KR102503163B1 - 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 ingredient. Since it was confirmed that production of nitric oxide (NO) was inhibited by dead cells of Coccus faecalis EF-2001 and expression of iNOS and COX-2 proteins were suppressed, Enterococcus faecalis of the present invention, its culture medium or its dead cells It can be used as an active ingredient of a composition for preventing or treating inflammation.

Description

Composition for preventing or treating inflammatory disease containing Enterococcus faecalis as an active ingredient

The present invention relates to a composition for preventing or treating inflammatory diseases containing Enterococcus faecalis as an active ingredient, and more specifically, to a composition for preventing or treating inflammatory diseases comprising Enterococcus faecalis, its culture medium or its dead cells as an active ingredient It relates to a pharmaceutical composition for prevention or treatment.

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 use them to produce lactic acid, such as Enterococcus, Streptococcus, Lactobacillus, Leuconostoc, and Bifidobacteria ( Bifidobacteria) and Pediococcus.

Among them, Enterococcus genus widely exists in nature and uses carbohydrates aerobically. In general, Enterococcus genus is known to prevent damage caused by pathogenic microorganisms by in vivo antagonism or secreted antimicrobial substances.

Meanwhile, 70% of immunity is determined in the gut. The bacteria in our intestines help us break down and digest the food we eat and also train our immune system. On the other hand, it can cause cancer or diarrhea. Therefore, when the intestinal bacteria are well balanced, the intestines are healthy and the body is healthy. Recent studies have also revealed that obesity and various incurable diseases are deeply related to these intestinal bacteria and intestinal immunity.

Inflammation is an important immune response that protects against damage or infection by eliminating harmful factors and repairing cells and tissues. However, excessive acute or chronic inflammation can lead to 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 treatment. The outer membrane of Gram-negative bacteria, known as lipopolysaccharide (LPS), is a potent pro-inflammatory endotoxin. LPS stimulation stimulates the production of inducible nitric oxide synthase (iNOS)-derived nitric oxide (NO) and cyclooxygenase-2 (COX-2)-derived prostaglandin E2 (PGE2) in macrophages and microglia. It can induce the production of inflammatory mediators.

Nitric oxide (NO) is an inflammatory molecule produced by inducible nitric oxide synthase (iNOS), and an excessive increase in iNOS activity or production of nitric oxide is the etiology of various inflammatory diseases. In addition, since prostaglandin E2 (PGE2) synthesized by cyclooxygenase-2 (COX-2) enzyme is an important mediator of inflammatory symptoms such as fever and pain, inhibition of the production of these inflammatory mediators can be used to treat various inflammatory diseases. useful for

For example, dexamethasone, which is well known to inhibit the production of nitric oxide and the expression of iNOS, is used as a powerful anti-inflammatory agent, but has many side effects as a steroid drug, so there is a problem that side effects are large compared to effects.

Accordingly, the present inventors have tried to develop an anti-inflammatory agent with fewer side effects, and as a result, Enterococcus faecalis with lactic acid bacteria faecalis ) EF-2001 killed cells were treated and it was confirmed that the production of nitric oxide (NO) was inhibited and the expression of iNOS and COX-2 proteins was inhibited in macrophages that induced an inflammatory response. The present invention was completed by revealing that Callis EF-2001, its culture medium or its dead cells 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, 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, Timothy Hla (2004) Cyclooxygenase Isozymes: The Biology of Prostaglandin Synthesis and Inhibition. Pharmacol Rev 56(3): 387-437. Ozaki T, Habara K, Matsui K, Kaibori M, Kwon AH, Ito S, Nishizawa M, Okumura T (2010) Dexamethasone inhibits the induction of iNOS gene expression through destabilization of its mRNA in proinflammatory cytokine-stimulated hepatocytes. Shock 33(1): 64-69. Eun-Ju Choi, Masahiro Iwasa, Kwon-Il Han, Wan-Jae Kim, Yujiao Tang, Young Joung Hwang, Jeong Ryong Chae, Weon Cheol Han, Yu-Su Shin, Eun-Kyung Kim (2016) Heat-Killed Enterococcus faecalis EF-2001 Ameliorates Atopic Dermatitis in a Murine Model. Nutrients 8, 146.

