KR102069807B1 - Composition for preventing or treating immune diseases comprising mixture of lactic acid bacteria - Google Patents

Abstract

The present invention relates to compositions for the prevention and treatment of immune diseases, including lactic acid bacteria mixtures and various applications thereof. The composition of the present invention is a mixture of probiotics or prebiotics in the most effective ratio, and inhibits the expression of inflammatory cytokines and cartilage destruction factors when compared with conventional anti-inflammatory agents in vitro or in vivo. It is effective. In addition, cartilage protection factor and anti-inflammatory cytokine expression is increased, and cartilage protection effect was found to be excellent in the immune disease animal model, it can be usefully used in the related pharmaceutical and dietary supplement industry.

Description

Composition for preventing or treating immune diseases comprising mixture of lactic acid bacteria}

The present invention relates to compositions for the prevention and treatment of immune diseases, including lactic acid bacteria mixtures and various applications thereof.

Immune disorders are diseases in which components of the mammalian immune system cause, mediate or otherwise contribute to mammalian pathologies, 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 to host invasion by foreign substances or harmful stimuli. 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 preparations; It may be an immune response, such as an allergic and autoimmune response, or a condition associated with oxidative stress.

Inflammation is characterized by pain, redness, swelling, fever and ultimate loss of function of the infected area. These symptoms are the result of a series of complex interactions between cells of the immune system. The response of the cell results in an interactive network of different groups of inflammatory mediators: proteins (eg cytokines, enzymes (eg proteases, peroxidases), major basic proteins, adhesion molecules (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) However, most of these mediators of inflammation are also regulators of normal cellular activity, so the lack of an inflammatory response leads to uncontrolled damage (ie, infection) of the host and, in part, due to chronic inflammation. Overproduction of several of the above-mentioned mediators results in inflammatory diseases that are mediated, and one of the autoimmune diseases, one of which is the immune system, Attacks the organs of the kidney and causes spontaneous reactions, which are due to the recognition of auto-antigens by T lymphocytes, which leads to humoral (autoantigen production) and cellular phases (lymphocytes and macrophages). Increases in phagocytic cytotoxic activity).

Probiotics are probiotics that are distributed as dominant bacteria in the intestines of various microorganisms and promote the growth of beneficial bacteria in the body. They are responsible for making fiber and complex proteins break down in the digestive system of the human body to make important nutrients. do. In addition, it maintains the pH of the intestinal acid to inhibit the reproduction of harmful bacteria such as Escherichia coli and Chlostridum sp., Improves diarrhea and constipation, and plays a role in vitamin synthesis and lowering blood cholesterol.

Probiotics, in particular, have a strong ability to bind mucous membranes and epithelial cells of the intestine, which greatly aids the work of the suit. In addition, probiotics promote the proliferation of macrophages to enhance macrophage macrophage cognitive and bactericidal ability against harmful bacteria.

Therefore, the present inventors mixed the plant extract, coenzyme or trace element of prebiotics with Lactobacillus spp., Bifidobacterium strain or Streptococcous strain among probiotics. Increasing the secretion of immune-related substances to induce immunity enhancement, so that the present invention was confirmed to be effective in the prevention and treatment of related immune diseases.

An object of the present invention comprises a first composition comprising Lactobacillus acidophilus ( Lactobacillus Acidophilus ); A second composition comprising Lactobacillus sp. Strain, Bifidobacterium strain and Streptococcous strain; And a third composition comprising a trace element and Rosavin (Rosavin); comprising at least one selected from the group consisting of as an active ingredient, it can provide a pharmaceutical composition for the prevention or treatment of immune diseases.

In addition, an object of the present invention is a Lactobacillus (Lactobacillus) strain, Bifidobacterium strain (Bifidobacterium) strain and Streptococcus genus (Steptococcous) comprising a second composition; Or a fourth composition comprising a trace element and a coenzyme; may provide a pharmaceutical composition for the prevention or treatment of immune diseases, including as an active ingredient.

In addition, an object of the present invention is a fifth composition comprising a strain of the genus Lactobacillus (Lactobacillus); Or a sixth composition comprising a trace element and Rosavin (Rosavin); may provide a pharmaceutical composition for the prevention or treatment of immune diseases, including as an active ingredient.

In addition, an object of the present invention comprises a first composition comprising Lactobacillus acidophilus ( Lactobacillus Acidophilus ); A second composition comprising Lactobacillus sp. Strain, Bifidobacterium strain and Streptococcous strain; And a third composition comprising a trace element and Rosavin (Rosavin); comprising at least one selected from the group consisting of as an active ingredient, it can provide a health functional food composition for the prevention or improvement of immune diseases.

In addition, an object of the present invention is a Lactobacillus (Lactobacillus) strain, Bifidobacterium strain (Bifidobacterium) strain and Streptococcus genus (Steptococcous) comprising a second composition; Or a fourth composition comprising a trace element and a coenzyme; may provide a health functional food composition for the prevention or improvement of immune diseases, including as an active ingredient.

In addition, an object of the present invention is a fifth composition comprising a strain of the genus Lactobacillus (Lactobacillus); Or a sixth composition comprising a trace element and Rosavin (Rosavin); may provide a health functional food composition for the prevention or improvement of immune diseases, including as an active ingredient.

The present invention comprises a first composition comprising Lactobacillus acidophilus ( Lactobacillus Acidophilus ); A second composition comprising Lactobacillus sp. Strain, Bifidobacterium strain and Streptococcous strain; It provides a pharmaceutical composition for the prevention or treatment of immune diseases, comprising at least one selected from the group consisting of; and a third composition comprising a trace element and Rosavin (Rosavin).

In addition, the present invention is a Lactobacillus (Lactobacillus) strain, Bifidobacterium strain (Bifidobacterium) strain and Streptococcus genus (Steptococcous) comprising a second composition; Or a fourth composition comprising a trace element and a coenzyme; provides a pharmaceutical composition for the prevention or treatment of immune diseases, including as an active ingredient.

In addition, the present invention comprises a fifth composition comprising a strain of the genus Lactobacillus (Lactobacillus); Or a sixth composition comprising a trace element and Rosavin (Rosavin); Provides a pharmaceutical composition for the prevention or treatment of immune diseases, comprising as an active ingredient.

In addition, the present invention comprises a first composition comprising Lactobacillus acidophilus ( Lactobacillus Acidophilus ); A second composition comprising Lactobacillus sp. Strain, Bifidobacterium strain and Streptococcous strain; And a third composition comprising a trace element and rosavin (Rosavin). It provides a health functional food composition for preventing or ameliorating immune diseases, comprising at least one selected from the group consisting of active ingredients.

In addition, the present invention is a Lactobacillus (Lactobacillus) strain, Bifidobacterium strain (Bifidobacterium) strain and Streptococcus genus (Steptococcous) comprising a second composition; Or a fourth composition comprising a trace element and a coenzyme; provides a health functional food composition for the prevention or improvement of immune diseases comprising as an active ingredient.

In addition, the present invention comprises a fifth composition comprising a strain of the genus Lactobacillus (Lactobacillus); Or a sixth composition comprising a trace element and Rosavin (Rosavin); provides a health functional food composition for the prevention or improvement of immune diseases, comprising as an active ingredient.

The composition of the present invention is a mixture of probiotics or prebiotics in the most effective ratio, and inhibits the expression of inflammatory cytokines and cartilage destruction factors when compared with conventional anti-inflammatory agents in vitro or in vivo. It is effective. In addition, it enhances the expression of cartilage protectors and anti-inflammatory cytokines, and has the effect of simultaneously regulating Th17 and Treg cells, which can be usefully used in the related pharmaceutical and dietary supplement industries.

