WO2018004145A1 - Anti-inflammatory composition containing yeast-derived extracellular vesicle - Google Patents

Abstract

Disclosed in the present specification are an anti-inflammatory composition containing a yeast-derived extracellular vesicle as an active ingredient, and an anti-inflammatory composition containing, as an active ingredient, a food-derived extracellular vesicle containing yeast. According to one aspect, the yeast may be Saccharomyces cerevisiae, and the anti-inflammatory composition has the effect of suppressing or inhibiting the secretion of Tumor necrosis factor-α (TNF-α) or Interleukin-6 (IL-6) which are inflammation-related media.

Description

Anti-inflammatory composition comprising yeast-derived extracellular vesicles

Disclosed herein is an anti-inflammatory composition comprising an extracellular vesicle derived from yeast as an active ingredient.

Most animal cells have the ability to secrete extracellular vesicles of intracellular origin of varying size and composition, and these extracellular vesicles can contain any biological, including blood, urine, saliva, and cell cultures. Found in biological fluids (Loyer X, Vion AC, Tedgui A, Boulanger CM.Microvesicles as cell-cell messengers in cardiovascular diseases.Circ Res 2014; 114: 345-53, Ohno S, Ishikawa A, Kuroda M. Roles of exosomes and microvesicles in disease pathogenesis.Adv Drug Deliv Rev 2013; 65: 398-401).

Extracellular vesicles are membrane structure vesicles ranging in size from about 20 nm in diameter to about 2 μm in diameter, and are heterogeneous in size and composition, and include exosomes, ectosomes, and microvesicles. and many different species such as microvesicles, microparticles, and the like.

The different types of extracellular vesicles are of their origin, diameter, density at sucrose, shape, sedimentation rate, lipid composition, protein markers or secretion mode (i.e. signal (inductive) or spontaneous (constitutive)). ) And the like. Microvesicles, for example, are membrane vesicles with irregular shapes of about 100 to 1,000 nm, budding outward from the plasma membrane (derived from the plasma membrane) and having lipids including phosphatidylserine, markers containing integrin, selectin, CD40 ligand It is known. On the other hand, the exosomes are the smallest membrane vesicles having a cup shape of about 30 to 100 nm (<200 nm), which are germinated (originated from the endosomes) inside the late endosome, and are composed of CD63, CD9, tetraspanine, TSG101, It is known to have a marker comprising ESCRT, a lipid comprising cholesterol, sphingomyelin, ceramide, phosphatidylserine.

Extracellular vesicles reflect the state of the secreting cells of origin (donor cells), and exhibit a variety of biological activities, depending on which cells are secreted, and play an important role in cell-to-cell interactions by transferring genetic material and proteins between cells. .

Prokaryotic or eukaryotic cells are also known to secrete extracellular vesicles (Camussi, G., Deregibus, MC, Bruno, S., Cantaluppi, V., & Biancone, L. (2010). Exosomes / microvesicles as a mechanism of cell-to-cell communication.Kidney international, 78 (9), 838-848, Bang, Claudia, and Thomas Thum. "Exosomes: New players in cell-cell communication." The international journal of biochemistry & cell biology 44.11 ( 2012): 2060-2064. Kim, DK, Lee, J., Simpson, RJ, Lotvall, J., & Gho, YS (2015, April), EVpedia: A community web resource for prokaryotic and eukaryotic extracellular vesicles research. Seminars in cell & developmental biology (Vol. 40, pp. 4-7) .Academic Press, Kim, JH, Lee, J., Park, J., & Gho, YS (2015, April) .Gram-negative and Gram -positive bacterial extracellular vesicles.In Seminars in cell & developmental biology (Vol. 40, pp. 97-104) .Academic Press).

Conventionally, extracellular vesicles have been mainly used as biomarkers, and techniques for using extracellular vesicles for specific purposes using the efficacy of the extracellular vesicles themselves have not been developed.

