KR20200103219A - Composition for preventing or treating immune diseases comprising mixture of Microbiome - Google Patents

Abstract

The present invention provides a composition for preventing and treating immune diseases containing a microbiome mixture. The composition of the present invention is a mixture obtained by mixing a vitamin B complex and curcumin with a Lactobacillus sp. strain, which is microbiome, in the most effective ratio, so that the composition, compared to existing anti-inflammatory agents, has an effect of inhibiting the expression of inflammatory cytokines and bone destruction factors in vitro or in vivo. In addition, the composition increases the expression of anti-inflammatory cytokines, simultaneously regulates Th17 and Treg cells through STAT3 inhibition, and also effectively regulates B17 and Breg cells, thereby being able to be usefully used in pharmaceutical and health functional food industries.

Description

Composition for preventing or treating immune diseases comprising mixture of Microbiome}

The present invention provides a composition for preventing and treating immune diseases comprising a microbiome mixture.

Immune disease is a disease in which components of the mammalian immune system cause, mediate, or otherwise contribute to pathological conditions in mammals. In particular, inflammatory disorders are one of the most important health problems in the world. Inflammation is generally a localized protective response of body tissues against host invasion by foreign substances or harmful stimuli. The causes of inflammation include infectious causes such as bacteria, viruses and parasites; Physical causes such as burns or irradiation; Chemicals such as toxins, drugs or industrial agents; It may be an immune response such as an allergic and autoimmune reaction, or a condition associated with oxidative stress.

Inflammation is characterized by pain, redness, swelling, fever, and eventual loss of function in the affected area. These symptoms are the result of a series of complex interactions between cells in the immune system. Cellular responses result in a network of interactions of several groups of inflammatory mediators: proteins (e.g. cytokines, enzymes (e.g. proteases, peroxidase)), major basic proteins, adhesion molecules (e.g. ICAM, VCAM), lipid mediators (e.g. eicosanoids, prostaglandins, leukotriene, platelet activating factor (PAF)), reactive oxygen species (e.g. hydroperoxide, superoxide anion O2-, nitric oxide (NO), etc.) However, most of these mediators of inflammation are also modulators of normal cellular activity, so a lack of inflammatory response results in uncontrolled damage (ie infection) of the host, and thus partial damage due to chronic inflammation. As an inflammatory disease mediated by excessive production of several of the above-mentioned mediators, autoimmune disease, which is one of the immune diseases, is characterized in that the immune system attacks its own organs and causes a spontaneous response. These reactions are due to the recognition of auto-antigens by T lymphocytes, which induces both humoral (autoantigen production) and cellular (lymphocyte and macrophage cytotoxic activity increase) immune responses.

Microbiome refers to microorganisms that live in the human body, and this includes intestinal microorganisms. The number of microbiomes is more than twice the number of cells in a pure human body, and the number of genes is more than 100 times. Therefore, it is sometimes called the Second Genome because it is impossible to discuss human genes except for microorganisms. Microbiome is a field that can be widely used in the development of new drugs and research on treatments for incurable diseases, as it can analyze the principle of generating beneficial and harmful bacteria and the relationship between diseases. In addition, microbiome is used to develop food, cosmetics, and treatments. However, most researches on drug development using these are in the early stages, so it will take time to develop products.

Accordingly, the present inventors used a microbiome mixture composition in which a strain of the genus Lactobacillus, a strain of the genus Bifidobacterium, a vitamin B complex, and curcumin are mixed as an active ingredient among probiotics, as an active ingredient, the secretion of immune-related substances The present invention was completed by confirming that it is effective in preventing and treating related immune diseases because it increases to induce immunity enhancement.

An object of the present invention is to provide a pharmaceutical composition for preventing or treating immune diseases, comprising as an active ingredient at least one selected from the group consisting of a strain of the genus Lactobacillus, a vitamin B complex, and curcumin. have.

It is also an object of the present invention to provide a health functional food composition for preventing or improving immune diseases, comprising as an active ingredient at least one selected from the group consisting of Lactobacillus genus strain, vitamin B, and curcumin. .

In order to achieve the above object, the present invention comprises one or more selected from the group consisting of Lactobacillus genus strain, vitamin B complex, and curcumin as an active ingredient, a pharmaceutical for preventing or treating immune diseases The composition is provided.

In addition, the present invention provides a health functional food composition for preventing or improving immune diseases, including one or more selected from the group consisting of Lactobacillus genus strain, vitamin B, and curcumin as an active ingredient.

