KR101688224B1 - ID-CBT5101 having effect of preventing and treating arthritis and uses thereof - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the treatment and prevention of arthritis, and more particularly to a pharmaceutical composition comprising a biomarker of joint inflammation COX-2 and an inflammatory cytokine such as tumor necrosis factor-alpha (TNF- alpha) to prevent arthritis and alleviate the symptoms, and to a composition for preventing and treating arthritis, which comprises as an active ingredient, a fungus body of Clostridium butyricum IDCC 5101. The clostridium butyricum cells according to the present invention were found to be effective in preventing and treating arthritis by alleviating arthritic symptoms in a concentration-dependent manner in an animal model of arthritis.

Description

ID-CBT5101 < / RTI > having an arthritis prevention and treatment effect and an application thereof (ID-CBT5101)

The present invention relates to the use of Clostridium butyricum Tyndallizate (ID-CBT5101) and its use for preventing and treating arthritis. More specifically, the present invention relates to a biomarker of arthritis, COX-2 and inflammatory cytokine A composition for preventing and treating arthritis comprising tinctured Clostridium butyricum IDCC 5101 dead cells and an effective ingredient thereof, which contributes to prevent arthritis and alleviate symptoms by lowering the level of tumor necrosis factor-α (TNF-α) .

Rheumatoid arthritis is the most common inflammatory arthritis associated with synovial hyperplasia of the joints and bone and cartilage destruction is a symptom. Although the pathogenesis of rheumatoid arthritis is not clear, there are many reports that cyclin expression in synovial membrane models or synovial membranes in rheumatoid arthritis patients has been reported, and that inhibition of these cytokines prevents arthritis symptoms, physical findings, and joint destruction Cytokines are thought to play an important role in the onset and persistence of the disease. The pathogenesis of rheumatoid arthritis is unclear and various models have been proposed. The major mechanisms of rheumatoid arthritis are antigen - specific and antigen - nonspecific. In the case of antigen-specific mechanism, the causative T cell antigen is not known yet, but it is presumed that it is caused by virus or bacteria. Antigen presenting cell (APC) is a class II major histochmpatibility complex, MHC) to provide T cells with an arthritogenic peptide to activate T cells and to stimulate macrophages by activating T cells cross-reacting with self-antigens, It starts.

The development of arthritis of the antigen-nonspecific mechanism is accompanied by tumor necrosis factor-α (TNF-α) produced from macrophages activated and activated by synovial macrophages by nonspecific stimulation such as repeated infections, tissue damage, allergies, And granulocyte-macrophage colony-stimulating factor (GM-CSF) induced differentiation of dendritic cells. Differentiated dendritic cells function as powerful antigen-presenting cells and transmit autoantigen to autoreactive T cells T cells are stimulated and arthritis begins. Cytokines produced by synovial membrane after arthritis induction and acting as paracrine or autocrine are known to be involved in the persistence and aggravation of chronic joint inflammation. The expression of cytokines such as TNF-α, IL-1β, IL-6, GM-CSF and IL-8 has been demonstrated in rheumatoid arthritis and synovial fluid. β and IL-1 receptor antagonist (IL-1Ra) and soluble TNFR, but the production of these inflammatory regulators is not sufficient to neutralize the action of inflammatory cytokines . Therefore, it can be assumed that rheumatoid arthritis will persist and worsen due to the imbalance in the production of inflammatory substances including inflammatory cytokines and antiinflammatory substances. There have been efforts to treat and ameliorate the inflammatory autoimmune disease, rheumatoid arthritis, through intestinal immunization, and there have been studies using probiotics such as lactic acid bacteria to control the immune system.

In a mouse model of rheumatoid arthritis, consumption of Lactobacillus casei was effective in inhibiting arthritis (Life Science, 1998; 63 (8): 635-644) and in an animal model of arthritis using Lewis rat, It has been reported that ingestion of Bacillus rhamnosus GG is effective in inhibiting arthritis (Journal of Nutrition, 2004; 134 (8); 1964-1969). In previous studies, it has been known that the efficacy of lactic acid bacteria after ingestion of live cells is that a certain number of live bacteria should be attached to the intestines to maintain immunostimulation at a certain level. However, lactic acid bacteria are killed by stomach acid and bile acid attack while passing through the gastrointestinal tract, and tend to be below the concentration that can stimulate the intestinal immune system. Therefore, overdose of lactic acid bacteria in accordance with the focus on the ability to adhere lowers the economic efficiency.

In addition, most of the patients with rheumatoid arthritis have a problem that the administration of NSAID-based anti-inflammatory drugs or antibiotics in order to reduce the pain response due to arthritis is limited to achieve the desired purpose because the drug is killed by the combination of lactic acid bacteria and live bacteria .

