Composition for preventing, improving, or treating non-alcoholic fatty liver disease, comprising extracellular vesicles derived from the genus Roseburia or Bifidobacterium

Abstract

The present invention relates to a composition for preventing, improving, or treating non-alcoholic fatty liver disease (NAFLD) comprising Roseburia spp., Bifidobacterium spp., or extracellular vesicles (EVs) derived therefrom. The Roseburia spp., Bifidobacterium spp., or EVs derived therefrom of the present invention can restore liver function, improve hepatic steatosis and inflammation, and inhibit hepatic fibrosis, and therefore may be provided as a composition for preventing, improving, or treating NAFLD.

Description

[Technical field]

[0001] The present invention relates to a composition for preventing, ameliorating, or treating non-alcoholic fatty liver disease (NAFLD), comprising a Roseburia spp. strain, a Bifidobacterium spp. strain, or extracellular vesicles (EVs) derived therefrom.

[0002] The present invention claims priority based on Korean Patent Application No. 10-2022-0008555, filed on January 20, 2022, and Korean Patent Application No. 10-2023-0006808, filed on January 17, 2023, and all contents disclosed in the specification and drawings of said application are incorporated by reference into this application. [Background technology]

[0003] Background technology

[0004] The liver is an organ that plays a central role in metabolism, and abnormal liver function causes problems in the overall nutrient metabolism of an individual. Among liver diseases, fatty liver refers to the phenomenon in which neutral fat, which does not exist in normal cells, is abnormally deposited in liver cells. Approximately 5% of a normal liver is composed of adipose tissue, which consists of neutral fat, fatty acids, phospholipids, cholesterol, and cholesterol esters. When fatty liver begins to develop, most of the fat components are replaced by neutral fat, and if the amount of neutral fat is 5% or more of the total liver weight, fatty liver is diagnosed. When fatty liver worsens and the fat mass in the liver cells grows, important components of the cells, such as the nucleus, are pushed to one side, and the liver cells expanded by the accumulated fat compress the surrounding microvessels and lymph nodes, causing problems in the circulation of blood and lymph in the liver. As a result, the liver cells cannot receive an adequate supply of oxygen and nutrients, and liver function declines.

[0005] Fatty liver can be divided into alcoholic fatty liver caused by excessive drinking and non-alcoholic fatty liver. Non-alcoholic fatty liver disease (NAFLD) is a metabolic liver disease resulting from metabolic dysregulation, and is a disease caused by fat accumulation in the liver that is not related to alcohol consumption. NAFLD includes a variety of diseases ranging from simple steatosis to inflammation-related steatohepatitis and cirrhosis (Brunt EM, 2001), but it can also occur due to fatty deposition in liver cells caused by obesity or insulin resistance, diabetes, or genetic causes. In particular, unlike simple fatty liver, which has a good clinical prognosis, non-alcoholic steatohepatitis (NASH) is a progressive liver disease that has a high probability of progressing to cirrhosis and liver cancer, so timely and appropriate treatment is required (Angulo P., Hepatology, 2010; 51: 373-375).

[0006] As the number of patients with non-alcoholic fatty liver disease continues to increase, there is an increasing need to develop drugs to prevent, improve, and treat the disease. However, there are still no drugs approved as pharmacological treatments for NAFLD, and there is a need to develop a treatment that can fundamentally treat NAFLD. Summary of the Invention [Problem to be solved by the invention]

[0007] Detailed Description of the Invention technical challenges

[0008] The present inventors have made extensive efforts to find a drug having a more effective effect than conventional therapeutic agents for non-alcoholic fatty liver disease, and have confirmed that Roseburia spp. strains, Bifidobacterium spp. strains, or extracellular vesicles (EVs) derived therefrom exhibit excellent preventive, ameliorative, and therapeutic effects against NAFLD, thereby completing the present invention.

[0009] Therefore, an object of the present invention is to provide a pharmaceutical composition for preventing or treating non-alcoholic fatty liver disease, comprising at least one selected from the group consisting of extracellular vesicles derived from Roseburia strains and extracellular vesicles derived from Bifidobacterium strains as an active ingredient.

[0010] Another object of the present invention is to provide a kit for preventing or treating non-alcoholic fatty liver disease, comprising at least one selected from the group consisting of extracellular vesicles derived from Roseburia strains and extracellular vesicles derived from Bifidobacterium strains.

[0011] Yet another object of the present invention is to provide a method for preventing or treating non-alcoholic fatty liver disease, comprising administering to an individual in need thereof at least one selected from the group consisting of extracellular vesicles derived from Roseburia strains and extracellular vesicles derived from Bifidobacterium strains.

[0012] Yet another object of the present invention is to provide a food composition for preventing or ameliorating non-alcoholic fatty liver disease, comprising at least one selected from the group consisting of extracellular vesicles derived from Roseburia strains and extracellular vesicles derived from Bifidobacterium strains.

[0013] Yet another object of the present invention is to provide a feed composition for preventing or ameliorating non-alcoholic fatty liver disease, comprising at least one selected from the group consisting of extracellular vesicles derived from Roseburia strains and extracellular vesicles derived from Bifidobacterium strains.

[0014] However, the technical problems that the present invention aims to solve are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by a person having ordinary skill in the art to which the present invention pertains from the following description. [Means for solving the problem]

[0015] Problem solving means

[0016] The present invention provides a pharmaceutical composition for preventing or treating non-alcoholic fatty liver disease, comprising at least one selected from the group consisting of extracellular vesicles derived from Roseburia spp. strains and extracellular vesicles derived from Bifidobacterium spp. strains as an active ingredient.

[0017] The present invention also provides a food composition for preventing or ameliorating non-alcoholic fatty liver disease, comprising as an active ingredient at least one selected from the group consisting of extracellular vesicles derived from Roseburia strains and extracellular vesicles derived from Bifidobacterium strains.

[0018] The present invention also provides a feed composition for preventing or ameliorating non-alcoholic fatty liver disease, comprising as an active ingredient at least one selected from the group consisting of extracellular vesicles derived from Roseburia strains and extracellular vesicles derived from Bifidobacterium strains.

[0019] The present invention also provides a kit for preventing, ameliorating, or treating non-alcoholic fatty liver disease, comprising at least one selected from the group consisting of extracellular vesicles derived from Roseburia strains and extracellular vesicles derived from Bifidobacterium strains.

[0020] Furthermore, the present invention provides a method for preventing or treating non-alcoholic fatty liver disease, comprising administering to an individual in need thereof at least one selected from the group consisting of extracellular vesicles derived from Roseburia strains and extracellular vesicles derived from Bifidobacterium strains.

[0021] Furthermore, the present invention provides use of at least one selected from the group consisting of extracellular vesicles derived from Roseburia strains and extracellular vesicles derived from Bifidobacterium strains for the prevention or treatment of non-alcoholic fatty liver disease.

[0022] Furthermore, the present invention provides use of at least one selected from the group consisting of extracellular vesicles derived from a Roseburia strain and extracellular vesicles derived from a Bifidobacterium strain for the manufacture of a therapeutic agent for non-alcoholic fatty liver disease.

[0023] In one embodiment of the present invention, the extracellular vesicles may have an average diameter of 30 nm to 200 nm, but is not limited thereto.

[0024] In yet another embodiment of the present invention, the extracellular vesicles derived from a Roseburia strain may be naturally or artificially secreted from a Roseburia strain, but are not limited thereto.

[0025] In yet another embodiment of the present invention, the extracellular vesicles derived from a Bifidobacterium strain may be naturally or artificially secreted from a Bifidobacterium strain, but are not limited thereto.

[0026] In yet another embodiment of the present invention, the Roseburia strain may be at least one selected from the group consisting of Roseburia intestinalis, Roseburia faecies, Roseburia hominis, and Roseburia inulinivorans, but is not limited thereto.

[0027] In yet another embodiment of the present invention, the Bifidobacterium strain may be at least one selected from the group consisting of Bifidobacterium longum, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium lactis, and Bifidobacterium adolescentis, but is not limited thereto.