The composition of the present invention of the present invention is a mixture of vitamin B complex and curcumin in the most effective ratio in the microbiome Lactobacillus genus strain, in vitro or in vivo, when compared with conventional anti-inflammatory agents, inflammatory cytokines It has an effect of inhibiting expression and expression of bone destruction factors. In addition, it increases the expression of anti-inflammatory cytokines, simultaneously regulates Th17 and Treg cells through STAT3 inhibition, and effectively regulates B17 and Breg cells, so it can be usefully used in related pharmaceutical and health functional food industries. .

1 is a diagram confirming the effect of controlling B17 and Breg cells according to the treatment of the microbiome mixture composition (L.aci+VitB+Cur) of the present invention on mouse spleen cells.
2 is a diagram confirming the effect of controlling Th17 and Treg cells according to the treatment of the microbiome mixture composition (L.aci+VitB+Cur) of the present invention on human peripheral blood mononuclear cells.
3 is a diagram illustrating the effect of inhibiting osteoclast differentiation according to the treatment of the microbiome mixture composition (L.aci+VitB+Cur) of the present invention on human osteoclasts.
4 is a diagram illustrating the expression of each mRNA of TRAP, RANK, or cathepsin K according to the treatment of the microbiome mixture composition (L.aci+VitB+Cur) of the present invention in human osteoclasts.
5 is a diagram illustrating the effect of inhibiting Th17 according to treatment of the microbiome mixture composition (L.aci+VitB+Cur) of the present invention on human peripheral blood mononuclear cells.
6 is a diagram illustrating the effect of controlling Treg cells according to the treatment of the microbiome mixture composition (L.aci+VitB+Cur) of the present invention on human peripheral blood mononuclear cells.
7 is a diagram illustrating the effect of inhibiting STAT3 according to the treatment of the microbiome mixture composition (L.aci+VitB+Cur) of the present invention on human peripheral blood mononuclear cells.
Figure 8 is a diagram confirming the STAT3 inhibitory effect of the treatment of the microbiome mixture composition (L.aci + VitB + Cur) of the present invention on human peripheral blood mononuclear cells.
9 is a result of confirming the pain evaluation (Paw withdrawal threshold (PWT) and paw withdrawal latency (PWL)) according to the treatment of the microbiome mixture composition (L.aci+VitB+Cur) of the present invention in an osteoarthritis animal model.

The present invention provides a pharmaceutical composition for preventing or treating immune diseases, comprising as an active ingredient at least one selected from the group consisting of Lactobacillus genus strain, vitamin B complex, and curcumin.

The Lactobacillus genus strains are Lactobacillus Acidophilus, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus rhamnosus, helium (Lactobacillus Helveticus), Lactobacillus fermentum (Lactobacillus fermentum), Lactobacillus paracasei (Lactobacillus paracasei) and Lactobacillus salivarius (Lactobacillus salivarius) may be one or more selected from the group consisting of, but preferably If it is a philus (Lactobacillus Acidophilus), the vitamin B complex of the present invention, and curcumin (Curcumin) to achieve the purpose of showing an effect on the prevention or treatment of immune diseases by mixing, it is not limited thereto.

The strain can be used by culturing, and can be used in the form of a fermentation broth obtained by culturing in a culture medium, a concentrated fermentation broth, a dried product of the fermentation broth, a culture filtrate, a concentrated culture filtrate, or a dried product of the culture filtrate. It may be a culture filtrate containing or removing the strain after culturing. In addition, the formulation of the culture is not limited, and for example, may be a liquid or solid.

In the present invention, the term "culture" refers to growing microorganisms under appropriately artificially controlled environmental conditions.

The strain is capable of growing in a conventional medium, and contains nutrients required by the culture target, that is, the microorganism serving as a culture medium in order to cultivate a specific microorganism, and may be a mixture by adding a substance for a special purpose. The medium is also referred to as a culture medium or a culture medium, and is a concept including both natural medium, synthetic medium, or selective medium.

