KR20220091428A - A composition for improving, preventing and treating fatty liver comprising extracellular vesicles from Leuconostoc citreum - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating fatty liver comprising Leuconostoc citreum-derived extracellular vesicles as an active ingredient. Thereby, the composition of the present invention suppresses the accumulation of fat in hepatocytes, promotes the expression of Claudin 1, a tight junction gene, and strengthens the tight junction between enterocytes, thereby inhibiting the movement of inflammatory substances to the fatty liver. can be prevented, treated or ameliorated.

Description

A composition for improving, preventing or treating fatty liver comprising an extracellular vesicle derived from Leuconostoc citrium as an active ingredient TECHNICAL FIELD

The present invention relates to a composition for improving, preventing or treating fatty liver.

Fatty liver is a disease in which fat is accumulated in hepatocytes due to the ingestion of fat and consequent accumulation of fat in the liver, increase in synthesis, decrease in decomposition, and excessive alcohol intake. The number of patients with fatty liver is increasing rapidly due to a westernized high-fat, high-calorie diet and lack of exercise. If fat is continuously accumulated in the liver, it progresses to steatohepatitis accompanied by inflammation, and it progresses to necrosis or fibrosis of the liver tissue, which is known as a major cause of cirrhosis and liver cancer.

The fatty liver can be classified into alcoholic fatty liver disease (Alcoholic Steatosis, Alcoholic Fatty Liver Disease) and nonalcoholic fatty liver disease (NAFLD, Nonalcoholic Fatty Liver Disease) caused by chronic drinking, and non-alcoholic fatty liver disease is generally a two-stage disease can be distinguished as First, as the most common type, there is nonalcoholic simple fatty liver (NAFL, Nonalcoholic Fatty Liver), and secondly, there is nonalcoholic steatohepatitis (NASH, Nonalcoholic Steatohepatitis) as a type of severe disease. Such nonalcoholic steatohepatitis is a disease in which inflammation is induced in liver cells by fatty liver.

In particular, there is no approved treatment for nonalcoholic steatohepatitis, and the development of a treatment is urgently needed. Recently, as research on the microbiome is progressing, it is showing the improvement effect of microbial fatty liver or steatohepatitis. In addition, in the case of microorganism-derived extracellular ER, it is known that it can induce changes in the colony of intestinal microbes through interaction between microorganisms, and play an important role in many physiological and pathological situations through interaction with host cells. Accordingly, studies are being conducted to find substances for the prevention and treatment of fatty liver using microorganism-derived substances that have little toxicity and side effects.

Korean Patent No. 10-2032982

It is an object of the present invention to inhibit the movement of inflammatory substances by inhibiting fat accumulation in hepatocytes and strengthening the tight junction between enterocytes by promoting the expression of Claudin 1, a tight junction gene, to prevent or treat fatty liver. To provide a pharmaceutical composition.

Another object of the present invention is to inhibit the accumulation of fat in hepatocytes and promote the expression of Claudin 1, a close junction-related gene, to strengthen the close junction between enterocytes, thereby inhibiting the movement of inflammatory substances to prevent or improve fatty liver. to provide a composition, and a feed or feed additive.

According to one aspect of the present invention,

A pharmaceutical composition for preventing or treating fatty liver comprising Leuconostoc citreum-derived extracellular vesicles as an active ingredient is provided.

The fatty liver may be any one selected from alcoholic fatty liver (Alcoholic Fatty Liver), alcoholic steatohepatitis (ASH), nonalcoholic simple fatty liver (NAFL, Nonalcoholic Fatty Liver) and nonalcoholic steatohepatitis (NASH, Nonalcoholic Steatohepatitis). .

The pharmaceutical composition for preventing or treating fatty liver may be for inhibiting fatty acid accumulation in hepatocytes.

The pharmaceutical composition for preventing or treating fatty liver may be for promoting expression of a tight junction gene in enterocytes.

The tight junction gene may be Claudin 1.