[0028] In yet another embodiment of the present invention, the non-alcoholic fatty liver disease may be at least one selected from the group consisting of steatosis, fibrosis, non-alcoholic steatohepatitis, non-alcoholic fatty liver (NAFL), and cirrhosis, but is not limited thereto.

[0029] In yet another embodiment of the present invention, the composition according to the present invention may have at least one effect selected from the group consisting of, but not limited to:

[0030] i) Decreased serum aspartate aminotransferase (AST) levels or activity

[0031] ii) Decreased serum alanine aminotransferase (ALT) levels or activity

[0032] iii) Decreased serum total bilirubin (T-BIL) levels

[0033] iv) Reduction of steatosis in the liver

[0034] v) Reduction in inflammatory cell infiltration

[0035] vi) a reduction in the number of inflammatory foci, and

[0036] vii) Decreased number of myofibroblasts

[0037] In yet another embodiment of the present invention, the food composition may be, but is not limited to, a functional health food composition. Effect of the Invention

[0038] Effect of the Invention

[0039] The extracellular vesicles derived from the Roseburia or Bifidobacterium strains of the present invention were confirmed to restore liver function in an animal model of non-alcoholic fatty liver disease, reduce fat accumulation in the liver to improve steatosis, and suppress infiltration of inflammatory cells into liver tissue, as well as to reduce the expression of myofibroblasts and suppress liver fibrosis. Furthermore, it was shown that both the Roseburia and Bifidobacterium strain-derived extracellular vesicles, which are of the same species and different genera, exhibit strong anti-inflammatory effects. Thus, the Roseburia strain-derived extracellular vesicles and Bifidobacterium strain-derived extracellular vesicles can improve liver damage and dysfunction caused by fatty liver and exhibit strong anti-inflammatory and anti-fibrotic effects, and therefore may be used as a composition for preventing, improving, and / or treating non-alcoholic fatty liver disease.

[0040] BRIEF DESCRIPTION OF THE DRAWINGS [Brief description of the drawings]

[0041] [Figure 1] Figure 1 shows the particle size measurements of extracellular vesicles (EVs) derived from Roseburia intestinalis (Ri-EVs) and EVs derived from Bifidobacterium longum (Bl-EVs).

[0042] [Diagram 2] Figure 2 shows a schematic diagram of an animal model of non-alcoholic steatohepatitis (NASH) induced by feeding a methionine-choline deficient diet (MCD).

[0043] [Diagram 3] Figure 3 shows serum AST (aspartate aminotransferase), ALT (alanine aminotransferase), and serum T-BIL (total bilirubin) levels in a control group (Normal Ctrl) not fed a methionine-choline deficient diet (MCD) and not administered Ri-EV or Bl-EV, an experimental group not fed an MCD and administered Ri-EV (Normal Ri-EV) and Bl-EV (Normal Bl-EV), a control group (NASH animal model, NASH Ctrl) fed an MCD and not administered Ri-EV or Bl-EV, and an experimental group fed an MCD and administered Ri-EV (NASH Ri-EV) or Bl-EV (NASH Bl-EV).

[0044] [Figure 4] Figure 4 shows the results of staining liver tissue for histopathological analysis of liver tissue following administration of Ri-EV or Bl-EV, for the control group not fed MCD and not administered Ri-EV or Bl-EV (Normal Ctrl), the experimental group not fed MCD and administered Ri-EV (Normal Ri-EV) or Bl-EV (Normal Bl-EV), the control group fed MCD and not administered Ri-EV or Bl-EV (NASH Ctrl), and the experimental group fed MCD and administered Ri-EV (NASH Ri-EV) or Bl-EV (NASH Bl-EV).

[0045] [Diagram 5]Figure 5 shows the pathological scores for steatosis and the number of inflammation-related lesions in a control group (NASH Ctrl) fed MCD and not administered Ri-EV or Bl-EV, and in experimental groups fed MCD and administered Ri-EV (NASH Ri-EV) or Bl-EV (NASH Bl-EV).

[0046] [Figure 6] Figure 6 shows the number of myofibroblasts found in the liver fibrosis process in a control group (NASH Ctrl) fed MCD and not administered Ri-EV or Bl-EV, and in an experimental group fed MCD and administered Ri-EV (NASH Ri-EV) or Bl-EV (NASH Bl-EV), using alpha-smooth muscle actin (α-SMA) staining.

[0047] [Figure 7] Figure 7 shows a schematic diagram of an NASH animal model created by feeding MCD for 2 weeks (upper panel) or 4 weeks (lower panel). Mice fed MCT for 2 weeks were administered Ri-EV, Bl-EV, Ri-Cell (R. intestinalis cells), or Bl-Cell (B. longum cells) from the 3rd week, and mice fed MCT for 4 weeks were administered Ri-EV, Bl-EV, Ri-Cell (R. intestinalis cells), or Bl-Cell (B. longum cells) from the 5th week.

[0048] [Figure 8] Figure 8 shows the serum ALT and serum T-BIL levels in the untreated control group (Ctrl) not fed MCT and not administered EVs or cells, the NASH animal model (NASH Ctrl) fed MCT and not administered EVs or cells, and the experimental groups fed MCT and administered Ri-EVs (NASH Ri-EV), Ri-Cell (NASH Ri-Cell), Bl-EVs (NASH Bl-EV), or Bi-Cell (NASH BI-Cell) for one week.

[0049] [Figure 9]Figure 9 shows the results of measuring the amount of nitric oxide production to confirm the anti-inflammatory effect of treatment with EVs derived from Roseburia sp. strains (R. intestinalis, R. faecies, R. hominis, and R. inulinivorans) or EVs derived from Bifidobacterium sp. strains (B. bifidum, B. breve, B. lactis, B. adolescentis, and B. longum) after inducing an inflammatory environment by treating the cells with LPS in an in vitro environment mimicking Kuffer cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0050] Best Mode for Carrying Out the Invention

[0051] The present invention will be described in detail below. Meanwhile, each description and embodiment disclosed in the present invention may be applied to each other description and embodiment. That is, all combinations of various elements disclosed in the present invention belong to the scope of the present invention. In addition, the scope of the present invention is not considered to be limited by the specific description described below.

[0052] The present invention relates to a pharmaceutical composition for preventing or treating non-alcoholic fatty liver disease, comprising as an active ingredient at least one selected from the group consisting of extracellular vesicles (EVs) derived from Roseburia spp. strains and extracellular vesicles (EVs) derived from Bifidobacterium spp. strains. The present invention has been completed after confirming that Roseburia strains, Bifidobacterium strains, or extracellular vesicles derived therefrom can improve liver function and suppress liver inflammation and fibrosis in an animal model of non-alcoholic fatty liver disease, thereby exerting excellent therapeutic effects.

[0053] The composition according to the present invention may contain at least one selected from the group consisting of Roseburia spp. strains, their homogenates, culture broths, extracellular vesicles, and mixtures thereof, and Bifidobacterium spp. strains, their homogenates, culture broths, extracellular vesicles, and mixtures thereof. In one embodiment of the present invention, the composition according to the present invention may not contain a bacterial strain.

[0054] In the present invention, "strains of the genus Roseburia" include strains that are already known in the art as strains belonging to the genus Roseburia, or strains that will be newly known in the future. Roseburia is a Gram-positive bacterium that inhabits the human colon and is known to have the ability to decompose sugar and produce butyric acid. The Roseburia strain according to the present invention includes any strain that belongs to the genus Roseburia, and is not limited to a specific type, but may be at least one selected from the group consisting of Roseburia intestinalis, Roseburia faecies, Roseburia hominis, Roseburia inulinivorans, and Roseburia cecicola. Among these, Roseburia intestinalis is the first microorganism isolated from human feces and is known to produce metabolic products in the intestine to maintain energy homeostasis.

