KR20230112549A - Compositions for preventing, alleviating, or treating inflammatory diseases, comprising extracellular vesicles derived from Roseburia spp. or Bifidobacterium spp. - Google Patents

Abstract

The present invention relates to a composition for preventing, improving or treating inflammatory diseases, including extracellular vesicles (EV) derived from Roseburia spp. and/or Bifidobacterium spp. The extracellular vesicles of the present invention reduce the intestinal epithelial cell barrier and infiltration of immune cells in IBD patients, maintain the number of goblet cells, suppress the decrease in caecal weight and intestinal length associated with intestinal inflammation, suppress the secretion of inflammatory cytokines through changes in the intestinal flora, and restore the mucous secretion of goblet cells and the tight coupling ability of intestinal epithelial cells, thereby improving IBD. In addition, as a result of confirming the anti-inflammatory effect of EVs derived from homologous and heterogeneous strains corresponding to Roseburia genus strains or Bifidobacterium genus strains, it was confirmed that all of the EVs exerted a strong anti-inflammatory effect. Therefore, the strain of the present invention and the EV derived therefrom can be provided as a composition for preventing, improving, or treating various inflammatory diseases.

Description

Compositions for preventing, alleviating, or treating inflammatory diseases, comprising extracellular vesicles derived from Roseburia spp. or Bifidobacterium spp.}

The present invention relates to a composition for preventing, improving or treating inflammatory diseases, including Roseburia spp. strains, Bifidobacterium spp. strains, or extracellular vesicles (EVs) derived therefrom.

Inflammation refers to the pathological condition of an abscess formed by the invasion of external infectious agents (bacteria, fungi, viruses, various types of allergens). Specifically, when external bacteria invade and proliferate in a specific tissue, the leukocytes of the body recognize this and actively attack the proliferated external bacteria. During this process, the dead cells of the white blood cells generated are accumulated in the tissue invaded by the bacteria, and at the same time, the cell destruction of the invaded bacteria killed by the white blood cells dissolves into the invaded tissue to form an abscess. The treatment of abscesses due to inflammation can be promoted through anti-inflammatory action, which is an inflammatory treatment promoting action, such as inhibiting the proliferation of invading bacteria using antibacterial agents or activating macrophages that phagocytize foreign substances accumulated in abscesses to enhance the function of macrophages to digest and excrete the foreign substances.

In general, the inflammatory response is a biological defense reaction process for repairing and regenerating damage caused by invasion that causes organic changes in cells or tissues of a living body, and local blood vessels, various tissue cells of body fluids, and immune cells act in this reaction process. Normally, the inflammatory response induced by external invading bacteria is a defense system to protect the living body, but when an abnormally excessive inflammatory response is induced, various 　 diseases appear, and these 　 diseases are called inflammatory diseases. In inflammatory diseases, various inflammatory mediators secreted from target cells activated by external stimuli amplify and sustain inflammation, which can threaten the life of the human body. Inflammatory diseases include acute bronchitis, chronic bronchitis, acute bronchiolitis, chronic bronchiolitis, sepsis, septic shock, acute respiratory distress syndrome, multiple organ failure, and chronic obstructive pulmonary disease. Inflammatory bowel disease (IBD), one of the representative inflammatory diseases, is a disease that causes chronic inflammation in the gastrointestinal tract, shows a chronic course, gradually progresses, causes complications, and reduces physical function (SY Na and W Moon, Korean J Gastroenterol Vol. 71 No. 2, 61-68). Inflammatory bowel disease is mainly classified into two forms: ulcerative colitis (UC) and Crohn's disease (CD). Inflammatory bowel disease has no clear pathogenesis, and is presumed to be caused by a combination of genetic factors and environmental factors such as stress and drug use. Therefore, since each patient has a different etiology and a different course and prognosis, it is a disease that is difficult to cure.

The onset of many inflammation-related diseases is associated with the activation of macrophages and the consequent excessive production of inflammation-related factors, such as IL-1β and TNF-α. In addition, among the inducers of the inflammatory response, LPS (lipopolysaccharide) is a substance constituting the cell surface of Gram-negative bacteria, interacts with immune cells such as leukocytes, and is involved in the regulation of inflammatory-related cytokines. In particular, inflammatory cytokines such as IL-1βTNF-α and IL-6 play an important role in mediating the pathological progression of various infectious or inflammatory diseases. These inflammatory cytokines are produced by various immune and inflammatory cells and contribute to the recruitment of additional inflammatory cells to the site of injury resulting in tissue and organ damage.

Republic of Korea Patent Publication No. 10-2017-0033372

As a result of diligent efforts to find a drug having a superior effect than conventional therapeutic agents for inflammatory diseases, the inventors of the present invention have completed the present invention by confirming that extracellular vesicles (EVs) derived from Roseburia spp. strains and Bifidobacterium spp.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing or treating inflammatory diseases, comprising at least one selected from the group consisting of extracellular vesicles derived from strains of the genus Roseburia and extracellular vesicles derived from strains of the genus Bifidobacterium as an active ingredient.

Another object of the present invention is to provide a kit for preventing or treating inflammatory diseases, comprising at least one selected from the group consisting of extracellular vesicles derived from strains of the genus Roseburia and extracellular vesicles derived from strains of the genus Bifidobacterium as an active ingredient.

Another object of the present invention is to provide a method for preventing or treating inflammatory diseases, comprising administering at least one selected from the group consisting of extracellular vesicles derived from strains of the genus Roseburia and extracellular vesicles derived from strains of the genus Bifidobacterium to a subject in need thereof.

Another object of the present invention is to provide a food composition for preventing or improving inflammatory diseases, comprising at least one selected from the group consisting of extracellular vesicles derived from strains of the genus Roseburia and extracellular vesicles derived from strains of the genus Bifidobacterium.

Another object of the present invention is to provide a feed composition for preventing or improving inflammatory diseases, comprising at least one selected from the group consisting of extracellular vesicles derived from strains of the genus Roseburia and extracellular vesicles derived from strains of the genus Bifidobacterium.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

The present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases comprising, as an active ingredient, at least one selected from the group consisting of extracellular vesicles derived from Roseburia spp. strains and extracellular vesicles derived from Bifidobacterium spp. strains.

In addition, the present invention provides a food composition for preventing or improving inflammatory diseases, comprising at least one selected from the group consisting of extracellular vesicles derived from strains of the genus Roseburia and extracellular vesicles derived from strains of the genus Bifidobacterium as an active ingredient.

In addition, the present invention provides a feed composition for preventing or improving inflammatory diseases, comprising at least one selected from the group consisting of extracellular vesicles derived from a strain of the genus Roseburia and extracellular vesicles derived from a strain of the genus Bifidobacterium as an active ingredient.

In addition, the present invention provides a kit for preventing, improving, or treating inflammatory diseases, including at least one selected from the group consisting of extracellular vesicles derived from a strain of the genus Roseburia and extracellular vesicles derived from a strain of the genus Bifidobacterium.

In addition, the present invention provides a method for preventing or treating inflammatory diseases, comprising administering at least one selected from the group consisting of extracellular vesicles derived from strains of the genus Roseburia and extracellular vesicles derived from strains of the genus Bifidobacterium to a subject in need thereof.

In addition, the present invention provides a use for preventing or treating one or more inflammatory diseases selected from the group consisting of extracellular vesicles derived from strains of the genus Roseburia and extracellular vesicles derived from strains of the genus Bifidobacterium.

In addition, the present invention provides one or more uses selected from the group consisting of extracellular vesicles derived from a strain of the genus Roseburia and extracellular vesicles derived from a strain of the genus Bifidobacterium for the preparation of a therapeutic agent for inflammatory diseases.

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.

In another embodiment of the present invention, the extracellular vesicles derived from the strain of the genus Roseburia are naturally or artificially secreted from the strain of the genus Roseburia;

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

In another embodiment of the present invention, the strain of the genus Roseburia may be one or more selected from the group consisting of Roseburia intestinalis , Roseburia faecies , Roseburia hominis , and Roseburia inulinivorans , but is not limited thereto.