The leuconostoc citrium may be leuconostoc citreum WiKim0104 (Leuconostoc citreum WiKim0104) deposited with accession number KCCM12420P.

According to another aspect of the present invention,

There is provided a food composition for preventing or improving fatty liver comprising Leuconostoc citreum-derived extracellular vesicles as an active ingredient.

According to another aspect of the present invention,

A feed or feed additive for preventing or improving fatty liver comprising Leuconostoc citreum-derived extracellular vesicles as an active ingredient is provided.

According to another aspect of the present invention,

(A) culturing Leuconostoc citreum (Leuconostoc citreum);

(b) recovering the culture supernatant by centrifuging the culture medium in which the leuconostok citrium is cultured; and

(c) mixing and reacting the culture supernatant with a solution containing sodium chloride and an organic solvent, followed by centrifugation to obtain an extracellular vesicle pellet; and

(d) preparing a suspension by resuspending the extracellular vesicle pellet in an extracellular vesicle extraction buffer, and separating the extracellular vesicles from the suspension; a method for preparing a composition for preventing, treating or improving fatty liver is provided do.

The pharmaceutical composition for preventing or treating fatty liver of the present invention inhibits fat accumulation in hepatocytes and promotes the expression of Claudin 1, a tight junction gene, to strengthen tight junctions between intestinal cells, thereby moving inflammatory substances By inhibiting fatty liver, it is possible to prevent or treat fatty liver.

The food composition for preventing or improving fatty liver of the present invention, and feed or feed additive inhibit fat accumulation in hepatocytes and promote the expression of Claudin 1, a tight junction gene, to form a tight junction between intestinal cells. By strengthening it, it is possible to prevent or improve fatty liver by inhibiting the movement of inflammatory substances.

1 is an electron micrograph of an extracellular vesicle derived from Leukonostok citium isolated according to Example 1. FIG.
2 is an analysis result of the effect on fat accumulation in HepG2 cells according to Experimental Example 1.
3 is a result of close junction-related gene expression analysis in HT-29 cells according to Experimental Example 2.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings so that those of ordinary skill in the art can easily carry out the present invention. However, the following description is not intended to limit the present invention to specific embodiments, and when it is determined that detailed descriptions of related known technologies may obscure the gist of the present invention in describing the present invention, the detailed description thereof will be omitted. . The terminology used herein is used only to describe specific embodiments, and is not intended to limit the present invention. The singular expression includes the plural expression unless the context clearly dictates otherwise. In the present application, terms such as "comprise" or "have" are intended to designate that a feature, number, step, operation, element, or combination thereof described in the specification exists, and includes one or more other features or It should be understood that the possibility of the presence or addition of numbers, steps, acts, elements, or combinations thereof is not precluded in advance.

The pharmaceutical composition for preventing or treating fatty liver of the present invention includes Leuconostoc citreum-derived extracellular vesicles as an active ingredient.

In the present specification, the leukonostok citrium is not limited, but is preferably a leukonostok citrium derived from kimchi, and more preferably a leukonostok citrium deposited with accession number KCCM12420P.

Leukonostok citrium of the accession number KCCM12420P was deposited with the Korea Microorganism Conservation Center on December 14, 2018.

The fatty liver may be any one selected from alcoholic fatty liver (Alcoholic Fatty Liver), alcoholic steatohepatitis (ASH), nonalcoholic simple fatty liver (NAFL, Nonalcoholic Fatty Liver) and nonalcoholic steatohepatitis (NASH, Nonalcoholic Steatohepatitis). .

The pharmaceutical composition for preventing or treating fatty liver may be for inhibiting fatty acid accumulation in hepatocytes.

The pharmaceutical composition for preventing or treating fatty liver may be for promoting expression of a tight junction gene in enterocytes.

The tight junction gene may be Claudin 1.