The medium used for cultivation must meet the requirements of a specific strain in an appropriate manner while controlling temperature, pH, etc. in a conventional medium containing an appropriate carbon source, nitrogen source, amino acid, vitamin, and the like. As the carbon source that can be used, a mixed sugar of glucose and xylose is used as the main carbon source. In addition, sugars and carbohydrates such as sucrose, lactose, fructose, maltose, starch, cellulose, soybean oil, sunflower oil, castor oil, coconut Oils and fats such as oil, fatty acids such as palmitic acid, stearic acid, and linoleic acid, alcohols such as glycerol and ethanol, and organic acids such as acetic acid are included. These materials can be used individually or as a mixture. Examples of nitrogen sources that can be used include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; Amino acids such as glutamic acid, methionine, glutamine, and organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or its degradation products, skim soybean cake or its degradation products. I can. These nitrogen sources may be used alone or in combination. The medium may contain first potassium phosphate, second potassium phosphate, and corresponding sodium-containing salt as personnel.

Personnel that may be used include potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salt. In addition, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, and the like may be used as the inorganic compound. Finally, essential growth substances such as amino acids and vitamins can be used in addition to the above substances. In addition, precursors suitable for the culture medium may be used. The above-described raw materials may be added in a batch, fed-batch, or continuous manner to the culture during the culture process, but are not particularly limited thereto. Basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or acid compounds such as phosphoric acid or sulfuric acid can be used in an appropriate manner to adjust the pH of the culture.

The vitamin B complex is a subgroup of vitamins B1, B3, B5, B6, B9, and B12, preferably vitamins B1, B6, B9, and B12, and the mixing ratio thereof is 1:1:1:1, but is not limited thereto.

The strain of the genus Lactobacillus, vitamin B complex, and curcumin may be mixed at a mixing ratio of 1: 0.4: 50 to 1: 0.004: 5 to prepare a microbiome mixture composition of the present invention. If it is to achieve the purpose of showing an effect in the prevention or treatment of immune diseases by mixing with the strain of the genus Lactobacillus and curcumin, it is not limited thereto.

In an embodiment of the present invention, a "microbiome mixed composition" was prepared by mixing Lactobacillus acidophilus, vitamin B complex, and curcumin. The microbiome mixture composition inhibits the expression of inflammatory cytokines, expresses anti-inflammatory cytokines, and inhibits the expression of inflammation-related STAT3 to simultaneously regulate Th17 and Treg cells. In addition, B17 and Breg cells have the ability to regulate, there is an excellent effect of immune regulation ability. In addition, it inhibits the differentiation of osteoclasts by inhibiting the expression of the bone destruction mechanism factors TRAP (tartrate-resistant acid-phosphatase), RANKL (receptor activator of nuclear factor k appa-B ligand) and cathepsin K (cathepsin K). , It has excellent bone protection effect.

The immune disease may include an autoimmune disease or an inflammatory disease.

In the present invention, the term "autoimmune disease" refers to an unresponsiveness of a living body to an autoantigen as immunologic unresponsiveness or tolerance, and if a problem arises in inducing or maintaining such self-tolerance An immune response occurs against the self-antigen, and as a result, a phenomenon that attacks the own tissue occurs, which means a disease caused by this process.

In the present invention, the term "inflammatory disease" is TNF-α (tumor necrosis factor-α), IL secreted by immune cells such as macrophages by excessively promoting the human immune system due to harmful stimulation such as inflammation inducing factor or irradiation. -1 (interleukin-1), IL-6, prostaglandin (prostagladin), leukotriene (luecotriene) or nitric oxide (nitric oxide, NO) is a disease caused by an inflammatory substance (inflammatory cytokine) .

The immune diseases include osteoarthritis, rheumatoid arthritis, ankylosing spondylitis, spondyloarthritis, inflammatory bowel disease, psoriatic arthritis, gout, bacterial arthritis, childhood rheumatoid arthritis, lupus, scleroderma, multiple sclerosis, fibromyalgia, multiple myositis, dermatitis, Behcet's disease, Reiter Syndrome, Lyme Arthritis, Adhesive Arthritis, Frozen Shoulder, Tendon Synovitis, Elbow Head Positis, DeQuavein Tendon Synovialitis, Recurrent Rheumatoid, Multiple Rheumatoid Pain, Adult Still's Disease, Autoimmune Hemocytopenia, Autoimmune Myocarditis, Atopic Dermatitis , Asthma, primary cirrhosis, Goodfighter syndrome, autoimmune meningitis, Sjogren syndrome, Addison's disease, alopecia areata, autoimmune hepatitis, autoimmune mumps, Crohn's disease, insulin-dependent diabetes, dystrophic vesicular epidermolysis, epididymitis, glomerular Nephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, sarcoidosis, skin sclerosis, spondyloarthrosis, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, mitochondria At least one selected from the group consisting of related syndromes and ulcerative colitis, but is not limited thereto.

