Composition for preventing, ameliorating or treating intestinal fibrosis comprising zeolite as effective component

Abstract

The present invention relates to a composition for the prevention, improvement, or treatment of intestinal fibrosis comprising zeolite as an active ingredient. The zeolite according to the present invention is expected to be useful as a composition for the prevention, improvement, or treatment of intestinal fibrosis because it has excellent effects in reducing the thickness of intestinal tissue and the accumulation of collagen in intestinal tissue in an animal model of intestinal fibrosis induced by DSS (Dextran sulfate sodium), reducing the expression of intestinal fibrosis-related genes, and reducing the expression of α-SMA (α-smooth muscle actin) protein in a cell model of intestinal fibrosis induced by TGF-β.

Description

Technology Field

[0001] The present invention relates to a composition for the prevention, improvement, or treatment of intestinal fibrosis comprising zeolite as an active ingredient. Background Technology

[0002] Intestinal fibrosis is a disease caused by chronic and recurrent enteritis, leading to intestinal stenosis and obstruction. Although the exact cause of intestinal fibrosis has not yet been clearly identified, it is known that when intestinal microorganisms invade due to the destruction of the intestinal mucosa caused by genetic, environmental, or abnormal immune factors, the activation of intestinal immune cells progresses; this excessive activation leads to abnormal abnormalities in the immune cell regulatory system, progressing to chronic inflammation, and if such intestinal inflammation occurs continuously and repeatedly, intestinal fibrosis develops.

[0003] Currently, there are no reports of drugs that can cure intestinal fibrosis, but commonly prescribed medications include 5-aminosalicylic acid, adrenocortical steroids, immunomodulators (thiopurines), and anti-TNFα biological agents, which are prescribed and used in stages depending on the severity of symptoms; however, various side effects have been reported for these drugs. Therefore, there is a need for research on the development of treatments for intestinal fibrosis, which causes a decrease in quality of life and increases the mortality rate of patients with severe intestinal disease.

[0004] Meanwhile, Korean Registered Patent No. 2417640 discloses a "composition for the prevention or treatment of fibrotic diseases comprising a thiourea derivative" and Korean Registered Patent No. 1871949 discloses a "method for manufacturing an intestinal fibrosis cell model," but there is no description of a composition for the prevention, improvement, or treatment of intestinal fibrosis comprising the zeolite of the present invention as an active ingredient. The problem to be solved

[0005] The present invention was derived from the above-mentioned needs, and the inventors confirmed that in an animal model of intestinal fibrosis induced by DSS (Dextran sulfate sodium), zeolite reduces the thickness of intestinal tissue and the accumulation of collagen within the intestinal tissue, and reduces the expression level of genes related to intestinal fibrosis, and completed the present invention by confirming that it reduces the expression level of α-SMA (α-smooth muscle actin) protein in a cell model of intestinal fibrosis induced by TGF-β. means of solving the problem

[0006] To solve the above problem, the present invention provides a health functional food composition for the prevention or improvement of intestinal fibrosis comprising zeolite as an active ingredient.

[0007] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of intestinal fibrosis comprising zeolite as an active ingredient.

[0008] In addition, the present invention provides a veterinary composition for the prevention or treatment of intestinal fibrosis comprising zeolite as an active ingredient.

[0009] In addition, the present invention provides a feed additive for the prevention or improvement of intestinal fibrosis comprising zeolite as an active ingredient. Effects of the invention

[0010] The zeolite according to the present invention is expected to be useful as a composition for the prevention, improvement, or treatment of intestinal fibrosis because it has excellent effects in reducing the thickness of intestinal tissue and the accumulation of collagen in intestinal tissue in an animal model of intestinal fibrosis induced by DSS (Dextran sulfate sodium), reducing the expression of intestinal fibrosis-related genes, and reducing the expression of α-SMA (α-smooth muscle actin) protein in a cell model of intestinal fibrosis induced by TGF-β. Brief explanation of the drawing