A tight junction is a type of cell junction that limits the movement of substances between connected cells, and is wound around the cell in the form of a band. A tight junction can be formed by partially fusion of the cell membrane of an adjacent cell by a protein called claudin and occludin. It is mainly present in humans and vertebrates, and it can also play a role in preventing harmful substances in the blood from reaching the cerebrospinal fluid by creating a blood-brain barrier.

As used herein, the term "extracellular vesicle" refers to a vesicle having a lipid bilayer structure with a diameter ranging from 20 to 1,000 nm that is secreted into the extracellular environment through the fusion of polycystic bodies and plasma membranes in various cells.

According to a specific embodiment of the present invention, the average diameter of the extracellular vesicles produced by the method of the present invention may be in the range of 20 to 500 nm, preferably 50 to 300 nm, more preferably 100 to 200 nm. . Extracellular vesicles having a microdiameter within this range are called exosomes.

Hereinafter, a method for preparing a pharmaceutical composition for preventing or treating fatty liver comprising Leuconostoc citreum-derived extracellular vesicles as an active ingredient will be described.

First, culturing Leuconostoc citreum (step a).

Leukonostok citrium can be obtained from the stock solution of the kimchi extract.

Next, the culture supernatant is recovered by centrifugation of the culture medium in which the Leukonostok citrium is cultured (step b).

Thereafter, the culture supernatant is mixed with a solution containing sodium chloride and an organic solvent and reacted, followed by centrifugation to obtain an extracellular vesicle pellet (step c).

The culture supernatant and the solution containing sodium chloride and the organic solvent may be mixed in a volume ratio of 1:0.5 to 1:1.5, preferably in a volume ratio of 1:0.8 to 1:1.2, more preferably the same amount can be mixed with

The organic solvent may be polyethylene glycol.

The solution containing sodium chloride and the organic solvent preferably has a sodium chloride concentration of 0.5 to 1 M.

After the reaction is mixed with the solution containing the sodium chloride and the organic solvent, centrifugation may be performed, and then the mixing and centrifugation may be repeated.

Finally, the extracellular vesicle pellet is resuspended in extracellular vesicle extraction buffer to prepare a suspension, and the extracellular vesicles are isolated from the suspension (step d).

It is preferable to use the extracellular vesicle extraction buffer (Phosphate-buffered saline).

On the other hand, in the present specification, the term 'comprising as an active ingredient' means including an amount sufficient to achieve the efficacy or activity of leukonostok citrium-derived extracellular vesicles. In one embodiment of the present invention, the leukonostok citrium-derived extracellular vesicles in the composition of the present invention are, for example, 0.001 mg/kg or more, preferably 0.1 mg/kg or more, more preferably 10 mg/kg or more. kg or more, even more preferably 100 mg/kg or more, even more preferably 250 mg/kg or more, and most preferably 1 g/kg or more. Leuconostok citrium-derived extracellular vesicles are natural products, and since there is no side effect to the human body even when administered in excess, the upper quantitative limit of the leuconostok citrium-derived extracellular vesicles contained in the composition of the present invention is selected and carried out by those skilled in the art within an appropriate range can do.

The pharmaceutical composition of the present invention may be prepared by using a pharmaceutically suitable and physiologically acceptable adjuvant in addition to the active ingredient, and the adjuvant includes an excipient, a disintegrant, a sweetener, a binder, a coating agent, a swelling agent, a lubricant, and a lubricant. agent or flavoring agent, etc. may be used.

The pharmaceutical composition may be preferably formulated as a pharmaceutical composition by including one or more pharmaceutically acceptable carriers in addition to the active ingredients described above for administration.

Formulations of the pharmaceutical composition may be granules, powders, tablets, coated tablets, capsules, suppositories, solutions, syrups, juices, suspensions, emulsions, drops or injectables. For example, for formulation in the form of a tablet or capsule, the active ingredient may be combined with an orally, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. In addition, if desired or required, suitable binders, lubricants, disintegrants and color-developers may also be included in the mixture. Suitable binders include, but are not limited to, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tracacanth or sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.