1 is a diagram confirming the expression of IL-17, IL-10 and IFN-r in peripheral mononuclear cells following lactic acid bacteria mixture 1 (complex 1) and lactic acid bacteria mixture plus 1 (complex 1P) treatment.
Figure 2 is a diagram confirming the expression of IL-17 according to Th17 regulatory capacity in peripheral mononuclear cells following lactic acid bacteria mixture plus 1 (complex 1P) treatment.
Figure 3 is a diagram confirming the expression of IL-10, IL-17 and TNF-a in splenocytes following lactic acid bacteria mixture 1 (complex 1) treatment.
Figure 4a is a diagram confirming the expression of TNF-α and IL-6 in human-derived joint cells following treatment with lactic acid bacteria mixture plus 1 (complex 1P) and celecoxib (celecoxib).
Figure 4b is a diagram confirming the expression level of MMP3 and TIMP3 mRNA in human-derived articular cells following lactic acid bacteria mixture plus 1 (complex 1P) and celecoxib (celecoxib) treatment.
Figure 5 is a diagram confirming the degree of pain relief in osteoarthritis animal model according to lactic acid bacteria mixture 1 (complex 1) and lactic acid bacteria mixture plus 1 (complex 1P) treatment.
Figure 6 is a comparison of cartilage destruction and cartilage volume in the osteoarthritis animal model according to the lactic acid bacteria mixture plus 1 (complex 1P) and celecoxib treatment.
Figure 7 is a comparison of cartilage damage in osteoarthritis animal models following treatment with lactic acid bacteria mixture plus 1 (complex 1P) and celecoxib.
8A and 8B show the expression of inflammatory and anti-inflammatory cytokines in osteoarthritis animal models following Lactobacillus mixture plus 1 (complex 1P) and celecoxib treatment.
9A and 9B are diagrams confirming the expression levels of MMP3 and TIMP3 in osteoarthritis animal models following Lactobacillus mixture plus 1 (complex 1P) and celecoxib treatment.
10 is a diagram confirming the improvement effect in the rheumatoid arthritis animal model according to the lactic acid bacteria mixture 1 (complex 1) and lactic acid bacteria mixture plus 1 (complex 1P) treatment.
FIG. 11 is a diagram showing an improvement effect in ankylosing spondylitis animal model following treatment with lactic acid mixture 1 (complex 1) and lactic acid mixture plus 1 (complex 1P).
12 is a diagram confirming the improvement effect in inflammatory bowel disease animal model according to lactic acid bacteria mixture 1 (complex 1) and lactic acid bacteria mixture plus 1 (complex 1P).
Figure 13 is a diagram confirming the expression of IL-17, IL-10 and IFN-r in mouse spleen cells following lactic acid bacteria mixture 2 (complex 2) and lactic acid bacteria mixture plus 2 (complex 2P) treatment.
Figure 14a is a diagram confirming the arthritis index and incidence in the arthritis animal model according to the lactic acid bacteria mixture plus 2 (complex 2P) treatment, Figure 14b is a diagram confirming the IgG, IgG1, IgG2a immunoglobulin inhibitory effect.
Figure 15a is a diagram confirming the destruction in the joint tissue arthritis animal model according to the lactic acid bacteria mixture plus 2 (complex 2P) treatment, Figure 15b is a diagram confirming the degree of inflammatory cell infiltration, bone damage and cartilage damage.
FIG. 16A is a diagram illustrating IHC staining of joint tissues in an arthritis animal model according to lactic acid bacteria mixture plus 2 (complex 2P) treatment, and FIG. It is the figure which confirmed the expression cell.
17A is a diagram confirming Th17 and Treg cells by confocal microscopy in an arthritis animal model according to lactic acid bacteria mixture plus 2 (complex 2P) treatment, and FIG. 17B is a diagram confirming Th17 cells, Treg cells, and pSTAT3 705 cell expression.
FIG. 18 is a diagram showing the effects of inflammatory cytokines IL-17 following treatment with lactic acid bacterium mixture 3 (complex 3) and the individual strains (Acidophilus, rhamnosus and paracasei) constituting the same.
19 is a diagram showing the effect of IL-10 expression according to lactic acid bacteria mixture 3 (complex 3) and lactic acid bacteria mixture plus 3 (complex 3P) treatment.
20 is a diagram confirming the inhibitory effect of Th17 and Th1 cells of lactic acid bacteria mixture 3 (complex 3) and lactic acid bacteria mixture plus 3 (complex 3P).
21 is a diagram confirming the effect of increasing the Treg and Th2 cells of lactic acid bacteria mixture 3 (complex 3) and lactic acid bacteria mixture plus 3 (complex 3P).
FIG. 22 is a diagram illustrating Th17 and Treg regulating effects of lactic acid bacteria mixture 3 (complex 3) and lactic acid bacteria mixture plus 3 (complex 3P). FIG.
FIG. 23 is a diagram showing pain relief in an osteoarthritis animal model following treatment with lactic acid bacteria mixture 3 (complex 3) and lactic acid bacteria mixture plus 3 (complex 3P).
24 is a view showing the control effect of cartilage destruction factor in osteoarthritis animal model according to lactic acid bacteria mixture 3 (complex 3) and lactic acid bacteria mixture plus 3 (complex 3P) treatment.

The present invention comprises a first composition comprising Lactobacillus acidophilus ( Lactobacillus Acidophilus ); A second composition comprising Lactobacillus sp. Strain, Bifidobacterium strain and Streptococcous strain; It provides a pharmaceutical composition for the prevention or treatment of immune diseases, comprising at least one selected from the group consisting of; and a third composition comprising a trace element and Rosavin (Rosavin).

Rosavin (Rosavin) is extracted from Hong Kyungcheon (Genus Rhodiola), a substance involved in the suppression of stress hormones. It may be represented by the formula (1) as follows. The honggyeongcheon is a perennial plant with dolnamul and grows in alpine rocks of 2000m above sea level such as Baekdusan, Nanglim, and Tibet. The root of this plant is mainly used as a health functional food material.

[Formula 1]

In addition, the present invention is a Lactobacillus (Lactobacillus) strain, Bifidobacterium strain (Bifidobacterium) strain and Streptococcus genus (Steptococcous) comprising a second composition; Or a fourth composition comprising a trace element and a coenzyme; provides a pharmaceutical composition for the prevention or treatment of immune diseases, including as an active ingredient.

In addition, the present invention comprises a fifth composition comprising a strain of the genus Lactobacillus (Lactobacillus); Or a sixth composition comprising a trace element and Rosavin (Rosavin); Provides a pharmaceutical composition for the prevention or treatment of immune diseases, comprising as an active ingredient.

There are two types of strains of the genus Lactobacillus, homo and hetero, which are not known to be pathogenic. Lactobacillus sp in the present invention include Lactobacillus ramno suspension (Lactobacillus rhamnosus), Lactobacillus know also the filler's (Lactobacillus acidophilus), Lactobacillus brevis (Lactobacillus brevis), Lactobacillus buchwi Neri (Lactobacillus buchneri), Lactobacillus Kasei (Lactobacillus casei), Lactobacillus Catena Pomeranian (Lactobacillus catenaforme), Lactobacillus cells lobby O suspension (Lactobacillus cellobiosus), Lactobacillus Cri spa Tuscan (Lactobacillus crispatus), Lactobacillus keoba Tuscan (Lactobacillus curvatus), Lactobacillus del bwiki (Lactobacillus delbrueckii ), Lactobacillus fermentum , Lactobacillus gasserii , Lactobacillus jensenii , Lactobacillus leichmanii , Lactobacillus minutus ( Lactobacillus minusus ), Lactobacillus minutus Tannum ( Lactobacillus p lantarum ), Lactobacillus rogosae , Lactobacillus salivarius and mixed strains thereof.

Preferably, the Lactobacillus genus strain Lactobacillus know even Phil Ruth (Lactobacillus Acidophilus), Lactobacillus casei (Lactobacillus casei), Lactobacillus Planta Room (Lactobacillus plantarum), Lactobacillus ramno Seuss (Lactobacillus rhamnosus), Lactobacillus Lactobacillus Helveticus, Lactobacillus fermentum, Lactobacillus paracasei and Lactobacillus salivarius selected from the group consisting of, but not limited thereto.

The Bifidobacterium genus strain is at least one selected from the group consisting of Bifidobacterium breve , Bifidobacterium bifidum and Bifidobacterium longum , It is not limited.

Streptococcus strain is characterized in that Streptococcus thermophilus ( Streptococcous thermophilus ), but is not limited thereto.

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

The term "culture" in the present invention means to grow microorganisms under environmental conditions that are appropriately artificially controlled.

The strain may be grown in a conventional medium, and include a nutrient required by the microorganism to be cultured, that is, the culture medium in order to cultivate a specific microorganism, and may be added and mixed with a substance for a special purpose. The medium may also be referred to as an incubator or a culture medium, and is a concept including all natural, synthetic, or selective media.

The medium used for cultivation should meet the requirements of the particular strain in a suitable manner while controlling the temperature, pH, etc. in a conventional medium containing a suitable carbon source, nitrogen source, amino acids, vitamins and the like. Carbon sources that can be used include mixed sugars of glucose and xylose as the main carbon source, and sugars and carbohydrates such as sucrose, lactose, fructose, maltose, starch and cellulose, soybean oil, sunflower oil, castor oil, coconut Oils such as oil and fats, fatty acids such as palmitic acid, stearic acid, linoleic acid, alcohols such as glycerol, ethanol, organic acids such as acetic acid. These materials can be used individually or as a mixture. Nitrogen sources that can be used include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, anmonium 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 product, skim soy cake or its degradation product Can be. These nitrogen sources may be used alone or in combination. The medium may include, as personnel, monopotassium phosphate, dipotassium phosphate and corresponding sodium-containing salts. Personnel that may be used include potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts. In addition, as the inorganic compound, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate and calcium carbonate may be used. Finally, essential growth substances such as amino acids and vitamins may be used in addition to the above substances.

In addition, suitable precursors to the culture medium may be used. The raw materials described above may be added batchwise, fed-batch or continuous in a suitable manner to the culture in the culture process, but is 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 trace element is one or more selected from the group consisting of zinc, copper, chromium, selenium, iron, boron, molybdenum, chlorine and manganese, and if it is to help boost the growth or activity of the probiotic strain of the present invention, This is not restrictive.

The composition may further include one or more selected from the group consisting of chicory extract, licorice extract and black rice bran extract, and is not limited thereto, as long as it is a plant extract for enhancing immunity.

As a suitable solvent for extracting the extract, any pharmaceutically acceptable organic solvent may be used, and water or organic solvent may be used. For example, anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms, propylene glycol including purified water, methanol, ethanol, propanol, isopropanol, butanol, etc. as an extraction solvent. Various solvents, such as butylene glycol, glycerin, acetone, ethyl acetate, butyl acetate, chloroform, diethyl ether, dichloromethane, hexane, ether, benzene, methylene chloride, and cyclohexane, can be used alone or in combination. Preferably water, lower alcohol, hexane, dichloromethane, ethyl acetate can be used, and more preferably water or lower alcohol can be used.

As the extraction method, any one of hot water extraction method, cold leaching extraction method, reflux cooling extraction method, solvent extraction method, steam distillation method, ultrasonic extraction method, elution method and compression method can be used. In addition, the desired extract may further be subjected to a conventional fractionation process, and may be purified using conventional purification methods. There is no limitation on the preparation method of the extract of the present invention, any known method may be used.

The coenzyme is Coenzyme M, Coenzyme Q, or Coenzyme B, preferably Coenzyme Q10, but is not limited thereto.