[0011] Figure 1 is a schematic diagram showing the experimental process to confirm the effect of zeolite administration in an animal model of intestinal fibrosis induced by DSS (dextran sulfate sodium). Figure 2 shows the results of quantifying the amount of collagen accumulated in the intestinal tissue by measuring the thickness of the intestinal tissue through H&E (Hematoxylin and eosin) staining and the amount of collagen accumulated in the intestinal tissue through Sirius Red staining after administering zeolite to an animal model of intestinal fibrosis. Control is the normal group, DSS is the animal model of intestinal fibrosis, DSS+ZO200 is the experimental group administered 200 mg / kg of zeolite to the animal model of intestinal fibrosis, DSS+ZO300 is the experimental group administered 300 mg / kg of zeolite to the animal model of intestinal fibrosis, and DSS+ZO400 is the experimental group administered 400 mg / kg of zeolite to the animal model of intestinal fibrosis. ### indicates that the thickness of intestinal tissue and the amount of collagen in the intestinal fibrosis animal model increased statistically significantly compared to the normal group, with p<0.001, and *, ** indicate that the thickness of intestinal tissue and the amount of collagen in the zeolite-treated group decreased statistically significantly compared to the intestinal fibrosis animal model, with * being p<0.05 and ** being p<0.01. Figure 3 shows the intestinal fibrosis-related gene (after administering zeolite to an animal model of intestinal fibrosis) Col3a1 , Mmp7 , Mmp9 This is the result of confirming the expression levels. Control is the normal group, DSS is an animal model of intestinal fibrosis, DSS+ZO200 is the experimental group administered 200 mg / kg of zeolite to the animal model of intestinal fibrosis, DSS+ZO300 is the experimental group administered 300 mg / kg of zeolite to the animal model of intestinal fibrosis, and DSS+ZO400 is the experimental group administered 400 mg / kg of zeolite to the animal model of intestinal fibrosis. #, ### are the results of the animal model of intestinal fibrosis compared to the normal group. Col3a1 , Mmp7 , Mmp9This indicates that gene expression levels increased statistically significantly, with # being p<0.05 and ### being p<0.001, and * and ** representing the zeolite-treated group compared to the intestinal fibrosis animal model. Col3a1 , Mmp7 , Mmp9 This indicates that the gene expression level decreased statistically significantly; * is p<0.05 and ** is p<0.01. Figure 4 shows the results of analyzing the expression levels of α-SMA (α-smooth muscle actin) protein after treating a cell model of intestinal fibrosis with zeolite. Con represents the normal group, and TGF-β represents the intestinal fibrosis-induced group. #### indicates that the expression level of α-SMA protein was statistically significantly increased in the intestinal fibrosis-induced group compared to the normal group, with p<0.0001, and *, ***, **** indicate that the expression level of α-SMA protein was statistically significantly decreased in the zeolite-treated group compared to the intestinal fibrosis-induced group, with * being p<0.05, *** being p<0.001, and **** being p<0.0001. Specific details for implementing the invention

[0012] To achieve the objective of the present invention, the present invention provides a health functional food composition for the prevention or improvement of intestinal fibrosis comprising zeolite as an active ingredient.

[0013] In the present invention, the zeolite is a general term for minerals in which anions formed by the combination of aluminum oxide and silicate oxide are combined with alkali metals and alkaline earth metals, and more precisely, it refers to aluminum silicate minerals.

[0014] In the health functional food composition of the present invention, the zeolite may preferably be natural zeolite, such as clinoptilolite or heulandite, and more preferably may be clinoptilolite.

[0015] The above zeolite may include one or more selected from silicon dioxide (SiO2), titanium dioxide (TiO2), aluminum oxide (Al2O3), iron oxide (Fe2O3), manganese oxide (MnO), calcium oxide (CaO), magnesium oxide (MgO), potassium oxide (K2O), sodium oxide (Na2O), and phosphorus pentoxide (P2O5), and preferably may include 60 to 70 parts by weight of silicon dioxide, 0.1 to 1 part by weight of titanium dioxide, 10 to 15 parts by weight of aluminum oxide, 0.1 to 1.5 parts by weight of iron oxide, 0.1 to 1 part by weight of manganese oxide, 0.1 to 2 parts by weight of calcium oxide, 0.1 to 1 part by weight of magnesium oxide, 0.1 to 3 parts by weight of potassium oxide, 0.1 to 3 parts by weight of sodium oxide, and 0.1 to 1 part by weight of phosphorus pentoxide, but is not particularly limited thereto. The above zeolite is characterized by a loss of ignition of 1.5% or less.

