WO2023153903A1 - Composition pour la prévention ou le traitement de la stéatose hépatique, comprenant, en tant que principe actif, enterococcus faecalis, sa solution de culture ou ses cellules mortes - Google Patents

Composition pour la prévention ou le traitement de la stéatose hépatique, comprenant, en tant que principe actif, enterococcus faecalis, sa solution de culture ou ses cellules mortes Download PDF

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WO2023153903A1
WO2023153903A1 PCT/KR2023/002085 KR2023002085W WO2023153903A1 WO 2023153903 A1 WO2023153903 A1 WO 2023153903A1 KR 2023002085 W KR2023002085 W KR 2023002085W WO 2023153903 A1 WO2023153903 A1 WO 2023153903A1
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enterococcus faecalis
fatty liver
dead cells
preventing
liver
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PCT/KR2023/002085
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English (en)
Korean (ko)
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김택중
이진호
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연세대학교 원주산학협력단
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Publication of WO2023153903A1 publication Critical patent/WO2023153903A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health

Definitions

  • 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.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • microorganisms of the genus Enterococcus exist widely in nature and use carbohydrates aerobically.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 .
  • 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.
  • 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.
  • FIG. 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
  • 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.
  • 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 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.
  • FIG. 9 is a diagram schematically illustrating the mechanism of inhibiting fatty liver of dead cells of Enterococcus faecalis EF-2001.
  • 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.
  • alcoholic fatty liver Alcoholic Fatty Liver
  • ASH alcoholic steatohepatitis
  • NAFL non-alcoholic simple fatty liver
  • NASH Nonalcoholic Steatohepatitis
  • NASH Nonalcoholic Steatohepatitis
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the Enterococcus faecalis may be Enterococcus faecalis EF-2001 ( Enterococcus faecalis EF-2001).
  • 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.
  • 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.
  • the suspension is, for example, Dead cell suspension obtained by heating at 80 to 115 ° C.
  • 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.
  • 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.
  • the dead cells may be prepared by the following methods, but are not limited thereto:
  • step 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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), ⁇ -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.
  • '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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Enterococcus faecalis its culture medium or its dead cells are added according to the present invention.
  • 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
  • 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.
  • 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.
  • 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.
  • natural flavoring agents thaumatin, stevia extract (eg rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can advantageously be used. .
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Example 1 Enterococcus faecalis EF-2001 ( Enterococcus faecalis EF-2001) Manufacturing dead cells
  • Enterococcus faecalis EF-2001 live bacteria 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.
  • DW dry weight
  • AICAR 5-aminoimidazole-4-carboxamide-1- ⁇ -D-ribofuranoside
  • AMPK activator an AMPK activator
  • compound C an AMPK inhibitor
  • mice 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).
  • ALT aspartate aminotransferase
  • AST aspartate transaminase
  • 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 .
  • 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.
  • OA oleic acid
  • FL83B cells were treated with or without 25, 50, or 100 ⁇ g/ml of Enterococcus faecalis EF-2001 for 24 hours.
  • 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 ⁇ g/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.
  • PBS phosphate-buffered saline
  • formaldehyde Junsei Chemical, Japan
  • 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.
  • 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.
  • the primary antibodies (SREBP-1C, P-AMPK, AMPK, FAS, P-ACC, ACC, ATGL, P-HSL MGL, CD36 and ⁇ -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.
  • TBS Tris-buffered saline
  • PVDF polyvinylidene difluoride
  • Antibody treatment was performed with primary antibodies, and signal intensities were quantified using Image J software (NIH, USA).
  • 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).
  • 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.
  • FL83B cells were pretreated with 0.5 mM OA in a serum-free medium, and then treated with 25, 50 or 100 ⁇ g/ml of killed cells of Enterococcus faecalis EF-2001 for 24 hours.
  • the effect of dead cells of Enterococcus faecalis EF-2001 on hepatic lipid accumulation was examined from ORO staining.
  • 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.
  • the enterococcus faecalis EF-2001 killed cells 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.
  • Enterococcus faecalis of the present invention EF-2001 10 mg
  • a powder was prepared by mixing the above components and filling in airtight bags.
  • Enterococcus faecalis of the present invention EF-2001 0.1 mg
  • tablets were prepared by tableting according to a conventional tablet manufacturing method.
  • Enterococcus faecalis of the present invention EF-2001 0.1 mg
  • capsules were prepared by filling gelatin capsules according to a conventional capsule preparation method.
  • Enterococcus faecalis of the present invention EF-2001 1 mg
  • Enterococcus faecalis of the present invention EF-2001 0.15 mg
  • Enterococcus faecalis of the present invention EF-2001 1 mg
  • Vitamin A Acetate 70 ⁇ g
  • Vitamin B6 0.5 mg
  • Vitamin B12 0.2 ⁇ g
  • 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.
  • Enterococcus faecalis of the present invention EF-2001 1 mg
  • Health drink composition used in manufacturing.
  • 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.

