KR20190041864A - Composition for preventing or treating inflammatory disease comprising enterococcus faecalis - Google Patents

Abstract

The present invention relates to a composition for preventing or treating inflammatory diseases containing Enterococcus faecalis as an active component. As a result of treating dead cells of Enterococcus faecalis EF-2001 to macrophages and inducing an inflammatory reaction, it is confirmed that the production of nitric oxide (NO) is inhibited by dead cells of Enterococcus faecalis EF-2001 cells and the expression of iNOS and COX-2 protein, therefore, Enterococcus faecalis, a culture thereof, or dead cells thereof of the present invention can be usefully used as the active component of the composition for preventing or treating inflammatory diseases.

Description

TECHNICAL FIELD The present invention relates to a composition for preventing or treating an inflammatory disease containing enterococcus faecalis as an active ingredient,

The present invention relates to a composition for preventing or treating an inflammatory disease containing Enterococcus faecalis as an active ingredient, and more particularly to a composition for preventing or treating an inflammatory disease comprising Enterococcus faecalis , a culture solution thereof, Or a pharmaceutically acceptable salt thereof.

Recently, various roles of lactic acid bacteria have been studied, and the therapeutic efficacy of inflammatory diseases such as enteropathy and atopic dermatitis has been revealed.

Lactic acid bacteria are bacteria that decompose carbohydrates and make lactic acid by using them, and are useful as enterococci, Enterococcus, Streptococcus, Lactobacillus, Leuconostoc, Bifidobacteria Bifidobacteria, and Pediococcus.

Among them, enterococcus genus is widely found in nature and utilizes carbohydrates aerobically. In general, enterococcus is known to prevent damage by pathogenic microorganisms due to in vivo antagonism or secreted antimicrobial substances.

On the other hand, 70% of immunity is determined in the intestines. The germs in the intestines make it possible to break down and digest the food we eat, and it also serves to train the immune system. On the other hand, it causes cancer and diarrhea. Therefore, the intestinal bacterial balance must be well-balanced, the intestines are healthy and the body is healthy. Recent studies have also revealed that obesity and various intractable diseases are closely related to these intestinal bacteria and immunity.

Inflammation is an important immune response that protects against damage or infection by removing harmful elements and restoring cells and tissues. Excessive acute or chronic inflammation, however, can cause serious disorders such as arthritis, asthma, colitis, Parkinson's disease, Alzheimer's disease, and sepsis. In these disease processes, control of inflammation is essential for therapy. The outer membrane of Gram-negative bacteria, known as lipopolysaccharide (LPS), is a potent proinflammatory endotoxin. LPS stimulation was induced in macrophages and microglia cells, including iNOS-derived nitric oxide (NO) and cyclooxygenase-2 (COX-2) -induced prostaglandin E2 (PGE2) May induce the production of inflammatory mediators.

Nitric oxide (NO) is an inflammatory molecule produced by inducible nitric oxide synthase (iNOS). Excessive increase of iNOS activity or production of nitric oxide is a pathogen of various inflammatory diseases. Prostaglandin E2 (PGE2), which is synthesized by the cyclooxygenase-2 (COX-2) enzyme, is an important mediator of inflammatory symptoms such as fever and pain. Therefore, inhibition of the production of these inflammatory mediators .

For example, dexamethasone, which is known to inhibit nitric oxide production and iNOS expression, has been used as a potent anti-inflammatory agent. However, it has many side effects as a steroidal drug and thus has a problem of a large side effect against the effect.

Accordingly, the present inventors have made efforts to develop an anti-inflammatory agent with few side effects, and as a result, they have found that enterococcus faecalis It was confirmed that the production of nitric oxide (NO) was suppressed and the expression of iNOS and COX-2 protein was inhibited in the macrophages treated with EF-2001 microorganisms and the inflammatory reaction, Kalis EF-2001, a culture solution thereof, or a bacterium thereof can be used as an active ingredient of a composition for preventing or treating inflammation.