[0016] The above zeolite may be prepared with a size of 0.2 to 20 μm through dry and wet purification, preferably with a size of 1 to 10 μm, but is not limited thereto. The above zeolite is characterized by not containing heavy metals.

[0017] In the health functional food composition of the present invention, the intestinal fibrosis may be caused by continuous and repetitive inflammation of the intestine, but is not limited thereto.

[0018] The health functional food composition of the present invention is preferably prepared in any one formulation selected from powder, granules, pills, tablets, capsules, candies, syrups, and beverages, but is not limited thereto.

[0019] When the health functional food composition of the present invention is used as a food additive, the active ingredient may be added as is or used together with other foods or food ingredients, and may be used appropriately according to conventional methods. The amount of the active ingredient can be appropriately determined according to the purpose of use (improvement). Generally, when manufacturing food or beverages, the composition of the present invention is added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, relative to the raw material. However, in the case of long-term consumption for the purpose of health and hygiene or health control, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount greater than the above range.

[0020] There are no specific restrictions on the types of the above-mentioned foods. Examples of foods to which the above-mentioned active ingredients may be added include meat, sausage, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and include all health functional foods in the conventional sense.

[0021] When the health functional food composition of the present invention is used as a health beverage, it may contain various flavoring agents or natural carbohydrates as additional ingredients, as in conventional beverages. The aforementioned natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As sweeteners, natural sweeteners such as taumarin and stevia extract, or synthetic sweeteners such as saccharin or aspartame may be used. The proportion of the above natural carbohydrates is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 g of the composition of the present invention. The composition of the present invention may contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the composition of the present invention may contain fruit pulp for the production of natural fruit juices, fruit juice beverages, and vegetable beverages. These ingredients may be used independently or in combination. Although the proportion of these additives is not critical, the composition of the present invention is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight.

[0022] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of intestinal fibrosis comprising zeolite as an active ingredient.

[0023] In the pharmaceutical composition of the present invention, the zeolite and intestinal fibrosis are as described above.

[0024] The pharmaceutical composition of the present invention may further include, but is not limited to, pharmaceutically acceptable carriers, excipients, or diluents in addition to the active ingredient.

[0025] The pharmaceutical composition of the present invention may be prepared in any one formulation selected from capsules, powders, granules, tablets, suspensions, emulsions, syrups, and aerosols, but is not limited thereto.

[0026] Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are those commonly used in formulations and include, but are not limited to, saline solution, sterile water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, lactose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above components, antioxidants, buffers, bacteriostatic agents, diluents, surfactants, binders, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, or preservatives. Suitable dosages of the pharmaceutical composition of the present invention may be prescribed in various ways depending on factors such as the formulation method, mode of administration, patient's age, body weight, sex, pathological condition, diet, time of administration, route of administration, excretion rate, and response sensitivity. The route of administration of the pharmaceutical composition for the prevention or treatment of intestinal fibrosis of the present invention may be any generally accepted route as long as it can reach the target tissue. The pharmaceutical composition of the present invention may be administered via routes such as intramuscular administration, ophthalmic administration, intraperitoneal administration, intravenous administration, subcutaneous administration, intradermal administration, transdermal patch administration, oral administration, intranasal administration, intrapulmonary administration, and rectal administration, although it is not particularly limited thereto, depending on the purpose, and specifically may be administered via the route of oral administration.

[0027] In addition, the present invention provides a veterinary composition for the prevention or treatment of intestinal fibrosis comprising zeolite as an active ingredient.

