WO2023153903A1

WO2023153903A1 - Composition for preventing or treating fatty liver disease, comprising, as active ingredient, enterococcus faecalis, culture solution thereof or dead cells thereof

Info

Publication number: WO2023153903A1
Application number: PCT/KR2023/002085
Authority: WO
Inventors: 김택중; 이진호
Original assignee: 연세대학교 원주산학협력단
Priority date: 2022-02-11
Filing date: 2023-02-13
Publication date: 2023-08-17
Also published as: KR20230121587A

Abstract

The present invention relates to a composition for preventing or treating fatty liver disease, comprising Enterococcus faecalis as an active ingredient. Particularly, it has been identified that dead cells of Enterococcus faecalis EF-2001 prevent fatty liver disease and liver damage in experimental animals in which fatty liver disease is induced by high-fat feeding, reduce TG lipid accumulation, inhibit triglyceride droplet generation, increase lipase enzyme protein expression, induce activation of lipase enzymes such as ATGL and MGL, and increase AMPK phosphorylation so as to inhibit the SREBP-1c signaling pathway, and thus the Enterococcus faecalis, a culture solution thereof or dead cells thereof can be very effectively used as a pharmaceutical composition for preventing or treating fatty liver disease.

Description

A composition for preventing or treating fatty liver comprising Enterococcus faecalis, its culture medium or its dead cells as an active ingredient

The present invention relates to a composition for preventing or treating fatty liver comprising Enterococcus faecalis, its culture medium or dead cells thereof as an active ingredient.

The mortality rate of liver disease in Korea is very high at 23.5 per 100,000 population, the 1st cause of death in the 40s (41.1/100,000), the 2nd cause of death in the 50s (72.4/100,000), and the 3rd cause of death in the 30s. Liver disease is the leading cause of death among the middle-aged population in Korea.

Among the liver diseases, fatty liver refers to a phenomenon in which neutral fat, which does not exist in normal cells, is abnormally and excessively deposited in liver cells. A normal liver is composed of about 5% of adipose tissue, and triglycerides, fatty acids, phospholipids, cholesterol, and cholesterol esters are the main components of fat. Fatty liver is diagnosed when it is more than 5% of the liver weight. When fatty liver gets worse and the fat mass inside the liver cells grows, the important components of the cells, including the nucleus, are pushed to one side and the function of the liver cells deteriorates. As a result, the circulation of blood and lymph in the liver is impaired. When this happens, liver cells cannot receive oxygen and nutrients properly, and liver function deteriorates.

Non-alcoholic fatty liver disease (NAFLD) is defined as an accumulation of 5% or more of fatty acids in the parenchymal cells of the liver in the form of triglycerides, not alcohol-induced liver damage. Pathologically, it is classified into simple steatosis and steatohepatitis accompanied by inflammation. When left untreated for a long time, it can lead to serious liver diseases such as hepatitis, liver fibrosis, and cirrhosis. In Korea, the incidence of non-alcoholic liver disease is increasing due to lifestyle changes.

In addition, alcoholic fatty liver is caused by excessive drinking, and although there are differences depending on individual genetic characteristics and gender, liver diseases such as alcoholic fatty liver are highly likely to occur if alcohol is consumed in an amount of 80 g or more per day. In women, even lower doses increase the risk of alcoholic liver disease. In general, it can be calculated that about 10 g of alcohol is contained in 1 glass of soju, 1 glass of beer, 1 glass of liquor, and 1 hop of makgeolli. As for symptoms, most patients with alcoholic fatty liver disease, which is the mildest form, are asymptomatic, but mild hepatomegaly (a state in which the liver is larger than normal) may complain of mild tenderness in the right upper abdomen.

Until now, attempts have been made to restore liver function and prevent liver disease by improving liver levels by using the preventive action of natural ingredients such as milk thistle and black radish. Recently, probiotics and postbiotics materials using live and dead bacteria have been tried. (Republic of Korea Patent No. 10-2002-2840000) However, these drugs have not yet obtained sufficient results to improve the normal levels of aminotransferases in vivo.

In addition to this, drugs that act to improve liver function include Glycyrrhizin, Glycine, and Cysteine.

Glycyrrhizin has been reported to lower liver levels, restore liver function, and lower the rate of metastasis to liver cirrhosis in patients with hepatitis administered through clinical trials. It lowers liver levels to normal levels and is also effective for chronic hepatitis patients who do not respond to interferon. In addition, by controlling the concentration of cortisol, effects on chronic fatigue have been reported. However, because drug treatment accompanies high blood pressure, it can potentially act as another threat to patients with weak blood vessels or high blood pressure.

Glycine and cysteine, other ingredients, are known to promote the detoxification of drugs or toxic substances in the body by participating in liver detoxification along with glycyrrhizin.

In addition, Korean Patent Registration No. 10-2002-2840000 discloses pharmaceuticals for improving liver levels and preventing and treating liver diseases, including Lactobacillus plantarum extract, Lactobacillus casei extract, and Lactobacillus helveticus extract as active ingredients. composition is disclosed.

Meanwhile, microorganisms of the genus Enterococcus exist widely in nature and use carbohydrates aerobically. In general, bacteria such as microorganisms of the genus Enterococcus are known to prevent damage caused by pathogenic microorganisms by in vivo antagonism or secreted antibacterial substances. Among them, Enterococcus faecalis EF-2001 was identified through screening of the intestinal flora of a 2-year-old girl. The Enterococcus faecalis EF-2001 was killed by heat treatment and the cell components were recovered.