[0055] In the present invention, "strains of the genus Bifidobacterium" include strains that are already known in the art as belonging to the genus Bifidobacterium, or that will be newly known in the future. Bifidobacteria are Gram-positive anaerobic bacteria, and are one of the major types of bacteria that constitute the mammalian gastrointestinal tract microbial community. Some Bifidobacteria are also used as lactic acid bacteria. The Bifidobacterium strain according to the present invention is not limited to any particular species as long as it is a member of the genus Bifidobacterium, but is preferably at least one selected from the group consisting of Bifidobacterium longum, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium lactis, and Bifidobacterium adolescentis. Among these, Bifidobacterium longum is also known as bifidobacteria and is known to be effective in inhibiting the proliferation of Escherichia coli and enhancing intestinal motility and defecation.

[0056] In the present invention, the term "strain" includes not only live bacteria obtained from a culture such as a culture medium, but also any processed form of the strain known to those skilled in the art, including, but not limited to, crushed bacteria, dried products, frozen products, etc. In the present specification, the term "strain" may be used interchangeably with "bacteria", "cells", etc.

[0057] In the present invention, the term "disrupted product" refers to a product obtained by disrupting the cell walls of a strain by chemical treatment or by applying physical force to the strain.

[0058] In the present invention, the term "culture solution" refers to the entire medium containing the strain, its metabolites, excess nutrients, etc., obtained by culturing the strain for a certain period of time in a medium that can supply nutrients so that the strain can grow and survive in a test tube. The culture solution refers to a culture solution obtained by removing the bacteria from the culture solution obtained by culturing the strain. Meanwhile, the liquid from the culture solution from which the bacteria have been removed is also called "supernatant", and can be obtained by leaving the culture solution for a certain period of time and taking only the upper liquid excluding the part that has sunk to the bottom, removing the bacteria by filtration, or centrifuging the culture solution to remove the lower precipitate and taking only the upper liquid. The "bacterial cells" refers to the strain itself of the present invention, and includes the strain itself separated and selected from a biological sample or the like, or the strain separated from the culture solution in which the strain was cultured. The bacterial cells can be obtained by centrifuging the culture solution to take the part that has sunk to the bottom, or by leaving the culture solution for a certain period of time and removing the upper liquid since the bacterial cells sink to the bottom of the culture solution due to gravity. The culture medium may be referred to as a "culture supernatant," a "conditioned medium," or a "conditioned medium."

[0059] The culture medium may include, but is not limited to, a culture medium itself obtained by culturing a strain, an extract thereof, a concentrate thereof, or a freeze-dried product thereof, or a culture supernatant obtained by removing the strain from the culture medium, an extract thereof, a concentrate thereof, or a freeze-dried product thereof.

[0060] The culture medium may be one obtained by culturing the strain of the present invention in an appropriate medium (e.g., RCM (reinforced clostridial medium) medium) at any temperature of 10° C. to 50° C., 10° C. to 40° C., 20° C. to 50° C., 20° C. to 40° C., or 30° C. to 40° C. for a certain period of time, for example, 4 to 50 hours, 4 to 40 hours, 4 to 30 hours, 4 to 20 hours, or 10 to 20 hours, but is not limited thereto. The culture medium and culture conditions for culturing the strain of the present invention can be appropriately selected or modified by a person of ordinary skill in the art.

[0061] In one embodiment, the culture supernatant of the present invention is obtained by subjecting the above-mentioned strain culture solution to the step of removing the strain by a method such as centrifugation or filtration.

[0062] In the present invention, the term "concentrate" refers to the above-mentioned culture medium itself, or a culture medium obtained by concentrating a supernatant obtained by a method such as centrifugation or filtration.

[0063] In the present invention, the term "extract" means something extracted from the above-mentioned culture solution or a concentrate thereof, and may include an extract, a dilution or concentrate of the extract, a dried product or freeze-dried product obtained by drying the extract, or a crude or purified product thereof, or a fraction thereof.

[0064] In the present invention, the term "vesicle" refers to a particle secreted from a cell and released into the extracellular space, and may include a number of different species, such as exosomes, ectosomes, microvesicles, microparticles, and exosome-like vesicles. Extracellular vesicles (EVs) can reflect the state of the source cell (donor cell) from which they are secreted, and can exhibit various biological activities depending on the cell from which they are secreted, and can play an important role in intercellular interactions by transferring genetic material and proteins between cells. In addition, cell-derived substances including the vesicles can cause disease or stimulate immune cells to fight disease, and can help decompose and absorb substances that humans cannot digest through the metabolic process of microorganisms. The vesicles are divided into an inside and an outside by a membrane structure vesicle, and contain plasma membrane lipids, plasma membrane proteins, nucleic acids, and cytoplasmic components of the cell, and may be smaller than the original cell. The term "extracellular vesicles" may be used interchangeably with "extracellular vesicles" or may simply be called "endoplasmic reticulum" or "vesicles".

[0065] In particular, the vesicles derived from Gram-positive bacteria such as Roseburia strains and Bifidobacterium strains of the present invention may contain peptidoglycan and lipoteichoic acid, which are components of bacterial cell walls, in addition to proteins and nucleic acids.

[0066] In one embodiment of the present invention, the extracellular vesicles derived from a Roseburia strain may have a single lipid membrane structure, but is not limited thereto.

[0067] The vesicles according to the present invention may be naturally secreted from Roseburia or Bifidobacterium strains, or may be artificially produced (isolated) from bacteria through heat treatment, pressure treatment, etc., and may have a diameter of 30-200 nm, 30-150 nm, 30-100 nm, 30-80 nm, 40-200 nm, 50-200 nm, 50-100 nm, or 50-80 nm. In one embodiment of the present invention, the sizes of R. intestinalis-derived EVs and B. longum-derived EVs were measured using a zeta-sizer and found to have average particle sizes of 75 nm and 58 nm, respectively (FIG. 1).

[0068] Furthermore, the surface charge of the Roseburia extracellular vesicles according to the present invention may be, but is not limited to, -20 to 5 mV, -20 to 0 mV, -10 to 0 mV, -5 to 0 mV, -10 to -1 mV, or -5 to -1 mV.

[0069] The vesicles may be isolated by subjecting a culture solution containing a Roseburia or Bifidobacterium strain to at least one method selected from the group consisting of centrifugation, ultra-high speed centrifugation, extrusion, sonication, cell lysis, homogenization, freeze-thaw, electroporation, mechanical disintegration, chemical treatment, filtration through a filter, gel filtration chromatography, free-flow electrophoresis, and capillary electrophoresis, and may further include steps such as washing to remove impurities and concentrating the obtained vesicles.

[0070] The composition according to the present invention can have preventive, ameliorative, and / or therapeutic effects on non-alcoholic fatty liver disease.

[0071] As an example, the composition according to the present invention may have at least one effect selected from the following:

[0072] i) Decreased serum aspartate aminotransferase (AST) levels or activity

[0073] ii) Decreased serum alanine aminotransferase (ALT) levels or activity

[0074] iii) Decreased serum total bilirubin (T-BIL) levels

[0075] iv) Reduction of steatosis in the liver

[0076] v) Reduction in inflammatory cell infiltration

[0077] vi) Reduction in the number of inflammatory foci

[0078] vii) Decreased number of myofibroblasts

[0079] That is, the preventive, ameliorative, and / or therapeutic effect of the composition according to the present invention on NAFLD may be exerted by the effects i) to vii) above.

[0080] Regarding the effects of the composition of the present invention on i) reducing serum AST level or activity, ii) reducing serum ALT level or activity, and iii) reducing serum T-BIL level, the present inventors confirmed that when the extracellular vesicles derived from the Roseburia or Bifidobacterium strain of the present invention were administered to normal mice without disease, there was no change in serum AST, ALT, and T-BIL levels, which are indicators of liver function, whereas when the extracellular vesicles were administered to an animal model of non-alcoholic fatty liver disease, serum AST, ALT, and T-BIL levels were reduced compared to the untreated control group (Figure 3). The results indicate that the extracellular vesicles derived from the Roseburia or Bifidobacterium strain of the present invention restore liver function in non-alcoholic fatty liver disease without side effects.