Japanese Patent Publication No. 3151442

Kurt Lucas & Michael Maes (2013) Role of the Toll Like Receptor (TLR) Radical Cycle in Chronic Inflammation: Possible Treatments Targeting the TLR4 Pathway. Mol Neurobiol 48 (1): 190-204. McCartney-Francis, Janice B. Allen, Diane E. Mizel, Jorge E. Albina, Qiao-Wen Xie, Carl F. Nathan, and Sharon M. Wahl (1993) Suppression of arthritis by an inhibitor of nitric oxide synthase. J Exp Med 178 (2): 749-754. Samuelsson B, Morgenstern F, Jakobsson PJ (2007) Membrane prostaglandin E synthase-1: a novel therapeutic target. Pharmacol Rev 59 (3): 207-224. Daniel L. Simmons, Regina M. Botting, and Timothy Hla (2004) Cyclooxygenase Isozymes: The Biology of Prostaglandin Synthesis and Inhibition. Pharmacol Rev 56 (3): 387-437. (1999) Dexamethasone inhibits the induction of iNOS gene expression through its mRNA in proinflammatory cytokine-stimulated hepatocytes. Shock 33 (1): 64-69. Heat-Killed Enterococcus faecalis (2016) Heat-Killed Enterococcus faecalis (2016) Heat-Killed Enterococcus faecalis (2016) Heat-killed Enterococcus faecalis EF-2001 Ameliorates Atopic Dermatitis in a Murine Model. Nutrients 8, 146.

It is an object of the present invention to provide an enterococcus The present invention also provides a pharmaceutical composition for preventing or treating an inflammatory disease, comprising the culture medium of the present invention or a bacterium thereof as an active ingredient.

Another object of the present invention is to provide a health functional food for preventing or ameliorating an inflammatory disease comprising Enterococcus faecalis, a culture solution thereof or a bacterium cell thereof as an active ingredient.

In order to accomplish the above object, the present invention relates to an enterococcus The present invention also provides a pharmaceutical composition for preventing or treating an inflammatory disease, comprising the culture liquid or a bacterium thereof as an active ingredient.

The present invention also provides a health functional food for preventing or ameliorating an inflammatory disease comprising an Enterococcus faecalis, a culture solution thereof or a bacterium cell thereof as an active ingredient.

The present invention relates to a method for inhibiting the growth of enterococcus that faecalis) processing the EF-2001 four cells, and the generation of the result Enterococcus faecalis EF-2001 four nitrogen oxide by the cell (NO) to induce an inflammatory response is suppressed, and this inhibiting iNOS and the expression of COX-2 protein The enterococcus faecalis of the present invention, the culture thereof or the microbial cells thereof can be used as an effective ingredient of a composition for the prophylaxis or treatment of inflammatory diseases.

Fig. 1 shows the results of an experiment in which 10 μg / ml of LPS (Lipopolysaccharide) was treated or not treated with Enterococcus faecalis ) The amount of nitric oxide (NO) production in the macrophage RAW264.7 was determined according to the concentration of EF-2001 dead cells.
FIG. 2 is a graph showing the expression amount of iNOS protein in the macrophage cell line RAW264.7 according to the concentration of Enterococcus faecalis EF-2001 dead cells under 10 μg / ml of LPS treatment or no treatment.
FIG. 3 is a graph showing the expression level of COX-2 protein in the macrophage cell line RAW264.7 according to the concentration of Enterococcus faecalis EF-2001 cells under conditions of 10 μg / ml of LPS or no treatment.

Hereinafter, the present invention will be described in more detail.

The present invention relates to the use of Enterococcus The present invention also provides a pharmaceutical composition for preventing or treating an inflammatory disease, comprising the culture liquid or a bacterium thereof as an active ingredient.

In the present invention, the Enterococcus faecalis is preferably Enterococcus faecalis EF-2001, but is not limited thereto.

The Enterococcus faecalis EF-2001 is a lactic acid bacterium developed by BRM Laboratories, Inc. of Nihon Berumu Co., Ltd. It is preferable to suppress the production of nitric oxide and suppress the expression of iNOS or COX-2 Do.

In the present invention, the Enterococcus faecalis, the culture thereof or the microbial cells thereof may be commercially available or may be any of those produced by a known microbial cell production method, and it is not toxic and harmless to the human body.

Also, the culture solution means a culture product of cultured Enterococcus faecalis in a culture medium, a concentrated culture solution, a dried culture product, a cultured filtrate solution, a concentrated culture filtrate solution or a culture filtrate solution. , And may be a culture obtained by removing the strain after culturing.