Under this background, the present inventors have made efforts to develop a material with low side effects and excellent inhibitory effects on liver lipid accumulation and diet-induced fatty liver damage. As a result, dead cells of Enterococcus faecalis EF-2001 are found to Inhibits fatty liver and liver damage in laboratory animals induced with fatty liver, reduces TG lipid accumulation, inhibits neutral lipid droplet production, increases lipase enzyme protein expression, and induces activation of lipase enzymes such as ATGL and MGL AMPK phosphorylation was increased, and the effect of inhibiting the SREBP-1c signaling pathway through this was confirmed. Through this, the present invention was completed by revealing that the Enterococcus faecalis, its culture medium or its dead cells can be used as a composition or functional material for preventing or treating fatty liver.

An object of the present invention is to have a preventive or therapeutic effect on fatty liver, which can be used very usefully for controlling fatty liver induced by a high-fat diet . To provide a composition for preventing or treating fatty liver.

Another object of the present invention is to provide a method for preventing or treating fatty liver, comprising the step of administering a pharmaceutically effective amount of Enterococcus faecalis, its culture medium or its dead cells to a subject.

Another object of the present invention is to provide a use of Enterococcus faecalis, its culture medium or its dead cells for use as a composition for preventing or treating fatty liver.

An object of the present invention is achieved by providing a pharmaceutical composition for preventing or treating fatty liver containing at least one selected from the group consisting of Enterococcus faecalis , its culture medium and dead cells thereof as an active ingredient.

An object of the present invention is achieved by providing a health functional food composition for preventing or improving fatty liver containing at least one selected from the group consisting of Enterococcus faecalis, its culture medium and its dead cells as an active ingredient.

An object of the present invention is achieved by providing a food additive for preventing or improving fatty liver containing at least one selected from the group consisting of Enterococcus faecalis, its culture medium and dead cells thereof as an active ingredient.

An object of the present invention is to provide a method for treating fatty liver, comprising administering to a subject at least one selected from the group consisting of a pharmaceutically effective amount of Enterococcus faecalis, its culture medium, and its dead cells.

An object of the present invention is to provide a method for preventing or improving fatty liver, comprising administering to a subject at least one selected from the group consisting of a pharmaceutically effective amount of Enterococcus faecalis, its culture medium, and its dead cells.

An object of the present invention is to provide a use of any one or more selected from the group consisting of Enterococcus faecalis, its culture medium, and its dead cells for use as a pharmaceutical composition for preventing or treating fatty liver.

An object of the present invention is to provide one or more uses selected from the group consisting of Enterococcus faecalis, its culture medium, and its dead cells for use as a health functional food composition for preventing or improving fatty liver.

An object of the present invention is to provide one or more uses selected from the group consisting of Enterococcus faecalis, its culture medium, and its dead cells for use as a food additive for preventing or improving fatty liver.

In the present invention, Enterococcus faecalis EF-2001 ( Enterococcus faecalis EF-2001 ) dead cells inhibit fatty liver and liver damage in laboratory animals induced with fatty liver by high-fat diet, reduce TG lipid accumulation, and produce neutral lipid droplets Inhibiting, increasing lipase enzyme protein expression, inducing activation of lipase enzymes such as ATGL and MGL, increasing AMPK phosphorylation, and thus exhibiting the effect of inhibiting the SREBP-1c signal transduction pathway, the above Enterococcus faecalis, its culture medium or its dead cells can be used very usefully as an active ingredient of a composition for preventing or treating fatty liver.

1A of FIG. 1 is a view of liver tissue for measuring the weight and size of the liver of the experimental group in <Experimental Example 1> of the present invention, FIG. 1B is a diagram showing the weight of the liver tissue, and FIGS. 1C and 1D are It is a diagram showing the effect of dead cells of Enterococcus faecalis EF-2001 on GOT or GPT of the experimental group.

FIG. 2A of FIG. 2 is a diagram showing the effect on dead cells of Enterococcus faecalis EF-2001 of FL83B hepatocytes, in which lipid accumulation was induced by Oil red O staining, and enterococcus faecalis on the relative lipid accumulation of FL83B hepatocytes. It is a diagram showing the effect of dead cells of Coccus faecalis EF-2001. Data are presented as mean±SEM. (n=4); *p<0.05, **p<0.01 and ***p<0.001 vs. control group.

Figure 3 is a diagram showing the effect of Enterococcus faecalis EF-2001 dead cells on neutral lipid droplets caused by BODIPY 493/507 in FL83B hepatocytes.

Figure 4 is a diagram showing the effect of dead cells of Enterococcus faecalis EF-2001 on lipase in FL83B hepatocytes. Data are presented as mean±SEM. (n=3); *p<0.05, **p<0.01 and vs. control group.

5 is a diagram showing the effect of dead cells of Enterococcus faecalis EF-2001 on the AMPK signaling pathway in FL83B hepatocytes. Data are presented as mean±SEM. (n=3); *p<0.05, **p<0.01 and ***p<0.001 vs. control group.

Figure 6 is a diagram showing the effect of Enterococcus faecalis EF-2001 killed cells on the AMPK signaling pathway using AICAR in FL83B hepatocytes. Data are presented as mean±SEM. *p<0.05, **p<0.05 compared to OA group.

Figure 7 is a diagram showing the effect of Enterococcus faecalis EF-2001 killed cells on the AMPK signaling pathway using compound C in FL83B hepatocytes. Data are presented as mean±SEM. *p<0.05 compared to OA group.

8 is a diagram showing the effects of Enterococcus faecalis EF-2001 on lipase and AMPK signaling pathways in HFD-induced fatty liver. Data are presented as mean±SEM. (n=6); *p<0.05 vs. Control group (HFD induced fatty liver rats).

9 is a diagram schematically illustrating the mechanism of inhibiting fatty liver of dead cells of Enterococcus faecalis EF-2001.

