KR102231387B1 - A pharmaceutical composition for the treatment of colorectal disease comprising the fermentative products of Kalopanax septemlobus as an active ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition and a food composition for the treatment of colon disease, comprising the Eumnamu fermented product as an active ingredient. In the field of oriental medicine, the antibacterial and anti-inflammatory effects of Eumnamu have been disclosed, and technologies such as the use of Eumnamu extract to treat tuberculosis or acne have been developed, but the efficacy of Eumnamu related to colon disease has not been known.
Eumnamu, or Eumnamu fermented product of the present invention has a remarkable effect on treating colitis in an individual with colitis-induced colon disease. In particular, the fermented Eumnamu fermented product that has been fermented with basidiomycete mycelium or is bioconverted through additional enzymatic treatment has a more remarkable effect on the treatment of colon diseases than that of the unfermented Eumnamu plant, so it is expected to be widely used in the treatment of various colon diseases in the medical field. It is expected.

Description

{A pharmaceutical composition for the treatment of colorectal disease comprising the fermentative products of Kalopanax septemlobus as an active ingredient}

The present invention relates to a pharmaceutical composition for the treatment of inflammatory bowel disease comprising a bioconverted Eumnamu fermentation as an active ingredient.

Colon diseases include colon cancer, colitis, irritable growth syndrome, inflammatory growth disease, etc., and collectively refer to various diseases that occur in the large intestine. In modern times, the number of patients with bowel disease is increasing significantly due to the influence of frequent stress, westernized eating habits, and drinking. Colorectal cancer is a cancer that ranks second to third in cancer deaths, accounting for about 13% of all cancers, and in the United States, it is reported that about 50,310 people die of colorectal cancer annually and 136,830 new colon cancer patients occur. Has become. In addition, colitis is a state of inflammation in the large intestine and is caused by various causes.In Korea, the incidence rate increased rapidly from the 1980s, and the prevalence of ulcerative colitis in 2048 was 155.7 per 100,000 population, and the total number of ulcerative colitis patients. Is estimated to be between 67,300 and 74,900.

On the other hand, Eumnamu is a deciduous broad-leaved arboreous tree belonging to the Araceae family, and its scientific name is Kalopanax septemlobus. Depending on the region, they are also called Eommu, Eommok, and Gaulk. It is a large tree with a height of 25m and a diameter of 1m, and is a tree without clusters. It is distributed nationwide on the Korean peninsula, and is distributed in Far East Russia, southern Kuril, all over Japan and China. The area around 400-500m above sea level is the center. In the field of oriental medicine, the antibacterial and anti-inflammatory effects of Eumnamu have been disclosed, and “Use of Eumnamu extract to treat tuberculosis (Korean Patent Registration No. KR 10-1751604)”, and “Use of Eumnamu extract to treat acne (Korean Patent Laid-Open No. KR 2017-0032763)”. And the like were developed, but the efficacy of Eumnamu related to colon disease was not known.

Accordingly, the present invention is conceived to solve the problems of the prior art as described above, and relates to a pharmaceutical composition for treating colon diseases comprising a bioconverted Eumnamu fermentation as an active ingredient. The composition of the present invention may be used as a pharmaceutical/food composition for treatment, prevention or improvement of colon disease, in particular, colon inflammation.

The present invention is conceived to solve the problems of the prior art as described above, a pharmaceutical composition for treating colon diseases comprising fermented with basidiomycete hyphae or bioconverted through enzymatic treatment as an active ingredient It is about.

However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems that are not mentioned will be clearly understood by those of ordinary skill in the art from the following description.

Hereinafter, various embodiments described herein are described with reference to the drawings. In the following description, for a thorough understanding of the present invention, various specific details, such as specific forms, compositions and processes, etc. are set forth. However, certain embodiments may be practiced without one or more of these specific details, or with other known methods and forms. In other instances, well-known processes and manufacturing techniques have not been described in specific details in order not to unnecessarily obscure the present invention. Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, form, composition, or characteristic described in connection with the embodiment is included in one or more embodiments of the present invention. Thus, the context of “in one embodiment” or “an embodiment” expressed in various places throughout this specification does not necessarily represent the same embodiment of the invention. Additionally, particular features, forms, compositions, or properties may be combined in one or more embodiments in any suitable manner.

Unless otherwise defined in the specification, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

In one embodiment of the present invention, "colorectal disease" is a generic term for various diseases occurring in the large intestine, and preferably, colon cancer, colon polyps, colitis, ischemic bowel disease, dysentery, intestinal vascular dysplasia, diverticulosis, irritable growth syndrome , Inflammatory growth disease, more preferably colitis, but is not limited thereto.