In another embodiment of the present invention, the strain of the genus Bifidobacterium is Bifidobacterium longum, Bifidobacterium bifidum, Bifidobacterium breve , Bifidobacterium lactis , and Bifidobacterium adolescentis . It may be one or more selected from the group consisting of, but is not limited thereto.

In another embodiment of the present invention, the inflammatory disease is gastritis, gastric ulcer, duodenal ulcer, inflammatory skin disease, allergic 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 arthritis, chronic bronchiolitis, osteoarthritis, gout, spondyloarthropathy, ankylosing spondylitis, Reiter's syndrome, psoriatic arthropathy, enteropathic spondylitis, juvenile arthropathy, juvenile ankylosing spondylitis, reactive arthropathy, infectious arthritis, post-infectious arthritis, gonococcal arthritis, tuberculous arthritis, viral arthritis, fungal arthritis, syphilitic arthritis, Lyme disease, arthritis associated with vasculitic syndrome, polyarteritis nodosa, hypersensitivity vasculitis, Lou Gehrig Granulomatosis, polymyalgia rheumatica, joint cell arteritis, calcium crystal deposition arthropathy, pseudogout, non-articular rheumatism, bursitis, tenosynovitis, epicondylitis, neuropathic joint disease (charco and joint), hemorrhagic arthrosis (hemarthrosic), Henoch-Schönlein purpura, hypertrophic osteoarthropathy, multicentric reticulocytoma, surcoilosis, blood Pigmentosis, sickle cell disease, hemochromatosis, hyperlipoproteinemia, hypogammaglobulinemia, familial thalassemia fever, Behatt's disease, systemic lupus erythematosus, relapsing fever, psoriasis, multiple sclerosis, sepsis, septic shock, multiorgan dysfunction syndrome, acute respiratory distress syndrome, chronic obstructive pulmonary disease, acute lung injury, and broncho-pulmonary dysplasia dysplasia), but may be one or more selected from the group consisting of, but is not limited thereto.

In another embodiment of the present invention, the inflammatory bowel disease may be at least one selected from the group consisting of ulcerative colitis, Crohn's disease, Behcet's disease, collagenous colitis, lymphocytic colitis, ischemic colitis, hemorrhagic rectal ulcer, ileal pouchitis, and converting colitis, but is not limited thereto.

In another embodiment of the present invention, the composition according to the present invention may have one or more effects selected from the group consisting of, but not limited to:

i) reducing intestinal epithelial barrier damage;

ii) reduction of intestinal tissue infiltration of immune cells;

iii) inhibition of reduction in the number of goblet cells;

iv) inhibition of cecal weight loss;

v) inhibition of intestinal length reduction;

vi) inhibition of inflammatory cytokine production;

vii) promoting anti-inflammatory cytokine production;

viii) enhancement of mucus secretion by goblet cells;

ix) enhancing the compactness of intestinal epithelial cells; and

x) Increased number of Bifidobacterium in the intestine.

In another embodiment of the present invention, the food composition may be a health functional food composition, but is not limited thereto.

The present invention relates to a composition for preventing, improving or treating inflammatory diseases, including extracellular vesicles (EV) derived from Roseburia spp. and/or Bifidobacterium spp. The extracellular vesicles of the present invention have been shown to reduce intestinal epithelial cell barrier damage and immune cell infiltration in inflammatory bowel disease animal models, maintain the number of goblet cells, suppress a decrease in caecal weight and intestinal length due to enteritis, and inhibit secretion of inflammatory cytokines through changes in intestinal flora, as well as restore mucous secretion of goblet cells and the ability of intestinal epithelial cells to bind tightly. In particular, it was confirmed that all EVs derived from homologous and heterologous strains corresponding to Roseburia genus strains or Bifidobacterium genus strains exert strong anti-inflammatory effects. That is, the extracellular vesicles derived from the strain of the genus Roseburia or the genus Bifidobacterium according to the present invention can effectively inhibit inflammation and improve tissue damage or functional abnormality due to inflammation, and thus prevent, improve, and / or treat various inflammatory diseases. It can be provided as a composition for treatment.

1 is Roseburia intestinalis ) derived extracellular vesicles (extracellular vesicles, EV) (Ri-EV) Ultrathin-section TEM (left) and Cryo-TEM (right) photographs.
2 is a result of measuring the size and surface charge of Ri-EV.
Figure 3a is a result showing the particle concentration of Ri-EV extracted from R. intestinalis .
Figure 3b is Roseburia interiorlis derived extracellular vesicles (Ri-EV) and Bifidobacterium longum ( Bifidobacterium longum ) derived This is the result of measuring the particle size of EV (Bl-EV).
4 is a schematic diagram of an experiment of a dextran sulfate sodium salt colitis grade (DSS) induced inflammatory bowel disease (IBD) animal model.
5 is a diagram of H&E (Hematoxilin-Eosin) staining and AB (Alcian blue) staining of each intestinal tissue to observe the intestinal epithelial cell barrier of the control group, DSS-induced IBD animal model (DSS), R. intestinalis administration group (Ri-Cell), and Ri-EV administration group (Ri-EV).
6A to 6D are diagrams showing the results of measuring histological scores, the number of goblet cells (FIG. 6A), the weight of the cecum (FIG. 6B), the length of the intestine (FIG. 6C), and the disease activity index (FIG. 6D) for pathological analysis of the intestinal tissues of the control group, the DSS-induced IBD animal model, the R. intestinalis -administered group, and the Ri-EV-administered group.
7a to 7b show the results of measuring the expression levels (FIG. 7b) of IL (Interleukin)-1β in the intestinal tissue of the control group, the DSS-induced IBD animal model, the R. intestinalis- administered group, and the Ri-EV-administered group (Fig. 7a), Muc-2 (Mucin 2), ZO-1 (Zonula occludens-1) in order to confirm changes in intestinal inflammation level, mucous protein level, and tight junctions according to R. intestinalis or Ri-EV administration. is a diagram showing
8a and 8b are diagrams showing the results of measuring the intestinal microbial communities (FIG. 8a) and Shannon diversity (FIG. 8b) of a control group, a DSS-induced IBD animal model, an R. intestinalis- administered group, and a Ri-EV-administered group in order to confirm changes in the intestinal microbial community according to R. intestinalis or Ri-EV administration.
9 is a diagram showing the abundance of Bifidobacterium in the control group, the DSS-induced IBD animal model, the R. intestinalis administration group, and the Ri-EV administration group.
Figure 10 shows the amount of nitric oxide ( NO ) production, the amount of inflammatory cytokines (TNF-α, IL -6), and the anti-inflammatory cytokines ( IL-10) is a diagram showing the expression level measured.
Figure 11 is a Kuffer cell-simulated in vitro environment after inducing an inflammatory environment by treating LPS, Roseburia genus strains ( R. intestinalis, R. faecies, R. hominis, and R. inulinivorans ) derived EVs or Bifidobacterium genus strains (B. bifidum, B. breve, B. lactis, B. adolescentis, and B. longum ) The inflammation inhibitory effect according to the treatment of EVs produced by nitric oxide This is the result confirmed through measurement.

Hereinafter, examples will be described in detail to aid understanding of the present invention. However, the following examples are merely illustrative of the contents of the present invention, but the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.

The present invention relates to a pharmaceutical composition for the prevention or treatment of inflammatory diseases, including extracellular vesicles derived from Roseburia spp. and extracellular vesicles derived from Bifidobacterium spp. It was completed by confirming that it could be improved. Therefore, the extracellular vesicles of the present invention can be used as a composition for preventing, improving, and/or treating various inflammatory diseases.

The composition according to the present invention is Roseburia spp. strain, its lysate, its culture medium, its extracellular vesicles, and mixtures thereof; And it may include at least one selected from the group consisting of Bifidobacterium spp., a lysate thereof, a culture solution thereof, an extracellular vesicle thereof, and a mixture thereof. In one embodiment of the present invention, the composition according to the present invention may not contain a strain.