In the present invention, "treatment" is, unless otherwise stated, to reverse, alleviate, inhibit or prevent the disease or disease to which the term applies, or one or more symptoms of the disease or disease. As used herein, the term treatment refers to the act of treating when "treating" is defined as above. Thus, the "treatment" or "therapeutic regimen" of an immune disease in a mammal may include one or more of the following:

(1) inhibiting the growth of immune diseases, i.e., inhibiting their development,

(2) preventing the spread of immune diseases, that is, preventing metastasis,

(3) Alleviate immune diseases.

(4) preventing recurrence of immune diseases, and

(5) Palliating the symptoms of immune diseases.

The pharmaceutical composition of the present invention may further contain an adjuvant in addition to the active ingredient. Any of the adjuvants known in the art may be used without limitation, but, for example, Freund's complete adjuvant or incomplete adjuvant may be further included to increase its immunity.

The pharmaceutical composition according to the present invention may be prepared in a form in which an active ingredient is incorporated in a pharmaceutically acceptable carrier. Here, the pharmaceutically acceptable carrier includes carriers, excipients, and diluents commonly used in the pharmaceutical field. Pharmaceutically acceptable carriers that can be used in the pharmaceutical composition of the present invention are not limited thereto, but lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, Calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oils.

The pharmaceutical compositions of the present invention can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, or sterile injectable solutions according to a conventional method. .

In the case of formulation, it can be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are commonly used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations include at least one excipient, such as starch, calcium carbonate, sucrose, lactose, gelatin, in the active ingredient. It can be prepared by mixing and the like. Further, in addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, liquid solutions, emulsions, syrups, and other excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to water and liquid paraffin, which are commonly used diluents. I can. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, and suppositories. As the non-aqueous solvent and suspension, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like may be used. As a base for suppositories, witepsol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used.

The pharmaceutical composition according to the present invention can be administered to a subject by various routes. All modes of administration can be expected, for example, by oral, intravenous, intramuscular, subcutaneous, intraperitoneal injection.

The dosage of the pharmaceutical composition according to the present invention is selected in consideration of the age, weight, sex, and physical condition of the individual. It is obvious that the concentration of the active ingredient contained in the pharmaceutical composition can be selected in various ways depending on the subject, and is preferably included in the pharmaceutical composition at a concentration of 0.01 to 5,000 μg/ml. When the concentration is less than 0.01 μg/ml, pharmaceutical activity may not appear, and when the concentration exceeds 5,000 μg/ml, toxicity to humans may occur.

The pharmaceutical composition may be formulated in various oral or parenteral dosage forms.

Formulations for oral administration include, for example, tablets, pills, hard, soft capsules, solutions, suspensions, emulsifiers, syrups, and granules. These formulations include diluents (e.g., lactose, dextrose, water) in addition to the active ingredients. Krose, mannitol, sorbitol, cellulose and/or glycine), lubricants (eg, silica, talc, stearic acid and magnesium or calcium salts thereof and/or polyethylene glycol). In addition, the tablet may contain a binder such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidine, in some cases starch, agar, alginic acid Or disintegrants or boiling mixtures and/or absorbents, colorants, flavors and sweeteners such as sodium salts thereof. The formulation can be prepared by conventional mixing, granulating or coating methods.

In addition, a representative formulation for parenteral administration is a formulation for injection, and as a solvent for the formulation for injection, water, Ringer's solution, isotonic physiological saline or suspension may be mentioned. The sterile fixed oil of the injectable preparation can be used as a solvent or suspension medium, and any non-irritating fixed oil including mono-, di-glyceride can be used for this purpose.

In addition, the injectable formulation may use a fatty acid such as oleic acid.

In addition, the present invention provides a health functional food composition for preventing or improving immune diseases, including one or more selected from the group consisting of Lactobacillus genus strain, vitamin B, and curcumin as an active ingredient.

In addition to containing the active ingredient, the food composition of the present invention may contain various flavoring agents or natural carbohydrates as an additional ingredient, like a conventional food composition.

Examples of the above-described natural carbohydrates include monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, and the like; And polysaccharides, for example, common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. The above-described flavoring agents can be advantageously used as natural flavoring agents (taumatin), stevia extracts (eg, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.). The food composition of the present invention can be formulated in the same manner as the pharmaceutical composition and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gums, candy, ice cream, alcoholic beverages, vitamin complexes and health supplements, etc. There is this.