[0081] Regarding the effects of the composition of the present invention on iv) reducing steatosis, v) reducing inflammatory cell infiltration, and vi) reducing the number of inflammatory foci, the present inventors confirmed that when the extracellular vesicles of the present invention were administered to normal mice, no damage to liver tissue or inflammatory reaction was observed, whereas when the extracellular vesicles derived from Roseburia or Bifidobacterium strains were administered to non-alcoholic fatty liver disease animal models, steatosis and infiltration of inflammatory cells were significantly reduced at the lesion site of liver tissue, and the pathological score for steatosis and the number of inflammation-related foci were significantly reduced (Figure 5).The above results indicate that the extracellular vesicles derived from Roseburia or Bifidobacterium strains of the present invention suppress fat accumulation in the liver and exert anti-inflammatory effects.

[0082] Regarding the vii) effect of the composition of the present invention on reducing the number of myofibroblasts, the present inventors confirmed that the number of myofibroblasts found in the process of liver fibrosis was significantly reduced when extracellular vesicles derived from Roseburia or Bifidobacterium strains were administered to an animal model of non-alcoholic fatty liver disease (Figure 6).The above results indicate that the extracellular vesicles derived from Roseburia or Bifidobacterium strains of the present invention can suppress liver fibrosis caused by non-alcoholic fatty liver.

[0083] Furthermore, the present inventors isolated extracellular vesicles from various strains of the same genus belonging to the genus Roseburia or Bifidobacterium and confirmed their anti-inflammatory effects. As a result, it was confirmed that the extracellular vesicles of the above strains all exhibit strong anti-inflammatory effects. The above results indicate that the extracellular vesicles derived from the strains belonging to the genus Roseburia or Bifidobacterium exhibit excellent anti-inflammatory effects and therefore can be used as a therapeutic agent for non-alcoholic fatty liver.

[0084] In summary, the composition according to the present invention exerts anti-inflammatory and anti-fibrotic effects in the liver and can improve liver function, and therefore may be used for the prevention, improvement, and / or treatment of non-alcoholic fatty liver disease.

[0085] Meanwhile, the composition according to the present invention may be used for the prevention, amelioration, and / or treatment of "inflammatory diseases". In the present invention, the inflammatory disease includes, without limitation, any disease associated with inflammation or disease that can be ameliorated or treated by suppressing inflammation, and is not limited to a specific type, but is preferably gastritis, gastric ulcer, duodenal ulcer, inflammatory skin disease, allergic disease, Crohn's disease, irritable bowel syndrome, ulcerative colitis, inflammatory bowel disease, peritonitis, osteomyelitis, cellulitis, meningitis, encephalitis, pancreatitis, trauma-induced shock, bronchial asthma, cystic fibrosis, stroke, acute bronchitis, chronic bronchitis, acute bronchiolitis, chronic bronchiolitis, osteoarthritis, gout, spondyloarthropathy, ankylosing spondylitis, Reiter's syndrome, psoriatic arthropathy, intestinal spondylitis, juvenile arthropathy, juvenile ankylosing spondylitis, reactive arthropathy, infection Arthritis of the genital tract, post-infectious arthritis, gonococcal arthritis, tuberculous arthritis, viral arthritis, fungal arthritis, syphilitic arthritis, Lyme disease, arthritis associated with vasculitis syndromes, polyarteritis nodosa, hypersensitivity vasculitis, Rugeric's granulomatosis, polymyalgia rheumatica, articular cell arteritis, calcium crystal deposition arthropathy, caustic gout, non-rheumatoid arthritis, mucositis, tenosynovitis, epicondylitis (tennis elbow), neuropathic arthropathy (charco and joint, hemarthrosic, Henoch-Scholein purpura, hypertrophic osteoarthropathy, multicentric reticulohistiocytosis, sarcoilosis, hemochromatosis, sickle cell disease and other hemochromatopathies, hyperlipoproteinemia, hypogammaglobulinemia, familial Mediterranean fever, Behart's disease, systemic lupus erythematosus, relapsing fever, psoriasis, multiple sclerosis, sepsis, septic shock, multiple organ dysfunction syndrome, acute respiratory distress syndrome, chronic obstructive pulmonary disease, acute lung injury, and broncho-pulmonary dysplasia.

[0086] The composition according to the present invention may also be used for the purpose of preventing, improving, and / or treating "fibrotic disease." Fibrosis is a disease in which excessive fibrous connective tissue is formed in tissue, destroying normal tissue structure and causing hardening. In the present invention, the fibrosis includes, but is not limited to, liver fibrosis, pulmonary fibrosis, skin fibrosis, pancreatic fibrosis, systemic sclerosis, cardiac fibrosis, etc.

[0087] In addition, the composition according to the present invention may be used for the purpose of preventing, improving, and / or treating fatty liver disease. The fatty liver-related disease is not limited to a specific type, and may include any disease induced by excessive accumulation of fat in the liver, regardless of the cause. For example, the fatty liver-related disease includes alcoholic fatty liver and non-alcoholic fatty liver.

[0088] Most preferably, the compositions according to the present invention may be used for the purposes of preventing, ameliorating and / or treating non-alcoholic fatty liver disease.

[0089] In the present invention, the term "non-alcoholic fatty liver disease (NAFLD)" refers to a metabolic liver disease resulting from metabolic dysregulation, which is a disease caused by fat accumulation in the liver unrelated to alcohol consumption. As mentioned above, NAFLD is a series of diseases that, as the disease progresses, include simple steatosis, which has only excess accumulation of fat in hepatocytes, and NASH, which is accompanied by hepatocyte necrosis and inflammation and fibrosis in which the liver tissue hardens. NAFLD may include any disease that occurs due to accumulation of fat in the liver caused by causes other than alcohol, and is not limited to a specific type, but may include, for example, steatosis, fibrosis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver (NAFL), and cirrhosis. In addition, other liver diseases that develop due to the progression of NAFLD are not particularly limited and may be included in the scope of the present application. The term "fibrosis" may be used interchangeably with "fibrosis."

[0090] The content of the strain, lysate, culture medium and / or extracellular vesicles in the composition of the present invention can be appropriately adjusted depending on the symptoms of the disease, the degree of progression of the symptoms, the condition of the patient, etc., and may be, for example, 0.0001 to 99.9% by weight, or 0.001 to 50% by weight based on the total composition weight, but is not limited thereto. The content ratio is a value based on the dry amount after removing the solvent.

[0091] The pharmaceutical composition according to the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions, such as at least one selected from the group consisting of diluents, binders, disintegrants, lubricants, adsorbents, moisturizers, film-coating materials and controlled release additives.

[0092] The pharmaceutical composition according to the present invention may be formulated by a conventional method into the form of a powder, granule, sustained-release granule, enteric-coated granule, liquid, eye drop, elucic, emulsion, suspension, spirit, troche, aromatic perfume, lemonade, tablet, sustained-release tablet, enteric-coated tablet, sublingual tablet, hard capsule, soft capsule, sustained-release capsule, enteric-coated capsule, pill, tincture, soft extract, dry extract, fluid extract, injection, capsule, perfusion solution, plaster, lotion, paste, spray, inhalant, patch, sterile injection, or external preparation such as aerosol, and the external preparation may have the form of a cream, gel, patch, spray, ointment, plaster, lotion, liniment, paste, or cataplasm.

[0093] Carriers, excipients and diluents that may be included in pharmaceutical compositions according to the invention include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginates, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, amorphous cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

[0094] When the formulation is made, it is usually prepared by using a diluent or excipient such as a filler, extender, binder, wetting agent, disintegrant, surfactant, etc.