In the present invention, " Roseburia spp. or the Roseburia genus strain" includes strains already known in the art or newly known in the future as strains belonging to the Roseburia genus. Roseburia is a Gram-positive bacterium that inhabits the human colon and is known to have glycolytic and butyrate-producing abilities. Roseburia genus strains according to the present invention are included without limitation as long as they correspond to the Roseburia genus and are not limited to specific types, but are preferably Roseburia intestinalis , Roseburia faecies , Roseburia hominis, Roseburia inulinivorans , and the like. It may be one or more selected from the group consisting of Ceburia cecicola . Among them, " Roseburia intestinalis " is a microorganism first isolated from human feces, and is known to maintain energy homeostasis by producing metabolites in the intestine.

In the present invention, " Bifidobacterium spp. or the genus Bifidobacterium strain" includes strains already known in the art or newly known in the future as strains belonging to the genus Bifidobacterium. Bifidobacterium is a Gram-positive anaerobic bacterium, and is one of the main types of bacteria constituting the gastrointestinal tract microflora of mammals. Some Bifidobacterium are also used as lactobacilli. Bifidobacterium strains according to the present invention are included without limitation as long as they correspond to the genus Bifidobacterium and are not limited to specific types, but are preferably Bifidobacterium longum , Bifidobacterium bifidum, Bifidobacterium breve , Bifidobacterium lactis , and Bifidobacterium. It may be at least one selected from the group consisting of Bifidobacterium adolescentis . Among them, “ Bifidobacterium longum” is also named as Bifidobacteria, and is known to be effective in inhibiting the growth of Escherichia coli and enhancing intestinal motility and defecation activity.

In the present invention, the term "strain" includes not only live cells obtained from cultures such as culture media, but also any processed forms of strains known to those skilled in the art, for example, cell lysates, dried products, frozen products, etc., but is not limited thereto. In the present specification, the term “strain” may be used interchangeably with “bacteria” and “cells”.

As used herein, the term "lysate" may refer to a product obtained by crushing the cell wall of a strain by applying chemical treatment or physical force to the strain.

In the present invention, the term "culture medium" is obtained by culturing the strain for a certain period of time in a medium capable of supplying nutrients so that the strain can grow and survive in vitro. In addition, the culture solution may mean a culture solution obtained by removing the cells from the cell culture solution obtained by culturing the strain. On the other hand, the liquid from which the cells are removed from the culture solution is also called "supernatant", and the culture solution is left for a certain period of time to take only the liquid of the upper layer except for the part sunk in the lower layer, or the cells are removed through filtration. The "cell" refers to the "strain" itself of the present invention, and includes the "strain" itself separated and selected from biological samples, etc., or the "strain" separated from the culture medium in which the "strain" is cultured. The cells can be obtained by centrifuging the culture solution and taking the part that has sunk in the lower layer, or it can be obtained by leaving it for a certain period of time and then removing the upper liquid as it sinks to the lower layer of the culture medium by gravity. The culture medium may be used interchangeably with "culture supernatant", "conditioned medium" or "conditioned medium".

The culture medium itself obtained by culturing the strain, its extract, its concentrate, or its lyophilized product; Or a culture supernatant obtained by removing the strain from the culture medium, an extract thereof, a concentrate thereof, or a lyophilisate thereof; may include, but is not limited thereto.

The culture medium is prepared by cultivating the strain of the present invention in an appropriate medium (eg, reinforced clostridial medium (RCM) 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 40 hours. It may be obtained by culturing for 20 hours, or 10 to 20 hours, but is not limited thereto. Culture medium and culture conditions for culturing the strain of the present invention can be appropriately selected or modified by those skilled in the art.

In one embodiment, the culture supernatant of the present invention can be obtained by removing the strain from the strain culture medium described above by centrifugation, filtration, or the like.

In the present invention, the term "concentrate" may be obtained by concentrating the above-described culture medium itself or the supernatant obtained by centrifugation, filtration, or the like.

In the present invention, the term "extract" means an extract from the aforementioned culture medium or a concentrate thereof, and may include an extract, a diluent or concentrate of the extract, a dried or lyophilized product obtained by drying the extract, or a crude or purified product thereof, or a fraction obtained by fractionating them.

In the present invention, the term "vesicle" refers to particles secreted from cells 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 secreting cell of origin (donor cell), exhibit various biological activities depending on which cell they are secreted from, and play an important role in cell-to-cell interactions by transferring genetic material and proteins between cells. In addition, cell-derived substances including the endoplasmic reticulum cause diseases or stimulate immune cells to fight against diseases, and have an effect of helping to decompose and absorb substances that humans cannot digest through the metabolic process of microorganisms. The endoplasmic reticulum is a membrane-structured endoplasmic reticulum, which is divided into an inside and an outside, has a plasma membrane lipid, a plasma membrane protein, a nucleic acid, and a cytoplasmic component of the cell, and may be smaller in size than the original cell. The term "extracellular endoplasmic reticulum" may be used interchangeably with "extracellular endoplasmic reticulum", and may be briefly referred to as "endosicles" or "vesicles".

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

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

Vesicles according to the present invention can be produced during in vitro cultivation of strains. Specifically, the vesicles according to the present invention may be naturally secreted from strains of the genus Roseburia and / or strains of the genus Bifidobacterium or artificially produced (separated) through heat treatment, pressure treatment, etc. to bacteria, 30 to 200 nm, 30 to 150 nm, 30 to 100 nm, 30 to 80 nm, 40 to 200 nm, 50 to 200 nm, 50 to 100 nm, or may have a diameter of 50 to 80 nm. In one embodiment of the present invention, as a result of measuring the sizes of R. intestinalis- derived EV and B. longum- derived EV using a zeta-sizer, it was confirmed that each had an average particle size of about 76 nm (FIG. 1).

In addition, the extracellular vesicles according to the present invention may have a surface charge of -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, but is not limited thereto.

The vesicles may be separated by applying one or more methods selected from the group consisting of centrifugation, ultra-high-speed centrifugation, extrusion, sonication, cell lysis, homogenization, freeze-thaw, electroporation, mechanical disassembly, chemical treatment, filtration with a filter, gel filtration chromatography, free-flow electrophoresis, and capillary electrophoresis to a culture solution containing a Roseburia genus strain and/or a Bifidobacterium genus strain. In addition, processes such as washing to remove impurities and concentration of the obtained vesicles may be further included.

The composition according to the present invention may have preventive, ameliorative, and/or therapeutic effects on inflammatory diseases.

For example, the composition according to the present invention may have one or more effects selected from among the following:

i) reducing intestinal epithelial barrier damage;

ii) reduced intestinal tissue infiltration of immune cells;

iii) inhibition of reduction in the number of goblet cells;

iv) inhibition of cecal weight loss;

v) inhibition of intestinal length reduction;

vi) inhibition of inflammatory cytokine production;

vii) promoting anti-inflammatory cytokine production;

viii) enhancement of mucus secretion by goblet cells;

ix) promoting the compaction of intestinal epithelial cells; and

x) Increased number of Bifidobacterium in the intestine.