In one embodiment of the present invention, the term "basidiomycete" is a fungus that becomes a cell called basidiomycete as a result of sexual reproduction as a subfamily of fungi and creates spores. It cannot make nutrients by itself, so it is attached to other organisms and is parasitic, and many are known as mushrooms. Shiitake mushrooms, Pseudomonas mushrooms, Chaga mushrooms, Maitake mushrooms, Wood ear mushrooms, Snowflake mushrooms and skirt mushrooms are representative. Basils are densely formed on the wrinkled side of mature mushrooms. Most of basidiomycete mushrooms live by-products in the fluids of higher plants, and play an indispensable role in the biological circulation of substances by decomposing cellulose and lignin in particular, but they also form mycorrhizal roots at the roots of trees to live in common. There are also many. There are other types that are considered important as edible bacteria or that need attention as poisonous mushrooms.

In one embodiment of the present invention, "hyphae" is a vegetative cell of a filamentous fungus (fungus). Functional differentiation, such as nutritional or physiological functions, can also be seen. In terms of morphology, the presence or absence of a septum is used for classification, and it develops in the germination tube of spores and elongates by apical growth. A group of branched hyphae is called mycelium. Depending on their physiological function, they are divided into basal hyphae that elongate as they enter the surface or inside of the medium or host, and parasitic hyphae that elongate in the air. Parasitic hyphae depend on the transfer of nutrients from the basal hyphae, and some of them differentiate into spore-forming organs. If there are organic substances and environmental conditions such as suitable humidity and temperature are good, various mycelial structures are formed. The diaphragm of the hyphae of ascytic fungi is simple, but in basidiomyces, it can be seen that they form a clamp connection. In fungi, it is branched into several branches and tangled like a thread. In mushrooms, it aggregates to form fruiting bodies. Because of the lack of chlorophyll, photosynthesis is not possible, and it is attached to host organisms and absorbs nutrients. In the case of basidiomycete, which is a mushroom, a hyphae germinated from a spore has one nucleus, and when it fuses with another hyphae, one cell has two nuclei. A hypha with one nucleus is called a primary hyphae, and a hyphae with two nuclei is called a secondary hyphae. In general, the cell wall of the hyphae is mainly composed of chitin or hemicellulose, and the two are sometimes combined.

In the present specification, the basidiomycete hyphae can be specifically selected and used from hyphae such as medicinal and edible basidiomycete shiitake mushrooms or physalis mushrooms, chaga mushrooms, Maitake mushrooms, tree ear mushrooms, snowflake mushrooms, and skirt mushrooms, These basidiomycete hyphae are the Korea Microbial Conservation Center (KCCM), the Korea Research Institute of Bioscience and Biotechnology Life Resource Center (KCTC), the Korea Cell Line Bank (KCLB), the Korea Agricultural Microbial Resource Center (KACC), and the American Standard Strain Culture Recorded Preservation Center (ATCC). The strain can be distributed and used by such institutions.

In one embodiment of the present invention, the term "umnamu" is a deciduous broad-leaved arboreous tree belonging to the Araliaceae family, and its scientific name is Kalopanax septemlobus. Depending on the region, they are also called Eommu, Eommok, and Gaulk. It reaches 25m in height, and the branch has thick and spread thorns. The leaves are alternate and round, the edges are deeply divided into 5-9 pieces, and there are serrations on the forked pieces, and the petiole is longer than the leaves. The flower blooms in early August, is yellow-green, and hangs on a double-mountainous inflorescence. There are 5 petals and stamens each, and 2 styles. Fruits are drupe, round, and ripen black in mid-September-October. It is distributed nationwide on the Korean peninsula, and is distributed in Far East Russia, southern Kuril, all over Japan and China. Magnificus (var. magnificus) with dense hairs on the back of leaves, var. maximowiczii (var. maximowiczii) with densely divided leaves and white hairs on the back. I see all of them as the tinnitus of Eumnamu.

In one embodiment of the present invention, "bioconversion" refers to converting an added substance into a chemically modified form using the physiological function of an organism.