The trace elements, coenzymes and plant extracts can function the same as "prebiotics" in the present invention, "prebiotics" is used by the useful microorganisms in the large intestine growth or activity of the microorganisms By means of non-digestible food ingredients that have a beneficial effect on the host health. In order for food ingredients to meet the conditions of prebiotics, they must not be digested or absorbed from the upper part of the gastrointestinal tract, selectively activate useful bacteria such as Bifidobacteria among colonic microorganisms, and inhibit harmful bacteria such as pathogens.

In one embodiment of the present invention, the probiotic strain of the present invention comprises one or more selected from the group consisting of Lactobacillus genus strain, Bifidobacterium strain and Streptococcous strain The mixture of only lactic acid bacteria was named "lactic acid bacteria mixture" and according to the method of Preparation Example 1, Preparation Example 2 and Preparation Example 3, lactic acid bacteria mixture 1 (complex 1), lactic acid bacteria mixture 2 (complex 2) and lactic acid bacteria mixture 3 ( complex 3). In addition, the trace elements, plant extracts and coenzymes that can function as prebiotics in the lactic acid bacteria mixture was named "lactic acid bacteria mixture plus" and according to the method of Preparation Example 1, Preparation Example 2 and Preparation Example 3 1 (complex 1P), lactic acid bacteria mixture plus 2 (complex 2 P) and lactic acid bacteria mixture plus 3 (complex 3P).

The lactic acid bacteria mixture 1 (complex 1) may be prepared by mixing Lactobacillus acidophilus (Lactobacillus acidophilus), the first composition, 12 kinds of lactic acid bacteria in a ratio of 1: 1 (Table 1). In addition, the lactic acid bacterium mixture plus 2 (complex 2 P) includes a third composition of zinc oxide, copper, chicory extract, licorice extract, black rice bran extract and red ginseng extract (rosavine) in the lactic acid bacteria mixture 1 (complex 1) It can be manufactured (Table 2).

The second composition comprises 2-8% by weight relative to the total weight of the second composition Lactobacillus know also required Ruth (Lactobacillus Acidophilus), 6 ~ 12% by weight of Lactobacillus casei (Lactobacillus casei), 17 ~ 23% by weight of Lactobacillus Lactobacillus plantarum , 5-11 wt% Bifidobacterium breve , 14-21 wt% Lactobacillus rhamnosus , 3-9 wt% Bifidobacterium bifido ( Bifidobacterium bifidum , 1-6 wt% Lactobacillus Helveticus , 2-8 wt% Bifidobacterium longum , 1-7 wt% Streptococcus thermophilus , 9-15 wt% Lactobacillus fermentum , 2-8 wt% Lactobacillus paracasei , and 3-9 wt% Lactobacillus salivarius .

The lactic acid bacterium mixture 2 (complex 2) is composed of 12 kinds of lactic acid bacteria of the second composition, lactic acid bacteria mixture plus 2 (complex 2P) can be prepared by including the fourth composition zinc and coenzyme Q10 in the second composition ( Table 3). Lactobacillus mixture 2 (complex 2) and zinc and coenzyme Q10 may be prepared in a ratio of 1: 3 to 7: 8 to 12, but may be preferably mixed in a ratio of 1: 5: 10, and may be used for enhancing immune and If the purpose is to achieve the prevention and treatment of immune diseases, it is not limited thereto (Table 4).

The lactic acid bacteria mixture 3 (complex 3) is composed of three kinds of lactic acid bacteria of the fifth composition (Table 5, lactic acid bacteria mixture plus 3 (complex 3P) is a composition containing the sixth composition zinc and hongkyungcheon extract derived from rosavin (Table 6), and the mixing ratio thereof is not limited thereto, as long as the purpose is to achieve immune boosting and prevention and treatment of immune diseases.

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

In the present invention, the term "autoimmune disease" refers to immunological unresponsiveness or tolerance of the body to autoantigens, and when problems arise in inducing or maintaining such self tolerance The immune response to the autoantigen occurs, which causes the attack of his tissue, which means a disease caused by this process.

As used herein, the term "inflammatory disease" refers to TNF-α (tumor necrosis factor-α), IL secreted from immune cells such as macrophages by excessively promoting the human immune system due to harmful stimuli such as inflammation-inducing factors or irradiation. Refers to diseases caused by proinflammatory substances (inflammatory cytokines) such as interleukin-1, IL-6, prostaglandin, luecotriene or nitric oxide (NO) .

The immune diseases include osteoarthritis, rheumatoid arthritis, ankylosing spondylitis, spondyloarthropathies, 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, Adhesion arthritis, Fifty shoulders, Tendon synoviitis, Elbow pochitis, Dquavane tendon lubricitis, Recurrent maturation, Rheumatoid polymyalgia, Adult type Still's disease, Autoimmune cytopenia, Autoimmune myocarditis, Atopic dermatitis , Asthma, Primary cirrhosis, Goodfiction syndrome, Autoimmune meningitis, Sjogren's syndrome, Addison's disease, Alopecia areata, Autoimmune hepatitis, Autoimmune mumps, Crohn's disease, Insulin-dependent diabetes mellitus, Epididymal bullous epidermal detachment, Epididymitis, Glomeruli Nephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, multiple sclerosis At least one selected from the group consisting of myasthenia gravis, vulgaris, psoriasis, rheumatic fever, sarcoidosis, scleroderma, spondyloarthropathy, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, mitochondrial related syndrome and ulcerative colitis It is not limited.

In one embodiment of the invention, the composition may inhibit the expression of inflammatory cytokines, increase the expression of anti-inflammatory cytokines, inhibit the expression of matrix metalloproteinase 3 (MMP3), a cartilage tissue destruction factor In addition, cartilage regeneration factor TIMP3 (Metalloproteinase inhibitor 3) can increase the expression.

As used herein, unless otherwise indicated, the term "treatment" means to reverse, alleviate, inhibit the progression of, or prevent the disease or condition to which the term applies, or one or more symptoms of the disease or condition. As used herein, the term treatment refers to the act of treating when “treating” is defined as above. Thus, "treatment" or "therapy" of an immune disease in a mammal may include one or more of the following:

(1) inhibits the growth of an immune disease, i.e. arrests its development,

(2) preventing the spread of immune diseases, ie preventing metastasis,

(3) alleviates immune diseases.

(4) prevent the recurrence of immune diseases, and

(5) Palliating the symptoms of an immune disease.

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

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

The pharmaceutical compositions of the present invention may be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, oral dosage forms, external preparations, suppositories, or sterile injectable solutions, respectively, according to conventional methods. .

When formulated, it may be prepared using conventional diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid form preparations contain at least one excipient such as starch, calcium carbonate, sucrose, lactose, gelatin in the active ingredient. It can be prepared by mixing. In addition to simple excipients, lubricants such as magnesium stearate, talc can also be used. Liquid preparations for oral administration include suspensions, solvents, emulsions, and syrups.In addition to commonly used diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. Can be. Formulations for parenteral administration include sterile aqueous solutions, water-insoluble solvents, suspensions, emulsions, lyophilized preparations and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can 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, physical condition, etc. of the individual. Obviously, the concentration of the active ingredient included in the pharmaceutical composition can be variously selected according to the object, and preferably, the pharmaceutical composition is included in a concentration of 0.01 to 5,000 ㎍ / ml. If the concentration is less than 0.01 μg / ml, the pharmaceutical activity may not appear, and when the concentration is more than 5,000 μg / ml, the human body may be toxic.

The pharmaceutical compositions can be formulated in a variety of oral or parenteral dosage forms.

Formulations for oral administration include, for example, tablets, pills, hard capsules, soft capsules, solutions, suspensions, emulsifiers, syrups, granules, etc. These formulations may contain diluents (e.g., lactose, dextrose, water, etc.) in addition to the active ingredients. Cros, mannitol, sorbitol, cellulose and / or glycine), glidants such as silica, talc, stearic acid and its magnesium or calcium salts and / or polyethylene glycols. The tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidine, optionally starch, agar, alginic acid. Or disintegrating or boiling mixtures such as sodium salts thereof and / or absorbents, colorants, flavors and sweeteners. The formulations may be prepared by conventional mixing, granulating or coating methods.

Representative of parenteral formulations are also injectable preparations, which may include water, Ringer's solution, isotonic saline or suspensions as solvents for injectable preparations. Sterile fixed oils of the injectable preparations may be used as solvents or suspending media and any non-irritating fixed oil may be used for this purpose, including mono- and diglycerides.

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

In general, the composition of the present invention may use a coating performed in the manufacture of a pharmaceutical, and may use a mixed solvent in consideration of the solubility and coating property of the coating base. The preparation of the capsule may include an enteric material for lactic acid bacteria to be safely mounted in the intestine by minimizing the influence of body fluids. In addition, in the case of an enteric coating which should have acid resistance, it can be maintained without disintegration (humidification) of the capsule for 2 hours at pH 1.2, which is an acid resistance condition of a general health functional food, and conditions such as coating conditions and coating amount are not limited. . In addition, it can be prepared so that there is no reduction in the number of lactic acid bacteria and residual solvents while maintaining the quality for disintegration.

In addition, the present invention comprises a first composition comprising Lactobacillus acidophilus ( Lactobacillus Acidophilus ); A second composition comprising Lactobacillus sp. Strain, Bifidobacterium strain and Streptococcous strain; And a third composition comprising a trace element and rosavin (Rosavin). It provides a health functional food composition for preventing or ameliorating immune diseases, comprising at least one selected from the group consisting of active ingredients.

In addition, the present invention is a Lactobacillus (Lactobacillus) strain, Bifidobacterium strain (Bifidobacterium) strain and Streptococcus genus (Steptococcous) comprising a second composition; Or a fourth composition comprising a trace element and a coenzyme; provides a health functional food composition for the prevention or improvement of immune diseases comprising as an active ingredient.

