ingredient on intestinal wall permeability, paracellular permeability was examined using FITC-dextran.

[0063] Specifically, 5 × 10 Caco-2 cells were placed on a transwell insert. 4 The upper layer was seeded with cells at a concentration of 1000 cells / well, and the lower layer was cultured with 600 μL of medium without cells. The medium was changed every two days and cultured for three weeks to allow for polarization. After polarization, the upper layer containing the cells was pretreated with IFN-γ (10 ng / ml) for 24 hours, treated with allulose at concentrations of 5, 10, or 20 mM for one hour, and then treated with TNF-α (10 ng / ml) for six hours. The transwell was treated with FITC-dextran (100 μg / ml) diluted in medium and incubated for four hours. Paracellular permeability was measured using fluorescence absorbance at excitation 488 nm and emission 535 nm.

[0064] Based on the experimental results, the fluorescence intensity increased by IFN-γ / TNF-α through FITC-dextran (4 kD) in polarized Caco-2 cells was shown in Figure 9, which is a graph showing the increase in fluorescence intensity by allulose.

[0065] Example 7: Evaluation of the ROS scavenging ability of allulose In Caco-2 cells where inflammation was induced by LPS, we confirmed that accumulated ROS was reduced by allulose via the DCFH-DA fluorescent probe, thereby reducing intestinal permeability.

[0066] Caco-2 cells were cultured in a 96-well plate at 5 × 10 4 Cells were seeded at a cell / well concentration and cultured. Then, LPS (1 μg / ml) and allulose (5 mM, 10 mM, or 20 mM) were co-treated and cultured for 12 hours at 5% CO2 and 37°C. After removing the culture supernatant, DCF-DA was added to the medium at a concentration of 20 μM and cultured in an incubator for 30 minutes. After incubation, the cells were washed with PBS and ROS production was measured using fluorescence absorbance at excitation 488 nm and emission 535 nm.

[0067] FIG. 10 is a graph showing that allulose reduced ROS accumulated through the DCFH-DA fluorescent probe in Caco-2 cells in which inflammation was induced by LPS, resulting in a decrease in intestinal permeability, as shown in Example 7.

[0068] Example 8: Allulose increases the expression of tight junction factors Caco-2 cells were plated in a 24-well plate at 2 × 10 5Cells were cultured at a concentration of 1000 cells / well. The medium was changed every two days, and cells were treated with 5, 10, or 20 mM allulose for 6 hours. Then, cells were treated with LPS (1 μg / ml) and incubated for 12 hours. RNA was extracted from the cells using Trizol, cDNA was synthesized using a cDNA synthesis kit, and gene expression was confirmed using primers tailored to each gene via qRT-PCR. The PCR conditions used were denaturation at 95°C for 15 seconds, annealing at 62°C for 30 seconds, and extension at 72°C for 1 minute. Proteins were extracted from the cultured cells using RIPA lysis buffer for protein expression analysis. The extracted proteins were pretreated at 95°C for 5 minutes and then separated by SDS-PAGE. After transferring the proteins to a PVDF membrane, the membrane was blocked with 5% (w / v) BSA solution for 1 hour, and then incubated with primary antibodies for each factor at a 1:1000 dilution in 5% (w / v) BSA solution at 4°C for 1 day. The membrane was then incubated with secondary antibodies in Tris-buffered saline containing 0.1% (v / v) Tween 20 at room temperature for 1 hour. Bands were detected using the LAS 4000 system, and expression densities were calculated using image J software to determine expression levels.

[0069] The results of the above experiment show that in Caco-2 cells where inflammation was induced by LPS, the expression levels of mRNA and protein of ZO-1 and occludin, which are tight junction factors that are decreased by inflammation, increased. These results are shown in Figure 11. In Caco-2 cells where inflammation was induced by LPS, the expression levels of mRNA and protein of ZO-1 and occludin, which are tight junction factors that are decreased by inflammation, increased with allulose treatment. This indicates that allulose improves intestinal permeability, i.e., tight junctions, which were damaged by inflammation.

[0070] Example 9: Evaluation of the toxicity of allulose Human intestinal epithelial cells, HT-29 cells and Caco-2 cells, were treated with allulose at concentrations of 5, 10, and 20 mM, respectively, and then cultured for 24 hours, after which an MTT test was performed.