The dead cells may be prepared by heat-treating the corresponding live cells or treating them with formalin or other bactericides, and the dead cells may be substantially dead. The bacterial cells are cultured by a conventional method, and the obtained strains are washed, centrifugally dehydrated, washed and dehydrated repeatedly, if necessary, suspended in distilled water, physiological saline or the like, A suspension of the cell suspension obtained by heating at 80 to 115 캜 for 30 minutes to 3 seconds or a dried product thereof or a suspension of the cell suspension obtained by irradiating the cell suspension with a gamma ray or a neutron beam. The drying method of the dead cell suspension is not particularly limited as long as it is a known drying method, and examples thereof include spray drying and freeze drying. In some cases, the enzyme treatment, the surfactant treatment, the crushing and crushing treatment can be carried out before or after the sterilization treatment by heating or before or after the drying treatment, and those obtained by such treatment are also included in the dead cells of the present invention do. In addition, the dead cells can be produced by the following method, but are not limited thereto:

1) seed culture of Enterococcus faecalis followed by culture at pH 5.0-8.0 at 20-40 ° C;

2) heat-treating the cultured Enterococcus faecalis cultivated in step 1) at a temperature of 70 to 130 ° C for 5 to 30 minutes, followed by drying and pulverizing.

In the present invention, the inflammatory disease is selected from the group consisting of arthritis, rhinitis, hepatitis, keratitis, gastritis, enteritis, nephritis, bronchitis, pleurisy, peritonitis, spondylitis, pancreatitis, inflammatory pain, urethritis, cystitis, burn inflammation, periodontitis, gingivitis or degenerative nerve inflammation But is not limited thereto.

In a specific example of the present invention, the present inventors produced Enterococcus faecalis EF-2001 dead cells. In order to investigate the anti-inflammatory effect of the dead cells, LPS was treated to induce inflammation, It was confirmed that the production of NO x was inhibited by the cells of Enterococcus faecalis EF-2001 and the expression of i-NOS and COX-2 protein was inhibited. Therefore, The falciparum, its culture or its microbial cells may be used as an effective ingredient of a composition for preventing or treating inflammation or a health functional food.

The composition of the present invention may contain the strain as an active ingredient in an amount of 10 ⁶ to 10 ¹³ cfu / g, based on the total weight of the composition, or may include culture or dead cells having an equivalent number of live cells. The effective amount of the enterococcus faecalis of the present invention which is the main component of the present invention, the culture solution thereof or the microbial cells thereof may be selected from one or two or more kinds of pharmaceutically acceptable conventional carriers or one or more additives, ≪ / RTI >

The carrier may be selected from one or more selected from among diluents, lubricants, binders, disintegrants, sweeteners, stabilizers and preservatives. Examples of the additives include one or more of flavorings, vitamins and antioxidants Can be used.

In the present invention, the carrier and the additive may be any pharmaceutically acceptable ones. Specific examples of the diluent include lactose monohydrate, trehalose, cornstarch, soybean oil, Microcrystalline cellulose or D-mannitol may be used as the lubricant and magnesium stearate or talc may be used as the lubricant. The binder may be polyvinyl pyrolidone (PVP) or And hydroxypropylcellulose (HPC). The disintegrant is preferably selected from carboxymethylcellulose calcium (Ca-CMC), sodium starch glycollate, polacrylin potassium or cross-linked polyvinylpyrrolidone And the sweetening agent is selected from white sugar, fructose, sorbitol or aspartame. Examples of the stabilizer include sodium carboxymethylcellulose sodium (Na-CMC), beta-cyclodextrin (beta -cyclodextrin) white bee's wax or xanthan gum and the preservative may be selected from the group consisting of methyl p-hydroxy benzoate, methylparaben, propyl p-hydroxybenzoate, propylparaben, or potassium sorbate potassium sorbate, but is not limited thereto.

The pharmaceutical compositions of the present invention may be administered to a patient in a single dose and may be administered by a fractionated treatment protocol in which multiple doses are administered over a prolonged period of time. The term "pharmaceutically effective amount" as used herein refers to an amount that exhibits a reaction higher than that of the negative control, and preferably refers to an amount sufficient to prevent or treat an inflammatory disease. In addition, the pharmaceutically effective amount may be appropriately changed according to various factors such as the disease and its severity, the patient's age, body weight, health condition, sex, administration route and treatment period.

The composition of the present invention can be variously formulated according to the route of administration by a method known in the art together with the pharmaceutically acceptable carrier. The term " pharmaceutically acceptable " as used herein means a non-toxic composition which is physiologically acceptable and which, when administered to humans, does not inhibit the action of the active ingredient and does not normally cause an allergic reaction such as gastrointestinal disorder, dizziness, . The composition of the present invention can be variously formulated according to the route of administration by a method known in the art together with the pharmaceutically acceptable carrier. Routes of administration include, but are not limited to, oral or parenteral administration.

The present invention also relates to the use of Enterococcus The present invention provides a health functional food for preventing or ameliorating an inflammatory disease, comprising the culture liquid of the present invention or a bacterium thereof as an active ingredient.

In the present invention, the Enterococcus faecalis is preferably Enterococcus faecalis EF-2001, but is not limited thereto.