Accordingly, the inventors of the present invention have completed a tyndallization method capable of maximizing the anti-inflammatory action of Clostridium butyricum IDCC 5101 in order to solve the administration problems of the above-mentioned prior studies, The inventors of the present invention have completed the present invention by confirming that the dead cells of tidium butyricum IDCC 5101 exhibit arthritis-mitigating effect in a concentration-dependent manner in an animal model test of rheumatoid arthritis.

Accordingly, an object of the present invention is to provide Clostridium butyricum IDCC 5101 strain (accession number: KCTC12738BP) having an arthritis preventive and therapeutic effect.

Another object of the present invention is to provide a composition for preventing and treating arthritis, which comprises the strain and a culture solution of the strain as an active ingredient.

Another object of the present invention is to provide a bacterium of T. lucidium butyricum IDCC 5101 (accession number: KCTC12738BP) having an arthritis preventive and therapeutic effect.

Another object of the present invention is to provide a composition for the prevention and treatment of arthritis which comprises the microbial cells as an active ingredient.

Another object of the present invention is to provide a composition for preventing and treating arthritis, comprising as active ingredients Clostridium butyricum IDCC 5101 strain (Accession No .: KCTC12738BP) and a culture solution of the strain.

Another object of the present invention is to provide a method for culturing Clostridium butyricum strain, comprising: (a) anaerobically culturing Clostridium butyricum strain IDCC 5101; (B) culturing the cells and culture medium at a temperature of from 111 to 150 DEG C at a pressure of 0.6 to 1.3 kgf / cm < ² > to obtain an anti-arthritic anti-arthritic effect. And a method for producing the microorganism.

In order to solve the above-mentioned problems, the present invention provides Clostridium butyricum IDCC 5101 strain (accession number: KCTC12738BP) having an arthritis prevention and treatment effect.

In order to accomplish another object of the present invention, the present invention provides a composition for preventing and treating arthritis, comprising the strain and the culture medium of the strain as an active ingredient.

In order to accomplish another object of the present invention, the present invention provides a bacterium of Tincta clostridium butyricum IDCC 5101 (accession number: KCTC12738BP) having an arthritis preventive and therapeutic effect.

In order to accomplish another object of the present invention, the present invention provides a composition for preventing and treating arthritis comprising the microbial cells as an active ingredient.

In order to accomplish another object of the present invention, there is provided a composition for preventing and treating arthritis, comprising as active ingredients Clostridium butyricum IDCC 5101 strain (accession number: KCTC12738BP) and a culture solution of said strain.

Another object of the present invention is to provide a method for culturing Clostridium butyricum strain, comprising: (a) anaerobically culturing Clostridium butyricum strain IDCC 5101; (B) culturing the cells and culture medium at a temperature of from 111 to 150 DEG C at a pressure of 0.6 to 1.3 kgf / cm < ² > to obtain an anti-arthritic anti-arthritic effect. A method for producing a microorganism is provided.

Hereinafter, the present invention will be described in detail.

The present invention provides Clostridium butyricum IDCC 5101 (accession number: KCTC12738BP) having an arthritis preventive and therapeutic effect.

The present invention also provides a composition for preventing and treating arthritis, which comprises the above-mentioned strain and a culture solution of the strain as an active ingredient.

In the present invention, the composition may include any type of composition including Clostridium butyricum IDCC 5101 (accession number: KCTC12738BP), and preferably the composition may be a food composition or a pharmaceutical composition, no.

 When the composition is a food composition, it may be added as it is or may be used together with other food or food ingredients, and may be suitably used according to conventional methods.

As used herein, the term " food " refers to any product, including but not limited to meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, ice creams, Beverages, and vitamin complexes, all of which include foods in a conventional sense.

A health functional food may be included in the food composition of the present invention. The term "health functional food " as used in the present invention refers to a food prepared and processed in the form of tablets, capsules, powders, granules, liquids and rings using raw materials and components having useful functions in the human body. Here, 'functional' refers to the structure and function of the human body to obtain nutritional effects and obtain useful effects for health use such as physiological action. The health functional food of the present invention can be prepared by a method commonly used in the art and can be prepared by adding raw materials and ingredients that are conventionally added in the art.

In addition, the formulations of the above health functional foods may also be manufactured without limitations as long as they are acceptable as health functional foods. The composition for food of the present invention can be manufactured in various forms, and unlike general pharmaceuticals, it has the advantage that there is no side effect that may occur when a drug is used for a long period of time, and is excellent in portability, Can be ingested as an adjunct to improve the effectiveness of arthritis treatment.

In addition, the food composition of the present invention can be used as a food composition containing various nutrients, vitamins, electrolytes, flavors, colorants, pectic acids and salts thereof, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, , A carbonating agent used in carbonated drinks, and the like. It may also contain flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components may be used independently or in combination. Although the ratio of such additives is not critical, it is generally, but not exclusively, selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the food composition of the present invention.