An object of the present invention is Enterococcus faecalis ( Enterococcus faecalis ), to provide a pharmaceutical composition for preventing or treating inflammatory diseases comprising its culture medium or dead cells thereof as an active ingredient.

Another object of the present invention is to provide a health functional food for preventing or improving inflammatory diseases comprising Enterococcus faecalis, its culture medium or its dead cells as an active ingredient.

In order to achieve the above object, the present invention Enterococcus faecalis ( Enterococcus faecalis ), it provides a pharmaceutical composition for preventing or treating inflammatory diseases comprising its culture medium or dead cells thereof as an active ingredient.

In addition, the present invention provides a health functional food for preventing or improving inflammatory diseases comprising Enterococcus faecalis, its culture medium or its dead cells as an active ingredient.

The present invention relates to macrophages Enterococcus faecalis ( Enterococcus faecalis ) EF-2001 dead cells were treated and an inflammatory response was induced, and as a result, the production of nitric oxide (NO) was inhibited by the dead cells of Enterococcus faecalis EF-2001, and the expression of iNOS and COX-2 proteins was suppressed. As confirmed, the Enterococcus faecalis of the present invention, its culture medium or its dead cells can be used as an active ingredient of a composition for preventing or treating inflammatory diseases.

1 is LPS (Lipopolysaccharide) 10 μg / ml treated or untreated, Enterococcus faecalis ( Enterococcus faecalis ) EF-2001 It is a diagram confirming the amount of nitric oxide (NO) production in the macrophage cell line RAW264.7 according to the concentration of dead cells.
2 is a diagram confirming the iNOS protein expression level in the macrophage cell line RAW264.7 according to the concentration of dead cells of Enterococcus faecalis EF-2001 under conditions of 10 μg/ml LPS treatment or no treatment.
Figure 3 is a diagram confirming the COX-2 protein expression level in the macrophage cell line RAW264.7 according to the concentration of dead cells of Enterococcus faecalis EF-2001 under conditions of 10 μg/ml LPS treatment or no treatment.

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

The present invention is Enterococcus faecalis ( Enterococcus faecalis ), it provides a pharmaceutical composition for preventing or treating inflammatory diseases comprising its culture medium or dead cells thereof as an active ingredient.

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

In addition, the Enterococcus faecalis EF-2001 is a lactic acid bacterium developed by the BRM Research Institute of Nihon Berumu Co., Ltd., preferably suppressing the production of nitric oxide, and inhibiting the expression of iNOS or COX-2. do.

In the present invention, the Enterococcus faecalis, its culture medium or its killed cells may be used either commercially available or prepared by a known method for preparing dead cells, and is non-toxic and harmless to the human body.

In addition, the culture solution refers to a culture solution obtained by culturing Enterococcus faecalis in a culture medium, a concentrated culture solution, a dried culture solution, a culture filtrate, a concentrated culture filtrate, or a dried product of the culture filtrate, including the strain , It may be a culture medium from which strains are removed after culturing.

In addition, the dead cells may be prepared by heat-treating corresponding live cells or treating them together with formalin or other fungicides, and dead cells may be used even if they are substantially dead. In addition, after washing the strain obtained by culturing the dead cell body by a conventional method, centrifuging and repeating washing and dehydration as necessary, it is suspended in distilled water, physiological saline, etc., and the suspension is, for example, Dead cell suspension obtained by heating at 80 to 115 ° C. for 30 minutes to 3 seconds or a dried product thereof, or killed cell suspension obtained by irradiating the dead cell suspension with gamma rays or neutron rays, or a dried product thereof. The method for drying the dead cell suspension is not particularly limited as long as it is a known drying method, but spray drying, freeze drying and the like can be exemplified. In some cases, enzyme treatment, surfactant treatment, grinding/pulverization treatment may be performed before or after the sterilization treatment by heating or the like, or before or after the drying treatment, and those obtained by these treatments are also included in the dead cells of the present invention. do. In addition, the dead cells may be prepared by the following methods, but are not limited thereto:

1) After seed culture of Enterococcus faecalis, main culture at pH 5.0 ~ 8.0 and temperature of 20 ~ 40 ℃;

2) Step of drying and pulverizing Enterococcus faecalis main cultured in step 1) after heat treatment at 70 to 130 ° C for 5 to 30 minutes.

In the present invention, the inflammatory disease is arthritis, rhinitis, hepatitis, keratitis, gastritis, enteritis, nephritis, bronchitis, pleurisy, peritonitis, spondylitis, pancreatitis, inflammatory pain, urethritis, cystitis, burn inflammation, periodontitis, gingivitis, or degenerative neuroinflammation. It may, but is not limited thereto.

In a specific embodiment of the present invention, the present inventors prepared dead cells of Enterococcus faecalis EF-2001, and in order to determine the anti-inflammatory effect of the dead cells, the dead cells were treated with LPS to induce an inflammatory response. As a result of the treatment, it was confirmed that the production of nitric oxide (NO) was suppressed by the dead cells of Enterococcus faecalis EF-2001 and the expression of i-NOS and COX-2 proteins was suppressed. Faecalis, its culture medium or its dead cells can be used as an active ingredient of a composition for preventing or treating inflammation, or a health functional food.

The composition of the present invention may include a 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 cultures or dead cells having an equivalent number of viable cells. In addition, by selecting one or two or more pharmaceutically acceptable conventional carriers or one or two or more additives in an effective amount of the main ingredient, Enterococcus faecalis, its culture medium or dead cells of the present invention, composition can be made.

One or two or more carriers may be selected from diluents, lubricants, binders, disintegrants, sweeteners, stabilizers, and preservatives, and one or two or more additives may be selected from among flavoring agents, vitamins, and antioxidants. and can be used.

In the present invention, all pharmaceutically acceptable carriers and additives may be used, and specifically, diluents include lactose monohydrate, trehalose, cornstarch, and soybean oil. , Microcrystalline cellulose or mannitol (D-mannitol) is preferable, magnesium stearate or talc is preferable as a lubricant, and polyvinyl pyrrolidone (PVP) or polyvinyl pyrolidone (PVP) or It is preferable to select from hydroxypropylcellulose (HPC). In addition, the disintegrant is preferably selected from carboxymethylcellulose calcium (Ca-CMC), sodium starch glycolate, polacrylin potassium or cross-linked polyvinylpyrrolidone. The sweetener is selected from white sugar, fructose, sorbitol, or aspartame, and the stabilizer is sodium carboxymethylcellulose (Na-CMC: carboxymethylcellulose sodium), β-cyclodextrin, and white lead. It is selected from (white bee's wax) or xanthan gum, and as a preservative, methyl p-hydroxy benzoate (methylparaben), propyl p-hydroxybenzoate (propylparaben), or potassium sorbate ( potassium sorbate), but is not limited thereto.

The pharmaceutical composition of the present invention may be administered to a patient as a single dose, or may be administered by a fractionated treatment protocol in which multiple doses are administered over a long period of time. In the above, 'pharmaceutically effective amount' refers to an amount that exhibits a higher response than that of the negative control group, and preferably refers to an amount sufficient to prevent or treat inflammatory diseases. In addition, the pharmaceutically effective amount may be appropriately changed depending on various factors such as the disease and its severity, the patient's age, weight, health condition, sex, administration route and treatment period.