That is, the effects of preventing, improving, and/or treating inflammatory diseases of the composition according to the present invention may be exerted by the effects of i) to x).

i) reduction of intestinal epithelial barrier damage; ii) reduced immune cell infiltration; iii) Regarding the effect of inhibiting the decrease in the number of goblet cells, in one embodiment of the present invention, as a result of experiments using an inflammatory bowel disease (IBD) animal model, the IBD animal model had intestinal epithelial cell barrier damage, immune cells were infiltrated, and the number of goblet cells responsible for mucus production was reduced, whereas in the Ri-EV-administered group of the present invention, intestinal epithelial cell barrier damage was improved, immune cell infiltration was reduced, and the number of goblet cells was maintained at a level similar to that of the normal control group. In particular, when Ri-EV was administered, the histological score due to IBD significantly decreased, and it was confirmed that intestinal damage and functional abnormality due to IBD were improved (FIGS. 5 and 6a).

iv) inhibition of caecal weight loss; v) Regarding the intestinal length reduction inhibitory effect, in another embodiment of the present invention, the caecal weight and intestinal length were significantly reduced due to intestinal inflammation in the IBD animal model compared to the normal control group, whereas the R. intestinalis or Ri-EV administered group suppressed the reduction in caecal weight and intestinal length, and in particular, the effect of the caecum and intestinal recovery / improvement of the extracellular endoplasmic reticulum was more excellent than that of the strain itself (Figs. 6b, 6c). The disease activity index also decreased when R. intestinalis or Ri-EV was administered, and it was confirmed that the disease activity was the lowest in the Ri-EV-administered group (FIG. 6d). The above results show that the extracellular vesicles according to the present invention can improve histological changes in the cecum or intestine caused by intestinal inflammation.

vi) inhibition of inflammatory cytokine secretion by the composition of the present invention; vii) promoting anti-inflammatory cytokine production; viii) enhancement of mucus secretion by goblet cells; ix) Regarding the effect of enhancing the tight junction of intestinal epithelial cells, in another embodiment of the present invention, the expression of the inflammatory cytokine IL-1β increased in the IBD animal model compared to the normal control group, and mucous proteins and tight junction. As the expression of Muc-2 and ZO-1 decreased, it was confirmed that intestinal inflammation increased and mucus secretion and tight junction were weakened. On the other hand, in the R. intestinalis or Ri-EV-administered group, the expression of IL-1β was decreased, and the expression of Muc-2 and ZO-1 was increased. In particular, such intestinal anti-inflammatory effect and mucus secretion ability and recovery ability of dense bonding were found to be more excellent than the extracellular vesicles compared to strains. In addition, as a result of confirming their anti-inflammatory effect by isolating extracellular vesicles from various homologous and heterogeneous strains of the genus Roseburia and strains of the genus Bifidobacterium, it was confirmed that both of the extracellular vesicles suppressed the production of inflammatory factors and promoted the production of anti-inflammatory cytokines to exert excellent anti-inflammatory effects (FIGS. 10 and 11).

In relation to x) the effect of increasing the number of bifidobacterium in the intestine, in another embodiment of the present invention, in the IBD animal model compared to the control group, changes in the intestinal microbial community, such as an increase in the phylum of Proteobacteria and Deferribacteres , were conspicuously observed (Figs. The number of Pidobacterium was increased, and in particular, the number of Bifidobacterium was significantly increased in the Ri-EV-administered group than in the R. intestinalis -administered group (FIG. 9). From this, it can be seen that the administration of the roseburia strain-derived extracellular vesicles according to the present invention induces changes in the intestinal microflora according to the growth induction of Bifidobacterium in the intestinal environment of IBD, thereby inhibiting the secretion of inflammatory cytokines, and recovering mucus secretion of goblet cells and the ability of intestinal epithelial cells to bind tightly, thereby improving IBD.

In summary, the composition according to the present invention can reduce intestinal epithelial cell barrier damage due to inflammation and infiltration of immune cells into the intestine, maintain the number of goblet cells at a normal level, suppress the decrease in caecal weight and intestinal length due to intestinal inflammation, suppress the secretion of inflammatory cytokines through changes in intestinal flora, and restore mucus secretion of goblet cells and the ability of intestinal epithelial cells to bind tightly, and thus can be used for the prevention, improvement, and/or treatment of inflammatory diseases.

Meanwhile, the composition according to the present invention may be used for preventing, improving, and/or treating “inflammatory diseases”. In the present invention, inflammatory diseases include without limitation any disease related to inflammation or a disease that can be improved/treated by suppressing inflammation, and is not limited to specific types, 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, enteropathic spondylitis, juvenile arthropathy, juvenile ankylosing spondylitis, reactive arthropathy, infectious arthritis, post-infectious arthritis, gonococcal arthritis, tuberculosis Arthritis, viral arthritis, fungal arthritis, syphilitic arthritis, Lyme disease, arthritis associated with vasculitic syndrome, polyarteritis nodosa, hypersensitivity vasculitis, amyotrophic granulomatosis, polymyalgia rheumatica, articular cell arteritis, calcium crystal deposition arthropathy, pseudogout, non-articular rheumatism, bursitis, tenosynovitis, epicondylitis (tennis elbow), neuropathic joint disease (charco and joint), hemar throsic), Henoch-Schenlein purpura, hypertrophic osteoarthropathy, multicentric reticuloid histiocytoma, surcoilosis, hemochromatosis, sickle cell disease and other hemoglobinopathy, hyperlipoproteinemia, hypogammaglobulinemia, familial thalassemia fever, Behart's disease, systemic lupus erythematosus, relapsing fever, psoriasis, multiple sclerosis, sepsis, septic shock, multiple bowel It may be selected from respiratory dysfunction syndrome, acute respiratory distress syndrome, chronic obstructive pulmonary disease, acute lung injury, broncho-pulmonary dysplasia, and the like.

In the present invention, the term "inflammatory bowel disease (IBD)" means a disease caused by chronic inflammation in the gastrointestinal tract. Inflammatory bowel disease refers to a disease in which chronic inflammation occurs in the intestine, and has recurrent abdominal pain, diarrhea, bloody stools, weight loss, and the like as major symptoms. Specifically, it collectively refers to chronic digestive diseases in which inflammation in the intestines lasts for 6 months or more and becomes chronic. Accordingly, infectious enteritis such as bacterial, viral, amebic, and tuberculosis enteritis, ischemic bowel disease, and radiation-induced enteritis may be included therein. In the present invention, inflammatory bowel disease includes without limitation any disease related to intestinal inflammation or a disease that can be improved/treated by suppressing inflammation, and is not limited to specific types, but is preferably one or more selected from the group consisting of ulcerative colitis, Crohn's disease, Behçet's disease, collagenous colitis, lymphocytic colitis, ischemic colitis, hemorrhagic rectal ulcer, ileal pouchitis, and converting colitis.

Preferably, the inflammatory bowel disease according to the present invention may be selected from ulcerative colitis (UC), Crohn's disease (CD), and Behcet's disease. "Ulcerative colitis" means a chronic inflammatory disease in the large intestine, and according to its scope, it can be further divided into proctitis, left colitis, and extensive colitis. "Crohn's disease" refers to a disease in which inflammation occurs throughout the digestive tract, from the mouth to the anus. However, the inflammatory site is not always continuous, and may exist separately in several places. About 1/3 of patients with Crohn's disease have inflammation only in the small intestine, 1/3 have inflammation only in the large intestine, and 1/3 have chronic inflammation in both the large intestine and small intestine. In particular, the most common case where inflammation occurs in the area where the end of the small intestine and the large intestine meet. Ulcerative colitis is inflamed only in the mucosal layer of the intestine, whereas Crohn's disease is characterized by an inflammatory reaction that invades all layers of the intestinal wall, including the mucosal, submucosal, muscular and serosal layers. “Behçet’s disease” is a chronic and repetitive systemic disease in which inflammation invades various organs such as the skin, mucous membranes, eyes, intestines, joints, genitourinary system, and the causal system. In 3 to 5% of patients with Behcet's disease, inflammation occurs in the small or large intestine, and inflammation most commonly occurs at the site where the end of the small intestine and the large intestine are connected.

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

The pharmaceutical composition according to the present invention may further include suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions. The excipient may be, for example, one or more selected from the group consisting of a diluent, a binder, a disintegrant, a lubricant, an adsorbent, a moisturizer, a film-coating material, and a controlled release additive.