In addition, the food composition includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring and natural flavoring agents, coloring agents and heavy weight agents (cheese, chocolate, etc.), pectic acid and salts thereof, Alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonates used in carbonated beverages, and the like may be contained. In addition, the food composition of the present invention may contain flesh for the production of natural fruit juice and fruit juice beverages and vegetable beverages.

The functional food composition of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, and pills. In the present invention, the term'health functional food composition' refers to a food manufactured and processed using raw materials or ingredients having useful functions for the human body pursuant to Health Functional Food Act No.6727, and with respect to the structure and function of the human body. It refers to ingestion for the purpose of obtaining useful effects for health purposes such as controlling nutrients or physiological effects. The health functional food of the present invention may contain ordinary food additives, and whether it is suitable as a food additive is determined according to the general rules and general test methods for food additives approved by the Food and Drug Administration, unless otherwise specified. It is judged according to standards and standards. Examples of items listed in the'Food Additives Code' include chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; Natural additives such as reduced pigment, licorice extract, crystalline cellulose, high color pigment, and guar gum; Mixed preparations, such as a sodium L-glutamate preparation, an alkali additive for noodles, a preservative preparation, and a tar color preparation, etc. are mentioned. For example, in the health functional food in the form of a tablet, a mixture obtained by mixing the active ingredient of the present invention with an excipient, a binder, a disintegrant, and other additives is granulated by a conventional method, and then a lubricant, etc. The mixture can be directly compression molded. In addition, the health functional food in the form of a tablet may contain a mating agent or the like, if necessary. Among the health functional foods in the form of capsules, hard capsules can be prepared by filling a mixture of the active ingredient of the present invention with additives such as excipients in a conventional hard capsule, and the soft capsules contain the active ingredient of the present invention with additives such as excipients. The mixture mixed with can be prepared by filling a capsule base such as gelatin. The soft capsules may contain a plasticizer such as glycerin or sorbitol, a colorant, a preservative, and the like, if necessary. Ring-shaped health functional foods can be prepared by molding a mixture of the active ingredient of the present invention, excipients, binders, disintegrants, etc. by conventionally known methods, and can be coated with white sugar or other coating agents if necessary, Alternatively, the surface may be coated with a material such as starch or talc. Health functional foods in the form of granules can be prepared in granular form by a mixture of the excipients, binders, disintegrants, etc. of the active ingredients of the present invention by a known method, and if necessary, may contain flavoring agents, flavoring agents, etc. I can.

The present invention will be described in more detail through the following examples. However, the following examples are only for embodiing the contents of the present invention and the present invention is not limited thereto.

<Production Example 1> Preparation of the microbiome mixed composition of the present invention

Lactobacillus acidophilus ( L. acidophilus ), vitamin B complex and curcumin (curcumin) were mixed to prepare a "microbiome mixed composition".

To prepare the microbiome mixture composition, vitamin B complex was used as vitamins B1, B6, B9, and B12 (each 0.1-10mg/kg), which was mixed in a ratio of 1:1:1:1. The composition was L. acidophilus (2X10*11 CFU/g) 100mg/kg, curcumin 500mg/kg-5000mg/kg, and vitamin B complex 0.4-40mg/kg. The Lactobacillus acidophilus, vitamin B complex, and curcumin were mixed in a mixing ratio of 1: 0.4: 50 to 1: 0.004: 5.

<Example 1> B17 and Breg cell control effect by treatment of the microbiome mixture composition of the present invention

Lactobacillus acidophilus of the present invention ( L. acidophilus ), vitamin B complex and curcumin (curcumin) was named as "microbiome mixed composition ( L. Acidophilus + VitB + Cur)". Among regulatory B cells, B17 is specialized by a cell signaling protein called interleukin-17 (IL-17), and Breg (Regulatory B cells) is interleukin-10 (interleukin-10). IL-10)).

Specifically, normal mice (c57bl/6, male, 9 weeks old, obtained from Orient Bio) were sacrificed to obtain spleens. After grinding the spleen using a slide, the RBC was removed using an ACK buffer, and then spleen cells were obtained. Induce stimulation with antiCD3 (0.5ug/ml) or LPS (100ug/ml) in the mouse spleen cells, and Microbiome mixed composition treatment group (10 ug/ml); L. Acidophilus alone group (10 ug/ml); VitB complex alone group (B1 20uM, B6 20uM, B9 20uM, B12 20uM, so a total of 80 uM) and curcumin (1uM) alone group were placed. In order to confirm the regulatory effect of B17 cells and Breg cells according to each treatment group, flow cytometry analysis was performed.