The composition of the present invention can be formulated in various ways according to the route of administration by a method known in the art together with the pharmaceutically acceptable carrier. In the above, "pharmaceutically acceptable" means a non-toxic composition that is physiologically acceptable and does not inhibit the action of the active ingredient when administered to humans and does not usually cause allergic reactions such as gastrointestinal disorders and dizziness or similar reactions. says The composition of the present invention can be formulated in various ways according to the route of administration by a method known in the art together with the pharmaceutically acceptable carrier. The route of administration is not limited thereto, but may be administered orally or parenterally.

In addition, the present invention is Enterococcus faecalis ( Enterococcus faecalis ), providing a health functional food for preventing or improving inflammatory diseases containing its culture medium or its dead cells as an active ingredient.

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

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

In the present invention, the inflammatory disease is arthritis, rhinitis, hepatitis, keratitis, gastritis, enteritis, nephritis, bronchitis, pleurisy, peritonitis, spondylitis, pancreatitis, inflammatory pain, urethritis, cystitis, burn inflammation, periodontitis, gingivitis, or degenerative neuroinflammation. It may, but is not limited thereto.

Examples of the health functional food of the present invention include folk remedies for the purpose of improving intestinal function, such as tea, jelly, juice, extract, and beverage. Thus, the health functional food of the present invention processed in various forms can be easily taken and stored for a long period of time without side effects on the human body.

When the Enterococcus faecalis of the present invention, its culture medium or its dead cells is used as a food additive, the strain, its culture medium or its dead cells may be added as is or used together with other foods or food ingredients, and according to a conventional method can be used appropriately. The mixing amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health or therapeutic treatment). In general, when preparing food or beverage, it is added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, based on 100 parts by weight of the strain, its culture medium or its dead cell raw material of the present invention. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.

There is no particular limitation on the type of food. Examples of foods to which the strain of the present invention, its culture medium or dead cells thereof can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, chewing gum, ice snacks, ice cream, Dairy products including milk, milk substitutes, cream, butter, buttermilk, yogurt, yogurt, and cheese, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, all of which are health foods in a conventional sense. include

The health beverage composition of the present invention may contain various flavoring agents or natural carbohydrates as additional components like conventional beverages. The aforementioned natural carbohydrates include 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. As the sweetener, natural sweeteners such as thaumatin and stevia extract or synthetic sweeteners such as saccharin and aspartame may be used. 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, culture medium or dead cells of the present invention.

In addition to the above, the strain of the present invention, its culture medium or its dead cells are various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH regulators, stabilizers , preservatives, glycerin, alcohol, carbonation agents used in carbonated beverages, and the like. In addition, the strain of the present invention, its culture medium or its dead cells may contain fruit flesh for the production of natural fruit juice, fruit juice beverages and vegetable beverages. These components may be used independently or in combination. The ratio of these additives is not very important, but is generally selected in the range of 0.001 to 0.1 parts by weight per 100 parts by weight of the strain or its culture medium of the present invention.

Hereinafter, the present invention will be described in detail by examples and experimental examples.

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

< Example 1> Enterococcus faecalis ( Enterococcus faecalis ) EF -2001 dead cells manufacturing

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

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

< Experimental example 1> Enterococcus faecalis EF -2001 to dead cells Confirmation of inhibition of nitric oxide production by

Nitric oxide (NO) mediates important physiological functions such as vascular homeostasis and apoptosis induction under normal physiological conditions, but large amounts of nitric oxide kill normal cells and induce inflammation, resulting in acute or chronic inflammation. It acts as a substance that causes disease. Therefore, the action of weakening the production of nitric oxide can be judged as an anti-inflammatory action.

Therefore, in order to examine the anti-inflammatory effect of the dead cells of Enterococcus faecalis EF-2001, the macrophage cell line was treated with the dead cells of Enterococcus faecalis prepared in <Example 1> and the inflammatory response was induced. Changes in production of nitric oxide were confirmed by ELISA.