The pharmaceutical compositions according to the present invention are powders, granules, sustained-release granules, enteric granules, solutions, eye drops, elsilic agents, emulsions, suspensions, spirits, troches, perfumes, limonadese, tablets, sustained-release tablets, enteric-coated tablets, sublingual tablets, hard capsules, soft capsules, sustained-release capsules, enteric capsules, pills, tinctures, and soft drinks, respectively, according to conventional methods. It can be formulated and used in the form of external preparations such as extracts, dry extracts, fluid extracts, injections, capsules, irrigation solutions, warning agents, lotions, pastes, sprays, inhalants, patches, sterilized injection solutions, or aerosols.

Carriers, excipients and diluents that may be included in the pharmaceutical composition according to the present invention include lactose, dextrose, sucrose, oligosaccharide, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, and propylhydroxy. hydroxybenzoate, talc, magnesium stearate and mineral oil.

When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Corn starch, potato starch, wheat starch, lactose, white sugar, glucose, fructose, D-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, calcium monohydrogen phosphate, calcium sulfate, sodium chloride, sodium hydrogen carbonate, purified lanolin, microcrystalline cellulose, dextrin, sodium alginate, methyl cellulose, carboxymethyl cellulose sodium, kaol excipients such as phosphorus, urea, colloidal silica gel, hydroxypropyl starch, hydroxypropylmethylcellulose (HPMC) 1928, HPMC 2208, HPMC 2906, HPMC 2910, propylene glycol, casein, calcium lactate, and Primogel; Gelatin, gum arabic, ethanol, agar powder, cellulose phthalate acetate, carboxymethylcellulose, calcium carboxymethylcellulose, glucose, purified water, sodium caseinate, glycerin, stearic acid, sodium carboxymethylcellulose, sodium methylcellulose, methylcellulose, microcrystalline cellulose, dextrin, hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, purified shellac, starch starch, hydroxypropylcellulose, hydroxypropylmethyl Binders such as cellulose, polyvinyl alcohol, and polyvinylpyrrolidone may be used, and hydroxypropyl methyl cellulose, corn starch, agar powder, methyl cellulose, bentonite, hydroxypropyl starch, sodium carboxymethyl cellulose, sodium alginate, calcium carboxymethyl cellulose, calcium citrate, sodium lauryl sulfate, silicic anhydride, 1-hydroxypropyl cellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde disintegrants such as hydrotreated casein and gelatin, alginic acid, amylose, guar gum, sodium bicarbonate, polyvinylpyrrolidone, calcium phosphate, gelled starch, gum arabic, amylopectin, pectin, sodium polyphosphate, ethyl cellulose, sucrose, magnesium aluminum silicate, di-sorbitol liquid, light anhydrous silicic acid; Calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopod, kaolin, petrolatum, sodium stearate, cacao butter, sodium salicylate, magnesium salicylate, polyethylene glycol (PEG) 4000, PEG 6000, liquid paraffin, hydrogenated soybean oil (Lubri wax), aluminum stearate, Lubricants such as zinc stearate, sodium lauryl sulfate, magnesium oxide, macrogol, synthetic aluminum silicate, silicic anhydride, higher fatty acid, higher alcohol, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, light anhydrous silicic acid; can be used.

As the additives for the liquid formulation according to the present invention, 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, carr boxymethylcellulose sodium and the like can be used.

In the syrup according to the present invention, a solution of white sugar, other sugars, or a sweetener may be used, and aromatics, coloring agents, preservatives, stabilizers, suspending agents, emulsifiers, thickeners, etc. may be used as necessary.

Purified water may be used in the emulsion according to the present invention, and emulsifiers, preservatives, stabilizers, fragrances, etc. may be used as needed.

Acacia, tragacantha, methyl cellulose, carboxymethyl cellulose, carboxymethyl cellulose sodium, microcrystalline cellulose, sodium alginate, hydroxypropylmethyl cellulose (HPMC), HPMC 1828, HPMC 2906, HPMC 2910, etc. suspending agents may be used in the suspension agent according to the present invention, and surfactants, preservatives, stabilizers, colorants, and fragrances may be used if necessary.

Injections according to the present invention include distilled water for injection, 0.9% sodium chloride injection, IV injection, dextrose injection, dextrose + sodium chloride injection, PEG (PEG), lactated IV injection, ethanol, propylene glycol, solvents such as non-volatile oil-sesame oil, cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, benzene benzoate; solubilizing agents such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidine, propylene glycol, twins, nijuntinamide, hexamine, and dimethylacetamide; buffers such as weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, albumin, peptone, and gums; tonicity agents such as sodium chloride; Stabilizers such as sodium bisulfite (NaHSO ₃ ) carbon dioxide gas, sodium metabisulfite (Na ₂ S ₂ O ₅ ), sodium sulfite (Na ₂ SO ₃ ), nitrogen gas (N ₂ ), ethylenediaminetetraacetic acid; Sulfating agents such as sodium bisulfide 0.1%, sodium formaldehyde sulfoxylate, thiourea, ethylenediamine disodium tetraacetate, acetone sodium bisulfite; analgesics such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; Suspending agents such as Siemesis sodium, sodium alginate, Tween 80, aluminum monostearate may be included.

The suppository according to the present invention includes cacao butter, lanolin, witapsol, polyethylene glycol, glycerogelatin, methylcellulose, carboxymethylcellulose, a mixture of stearic acid and oleic acid, Subanal, cottonseed oil, peanut oil, palm oil, cacao butter + cholesterol, lecithin, lanet wax, glycerol monostearate, twin or span, Imhausen, monolen (monosteol) Propylene Glycol Phosphate), Glycerin, Adeps Solidus, Buytyrum Tego-G, Cebes Pharma 16, Hexalide Base 95, Cotomar, Hydrocote SP, S-70-XXA, S-70-XX75 (S-70-XX95), Hydrocote (Hydr okote) 25, Hydrocote 711, Idropostal, Massa estrarium (A, AS, B, C, D, E, I, T), Massa-MF, Masupol, Masupol-15, Neosupostal-N, Paramound-B, Suposiro (OSI, OSIX, A, B, C, D, H, L), suppository type IV (AB, B , A, BC, BBG, E, BGF, C, D, 299), Supostal (N, Es), Wecobi (W, R, S, M, Fs), testosterone triglyceride bases (TG-95, MA, 57).

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations are prepared by mixing the extract with at least one or more excipients, for example, starch, calcium carbonate, sucrose or lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

Liquid preparations for oral administration include suspensions, solutions for oral administration, emulsions, syrups, etc. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents.

The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level is the type of patient's disease, severity, drug activity, sensitivity to the drug, administration time, administration route and discharge rate, treatment period, factors including concurrently used drugs, and other factors well known in the medical field.

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 single or multiple times. Considering all of the above factors, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by a person skilled in the art to which the present invention belongs.

The pharmaceutical composition of the present invention can be administered to a subject by various routes. All modes of administration can be envisaged, for example, oral administration, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, paraspinal space (intrathecal) injection, sublingual administration, buccal mucosal administration, rectal insertion, vaginal insertion, ocular administration, ear administration, nasal administration, inhalation, spraying through the mouth or nose, dermal administration, transdermal administration, and the like.

The pharmaceutical composition of the present invention is determined according to the type of drug as an active ingredient together with various related factors such as the disease to be treated, the route of administration, 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 patient's age, sex, and weight, and is generally 0.001 to 150 mg, preferably 0.01 to 100 mg per 1 kg of body weight, administered daily or every other day, or 1 to 3 times a day. However, since it may increase or decrease depending on the route of administration, severity of disease, sex, weight, age, etc., the dosage is not limited to the scope of the present invention in any way.

In the present invention, "individual" means a subject in need of treatment of a disease, and more specifically, a mammal such as a human or non-human primate, mouse, rat, dog, cat, horse, and cow. It means.

In the present invention, "administration" means providing a given composition of the present invention to a subject by any suitable method.

In the present invention, "prevention" means any action that inhibits or delays the onset of a desired disease, and "treatment" means any action that improves or beneficially changes a desired disease and its associated metabolic abnormalities by administration of the pharmaceutical composition according to the present invention, and "improvement" means any action that reduces a parameter related to a desired disease, for example, the severity of symptoms, by administration of the composition according to the present invention.