In one embodiment of the present invention, "fermentation" refers to a process in which a microorganism decomposes organic matter using an enzyme possessed by it. Fermentation is a phenomenon in which organic substances are converted into simple compounds by enzyme action to release free energy, but in general, it refers to a phenomenon in which microorganisms decompose organic substances and accumulate metabolites. In other words, alcohol fermentation in which yeast decomposes sugar anaerobicly into ethyl alcohol and carbon dioxide, and lactic acid fermentation in which lactic acid bacteria anaerobicly decompose sugar into lactic acid. A phenomenon in which alcohol is oxidized to acetic acid by use, and a phenomenon in which molds oxidize glucose to gluconic acid using oxygen in the air are also called acetic acid fermentation, gluconic acid fermentation, and the like, respectively. In addition, these are divided into anaerobic fermentation and aerobic fermentation (oxidative fermentation), and other anaerobic fermentations other than those mentioned above include glycerol fermentation, acetone fermentation, butanol fermentation, 2, 3-butylene glycol fermentation, butyric acid fermentation. , Propionic acid fermentation, methane fermentation, etc., and aerobic fermentation include citric acid fermentation, itaconic acid fermentation, yeast acid fermentation, 2-ketoglutaric acid fermentation, oxalic acid fermentation, fumaric acid fermentation, sorbose fermentation, and the like. In addition, the production of amino acids, vitamins and antibiotics by microorganisms is also called glutamic acid fermentation, riboflavin fermentation, penicillin fermentation, etc., but it is not a very good name. In principle, the name of the fermentation action is called the product, but sometimes the name of the substrate is given by cellulose fermentation, pectin fermentation, etc.

In one embodiment of the present invention, the pharmaceutical composition means a composition administered for a specific purpose. For the purposes of the present invention, the pharmaceutical composition of the present invention is used to treat colon disease, preferably colitis, and may include a composition for this and a pharmaceutically acceptable carrier, excipient, or diluent. The pharmaceutical composition according to the present invention contains 0.1 to 100% by weight of an active ingredient corresponding to the pharmaceutical composition of the present invention based on the total weight of the composition. Carriers, excipients and diluents that may be included in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate. , Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

In one embodiment of the present invention, the food composition is variously used as a food composition for improving colon disease, preferably colitis, and the food composition comprising the composition of the present invention as an active ingredient is a variety of foods, for example , Beverages, gum, tea, vitamin complexes, powders, granules, tablets, capsules, snacks, rice cakes, bread, etc. can be prepared in the form. Since the food composition of the present invention is composed of natural foods and fermented products with little toxicity and side effects, it can be safely used even when taken for a long time for prophylactic purposes. When the composition of the present invention is included in the food composition, the amount may be added in a proportion of 0.1 to 100% of the total weight. Here, when the food composition is prepared in the form of a beverage, there is no particular limitation other than containing the food composition in the indicated ratio, and various flavoring agents or natural carbohydrates, etc. may be included as an additional component, such as a normal beverage. That is, as natural carbohydrates, monosaccharides such as glucose, disaccharides such as fructose, and common sugars such as sucrose and polysaccharides, dextrins, cyclodextrins, and sugar alcohols such as xylitol, sorbitol, and erythritol are included. can do. Examples of the flavoring agent include natural flavoring agents (taumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.). The food composition of is various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, It may contain stabilizers, preservatives, glycerin, alcohols, carbonates used in carbonated beverages, etc. These ingredients may be used independently or in combination The proportion of these additives is not so important, but 100% by weight of the composition of the present invention It is generally selected in the range of 0.1 to 100 parts by weight per part.

In one embodiment of the present invention, the steps of (a) cultivating Eumnamu powder obtained by powdering Eumnamu into a liquid culture medium, treating with a cell wall degrading enzyme, and sterilizing to prepare a liquid Eumnamu medium; (b) performing a biotransformation fermentation process of inoculating basidiomycete mycelium into the culture medium of step (a) with basidiomycete mycelium for cultivation and fermentation; And (c) producing a bioconverted product having improved colon disease treatment efficacy through a bioconversion enzyme treatment process of treating fibrinolytic enzyme from the fermented product produced by the biotransformation fermentation process of step (b). It provides a method for producing a fermented Eumnamu, wherein the fermented Eumnamu provides a method of fermented Eumnamu, wherein the fermented Eumnamu is the bark of Eumnamu, and the method of manufacturing a fermented Eumnamu is provided.