In addition, the food composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. The carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. The ratio of the natural carbohydrate may be generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 mL of the composition of the present invention, but is not limited thereto.

The amount of Clostridium butyricum IDCC 5101 in the food composition of the present invention can be suitably determined according to its intended use (prevention, health or therapeutic treatment).

Generally, the Clostridium butyricum IDCC 5101 of the present invention is used in the production of food or beverage in an amount of 1 x 10 ⁸ to 1 x 10 ¹⁰ (bacterium) of the present invention per 1 g (or ml) And is preferably added in an amount of 5 x 10 ⁸ to 5 x 10 ⁹ . In addition, the daily intake of the food composition of the present invention is 1 × 10 ⁵ to 1 × 10 ¹⁰ CFU / kg, preferably 1 × 10 ⁶ to 1 × 10 ⁸ CFU / kg, , Ingestion may be ingested once a day or divided into several doses, but is not limited thereto.

The effective dose of the strain in the food composition of the present invention can be used in accordance with the effective dose of the pharmaceutical composition, but may be less than the above range for health and hygiene purposes or long-term intake for health control purposes, It is clear that the component can be used in an amount of more than the above range since there is no problem in terms of safety.

In the present invention, when the composition is a pharmaceutical composition, it may further include pharmaceutically acceptable additives in addition to Clostridium butyricum IDCC5101. Examples of the pharmaceutically acceptable additives include starch, gelatinized starch, microcrystalline cellulose, lactose , Corn starch, powdered cellulose, hydroxypropylcellulose, opaques, sodium starch glycolate, carnauba wax, synthetic starch, corn starch, calcium phosphate, calcium hydrogen phosphate, lactose, mannitol, Aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, white sugar, dextrose, sorbitol and talc may be used. The pharmaceutically acceptable additives according to the present invention are preferably included in the composition in an amount of 0.1 to 90 parts by weight, but are not limited thereto.

In addition, the composition of the present invention can be administered in various formulations of oral and parenteral administration at the time of actual clinical administration. In the case of formulation, a diluent such as a filler, an extender, a binder, a wetting agent, a disintegrant, . ≪ / RTI >

Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, lactose Or gelatin. In addition to simple excipients, lubricants such as magnesium stearate talc may also be used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions and syrups, and various excipients such as wetting agents, sweetening agents, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are commonly used simple diluents .

Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the non-aqueous solvent and the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

On the other hand, injecting agents may include conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, preservatives and the like.

In addition, the therapeutic compositions of the present invention may be formulated with any physiologically acceptable carrier, excipient or stabilizer (Remington: The Science and Practice of Pharmacy, l9th Edition, Alfonso, R., ed., Mack Publishing Co. : 1995)). Acceptable carriers, excipients or stabilizers are nontoxic to the recipient at the dosages and concentrations employed and include buffer solutions such as phosphoric acid, citric acid and other organic acids; Antioxidants including ascorbic acid; Low molecular weight (less than about 10 residues) polypeptides; Proteins, such as serum albumin, gelatin or immunoglobulins; Hydrophilic polymers such as polyvinylpyrrolidone; Amino acids such as glycine, glutamine, asparagine, arginine or lysine; Monosaccharides, disaccharides, and other carbohydrates including glucose, mannose or dextrin; Chelating agents such as EDTA; Sugar alcohols such as mannitol or sorbitol; Salt-forming counterions such as sodium; And / or non-ionic surfactants such as tween, pluronics or polyethylene glycol (PEG).

 The dosage of the pharmaceutical composition of the present invention to the human body may vary depending on the patient's age, body weight, sex, dosage form, health condition and disease severity, and is generally 0.01 to 100 mg / kg / day, 0.1 to 50 mg / kg / day, and more preferably 5 to 10 mg / kg / day. It may also be administered at a fixed interval according to the judgment of a doctor or pharmacist.

In the present invention, the arthritic condition includes osteoarthritis, rheumatoid arthritis, lupus arthritis, reactive arthritis, psoriatic arthritis and the like, preferably rheumatoid arthritis or degenerative arthritis. But is not limited thereto.

The present invention also provides a bacterium of tincta clostridium butyricum IDCC 5101 (accession number: KCTC12738BP) having an arthritis preventive and therapeutic effect.

The most commonly used method in the sterilization process is sterilization at 121 ° C for 15 minutes at high temperature and high pressure. However, a method that can be used to sterilize a subject that can not withstand sterilization under high-temperature and high-pressure conditions is the tin-meal sterilization method. As used herein, the term " tin metarization " means a condition in which the bacteria have been killed by the tin-meal sterilization method commonly used in the art.