Inflammation, on the other hand, is a local or systemic defense mechanism against damage or infection of cells and tissues. Inflammation is primarily caused by a chain of biological reactions that occur by the direct response of numerous humoral mediators that make up the immune system or by stimulating local or systemic effector systems. Mediators involved in the inflammatory response include inflammatory cells such as immune cells, macrophage, neutrophil, eosinophil, mast cell, and cytokines secreted from these cells.

Inflammatory diseases are particularly characterized by the secretion of inflammatory cytokines, resulting in imbalances in tissue damage and healing. The major inflammatory cytokines known to date are the tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), IL-6, and IL-8 produced by macrophages and monocyte cells. Etc. At present, various studies have been made to suppress the inflammatory cytokines in order to treat inflammatory diseases caused by the imbalance of such inflammatory cytokines. Korean Patent Publication No. 10-2015-0053034 is a prior art document regarding anti-inflammatory compositions.

In one aspect, the present disclosure aims to provide an anti-inflammatory composition comprising an extracellular vesicle derived from yeast as an active ingredient.

In another aspect, the present disclosure aims to provide an anti-inflammatory composition comprising an extracellular vesicle derived from food, including yeast as an active ingredient.

In another aspect, the present disclosure aims to provide a method for separating extracellular vesicles to separate the extracellular vesicles in high yield.

In one aspect, the technology disclosed herein provides an anti-inflammatory composition comprising an extracellular vesicle derived from yeast as an active ingredient.

In another aspect, the technology disclosed herein provides an anti-inflammatory composition comprising as an active ingredient extracellular vesicles derived from food, including yeast.

In an exemplary embodiment, the yeast may be Saccharomyces cerevisiae .

In an exemplary embodiment, the extracellular vesicles may have a diameter of 20 to 200 nm.

In an exemplary embodiment, the extracellular vesicles may be separated by density gradient ultracentrifugation.

In an exemplary embodiment, the extracellular vesicles may have a floating density of 1.08 to 1.19 g / mL in iodixanol.

In an exemplary embodiment, the extracellular vesicles may be separated by size exclusion chromatography.

In an exemplary embodiment, the extracellular vesicles derived from food comprising the yeast may be extracellular vesicles derived from beer.

In one exemplary embodiment, the anti-inflammatory composition inhibits or inhibits the secretion of tumor necrosis factor-α (TNF-α) or Interleukin-6 (IL-6), an inflammation-related mediator. It may be.

In one exemplary embodiment, the anti-inflammatory composition may be an anti-inflammatory activity against inflammatory skin diseases.

In an exemplary embodiment, the inflammatory skin disease may be at least one selected from the group consisting of acne, contact dermatitis, seborrheic dermatitis, atopic dermatitis and psoriasis.

In an exemplary embodiment, the anti-inflammatory composition may be a pharmaceutical composition, cosmetic composition or food composition.

In another aspect, the techniques disclosed herein include a method of preparing the extracellular vesicles, comprising: culturing a yeast; Centrifuging the culture solution to remove residue and filtering the supernatant; It provides a method for producing extracellular vesicles comprising the step of ultracentrifuging the filtrate and then collecting the pellets to separate the iodixanol density gradient ultracentrifugation.

In one exemplary embodiment, the filtration may be to filter with a 0.2 to 0.5 ㎛ filter.

In an exemplary embodiment, the ultracentrifugation may be performed at 100,000 × g or more.

In an exemplary embodiment, after the iodixanol density gradient ultracentrifugation, extracellular vesicles can be obtained from fractions having a floating density of 1.08 to 1.19 g / mL in iodixanol.

In one aspect, the technology disclosed herein has the effect of providing an anti-inflammatory composition comprising an extracellular vesicle derived from yeast as an active ingredient.

In another aspect, the technique disclosed herein has the effect of providing an anti-inflammatory composition comprising as an active ingredient extracellular vesicles derived from foods including yeast.