In the composition formulated as a liquid solution, acceptable pharmaceutical carriers are sterile and biocompatible, and include saline, sterile water, Ringer's solution, buffered saline, albumin injection, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostats may be added as needed. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to form an injectable formulation such as an aqueous solution, suspension, emulsion, etc., pills, capsules, granules or tablets.

The pharmaceutical composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, it may be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, etc., preferably oral administration.

A suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, administration mode, age, weight, pathological condition, food, administration time, route of administration, excretion rate, and response sensitivity of the patient, usually skilled A trained physician can readily determine and prescribe an effective dosage for the desired treatment or prevention. According to a preferred embodiment of the present invention, the daily dose of the pharmaceutical composition of the present invention is 0.001-10 g/kg.

The pharmaceutical composition of the present invention is prepared in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person of ordinary skill in the art to which the present invention pertains. Alternatively, it may be prepared by being introduced into a multi-dose container. At this time, the formulation may be in the form of a solution, suspension, or emulsion in oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet or capsule, and may additionally include a dispersant or stabilizer.

The present invention provides a food composition for preventing or improving fatty liver comprising Leuconostoc citreum-derived extracellular vesicles as an active ingredient.

The leuconostok citrium is not limited, but is preferably a leukonostok citrium derived from kimchi, and more preferably a leuconostok citrium deposited under the accession number KCCM12420P.

The content of the leuconostok citrium-derived extracellular vesicles is the same as the description in the pharmaceutical composition for preventing or treating fatty liver comprising the above-described leuconostok citrium-derived extracellular vesicles as an active ingredient. shall refer to.

The food composition according to the present invention may be formulated in the same manner as the pharmaceutical composition and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, alcoholic beverages, sweets, diet bars, dairy products, meat, chocolate, pizza, ramen, other noodles, gums, ice cream, vitamin complexes, health supplements etc.

The food composition of the present invention may include not only the active ingredients, but also ingredients commonly added during food production, for example, proteins, carbohydrates, fats, nutrients, seasonings and flavoring agents. Examples of the above-mentioned carbohydrates include monosaccharides such as glucose, fructose and the like; disaccharides such as maltose, sucrose, oligosaccharides and the like; and polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin, and the like, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents, natural flavoring agents [taumatine, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used. For example, when the food composition of the present invention is prepared as a drink or beverage, citric acid, high fructose, sugar, glucose, acetic acid, malic acid, fruit juice, and various plant extracts may be additionally included in addition to the active ingredient of the present invention.

The present invention provides a health functional food comprising a food composition for preventing or improving fatty liver comprising the leuconostok citrium-derived extracellular vesicles as an active ingredient. Health functional food is a food prepared by adding Leukonostok citrium-derived extracellular vesicles to food materials such as beverages, teas, spices, gum, and confectionery, or by encapsulating, powdering, or suspension, etc. It means to bring a specific effect, but unlike general drugs, it has the advantage of not having side effects that may occur when taking the drug for a long period of time by using food as a raw material. The health functional food of the present invention obtained in this way is very useful because it can be ingested on a daily basis. The amount of leukonostok citrium-derived extracellular vesicles added in such health functional food varies depending on the type of health functional food and cannot be defined uniformly, but it can be added within a range that does not impair the original taste of the food. , It is usually in the range of 0.01 to 50% by weight, preferably 0.1 to 20% by weight, based on the target food. In addition, in the case of a health functional food in the form of pills, granules, tablets or capsules, it is usually added in an amount of 0.1 to 100% by weight, preferably 0.5 to 80% by weight. In one embodiment, the health functional food of the present invention may be in the form of a pill, tablet, capsule or beverage.

The leukonostok citrium-derived extracellular vesicles according to the present invention or a composition comprising the same as an active ingredient may be used as a feed additive or feed.