In addition, the present invention provides a kit for preventing, improving, or treating inflammatory diseases, including the composition according to the present invention.

The kit according to the present invention is not limited in specific form as long as it can perform the purpose of prevention, improvement, or treatment of inflammatory diseases, and preparation of the composition according to the present invention (eg, culturing of strains, isolation of extracellular vesicles, etc.), It may include arbitrary components and devices for storage, administration, etc. without limitation. In addition, the kit may include a description describing the general description of the composition of the present invention, such as how to use it, how to store it, and how to administer it.

In addition, the present invention provides a food composition for preventing or improving inflammatory diseases, comprising at least one selected from the group consisting of extracellular vesicles derived from strains of the genus Roseburia and extracellular vesicles derived from strains of the genus Bifidobacterium as an active ingredient. The food composition includes a health functional food composition.

When using the strain, lysate, culture medium, and / or extracellular vesicles of the present invention as a food additive, the strain, extracellular vesicles, etc. may be added as is or used together with other foods or food ingredients, and may be appropriately used according to conventional methods. 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 mixing amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health or therapeutic treatment). In general, when preparing food or beverage, 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 control, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount greater than the above range.

There is no particular limitation on the type of food. Examples of foods to which the substance can be added include meat, sausages, bread, chocolates, candies, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages and vitamin complexes, and includes all health functional foods in the usual sense.

The health beverage composition according to the present invention may contain various flavoring agents or natural carbohydrates as additional components, like conventional beverages. The aforementioned natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrins and cyclodextrins, and sugar alcohols such as xylitol, sorbitol and erythritol. As the sweetener, natural sweeteners such as thaumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame may be used. The proportion of the natural carbohydrate is generally about 0.01-0.20 g, or about 0.04-0.10 g per 100 mL of the composition of the present invention.

In addition to the above, the composition of the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, and the like. In addition, the composition of the present invention may contain fruit flesh for preparing natural fruit juice, fruit juice beverages and vegetable beverages. These components may be used independently or in combination. The ratio of these additives is not critical, but is generally selected in the range of 0.01-0.20 parts by weight per 100 parts by weight of the composition of the present invention.

In the present specification, "health functional food" is the same term as food for special health use (FoSHU), and refers to medical and medically effective foods that are processed to efficiently display bioregulatory functions in addition to nutritional supply. In order to obtain useful effects for preventing or improving inflammatory diseases, the food can be prepared in various forms such as tablets, capsules, powders, granules, liquids, and pills.

The health functional food of the present invention can be prepared by a method commonly used in the art, and can be prepared by adding raw materials and components commonly added in the art during the preparation. In addition, unlike general drugs, there is an advantage in that there is no side effect that may occur when taking a drug for a long time by using food as a raw material, and it can be excellent in portability.

In addition, the present invention provides a feed composition for preventing or improving inflammatory diseases, comprising at least one selected from the group consisting of extracellular vesicles derived from a strain of the genus Roseburia and extracellular vesicles derived from a strain of the genus Bifidobacterium as an active ingredient.

The term "feed" of the present invention refers to any natural or artificial diet, one-meal meal, etc., or a component of the one-meal meal for animals to eat, ingest, and digest.

The strain, lysate, culture medium, and/or extracellular vesicles of the present invention included in the feed composition of the present invention may vary depending on the purpose and conditions of use of the feed, for example, 0.01 to 100% by weight, more specifically, 1 to 80% by weight relative to the total weight of the livestock feed composition, but is not limited thereto.

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

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

The feed additive may be administered to animals alone or in combination with other feed additives in an edible carrier. In addition, the feed additives can be easily administered to animals as a top dressing, directly mixed with animal feed, or in an oral formulation separate from feed. When the feed additive is administered separately from animal feed, it can be prepared as an immediate release or sustained release formulation by combining it with a pharmaceutically acceptable edible carrier, as is well known in the art. Such edible carriers can be solid or liquid, for example corn starch, 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, capsule, powder, troche or sugar-containing tablet or top dressing in a microdispersible 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 formulation.

In addition, the feed and feed additives may contain auxiliary agents, such as preservatives, stabilizers, wetting agents or emulsifying agents, solution accelerators, and the like. The feed additive may be used by adding it to an animal's feed by steeping, 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 not only for pigs, cows, horses, sheep, rabbits, goats, rodents and laboratory rodents such as rats, hamsters, and guinea pigs, but also for companion animals (eg, dogs and cats). It can also be used for chickens, turkeys, ducks, geese, pheasants, and quail as the poultry, and it can be used for carp, crucian carp, and trout as the fish, but is not limited thereto.

Hereinafter, examples will be described in detail to aid understanding of the present invention. However, the following examples are merely illustrative of the contents of the present invention, but the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.

[Example]

Example 1. Roseburia internalis ( Roseburia intestinalis ) or Bifidobacterium longum ( Bifidobacterium longum ) extraction and size analysis of extracellular vesicles (EV) (Ri-EV, Bl-EV)

To extract EVs from R. intestinalis and B. longum , respectively, R. intestinalis KCTC 15746 and B. longum KCTC 3249 were cultured for 18 hours in an anaerobic chamber at 37°C using reinforced clostridial medium (RCM) medium (MB cell, MB-R1602). After centrifuging the culture medium at 2000 × g for 20 minutes, the supernatant was collected, and cell debris and waste products in the culture medium were removed using a 0.22 μm filter. EVs were extracted from the culture medium through a 300 kDa molecular weight Tangential Flow Filtration (TFF) membrane filter-based TFF system, diluted in phosphate-buffered saline (PBS) and purified, and finally R. intestinalis- derived EV (Ri-EV) and B. longum- derived EV (Bl-EV) dispersed in PBS were extracted.

Next, the physicochemical properties of Ri-EV were confirmed through electron microscopy (ultrathin-section TEM, cryo-TEM) and nanoparticle tracking analyzer. As a result, it was confirmed through ultrathin-section TEM that R. intestinalis produced EVs during in vitro culture, and through cryo-TEM it was confirmed that the EVs had a single lipid membrane structure (FIG. 1). The extracted Ri-EV exhibited a size of 76±10 nm and a surface charge of -4±1 mV (Fig. 2), and was extracted at a concentration of 4.8 × 10 ¹⁰ ± 5.9 × 10 ⁷ particles/ml (Fig. 3a).

In addition, as a result of measuring the sizes of Ri-EV and Bl-EV using a zeta-sizer, it was confirmed that they had average particle sizes of 75 nm and 58 nm, respectively (FIG. 3b).

Example 2. Anti-inflammatory effect evaluation of Ri-EV administration in DSS (dextran sulfate sodium salt colitis grade) induced inflammatory bowel disease (IBD) animal model

An IBD animal model was constructed by providing normal C57BL/6 mice with drinking water containing DSS, which is known to cause IBD, at a concentration of 2.5% [v/v] for one week (FIG. 4). To the experimental group, R. intestinalis strain or Ri-EV extracted in Example 1 was orally administered on the basis of total protein of 200 μg/mouse/day on days 1, 3, 5, and 7 during a week of DSS injection. On the 10th day of the experiment, the mice were sacrificed to confirm the anti-inflammatory effect of the EV administration. Normal mice not administered with DSS were used as a control group.

First, for pathological analysis of intestinal tissue, the intestines of mice were excised, fixed in 4% neutral buffered formalin, embedded in paraffin, and tissue sections having a thickness of 4 μm were prepared. After performing H&E (Hematoxilin-Eosin) staining and AB (Alcian blue) staining on the tissue sections, they were observed under an optical microscope.

Next, cecum weight and intestinal length related to intestinal inflammation were measured.