In addition, the present invention comprises a fifth composition comprising a strain of the genus Lactobacillus (Lactobacillus); Or a sixth composition comprising a trace element and Rosavin (Rosavin); provides a health functional food composition for the prevention or improvement of immune diseases, comprising as an active ingredient.

The food composition of the present invention may contain, as an additional ingredient, various flavors or natural carbohydrates, and the like, in addition to the extract as an active ingredient, as in a conventional food composition.

Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. The aforementioned flavoring agents can advantageously be used natural flavoring agents (tautin), stevia extracts (for example rebaudioside A, glycyrzin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.). The food composition of the present invention may be formulated in the same manner as the pharmaceutical composition, 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. There is this.

In addition to the extract as an active ingredient, the food composition may include various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic and natural flavors, coloring and neutralizing agents (such as cheese, chocolate), pectic acid and salts thereof, Alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks and the like. In addition, the food composition of the present invention may contain fruit flesh for the production of natural fruit juice and fruit juice beverage and vegetable beverage.

The functional food composition of the present invention can be prepared and processed in the form of tablets, capsules, powders, granules, liquids, pills and the like. In the present invention, the "health functional food composition" refers to a food manufactured and processed using raw materials or ingredients having functional properties useful for the human body according to Act No. 6767 of the Health Functional Food Act. Ingestion is intended to obtain useful effects for health purposes such as nutrient control or physiological effects. The health functional food of the present invention may include a conventional food additive, and the suitability as a food additive is related to the item according to the General Regulations and General Test Act of the Food Additives approved by the Food and Drug Administration, unless otherwise specified. Judging by the standards and standards. Examples of the items listed in the 'Food Additive Revolution' include chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid and cinnamic acid; Natural additives such as dark blue, licorice extract, crystalline cellulose, high amount of pigment and guar gum; Mixed preparations, such as a sodium L- glutamate preparation, a noodle addition alkali agent, a preservative preparation, and a tar pigment preparation, etc. are mentioned. For example, the health functional food in the form of tablets may be granulated in a conventional manner by mixing the active ingredient of the present invention with excipients, binders, disintegrants and other additives, and then compression-molded with a lubricant or the like, or The mixture can be compression molded directly. In addition, the health functional food in the form of tablets may contain a mating agent or the like as necessary. Hard capsules among the health functional foods in the form of capsules may be prepared by filling a conventional hard capsule with a mixture of the active ingredient of the present invention and additives such as excipients, and the soft capsule agent may include the active ingredient of the present invention as an additive such as an excipient. It can be prepared by mixing the mixture with a capsule base such as gelatin. The soft capsule agent may contain a plasticizer such as glycerin or sorbitol, a colorant, a preservative, and the like, as necessary. The health functional food of the cyclic form may be prepared by molding a mixture of the active ingredient and the excipient, the binder, the disintegrant and the like of the present invention by a known method, and may be avoided with sucrose or other epidermis, if necessary, Alternatively, the surface may be coated with a material such as starch or talc. The health functional food in the form of granules may be prepared by granulating a mixture of an excipient, a binder, a disintegrant, and the like of the active ingredient of the present invention in a known manner, and may contain a flavoring agent, a coagulant, etc. as necessary. Can be.

In addition, the present invention comprises a first composition comprising Lactobacillus acidophilus ( Lactobacillus Acidophilus ); A second composition comprising Lactobacillus sp. Strain, Bifidobacterium strain and Streptococcous strain; And a third composition comprising a trace element and rosavin (Rosavin). It provides an immune enhancing feed composition comprising at least one selected from the group consisting of active ingredients.

In addition, the present invention is a Lactobacillus (Lactobacillus) strain, Bifidobacterium strain (Bifidobacterium) strain and Streptococcus genus (Steptococcous) comprising a second composition; Or a fourth composition comprising a trace element and a coenzyme; provides an immune enhancing feed composition comprising as an active ingredient.

In addition, the present invention comprises a fifth composition comprising a strain of the genus Lactobacillus (Lactobacillus); Or a sixth composition comprising a trace element and Rosavin (Rosavin); provides an immune enhancing feed composition comprising as an active ingredient.

The feed composition of the present invention can be expected to improve the health of the animal body, improve the weight gain and meat quality of the livestock, and increase the acid flow rate and immunity. Feed composition of the present invention can be prepared in the form of fermented feed, compound feed, pellet form and silage. The fermented feed may be prepared by fermenting an organic material by adding a composition, various microorganisms or enzymes of the present invention, and a compound feed may be prepared by mixing various kinds of general feed and the composition of the present invention. Pellet-type feed may be prepared by applying heat and pressure to the blended feed, etc. in a pellet machine, silage can be prepared by fermenting the cheonye feed with the microorganism according to the present invention. The wet fermented feed collects and transports organic materials such as food waste, mixes excipients for sterilization and moisture control at a certain ratio, and then ferments at a temperature suitable for fermentation for at least 24 hours so that the water content is about 70%. It can be prepared by adjusting. Fermented dry fertilizer can be prepared by adjusting the wet fermented feed to further contain a moisture content of about 30% to 40% through the drying process.

Feed composition of the present invention may further comprise a component added to the conventional feed. One example of the ingredients added to the feed may include grain powder, meat powder, legumes and the like. The grain powder may be used at least one selected from rice flour, flour, barley flour, and corn flour. The meat powder may be a meat powder powdered at least one selected from chicken, beef, pork, and ostrich meat. In the above beans can be used one or more selected from soybeans, kidney beans, peas, and black beans.

The feed composition of the present invention may be added to any one or more selected from nutrients and minerals to increase the nutritional properties of the feed in addition to cereal powder, meat powder, and soybean, which are added to the conventional feed mentioned above. It may include one or more selected from antifungal agents, antioxidants, anticoagulants, emulsifiers, and binders to prevent the degradation of.

The present invention will be described in more detail with reference to the following examples. However, the following examples are only intended to embody the contents of the present invention, and the present invention is not limited thereto.

Preparation Example 1 Preparation of Lactic Acid Bacteria Mixture 1 (complex 1) and Lactic Acid Bacteria Mixture Plus 1 (complex 1P)

For the preparation of lactic acid bacteria mixture 1 (complex 1) and lactic acid bacteria mixture plus 1 (complex 1P), each of the lactic acid bacteria was purchased from CTC Bio. Specifically, the lactic acid bacteria mixture 1 (complex 1) is Lactobacillus acidophilus as the first composition (lactic acid bacteria), and Lactobacillus genus strain as the second composition (12 species lactic acid bacteria), BP It was configured to include the genus Bifidobacterium strain and Streptococcous strain, and was prepared by mixing the first composition and the second composition in a ratio of 1: 1 (Table 1).

In addition, lactic acid bacteria mixture plus 1 (complex 1P) is 30 mg of zinc oxide, 2 mg of copper (copper sulfate), 150 mg of cichorium intybu s extract, licorice (Genus Glycyrrhiza) as a third composition in the lactic acid bacteria mixture 1 (complex 1) 100 mg of extract, 90 mg of Black Rice Bran extract, and 200 mg of Rosavin extracted from Genus Rhodiola were prepared (Table 2).

Lactobacillus mixture 1 Strain name weight% ratio First composition (1 type of lactic acid bacteria) Lactobacillus Acidophilus One Second composition (12 kinds of lactic acid bacteria) Lactobacillus Acidophilus 5 One Lactobacillus casei 9 Lactobacillus plantarum 20 Lactobacillus rhamnosus 17 Lactobacillus Helveticus 3 Lactobacillus fermentum 12 Lactobacillus paracasei 5 Lactobacillus salivarius 6 Bifidobacterium breve 8 Bifidobacterium bifidum 6 Bifidobacterium longum 5 Streptococcous thermophilus 4

Lactobacillus Mixture Plus 1 (complex 1P) Strain Name / Component weight(%) First composition (1 type of lactic acid bacteria) Lactobacillus Acidophilus 20.472 Second composition (12 kinds of lactic acid bacteria) Lactobacillus Acidophilus 20.472 Lactobacillus casei Lactobacillus plantarum Lactobacillus rhamnosus Lactobacillus Helveticus Lactobacillus fermentum Lactobacillus paracasei Lactobacillus salivarius Bifidobacterium breve Bifidobacterium bifidum Bifidobacterium longum Streptococcous thermophilus Article 3 Composition Zinc oxide 4.167 Copper (copper sulfate)  0.889 Chicory Extract 15 Licorice Extract 10 Black Rice Extract 9 Honggyeongcheon Extract (Rosabin) 20

Example 1. Confirmation of Inflammatory and Anti-Inflammatory Cytokine Expression in Human Peripheral Blood Monocytes

1-1. Expression of IL-17, IL-10 and IFN-r in Peripheral Monocyte Cells

Inflammatory cytokines IL-17 and IFN-r and anti-inflammatory cytokine IL-10 expression following lactic acid mixture 1 (complex 1) and lactic acid mixture plus 1 (complex 1P) treatment were identified.

Specifically, after isolation of CD4 T cells from healthy human peripheral blood monocytes, lactic acid mixture 1 (complex 1) and lactic acid mixture plus 1 (complex 1P) prepared in Preparation Example 1 under antiCD3 stimulation conditions (0.5 ug / ml). Were treated with 10 ug / ml each. After incubation for 3 days, ELISA was performed using the supernatant separated by centrifugation. After coating the 96-well plate for 2 hours at room temperature using the first capture ab (anti-mouse IL-6, IL-10, TNF-a), the non-specific reaction was blocked using a blocking buffer. After the blocking process, the standard and the sample were put in a plate and washed with a wash buffer after the reaction for 2 hours. Standard (recombinant: anti-mouse IL-6, IL-10, TNF-α) and the sample was added and washed for 2 hours after using the wash buffer. After washing, the detection ab (anti-mouse IL-17, IL-10, IFN-r) was added, and after 2 hours, the reaction was washed and developed.