When used as a feed additive, the composition may be 20 to 90% highly concentrated or prepared in powder or granular form. The feed additives include organic acids such as citric acid, humic acid, adipic acid, lactic acid, and malic acid, phosphates such as sodium phosphate, potassium phosphate, acid pyrophosphate, polyphosphate (polyphosphate), polyphenol, catechin, alpha-tocopherol, rosemary Extract, vitamin C, green tea extract, licorice extract, chitosan, tannic acid, any one or more of natural antioxidants such as phytic acid may be further included. When used as feed, the composition may be formulated in the form of a conventional feed, and may include common feed ingredients together.

The feed additive and feed include grains such as milled or crushed wheat, oats, barley, corn and rice; plant protein feeds, such as feeds based on rape, soybean, and sunflower; animal protein feeds such as blood meal, meat meal, bone meal and fish meal; Sugar and dairy products, for example, may further include dry ingredients made of various powdered milk and whey powder, and may further include nutritional supplements, digestion and absorption enhancers, growth promoters, and the like.

The feed additive may be administered alone or in combination with other feed additives in an edible carrier. In addition, the feed additive can be easily administered to the animal as a top dressing, directly mixing them with animal feed, or in an oral formulation separate from the feed. When the feed additive is administered separately from animal feed, it may be combined with a pharmaceutically acceptable edible carrier as well known in the art, and may be prepared into an immediate release or sustained release formulation. Such edible carriers may be solid or liquid, for example cornstarch, lactose, sucrose, soybean flakes, peanut oil, olive oil, sesame oil and propylene glycol. When a solid carrier is used, the feed additive may be a tablet, a capsule, a powder, a troche or a sugar-containing tablet, or a top dressing in a microdispersed form. When a liquid carrier is used, the feed additive may be in the form of a gelatin soft capsule, or a syrup, suspension, emulsion, or solution.

In addition, the feed additives and feed may contain auxiliary agents, for example, preservatives, stabilizers, wetting or emulsifying agents, solution accelerators, and the like. The feed additive may be used by adding to animal feed by immersion, spraying, or mixing.

The feed or feed additive of the present invention can be applied to a number of animal diets including mammals, poultry and fish.

As the mammal, it can be used for pigs, cattle, sheep, goats, laboratory rodents, and laboratory rodents, as well as pets (eg, dogs, cats), etc., as the poultry, chickens, turkeys, ducks, geese, pheasants, and quail, and the like, and may be used as the fish such as trout, but is not limited thereto.

In addition, the present invention provides a use of the leukonostok citrium-derived extracellular vesicles for the manufacture of a medicament or food for preventing, treating or improving fatty liver. As described above, the leukonostok citrium-derived extracellular vesicles can be used for the treatment or improvement of fatty liver.

In addition, the present invention provides a method for improving, preventing or treating fatty liver comprising administering to a mammal an effective amount of an extracellular vesicle derived from leukonostok citrium.

As used herein, the term “mammal” refers to a mammal that is the subject of treatment, observation or experimentation, preferably a human.

As used herein, the term "effective amount" refers to the amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human as conceived by a researcher, veterinarian, physician or other clinician, which is the disease in question. or an amount that induces alleviation of the symptoms of the disorder. The effective amount and frequency of administration for the active ingredient of the present invention may vary depending on the desired effect. Therefore, the optimal dosage to be administered can be easily determined by those skilled in the art, and the type of disease, the severity of the disease, the content of active ingredients and other components contained in the composition, the type of formulation, and the age, weight, and general health of the patient It can be adjusted according to various factors including state, sex and diet, administration time, administration route and secretion rate of the composition, treatment period, and concurrently used drugs. In the prevention, treatment or improvement method of the present invention, for adults, it is preferable to administer the extract at a dose of 0.001 g/kg to 10 g/kg when administered once to several times a day.

In the treatment method of the present invention, the composition comprising leuconostok citrium-derived extracellular vesicles as an active ingredient is administered by oral, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, topical, or intradermal routes. It can be administered in a conventional manner.