Next, in order to confirm the ability of Ri-EV to regulate inflammation, mucous proteins, and tight junctions in the intestinal environment, the expression levels of IL (Interleukin) -1β, Muc-2 (Mucin 2), and ZO-1 (Zonula occludens-1) in the previously obtained intestinal tissue were confirmed by qRT-PCR. After dissolving the intestinal tissue in Trizol solution (Life Technologies, CA, USA), total RNA was isolated from the tissue using the chloroform-isopropanol method according to the manufacturer's analysis method. The cDNA of the isolated sample was synthesized using a high-capacity cDNA synthesis kit (Invitrogen, CA, USA) and amplified using a TaqMan gene-specific probe (Thermo Fisher Scientific) with a 7500 Real-Time PCR System (Applied Biosystems, CA, USA).

As a result, as shown in FIG. 5, the intestinal epithelial cell barrier was damaged, immune cells were infiltrated, and the number of goblet cells responsible for mucus production was reduced in the DSS-induced IBD animal model compared to the control group not administered with DSS. On the other hand, in the Ri-EV-administered group, intestinal epithelial cell barrier damage and immune cell infiltration were reduced, and the number of goblet cells was maintained similar to that of the normal group. In particular, in the Ri-EV-administered group, it was confirmed that the histological score due to the onset of IBD was significantly reduced, and IBD was improved (FIG. 6a).

In addition, in the DSS-induced IBD animal model compared to the control group, the caecal weight and intestinal length related to intestinal inflammation were significantly reduced, whereas the R. intestinalis or Ri-EV-administered group suppressed the caecal weight and intestinal length reduction. In particular, the Ri-EV-administered group significantly suppressed the caecal weight and intestinal length reduction compared to the R. intestinalis- administered group (Figs. 6b and 6c). The disease activity index was also confirmed to be the lowest in the Ri-EV administration group (FIG. 6d).

In addition, in the DSS-induced IBD animal model compared to the control group, the expression of IL-1β, an inflammatory cytokine that induces inflammation, increased, and the expression of Muc-2 and ZO-1 related to mucus protein and tight junction decreased. It was confirmed that inflammation increased and mucus secretion and tight junction were weakened. On the other hand, the R. intestinalis or Ri-EV administration group showed a decrease in the expression of IL-1β compared to the IBD animal model (FIG. 7a) and an increase in the expression of Muc-2 and ZO-1 (FIG. 7b). In particular, the Ri-EV-administered group showed a significant decrease in the expression of IL-1β and an increase in the expression of Muc-2 and ZO-1, compared to the R. intestinalis- administered group. The above results show that administration of Ri-EV can effectively reduce intestinal inflammation as well as restore mucus secretion ability and tight coupling, and that this effect is superior to administration of the strain itself.

Example 3. Evaluation of intestinal microbiota change according to Ri-EV administration in DSS-induced IBD animal model

In order to confirm the changes in the intestinal flora and the anti-inflammatory effect according to Ri-EV administration in the IBD animal model, the DSS induction of Example 2 16S ribosomal RNA gene sequencing analysis was performed using the QIAamp Power Fecal Pro DNA Kit (QIAGEN, Germany) for fecal samples obtained from IBD animal models.

As a result, as shown in FIG. 8a, in the DSS-induced IBD animal model compared to the control group not administered with DSS, changes in the intestinal microbial community, such as an increase in the phylum of Proteobacteria and Deferribacteres , were conspicuously observed. In addition, it was confirmed that the Shannon diversity value in the Ri-EV-administered group increased significantly compared to the other groups, indicating that various changes in the flora occurred in the sample (FIG. 8b). In particular, as shown in FIG. 9, the number of Bifidobacterium known as bacteria contributing to the alleviation of IBD in the change of intestinal bacteria increased in the R. intestinalis or Ri-EV-administered group, and the Ri-EV-administered group significantly increased than the R. intestinalis -administered group. From this, it can be seen that Ri-EV administration improves IBD by suppressing the secretion of inflammatory cytokines through changes in the intestinal microflora caused by the induction of growth of Bifidobacterium in the intestinal environment of IBD, and restoring mucus secretion of goblet cells and the ability of intestinal epithelial cells to bind tightly.

Example 4. Roseburia spp. Anti-inflammatory activity assay for strain-derived extracellular vesicles

In this Example, it was confirmed whether EVs of other strains of the genus Roseburia, in addition to EVs of R. intestinalis extracted through the process of Example 1, could exert a therapeutic effect on IBD.

Specifically, in addition to Roseburia interioris (Ri), three strains of the genus Roseburia (Roseburia faecies (Rfa, KCTC 15739), Roseburia hominis (Rho, KCTC 5845), Roseburia inulinivorans (Rin, DSM 1) 6841)), respectively, after extracting extracellular vesicles, their anti-inflammatory activity was analyzed. EVs were extracted in the same manner as in Example 1.

In order to evaluate the anti-inflammatory activity of each EV, the amount of nitric oxide (NO) production, which is an inflammatory indicator, was measured, and the expression levels of inflammatory cytokines and anti-inflammatory cytokines were also measured, and the results are shown in FIG. 10 .

Specifically, the amount of nitric oxide production was measured using mouse macrophages. Mouse macrophage Raw 264.7 cells were cultured at 37°C in the presence of 5% CO ₂ in RPMI1640 culture medium containing 10% fetal bovine serum (FBS) and 1% antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin). Thereafter, 1ml was dispensed into a 12-well plate at a concentration of 5×10 ⁵ cells/well, and incubated at 37° C. for 24 hours in a CO2 incubator. To induce inflammation, the wells were treated with a medium supplemented with 10 μg/ml of lipopolysaccharide (LPS), followed by further incubation for 4 hours, and the vesicles were treated by adding 2 mg based on the total protein amount to the medium, and then cultured at 37° C. for 16 hours. Thereafter, 50 μl of the well supernatant and 50 μl of Griess reagent were mixed and reacted at room temperature for 10 minutes, and then the absorbance was measured at 540 nm using a plaid reader to confirm the production of nitric oxide.

Expression levels of inflammatory cytokines and anti-inflammatory cytokines were also measured using LPS-treated Raw264.7 cells. Raw264.7 cells treated with LPS in the same manner as above were treated with 2 mg of the endoplasmic reticulum based on the total protein amount, and then cultured at 37° C. for 16 hours. Thereafter, the protein expression levels of TNF-α, IL-6, and IL-10 in the cells were measured for absorbance at 450 nm using an ELISA kit (R&D Systems, US) according to the manufacturer's protocol.

As a result, as shown in FIG. 10, when the inflammation-induced mouse macrophages were treated with the homologous and heterogeneous Roseburia genus strain-derived extracellular vesicles, the rest of the Roseburia genus strain-derived extracellular vesicles except for Rin-derived EV (Rin-EV) It was confirmed that the production of nitric oxide was significantly reduced. In addition, in the case of the expression level of inflammatory cytokines and anti-inflammatory cytokines, all of the extracellular vesicles derived from Roseburia genus strains used as experimental groups (hereinafter, Ri-EV, Rho-EV, Rfa-EV, and Rin-EV) were significantly compared to the control group. It was confirmed that TNF-α and IL-6 were effectively reduced as well as IL-10 was increased.

Through analysis of the anti-inflammatory activity of the Roseburia strains among all species, it was confirmed that all the extracellular vesicles derived from all Roseburia genus strains used in the experiment exhibited excellent anti-inflammatory effects, and therefore could be used as a composition for preventing, improving or treating inflammatory bowel disease.

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

Following Example 4, it was confirmed whether EVs derived from strains corresponding to the genus R. intestinalis or B. longum could exert an anti-inflammatory effect.

Specifically, four strains of the genus Roseburia (Roseburia faecies, KCTC 15739), Roseburia hominis (KCTC 5845), Roseburia inulinivorans (DSM 16841)), and five strains of the genus Bifidobacterium (Bifidobacterium Extracellular vesicles were extracted from Bifidobacterium bifidum (KCTC 3281), Bifidobacterium breve (KCTC 3220), Bifidobacterium lactis (KCTC 5854), and Bifidobacterium adolescentis (KCTC 3216), respectively. Anti-inflammatory activity was assayed. EVs were extracted in the same manner as in Example 1.