Specifically, 5 x 10 ⁴ of the macrophage cell line RAW264.7 were seeded into a 24-well-plate, and then cultured for 24 hours at 37°C and 5% CO ₂ in DMEM medium containing 10% FBS. . In addition, the concentration of dead cells of Enterococcus faecalis EF-2001 prepared in <Example 1> was diluted with DMEM medium so that the concentrations were 50, 100, 250 and 500 μg/ml. Then, the cells cultured for 24 hours were treated with dead cells of Enterococcus faecalis EF-2001 diluted at each concentration, and after 30 minutes, treated with 10 μg/ml of LPS, followed by DMEM medium containing 10% FBS. and incubated for 18 hours at 37°C and 5% CO2. Then, ELISA was performed using a NO ELISA kit (Invitrogen, US) according to the manufacturer's procedure to measure the degree of nitric oxide production.

As a control, RAW264.7 cultured without LPS and dead cells of Enterococcus faecalis EF-2001 was used.

As a result, as shown in FIG. 1, in the case of macrophages treated with only LPS, nitric oxide production was increased by LPS, whereas in the case of macrophages treated with both LPS and dead cells of Enterococcus faecalis EF-2001, Enterococcus It was confirmed that the production of nitric oxide decreased in a concentration dependent manner according to the concentration of dead cells of Faecalis EF-2001.

< Experimental example 2> Enterococcus faecalis EF -2001 to dead cells Confirmation of suppression of protein expression of inflammation-related factors by

In order to examine the anti-inflammatory effect of dead cells of Enterococcus faecalis EF-2001, the macrophage cell line was treated with dead cells of Enterococcus faecalis prepared in <Example 1>, and after inducing an inflammatory response, inflammation-related factors Changes in protein expression of phosphorus iNOS and COX-2 were confirmed by Western blotting.

Specifically, the control macrophage cell line, the macrophage cell line treated with LPS and dead cells of Enterococcus faecalis EF-2001 were each recovered in the same manner as described in <Experimental Example 1>. Then, the recovered cells were lysed using RIPA buffer rich in protease inhibitor (Sigma) and phosphatase inhibitor coctail 2,3 (Sigma) to isolate proteins. The protein lysate (30 μg) of the cells was electrophoresed by SDS-PAGE and then transferred to a PVDF membrane (Immobilon-P). Then, in order to confirm the expression changes of iNOS and COX-2 proteins, the primary antibody was treated with an anti-iNOS antibody and an anti-COX-2 antibody, and then an HRP-conjugated secondary antibody was attached to the primary antibody attached to the membrane. , This was confirmed using ECL (GE Healthcare-Life Sciences). Images were taken using an SRX-101A Medical Film Processor (Konica Minolta).

As a result, as shown in FIGS. 2 and 3 , it was confirmed that the expression levels of iNOS and COX-2 proteins increased by the LPS treatment were decreased by the treatment of dead cells of Enterococcus faecalis EF-2001.

Therefore, through the above results, it was confirmed that the dead Enterococcus faecalis EF-2001 cell body of the present invention inhibits the production of nitric oxide (NO) and inhibits the expression of i-NOS and COX-2 proteins. Enterococcus faecalis EF-2001 of the present invention, its culture medium or its dead cells can be used as an active ingredient of a composition for preventing or treating inflammation.

Claims (9)

An anti-inflammatory pharmaceutical composition comprising dead cells of Enterococcus faecalis EF-2001 as an active ingredient,
Wherein the anti-inflammatory is to inhibit the production of nitric oxide and inhibit the expression of iNOS and COX-2, the composition.
delete delete delete The anti-inflammatory pharmaceutical composition according to claim 1, wherein the dead cells are killed by heat-treating Enterococcus faecalis EF-2001 at a temperature of 70 to 130 ° C for 5 to 30 minutes.
delete As an anti-inflammatory health functional food containing dead cells of Enterococcus faecalis EF-2001 as an active ingredient,
The anti-inflammatory is to inhibit the production of nitric oxide, and inhibit the expression of iNOS and COX-2, health functional food. delete delete

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