Hereinafter, the present invention will be described in more detail through examples, etc., but the scope and contents of the present invention cannot be construed as being reduced or limited by the following examples. In addition, based on the disclosure of the present invention including the following examples, it is clear that a person skilled in the art can easily practice the present invention for which specific experimental results are not presented. It goes without saying that it falls within the scope of the claims.

In addition, the experimental results presented below describe only the representative experimental results of the Examples and Comparative Examples, and the respective effects of various embodiments of the present invention that are not explicitly presented below will be described in detail in the corresponding part.

[Example]

Example 1: Isolation of Leukonostok Citrium-derived Extracellular Vesicles

(1) strain culture

The Leukonostok Citrium Wikim0104 (Accession No. KCCM12420P) strain was tested with the permission of the Korea Food Research Institute, the depositor. 0.1% of the received leuconostok citrium strain was inoculated into 30 ml of MRS liquid medium and cultured at 30° C. for 18 hours. After incubation, centrifuged at 3000 rpm for 10 minutes, the culture medium was stored separately for nano-vesicle separation, and the cells were washed three times with a PBS (phosphate buffered saline) solution to remove the remaining medium components.

(2) Isolation of extracellular vesicles

The culture solution obtained after culturing the strain and 1M NaCl solution containing 16% PEG 6000 were mixed in equal amounts and reacted at 4°C for 15 hours. Then, the reacted solution was centrifuged at 10,000 g and 4° C. for 20 minutes to obtain an extracellular vesicle pellet. The nano-ER pellet was resuspended in 0.5M NaCl solution containing 5% PEG6000 and washed, followed by centrifugation at 11,000 rpm at 4°C for 20 minutes. Thereafter, the supernatant was discarded and the pellet was resuspended in 1X PBS to finally separate the leukonostok citrium-derived extracellular vesicles. An electron microscope image of the isolated leuconostok citrium-derived extracellular vesicles is shown in FIG. 1 .

Comparative Example 1: Strain culture solution

A leukonostok citrium strain culture solution in (1) of Example 1 was prepared .

Comparative Example 2: Leukonostok citrium strain

A strain of leukonostok citrium (accession number KCCM12420P) cultured according to (1) of Example 1 was prepared.

Comparative Example 3: Particles derived from culture medium

In the MRS liquid medium in which the Leukonostok citrium strain was not cultured, the particles were separated in the same manner as the extracellular vesicle separation method in (2) of Example 1.

[Experimental example]

Experimental Example 1: Analysis of the effect on fat accumulation in HepG2 cells

An in vitro enterocyte-hepatocyte co-culture experiment using a transwell was devised to resemble the gut-liver axis. HT-29 cells, which are human-derived colorectal cancer cells, and HepG2 cells, which are human-derived liver cancer cells, were prepared using RPMI-1640 medium supplemented with penicillin/streptomycin and 10% fetal bovine serum (FBS) at 37° C., 5% CO ₂ condition. It was prepared by culturing in HT-29 cells were plated in the upper chamber of a 12-well plate Transwell chamber (4 μm pore size), and a cover slip was put in the lower chamber, and 2X10 ⁵ HepG2 cells were plated thereon. After 24 hours, HepG2 cells in the lower chamber were treated with 250 μM of oleic acid and palmitic acid in serum free media for 24 hours to induce adipogenesis and accumulation in hepatocytes. .

Leukonostok citrium-derived extracellular vesicles isolated according to Example 1 in the upper chamber, the bacterial culture medium of Comparative Example 1, the Leukonostok citrium strain of Comparative Example 2 (1X10 ⁷ CFU), the MRS medium of Comparative Example 3 Each particle was treated for 24 hours. Thereafter, the upper chamber was removed, and the cells in the lower chamber were washed twice with 1X PBS and then fixed with 4% paraformaldehyde at room temperature for 15 minutes. After washing twice with PBS, it was reacted at 37° C. for 15 minutes with 5 μg/ml of BODIPY dye for fat staining. Next, after washing twice with PBS, the cell nucleus was stained with DAPI (4',6-diamidino-2-phenylindole) to observe the accumulation of fatty acids under a fluorescence microscope, and the results are shown in FIG. 2 .