The inventors of the present invention have found that when trastamylating Clostridium butyricum, excellent arthritis preventive and therapeutic effects are exhibited, and since the extractability is different from the cells of the substance exhibiting the anti-inflammatory effect according to the tincture conditions, The antitumor effect was optimized by evaluating the antinflammatory effects of the thymidylation temperature condition and the pressurization condition in order to produce dead cells of T. dolomatis Clostridium butyricum IDCC 5101 which was optimized for prevention and treatment.

That is, according to one embodiment of the present invention, the present inventors maintained the temperature of the crystallization temperature at 90 ° C, 100 ° C, 110 ° C and 121 ° C respectively for 10 minutes, cooled again to 50 ° C, The mixture was allowed to stand for 5 minutes under pressure, followed by cooling to 40 DEG C to obtain trimellitium butyricum (Example 2).

According to another embodiment of the present invention, the anti-inflammatory effect of tinctured Clostridium butyricum at the respective temperature conditions was evaluated through COX-2 inhibition. As a result, it was found that Clostridium butyricum, The effect was most excellent (Example 3). The cells that had been killed by clathridinium butyricum at 90 ° C, 100 ° C, and 110 ° C were not excellent in COX-2 inhibition as compared to Clostridium butyricum cells that had been tinctured at 121 ° C. This is probably due to the formation of favorable conditions in which Clostridium butyricum is fragmented and becomes smaller in size and sterilized under conditions of high pressure and high temperature, and a substance exhibiting an anti-inflammatory effect can be reacted.

 As a result of evaluating the influences of inflammatory cytokine and anti-inflammatory cytokine secretion of macrophages of Clostridium butyricum IDCC 5101 cells cultured at 121 ° C, it was found that the tinctured Clostridium butyricum IDCC 5101 was a positive control substance It was confirmed that it acts to directly inhibit the secretion of inflammatory cytokine TNF-a as in dexamethasone (Example 4). In addition, the administration of the tinctured Clostridium butyricum IDCC 5101 cells to the arthritis animal model for 4 weeks showed that the arthritic symptoms were significantly alleviated from the 6th day after arthritis induction, and the positive control (Example 5), which was 1.5 times more effective than the green lipped mussel extract-treated group. In addition, the hind paws of the experimental animals were evaluated for joint pathology. As a result, the synovial membrane proliferation and infiltration of inflammatory cells were markedly decreased in the group treated with the trinitized Clostridium butyricum IDCC 5101 cells It was confirmed that the effect of alleviating the symptoms of arthritis was excellent (Example 5).

In the present invention, the vitrification means that the bacteria are killed at a temperature of 111 to 150 ° C, preferably 111 to 150 ° C, 112 to 150 ° C, 113 to 150 ° C, 114 to 150 ° C, 117-150 ° C, 118-150 ° C, 119-150 ° C, 111-145 ° C, 112-145 ° C, 113-145 ° C, 114-145 ° C, 115-145 ° C, 116-145 ° C, 117-145 ° C, 114-140 ° C, 115-140 ° C, 116-140 ° C, 117-140 ° C, 118-140 ° C, 118-145 ° C, 119-145 ° C, 111-140 ° C, 112-140 ° C, 113-140 ° C, 119-140 ° C, 111-135 ° C, 112-135 ° C, 113-135 ° C, 114-135 ° C, 115-135 ° C, 116-135 ° C, 117-135 ° C, 118-135 ° C, 111-130 ° C, 112-130 ° C, 113-130 ° C, 114-130 ° C, 115-130 ° C, 116-130 ° C, 117-130 ° C, 118-130 ° C, The cells are autoclaved at 125 ° C, 112 to 125 ° C, 113 to 125 ° C, 114 to 125 ° C, 115 to 125 ° C, 116 to 125 ° C or 117 to 125 ° C, and most preferably 118 to 125 ° C, But is not limited thereto.

On the other hand, when the temperature of the deguming is less than 111 DEG C, the flaky cells are not sufficiently segmented to form a favorable condition for exhibiting the anti-inflammatory effect, and when the temperature is above 150 DEG C, Since the active ingredient of the cells does not exhibit an anti-inflammatory effect due to denaturation, the setting of the temperature condition of thymolization is very important in the production of Clostridium butyricum ICDD 5101 dead cells showing anti-inflammatory effect.