In the method for preparing the blackberry fermentation, the cell wall degrading enzyme in step (a) may be at least one of cellulase, hemicellulase, pectinase, and glucanase-based cell wall degrading enzymes capable of degrading cell wall components, and , Basidiomycosis hyphae in the step (b) is selected from the group consisting of shiitake mushrooms, prickly pear mushrooms, chaga mushrooms, Maitake mushrooms, tree ear mushrooms, snowflake mushrooms, and skirt mushrooms, and the basidiomycete hyphae is 1% (v /v) to 20% (v/v), preferably 5% (v/v) to 15% (v/v), more preferably 8% (v/v) to 12% (v/v) The fibrinolytic enzyme in step (c) is selected from the group consisting of cellulase, hemicellulase, pectinase, and glucanase, but the fibrinolytic enzyme is 0.01 It may be added in an amount of% (v/v) to 1% (v/v), preferably 0.01% (v/v) to 0.1% (v/v), but is not limited thereto. In the present invention, the enzyme treatment process of steps (a) and (c) may be performed while stirring for 1 to 3 hours under a temperature condition of 50 to 60° C., but is not limited thereto. The colon disease is preferably selected from the group consisting of colon cancer, colon polyps, colitis, ischemic bowel disease, dysentery, intestinal vascular dysplasia, diverticulosis, irritable growth syndrome, and inflammatory growth disease, but is not limited thereto.

In another embodiment of the present invention, it provides a fermented Eumnamu prepared by the above method.

In another embodiment of the present invention, it provides a pharmaceutical composition for preventing or treating colon disease comprising the Eumnamu fermentation product as an active ingredient, and the colon disease is colon cancer, colon polyp, colitis, ischemic bowel disease, dysentery, It provides a pharmaceutical composition for preventing or treating bowel disease, which is selected from the group consisting of intestinal vascular dysplasia, diverticulosis, irritable growth syndrome, and inflammatory growth disease.

In another embodiment of the present invention, it provides a food composition for preventing or improving colon disease comprising the fermented Eumnamu as an active ingredient, and the colon disease is colon cancer, colon polyp, colitis, ischemic bowel disease, dysentery, It provides a food composition for preventing or improving bowel disease, which is selected from the group consisting of intestinal vascular dysplasia, diverticulosis, irritable growth syndrome, and inflammatory growth disease.

Hereinafter, the present invention will be described in detail step by step.

Eumnamu, or Eumnamu fermented product of the present invention has a remarkable effect on treating colitis in an individual with colitis-induced colon disease. In particular, the fermented Eumnamu fermented product that has been fermented with basidiomycete mycelium or is bioconverted through additional enzymatic treatment has a more remarkable effect on treating colon diseases compared to the unfermented Eumnamu original product, so it is expected to be widely used in the treatment of various colon diseases in the medical field. It is expected.

1 is a schematic diagram showing a process of obtaining a culture medium, a bioconversion process, and a bioconverted fermented powder according to an embodiment of the present invention.
2 is a graph showing the evaluation of cytokine secretion ability in macrophages of bioconverted Eumnamu fermentation according to an embodiment of the present invention.
3 is a result of treatment with TAK242, a selective inhibitor of TLR4 according to an embodiment of the present invention, completely inhibiting the activity of bioconverted Eumnamu fermentation, and confirming that the immunologically active active ingredient in Eumnamuth fermentation is a polysaccharide fraction. It is a graph.
4 is a graph confirming that neutrophil infiltration by LPS is inhibited when simultaneously treated with LPS according to an embodiment of the present invention. Figure 4 (a) evaluated the ability to induce intraperitoneal neutrophil infiltration according to the LPS intraperitoneal administration. (B) of Figure 4 evaluated the neutrophil infiltration induction ability and anti-LPS efficacy of the bioconverted Eumnamu fermentation polysaccharide fraction.
Figures 5a to 5c is a result showing the effect on the body weight, the length of the large intestine, and inflammation in the colon tissues of the diet of the raw material or fermented product of the inner bark in an acute colitis-induced individual according to an embodiment of the present invention.
Figures 6a to 6c are results showing the effect of reducing the inflammation in the body weight, the length of the large intestine and the inflammation in the colon tissue of the diet of the raw material or fermented product of the inner bark in chronic colitis-induced individuals according to an embodiment of the present invention.
7A to 7D are inflammatory cytokines TNF-α, IL-1β, IL-6, and IL- in the diet of the original product or fermented product of the inner bark in acute colitis-induced individuals according to an embodiment of the present invention. This is a result showing the effect on the activity change of 17.
8A to 8D are inflammatory cytokines TNF-α, IL-1β, IL-6, and IL- in the diet of the original product or fermented product of the inner bark in the chronic colitis-induced individual according to an embodiment of the present invention. This is a result showing the effect on the activity change of 17.

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

Throughout the specification, when a part "includes" a certain component, it means that other components may be further included rather than excluding other components unless specifically stated to the contrary.