In another aspect, the technology disclosed herein has the effect of providing a method for separating extracellular vesicles to separate the extracellular vesicles in high amounts.

1 shows a separation process of yeast-derived exosomes according to one test example of the present specification.

Figure 2 shows the protein content and nanoparticle number of the fraction and fractions fractionation method of the yeast-derived exosomes according to one test example of the present specification.

Figure 3 shows the analysis results of yeast derived exosomes isolated according to one test example of the present specification. Figure 3a is a form of yeast-derived exosomes, Figure 3b shows the size of the yeast-derived exosomes.

Figure 4 shows the separation process of beer-derived exosomes according to one test example of the present specification.

Figure 5 shows the analysis results of beer-derived exosomes isolated in accordance with one test example of the present specification. Figure 5a shows the distribution (boxed area) of the chromatogram of the Heineken-beer-derived exosomes, Figure 5b is the form of Heineken-beer-derived exosomes, Figure 5c shows the size of the Heineken-beer-derived exosomes.

Figure 6 shows the results of in vitro analysis of yeast-derived exosome efficacy isolated according to one test example of the present specification. Figure 6a shows the effect of yeast-derived exosomes on the secretion of TNF-α and IL-6, Figure 6b is a result of co-treatment of yeast-derived exosomes and LPS, Figure 6c shows the yeast-derived exosomes after induction of inflammatory response by LPS Post-treatment results are shown.

Figure 7 shows the results of in vitro analysis of beer-derived exosomes efficacy isolated in accordance with one test example of the present specification. Figure 7a shows the effect of beer-derived exosomes on IL-6 secretion, Figure 7b shows the results of inducing an inflammatory response with LPS after pre-treatment of beer-derived exosomes.

8a and 8b show the results of in vitro analysis of yeast-derived exosomes efficacy in human keratinocytes isolated according to one test example of the present specification.

Hereinafter, the present invention will be described in detail.

In one aspect, the technology disclosed herein provides an anti-inflammatory composition comprising an extracellular vesicle derived from yeast as an active ingredient.

In another aspect, the technology disclosed herein provides an anti-inflammatory composition comprising as an active ingredient extracellular vesicles derived from food, including yeast.

As used herein, the term "active ingredient" alone refers to a component that may exhibit the desired activity alone or together with a carrier having no activity.

As used herein, 'anti-inflammatory' means an effect of preventing, inhibiting, inhibiting, ameliorating or treating inflammation. 'Inflammation' refers to swelling caused by an increase in body fluids between tissue cells, hyperemia due to expansion of blood vessels, fever and blood vessels. Symptoms or signs such as fever due to dilatation, pain due to arachidonic acid metabolites, etc., and can be classified as acute, subacute, or chronic inflammatory diseases over time. Infectious, allergic, autoimmune, and toxic according to pathological physiology. , Metabolic and traumatic inflammatory diseases.

In an exemplary embodiment, the inflammation may include rhinitis, sinusitis, otitis media, nasopharyngitis, laryngitis, bronchitis, asthma, chronic obstructive pulmonary disease, bronchiectasis, bronchiolitis, pneumonia, pulmonary fibrosis and the like; Digestive system inflammatory diseases such as oral inflammation, esophagitis, gastritis, peptic ulcer, irritable growth syndrome, inflammatory growth disease, cholecystitis, cholangitis, pancreatitis and hepatitis; Skin inflammatory diseases such as acne, contact dermatitis, seborrheic dermatitis, atopic dermatitis and psoriasis; Cardiovascular inflammatory diseases such as endocarditis, myocarditis, pericarditis, vasculitis, arteriosclerosis, and sepsis; Endocrine inflammatory diseases such as thyroiditis, parathyroid acid, and diabetes; Urogenital inflammatory diseases such as glomerulonephritis, nephropathy, interstitial nephropathy, testicles, ovaries, endometritis and vaginitis; Musculoskeletal inflammatory diseases such as rheumatoid arthritis, spondyloarthropathy, osteoarthritis, gout, systemic lupus erythematosus, systemic sclerosis, myopathy, Sjogren's syndrome, Behcet's disease, and antiphospholipid syndrome; Vascular dementia, Alzheimer's disease, degenerative brain disease, depression, psychiatric inflammatory diseases such as schizophrenia may be selected from the group consisting of, but is not limited thereto.