Hereinafter, a preferred embodiment of the present invention and the physical properties of each component will be described in detail, which is the present invention. It is intended to be described in detail so that those skilled in the art can easily practice the invention, but this does not mean that the technical spirit and scope of the present invention are limited.

In the present invention, "fatty liver" is, for example, alcoholic fatty liver (Alcoholic Fatty Liver), alcoholic steatohepatitis (Alcoholic Steatohepatitis, ASH), alcoholic liver cirrhosis, non-alcoholic simple fatty liver (Nonalcoholic Fatty Liver, NAFL), non-alcoholic steatohepatitis ( Nonalcoholic Steatohepatitis (NASH) and a composite of symptoms presenting as a cluster of non-alcoholic liver cirrhosis.

Preferably, the fatty liver treated, improved or prevented by the present invention is alcoholic fatty liver, alcoholic steatohepatitis, alcoholic cirrhosis, nonalcoholic simple fatty liver, nonalcoholic steatohepatitis or nonalcoholic cirrhosis.

In the present invention, the term "prevention" means suppressing the occurrence of a disease or disease in an animal that has never been diagnosed as having the disease or disease, but is prone to such disease or disease. As used herein, the term “treatment” refers to (i) inhibition of a disease or development of a disease; (ii) alleviation of disease or illness; and (iii) the elimination of disease or disease.

The present invention provides a pharmaceutical composition for preventing or treating fatty liver comprising at least one selected from the group consisting of Enterococcus faecalis , its culture medium and dead cells thereof as an active ingredient.

In addition, the present invention provides one or more uses selected from the group consisting of Enterococcus faecalis, its culture medium, and its dead cells for use as a pharmaceutical composition for the prevention or treatment of fatty liver.

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 the present invention, the Enterococcus faecalis may be Enterococcus faecalis EF-2001 ( Enterococcus faecalis EF-2001).

In addition, the culture medium refers to a culture medium obtained by culturing Enterococcus faecalis EF-2001 in a culture medium, a concentrated culture medium, a dried culture medium, a dried culture filtrate, a concentrated culture filtrate or a dried culture filtrate. Including, it may be a culture solution from which strains are removed after culturing.

In addition, the dead cells may be prepared by heat-treating corresponding live cells, such as Enterococcus faecalis EF-2001 live cells, or by 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 cells by a conventional method, centrifuging, repeating washing and dehydration as necessary, suspending in distilled water, physiological saline, etc., 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) seed culture of Enterococcus faecalis EF-2001, followed by main culture at pH 4.0 to 9.0 and a temperature of 15 to 45° C., more preferably pH 5.0 to 8.0 at a temperature of 20 to 40° C.;

2) Heat treatment of Enterococcus faecalis EF-2001 viable cells main-cultured in step 1) at a temperature of 60 to 140 ° C for 1 to 40 minutes, more preferably at a temperature of 70 to 130 ° C for 5 to 30 minutes, followed by drying and pulverization step to do.

In a specific embodiment of the present invention, the present inventors found that dead cells of Enterococcus faecalis EF-2001 inhibited fatty liver and liver damage, reduced TG lipid accumulation, and neutral lipid droplets in laboratory animals induced with fatty liver on a high-fat diet. It was confirmed that it inhibits production, increases lipase enzyme protein expression, induces activation of lipase enzymes such as ATGL and MGL, increases AMPK phosphorylation, and inhibits the SREBP-1c signaling pathway through this, The Enterococcus faecalis, its culture medium or its dead cells can be used very usefully as an active ingredient of a pharmaceutical composition for preventing or treating fatty liver.

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 provides a health functional food composition for preventing or improving fatty liver, comprising Enterococcus faecalis, its culture medium or its dead cells as an active ingredient.

In addition, the present invention provides the use of Enterococcus faecalis, its culture medium or its dead cells for use as a health functional food composition for preventing or improving fatty liver.

In addition, the present invention provides a food additive for preventing or improving fatty liver comprising Enterococcus faecalis, its culture medium or its dead cells as an active ingredient.

In addition, the present invention provides the use of Enterococcus faecalis, its culture medium or its dead cells for use as a food additive for preventing or improving fatty liver.

In addition, the method for obtaining the Enterococcus faecalis, its culture medium and its dead cells is identical to that described in the pharmaceutical composition for preventing or treating fatty liver comprising the Enterococcus faecalis, its culture medium or its dead cells as an active ingredient, The specific description uses the above contents, and hereinafter, only the specific configuration of the health functional food will be described.

On the other hand, the present inventors have found that dead cells of Enterococcus faecalis EF-2001 inhibit fatty liver and liver damage, reduce TG lipid accumulation, inhibit neutral lipid droplet production, and lipase Since it was confirmed that there is an effect of increasing enzyme protein expression, inducing activation of lipase enzymes such as ATGL and MGL, increasing AMPK phosphorylation, and inhibiting the SREBP-1c signal transduction pathway through this, the Enterococcus faecalis, Its culture medium or its dead cells can be used very usefully as an active ingredient of a health functional food composition or food additive for preventing or improving fatty liver.

There is no particular limitation on the type of food to which Enterococcus faecalis, its culture medium or its dead cells are added according to the present invention. Examples of the food include drinks, meat, sausages, bread, biscuits, rice cakes, chocolates, candies, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, alcoholic beverages and vitamins There are complex drugs, etc., and includes all health foods in a conventional sense.

The mixing amount of Enterococcus faecalis according to the present invention, its culture medium, or its dead cells may be suitably determined depending on its purpose of use. In general, the amount of the Enterococcus faecalis, its culture medium or its dead cells in the health food may be added in an amount of 0.001 to 50% by weight of the total weight of the food. 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.