[0071] Specifically, HT-29 cells and Caco-2 cells were cultured in a 96-well plate at 5 × 10 4 The cells were dispensed at a concentration of 1 / well and cultured under 5% CO2 at 37°C. The medium was removed and 5 mg / ml MTT solution was added and incubated for 2 hours. 200 μl / well of dimethyl sulfoxide (DMSO) was added and incubated at room temperature for 10 minutes to dissolve formazan. The absorbance was measured at 590 nm to calculate cell activity (%), and the activity of cells with and without the sample was compared to determine cytotoxicity.

[0072] The results obtained from the above experiments show the cytotoxicity evaluation of allulose, and specifically, the toxicity evaluation at each allulose concentration is shown in Figure 12. Figure 12 shows the toxicity evaluation results for HT-29 cells and Caco-2 cells.

[0073] The toxicity evaluation results showed that allulose at concentrations of 5 mM, 10 mM, and 20 mM was not toxic. These results confirmed that allulose effectively protects intestinal wall cells.

Claims

1. A composition for preventing, improving, or treating leaky gut or leaky gut syndrome, comprising allulose as an active ingredient.

2. 2. The composition of claim 1, wherein the leaky gut is due to increased intestinal mucosal permeability, damage to the intestinal mucosa, or intestinal inflammation.

3. 2. The composition of claim 1, wherein the intestinal leakage is characterized by decreased expression of TLR4, increased expression of prostaglandin E2 and COX-2, decreased adhesion strength between intestinal epithelial cells, or increased expression of ZO-1 and occludin.

4. The composition according to claim 1, wherein the composition has one or more properties selected from the group consisting of allulose's ability to enhance intestinal mucosal immunity, anti-inflammatory ability against intestinal inflammation, intestinal mucosal protection ability, intestinal permeability improvement ability, and reactive oxygen species (ROS) scavenging ability.

5. The intestinal mucosal immunity-enhancing ability is achieved by regulating the expression of one or more factors related to TLR4 / NF-κB signaling and TLR4 / MAPK signaling that regulate inflammatory responses, preferably one or more factors related to TLR4 / NF-κB signaling selected from the group consisting of p50, p65, TNF-α, IL-6, IL-8, COX-2, TLR4, MyD88, IKKα, IKKβ, IKβα, IRAK4, and TRAF6, or one or more factors related to MAPK signaling selected from the group consisting of p38, p-p38, JNK, p-JNK, c-Jun, p-c-Jun, c-Fos, and p-c-Fos; The anti-inflammatory activity is characterized by decreasing or inhibiting the expression of one or more proteins selected from the group consisting of prostaglandin E2, a pro-inflammatory mediator, and COX-2, a core indicator of inflammatory bowel disease; The intestinal mucosa-protecting ability is achieved by decreasing or inhibiting the expression of one or more mucins selected from the group consisting of MUC2, MUC5AC, and MUC5B, which are mucins that protect the intestinal mucosa; The ability to improve intestinal permeability includes improving the increase in intestinal permeability induced by intestinal wall damage caused by inflammatory factors, improving the adhesion strength between intestinal epithelial cells, or increasing the expression of tight junction factors ZO-1 and occludin, or The composition according to claim 4, wherein the reactive oxygen species scavenging ability is an increase in the expression of one or more factors selected from the group consisting of PI3K, AKT, Nrf2, and HO-1, which are factors involved in Nrf2 / HO-1 signaling activated by ROS.

6. The composition of claim 1 , wherein the allulose is a TLR4 inhibitor or a TLR4 antagonist.

7. The composition of claim 6, wherein the allulose is a TLR4 inhibitor that regulates the expression of one or more factors involved in TLR4 / NF-κB signaling selected from the group consisting of p50, p65, TNF-α, IL-6, IL-8, COX-2, TLR4, MyD88, IKKα, IKKβ, IKβα, IRAK4, and TRAF6.

8. 2. The composition of claim 1, wherein the prevention, amelioration, or treatment of leaky gut or leaky gut syndrome is not due to the prebiotic properties of allulose that regulate the growth of intestinal microorganisms.

9. The composition according to claim 1, wherein the allulose is contained in the composition in a total dosage of 1 g to 60 g per day based on a 60 kg adult.

10. The composition of claim 1, wherein the allulose is provided as a sugar syrup containing allulose.

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