In addition, in the present invention, the tin metarization means that bacteria are killed under the pressurization condition, and the pressurization means that additional pressure is applied based on the atmospheric pressure of 1 atm. That is, the pressurization condition in the present invention means that a pressure of 0.1 to 1.3 kgf / cm ² is additionally applied based on the atmospheric pressure of 1 atm, preferably 0.2 to 1.3 kgf / cm ² , 0.3 ^{~ 1.3 kgf / cm 2, 0.4} ~ 1.3 kgf / cm 2, 0.5 ~ 1.3 kgf / cm 2, 0.6 ~ 1.3 kgf / cm 2, 0.7 ~ 1.3 kgf / cm 2, 0.8 ~ 1.3 kgf / cm 2, 0.2 ~ 1.2 ^{kgf / cm 2, 0.3 ~ 1.2} kgf / cm 2, 0.4 ~ 1.2 kgf / cm 2, 0.5 ~ 1.2 kgf / cm 2, 0.6 ~ 1.2 kgf / cm 2, 0.7 ~ 1.2 kgf / cm 2, or 0.8 ~ 1.2 kgf / cm < ² >, and most preferably 0.9 to 1.1 kgf / cm < ² >, but not limited thereto.

On the other hand, when the thymolization pressure is less than 0.1 kgf / cm < ² >, the flaky cells are not sufficiently segmented to provide a favorable condition for exhibiting the anti-inflammatory effect, ² , the active ingredients of the dead cells do not exhibit anti-inflammatory effects due to the denaturation of the active ingredients of the dead cells. Therefore, in the case of preparing clostridium butyricum ICDD 5101 dead cells showing the anti- It is very important.

In the present invention, the cell suspension of Clostridium difficile IDCC 5101 may be characterized in that it inhibits the expression of tumor necrosis factor-alpha (TNF-alpha) in macrophages, thereby having the effect of preventing and treating arthritis have.

Arthritis is a typical chronic inflammatory disease, which is caused by a number of cytokines in the body. Inflammatory reactions are caused by tumor necrosis factor-α (TNF-α). TNF-α is a pro-inflammatory cytokine that plays an important role in inflammatory diseases such as rheumatoid arthritis, psoriatic arthritis, Crohn's disease, psoriasis, and ankylosing spondylitis.

Clinically, overproduced TNF-α produces inflammatory mediators that stimulate macrophages in arthritic patients to amplify the inflammatory response and cause adhesion molecules to be expressed in vascular endothelial cells. And fibroblasts produce proteolytic enzymes that damage cartilage, bones, ligaments, and aggravate the disease. In addition, TNF-α increases the expression of adhesion molecules such as vascular cellular adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1) and E-selectin in vascular endothelial cells, TNF-α, TNF-α, and TNF-α in the synovial tissue of rheumatoid arthritis patients treated with anti-TNF-α antibody, the number of T lymphocytes decreased and the expression of VCAM- It is known that alpha plays an important role. In addition, TNF-α promotes the production of PG by increasing expression of secretory phospholipase A2 (sPLA2), cyclooxygenase-2 (COX-2) or microsomal PGE2 synthase (mPGES) involved in the production of prostaglandin It promotes the expression of inducible NO synthase (iNOS), an enzyme involved in the production of nitric oxide (NO), an inflammatory substance. In addition, TNF-α acts on synovial fibroblasts to promote cell proliferation, leading to synovial hyperplasia and fibrosis.

As described above, inhibiting the expression of TNF-a may be a key to the treatment strategy of arthritis. The inventors of the present invention have found that the bacterium of Clostridium butyricum IDCC 5101 inhibits the expression of TNF-a in macrophages (Example 4). It was confirmed that the compound of the present invention can be effectively used for the treatment of arthritis.

Meanwhile, Clostridium butyricum IDCC 5101 according to the present invention did not show a significant effect on IL-6 and IL-10 production ability of macrophages (results not shown), and inhibition of TNF- Of the patients.

Accordingly, the present invention provides a composition for the prevention and treatment of arthritis comprising the microbial cells as an active ingredient, and the composition of the present invention is as described above.

The present invention also provides a composition for preventing and treating arthritis, comprising as active ingredients Clostridium butyricum IDCC 5101 strain (Accession No. KCTC12738BP) and a culture solution of the strain.

The present composition has been described above.

In the present invention, the culture medium may be a culture medium itself in which Clostridium butyricum IDCC 5101 according to the present invention is cultured in a suitable culture medium, a filtrate (filtrate or centrifuged supernatant) from which the culture has been removed by filtration or centrifugation, A cell lysate obtained by ultrasonication of the culture broth or a lysozyme treatment of the culture broth, and preferably a supernatant after centrifugation, but is not limited thereto. In addition, the culture liquid may include both the concentrated liquid of the culture liquid and the dried material of the culture liquid.

The present invention also provides a composition, characterized in that the dead cells are vitellated.

The present invention also provides a composition characterized in that the vitrification is carried out by pressurization at 111 to 150 DEG C and 0.6 to 1.3 kgf / cm < ² >

The present invention also provides a method for producing a recombinant vector comprising: (a) anaerobically culturing Clostridium butyricum strain IDCC 5101; (B) culturing the cells and culture medium at a temperature of from 111 to 150 DEG C at a pressure of 0.6 to 1.3 kgf / cm < ² > to obtain an anti-arthritic anti-arthritic effect. A method for producing a microorganism is provided.