[0028] The veterinary composition of the present invention may further include suitable excipients and diluents according to conventional methods. Examples of excipients and diluents that may be included in the veterinary composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, cetanol, stearyl alcohol, liquid paraffin, sorbitan monostearate, polysorbate 60, methylparaben, propylparaben, and mineral oil. The veterinary composition according to the present invention may further include fillers, anticoagulants, lubricants, wetting agents, spices, emulsifiers, preservatives, etc., and the veterinary composition according to the present invention may be formulated using methods well known in the art to provide rapid, sustained, or delayed release of the active ingredient after administration to an animal, and the formulation may be in the form of powders, granules, tablets, capsules, suspensions, emulsions, solutions, syrups, aerosols, soft or hard gelatin capsules, suppositories, sterile injectable solutions, sterile topical preparations, etc.

[0029] The effective amount of the veterinary composition according to the present invention can be appropriately selected according to the individual animal. It may be determined based on the severity of the disease or condition, the sensitivity to the active ingredient of the present invention according to the individual's age, weight, health status or gender, the route of administration, the duration of administration, factors including other compositions combined with or used simultaneously with the composition, and other factors well known in the physiological or veterinary field.

[0030] In addition, the present invention provides a feed additive for the prevention or improvement of intestinal fibrosis comprising zeolite as an active ingredient.

[0031] The feed additive of the present invention corresponds to an auxiliary feed under the Feed Management Act. In the present invention, the term "feed" may refer to any natural or artificial prescribed food, single meal, etc., or the components of said single meal, intended for or suitable for animals to eat, consume, and digest. The types of said feed are not particularly limited, and feeds commonly used in the relevant technical field may be used. Non-limiting examples of said feed include plant-based feeds such as grains, root vegetables, food processing by-products, algae, fibers, pharmaceutical by-products, oils and fats, starches, meal, or grain by-products; and animal-based feeds such as proteins, inorganic substances, oils and fats, minerals, single-cell proteins, zooplankton, or food waste. These may be used individually or in a mixture of two or more types.

[0033] The present invention will be described in more detail below using manufacturing examples and embodiments. These manufacturing examples and embodiments are intended solely to explain the present invention more specifically, and it is obvious to those skilled in the art that the scope of the present invention is not limited by them.

[0035] Materials and Methods

[0036] 1. Establishment of an animal model of intestinal fibrosis

[0037] The intestinal fibrosis animal model used in the present invention was induced by continuous and repeated administration of DSS (dextran sulfate sodium). Specifically, an intestinal fibrosis animal model was established by sequentially providing 8-week-old C57BL / 6J mice with 1.75%, 2%, or 2.25% (w / v) DSS diluted with drinking water for 5 days, followed by providing regular drinking water for 10 days, repeating this cycle a total of 3 times (total 45 days) (Fig. 1).

[0039] 2. Administration of Zeolite

[0040] During the 45 days of the experiment, zeolite diluted in saline solution was orally administered daily at doses of 200 mg / kg, 300 mg / kg, or 400 mg / kg, respectively. The zeolite used in this experiment was clinoptilolite, a natural zeolite, and was prepared to a size of 10 μm or less through dry / wet purification.

[0042] Example 1. Histological analysis of the intestine in an animal model of intestinal fibrosis

[0043] After administering zeolite to an animal model of intestinal fibrosis, the thickness of the intestinal tissue was measured by H&E (Hematoxylin and eosin) staining, and the amount of collagen accumulated in the intestinal tissue was quantified by Sirius Red staining.

[0044] As a result, as disclosed in Figure 2, it was confirmed that the thickness of the intestinal tissue and the amount of accumulated collagen in the intestinal fibrosis-induced group increased compared to the normal group, while the thickness of the intestinal tissue and the amount of accumulated collagen in the zeolite-administered group decreased compared to the intestinal fibrosis-induced group.

[0045] Based on the above results, it was found that the intestinal fibrosis animal model of the present invention effectively induced intestinal fibrosis through repeated and continuous administration of DSS, and that the administration of zeolite had a therapeutic effect on intestinal fibrosis.