In one exemplary embodiment, the anti-inflammatory composition inhibits or inhibits the secretion of tumor necrosis factor-α (TNF-α) or Interleukin-6 (IL-6), an inflammation-related mediator. can do.

In another aspect, the techniques disclosed herein include administering to a subject in need thereof an extracellular vesicle derived from said yeast in an amount effective to prevent, ameliorate and / or treat inflammation. And / or provide a method of treatment.

In another aspect, the techniques disclosed herein comprise administering to a subject in need thereof an extracellular vesicle derived from a food comprising said yeast in an amount effective for preventing, ameliorating and / or treating inflammation. Prophylaxis, improvement and / or treatment methods are provided.

In another aspect, the techniques disclosed herein provide extracellular vesicles derived from said yeast for preventing, ameliorating and / or treating inflammation in a subject.

In another aspect, the techniques disclosed herein provide extracellular vesicles derived from food comprising said yeast for preventing, ameliorating and / or treating inflammation in a subject.

In another aspect, the technology disclosed herein provides a use for the preparation of an extracellular vesicle-containing composition derived from said yeast for preventing, ameliorating and / or treating inflammation in a subject.

In another aspect, the technology disclosed herein provides a use for preparing an extracellular vesicle-containing composition derived from a food comprising said yeast for preventing, ameliorating and / or treating inflammation in a subject.

In an exemplary embodiment, the extracellular vesicles may be applied or administered to the subject in the form of a pharmaceutical composition, cosmetic composition or food composition.

In an exemplary embodiment, the extracellular vesicles may be applied or administered to the skin or scalp of the subject.

In an exemplary embodiment, the yeast may be Saccharomyces cerevisiae .

As used herein, the term "extracellular vesicle" refers to a nano-sized extracellular vesicle secreted by cells and released into the extracellular space, wherein the extracellular vesicles are internal and external by a lipid bilayer consisting of cell membrane components. The cell membrane lipids and membrane proteins, genetic material and cytoplasmic components of the cell can be indirectly determined. In addition, extracellular vesicles bind to other cells and tissues to deliver membrane components, mRNAs, miRNAs, proteins (growth hormones, cytokines, etc.), and deliver these transporters to recipient cells for cell-cell communication. It acts as an intermediary extracellular carrier.

In an exemplary embodiment, the extracellular vesicles can be exosomes. Exosomes herein is the broadest concept that includes both exosomes and nano-sized endoplasmic reticulum structures and compositions whose similar vesicles.

In an exemplary embodiment, the extracellular vesicles may have a diameter of 20 to 200 nm. More specifically, the extracellular vesicles are at least 20 nm, at least 22 nm, at least 24 nm, at least 26 nm, at least 28 nm, at least 30 nm, at least 32 nm, at least 34 nm, at least 36 nm, at least 38 nm, 40 200 nm or less, 190 nm or less, 180 nm or less, 170 nm or less, 160 nm or less, 150 nm or less, 140 nm or more, 42 nm or more, 44 nm or more, 46 nm or more, 48 nm or more or 50 nm or more Or less, 130 nm or less, 120 nm or less, 110 nm or less, 100 nm or less, 95 nm or less, 90 nm or less, 85 nm or less, 80 nm or less, 75 nm or less, or 70 nm or less. For example, the extracellular vesicles may have a diameter of 20 to 150 nm, 30 to 150 nm, 30 to 100 nm, or 30 to 80 nm.