As a result, it was confirmed that the expression of the inflammatory cytokine IL-17 was regulated as the decrease in B17 cells was confirmed in the microbiome mixed composition treatment group of the present invention compared to each single treatment group. In addition, it was confirmed that the decrease in Breg cells was increased compared to other treatment groups alone, and thus the expression of the anti-inflammatory cytokine IL-10 could be regulated (FIG. 1).

<Example 2> Th17 and Treg cell regulation effect by treatment of the microbiome mixture composition of the present invention

Th17 cells (T helper 17 cells) induce the inflammatory cytokine IL-17, and Treg cells induce the anti-inflammatory cytokine IL-10. Therefore, it was confirmed whether the microbiome mixture composition of the present invention has the effect of regulating the Th17 and Treg cells.

Specifically, after separating CD4 T cells from healthy human peripheral blood mononuclear cells (huPBMC), under antiCD3 stimulation conditions (0.5 ug/ml), the microbiome mixture composition of the present invention (10 ug/ ml) was treated. Also L. Acidophilus alone group (10ug/ml); VitB complex alone group (B1 20uM, B6 20uM, B9 20uM, B12 20uM, so a total of 80 uM) and curcumin (1uM) alone group were placed. After incubation for 3 days, ELISA was performed using the supernatant separated by centrifugation. After coating the 96-well plate at room temperature for 2 hours using the first capture ab (anti-mouse IL-17, IL-10), non-specific reactions were blocked using a blocking buffer. After the blocking process, the standard and the sample were put into a plate, and after a reaction for two hours, they were washed using a wash buffer. Standard (recombinant: anti-mouse IL-17, IL-10) and sample were added, reacted for 2 hours, and washed using a wash buffer. After washing, detection ab (anti-mouse IL-17, IL-10) was added and reacted for 2 hours, followed by washing and color development.

As a result, it was confirmed that the microbiome mixture composition of the present invention reduced Th17 cells, thereby significantly reducing the induced expression of IL-17. In addition, by increasing the Treg cells, it was confirmed that the expression of IL-10 induced by this is increased compared to other treatment groups. Therefore, it was confirmed that the ability to regulate Th17/Treg cells simultaneously (FIG. 2 ).

<Example 3> The effect of inhibiting osteoclast differentiation by treatment with the microbiome mixture composition of the present invention

3-1. Identification of TRAP (tartrate-resistant acid-phosphatase) positive cells

In order to confirm the ability to regulate osteoclast differentiation by the treatment of the microbiome mixture composition of the present invention, RANKL and M-CSF were stimulated in human osteoclasts. Then, the microbiome mixed composition treatment group of the present invention; A group treated with VitB complex alone was placed and treated at the same time during the stimulation, and TRAP-stained cells were stained specifically for osteoclasts to examine TRAP-positive cells related to the regulation of osteoclast differentiation.

As a result, as a result of microscopic observation after TRAP staining and measurement of the number of cells after TRAP staining, it was confirmed that the number of TRAP-positive cells in the microbiome mixed composition treatment group of the present invention was significantly reduced (FIG. 3).

3-2. Confirmation of expression of Tartrate-resistant acid-phosphatase (TRAP), Receptor activator of nuclear factor kappa-B (RANK), or cathepsin K

In order to confirm the ability to regulate osteoclast differentiation by the treatment of the microbiome mixture composition of the present invention, RANKL and M-CSF were stimulated in human osteoclasts. Then, the microbiome mixed composition treatment group of the present invention; The VitB complex alone treatment group was placed and treated simultaneously at the time of the stimulation, and the expression of each mRNA of TRAP, RANK or RANKL related to the regulation of bone destructive differentiation was analyzed by a polymerase chain reaction device (Realtime PCR).

As a result, it was observed that the expression of each mRNA of TRAP, RANK, and cathepsin K was significantly reduced in the microbiome mixed composition treatment group of the present invention (Fig. 4).

Therefore, the microbiome mixture composition of the present invention inhibits TRAP, RANK, or cathepsin K, which is related to the regulation of osteoclast differentiation, so that the differentiation of osteoclasts induces inhibition, showing a protective effect of bone (cartilage). Confirmed.