Preferably, the bacterium preparation method of the step (b) is maintained at a temperature of 115 to 150 ° C. and 0.1 to 1.3 kgf / cm ² for 7 to 15 minutes, further cooled to 45 to 55 ° C., And keeping the mixture at a temperature of 35 to 45 DEG C for 3 to 7 minutes under pressure, thereby clustridium butyricum is vitrified.

The method of the present invention may be performed by isolating Clostridium butyricum IDCC 5101 from the culture broth to thereby solubilize only the bacterial cells. The culture medium containing the strain may be transformed into a biotinylated strain to kill the strain.

Preferably, the culture medium obtained by culturing Clostridium butyricum IDCC 5101 after the step (a) is centrifuged to separate the cells and the culture medium, a step of concentrating the culture to prepare a concentrate, and a step of preliminarily separating the concentrate The method may further comprise mixing one clostridium butyricum IDCC 5101 cell.

The tinctured Clostridium butyricum cells of the present invention can be used not only to lower the level of tumor necrosis factor-alpha (TNF-alpha), which is a biomarker of arthritis, COX-2 and inflammatory cytokine, Arthritis symptoms are alleviated in an animal model of arthritis which has received dihydrobutyricum IDCC 5101 cells.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a photograph showing a raw material of Tinarylated Clostridium butyricum IDCC 5101 with arthritis prevention and treatment efficacy.
FIG. 2 shows the results of a western blotting (T121: 121 ° C, 1.0 kgf / cm ² , T110: 110 ° C., 0.6 kgf / cm 2) inhibition effect of the inflammatory protein COX- tin in ² dalhwa, T100: 100 ℃, 0.1 tin in kgf / cm ² dalhwa, T90: tin dalhwa at ^{90 ℃, 0 kgf / cm 2} ).
Figure 3 shows the results of the effect of Tinadal Clostridium butyricum IDCC 5101 on TNF-a secretion as an inflammatory cytokine.
FIG. 4 shows the effect of cynthroid trinidium butyricum IDCC 5101 on IL-10 secretion, an anti-inflammatory cytokine.
FIG. 5 shows the results of alleviating arthritic symptoms in an animal model of arthritis of clindamycin tseud. Dalium butyricum IDCC 5101.
FIG. 6 shows the results of histopathological analysis of the inflammation-improving effect in an arthritic animal model administered with cynomolgus clostridium butyricum IDCC 5101. FIG.

Hereinafter, the present invention will be described in detail.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

≪ Example 1 >

Selection of a strain having excellent anti-inflammatory activity

The present inventors confirmed the anti-inflammatory activity of 50 immune-related probiotics possessed by Ildong Pharmaceutical in order to select strains having excellent anti-inflammatory effect against rheumatoid arthritis. The anti-inflammatory activity of Raw cell 264.7 cells was determined by COX-2 inhibition by treating live bacteria and Tindal samples of each probiotics. The COX-2 inhibitory effect of each sample was confirmed by western blotting. Among the 50 species of probiotic live bacteria and 50 kinds of tindahal, the highest anti-inflammatory effect was selected and finally T. dolindii butyricum IDCC 5101 was selected.

As a result, Clostridium butyricum IDCC 5101 showed the best anti-inflammatory activity, and deposited it in the Korean Microorganism Resource Center (Accession No .: KCTC12738BP).

≪ Example 2 >

Clostridium butyricum excellent anti-inflammatory effect Optimal tincture condition setting

Cells were sacrificed via thymarylation to produce tinctamycin clostridium butyricum IDCC 5101, which was optimized for arthritis prevention treatment. The anti - inflammatory effect of antimicrobials was optimized by evaluating the antimicrobial effects of the thymidylation temperature and pressurization conditions because of the different extractability of the antimicrobials from the cells.

Clostridium butyricum seedlings were cultivated in a CBT medium (glucose 2% (w / v), peptone 0.1% (w / v), skim milk 0.1% (w / v)) as sterilized tinctaryl production medium in an 800 L / And incubated for 16 hours anaerobically. As shown in the following Table 1, the temperature of the degassing temperature was maintained at 0 to 1.0 kgf / cm ² at 90 ° C, 100 ° C, 110 ° C and 121 ° C for 10 minutes, and then cooled to 50 ° C again. And maintained at a pressure of 0 to 1.0 kgf / cm ^{2 for} 5 minutes.

The tinctured Clostridium butyricum IDCC 5101 was concentrated 20 times under reduced pressure, mixed with an excipient corresponding to 1/10 of the initial culture solution, and then dried at 45 ° C for 20 hours to obtain a tinctured cloth tree To produce a diimbutyricum raw material.