According to this, it was confirmed that the accumulation of fatty acids was significantly inhibited in the treatment group of the leukonostok citrium-derived extracellular vesicles of Example 1, and in particular, compared to the group treated with the Leukonostok citrium strain of Comparative Example 2, fatty acid accumulation inhibition It was confirmed that the effect was remarkable.

Experimental Example 2: Analysis of tight junction-related gene expression in HT-29 cells

It is known that a decrease in the tight junction of enterocytes can induce nonalcoholic steatohepatitis by increasing the movement of inflammatory substances such as LPS (lipopolysaccharide) from the intestine. Real-time polymerase chain reaction (qPCR) was performed to determine the expression level of close junction genes in enterocytes following the treatment of the leukonostok citrium strain-derived extracellular vesicles.

Primer information and PCR test conditions are as follows.

Primer information

h.GAPDH (F) CTC TCT GCT CCT CCT GTT CG

h.GAPDH (R) TGA CTC CGA CCT TCA CCT TC

h.Claudin1 (F) TTG GGC TTC ATT CTC GCC TT

h.Claudin1 (R) GTC GCC GGC ATA GGA GTA AA

PCR conditions

Denaturation 95 °C/ 15 sec;

Annealing 60 ℃/30 sec

Extension 72 ℃/30 sec)

40 cycle

First, HT-29 cells, which are human-derived colorectal cancer cells, were plated on a 6-well plate, and when the cells were sufficiently grown, 1 μg/ml of LPS and 500 μM of palmitic acid were treated in serum-free media. Leukonostok citrium-derived extracellular vesicles of Example 1, the strain culture medium of Comparative Example 1, the Leukonostok citrium bacteria of Comparative Example 2 (1X10 ⁷ CFU), and the particles derived from the culture medium of Comparative Example 3, respectively, for close contact The expression level of the gene claudin 1 (Claudin 1) gene was measured, and the result is shown in FIG. 3 .

According to this, it was confirmed that the expression of claudin was significantly increased in the group treated with the citrium-derived nanovesicles of Example 1 compared to the strain culture treated group of Comparative Example 1. (*p=0.046 vs culture treated group/ *p=0.015 vs strain treated group).

Hereinafter, a formulation example of a composition comprising the powder of the present invention will be described, but the present invention is not intended to limit the present invention, but merely to describe it in detail.

Formulation Example 1. Preparation of powder

500 mg of extracellular vesicles of Example 1

Lactose 100 mg

talc 10 mg

The above ingredients are mixed and filled in an airtight bag to prepare a powder.

Formulation Example 2. Preparation of tablets

300 mg of extracellular vesicles of Example 1

100 mg cornstarch

Lactose 100 mg

2 mg magnesium stearate

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

Formulation Example 3. Preparation of capsules

200 mg of extracellular vesicles of Example 1

3 mg of crystalline cellulose

Lactose 14.8 mg

0.2 mg magnesium stearate

According to a conventional capsule preparation method, the above ingredients are mixed and filled in a gelatin capsule to prepare a capsule.

Formulation Example 4. Preparation of injection

600 mg of the extracellular vesicles of Example 1

mannitol 180 mg

2974 mg of sterile distilled water for injection

Na ₂ HPO _4, 12H ₂ O 26 mg

According to a conventional method for preparing injections, the content of the above components per 1 ampoule is prepared.

Formulation Example 5. Preparation of liquid formulation

4 g of extracellular vesicles of Example 1

10 g isomerized sugar

5 g of mannitol

Purified water appropriate amount

According to a conventional liquid preparation method, each component is added to purified water to dissolve, an appropriate amount of lemon flavor is added, the above components are mixed, purified water is added, the whole is adjusted to 100 g by adding purified water, and then filled in a brown bottle to sterilize to prepare a liquid.