As shown in FIG. 1, both lactic acid mixture 1 (complex 1) and lactic acid mixture plus 1 (complex 1P) treated groups showed significant decrease in IL-17 and IFN-r under antiCD3 stimulation conditions. In contrast, IL-10, an anti-inflammatory cytokine, was significantly increased in both lactic acid mixture 1 (complex 1) and lactic acid mixture plus 1 (complex 1P) treatment groups. In addition, it was confirmed that the expression increased more in the lactic acid bacteria mixture plus 1 (complex 1P) than lactic acid bacteria mixture 1 (complex 1).

1-2. Expression of IL-17 According to Th17 Regulation in Peripheral Monocyte Cells

In addition, Th17 cells (T helper 17 cells) induced the inflammatory cytokine IL-17, and confirmed the expression of Th17 cells and thus IL-17 according to the lactic acid bacteria mixture plus 1 (complex 1P) treatment.

CD4 T cells were isolated from healthy human peripheral monocytes and then treated with lactic acid bacteria mixture plus 1 (complex 1P) prepared in Preparation Example 10 under antiCD3 stimulation conditions (0.5 ug / ml). The cells cultured for 3 days were analyzed by flow cytometry to confirm the expression of Th17 cells and IL-17.

As shown in Figure 2, it was confirmed that IL-17 expression is significantly inhibited compared to the control group as the number of Th17 cells is reduced.

Example 2. Confirmation of Inflammatory and Anti-Inflammatory Cytokine Expression in Splenocytes

Spleens were obtained at the expense of normal mice (c57bl / 6, male, 9 weeks old, obtained from Orient Bio). The spleens were changed using a slide, and then splenocytes were obtained after removing RBC using an ACK buffer. The splenocytes were treated with lactic acid bacteria mixture 1 (complex 1) at various concentrations (10 ng / ml and 100 ng / ml) at the same time as the anti-CD3 stimulation alone, followed by sup (supernatant) for 3 days under the same conditions as in Example 1. ) Was obtained. In the obtained sup, ELISA was performed on IL-17 and IL-10.

Furthermore, sup was obtained after incubation for 3 days after treatment of lactic acid bacteria mixture 1 (complex 1) at 100 ng / ml simultaneously with LPS stimulation alone. ELISA was performed using the obtained sup. After the coating was carried out for 2 hours at room temperature using a first capture ab (anti-mouse IL-6, IL-10, TNF-a), the non-specific reaction was blocked using a blocking buffer. After the blocking process, the standard and the sample were put in a plate and washed with a wash buffer after the reaction for 2 hours. Standard (recombinant: anti-mouse IL-6, IL-10, TNF-α) and the sample was added and washed for 2 hours after using the wash buffer. After washing, the detection ab (anti-mouse IL-6, IL-10, TNF-a) was added thereto, and the reaction was followed by color development after washing for 2 hours.

As shown in FIG. 3, the inflammatory cytokines IL-17 and TNF-a were significantly reduced by the lactic acid bacterium mixture 1 (complex 1) under both the antiCD3 stimulation condition and the LPS condition, and the anti-inflammatory cytokine IL-10 was It was confirmed to increase.

Example 3. Determination of joint tissue destruction and regenerative factors and expression of inflammatory cytokines in human-derived joint cells

In vitro experiments pre-treated with human-derived Chondrocytes for 24 hours to stabilize cells, stimulate IL-1b to promote cell activity, and at the same time treat lactic acid mixture plus 1 (complex 1P and celecoxib) After culturing for 24 hours, RNA was obtained from the cells, and mRNA expression of MMP3 (matrix metalloproteinase 3), which is a cartilage tissue destruction factor, and Metalloproteinase inhibitor 3 (TIMP3), a cartilage regeneration factor, was confirmed by real time PCR.

As shown in FIGS. 4A and 4B, expression of inflammatory cytokines TNF-α and IL-6 was significant in the lactic acid bacteria mixture plus 1 (complex 1P) treated group of the present invention compared to the control and celecoxib. It was confirmed that the inhibition.

In addition, as shown in Figures 4c and 4d, mRNA expression of TIMP3, a cartilage regeneration factor, was significantly expressed in the lactic acid bacterium mixture plus 1 (complex 1P) treatment group of the present invention compared to the control and celecoxib. It was confirmed that this was suppressed.

Thus, the lactic acid bacteria mixture 1 (complex 1) and the lactic acid bacteria mixture plus 1 (complex 1P) of the present invention inhibit inflammatory cytokine expression and cartilage destruction factor expression in comparison with celecoxib in vitro, as well as cartilage protectors and It has been shown to increase anti-inflammatory cytokine expression.

Example 4 Osteoarthritis (Osteoarthritis) Treatment Effect

4-1. Osteoarthritis Treatment Effect Using Osteoarthritis Animal Model

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

Lactobacillus Mixture 1 (complex 1) and Lactobacillus Mixture Plus 1 (complex 1P) were orally administered 500 mg per rat daily from the day after MIA induction.

For measuring pain in rats, a dynamic planter esthesiometer (UgoBasile, Comerio, Itaily) was used. Place the stimulator on the bottom of the rat, adjust the 0.5mm thick plastic stimulating microfilament to the side of the hind limb, and operate the machine.The stimulation needle will gradually press the force while pressing the posterior metastasis at a constant speed and force. Increasing the time (sec) and the force applied (g) until the rat was unable to withstand the stimulus and released the foot was measured. Each measurement was carried out three times and averaged, and the results are shown in Fig. 5 (A, B, D and E in Fig. 5).

Incapacitance meter (IITC, Victory Blvd Woodland Hills, CA, USA) was used to measure the weight bearing of the rats, and the results are shown in FIG. 5 (C and F of FIG. 5).

As shown in Figure 5, both pain and weight bearing can be seen to show an excellent osteoarthritis treatment effect compared to the control group in both lactic acid mixture 1 (complex 1) and lactic acid mixture plus 1 (complex 1P) treatment group, in particular, lactic acid bacteria mixture plus More effective was observed in the 1 (complex 1P) group.

4-2. Comparison of Cartilage Breakdown and Cartilage Volume between Lactic Acid Bacteria Mixture Plus 1 and Conventional Therapeutics

To compare the osteoarthritis treatment effect of Lactobacillus mixture plus 1 (complex 1P) and celecoxib, which is used as an anti-inflammatory agent, cartilage destruction and cartilage volume of osteoarthritis animal models were measured.

Specifically, the animal model of MIA-induced osteoarthritis was used as the method of 4-1. Lactobacillus mixture plus 1 (complex 1P) or celecoxib were administered orally at 500 mg per rat daily from the day after MIA-induction. Femur and Tibia of rats were collected and stained with Indian ink, and then the degree of cartilage destruction and cartilage volume were analyzed by micro-CT image.

As shown in FIG. 6A, in the MIA-induced osteoarthritis animal model, the lactobacillus mixture plus 1 (complex 1P) of the present invention was found to significantly reduce cartilage destruction compared to the control group. It was also confirmed that cartilage destruction was further reduced compared to celecoxib.

As shown in B of FIG. 6, as a result of measuring the volume of cartilage, it was confirmed that the volume of cartilage was increased in the lactic acid bacteria mixture plus 1 (complex 1P) treated group when compared with the control group and celecoxib.

4-3. Comparison of Cartilage Damage between Lactobacillus Mixture Plus 1 and Conventional Therapeutics

In order to compare the osteoarthritis treatment effect of Lactobacillus mixture plus 1 (complex 1P) and celecoxib, a conventional anti-inflammatory agent, the degree of inhibition of cartilage damage in the osteoarthritis animal model was confirmed.

Specifically, the animal model of MIA-induced osteoarthritis was used as the method of 4-1. Celecoxib or Lactobacillus mixture plus 1 (complex 1P) was orally administered 500 mg / rat per rat daily from the day following MIA-induction. In order to confirm the degree of cartilage damage in each treatment group, H & E staining was performed, and the total Mankin score, OARSI score and structure of each group were confirmed.

As shown in Figure 7, compared to the control and celecoxib (celecoxib), the lactic acid bacteria mixture plus 1 (complex 1P) of the present invention confirmed that the index of Mankin score, OARSI score and structure significantly lower, cartilage It was confirmed that destruction was inhibited and cartilage was protected.

4-4. Comparison of Inflammatory or Anti-Inflammatory Cytokine Expression in Lactobacillus Mixture Plus 1 and Conventional Therapeutics

In order to compare the therapeutic effects of osteoarthritis of leucoxib with complex lactic acid complex plus 1 (complex 1P), the expression of inflammatory cytokines TNF-a and IL-6 in the joint tissue of animal models of osteoarthritis , The expression of the anti-inflammatory cytokine IL-10 was confirmed.

Specifically, the animal model of MIA-induced osteoarthritis was used as the method of 4-1. Lactobacillus mixture plus 1 (complex 1P) or celecoxib were administered orally at 500 mg / rat per rat daily from the day following MIA-induction. Thereafter, each treatment group was stained with IHC to confirm expression of TNF-a, IL-6, and IL-10.

As shown in Figures 8a and 8b, the lactic acid bacteria mixture plus 1 (complex 1P) treatment group of the present invention significantly inhibited the expression of inflammatory cytokines TNF-a and IL-6 compared to the control group, and also with celecoxib In comparison, it was confirmed to inhibit TNF-a and IL-6. In addition, it was confirmed that significantly increased the expression of anti-inflammatory cytokine IL-10.