The health functional beverage composition of the present invention is not particularly limited in other ingredients except for containing the Enterococcus faecalis, its culture medium or its dead cells as essential components in the indicated ratio, and various flavors or natural ingredients like conventional beverages. Carbohydrates and the like may be contained as additional components. Examples of the aforementioned natural carbohydrates include monosaccharides such as glucose, fructose, and the like; disaccharides such as maltose, sucrose and the like; and polysaccharides such as conventional sugars such as dextrins, cyclodextrins, and the like, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (thaumatin, stevia extract (eg rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can advantageously be used. .

In addition to the above, the food or food additive of the present invention is various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and enhancers (cheese, chocolate, etc.), pectic acid and its salts, Alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, and the like may be contained. In addition, the Enterococcus faecalis of the present invention, its culture medium or its dead cell body may contain fruit flesh for producing 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 so critical, but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of Enterococcus faecalis of the present invention, its culture medium or dead cells thereof.

In addition, the present invention provides a method for preventing or improving fatty liver, comprising the step of administering a pharmaceutically effective amount of Enterococcus faecalis, its culture medium or its dead cells to a subject.

In addition, the present invention provides a method for treating fatty liver, comprising the step of administering a pharmaceutically effective amount of Enterococcus faecalis, its culture medium or its dead cells to a subject.

The Enterococcus faecalis, its culture medium or its dead cells, and fatty liver are the same as the description of the pharmaceutical composition for preventing or treating fatty liver, and the specific description is incorporated herein by reference.

On the other hand, the present invention relates to a composition for preventing or treating fatty liver comprising Enterococcus faecalis as an active ingredient, and specifically, an experiment in which dead cells of Enterococcus faecalis EF-2001 were induced with a high-fat diet Inhibits fatty liver and liver damage in animals, reduces TG lipid accumulation, inhibits neutral lipid droplet production, increases lipase enzyme protein expression, induces activation of lipase enzymes such as ATGL and MGL, and increases AMPK phosphorylation And since it was confirmed that there is an effect of inhibiting the SREBP-1c signal transduction pathway through this, Enterococcus faecalis of the present invention, its culture medium or its dead cells can be usefully used for the prevention, improvement or treatment of fatty liver.

Hereinafter, the present invention will be described in detail by Examples, Experimental Examples and Manufacturing Examples.

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

<실시예 1> 엔테로코커스 패칼리스 EF-2001(<Example 1> Enterococcus faecalis EF-2001 ( Enterococcus faecalisEnterococcus faecalis EF-2001) 사균체 제조 EF-2001) Manufacturing dead cells

Enterococcus faecalis EF-2001 live bacteria (Korea Berm Co., Ltd., Korea) are aerobically or anaerobically cultured in a medium used for general lactobacillus culture, and after pre-culture, pH 5.0 to 8.0, while maintaining 20 to 40 ° C. The main culture was performed by culturing for 3 days to reach a dry weight (DW) of 7.5 × 10 ¹² cfu/g or more. Then, the cells were killed by heat treatment at 70 to 130 ° C. for 5 to 30 minutes, and then the cells were separated and collected by a continuous centrifugal machine, and then lyophilized and powdered.

<실시예 2> 화학 시약<Example 2> Chemical Reagent

5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), an AMPK activator, and compound C, an AMPK inhibitor, were obtained from Sigma - Purchased from Aldrich (USA).

<실시예 3> 고지방식이로 유도한 지방간 실험동물 제작<Example 3> Production of experimental animals with fatty liver induced by high-fat diet

24 3-week-old male SD (Sprague-Dawley) mice (Orient Bio Co., Ltd., Korea) were purchased, and after giving an adaptation period for 1 week, randomly divided into 4 groups of 6 mice each according to the diet [Table 1] shared with 5L79 (LabDiet, USA) was provided as a standard diet (Standard diet, SD) to the normal group, and D12492 (Research Diets, USA) was provided to the three high fat diet (HFD) groups for 6 weeks, Distilled water and 3 mg/kg or 30 mg/kg of dead cells of Enterococcus faecalis EF-2001 prepared in <Example 1> were orally administered to each experimental group once a day for 6 weeks (FIG. 1). All experimental procedures were approved by the Yonsei University Animal Laboratory Management Committee and were performed in accordance with the approved guidelines (YWCI-202102-003-01).

실험군experimental group	식이diet	투여한 시료sample administered
정상군 (SD)Normal group (SD)	표준식이standard diet	6주간 증류수 1일 1회 경구투여Oral administration of distilled water once a day for 6 weeks
고지방식이군 (HFD)high fat diet (HFD)	고지방식이high fat diet	6주간 증류수 1일 1회 경구투여Oral administration of distilled water once a day for 6 weeks
고지방식이+EF-2001 3 ㎎/㎏ 투여군 (HFD-EF-2001 (3 ㎎/㎏))High-fat diet + EF-2001 3 mg/kg administration group (HFD-EF-2001 (3 mg/kg))	고지방식이high fat diet	6주간 엔테로코커스 패칼리스 EF-2001 사균체 3 ㎎/㎏ 1일 1회 경구투여Enterococcus faecalis EF-2001 killed cells 3 mg/kg orally once a day for 6 weeks
고지방식이+EF-2001 30 ㎎/㎏ 투여군 (HFD-EF-2001 (30 ㎎/㎏))High-fat diet + EF-2001 30 mg/kg administration group (HFD-EF-2001 (30 mg/kg))	고지방식이high fat diet	6주간 엔테로코커스 패칼리스 EF-2001 사균체 30 ㎎/㎏ 1일 1회 경구투여Enterococcus faecalis EF-2001 killed cells 30 mg/kg orally once a day for 6 weeks

<실시예 4> 혈청학적 분석<Example 4> Serological analysis

On the last day of orally administering the sample to each of the experimental groups of <Example 3> for 6 weeks, cardiac blood was collected under ether anesthesia, and serum samples obtained from blood collected from experimental animals were fractionated. ALT) and aspartate aminotransferase (Aspartate transaminase, AST) were used for each assay kit (Asan Pharmaceutical, Korea) according to the manufacturer's protocol to determine the activity of liver enzymes.