Example 1. Bioconversion process of fermentation and enzyme treatment

Example 1-1. Pre-treatment work

First, the bark was separated into outer bark and inner bark, and after measuring the degree of contamination of mold in the raw material state for pretreatment from the original material to powdering, washing was performed to remove foreign matter and contaminants from the natural material. The water washing operation was carried out through a total of three steps: ① air washing to remove foreign matter and mold spores, ② water washing to remove residual pesticides, and ③ alcohol (Et-OH) to remove contamination of microorganisms, etc. The raw material was washed with water through the washing step. After washing and cutting, the raw materials were dried in a hot air dryer capable of mass drying, and then coarse and finely pulverized to perform powdering of each natural raw material.

Example 1-2. Acquisition of fermented powder of black tree

Eumnamu, which has been removed from foreign matter and fungal contamination, was subjected to enzyme treatment and heat treatment sterilization for liquid culture medium. As enzymes, cell wall degrading enzymes containing cellulose and hemicellulose hydrolase, pectinase, and glucosidase, which can decompose cell wall components in bark, root skin, roots, leaves, etc., were used, and the above After the enzyme was added, it was reacted at 60° C. for 1 hour, and sterilized at high temperature for 30 minutes to perform culture (fermentation) culture of Eumnamu.

In this case, commercially available enzyme products can be used. In this case, it was carried out in an appropriate amount (optimal amount suggested in the product manual for each enzyme product) and under appropriate conditions (appropriate temperature and pH suggested in the product manual for each enzyme product).

Then, separately cultured shiitake mushroom mycelium was added to the culture (fermentation) medium. Shiitake mushroom mycelium seed culture solution was inoculated with 10% (v/v) and cultured for 2 to 10 days until the concentration of the residual carbon source was depleted below a certain concentration under conditions of 28 to 30°C and pH 4.5 to 7. In addition to the above shiitake mycelia, you can select any one of mycelia from medicinal and edible basidiomycete fungi, such as Pseudomonas mushrooms, Chaga mushrooms, Maitake mushrooms, Wood ear mushrooms, Snowflake mushrooms, and skirt mushrooms. Preservation Center (KCCM), Korea Research Institute of Bioscience and Biotechnology Life Resource Center (KCTC), Korea Cell Line Bank (KCLB), Korea Agricultural Microbiological Resource Center (KACC), and American Standard Strain Culture Records Preservation Center (ATCC) You can use it.

Enzymatic treatment of the bioconversion process for the fermentation is performed by using various enzymes such as fibrinolytic enzymes, that is, cellulase, hemicellulase, pectinase, and glucanase after hot water extraction. It was carried out by adding the enzyme in the optimal amount suggested in the product manual. As enzymes, commercially available enzymes, namely, fibrinolytic enzymes, Viscozyme (complex derived from Aspergillus), cellulase (from Aspergillus niger), piltrase (complex derived from Tricoderma reesei), and rapidase (Rapidase: Aspergillus niger-derived complex) and Sumizyme (complex containing pectinase derived from Aspergillus niger) were added at the recommended concentration (0.05% v/v) suggested in the product manual of each enzyme, and at 50~60℃ Enzyme/substrate reaction was performed while rotating at 250 rpm for 1 to 3 hours.

The blackberry fermented product produced by the bioconversion process was subjected to enzyme inactivation and sterilization at 90°C for 1 hour, and then freeze-dried and powdered. In addition, the powder of Eumnamu fermentation product having the above immunological activity is dissolved in 20 times of water and centrifuged at 10,000 rpm for 30 minutes to remove insoluble residues, and the supernatant is freeze-dried to obtain a water-soluble powder, or 5 times of ethanol is added to the supernatant. The mixture was added to induce precipitation at 4° C. for more than 24 hours, and the precipitate was lyophilized to obtain a powder of a polymeric polysaccharide fraction having immunological activity. Fig. 1 shows the culture mediumization, bioconversion process, and bioconversion fermentation powder (polysaccharide fraction) obtained above.

Example 2. In vitro efficacy evaluation of bioconverted Eumnamu fermentation

Example 2-1. Cell culture

The RAW264.7 cell line used in the experiment was purchased from ATCC (Manassas, VA). DMEM medium was purchased and used from Welgene (Gyeongsan, Korea), and both fetal bovine serum (FBS) and antibiotics added to the medium were purchased and used from Thermo Fisher Scientific (Waltham, MA), and contained _{5% CO 2} It was incubated in an air condition with a saturated humidity of 37°C.

Example 2-2. Cytokine measurement in RAW264.7 cell line

The RAW264.7 cell line was inoculated into a 24-well plate at 2×10 ⁵ cells/well and cultured overnight, and each sample was diluted according to the concentration and treated. After 16 hours, the supernatant was recovered, and TNF-α, IL-6, IL-10, and IFN-β were measured using an ELISA kit (mouse quantikine ELISA kit, R&D systems, Minneapolis, MN).