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Abstract

La présente invention concerne une composition pour la prévention ou le traitement de la stéatose hépatique, comprenant Enterococcus faecalis en tant que principe actif. En particulier, il a été identifié que des cellules mortes d'Enterococcus faecalis EF-2001 empêchent une stéatose hépatique et une lésion hépatique chez des animaux expérimentaux dans lesquels la stéatose hépatique est induite par une alimentation à haute teneur en matières grasses, réduisent l'accumulation de lipides TG, inhibent la génération de gouttelettes de triglycéride, augmentent l'expression de protéine enzymatique de lipase, induisent l'activation d'enzymes lipases telles que ATGL et MGL, et augmentent la phosphorylation d'AMPK de sorte à inhiber la voie de signalisation SREBP-1c, et ainsi l'Enterococcus faecalis ; une solution de culture de celle-ci ou des cellules mortes de celle-ci peuvent être très efficacement utilisées en tant que composition pharmaceutique pour prévenir ou traiter une stéatose hépatique.
PCT/KR2023/002085 2022-02-11 2023-02-13 Composition pour la prévention ou le traitement de la stéatose hépatique, comprenant, en tant que principe actif, enterococcus faecalis, sa solution de culture ou ses cellules mortes WO2023153903A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104415062A (zh) * 2013-08-27 2015-03-18 弘光科技大学 使用包含有4株乳酸菌菌株的混合物来预防和/或缓解酒精性肝病变
KR101508586B1 (ko) * 2013-10-21 2015-04-08 한국식품연구원 비만억제능이 우수한 신규한 엔테로코커스 휘칼리스 md366 균주
KR101873193B1 (ko) * 2010-06-08 2018-07-02 아사히 그룹 홀딩스 가부시키가이샤 지질 대사 개선제
KR20210075916A (ko) * 2019-10-24 2021-06-23 (주)닥터티제이 엔테로코커스 패칼리스를 유효성분으로 함유하는 비만 또는 비만으로부터 유도된 대사증후군의 예방 또는 치료용 조성물
CN113164530A (zh) * 2018-11-06 2021-07-23 中国科学院动物研究所 粪肠球菌的用途

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101873193B1 (ko) * 2010-06-08 2018-07-02 아사히 그룹 홀딩스 가부시키가이샤 지질 대사 개선제
CN104415062A (zh) * 2013-08-27 2015-03-18 弘光科技大学 使用包含有4株乳酸菌菌株的混合物来预防和/或缓解酒精性肝病变
KR101508586B1 (ko) * 2013-10-21 2015-04-08 한국식품연구원 비만억제능이 우수한 신규한 엔테로코커스 휘칼리스 md366 균주
CN113164530A (zh) * 2018-11-06 2021-07-23 中国科学院动物研究所 粪肠球菌的用途
KR20210075916A (ko) * 2019-10-24 2021-06-23 (주)닥터티제이 엔테로코커스 패칼리스를 유효성분으로 함유하는 비만 또는 비만으로부터 유도된 대사증후군의 예방 또는 치료용 조성물

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