Further, the Enterococcus faecalis EF-2001 preferably suppresses the production of nitric oxide, and preferably inhibits the expression of iNOS or COX-2.

In the present invention, the inflammatory disease is selected from the group consisting of arthritis, rhinitis, hepatitis, keratitis, gastritis, enteritis, nephritis, bronchitis, pleurisy, peritonitis, spondylitis, pancreatitis, inflammatory pain, urethritis, cystitis, burn inflammation, periodontitis, gingivitis or degenerative nerve inflammation But is not limited thereto.

The health functional foods of the present invention include, for example, folk remedies for the purpose of improving bowel function such as tea, jelly, juice, extract, and beverage. The health functional food of the present invention processed in various forms as described above is easy to take and can be stored for a long time without adverse effects on the human body.

When the enterococcus faecalis of the present invention, its culture or its dead cells is used as a food additive, the strain, its culture or its dead cells may be directly added or used together with other food or food ingredients, Can be suitably used. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment). In general, when the food or beverage is produced, it is added in an amount of not more than 15 parts by weight, preferably not more than 10 parts by weight, based on 100 parts by weight of the strain, the culture liquid of the present invention or the raw cell material thereof. However, in the case of long-term consumption intended for health and hygiene purposes or health control purposes, the amount may be less than the above range, and there is no problem in terms of safety. Therefore, the active ingredient may be used in an amount of more than the above range.

There is no particular limitation on the kind of the food. Examples of the food to which the strain, the culture of the present invention or the microbial cells of the present invention can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, ice confection, ice cream, Milk, milk substitutes, cream, butter, dairy products including buttermilk, yogurt, yoghurt, cheese, various soups, beverages, tea, drinks, alcoholic drinks and vitamin complexes. .

The health beverage composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. The above-mentioned natural carbohydrates are sugar saccharides such as monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. 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 is generally 0.001 to 1.0 g, preferably about 0.01 to 1.0 g per 100 ml of the strain, the culture liquid of the present invention or the bacterium of the present invention.

The strain of the present invention, its culture or its microbial cells can be used for various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickening agents, pH adjusting agents, , Preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the strain of the present invention, its culture or its dead cells may contain flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not critical, but it is common that the strain or culture of the present invention is selected in the range of 0.001 to 0.1 part by weight per 100 parts by weight.

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples.

However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the present invention is not limited to the following Examples and Experimental Examples.

< Example  1> Enterococcus Lacalis ( Enterococcus faecalis ) EF -2001 Dead cells  Produce

Enterococcus faecalis ) EF-2001 dead cells were prepared as follows.

Specifically, Enterococcus faecalis EF-2001 live cells (Nihon BRM Co. Ltd, Japan) were aerobically or anaerobically cultured in a medium used for culturing general lactic acid bacteria, followed by pre-culture and maintained at pH 5.0 to 8.0 and 20 to 40 ° C And cultured for 1 to 3 days to reach the number of cells of 7.5 × 10 ¹² cfu / g or more based on dry weight (DW). Then, it was heat-treated at 70 to 130 ° C for 5 to 30 minutes and then quenched. After heat treatment, the cells were separated and recovered by a continuous centrifuge, followed by lyophilization and pulverization.

< Experimental Example  1> Enterococcus Lacalis EF -2001 On dead cells  Confirmation of inhibition of nitric oxide production by

Nitric oxide (NO) mediates important physiological functions such as homeostasis of blood vessels and induction of apoptosis in a normal physiological state, but a large amount of nitric oxide kills normal cells and induces inflammation, resulting in acute or chronic inflammation It acts as a cause of disease. Therefore, the action of weakening the production of nitric oxide can be judged as anti-inflammatory action.

In order to examine the anti-inflammatory effect of Enterococcus faecalis EF-2001 cells, the macrophage cells were treated with the Enterococcus faecalis EF-2001 cells prepared in Example 1 to induce an inflammatory reaction Changes in nitric oxide production were confirmed by ELISA.

Specifically, 5 x 10 ⁴ macrophage cell lines RAW264.7 were dispensed into a 24-well plate and cultured in DMEM medium containing 10% FBS at 37 ° C and 5% CO _{2 for} 24 hours . In addition, the cells were diluted with DMEM medium to a concentration of 50, 100, 250 and 500 占 퐂 / ml of the Enterococcus faecalis EF-2001 cells prepared in Example 1, Then, the cells cultured for 24 hours were treated with the cells of Enterococcus faecalis EF-2001 diluted by the respective concentrations, treated with 10 μg / ml of LPS for 30 minutes, and then cultured in DMEM medium containing 10% FBS At 37 < 0 > C, 5% CO2 for 18 hours. Then, the degree of nitric oxide production was measured by performing ELISA according to the manufacturer's procedure using a NO ELISA kit (Invitrogen, US).