4-5. Comparison of the Expression of Cartilage Protectors or Cartilage Destruction Factors in Complex 1P

In order to compare the effectiveness of osteoarthritis treatment of leucoxib (complex 1P) and celecoxibib, a conventional anti-inflammatory agent, the expression of cartilage tissue destruction factor MMP3 and cartilage regeneration factor TIMP3 in joint tissues of osteoarthritis animal model Was compared.

Specifically, the animal model of MIA-induced osteoarthritis was used as the method of 4-1. Lactobacillus mixture plus 1 (complex 1P) or celecoxib were administered orally at 500 mg per rat daily from the day after MIA-induction. Thereafter, each treatment group was stained with IHC, and expression of MMP3 and TIMP3 was confirmed by real time PCR.

9A and 9B, the lactic acid bacteria mixture plus 1 (complex 1P) of the present invention significantly inhibits MMP3, a cartilage tissue destruction factor, and inhibits the expression of TIMP3, a cartilage regeneration factor, compared to the control and celecoxib. It was confirmed to increase.

Therefore, it was confirmed that the lactic acid bacteria mixture 1 (complex 1) and the lactic acid bacteria mixture plus 1 (complex 1P) of the present invention had excellent pain relief effect in vivo. In addition, compared to the conventional anti-inflammatory celecoxib, inhibits cartilage destruction and damage, increases cartilage volume, inhibits the expression of inflammatory cytokines and cartilage destruction factors, and increases cartilage protective factor and anti-inflammatory cytokine expression. Confirmed. The above results suggest that the lactic acid bacteria mixture 1 (complex 1) and the lactic acid bacteria mixture plus 1 (complex 1P) of the present invention have an excellent effect on the prevention and treatment of osteoarthritis.

Example 5 Rheumatoid Arthritis Treatment Effect

Rheumatoid arthritis was induced by first and second injections of type II collagen into normal DBA1 / J mice to evaluate the therapeutic efficacy of rheumatoid arthritis of complex lactic acid mixture 1 (complex 1) and lactic acid bacteria mixture plus 1 (complex 1P). . Group segregation was divided by the average Arthritis Index 21 days after induction of rheumatoid arthritis.

100 mg of lactic acid bacteria mixture 1 (complex 1) and lactic acid bacteria mixture plus 1 (complex 1P) per mouse were respectively inoculated, and three observers who did not know the contents of the experiment, starting at the first inoculation, had a stomach severity of arthritis twice a week. Was evaluated by observation. The arthritis evaluation was based on the sum of the scores according to the following scales, and the average among the observers. The scores and criteria according to the arthritis evaluation are as follows.

0 points: There is no edema or swelling.

1 point: Mild swelling and redness limited to the foot or ankle joint

2 points: Mild swelling and redness from the ankle joint to the metatarsal

3 points: moderate swelling and redness from the ankle joint to the ankle bone

4 points: swelling and redness from ankle to leg

As shown in FIG. 10, both the arthritis index and the incidence showed improved effects in the lactic acid mixture 1 (complex 1) and lactic acid mixture plus 1 (complex 1P) treatment groups, and were more effective in the lactic acid mixture plus 1 (complex 1P). Was observed to be good.

Example 6 Therapeutic Effect of Ankylosing Spondylitis (AS)

To evaluate the therapeutic efficacy of ankylosing spondylitis of lactic acid mixture 1 (complex 1) and lactic acid bacteria mixture plus 1 (complex 1P) of the present invention, normal SKG mice were orally administered Curdlan 3 mg / kg and 6 mg / k intraperitoneally. Lactobacillus Mixture 1 (complex 1) and Lactobacillus Mixture Plus 1 (complex 1P) were orally administered 100 mg daily to mice one week after induction of ankylosing spondylitis.

The AS Scoring index was evaluated by scoring a total of 0.1 points for each swollen toe of the mouse and a total of 6 points for ankle swelling.

As shown in FIG. 11, it can be seen that the ankylosing spondylitis index was alleviated in the lactic acid mixture 1 (complex 1) and lactic acid mixture plus 1 (complex 1P) treatment groups, and particularly in the lactic acid mixture mixture 1 (complex 1P) treatment group. Ankylosing spondylitis was almost completely cured.

In addition, in order to examine immunoglobulins in the serum of the mouse, IgG in the serum was measured using the ELISA technique and the results are shown in FIG. 11. IgG also showed a significant inhibitory effect on IgG in the lactic acid mixture 1 (complex 1) and lactic acid mixture plus 1 (complex 1P) treatment group, especially in the lactic acid mixture plus 1 (complex 1P) treatment group. It was.

Example 7 Inflammatory Bowel Disease (IBD) Treatment Effect

In order to confirm the therapeutic effect of the lactic acid bacteria mixture 1 (complex 1) and lactic acid bacteria mixture plus 1 (complex 1P) of the present invention, mice were freely fed 3% DSS for 4 days and general day 5 to induce inflammatory bowel disease. The cells were replaced with water and lactic acid mixture 1 (complex 1) and lactic acid bacteria mixture plus 1 (complex 1P) were orally administered 200 ul (100 mg / mice) daily from day 3 until the end of the experiment. The mice were weighed every day for the duration of the experiment, and at the end of the experiment the mice were sacrificed to determine the intestinal length.

As can be seen in Figure 12, the weight loss was observed initially in both the lactic acid mixture 1 (complex 1) and lactic acid mixture plus 1 (complex 1P) treatment group, but since the mid-term to confirm that the weight loss effect is reduced compared to the control group Could. Intestinal length also showed longer intestinal length in both lactic acid mixture 1 and complex 1P treated groups compared to the control group, especially in the lactic acid mixture plus 1 complex group. It was observed that the length of the field was almost similar to that of the type.

Preparation Example 2 Preparation of Lactic Acid Bacteria Mixture 2 (complex 2) and Lactic Acid Bacteria Mixture Plus 2 (complex 2P)

For the preparation of lactic acid bacteria mixture 2 (complex 2) and lactic acid bacteria mixture plus 2 (complex 2P), each of the lactic acid bacteria was purchased from CTC Bio. Specifically, the lactic acid bacteria mixture 2 (complex 2) was configured to include the genus Lactobacillus strain, the Bifidobacterium strain, and the Streptococcous strain as the second composition (12 kinds of lactic acid bacteria). (Table 3).

In addition, lactic acid bacteria mixture plus 2 (complex 2P) was configured to include zinc and coenzyme Q10 as a fourth composition in the lactic acid bacteria mixture 2 (complex 2). Lactobacillus mixture 2 (complex 2) and zinc and coenzyme Q10 were prepared by mixing in a ratio of 1: 5: 10 (Table 4).

Lactic acid bacteria mixture 2 (complex 2) Strain name weight% Second composition (12 kinds of lactic acid bacteria) Lactobacillus Acidophilus 5 Lactobacillus casei 9 Lactobacillus plantarum 20 Lactobacillus rhamnosus 17 Lactobacillus Helveticus 3 Lactobacillus fermentum 12 Lactobacillus paracasei 5 Lactobacillus salivarius 6 Bifidobacterium breve 8 Bifidobacterium bifidum 6 Bifidobacterium longum 5 Streptococcous thermophilus 4

Lactobacillus Mixture Plus 2 (complex 2P) Strain name weight% ratio Second composition (12 kinds of lactic acid bacteria) Lactobacillus Acidophilus 5 One Lactobacillus casei 9 Lactobacillus plantarum 20 Lactobacillus rhamnosus 17 Lactobacillus Helveticus 3 Lactobacillus fermentum 12 Lactobacillus paracasei 5 Lactobacillus salivarius 6 Bifidobacterium breve 8 Bifidobacterium bifidum 6 Bifidobacterium longum 5 Streptococcous thermophilus 4 Article 4 Compositions zinc 5 Coenzyme Q10 50

Example 8. Confirmation of Inflammatory and Anti-Inflammatory Cytokine Expression in Mouse Spleen Cells

8-1. Expression of IL-17, IL-6, IFN-r and IL-10

Inflammatory cytokines IL-17, IL-6 and IFN-r and anti-inflammatory cytokine IL-10 expression following lactic acid mixture 2 (complex 2) and lactic acid mixture plus 2 (complex 2P) treatment were identified.

After isolation of CD4 T cells from normal mouse spleen, lactic acid mixture 2 (10 ug / ml) and lactic acid mixture plus 2 (complex 2P) prepared in Preparation Example 2 under anti-CD3 stimulation conditions (0.5 ug / ml). ) (Lactic acid bacteria mixture 2 100 ug / ml, zinc 50 uM, coenzyme Q10 10 uM), respectively. After culturing for 3 days, ELISA was performed using the supernatant separated by centrifugation. After coating the 96well plate at room temperature for 2 hours using the first capture ab (anti-mouse IL-17, IL-6, IFN-r and IL-10), blocking nonspecific reaction using blocking buffer I was. After the blocking process, the standard and the sample were put in a plate and washed with a wash buffer after the reaction for 2 hours. Standard (recombinant: IL-17, IL-6, IFN-r and IL-10) and the sample was added and washed for 2 hours after using the wash buffer. After washing, the detection ab (anti-mouse IL-17, IL-6, IFN-r, and IL-10) was added thereto, and then washed and developed for 2 hours.