[0095] Additives for the tablets, powders, granules, capsules, pills and lozenges according to the present invention include excipients such as corn starch, potato starch, wheat starch, lactose, sucrose, glucose, fructose, D-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, calcium hydrogen phosphate, calcium sulfate, sodium chloride, sodium bicarbonate, purified lanolin, microcrystalline cellulose, dextrin, sodium alginate, methylcellulose, sodium carboxymethylcellulose, kaolin, urea, colloidal silica gel, hydroxypropyl starch, hydroxypropylmethylcellulose (HPMC) 1928, HPMC 2208, HPMC 2906, HPMC 2910, propylene glycol, casein, calcium lactate and Primozel, gelatin, gum arabic, ethanol, agar powder, cellulose acetate phthalate, carboxymethylcellulose, calcium carboxymethylcellulose, glucose, purified water, casein sodium, Binders such as cellulose, glycerin, stearic acid, sodium carboxymethylcellulose, sodium methylcellulose, methylcellulose, microcrystalline cellulose, dextrin, hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, purified shellac, starch paste, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl alcohol, and polyvinylpyrrolidone may be used, and hydroxypropyl methylcellulose, corn starch, agar powder, methylcellulose, bentonite, hydroxypropyl starch, sodium carboxymethylcellulose, sodium alginate, calcium carboxymethylcellulose, calcium citrate, sodium lauryl sulfate, anhydrous silicic acid, 1-hydroxypropyl cellulose, dextran, ion exchange resins, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginic acid, amylose, guar gum (Guargum), baking soda, polyvinylpyrrolidone, calcium phosphate, gelling starch, gum arabic, amylopectin, pectin, sodium polyphosphate, ethylcellulose, white sugar, magnesium aluminum silicate, D-sorbitol liquid, disintegrants such as hard anhydrous silicic acid, calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopodium, kaolin, petrolatum, sodium stearate, cacao butter, sodium salicylate, magnesium salicylate, polyethylene glycol (PEG) 4000, PEG 6000, liquid paraffin, hydrogenated soybean oil (Lubri Lubricants such as wax, aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, Macrogol, synthetic aluminum silicate, anhydrous silicic acid, higher fatty acids, higher alcohols, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, and hard anhydrous silicic acid may be used.

[0096] Examples of additives that may be used in the liquid preparation according to the present invention include water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, sucrose monostearate, polyoxyethylene sorbitol fatty acid esters (twin esters), polyoxyethylene monoalkyl ethers, lanolin ethers, lanolin esters, acetic acid, hydrochloric acid, aqueous ammonia, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethyl cellulose, and sodium carboxymethylcellulose.

[0097] The syrup according to the present invention may contain a solution of white sugar, other sugars or sweeteners, and may contain flavorings, coloring agents, preservatives, stabilizers, suspending agents, emulsifiers, thickening agents, etc., as necessary.

[0098] The emulsion of the present invention may contain purified water, and may contain emulsifiers, preservatives, stabilizers, fragrances, etc., as required.

[0099] The suspension according to the present invention may contain a suspending agent such as acacia, tragacanth, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose (HPMC), HPMC1828, HPMC2906, or HPMC2910, and may also contain a surfactant, a preservative, a stabilizer, a coloring agent, or a fragrance, as necessary.

[0100] The injections according to the present invention may contain distilled water for injection, 0.9% sodium chloride injection, Ringer's injection, dextrose injection, dextrose + sodium chloride injection, PEG, lactated Ringer's injection, ethanol, propylene glycol, non-volatile oils - sesame oil, cottonseed oil, peanut oil, soybean oil, corn oil, solvents such as ethyl oleate, isopropyl myristate, and benzene benzoate, solubilizers such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidine, propylene glycol, twines, nidiotinamide, hexamine, and dimethylacetamide, weak acids and their salts (acetic acid and sodium acetate), and weak bases and their salts (ammonia and ammonium acetate). It may also contain buffers such as organic compounds, proteins, albumin, peptones, and gums, isotonicity agents such as sodium chloride, stabilizers such as sodium hydrogen sulfite (NaHSO3), carbon dioxide gas, sodium metabisulfite (Na2S2O5), sodium sulfite (Na2SO3), nitrogen gas (N2), and ethylenediaminetetraacetic acid, sulfating agents such as sodium bisulfide 0.1%, sodium formaldehyde sulfoxylate, thiourea, disodium ethylenediaminetetraacetate, and acetone sodium bisulfite, soothing agents such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate, and suspending agents such as sodium CMC, sodium alginate, Tween 80, and aluminum monostearate.

[0101] Suppositories according to the invention may contain any of the following ingredients: cocoa butter, lanolin, witepsol, polyethylene glycol, glycerogelatin, methylcellulose, carboxymethylcellulose, mixture of stearic and oleic acids, Subanal, cottonseed oil, peanut oil, coconut oil, cocoa butter + cholesterol, lecithin, lanet wax, glycerol monostearate, Tween or Span, Imhausen, Monolen (propylene glycol monostearate), glycerin, Adeps solidus, Buytyrum Tego-G, Cebes Pharma 16, etc. 16), Hexalide Base 95, Cotomar, Hydrocoat SP, S-70-XXA, S-70-XX75 (S-70-XX95), Hydrokote 25, Hydrocoat 711, Idropostal, Massa Estralium Bases such as estrarium, A, AS, B, C, D, E, I, T), Masa-MF, Maspol, Maspol-15, Neospostal-En, Paramound-B, Sporsilo (OSI, OSIX, A, B, C, D, H, L), suppository base type IV (AB, B, A, BC, BBG, E, BGF, C, D, 299), Spostal (N, Es), Wecoby (W, R, S, M, Fs), and Tezeta triglyceride base (TG-95, MA, 57) may be used.

[0102] The solid preparation for oral administration includes tablets, pills, powders, granules, capsules, etc., and such solid preparations are prepared by mixing the extract with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium styrenate talc are also used.

[0103] Liquid preparations for oral administration include suspensions, liquid preparations, emulsions, syrups, etc., and may contain various excipients such as wetting agents, sweeteners, flavorings, preservatives, etc. in addition to water and liquid paraffin, which are commonly used simple diluents. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc.

[0104] The pharmaceutical composition according to the present invention is administered in a pharmacologic effective amount. In the present invention, the term "pharmacologic effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to any medical treatment, and the effective dose level may be determined by factors including the type and severity of the patient's disease, the activity of the drug, sensitivity to the drug, administration time, administration route and excretion rate, treatment duration, concurrently used drugs, and other factors well known in the medical field.

[0105] The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or in multiple doses. Taking into consideration all of the above factors, it is important to administer an amount that provides maximum effect at a minimum amount without side effects, which may be easily determined by a person skilled in the art to which the present invention pertains.

[0106] The pharmaceutical compositions of the present invention may be administered to an individual by a variety of routes, including, for example, oral ingestion, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, paraspinal space (intradural) injection, sublingual administration, buccal administration, rectal insertion, vaginal insertion, ocular administration, aural administration, nasal administration, inhalation, spraying through the mouth or nose, dermal administration, transdermal administration, and the like, although all modes of administration are contemplated.

[0107] The pharmaceutical composition of the present invention is determined by the type of drug as an active ingredient, along with various related factors such as the disease to be treated, the administration route, the age, sex, weight and severity of the disease of the patient. Specifically, the effective amount of the composition according to the present invention may vary depending on the age, sex and weight of the patient, and is generally 0.001 to 150 mg, preferably 0.01 to 100 mg per kg of body weight, administered daily or every other day, or administered in 1 to 3 divided doses per day. However, since the amount may be increased or decreased depending on the administration route, severity of the disease, sex, weight, age, etc., the above-mentioned dosage does not limit the scope of the present invention in any way.

[0108] In the present invention, the term "individual" refers to a subject in need of treatment for a disease, and more specifically, refers to mammals such as humans or non-human primates, mice, rats, dogs, cats, horses, and cows.

[0109] In the present invention, "administration" means providing a given composition of the present invention to an individual by any suitable method.