<Example 4> Th17 inhibition effect by treatment of the microbiome mixture composition of the present invention

L. Acidophilus alone group (10 ug/ml) in healthy human peripheral blood mononuclear cells (huPBMC); VitB complex alone group (B1 20uM, B6 20uM, B9 20uM, B12 20uM, so a total of 80 uM) and curcumin (1uM) alone group were placed. In order to confirm the inhibitory effect of Th17 cells according to each treatment group, flow cytometry analysis was performed.

As a result, the microbiome mixed composition treatment group of the present invention confirmed that the decrease of Th17 cells was confirmed compared to each single treatment group, and thus the expression of the inflammatory cytokine IL-17 was regulated, and it was confirmed that the Th17 inhibitory effect was excellent ( Fig. 5).

<Example 5> Confirmation of the effect of maintaining Tregs by treating the microbiome mixture composition of the present invention

L. Acidophilus alone group (10 ug/ml) in healthy human peripheral blood mononuclear cells (huPBMC); VitB complex alone group (B1 20uM, B6 20uM, B9 20uM, B12 20uM, so a total of 80 uM) and curcumin (1uM) treatment groups were placed. In order to confirm the inhibitory effect of Treg cells according to each treatment group, it was analyzed by Foxp3 FITC and CD25 APC.

As a result, it was confirmed that the treatment group of the microbiome mixture composition of the present invention has superior maintenance effect of Treg cells than each treatment group alone (FIG. 6).

<Example 6> Confirmation of STAT3 inhibitory effect by treatment of the microbiome mixture composition of the present invention

6-1. Confirmation of STAT3 cells through flow cytometry analysis

In healthy human peripheral blood mononuclear cell cells (huPBMC), also L. Acidophilus alone group (10 ug/ml) of the present invention; VitB complex alone group (B1 20uM, B6 20uM, B9 20uM, B12 20uM, so a total of 80 uM) and curcumin (1uM) alone group; L. Acidophilus + VitB complex treatment, reaction for 10 minutes, and stimulation of inflammatory cytokine IL-21 (10 ng/mL) for 1 hour, and then cells were obtained. Thereafter, flow cytometry analysis was performed to confirm the modulating effect of STAT3 according to each treatment group.

As a result, it was confirmed that the microbiome mixed composition treatment group of the present invention inhibited STAT3 more than twice as compared to the L. Acidophilus + VitB complex treatment group (FIG. 7).

6-2. Confirmation of p-STAT3 inhibition through Western blot

Under the conditions of stimulation of the inflammatory cytokine IL-21 (10 ng/mL) on healthy human peripheral blood mononuclear cell cells (huPBMC), the present invention also L. Acidophilus alone group (10 ug/ml); VitB complex alone group (B1 20uM, B6 20uM, B9 20uM, B12 20uM, so a total of 80 uM) and curcumin (1uM) alone group; L. Acidophilus + VitB complex treatment, reaction for 10 minutes, and stimulation of inflammatory cytokine IL-21 (10 ng/mL) for 1 hour, and then cells were obtained. Thereafter, expression of p-STAT3 (727) and T-STAT3, which are related to Th17 activity, was confirmed through Western blot.

As a result, it was confirmed that p-STAT3 was significantly suppressed in the microbiome mixed composition-treated group of the present invention compared to other groups (FIG. 8).

<Example 7> The effect of treating osteoarthritis in an osteoarthritis animal model by treatment with the microbiome mixture composition of the present invention

Monosodium Iodoacetate (MIA, I2512, Sigma, Poole, UK) was dissolved in saline for injection at a concentration of 60 mg/ml and prepared on the day of the start of the experiment (day 0). At the start of the experiment, rats (Wistar rat, 6 weeks old, 180g~220g) were given respiratory anesthesia using Isoflurane, and then MIA was placed into the right knee joint using a gauge 0.5 inch needle (model 750; Hamiltion Companym, Reno, NV). Osteoarthritis (MIA) was induced by injecting 50ul (MIA 3mg/body) via infrapatellar ligament.

The microbiome mixture composition of the present invention (Curcumin 185mg/kg + L. Acidophilus 500mg/kg + VitaminB complex (B1:1mg/kg + B6:1mg/kg + B9:1mg/kg + B12:1mg/kg), so the total 4mg/kg) treatment group; 30mg/Kg celecoxib treatment group; L. Acidophilus + VitB treatment group; curcumin alone group, each treatment was administered orally per rat every day from the day after MIA induction, 0, 7, 14 and The progress of 21 days was confirmed.