As shown in FIG. 13, the expression of the inflammatory cytokines IL-17, IFN-r and IL-6 was reduced in the lactic acid mixture 2 (complex 2) and lactic acid mixture plus 2 (complex 2P) treatment groups as compared to the control group, It was confirmed that the anti-inflammatory cytokine IL-10 is increased. In addition, in the lactic acid bacteria mixture plus 2 (complex 2P) treatment group, it was confirmed that the expression control ability of the inflammatory or anti-inflammatory cytokines more significantly.

Example 9 Confirmation of Arthritis Treatment Effect of Lactic Acid Bacteria Mixture Plus 2 (complex 2P)

9-1. Arthritis Index, Incidence and Immunoglobulin Identification in Arthritis Animal Models

To determine arthritis score (%) and incidence (%) in arthritis animal models, normal DBA1 / J mice were injected with type II collagen 100ug / CFA (Complete Freund's Adjuvant) subcutaneously (Day) 2 weeks after induction, type II collagen 100ug / IFA (incomplete Freund's Adjuvant) was reinjected into the tail subcutaneously (Day 14). One week after arthritis induction, lactic acid bacteria mixture plus 2 (complex 2P) (lactic acid bacteria mixture 2 50 mg / kg, zinc 1 mg / kg, CoQ10 5 mg / kg) was orally administered daily. The arthritis index and incidence were then analyzed for 0-56 days.

In addition, in order to confirm the expression of immunoglobulin in the arthritis animal model, serum was separated from blood of each group 56 days after the onset of arthritis, and IgG, IgG1, and IgG2a immunoglobulins were analyzed from the separated serum by ELISA. After coating the 96-well plate for 2 hours using a first capture ab (anti-mouse IgG, IgG1, IgG2a) at room temperature, the non-specific reaction was blocked using a blocking buffer. After the bloicking process, the standard and the sample were put in a plate and washed with a wash buffer after 2 hours of reaction. Standard (recombinant: IgG, IgG1, IgG2a) and the sample was added and washed for 1 hour after using the wash buffer. After washing, detection ab (anti-mouse IgG, IgG1, IgG2a) was added, and after 1 hour of reaction, washing and color development were performed.

As shown in FIG. 14A, as a result of confirming the arthritis index and the incidence rate, it was confirmed that the arthritis index and the incidence rate significantly decreased over time in the lactic acid bacteria mixture plus 2 (complex 2P) treatment group compared to the control group. .

In addition, as shown in Figure 14b, it was confirmed that IgG, IgG1, IgG2a immunoglobulin significantly inhibited the expression in the lactic acid bacteria mixture plus 2 (complex 2P) treatment group of the present invention compared to the control.

Example 10 Confirmation of the Arthritis Destruction and Inhibition of Inflammatory Cells by Lactic Acid Bacteria Mixture Plus 2 in Complex Animal Model

On the 56th day after the arthritis induction of Example 9, the hind paws of each mouse group were fixed with 10% formalin, decalcified from bone, and blocks were made of paraffin. From this, a joint section (7um) was prepared and stained with hematoxylin and eosin. In addition, toluidin blue and safranin O were stained to confirm the degree of cartilage destruction. The degree of destruction in the joint tissue was confirmed. In addition, the degree of infiltration of inflammatory cells, the degree of bone damage and the degree of cartilage damage were analyzed.

As shown in Figure 15a, it was confirmed that the destruction in the joint tissue in the lactic acid bacteria mixture plus 2 (complex 2P) treatment group of the present invention is suppressed.

In addition, as shown in Figure 15b, compared with the control group, the infiltration of inflammatory cells in the lactic acid bacteria mixture plus 2 (complex 2P) treatment group is inhibited, confirmed that the degree of bone damage and cartilage damage significantly lower It was.

Example 11 Confirmation of Inflammatory Cytokine Inhibitory Effect of Lactobacillus Mixture Plus 2 (complex 2P) in Arthritis Animal Models

On day 56 after arthritis induction, the hind paws of each group of mice were fixed with 10% formalin, decalcified from bone and prepared with paraffin. From this, a joint section (7um) was prepared and inflammatory cytokine expression in the joint tissue was analyzed using IHC. Each cytokine expressing cell was analyzed with each antibody of TNF-α, IL-17, IL-6, IL-1β, and VEGF.

As shown in FIGS. 16A and 16B, it was confirmed that TNF-α, IL-17, IL-6, IL-1β, and VEGF inflammatory cytokine expressing cells were significantly reduced in the Lactobacillus mixture plus 2 (complex 2P) treatment group. .

Example 12 Confirmation of Simultaneous Regulation of Th17 and Treg Cells of Lactic Acid Bacteria Mixture Plus 2 (complex 2P)

Mouse spleen tissues were confirmed to co-regulate Th17 and Treg cells following Lactobacillus mixture plus 2 (complex 2P) treatment. Specifically, in order to confirm Th17 cell regulation ability, it was analyzed by CD4 FITC and IL-17 PE. In addition, Treg cell regulation was analyzed by CD4 Pe, Foxp3 FITC and CD25 APC. Thereafter, the expression levels of each Th17 cell and Treg cell were confirmed by confocal microscopy.

As shown in FIGS. 17A and 17B, it was confirmed that pSTAT3 705 cells were decreased, T17 cells were decreased, and Treg cells were increased in the lactic acid mixture plus 2 (complex 2P) treated spleen sections. Thus, it was confirmed that coexistence of Th17 / Treg cells was present.

Therefore, the lactic acid bacteria mixture 2 (complex 2) and the lactic acid bacteria mixture plus 2 (complex 2P) of the present invention inhibit the expression of inflammatory cytokines in vitro and increase the expression of anti-inflammatory cytokines, and thus have an immune enhancing effect. It was confirmed that there is. In particular, the Lactobacillus mixture plus 2 (complex 2P) significantly inhibits arthritis index, incidence and immunoglobulin expression in vivo and induces decreased inflammatory cell infiltration, reduced bone and cartilage damage. In addition, inflammatory cytokines are inhibited and Th17 / Treg cell co-regulation is present, suggesting that they are effective in the prevention and treatment of related immune diseases.

Preparation Example 3 Preparation of Lactic Acid Bacteria Mixture 3 (complex 3) and Lactic Acid Bacteria Mixture Plus 3 (Compplex 3P)

For the preparation of lactic acid bacteria mixture 3 (complex 3) and lactic acid bacteria mixture plus 3 (complex 3P), each of the lactic acid bacteria was purchased from CTC Bio. Specifically, lactic acid bacteria mixture 3 (complex 2) was configured to include the genus Lactobacillus (genus) as a fifth composition (three kinds of lactic acid bacteria) (Table 5).

In addition, lactic acid bacteria mixture plus 3 (complex 3P) was configured to include Rosavin (Rosavin) derived from zinc and Honggyeongcheon extract as a sixth composition in the lactic acid bacteria mixture 3 (complex 3) (Table 6).

Lactic acid bacteria mixture 3 (complex 3) Strain name ratio Article 5 composition (3 types of lactic acid bacteria)
Lactobacillus Acidophilus One Lactobacillus rhamnosus One Lactobacillus paracasei One

Lactobacillus Mixture Plus 3 (complex 3P) Strain Name / Component Article 5 composition (3 types of lactic acid bacteria) Lactobacillus Acidophilus 10ug / ml Lactobacillus rhamnosus 10ug / ml Lactobacillus paracasei 10ug / ml Article 6 Composition zinc 10 uM Rosabin from Honggyeongcheon extract 20ug / ml

Example 13. Confirmation of Inflammatory Cytokine Expression of Lactic Acid Bacteria Mixture 3 (complex 3) or Each Strain

Expression of IL-17 following Treatment with Lactic Acid Bacteria Mixture 3 and L. Acidophilus , L. rhamnosus or L. paracasei It was confirmed.

Specifically, lactic acid bacterium mixture 3 prepared in Preparation Example 3 (complex 3) and lactobacillus acidophyl under antiCD3 stimulation conditions (0.5 ug / ml) in healthy human peripheral blood mononuclear cells (huPBMC). Each of L. Acidophilus , L. rhamnosus or L. paracasei was treated with 10 ug / ml, respectively. After incubation for 3 days, ELISA was performed using the supernatant separated by centrifugation. After coating the 96-well plate for 2 hours at room temperature using the first capture ab (anti-mouse IL-17), the non-specific reaction was blocked using a blocking buffer. After the blocking process, the standard and the sample were put in a plate and washed with a wash buffer after the reaction for 2 hours. Standard (recombinant: anti-mouse IL-17) and the sample was added and washed for 2 hours after using the wash buffer. After washing, the detection ab (anti-mouse IL-17) was added, and after 2 hours, the reaction was washed and developed.

As shown in FIG. 18, it was confirmed that the lactic acid mixture 3 (complex 3) significantly inhibited the inflammatory cytokine IL-17 as compared to the individual strains constituting the lactic acid bacteria mixture 3 (complex 3).

Example 14. Confirmation of Anti-Inflammatory Cytokine Expression of Lactic Acid Bacteria Mixture 3 and Complex 3P

In order to confirm anti-inflammatory cytokine expression in healthy human peripheral blood mononuclear cells and mouse splenocytes, human peripheral blood mononuclear cells were prepared under the conditions of Example 11 above. In addition, spleen cells of mice were sacrificed to normal mice (c57bl / 6, male, 9 weeks old, obtained from Orient Bio) to obtain a spleen. The spleens were changed using a slide, and then splenocytes were obtained after removing RBC using an ACK buffer.

10 ug / ml of lactic acid bacterium mixture 3 (complex 3) and lactic acid bacteria mixture plus 3 (complex 3P) lactic acid bacteria, respectively, at the same time as the anti-CD3 0.05ug / ml stimulation alone conditions in human peripheral blood mononuclear cells and mouse splenocytes After treatment with / ml, Rosavin 20ug / ml, zinc 10uM sup (supernatant) was obtained after 3 days of incubation. ELISA was performed on IL-10 at the sup obtained.