[0110] In the present invention, "prevention" refers to any action that suppresses or delays the onset of a desired disease, "treatment" refers to any action that improves or beneficially alters the symptoms of a desired disease and associated metabolic disorders by administering a pharmaceutical composition according to the present invention, and "improvement" refers to any action that reduces parameters related to a target disease, such as the severity of symptoms, by administering a composition according to the present invention.

[0111] The present invention also provides a kit for preventing, ameliorating, or treating non-alcoholic fatty liver disease, comprising the composition according to the present invention.

[0112] The kit according to the present invention is not limited to a specific form as long as it can achieve the purpose of preventing, improving, or treating non-alcoholic fatty liver disease, and may include any components and devices for producing the composition according to the present invention (e.g., culturing a strain, isolating extracellular vesicles, etc.), storing, administering, etc. In addition, the kit may include an instruction manual that describes the characteristics, usage method, storage method, dosage, etc. of the composition of the present invention.

[0113] The present invention also provides a food composition for preventing or ameliorating non-alcoholic fatty liver disease, comprising at least one selected from the group consisting of extracellular vesicles derived from Roseburia strains and extracellular vesicles derived from Bifidobacterium strains as an active ingredient. The food composition includes a functional health food composition.

[0114] When the strain, lysate, culture solution, and / or extracellular vesicles of the present invention are used as food additives, the strain, extracellular vesicles, etc. may be added as is or together with other foods or food ingredients, or may be used appropriately by a conventional method. The content of the active ingredient in the food composition may be appropriately determined depending on the purpose of use (prevention, improvement, or therapeutic treatment). The amount of the active ingredient mixed may be appropriately determined depending on the purpose of use (prevention, health, or therapeutic treatment). In general, when producing a food or beverage, the strain, extracellular vesicles, etc. of the present invention may be added in an amount of 15% by weight or less, or 10% by weight or less, based on the raw material. However, in the case of long-term intake for the purpose of health and hygiene or health regulation, 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 greater than the above range.

[0115] The type of the food is not particularly limited. Examples of the food to which the substance can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, drinking water, tea, energy drinks, alcoholic beverages, and vitamin complexes, and include all health functional foods in the usual sense.

[0116] The health drink composition according to the present invention may contain various flavorings or natural carbohydrates as additional ingredients, like ordinary drinks. The above-mentioned natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As sweeteners, natural sweeteners such as thaumatin and stevia extract, and synthetic sweeteners such as saccharin and aspartame may be used. The proportion of the natural carbohydrates is generally about 0.01 to 0.20 g, or about 0.04 to 0.10 g per 100 mL of the composition of the present invention.

[0117] In addition to the above, the composition of the present invention may contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated drinks, etc. In addition, the composition of the present invention may contain fruit pulp for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These ingredients may be used independently or in combination. The proportion of such additives is not very important, but is generally selected from the range of 0.01 to 0.20 parts by weight per 100 parts by weight of the composition of the present invention.

[0118] In this specification, the term "health functional food" is the same as food for special health use (FoSHU), and refers to food with high medical and therapeutic effects that is processed to efficiently exert a bioregulatory function in addition to providing nutrition, and the food may be manufactured in various forms such as tablets, capsules, powder, granules, liquid, pills, etc. to obtain useful effects in preventing or improving NAFLD.

[0119] The health functional food of the present invention can be manufactured by a method commonly used in the art, and may be manufactured by adding raw materials and ingredients commonly added in the art. Also, unlike general medicines, it has the advantage of being free of side effects that may occur when taking medicines for a long period of time, as it is made from food as a raw material, and is highly portable.

[0120] The present invention also provides a feed composition for preventing or ameliorating non-alcoholic fatty liver disease, comprising as an active ingredient at least one selected from the group consisting of extracellular vesicles derived from Roseburia strains and extracellular vesicles derived from Bifidobacterium strains.

[0121] The term "feed" in the present invention refers to any natural or artificial diet, meal, etc., or components of said meal, which are intended or suitable for an animal to eat, ingest, and digest, and the feed containing the graphene nanoparticles of the present invention as an active ingredient can be produced in various forms of feed known in the art, and specifically may include concentrated feed, roughage, and / or special feed.

[0122] The amount of the bacterial strain, homogenate, culture medium, and / or extracellular vesicles of the present invention contained in the feed composition of the present invention may vary depending on the purpose and conditions of use of the feed, and may be, for example, 0.01 to 100 wt %, more specifically 1 to 80 wt %, based on the total weight of the livestock feed composition, but is not limited thereto.

[0123] When the composition is produced as a feed additive, the composition may be produced in the form of a highly concentrated liquid (20 to 90%), powder, or granules. The feed additive may further contain any one or more of organic acids such as citric acid, fumaric acid, adipic acid, lactic acid, malic acid, etc., phosphates such as sodium phosphate, potassium phosphate, acid pyrophosphate, and polyphosphates (polymerized phosphates), and natural antioxidants such as polyphenols, catechins, α-tocopherol, rosemary extract, vitamin C, green tea extract, licorice extract, chitosan, tannic acid, and phytic acid. When produced as a feed, the composition may be formulated in the form of a normal feed and may contain normal feed ingredients.

[0124] The feed and feed additives may further include grains such as crushed or crushed wheat, oats, barley, corn, and rice, vegetable protein feeds such as feeds mainly composed of rapeseed, soybeans, and sunflowers, animal protein feeds such as blood meal, meat and bone meal, bone meal, and fish meal, sugar, and dairy products such as various types of milk powder and whey powder, and may further include nutritional supplements, digestion and absorption enhancers, growth promoters, and the like.

[0125] The feed additives may be administered to animals alone or in combination with other feed additives in an edible carrier. The feed additives may also be readily administered to animals as a top dressing, or mixed directly into these animal feeds, or in an oral dosage form separate from the feed. When the feed additives are administered separately from the animal feed, they may be combined with pharma- ceutically acceptable edible carriers and made into immediate or sustained release formulations, as is well known in the art. These edible carriers may be solid or liquid, such as corn starch, lactose, sucrose, soybean flakes, peanut oil, olive oil, sesame oil, and propylene glycol. When solid carriers are used, the feed additives may be in the form of tablets, capsules, powders, lozenges, or lozenges or top dressings in microdispersible form. When liquid carriers are used, the feed additives may be in the form of gelatin soft capsules, or syrups, suspensions, emulsions, or solutions.

[0126] The feed and feed additives may also contain auxiliary agents, such as preservatives, stabilizers, wetting agents, emulsifiers, solution promoters, etc. The feed additives may be used by acupuncture, spraying, or mixing and adding to animal feed.

[0127] The feed or feed additive of the present invention may be applied to the diets of many animals including mammals, poultry and fish.

[0128] The mammals may include pigs, cows, horses, sheep, rabbits, goats, rodents, and laboratory rodents such as mice, hamsters, and guinea pigs, as well as pet animals (e.g., dogs and cats). The poultry may include chickens, turkeys, ducks, geese, pheasants, and quails. The fish may include carp, crucian carp, and trout, but are not limited thereto.

[0129] MODE FOR CARRYING OUT THEINVENTION

[0130] In order to facilitate understanding of the present invention, the present invention will be described in detail with reference to the following examples. However, the following examples are merely illustrative of the contents of the present invention, and the scope of the present invention is not limited to the following examples. The examples of the present invention are provided to more completely explain the present invention to those having average knowledge in the art.

[0131] (blank) EXAMPLES

[0132] [Example]

[0133] Example 1. Extraction and size analysis of extracellular vesicles (EVs) (Ri-EVs, Bl-EVs) from Roseburia intestinalis or Bifidobacterium longum

[0134] To extract EVs from R. intestinalis and B. longum, R. intestinalis KCTC 15746 and B. longum KCTC 3249 were cultured in reinforced clostridial medium (MB cell, MB-R1602) in an anaerobic chamber at 37°C for 18 hours. The cultures were centrifuged at 2000×g for 20 minutes, and the supernatant was collected and 0.22 μm filters were used to remove cell debris and waste products from the culture medium. EVs were extracted from each culture through a 300 kDa molecular weight TFF (tangential flow filtration) membrane filter-based TFF system, diluted and purified with phosphate-buffered saline (PBS), and finally, R. intestinalis-derived EVs (Ri-EVs) and B. longum-derived EVs (Bl-EVs) dispersed in PBS were extracted.