≪ Example 3 >

COX-2 Inhibitory Effect of Tinized Clostridium Butyricum on Different Conditions

The COX-2 inhibitory effect was measured to determine the anti-inflammatory action of the tinylated Clostridium butyricum IDCC 5101 for each condition, and the final process was selected. The COX-2 inhibitory effect measurement method is as follows. Prepare Raw 264.7 cells to be 2.5 x 10 ⁵ cells / ml, make a final concentration of 5.0 x 10 ⁵ cells / well in a 6-well plate at 2 ml / well, and incubate overnight. Each well was treated with 20 μl each of ID-CBT5101 (4 μl / LPS (2 μg / ml concentration) and 1/5 dilution) and then incubated overnight.

At the end of incubation, protein was quantitated by BCA method, separated by 10% SDS-polycrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membrane (Milipore, USA) at 100V for 120 min. At the end of the transfection, the membrane was separated, blocked with blocking solution (20 ml of 0.1% TBST buffer containing 5% skim milk), blocked with shaking for 30 minutes to 1 hour, washed with 0.1% TBST buffer, And reacted with primary antibodies (COX-2 and β-actin). (1: 5000, Cell Signaling, USA) at 4 ° C in the presence of COX-2 antibody (1: 1000, Cell Signaling, USA). Subsequently, the cells were washed with 0.1% TBST buffer and reacted with a secondary antibody (anti-rabbit polyclonal antibody) for 1 hour and 30 minutes. After washing with 0.1% TBST buffer, the membrane was placed on an OHP film, soaked with a western-one substrate, and protein expression was detected using Chemi Dox to compare the degree of inhibition.

The results are shown in Table 1 and FIG.

As shown in Table 1 and FIG. 2, Clostridium butyricum IDCC 5101, which was citrated at 121 ° C and 1.0 kgf / cm ² , had the best COX-2 inhibitory effect. This is probably due to the formation of favorable conditions for clostridium butyricum to be fragmented and to be small in size and to react with macrophages after sterilization of the cells under high pressure and high temperature conditions.

<Example 4>

Assessment of anti-inflammatory effects by cytokines modulation

Raw 264.7 cells cultured to confirm the effect on inflammatory cytokines and anti-inflammatory cytokines of T121-derived T121 samples obtained in Example 1 were recovered using a scrapper or trypsinize, and then transferred to a 15 ml falcon tube And centrifuged at 500 rpm for 5 minutes. After removal of the supernatant, 1 ml of complete media was added and resuspended to count the number of cells per ml. 250,000 / ml. 2 ml of diluted cells in each well of a 6-well plate was added and incubated overnight. The medium of each well of the 6-well plate was washed and then washed with PBS. After 2 ml of fresh medium was dispensed, each well was treated with 4 μl of LPS (final LPS concentration was 2 μl / ml), and 1 mM dexamethasone was added to the wells in a positive control (20 μl Final concentration 10 uM / ml). The remaining wells were treated with 10% of PBS and a 1/50 dilution of T121 thymarylated Clostridium butyricum IDCC 5101 (ID-CBT5101) as a negative control, followed by overnight incubation for 24 hours. The culture broth of each well was transferred to a 15 ml falcon tube and centrifuged at 9,500 rpm for 5 minutes. The supernatant of each tube was collected separately and used for cytokine experiments. An ELISA kit (R & D system) was used to confirm anti-inflammatory activity through inflammatory cytokine TNF-α and anti-inflammatory cytokine IL-10.

The results are shown in Fig.

As shown in FIG. 3, Clostridium butyricum IDCC 5101 (ID-CBT5101) cells minced at 121 ° C-1.0 kgf / cm ² inhibited the inflammatory cytokine TNF-α as well as the positive control substance dexamethasone . In contrast, it was confirmed that the secretion amount of IL-10, which is a cytokine showing an anti-inflammatory mechanism, could not be increased (not shown). Thus, the fungus body of Tinarylated Clostridium butyricum IDCC 5101 (ID-CBT5101) And it was confirmed that it is effective for relieving inflammation.

&Lt; Example 5 >

Evaluation of anti-inflammatory efficacy in arthritic animal models

<5-1> Arthritis animal model established

Six week old male Lewis rats (Orient Bios) were purchased and used after a week of purifying period. The animals were kept in constant temperature (22 ± 2 ℃) and humidity (50 ~ 60%) and were kept in Gwangju (08: 00 ~ 20: 00) Respectively. The animals were divided into three or two groups in poly-sulfone cage and the feed and water were fed for 24 hours in a free-diet. For each test group, 9 animals were used. Four groups, total 36 animals, were used in the vehicle, positive control group (100 mg / kg as green leaf mussel extract oil), ID-CBT5101 5 × 10 ⁸ , and 5 × 10 ⁹ CFU / rat. To induce arthritis, 100 μl of Complete Freund`s Adjuvant (CFA) (M. tuberculosis 10 mg / ml in mineral oil) purchased from Chondrex was administered intradermally to the base of rat tail. Arthritis symptoms began to appear on the 11th day after administration and arthritis symptoms appeared in all rats on the 13th day.