As a control, RAW 264.7, which was cultured without treatment of LPS and Enterococcus faecalis EF-2001 cells, was used.

As a result, as shown in Fig. 1, in the case of macrophages treated with LPS alone, the production of nitrogen oxide was increased by LPS, whereas in the case of macrophages treated with both LPS and enterococcus faecalis EF-2001 cells, It was confirmed that the production of nitric oxide was decreased in a concentration-dependent manner according to the concentration of Pasolis EF-2001 cells.

< Experimental Example  2> Enterococcus Lacalis EF -2001 On dead cells  Inhibition of protein expression by inflammatory factors

To examine the anti-inflammatory effect of Enterococcus faecalis EF-2001 cells, the macrophage cells were treated with the Enterococcus faecalis EF-2001 cells prepared in Example 1, and the inflammatory reaction was induced. The expression of iNOS and COX-2 was determined by Western blotting.

Specifically, each of the macro cell lines treated with the control macrocyclic cell line, LPS, and Enterococcus faecalis EF-2001 cells were recovered by the same method as described in the <Experimental Example 1>. Then, the recovered cells were dissolved by using a RIPA buffer rich in protease inhibitor (Sigma) and a phosphatase inhibitor cocktail (Phosphatase inhibitor coctail 2,3, Sigma) to separate proteins. The protein lysate (30 μg) of the cells was electrophoresed by SDS-PAGE and then transferred to a PVDF membrane (Immobilon-P). Next, in order to confirm the change of expression of iNOS and COX-2 protein, anti-iNOS antibody and anti-COX-2 antibody were treated and reacted with the primary antibody, and HRP-conjugated secondary antibody was added to the primary antibody attached to the membrane , Which was confirmed using ECL (GE Healthcare-Life Sciences). Images were recorded using the SRX-101A Medical Film Processor (Konica Minolta).

As a result, as shown in Fig. 2 and Fig. 3, it was confirmed that the expression amount of iNOS and COX-2 protein increased by LPS treatment was decreased by treatment with Enterococcus faecalis EF-2001 microbial cells.

Therefore, it was confirmed from the above results that the Enterococcus faecalis EF-2001 cells of the present invention have an effect of inhibiting the production of NO and inhibiting the expression of i-NOS and COX-2 protein, The Enterococcus faecalis EF-2001 of the present invention, a culture thereof or a bacterium thereof can be used as an effective ingredient of a composition for preventing or treating inflammation.

Claims (9)

Enterococcus faecalis), its culture broth, and its use inflammatory disease preventing or treating a pharmaceutical composition comprising at least one selected from the group consisting of bacterial cells as an active ingredient.
The pharmaceutical composition according to claim 1, wherein the Enterococcus faecalis is Enterococcus faecalis EF-2001.
The pharmaceutical composition according to claim 2, wherein the Enterococcus faecalis EF-2001 inhibits the production of nitric oxide.
The pharmaceutical composition according to claim 2, wherein the Enterococcus faecalis EF-2001 inhibits the expression of iNOS or COX-2.
The pharmaceutical composition for preventing or treating inflammatory diseases according to claim 1, wherein the microorganism is selected from the group consisting of Enterococcus faecalis, which is heat-treated at 70 to 130 ° C for 5 to 30 minutes.

The method of claim 1, wherein the inflammatory disease is selected from the group consisting of arthritis, rhinitis, hepatitis, keratitis, gastritis, enteritis, nephritis, bronchitis, pleurisy, peritonitis, spondylitis, pancreatitis, inflammatory pain, urethritis, cystitis, burn inflammation, periodontitis, &Lt; / RTI &gt; inflammatory disease or inflammatory disease.
An enterococcus faecalis, a culture thereof, and a bacterium thereof, as an active ingredient.
The health functional food according to claim 7, wherein the Enterococcus faecalis is Enterococcus faecalis EF-2001.
The method of claim 7, wherein the inflammatory disease is selected from the group consisting of arthritis, rhinitis, hepatitis, keratitis, gastritis, enteritis, nephritis, bronchitis, pleurisy, peritonitis, spondylitis, pancreatitis, inflammatory pain, urethritis, cystitis, burn inflammation, periodontitis, Inflammatory diseases, inflammatory diseases or inflammatory diseases.

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