[0135] The size of Ri-EVs and Bl-EVs was measured using a zeta-sizer and was confirmed to have an average particle size of 75 nm and 58 nm, respectively (Figure 1).

[0136] (blank)

[0137] Example 2. Evaluation of the effect of EV administration on liver function recovery in an animal model of non-alcoholic steatohepatitis (NASH)

[0138] Non-alcoholic fatty liver disease (NAFLD) is a group of diseases that progresses through a series of stages, including simple steatosis, which is characterized by excessive accumulation of fat in liver cells, and non-alcoholic steatohepatitis (NASH), which is characterized by liver cell necrosis, inflammation, and fibrosis (Brunt EM, 2001). Therefore, an NASH animal model was used to evaluate whether or not liver function was restored by administration of Ri-EVs and Bl-EVs extracted in Example 1. The process of creating the NASH animal model is shown in Figure 2.

[0139] A NASH animal model was created by feeding C57BL / 6 mice a methionine-choline deficient diet (MCD) for 4 weeks. The experimental group was orally administered Ri-EVs or Bl-EVs at a total protein level of 200 μg / mouse / day three times a week for 4 weeks while feeding the MCD. This was compared with a control group (NASH animal model) that was fed the MCD but did not receive Ri-EVs or Bl-EVs.

[0140] In addition, normal mice not fed an MCD were orally administered Ri-EVs or Bl-EVs at a total protein dose of 200 μg / mouse / day three times a week for four weeks, and these were compared with a control group not fed an MCD and not administered Ri-EVs or Bl-EVs.

[0141] For blood biochemistry analysis to evaluate liver function, mice were sacrificed 4 weeks after the start of the experiment and serum was collected. Blood biochemistry analysis was performed by requesting T&P Bio Co., Ltd. to perform liver function tests. Serum AST (aspartate aminotransferase) and ALT (alanine aminotransferase) activities were measured using Reitman-Frankel colorimetric kits (Asan Pharmaceutical, Korea) according to the manufacturer's protocol, and serum T-BIL (total bilirubin) was quantified using a Jendrassik-Grof enzymatic assay kit (BioAssay Systems, Hayward, CA, USA) according to the manufacturer's protocol.

[0142] As a result, as shown in Figure 3, when Ri-EV or Bl-EV were administered to normal mice not fed MCD, there was no change in serum AST, ALT, and T-BIL levels, which are indicators of liver function, compared to the control group not fed MCD and not administered Ri-EV or Bl-EV. On the other hand, when Ri-EV or Bl-EV were administered to NASH animal models, serum AST, ALT, and T-BIL levels were significantly reduced compared to NASH animal models not administered EV. This confirmed that Ri-EV and Bl-EV administration reduced serum AST, ALT, and T-BIL levels in NASH animal models without side effects, restoring liver function.

[0143] (blank)

[0144] Example 3. Histological evaluation of liver tissue following EV administration in NASH animal models

[0145] For histopathological analysis of liver tissues following administration of Ri-EVs and Bl-EVs extracted in Example 1, livers were excised from the NASH animal model of Example 2 and the tissues were stained. Specifically, the excised livers were fixed in 4% neutral buffered formalin and embedded in paraffin, and then 4 μm-thick tissue sections were prepared, stained with H&E (Hematoxilin-Eosin) and α-SMA (alpha-smooth muscle actin) to detect myofibroblasts found in the process of liver fibrosis, and observed under an optical microscope.

[0146] (blank)

[0147] As a result, as shown in Figure 4, when Ri-EVs or Bl-EVs were administered to normal mice not fed an MCD, no damage to liver tissue or inflammatory reactions were observed. On the other hand, when Ri-EVs or Bl-EVs were administered to NASH animal models, it was confirmed that steatosis and infiltration of inflammatory cells were significantly reduced in the lesions of liver tissue.

[0148] In addition, as shown in Figure 5, when Ri-EVs or Bl-EVs were administered to NASH animal models, the pathological score for steatosis and the number of inflammation-related foci (foci) were significantly reduced, and as shown in Figure 6, when Ri-EVs or Bl-EVs were administered to NASH animal models, the number of myofibroblasts found during the liver fibrosis process was significantly reduced.

[0149] As a result, it was confirmed that administration of Ri-EVs and Bl-EVs reduced steatosis and inflammatory cell infiltration in NASH animal models without side effects, reduced the pathological score for steatosis and the number of inflammation-related lesions, and suppressed the expression of myofibroblasts associated with liver fibrosis, thereby suppressing the deterioration of liver function. In other words, the above results indicate that the Ri-EVs and Bl-EVs of the present invention can effectively improve liver inflammation, fat accumulation, and fibrosis caused by NASH.

[0150] (blank)

[0151] Example 4. Evaluation of the NASH therapeutic efficacy of strains and EVs in NASH animal models

[0152] The preventive effect of Ri-EV and Bl-EV against NASH was confirmed through Example 2, and in this Example, the therapeutic efficacy of the Roseburia and Bifidobacterium strains themselves (i.e., cells) and EVs derived from them against NASH was compared. For this purpose, according to the process shown in FIG. 7, C57BL / 6 mice were fed MCD for 2 weeks and 4 weeks, respectively, to prepare NASH animal models. For the experimental groups, Ri-EV, Bl-EV, Ri-Cell (R. intestinalis cells), or Bl-Cell (B. longum cells) were orally administered at 200 μg / mouse / day based on total protein amount once a day for a total of 1 week to mice fed MCD for 2 and 4 weeks, respectively, from the 3rd and 5th weeks. This was compared with a control group (NASH animal model) that was fed MCT and did not receive the sample group.

[0153] For blood biochemistry analysis to evaluate liver function, the mice were sacrificed one week after the start of administration of the sample group, and blood biochemistry analysis was requested to T&P Bio Co., Ltd.

[0154] As a result, in the case of the NASH animal model created by feeding MCD for 2 weeks as shown in Figure 8, it was confirmed that the ALT value was significantly reduced in the Ri-EV, Ri-Cell, Bl-EV, and Bl-Cell administration groups compared to the control group that was not administered with any sample group (cells or EV), and the degree of reduction in the ALT value was greater in the remaining sample groups except for Bl-Cell. In the case of T-BIL (Total Bilirubin), it was confirmed that the Ri-EV and Bl-EV administration groups significantly reduced the T-BIL value compared to the control group that was not administered with any sample group, and in particular, the reduction was greater than that of each cell administration group (Ri-Cell or Bl-Cell administration group). Of these, it was confirmed that the Ri-EV administration group had a significantly greater degree of reduction in T-BIL compared to the control group compared to the Bl-EV administration group.

[0155] In a NASH animal model created by feeding MCD for 4 weeks, the ALT and T-BIL levels in the cell-administered group were no different from those in the control group not administered the sample, but they were significantly reduced in the Ri-EV- or BL-EV-administered groups.

[0156] The above results indicate that administration of the Ri-EVs and BL-EVs of the present invention can reduce ALT and T-BIL levels and restore liver function in NASH animal models, and that such a therapeutic effect for NASH is much more effective than direct administration of the EV cells.

[0157] (blank)

[0158] Example 5. Analysis of anti-inflammatory activity of extracellular vesicles derived from Roseburia spp. and Bifidobacterium spp. strains

[0159] In this example, in addition to the EVs from R. intestinalis and B. longum extracted through the process of Example 1, it was confirmed whether the EVs from other Roseburia and Bifidobacterium strains could also have a therapeutic effect against NASH.