Formulation Example 6. Preparation of granules

1,000 mg of extracellular vesicles of Example 1

appropriate amount of vitamin mixture

70 μg vitamin A acetate

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

0.5 mg of vitamin B6

0.2 μg of vitamin B12

Vitamin C 10 mg

Biotin 10 μg

Nicotinamide 1.7 mg

50 μg of folic acid

Calcium pantothenate 0.5 mg

Mineral mixture appropriate amount

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

100 mg of calcium carbonate

Magnesium chloride 24.8 mg

The composition ratio of the vitamin and mineral mixture is relatively suitable for granules in a preferred embodiment, but the mixing ratio may be arbitrarily modified. It can be prepared and used in the preparation of a health functional food composition according to a conventional method.

Formulation Example 7. Preparation of functional beverage

1,000 mg of extracellular vesicles of Example 1

1,000 mg citric acid

100 g of oligosaccharides

2 g of plum concentrate

1 g taurine

Add purified water to total 900 mL

After mixing the above ingredients according to the usual health drink manufacturing method, after stirring and heating at 85 ° C for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed and sterilized, then refrigerated. It is used to prepare the functional beverage composition of the present invention.

Although the composition ratio is prepared by mixing ingredients suitable for relatively favorite beverages in a preferred embodiment, the mixing ratio may be arbitrarily modified according to regional and national preferences such as demand class, demanding country, and use.

Korea Microorganism Conservation Center KCCM12420P 20181214

<110> LISCure Biosciences <120> A composition for improving, preventing and treating fatty liver comprising extracellular vesicles from Leuconostoc citreum <130> HPC-10416 <150> KR 2020-0181542 <151> 2020-12-23 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> h.GAPDH F <400> 1 ctctctgctc ctcctgttcg 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH R <400> 2 tgactccgac cttcaccttc 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Claudin F <400> 3 ttgggcttca ttctcgcctt 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Claudin R <400> 4 gtcgccggca taggagtaaa 20

Claims (9)

A pharmaceutical composition for preventing or treating fatty liver comprising Leuconostoc citreum-derived extracellular vesicles as an active ingredient. The method of claim 1,
The fatty liver is characterized in that any one selected from alcoholic fatty liver (Alcoholic Fatty Liver), alcoholic steatohepatitis (ASH), nonalcoholic simple fatty liver (NAFL, Nonalcoholic Fatty Liver) and nonalcoholic steatohepatitis (NASH, Nonalcoholic Steatohepatitis) A pharmaceutical composition for preventing or treating fatty liver. The method of claim 1,
The pharmaceutical composition for preventing or treating fatty liver is a pharmaceutical composition for preventing or treating fatty liver, characterized in that it is for inhibiting fatty acid accumulation in hepatocytes. The method of claim 1,
The pharmaceutical composition for preventing or treating fatty liver is a pharmaceutical composition for preventing or treating fatty liver, characterized in that it is for promoting expression of a tight junction gene in enterocytes. The method of claim 1,
The tight junction (tight junction) gene is a fatty liver prevention or treatment pharmaceutical composition, characterized in that Claudin 1. The method of claim 1,
The leuconostoc citrium is a pharmaceutical composition for preventing or treating fatty liver, characterized in that it is leuconostoc citreum WiKim0104 (Leuconostoc citreum WiKim0104) deposited with accession number KCCM12420P. A food composition for preventing or improving fatty liver comprising Leuconostoc citreum-derived extracellular vesicles as an active ingredient. A feed or feed additive for preventing or improving fatty liver comprising Leuconostoc citreum-derived extracellular vesicles as an active ingredient. (a) culturing Leuconostoc citreum;
(b) recovering the culture supernatant by centrifuging the culture medium in which the leuconostok citrium is cultured; and
(c) mixing and reacting the culture supernatant with a solution containing sodium chloride and an organic solvent, followed by centrifugation to obtain an extracellular vesicle pellet; and
(d) preparing a suspension by resuspending the extracellular vesicle pellet in an extracellular vesicle extraction buffer, and isolating the extracellular vesicles from the suspension;

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