<실시예 5> FL83B 세포의 배양 및 유도 지방간 세포<Example 5> Culture and induced fatty liver cells of FL83B cells

FL83B cells purchased from the American Type Culture Collection (ATCC) were supplemented with 10% Fetal bovine serum (FBS), 1% Penicillin and 1% Streptomycin (Sigma-Aldrich, USA). Maintained in F12K medium, FL83B cells were cultured in an incubator at 37° C. with CO ₂ .

Then, FL83B cells were inoculated in complete medium for 24 hours, and then cultured with 0.5 mM oleic acid (OA) for 48 hours to induce lipid accumulation. In addition, in order to analyze the experimental results, FL83B cells were treated with or without 25, 50, or 100 μg/ml of Enterococcus faecalis EF-2001 for 24 hours.

<실시예 6> FL83B 간세포의 오일 레드 O(Oil red O, ORO) 염색<Example 6> Oil red O (ORO) staining of FL83B hepatocytes

Enterococcus faecalis EF-2001 dead cells prepared by the method described in <Example 1> were differentiated into a differentiation induction medium at concentrations of 0, 25, 50, 100, and 250 μg/ml at 100% cell confluence. induction medium) to induce lipid accumulation in FL83B cells. Then, the FL83B cells were washed with phosphate-buffered saline (PBS), fixed with 3.7% formaldehyde (Junsei Chemical, Japan), and stained with 60% ORO diluted in distilled water. Quantification of lipid accumulation was obtained by concentration after treatment with 100% isopropanol in each well treated with dead cells of Enterococcus faecalis EF-2001, and 490 nm microplate measurement (Molecular Devices, USA) collected by performing Results are graphed and the percentage of ORO staining was relative to the percentage of untreated control cells representing the percentage of lipid droplets in stained cells.

<실시예 7> 웨스턴 블롯 분석(Western blot analysis)<Example 7> Western blot analysis

Liver tissues of experimental animals induced with fatty liver by HFD of <Example 3> were used, or FL83B cells were treated with dead cells of Faecalis EF-2001, and lysis buffer was used at an appropriate stage of liver lipid accumulation. (iNtRON Biotechnology, Korea) was added, followed by sonication, and the Bradford assay (Bio-Rad, USA) was used for protein quantification and Western blotting. The ratio of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was determined according to the molecular weight of the protein to be identified, and electrophoresis was performed at 100V for about 2 hours. Then, the primary antibodies (SREBP-1C, P-AMPK, AMPK, FAS, P-ACC, ACC, ATGL, P-HSL MGL, CD36 and β-actin) were incubated overnight at 4°C at a ratio of 1:2500. The cells were washed three times for 10 minutes in Tris-buffered saline (TBS) containing tween 20, and a secondary antibody was added at a ratio of 1:5000 for 2 hours at room temperature. Transcribed protein bands on polyvinylidene difluoride (PVDF) membranes were visualized by inducing an enhanced chemiluminescence response using a LAS 4000 system (GE Healthcare, UK).

Antibody treatment was performed with primary antibodies, and signal intensities were quantified using Image J software (NIH, USA).

<실시예 8> 공초점 현미경<Example 8> Confocal microscopy

FL83B hepatocytes were cultured and differentiated in a 3 cm cover glass plate (Mattek Corp, USA) and treated with each dose of dead cells of Enterococcus faecalis EF-2001. Then, to facilitate observation of nuclei, the fluorescent dye DAPI diluted in PBS was fixed with paraformaldehyde at room temperature, incubated for 10 minutes, and cells were fixed with paraformaldehyde to visualize triglycerol. while incubating with the fluorescent BODYPY 493/503 dye GFP (Thermo fisher scientific, USA) diluted in PBS for 30 minutes at room temperature. GFP expression was visualized with an LSM710 confocal microscope (Carl Zeiss, Germany).

<실시예 9> 통계분석<Example 9> Statistical analysis

Experimental results were expressed as mean ± standard error (SE). Analysis of variance and paired or unpaired t-tests were performed as appropriate for statistical analysis. A p value < 0.05 was considered statistically significant, and all experiments were performed at least three times.

<실험예 1> 엔테로코커스 패칼리스 EF-2001 사균체 투여의 지방간 조직 및 간 손상 예방 효과 확인<Experimental Example 1> Confirmation of preventive effect of fatty liver tissue and liver damage of Enterococcus faecalis EF-2001 dead cell administration

In order to establish the HFD-induced fatty liver model in <Example 3>, male mice were divided into SD or HFD groups, and enterococcus faecalis EF-2001 dead cells in distilled water or water were orally administered daily according to each dose. . The HFD group was subcategorized into distilled water, 3 mg/kg or 30 mg/kg Enterococcus faecalis EF-2001 group to evaluate the effect of dead cells of Enterococcus faecalis EF-2001 on fatty liver-induced experimental animals. The effect of ingestion of dead cells of Enterococcus faecalis EF-2001 on HFD-induced elevation in alcoholic fatty liver disease was investigated.