For the measurement of pain in rats, a Dynamic planter esthesiometer (UgoBasile, Comerio, Itaily) was used. Place the stimulator on the lower part of the rat, adjust the 0.5mm-thick plastic stimulating microfilament so that it is positioned on the hind limb, and operate the machine, while the stimulating needle presses the procimal metatasal at a constant speed and force, gradually increasing the pressure. The time (sec) and applied force (g) were measured until the rat could not withstand the stimulus and took off its feet. Each measurement was made three times and an average was given.

As a result, as a result of confirming the degree of pain through pain evaluation (Paw withdrawal threshold (PWT) and paw withdrawal latency (PWL)), the microbiome mixture composition (L.aci+VitB+Cur) treatment group of the present invention was Celecok. Compared with the sieve treatment group, the effect of reducing pain appeared as time passed, and it was confirmed that the treatment effect for osteoarthritis was excellent (FIG. 9).

Claims (11)

Lactobacillus (Lactobacillus) genus strain, vitamin B complex, and containing one or more selected from the group consisting of curcumin (Curcumin) as an active ingredient, a pharmaceutical composition for preventing or treating immune diseases. The method of claim 1, wherein the Lactobacillus genus strain is Lactobacillus Acidophilus, Lactobacillus casei, Lactobacillus plantarum, and Lactobacillus rhamnosus. , Lactobacillus Helveticus (Lactobacillus Helveticus), Lactobacillus fermentum (Lactobacillus fermentum), Lactobacillus paracasei (Lactobacillus paracasei) and Lactobacillus salivarius (Lactobacillus salivarius) one or more selected from the group consisting of . The composition of claim 1, wherein the vitamin B complex is at least one selected from the group consisting of vitamins B1, B3, B5, B6, B9 and B12 subgroups. The composition of claim 3, wherein the vitamin B complex is mixed in the same ratio. The composition of claim 1, wherein the Lactobacillus sp. strain, vitamin B complex, and curcumin are mixed in a mixing ratio of 1: 0.4: 50 to 1: 0.004: 5. The method of claim 1, wherein the immune disease is osteoarthritis, rheumatoid arthritis, ankylosing spondylitis, spondyloarthritis, inflammatory bowel disease, psoriatic arthritis, gout, bacterial arthritis, childhood rheumatoid arthritis, lupus, scleroderma, multiple sclerosis, fibromyalgia, multiple myositis, Dermatitis, Behcet's disease, Reiter's syndrome, Lyme arthritis, adhesive arthritis, frozen shoulder, tendon synovitis, elbow head pouchitis, dequeuvain tendon synovitis, recurrent rheumatism, rheumatoid multiple muscle pain, adult Still's disease, autoimmune hemocytopenia, Autoimmune myocarditis, atopic dermatitis, asthma, primary cirrhosis, Goodfighter syndrome, autoimmune meningitis, Sjogren syndrome, Addison's disease, alopecia areata, autoimmune hepatitis, autoimmune mumps, Crohn's disease, insulin-dependent diabetes, dystrophic blisters Epidermal detachment, epididymitis, glomerulonephritis, Graves disease, Guillain-Barre syndrome, Hashimoto's disease, hemolytic anemia, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, sarcoidosis, skin sclerosis, spondyloarthrosis, thyroiditis, vasculitis, vitiligo, mucus The composition is one or more selected from the group consisting of several species, pernicious anemia, mitochondrial-related syndrome and ulcerative colitis. The composition of claim 1, wherein the composition inhibits the expression of inflammatory cytokines and increases the expression of anti-inflammatory cytokines. The composition of claim 1, wherein the composition controls Th17 and Treg cells simultaneously by inhibiting the expression of inflammation-related STAT3. The composition of claim 1, wherein the composition has the ability to regulate B17 and Breg cells. According to claim 1, By inhibiting the expression of the bone destruction mechanism factor TRAP (tartrate-resistant acid-phosphatase), RANKL (Receptor activator of nuclear factor k appa-B ligand) and cathepsin K (cathepsin K) To inhibit differentiation, the composition. Lactobacillus (Lactobacillus) genus strain, vitamin B, and containing one or more selected from the group consisting of curcumin as an active ingredient, a health functional food composition for preventing or improving immune diseases.

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