Example 2-3. Proven as a TLR4 agonist of bioconverted blackberry fermentation

In order to confirm the macrophage activation effect of the original Eumnamu plant and the bioconverted Eumnamu fermentation according to the present invention, each material is treated at different concentrations in the RAW264.7 cell line to obtain TNF-α, IL-6, IL-10, and IFN-β. The expression level was confirmed by ELISA method. As a result, it was confirmed that the increase in cytokine expression in a pattern similar to that of LPS used as a positive control when the bioconverted Eumnamu fermentation according to the present invention was treated, and an increase in activity of at least 100 times or more compared to the original Eumnamu were observed. The results are shown in FIG. 2.

Example 2-4. Proof of active ingredient in bioconverted blackberry fermentation

In order to confirm the innate immune activity efficacy of Eumnamu fermentation, the ability to secrete cytokines and secretion patterns in the macrophage cell line RAW264.7 cells were investigated, and the site of action (specific receptor) was inferred through the secretion pattern and confirmed. For this purpose, the site of action was confirmed by treating an inhibitor for the inferred specific receptor to check whether signal transduction was blocked.

As a result of examining the cytokine secretion pattern, TNF-α, IL-6, IL-10 and IFN-β were secreted in the same way as LPS, and IL-1β, IL-4, IL-5, IL-12p70 and IFN-α Was not secreted. LPS is well known as a representative ligand recognized by TLR4, and through this, it could be inferred that the Eumnamu fermentation would be recognized by TLR4 and mediate the signal.

As the treatment of Eumnamu fermentation effectively induces the production of four cytokines in macrophage cell lines, it was attempted to confirm the active ingredient in Eumnamu fermentation. The polysaccharide fraction in the Eumnamu fermentation product was isolated and purified and treated on a macrophage cell line to measure the expression levels of four cytokines, and treated at a concentration of 1/100 of the Eumnamu fermentation product in consideration of the recovery rate of the polysaccharide fraction. As a result, even when only the polysaccharide fraction was treated, cytokines similar to those treated with Eumnamu fermentation were produced, and the active ingredient of Eumnamu fermentation was confirmed to be a polysaccharide.

In addition, as a result of measuring the amount of cytokines by treatment with TAK242, a specific inhibitor of TLR4, it was confirmed that the production of all four cytokines induced by treatment of the Eumnamu fermentation product and the polysaccharide fraction during TAK242 treatment was completely inhibited. . Therefore, it was confirmed that Eumnamu fermentation and polysaccharide fractions of Eumnamu fermentation are TLR4 agonists that bind to TLR4 and induce activation of macrophages. The results are shown in FIG. 3.

As a result of investigating the cytokine secretion pattern related to immunological activity in macrophages, it was confirmed that it induces very high expression of TNF-α, IL-6, IL-10 and IFN-β, among which IFN-β Is a type I interferon that has antibacterial and antiviral effects, and is a representative cytokine that induces Th1 immune responses by contributing to the differentiation of Th1 cells. While inducing Th1 immune responses, it has an immunomodulatory function that suppresses Th2 and Th17 immune responses. It is a cytokine.

Therefore, Eumnamu fermentation is a Th1 adjuvant of TLR4 agonist that can specifically activate Th1 immune response, and it is a substance that has a mechanism different from general anti-inflammatory drugs that inhibit the production _{of PGE 2 and NO in macrophages.} Could be evaluated.

Example 3. Verification of LPS competitive inhibitors of bioconverted blackberry fermentation in vivo experiment

Example 3-1. Experimental animals

As for the mice used in the experiment, 6-week-old female BALB/c mice were purchased from Orient Bio (Seongnam, Korea), and they were reared by supplying sufficient water and feed while maintaining the room temperature at 22±2°C before use in the experiment. .

Example 3-2. Evaluation of the ability to induce neutrophil infiltration and anti-LPS efficacy in the abdominal cavity Animal experiments

To measure the LPS-induced neutrophil induction ability, mice were randomly separated into 10 mice per group, and LPS (SIGMA, St. Louis, MO) and each material were injected at different concentrations into the abdominal cavity after an adaptive diet for 1 week. After 6 hours, the mice were sacrificed, the abdominal cavity was washed with PBS to recover the cells infiltrated into the abdominal cavity, and the number of neutrophils was counted through Kimja staining.