As a result, as shown in FIG. 1, in the experimental animals fed the HFD, white and enlarged livers were observed along with fat accumulation. Among the HFD group, the appearance of the liver with accumulated light-tone fat in the experimental animals administered with dead cells of Enterococcus faecalis EF-2001 was similar to the experimental animals in the HFD group, but the size of the liver was similar to that of the experimental animals in the SD group. Confirmed. All of the experimental animals in the HFD group to which 3 mg/kg or 30 mg/kg of dead cells of Enterococcus faecalis EF-2001 were administered showed a significant decrease in liver tissue weight, and the weight of fatty liver tissue in the HFD group was lower than that in the SD group. Although higher in experimental animals, it was confirmed that administration of dead cells of Enterococcus faecalis EF-2001 significantly reduced HFD-induced fatty liver tissue. In addition, both GOT and GPT levels were found to increase significantly in the HFD group, and GOT and GPT levels recovered in both groups administered with 3 mg/kg or 30 mg/kg of dead cells of Enterococcus faecalis EF-2001. It was found that HFD-induced liver damage was significantly reduced.

From the above results, it was confirmed that ingestion of dead cells of Enterococcus faecalis EF-2001 down-regulated fatty liver induction and liver damage caused by HFD.

<실험예 2> 엔테로코커스 패칼리스 EF-2001 사균체의 트라이글리세라이드(Triglyceride, TG) 지질 축적의 감소 효과 확인<Experimental Example 2> Confirmation of the effect of reducing triglyceride (TG) lipid accumulation of dead cells of Enterococcus faecalis EF-2001

In <Example 5>, FL83B cells were pretreated with 0.5 mM OA in a serum-free medium, and then treated with 25, 50 or 100 μg/ml of killed cells of Enterococcus faecalis EF-2001 for 24 hours. 6>, the effect of dead cells of Enterococcus faecalis EF-2001 on hepatic lipid accumulation was examined from ORO staining.

As a result, as shown in FIG. 2, significantly increased lipid accumulation was confirmed in OA-treated FL83B cells compared to non-OA-treated cells, and 50 or 100 μg/ml of Enterococcus faecalis EF-2001 killed cells was found to significantly reduce OA-induced lipid accumulation in FL83B cells.

<실험예 3> 엔테로코커스 패칼리스 EF-2001 사균체의 중성 지질 방울 억제 효과 확인 <Experimental Example 3> Confirmation of neutral lipid drop inhibition effect of dead cells of Enterococcus faecalis EF-2001

In <Example 5>, 25, 50 or 100 μg/ml of dead cells of Enterococcus faecalis EF-2001 were co-administered in OA-induced FL83B cells to investigate how adipogenesis and lipolysis were carried out in lipid accumulation. Focus microscopy was performed.

As a result, as shown in Figure 3, lipid synthesis increased in the control group 48 hours after OA induction, but in the group treated with dead cells of Enterococcus faecalis EF-2001, intracellular neutral lipid droplets were reduced for up to 24 hours. .

From the above results, it was confirmed that dead cells of Enterococcus faecalis EF-2001 inhibited lipid accumulation in hepatocytes in a dose-dependent manner.

<실험예 4> 엔테로코커스 패칼리스 EF-2001 사균체의 리파아제 효소 단백질 발현 증가 효과 확인<Experimental Example 4> Enterococcus faecalis EF-2001 confirmed effect of increasing lipase enzyme protein expression of dead cells

After OA induction in FL83B cells in <Example 5>, the expression of adipose triglyceride lipase (ATGL) and monoacylglycerol (MGL) was increased within 24 hours of treatment with dead cells of Enterococcus faecalis EF-2001. Observed. Accordingly, cells were harvested at the time of induction of expression of each lipase protein and Western blotting was performed.

As a result, as shown in FIG. 4 , treatment of dead cells of Enterococcus faecalis EF-2001 was found to increase expression of lipase enzyme in a dose-dependent manner. ATGL, an early lipolytic enzyme protein, showed increased expression in cells treated with dead cells of Enterococcus faecalis EF-2001, and it was confirmed that the expression of ATGL increased for 6 hours in FL83B cells induced by lipolysis. .

<실험예 5> 엔테로코커스 패칼리스 EF-2001 사균체의 AMPK 및 SREBP 신호 전달 경로 발현 관련 효과 확인<Experimental Example 5> Confirmation of AMPK and SREBP signaling pathway expression-related effects of dead cells of Enterococcus faecalis EF-2001

To investigate the mechanism of lipolysis induced by dead cells of Enterococcus faecalis EF-2001, the effect of dead cells of Enterococcus faecalis EF-2001 on the AMPK and SREBP signaling pathways was investigated. In OA-induced FL83B hepatocytes, AMPK and ACC, proteins related to the AMPK signaling pathway, and SREBP-1c and FAS, proteins related to lipid synthesis, were compared. Cells were harvested at the time point when the expression of each AMPK signaling protein was induced, and Western blotting was performed.

As a result, as shown in FIG. 5, it was confirmed that the treatment of dead cells of Enterococcus faecalis EF-2001 significantly increased the expression of phosphorylated AMPK and ACC in a dose-dependent manner, and in the OA-induced hepatic lipid accumulation signal. SREBP-1c and FAS expression levels increased. In contrast, it was confirmed that the treatment of dead cells of Enterococcus faecalis EF-2001 dose-dependently reduced the expression of SREBP-1c and FAS.

From the above results, it was confirmed that hepatic lipid degradation by dead cells of Enterococcus faecalis EF-2001 in the lipogenesis of OA-induced FL83B cells is due to the activation of the AMPK signaling pathway. In addition, the above results indicate that liver lipid accumulation was downregulated by the treatment of dead cells of Enterococcus faecalis EF-2001 during the lipolysis step, and expression of lipolytic proteins increased in a dose-dependent manner of dead cells of Enterococcus faecalis EF-2001. confirmed through.