As shown in FIG. 19, it was confirmed that the anti-inflammatory cytokine IL-10 was increased in the lactic acid mixture 3 (complex 3) and lactic acid mixture plus 3 (complex 3P) treatment groups compared to the control group. In particular, it was confirmed that IL-10 expression was significantly increased in the lactic acid bacteria mixture plus 3 (complex 3P) treatment group in both cells.

Example 15 Inhibitory Effect of Th17 and Th1 Cells on Lactic Acid Bacteria Mixture 3 and Complex 3P

Peripheral blood mononuclear cells were isolated from healthy human blood and cultured by treating Lactobacillus Mixture 3 (complex 3) and Lactobacillus Mixture Plus 3 (complex 3P), respectively, under Th17 or Th1 differentiation conditions. Thereafter, flow cytometry analysis was performed to identify Th17 and Th1 cells.

As shown in FIG. 20, it was confirmed that Th17 and Th1 cells were reduced in the lactic acid bacteria mixture plus 3 (complex 3P) treatment group. In addition, it was confirmed that IL-17 and IFN-r induced in each cell were also inhibited.

Example 16. Treg and Th2 Cell Increasing Effects of Lactic Acid Bacteria Mixture 3 and Complex 3P

CD4 + T cells were isolated from healthy human peripheral blood mononuclear cells and cultured by treating Lactobacillus Mixture 3 (complex 3) and Lactobacillus Mixture Plus 3 (complex 3P), respectively, under differentiation conditions of Treg and Th2 cells. Thereafter, flow cytometry analysis was performed to identify Treg and Th2 cells.

As shown in FIG. 21, it was confirmed that Treg and Th2 cells were increased in the lactic acid bacteria mixture plus 3 (complex 3P) treatment group.

Example 17 Confirmation of Th17 and Treg Regulatory Effects of Lactic Acid Bacteria Mixture 3 and Complex 3P

Mouse spleen cells were stimulated with 0.5 ug / ml of antiCD3, and cultured with 10 lug / ml of lactic acid bacteria mixture 3 (complex 3) and lactic acid bacteria mixture plus 3 (complex 3P), 20 vin / ml Rosavin, and 10 uM zinc, respectively. Thereafter, flow cytometry analysis was performed to identify Th17 or Treg cells.

As shown in FIG. 22, in the lactic acid bacteria mixture plus 3 (complex 3P) treatment group, Th17 cells were decreased and Treg cells were increased, indicating that Th17 / Treg could be controlled simultaneously.

Example 18 Effect of Osteoarthritis (Osteoarthritis) Treatment

18-1. Osteoarthritis Treatment Effect Using Osteoarthritis Animal Model

Monosodium Iodoacetate (MIA, I2512, Sigma, Poole, UK) was dissolved at 60 mg / ml in saline for injection and prepared on the day of the experiment (day 0). Respiratory anesthesia was performed using Isoflurane in rats (Wistar rat, 6 weeks old, 180g ~ 220g) at the start of experiment, and then MIA was inserted into right knee joint using 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 lactobacillus mixture 3 (complex 3) and the lactobacillus mixture plus 3 (complex 3P) are each 125 mg / rat (Lactobacillus ecidophilus) + 125 mg / rat (Lactobacillus rhamnosus) + 125 mg / rat, per rat, from the day after MIA induction. rat (Lactobacillus paracasei) + was administered orally.

For measuring pain in rats, a dynamic planter esthesiometer (UgoBasile, Comerio, Itaily) was used. Place the stimulator on the bottom of the rat, adjust the 0.5mm thick plastic stimulating microfilament to the side of the hind limb, and operate the machine.The stimulation needle will gradually press the force while pressing the posterior metastasis at a constant speed and force. Increasing the time (sec) and the force applied (g) until the rat was unable to withstand the stimulus and released the foot was measured. Each measurement was carried out three times and averaged. The result was shown in FIG. 5 (A, B of FIG. 23). In addition, an incapacitance meter (IITC, Victory Blvd Woodland Hills, CA, USA) was used to measure the weight bearing of the rat, and the results are shown in FIG. 5 (FIG. 23C).

As shown in Figure 23, both pain and weight bearing can be seen to show an excellent osteoarthritis treatment effect compared to the control group in both lactic acid mixture 3 (complex 3) and lactic acid bacteria mixture plus 3 (complex 3P) treatment group, in particular, lactic acid bacteria mixture plus More effective was observed in group 3 (complex 3P).

18-2. Control of cartilage destruction factor in animal models of osteoarthritis

Expression of cartilage tissue destruction factors MMP1 and MMP3 was confirmed in the joint tissue of the animal model of osteoarthritis 18-1. Specifically, the animal model of MIA-induced osteoarthritis was used as the method of 16-1. Lactobacillus Mixture 3 (complex 3) and Lactobacillus Mixture Plus 3 (complex 3P) were orally administered 500 mg per rat daily from the day after MIA-induction. Thereafter, mRNA expression of MMP1 and MMP3 of each treatment group was confirmed by real time PCR.

As shown in Figure 24, in the lactic acid bacteria mixture plus 3 (complex 3P) treatment group of the present invention it was confirmed that the mRNA expression of MMP1 and MMP3 is significantly significantly inhibited to control the cartilage destruction factors associated with catabolism to protect joints It was.

Thus, the lactic acid bacterium mixture 3 and the complex 3P of the present invention also increased Treg and Th2 cells and inhibited Th17 and Th1 cells in vitro compared to the strain of the genus Lactobacillus alone. It was confirmed that the expression of inflammatory cytokines is suppressed and the expression of anti-inflammatory cytokines is increased. In addition, there is a co-regulation of Th17 and Tregs. In particular, Lactic Acid Bacteria Mixture Plus 3 (complex 3P) has a pain relief inhibitory and cartilage destruction factor control effect in vivo, suggesting that it is effective in the prevention and treatment of related immune diseases.

Claims (16)

First composition comprising Lactobacillus Acidophilus ( Lactobacillus Acidophilus ); And Lactobacillus know even Phil Ruth (Lactobacillus Acidophilus), Lactobacillus casei (Lactobacillus casei), Lactobacillus Planta Room (Lactobacillus plantarum), Lactobacillus ramno Seuss (Lactobacillus rhamnosus), Lactobacillus helveticus (Lactobacillus Helveticus), Lactobacillus Pere lactofermentum (Lactobacillus fermentum), Lactobacillus para casei (Lactobacillus paracasei), Lactobacillus salivarius (Lactobacillus salivarius), Bifidobacterium breather bracket (Bifidobacterium breve), Bifidobacterium Bifidobacterium Doom (Bifidobacterium bifidum), A pharmaceutical composition for the prevention or treatment of immune diseases, comprising a second composition comprising Bifidobacterium longum and Streptococcous thermophilus as an active ingredient,
The immune disease is a osteoarthritis, rheumatoid arthritis, ankylosing spondylitis and inflammatory bowel disease characterized in that the pharmaceutical composition for the prevention or treatment of immune diseases. The method of claim 1,
The composition comprises a third composition comprising zinc oxide, copper (copper sulfate), chicory ( Cicchorium intybu s) extract, licorice (Genus Glycyrrhiza) extract, black rice Bran extract, and Rosavin derived from Hong Kyungcheon extract Pharmaceutical composition for the prevention or treatment of immune diseases, further comprising. delete delete delete delete delete delete delete delete The method according to claim 1 or 2,
The composition inhibits the expression of inflammatory cytokines, pharmaceutical compositions for the prevention or treatment of immune diseases, characterized in that to increase the expression of anti-inflammatory cytokines. The method according to claim 1 or 2,
The composition of the present invention inhibits the expression of matrix metalloproteinase 1 (MMP1) and matrix metalloproteinase 3 (MMP3), which are cartilage tissue destruction factors, and prevents immune diseases, which are characterized by increasing the expression of cartilage regeneration factor TIMP3 (Metalloproteinase inhibitor 3). Or therapeutic pharmaceutical compositions. The method according to claim 1 or 2,
The composition is a pharmaceutical composition for the prevention or treatment of immune diseases, characterized in that to simultaneously control Th17 and Treg cells. First composition comprising Lactobacillus Acidophilus ( Lactobacillus Acidophilus ); And Lactobacillus know even Phil Ruth (Lactobacillus Acidophilus), Lactobacillus casei (Lactobacillus casei), Lactobacillus Planta Room (Lactobacillus plantarum), Lactobacillus ramno Seuss (Lactobacillus rhamnosus), Lactobacillus helveticus (Lactobacillus Helveticus), Lactobacillus Pere lactofermentum (Lactobacillus fermentum), Lactobacillus para casei (Lactobacillus paracasei), Lactobacillus salivarius (Lactobacillus salivarius), Bifidobacterium breather bracket (Bifidobacterium breve), Bifidobacterium Bifidobacterium Doom (Bifidobacterium bifidum), A health functional food composition for preventing or ameliorating immune diseases comprising a second composition comprising Bifidobacterium longum and Streptococcous thermophilus as an active ingredient,
The immune disease is osteoarthritis, rheumatoid arthritis, ankylosing spondylitis and inflammatory bowel disease, characterized in that the health functional food composition for preventing or improving immune disease, characterized in that selected from the group consisting of. The method of claim 14,
The composition comprises a third composition comprising zinc oxide, copper (copper sulfate), chicory ( Cicchorium intybu s) extract, licorice (Genus Glycyrrhiza) extract, black rice Bran extract, and Rosavin derived from Hong Kyungcheon extract Health functional food composition for the prevention or improvement of immune diseases, further comprising.
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