[0047] Example 2. Analysis of Intestinal Fibrosis-Related Gene Expression Levels in an Animal Model of Intestinal Fibrosis

[0048] After administering zeolite to an animal model of intestinal fibrosis, each animal was sacrificed on day 45, RNA was isolated from the separated intestinal tissue samples, and through reverse transcription and Q-PCR, the factors involved in intestinal fibrosis Col3a1 (collagen type III alpha 1), Mmp7 (matrix metalloproteinase 7), Mmp9 The relative mRNA expression levels of the (matrix metalloproteinase 9) gene were determined.

[0049] As a result, as disclosed in Fig. 3, the intestinal fibrosis-induced group compared to the normal group Col3a1 gene, Mmp7 gene, Mmp9 While gene expression levels increased, the zeolite-treated group compared to the intestinal fibrosis-induced group showed... Col3a1 gene, Mmp7 gene, Mmp9 It was confirmed that the gene expression level decreased.

[0051] Example 3. Analysis of α-SMA (α-smooth muscle actin) protein expression in an intestinal fibrosis cell model

[0052] It is known that an increase in the expression of α-SMA protein within intestinal tissue promotes the production of extracellular matrix such as collagen, leading to intestinal fibrosis.

[0053] After subculturing human intestinal myofibroblasts in a culture medium (High glucose DMEM, FBS 10%, Penicillin-Streptomycin 1%, L-glutamine, HEPES), 2×10⁶ cells were placed in a 384-well plate. 3 50 µl was inoculated per well. After 24 hours, the culture medium was changed to serum-free medium (High glucose DMEM, Penicillin-Streptomycin 1%, L-glutamine, HEPES) and cultured for an additional 24 hours. Subsequently, 5 ng / ml of TGF-β, a cytokine that induces intestinal fibrosis, was added along with zeolite at concentrations of 5 µg / ml, 10 µg / ml, 20 µg / ml, or 40 µg / ml, and cultured for 96 hours. Afterward, the samples were stained using antibodies against α-SMA protein, and the staining intensity was analyzed using the Harmony 4.9 program.

[0054] As a result, as disclosed in Figure 4, it was confirmed that the expression level of α-SMA protein increased in the intestinal fibrosis-induced group (TGF-β alone treatment group) compared to the normal group, while it decreased significantly in the zeolite treatment group (TGF-β and zeolite simultaneous treatment group).

Claims

Claim 1 A health functional food composition for the prevention or improvement of intestinal fibrosis comprising zeolite as an active ingredient, wherein the zeolite is characterized by reducing the expression of α-SMA (α-smooth muscle actin) protein in intestinal cells. Claim 2 A health functional food composition for the prevention or improvement of intestinal fibrosis according to claim 1, characterized in that the active ingredient is prepared in any one formulation selected from powder, granules, pills, tablets, capsules, candies, syrups, and beverages. Claim 3 A pharmaceutical composition for the prevention or treatment of intestinal fibrosis comprising zeolite as an active ingredient, wherein the zeolite is characterized by reducing the expression of α-SMA (α-smooth muscle actin) protein in intestinal cells. Claim 4 A pharmaceutical composition for the prevention or treatment of intestinal fibrosis according to paragraph 3, further comprising, in addition to the active ingredient, a pharmaceutically acceptable carrier, excipient, or diluent. Claim 5 A pharmaceutical composition for the prevention or treatment of intestinal fibrosis according to claim 3, characterized in that the composition is prepared in any one formulation selected from capsules, powders, granules, tablets, suspensions, emulsions, syrups, and aerosols. Claim 6 A veterinary composition for the prevention or treatment of intestinal fibrosis comprising zeolite as an active ingredient, wherein the zeolite is characterized by reducing the expression of α-SMA (α-smooth muscle actin) protein in intestinal cells. Claim 7 A feed additive for the prevention or improvement of intestinal fibrosis comprising zeolite as an active ingredient, wherein the zeolite is characterized by reducing the expression of α-SMA (α-smooth muscle actin) protein in intestinal cells.

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