<실험예 6> 엔테로코커스 패칼리스 EF-2001 사균체의 AMPK 표적 신호 전달 경로 관련 발현 증가 효과 확인<Experimental Example 6> Confirmation of the effect of increasing the expression of Enterococcus faecalis EF-2001 killed cells related to the AMPK target signaling pathway

In <Experimental Example 5>, it was confirmed that the treatment of dead cells of Enterococcus faecalis EF-2001 induces AMPK activity. Accordingly, an experiment was conducted to investigate the relationship between the expression of lipid-related biomarkers and the treatment of dead cells of Enterococcus faecalis EF-2001 according to the change in AMPK signal by targeting the AMPK activator (AICAR) and inhibitor (Compound C) of AMPK performed.

As a result, as shown in Figure 6, it was confirmed that AMPK phosphorylation increased in AICAR-treated FL83B cells. FL83B cells treated with AICAR and Enterococcus faecalis EF-2001 showed increased AMPK phosphorylation and ACC and ATGL expression.

In addition, as shown in FIG. 7, when FL83B cells were treated with AMPK inhibitor compound C and dead cells of Enterococcus faecalis EF-2001, AMPK phosphorylation and ACC expression were restored, and expression of ATGL, a lipase protein, was also recovered. It was confirmed that there was a significant difference in the degree of suppression of SREBP-1c expression in the group treated with dead cells of Enterococcus faecalis EF-2001.

<실험예 7> 엔테로코커스 패칼리스 EF-2001 사균체의 실험동물에서 AMPK 신호 전달 경로 관련 효과 확인<Experimental Example 7> Confirmation of AMPK signaling pathway-related effects in experimental animals of dead cells of Enterococcus faecalis EF-2001

The effect of dead cells of Enterococcus faecalis EF-2001 on the protein expression levels of the AMPK signaling pathway in the liver tissues of experimental animals fed SD and HFD was investigated. Experiments were also conducted during adipogenesis to investigate how treatment of dead cells of Enterococcus faecalis EF-2001 regulates the expression levels of lipolysis-related proteins such as p-AMPK, p-ACC and ATGL.

As a result, as shown in FIG. 8 , it was confirmed that p-AMPK protein expression during liver lipid accumulation was increased by the treatment of dead cells of Enterococcus faecalis EF-2001, and ACC phosphorylation was low in the HFD group. In addition, when 30 μg/ml of killed Enterococcus faecalis EF-2001 cells were orally administered to the HFD group, the protein expression level of SREBP-1c was lower than that of the HFD group not treated with killed Enterococcus faecalis EF-2001 cells. , and ATGL protein expression increased in the groups treated with 3 and 30 μg/kg of dead cells of Enterococcus faecalis EF-2001. However, there was no significant difference in MGL expression in the HFD group, and in contrast, it was confirmed that MGL was highly expressed in the SD group.

Through the above results, it can be seen that the dead cells of Enterococcus faecalis EF-2001 of the present invention regulate HFD-induced abnormal hepatic lipid accumulation in the liver.

Therefore, through the above results, the enterococcus faecalis EF-2001 killed cells according to the present invention inhibited fatty liver and liver damage, reduced TG lipid accumulation, and produced neutral lipid droplets in laboratory animals induced with fatty liver on a high-fat diet Inhibiting, increasing lipase enzyme protein expression, inducing activation of lipase enzymes such as ATGL and MGL, increasing AMPK phosphorylation, and thus inhibiting the SREBP-1c signaling pathway. Enterococcus faecalis, its culture medium or its dead cell body is a pharmaceutical composition for preventing or treating fatty liver and can be very useful for preventing, improving, and treating fatty liver caused by a high-fat diet.

Preparation examples for the composition of the present invention are also illustrated.

<제조예 1> 본 발명의 엔테로코커스 패칼리스 EF-2001을 유효성분으로 함유하는 약학적 제제의 제조<Preparation Example 1> Preparation of a pharmaceutical preparation containing Enterococcus faecalis EF-2001 of the present invention as an active ingredient

<1-1> 산제의 제조<1-1> Preparation of powder

Enterococcus faecalis of the present invention EF-2001 10 mg

1 g lactose

A powder was prepared by mixing the above components and filling in airtight bags.

<1-2> 정제의 제조<1-2> Manufacture of tablets

Enterococcus faecalis of the present invention EF-2001 0.1 mg

Corn Starch 100 mg

100 mg milk sugar

Magnesium stearate 2 mg

After mixing the above ingredients, tablets were prepared by tableting according to a conventional tablet manufacturing method.

<1-3> 캡슐제의 제조<1-3> Preparation of capsules

Enterococcus faecalis of the present invention EF-2001 0.1 mg

Corn Starch 100 mg

100 mg milk sugar

Magnesium stearate 2 mg

After mixing the above ingredients, capsules were prepared by filling gelatin capsules according to a conventional capsule preparation method.

<1-4> 환의 제조<1-4> Preparation of ring

Enterococcus faecalis of the present invention EF-2001 1 mg

lactose 1.5 g

1 g of glycerin

0.5 g xylitol

After mixing the above components, it was prepared to be 4 g per ring according to a conventional method.

<1-5> 과립의 제조<1-5> Preparation of granules

Enterococcus faecalis of the present invention EF-2001 0.15 mg

Glucose 200 mg

Starch 600 mg

After mixing the above components, 100 mg of 30% ethanol was added and dried at 60° C. to form granules, which were then filled into bags.