Example 3-2. Demonstrated competitive inhibitors of LPS from bioconverted blackberry fermentation

As a result of confirming the effect of intraperitoneal administration of LPS inducing neutrophil infiltration, it was confirmed that when 200 ng of LPS was injected, neutrophil invasion occurred to the maximum and remained constant thereafter. However, unlike LPS, the bioconverted polysaccharide fraction of Eumnamu fermentation did not induce intraperitoneal neutrophil invasion, unlike LPS, and it was confirmed that simultaneous treatment with LPS inhibited neutrophil infiltration by LPS. The results are shown in FIG. 4.

Unlike LPS, the bioconverted Eumnamu fermentation polysaccharide fraction does not induce intraperitoneal neutrophil infiltration, and rather, LPS-induced intraperitoneal neutrophil infiltration is inhibited by the bioconverted Eumnamu fermentation polysaccharide fraction, which is a competitive inhibitor for LPS. Proved. Therefore, the bioconverted Eumnamu fermentation product could be evaluated as a safe substance that does not induce an inflammatory reaction in the body, but rather a substance that can act competitively with LPS.

Example 4. Preparation of an animal model for colon disease and measurement of biological indicators

Example 4-1. Preparation of animal model for colon disease

The mice used in the experiment were 6-week-old male BALB/c mice were purchased from Orient Bio (Seongnam, Korea), and were reared by supplying sufficient water and feed while maintaining the room temperature at 22±2°C before use in the experiment. . Mice were randomly separated into 10 mice per group, and each material was administered on a diet for 2 weeks according to daily intakes of 10 mg/kg and 40 mg/kg after a 1-week adaptive diet. Thereafter, 2% dextran sulfate sodium salt (DSS), MP Biomedicals, Santa Ana, CA) was allowed to drink for 5 days to induce acute bowel inflammation. After 2 days, mice were sacrificed to acute colonization of each material. Inflammation relief effect was confirmed. In order to induce chronic bowel inflammation, 2% DSS was ingested for 5 days in mice fed with each material for 2 weeks, and then normal negative water was administered for 10 days to induce recovery, and 2% DSS was again ingested for 5 days for additional inflammation. The reaction was induced. Two days later, the mice were sacrificed to confirm the effect of alleviating chronic colon inflammation of each material.

Example 4-2. Measurement of biomarkers related to colon inflammation

In order to measure the length of the colon and measure the activity of myeloperoxidase in the colon tissue, the mice were sacrificed by inhaling _{CO 2 after induction of colon inflammation.} The colon was removed and the total length of the colon was measured, and 20 mg of colon tissue was taken into 500 μL of MPO assay buffer, homogenized, and centrifuged at 10,000 g for 10 minutes. Thereafter, the supernatant was taken and the myeloperoxidase peroxidation activity assay kit (BioVision, Milpitas, CA) was used to measure the myeloid cell type peroxidase activity present in the supernatant.

The amount of TNF-α in the colon tissue was measured using a TNF-α ELISA kit (mouse TNF-α quantikine ELISA kit, R&D systems, Minneapolis, MN), and the amount of IL-1β was measured using an IL-1β ELISA kit (mouse IL-1β). quantikine ELISA kit, R&D systems, Minneapolis, MN), and the amount of IL-6 was measured using an IL-6 ELISA kit (mouse IL-6 quantikine ELISA kit, R&D systems, Minneapolis, MN), and IL The amount of -17 was measured using an IL-17 ELISA kit (mouse IL-17 quantikine ELISA kit, R&D systems, Minneapolis, MN).

Example 5. Evaluation of the treatment effect of Eumnamu original products and bioconversion products for colitis

In order to confirm the effect of administration of each material on the alleviation of colitis, dextran sulfate sodium salt (DSS, 36,000-50,000 M.W.) was administered to the diet in a mouse model of colitis induced by drinking negatively, and the effect of reducing inflammation was evaluated. As for the colitis model using DSS, two acute inflammation models in which 2% DSS is ingested once for 5 days and a chronic inflammatory model in which 2% DSS is ingested twice for 5 days were used.In each model, the effect of each material was checked to prevent inflammation. To confirm the inhibitory effect and the mechanism of inhibition of inflammation.

Example 5-1. In the DSS-induced colon inflammation mouse model, the effect of reducing colon inflammation due to administration of the original Eumnamu and biotransformation products was confirmed.