In order to evaluate the anti-inflammatory activity of each EV, an in vitro environment mimicking Kupffer cells, one of the cells constituting liver tissue, was prepared using Raw264.7 cells, which are mouse macrophages, and the results of measuring the amount of nitric oxide (NO) produced by them are shown in FIG. 11. Specifically, mouse macrophage Raw 264.7 cells were cultured at 37° C. in the presence of 5% CO ₂ in RPMI1640 culture medium containing 10% fetal bovine serum (FBS) and 1% antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin). Thereafter, 1ml was dispensed into a 12-well plate at a concentration of 5×10 ⁵ cells/well, and incubated at 37° C. for 24 hours in a CO ₂ incubator. To induce inflammation, the wells were treated with a medium supplemented with 10 μg/ml of lipopolysaccharide (LPS), followed by further incubation for 4 hours, and the endoplasmic reticulum was added to the medium at a concentration of 200 μg based on the total protein amount, followed by incubation at 37° C. for 16 hours. Thereafter, 50 μl of the well supernatant and 50 μl of Griess reagent were mixed and reacted at room temperature for 10 minutes, and then the absorbance was measured at 540 nm using a plaid reader to confirm the production of nitric oxide.

As a result, it was confirmed that production of nitric oxide by LPS was effectively reduced significantly in all groups administered with the extracellular vesicles derived from the strain of Roseburia used as the experimental group, as shown in FIG. 11 . It was confirmed that the production of nitric oxide was effectively reduced even in the group administered with extracellular vesicles derived from a strain of the genus Bifidobacterium, and among them, the ability to inhibit the production of nitric oxide of Bbr-EV and Bla-EV was controlled at 200 μg or 50 μg of total protein.

The above results show that EVs of homogeneous strains corresponding to the genus Roseburia or the genus Bifidobacterium exert excellent anti-inflammatory effects. That is, from the above results, it was confirmed that extracellular vesicles derived from strains of the genus Roseburia and strains of the genus Bifidobacterium can be used for prevention, improvement, or treatment of various inflammatory diseases through strong anti-inflammatory effects.

As mentioned in the above embodiments, the derived from the roseburia strain in the present invention and the bifidobacterium -derived cell -derived cell -derived cell -derived cells, reducing the intestinal epithelial barrier damage due to inflammation, reducing the infiltration of immune cells, maintaining the number of compensation cells, suppressing the reduction of the cecum and intestinal length due to enteritis, and changing the intestinal bacteria through the change in the intestinal bacteria. It was found to inhibit the secretion of Cain and to restore the mucus secretion of compensation cells and the dense binding ability of the intestinal epithelial cells. In particular, it was confirmed that all extracellular vesicles derived from various homologous and heterogeneous strains of the genus Roseburia or the genus Bifidobacterium exhibit excellent anti-inflammatory effects. Therefore, the extracellular vesicles derived from strains of the genus Roseburia and the genus Bifidobacterium of the present invention not only exert a strong anticancer effect, but also improve histological damage or loss of function due to inflammation, and thus can be provided as a composition for the prevention, improvement, and/or treatment of various inflammatory diseases.

From the above description, those skilled in the art to which the present invention pertains will be able to understand that the present invention may be embodied in other specific forms without changing its technical spirit or essential features. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not limiting. The scope of the present invention should be construed as including all changes or modifications derived from the meaning and scope of the claims to be described later and equivalent concepts rather than the detailed description above are included in the scope of the present invention.

Claims (13)

A pharmaceutical composition for preventing or treating inflammatory diseases, comprising at least one selected from the group consisting of strain-derived extracellular vesicles derived from Roseburia spp. and extracellular vesicles derived from Bifidobacterium spp.
According to claim 1,
The extracellular vesicles are characterized in that the average diameter of 30 nm to 200 nm, the pharmaceutical composition.
According to claim 1,
The extracellular vesicles derived from the strain of the genus Roseburia are naturally or artificially secreted from the strain of the genus Roseburia;
The extracellular vesicles derived from the strain of the genus Bifidobacterium are naturally or artificially secreted from the strain of the genus Bifidobacterium, a pharmaceutical composition.
According to claim 1,
The Roseburia genus strain is at least one selected from the group consisting of Roseburia intestinalis , Roseburia faecies , Roseburia hominis , and Roseburia inulinivorans . Pharmaceutical composition.
According to claim 1,
The strain of the Bifidobacterium genus is at least one selected from the group consisting of Bifidobacterium longum , Bifidobacterium bifidum, Bifidobacterium breve , Bifidobacterium lactis , and Bifidobacterium adolescentis , about academic composition.
According to claim 1,
The inflammatory diseases include gastritis, gastric ulcer, duodenal ulcer, inflammatory skin disease, allergic 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 spine Inflammation, Reiter's syndrome, psoriatic arthropathy, enteropathic spondylitis, juvenile arthropathy, juvenile ankylosing spondylitis, reactive arthropathy, infectious arthritis, post-infectious arthritis, gonococcal arthritis, tuberculous arthritis, viral arthritis, fungal arthritis, syphilitic arthritis, Lyme disease, arthritis associated with vasculitis syndrome, polyarteritis nodosa, hypersensitivity vasculitis, Lou Gehrig's granulomatosis, polymyalgia rheumatica, articular cell arteritis, calcium Crystal deposition arthropathy, pseudogout, non-articular rheumatism, bursitis, tenosynovitis, epicondylitis, neuropathic joint disease (charco and joint), hemorrhagic joint disease (hemarthrosic), Henoch-Schenlein purpura, hypertrophic osteoarthropathy, multicentric reticulocytoma, surcoilosis, hemochromatosis, sickle cell disease, hemochromatosis, hyperlipoproteinemia, reduction Maglobulinemia, 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 (chronic obstructive pulmonary disease), acute lung injury, and at least one selected from the group consisting of broncho-pulmonary dysplasia, a pharmaceutical composition.
According to claim 6,
The inflammatory bowel disease is at least one selected from the group consisting of ulcerative colitis, Crohn's disease, Behçet's disease, collagenous colitis, lymphocytic colitis, ischemic colitis, hemorrhagic rectal ulcer, ileal pouchitis, and convertible colitis, a pharmaceutical composition.
According to claim 1,
Characterized in that the pharmaceutical composition has one or more effects selected from the group consisting of:
i) reducing intestinal epithelial barrier damage;
ii) reduction of intestinal tissue infiltration of immune cells;
iii) inhibition of reduction in the number of goblet cells;
iv) inhibition of cecal weight loss;
v) inhibition of intestinal length reduction;
vi) inhibition of inflammatory cytokine production;
vii) promoting anti-inflammatory cytokine production;
viii) enhancement of mucus secretion by goblet cells;
ix) promoting the compaction of intestinal epithelial cells; and
x) Increased number of Bifidobacterium in the intestine.
A kit for preventing or treating inflammatory diseases, comprising the pharmaceutical composition of any one of claims 1 to 8.
A method for preventing or treating inflammatory diseases, comprising administering at least one selected from the group consisting of extracellular vesicles derived from strains of the genus Roseburia and extracellular vesicles derived from strains of the genus Bifidobacterium to a subject in need thereof, wherein the subject is a non-human subject.
A food composition for preventing or improving inflammatory diseases, comprising at least one selected from the group consisting of extracellular vesicles derived from a strain of the genus Roseburia and extracellular vesicles derived from a strain of the genus Bifidobacterium as an active ingredient.
According to claim 11,
The food composition is a health functional food composition, food composition.
A feed composition for preventing or improving inflammatory diseases, comprising at least one selected from the group consisting of extracellular vesicles derived from strains of the genus Roseburia and extracellular vesicles derived from strains of the genus Bifidobacterium as an active ingredient.