&Lt; 5-2 > Evaluation of efficacy of Tin Dalla clostridium butyricum IDCC 5101

The test substance was prepared immediately before administration and was orally administered once a day. The administration period was 4 weeks from the day of administration of CFA. The positive control group, green leaf mussel extract oil, was administered at 100 mg / kg, and tinctamycin clostridium butyricum IDCC 5101 (ID-CBT5101) was administered at a dose of 5 × 10 ⁸ , 5 × 10 ⁹ CFU / ml / rat. After the administration of CFA, the clinical score of arthritis was measured twice a week from day 13. The measurement method is shown in Table 2 below. The score of each foot was between 0 and 4, and the sum of four foot scores was expressed as an arthritis clinical score.

The results are shown in Fig.

As shown in FIG. 4, the treatment effect of arthritis was dose-dependent in all of the experimental groups administered with clindrimodium tridymium butyricum IDCC 5101 (ID-CBT5101), and especially in the group administered with 5 x 10 ⁹ CFU It was confirmed that arthritic symptoms were significantly alleviated from day 7 (D20) after induction of arthritis (D13). After the end of administration, it was 1.5 times more effective in alleviating arthritic symptoms than the positive control substance, green lipped mussel extract .

<5-3> Evaluation of joint histopathology

Twenty-four hours after the last test substance administration, the animals were euthanized and the hind paws were removed. After fixed in 10% neutral formalin solution, it was dipped in a solution of 8% formic acid and 8% hydrochloric acid in a 1: 1 mixture. After completion of dewaxing, paraffin blocks were formed through general tissue processing and cut to a thickness of 5 μm to prepare tissue slides. After H & E staining, histopathology was evaluated by observation with an optical microscope. All test results were expressed as mean ± standard error. Comparisons between vehicle and test substance groups were performed using Student's t-test (using Microsoft Office Excel 2010) and statistically significant when the p-value was less than 5% .

As shown in FIG. 5, after histological evaluation of the hindlimb tissue slides, H & E staining was observed. As a result, synovial proliferation was more prominent in the vehicle-treated group than in the vehicle group in all of the group treated with Clostridium butyricum IDCC 5101 (ID-CBT5101) And inflammatory cell infiltration, and the degree of reduction was proportional to the degree of cilinical score reduction. The ID-CBT5101 5 × 10 ⁸ CFU group showed a slight decrease in synovial membrane proliferation and inflammatory cell infiltration compared to the vehicle group, while the ID-CBT 5 × 10 ⁹ CFU group showed positive control and ID - CTT5101 5 x 10 ⁸ CFU treated group showed better synovial membrane proliferation and inflammatory cell infiltration than the 5 × 10 ⁸ CFU treated group.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

The tinctured Clostridium butyricum cells of the present invention reduce the levels of COX-2, the biomarker of arthritis, and the inflammatory cytokine Itumor necrosis factor-alpha (TNF-a) It is industrially applicable in that it can be developed as an arthritis preventive and therapeutic agent by alleviating the symptoms of arthritis in an animal model of arthritis administered with RIKUM IDCC 5101 cells.

Korea Biotechnology Research Institute KCTC12738BP 20141230

Claims (9)

delete delete Microbial cells of T. dolastatinum butyricum IDCC 5101 (accession number: KCTC12738BP) having arthritis prevention and therapeutic effect.
4. The dead cells according to claim 3, wherein the vitrification is performed by pressurizing at 121 DEG C and 1.0 kgf / cm &lt; ² &gt;
A composition for prevention and treatment of arthritis, comprising the microorganism cell of claim 3 as an active ingredient.
A composition for preventing and treating arthritis, comprising as an active ingredient, histamalized clostridium butyricum strain IDCC 5101 (deposit number: KCTC12738BP) and a culture solution of said strain.
delete The composition according to claim 6, wherein the vitrification is performed by pressurizing at 121 ° C and 1.0 kgf / cm ² .
(a) anaerobically culturing Clostridium butyricum strain IDCC 5101 (accession number: KCTC12738BP);
(b) the having the cultured cells and arthritis prevention and treatment, comprising a culture medium disproportionated four presses in 121 ℃, 1.0 kgf / cm ² tin dalhwa Clostridium butyric rikum IDCC 5101 (Accession No: KCTC12738BP) .

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