Eumnamu original product and Eumnamu (bioconversion) product were fed for 2 weeks, and then DSS was ingested to induce colon inflammation, and as a result of measuring the length of the colon and myeloid cell type peroxidase activity in the colon tissue of the mouse. When administered, it recovered up to 16% of colon length and inhibited 12.6% of myeloid cell type peroxidase in colon tissue, and when administered Eumnamu (bioconversion) product, it recovered 24% of colon length and 16.6% of myeloid cell type peroxidation. It was confirmed that the inhibitory activity of the bioconversion product was higher than that of the original product due to the enzyme inhibitory effect. In addition, in the chronic inflammatory model, when Eumnamu (Bioconversion) products were administered, a maximum of 20% of the colon length recovery effect and 65.1% of myeloid cell type peroxidase inhibitory effects were shown, but when the Eumnamu (bioconversion) product was administered, a maximum of 56% of the colon length was recovered. And 80.6% of myeloid cell type peroxidase inhibitory effect was found to be shown, and it was confirmed that the Eumnamu (bioconversion) product has a better inhibitory effect on colon inflammation than the Eumnamu original product. The results are shown in FIGS. 5 and 6.

Example 5-2. Confirmation of inhibitory ability to produce inflammatory cytokines in colon tissue

By measuring the expression levels of TNF-α, IL-1β, IL-6, and IL-17 in the colon tissue, the effect of reducing colon inflammation of each material was confirmed. As a result, in the acute colorectal inflammation model, the original product showed up to 50% inhibitory activity against TNF-α, up to 11.5% inhibitory activity against IL-1β, and up to 7.5% inhibition against IL-6. Activity was shown, and the inhibitory activity against IL-17 was up to 20%, whereas in biotransformation products, 71.4%, 12.3%, and 21.6 were respectively against TNF-α, IL-1β, IL-6 and IL-17. % And 70% of inhibitory activity. In addition, in the chronic colorectal inflammation model, the original product has an inhibitory effect of up to 33.3%, 14.1%, 5.9% and 66.7%, respectively, against TNF-α, IL-1β, IL-6, and IL-17, and the biotransformation product is It was found to have an inhibitory effect of up to 55.6%, 23.4%, 25.6% and 83.3%, and it was confirmed that the inhibitory effect of biotransformants was high in both the acute and chronic bowel inflammation models. In particular, bioconversion products have been shown to have excellent inhibitory activity against IL-17, and it has been reported that if IL-17F is genetically deficient, colitis induced by DSS is suppressed (Yang XO, et al. Regulation of inflammatory responses by IL-17F. J. Exp. Med. 2008, 205, 1063-1075), and inhibition of IL-17A and IL-17F inhibited colitis (Leon P. McLean, et al. Combined blockade of IL- When referring to 17A and IL-17F may prevent the development of experimental colitis.Immunotherapy. 2013. 5(9)), it can be expected that the IL-17 inhibitory activity of biotransformants will have an effective inhibitory effect on colitis. .

As described above, specific parts of the present invention have been described in detail, and for those of ordinary skill in the art, it is clear that these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereto. Therefore, it will be said that the practical scope of the present invention is defined by the appended claims and their equivalents.

Claims (12)

(a) cultivating Eumnamu powder obtained by powdering Eumnamu into a liquid culture medium, treating with a cell wall degrading enzyme, and sterilizing to prepare a liquid Eumnamu medium;
(b) performing a biotransformation fermentation process of inoculating basidiomycete mycelium into the culture medium of step (a) with basidiomycete mycelium for cultivation and fermentation; And
(c) comprising the step of producing a bioconverted product having improved bowel disease treatment efficacy through a bioconversion enzyme treatment process of treating fibrinolytic enzyme from the fermented product produced by the biotransformation fermentation process of step (b). As a method of manufacturing a fermented blackberry,
The cell wall degrading enzyme includes cellulose and hemicellulose hydrolase, pectinase, and glucosidase capable of decomposing cell wall components,
The basidiomycete hyphae is shiitake mushroom hyphae,
The fibrinolytic enzyme is selected from the group consisting of cellulase, hemicellulase, pectinase, and glucanase,
The colon disease is that of colitis, the manufacturing method.
The method of claim 1,
The Eum tree is the bark of Eum tree, the method of manufacturing Eum tree fermentation.
The method of claim 2,
The Eum tree bark is the inner bark of Eum tree, the method of manufacturing Eum tree fermentation.
delete delete delete delete Made by the manufacturing method of claim 1, Eumnamu fermented product.
As a pharmaceutical composition for preventing or treating colon diseases, comprising the Eumnamu fermented product of claim 8 as an active ingredient,
The colon disease is that of colon inflammation, a pharmaceutical composition.
delete As a food composition for preventing or improving colon disease, comprising the fermented Eumnamu of claim 8 as an active ingredient,
The colon disease is that of colon inflammation, food composition. delete

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