<제조예 2> 본 발명의 엔테로코커스 패칼리스 EF-2001을 유효성분으로 함유하는 건강식품의 제조<Preparation Example 2> Preparation of health food containing Enterococcus faecalis EF-2001 of the present invention as an active ingredient

Enterococcus faecalis of the present invention EF-2001 1 mg

Appropriate amount of vitamin mixture

Vitamin A Acetate 70 μg

Vitamin E 1.0 mg

Vitamin 0.13 mg

Vitamin B2 0.15 mg

Vitamin B6 0.5 mg

Vitamin B12 0.2 μg

Vitamin C 10 mg

10 μg of biotin

Nicotinamide 1.7 mg

50 mg folic acid

Calcium Pantothenate 0.5 mg

Appropriate amount of mineral mixture

Ferrous sulfate 1.75 mg

Zinc Oxide 0.82 mg

Magnesium Carbonate 25.3 mg

Potassium Phosphate Monobasic 15 mg

Dibasic Calcium Phosphate 55 mg

Potassium citrate 90 mg

Calcium Carbonate 100 mg

Magnesium Chloride 24.8 mg

Although the composition ratio of the above vitamin and mineral mixture was prepared by mixing ingredients suitable for relatively healthy food in a preferred embodiment, the mixing ratio may be arbitrarily modified, and the above ingredients are mixed according to a normal health food manufacturing method, and then , Granules can be prepared and used in the preparation of health food compositions according to conventional methods.

<제조예 3> 본 발명의 엔테로코커스 패칼리스 EF-2001을 유효성분으로 함유하는 건강음료의 제조<Preparation Example 3> Preparation of a health drink containing Enterococcus faecalis EF-2001 of the present invention as an active ingredient

Enterococcus faecalis of the present invention EF-2001 1 mg

Citric Acid 1000 mg

100 g of oligosaccharides

2 g plum concentrate

1 g of taurine

Total 900 ml with purified water added

After mixing the above ingredients according to the usual health drink manufacturing method, stirring and heating at 85 ° C. for about 1 hour, the resulting solution is filtered and collected in a sterilized container, sealed and sterilized, and then refrigerated. Health drink composition used in manufacturing.

Although the composition ratio is a mixture of ingredients suitable for a relatively favorite beverage in a preferred embodiment, the mixing ratio may be arbitrarily modified according to regional and ethnic preferences such as the class of demand, the country of demand, and the purpose of use.

The present invention relates to a composition for preventing or treating fatty liver comprising Enterococcus faecalis as an active ingredient, and specifically, dead cells of Enterococcus faecalis EF-2001 are used in laboratory animals whose fatty liver is induced by a high-fat diet. Inhibits fatty liver and liver damage, reduces TG lipid accumulation, inhibits neutral lipid droplet production, increases lipase enzyme protein expression, induces activation of lipase enzymes such as ATGL and MGL, increases AMPK phosphorylation, Since the effect of inhibiting the SREBP-1c signal transduction pathway is excellent through this, it can be usefully used as a pharmaceutical composition for preventing or treating fatty liver.

Claims

Enterococcus faecalis ( Enterococcus faecalis ), a culture medium thereof, and a pharmaceutical composition for preventing or treating fatty liver comprising at least one selected from the group consisting of dead cells thereof as an active ingredient.
The pharmaceutical composition for preventing or treating fatty liver according to claim 1, wherein the Enterococcus faecalis is Enterococcus faecalis EF-2001.
The method of claim 1, wherein the killed cells are cultured at pH 4.0 to 9.0 and a temperature range of 15 to 45 ° C. after culturing Enterococcus faecalis as a seed, and then killed by heat treatment. Characterized in that, A pharmaceutical composition for preventing or treating fatty liver.
According to claim 3, wherein the heat treatment is characterized in that made for 1 to 40 minutes in the temperature range of 60 to 140 ℃, fatty liver prevention or treatment pharmaceutical composition.
The method of claim 1, wherein the fatty liver is alcoholic fatty liver (Alcoholic Fatty Liver), alcoholic steatohepatitis (ASH), alcoholic cirrhosis, nonalcoholic fatty liver (Nonalcoholic Fatty Liver (NAFL)), nonalcoholic steatohepatitis (Nonalcoholic Steatohepatitis, NASH) and non-alcoholic cirrhosis characterized in that any one or more selected from, a pharmaceutical composition for preventing or treating fatty liver.
The pharmaceutical composition for preventing or treating fatty liver according to claim 1, wherein the composition inhibits lipid accumulation in liver tissue.
The pharmaceutical composition for preventing or treating fatty liver according to claim 1, wherein the composition inhibits liver damage.
Enterococcus faecalis, a culture medium thereof, and a health functional food composition for preventing or improving fatty liver, comprising at least one selected from the group consisting of dead cells thereof as an active ingredient.
Enterococcus faecalis, a culture medium thereof, and a food additive for preventing or improving fatty liver containing at least one selected from the group consisting of dead cells thereof as an active ingredient.
A method for preventing or improving fatty liver comprising the step of administering to a subject any one or more selected from the group consisting of a pharmaceutically effective amount of Enterococcus faecalis its culture medium and dead cells thereof.
A method for treating fatty liver comprising administering to a subject at least one selected from the group consisting of a pharmaceutically effective amount of Enterococcus faecalis, its culture medium, and its dead cells.
Any one or more uses selected from the group consisting of Enterococcus faecalis, its culture medium, and its dead cells for use as a pharmaceutical composition for preventing or treating fatty liver.
Any one or more uses selected from the group consisting of Enterococcus faecalis, its culture medium, and its dead cells for use as a health functional food composition for preventing or improving fatty liver.
Any one or more uses selected from the group consisting of Enterococcus faecalis, its culture medium, and its dead cells for use as a food additive for preventing or improving fatty liver.