[0160] Specifically, four species of Roseburia spp. strains (Roseburia faecies, KCTC 15739, Roseburia hominis, KCTC 5845, and Roseburia inulinivorans, DSM 16841) and five species of Bifidobacterium spp. strains (Bifidobacterium bifidum, KCTC 3281, Bifidobacterium breve, KCTC 3220, Bifidobacterium lactis, KCTC 5854, Bifidobacterium adolescentis, KCTC 5856, and Bifidobacterium spp. strains, DSM 16841) were used. Extracellular vesicles were extracted from each of the strains, and their anti-inflammatory activity was analyzed. EVs were extracted in the same manner as in Example 1.

[0161] To evaluate the anti-inflammatory activity of each EV in non-alcoholic fatty liver disease, an in vitro environment replicating Kupffer cells, one of the cells that compose liver tissue, was created using mouse macrophages, Raw264.7 cells, and the amount of nitric oxide (NO) produced was measured. The results are shown in Figure 9. Specifically, mouse macrophages, Raw264.7 cells, were cultured at 37°C in the presence of 5% CO2 in RPMI1640 culture medium containing 10% fetal bovine serum (FBS) and 1% antibiotics (100 U / ml penicillin and 100 μg / ml streptomycin). Then, 5 × 10 5The cells were dispensed at a concentration of 1 ml per well and incubated in a CO2 incubator at 37°C for 24 hours. To induce inflammation, the wells were treated with medium containing 10 μg / ml lipopolysaccharide (LPS) and incubated for an additional 4 hours. The vesicles were added to the medium at a concentration of 200 μg of total protein and incubated at 37°C for 16 hours. Then, 50 μl of the well supernatant and 50 μl of Griess reagent were mixed and reacted at room temperature for 10 minutes, and the amount of nitric oxide produced was confirmed by measuring the absorbance at 540 nm using a plate reader.

[0162] As a result, it was confirmed that LPS-induced nitric oxide production was significantly and effectively reduced in all groups administered extracellular vesicles derived from Roseburia strains, which were used as experimental groups, as shown in Figure 9. It was also confirmed that nitric oxide production was effectively reduced in the group administered extracellular vesicles derived from Bifidobacterium strains, and it was confirmed that the ability of Bbr-EV and Bla-EV to suppress nitric oxide production was regulated at a total protein amount of 200μg or 50μg.

[0163] The above results indicate that EVs from different strains of the same genus, Roseburia or Bifidobacterium, have excellent anti-inflammatory effects. That is, the above results confirm that extracellular vesicles derived from Roseburia and Bifidobacterium strains can be used to prevent, improve, or treat non-alcoholic fatty liver disease through their strong anti-inflammatory effects.

[0164] (blank)

[0165] As seen from the above examples, the extracellular vesicles derived from Roseburia strains and Bifidobacterium strains of the present invention were confirmed to restore liver function in NASH animal models, reduce fat accumulation in the liver to improve steatosis, and suppress infiltration of inflammatory cells into liver tissue, thereby exerting anti-inflammatory effects, as well as reducing the expression of myofibroblasts to suppress liver fibrosis. Furthermore, it was shown that both the extracellular vesicles derived from Roseburia strains and Bifidobacterium strains, which are of the same species, exert a strong anti-inflammatory effect. In other words, the extracellular vesicles derived from Roseburia strains and Bifidobacterium strains can improve liver damage and dysfunction caused by fatty liver and exert strong anti-inflammatory and anti-fibrotic effects, and therefore may be used as a composition for preventing, improving, and / or treating non-alcoholic fatty liver disease.

[0166] (blank)

[0167] From the above description, a person skilled in the art to which the present invention belongs can understand that the present invention can be embodied in other specific forms without changing the technical idea or essential features of the present invention. In this regard, the above-described embodiments should be understood to be illustrative and not limiting in all respects. The scope of the present invention should be interpreted as including all modifications or variations derived from the meaning and scope of the claims described below from the above detailed description, and their equivalent concepts. [Industrial Applicability]

[0168] Industrial Applicability

[0169] The extracellular vesicles derived from the Roseburia or Bifidobacterium strains of the present invention were confirmed to restore liver function in an animal model of non-alcoholic fatty liver disease, reduce fat accumulation in the liver to improve steatosis, and suppress infiltration of inflammatory cells into liver tissue, as well as to reduce the expression of myofibroblasts and suppress liver fibrosis. Furthermore, it was shown that both the Roseburia and Bifidobacterium strain-derived extracellular vesicles, which are of the same species and different genera, exhibit strong anti-inflammatory effects. Thus, the Roseburia strain-derived extracellular vesicles and Bifidobacterium strain-derived extracellular vesicles can improve liver damage and dysfunction caused by fatty liver and exhibit strong anti-inflammatory and anti-fibrotic effects, and therefore may be used as a composition for preventing, improving, and / or treating non-alcoholic fatty liver disease.

Claims

1. A pharmaceutical composition for preventing or treating non-alcoholic fatty liver disease, comprising at least one active ingredient selected from the group consisting of extracellular vesicles derived from Roseburia spp. strains and extracellular vesicles derived from Bifidobacterium spp. strains.

2. The pharmaceutical composition of claim 1, wherein the extracellular vesicles have an average diameter of 30 nm to 200 nm.

3. The extracellular vesicles derived from a Roseburia strain are naturally or artificially secreted from a Roseburia strain, The pharmaceutical composition according to claim 1, wherein the extracellular vesicles derived from a Bifidobacterium strain are naturally or artificially secreted from a Bifidobacterium strain.

4. 2. The pharmaceutical composition according to claim 1, wherein the Roseburia strain is at least one selected from the group consisting of Roseburia intestinalis, Roseburia faeces, Roseburia hominis, and Roseburia inulinivorans.

5. 2. The pharmaceutical composition according to claim 1, wherein the Bifidobacterium strain is at least one selected from the group consisting of Bifidobacterium longum, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium lactis, and Bifidobacterium adolescentis.

6. 2. The pharmaceutical composition of claim 1, wherein the non-alcoholic fatty liver disease is at least one selected from the group consisting of fatty liver, fibrosis, non-alcoholic steatohepatitis, non-alcoholic fatty liver (NAFL), and cirrhosis.

7. The pharmaceutical composition according to claim 1, characterized in that it has at least one effect selected from the group consisting of the following: i) a decrease in serum aspartate aminotransferase (AST) levels or activity ii) a decrease in serum alanine aminotransferase (ALT) levels or activity iii) Reduction in serum total bilirubin (T-BIL) levels iv) Reduction of fatty liver v) Reduction of inflammatory cell infiltration vi) Reduction in the number of inflammatory lesions vii) Reduction in myofibroblast numbers

8. A kit for preventing or treating non-alcoholic fatty liver disease, comprising the pharmaceutical composition according to any one of claims 1 to 7.

9. A method for preventing or treating non-alcoholic fatty liver disease, comprising administering to an individual in need thereof at least one selected from the group consisting of extracellular vesicles derived from a Roseburia strain and extracellular vesicles derived from a Bifidobacterium strain.

10. A food composition for preventing or ameliorating non-alcoholic fatty liver disease, comprising as an active ingredient at least one selected from the group consisting of extracellular vesicles derived from a Roseburia strain and extracellular vesicles derived from a Bifidobacterium strain.

11. The food composition according to claim 10, wherein the food composition is a functional health food composition.

12. A feed composition for preventing or ameliorating non-alcoholic fatty liver disease, comprising as an active ingredient at least one selected from the group consisting of extracellular vesicles derived from a Roseburia strain and extracellular vesicles derived from a Bifidobacterium strain.

13. 2. Use of at least one selected from the group consisting of extracellular vesicles derived from a Roseburia strain and extracellular vesicles derived from a Bifidobacterium strain for the prevention or treatment of non-alcoholic fatty liver disease.

14. 1. Use of at least one selected from the group consisting of extracellular vesicles derived from a Roseburia strain and extracellular vesicles derived from a Bifidobacterium strain for the manufacture of a therapeutic agent for non-alcoholic fatty liver disease.

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