In an exemplary embodiment, the extracellular vesicles may be chemically or physically modified with, for example, membrane components to efficiently perform the desired function in the target cell. For example, the membrane component of the extracellular vesicles may be modified by a chemical method using a thiol group (-SH) or an amine group (-NH ₂ ), or the target-inducing substance, cell membrane fusion material, or polyethylene glycol may be added to the extracellular vesicles. By chemically bonding may further comprise a component other than the membrane.

In an exemplary embodiment, the extracellular vesicles are ultracentrifugation, differential centrifugation, equilibrium density centrifugation, density gradient, filtration, dialysis (dialysis) and free-flow electrophoresis can be separated using one or more methods selected from the group consisting of, but the separation method of extracellular vesicles is not limited thereto.

Density gradient is the most used method for distinguishing materials having different densities, the extracellular vesicles according to the present specification can be separated through the density gradient is divided density. Specific examples of this method may be performed using a density gradient separation material such as ficoll, glycerol, sucrose, cesium chloride, and iodixanol. It is not limited. In one aspect, density gradients may be used with ultracentrifugation and the like. In another aspect, gel filtration or ultrafiltration may be used to select extracellular vesicles. In another aspect, dialysis may be used instead of filtration to remove small molecules. In another aspect, free flow electrophoresis can be used.

In an exemplary embodiment, the yeast-derived extracellular vesicles may be separated by density gradient ultracentrifugation.

In an exemplary embodiment, the yeast-derived extracellular vesicles have a suspended density in iodixanol of 1.08 to 1.19 g / mL, more specifically 1.08 g / mL or more, 1.09 g / mL or more, 1.10 g / mL or greater, 1.11 g / mL or greater, or 1.12 g / mL or greater and 1.19 g / mL or smaller, 1.18 g / mL or smaller, 1.17 g / mL or smaller, 1.16 g / mL or smaller, or 1.15 g / mL or smaller. . At this time, iodixanol is mixed in the same amount of 5%, 10%, 30% 40%, 50% concentration of iodixanol. The suspended density refers to the density measured by the density gradient centrifugal method.

In an exemplary embodiment, the extracellular vesicles derived from foods including the yeast may be separated by size exclusion chromatography.

In an exemplary embodiment, the food comprising yeast may be one or more selected from the group comprising bread, beer, wine and rice wine.

In an exemplary embodiment, the anti-inflammatory composition may be a lyophilized formulation. The composition may be a lyophilized formulation in a sealed packaging or ready-to-use sealed package.

The present disclosure also includes a composition comprising the extracellular vesicles as an active ingredient and having a lyophilized formulation; It provides an anti-inflammatory kit comprising; and sterile water or purified water. The kit may be contained in a sealed packaging material or packaging container ready-to-use.

According to an exemplary embodiment, the composition may be a pharmaceutical composition.

The pharmaceutical composition may further contain, in addition to the extracellular vesicles, preservatives, stabilizers, hydrating or emulsifying accelerators, pharmaceutical auxiliaries such as salts and / or buffers for the control of osmotic pressure, and other therapeutically useful substances. It can be formulated into various oral or parenteral dosage forms according to the invention.

The oral dosage forms include, for example, tablets, pills, hard and soft capsules, solutions, suspensions, emulsifiers, syrups, powders, powders, fine granules, granules, pellets, and the like, and these formulations include surfactants in addition to active ingredients. , Diluents (eg lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and glycine), glidants (eg silica, talc, stearic acid and its magnesium or calcium salts and polyethylene glycols). . Tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and polyvinylpyrrolidine, optionally starch, agar, alginic acid or its sodium salt Pharmaceutical additives such as disintegrants, absorbents, colorants, flavors, and sweeteners. The tablets can be prepared by conventional mixing, granulating or coating methods.

In addition, the parenteral dosage form may be a transdermal dosage form, for example, an injection, drop, ointment, lotion, gel, cream, spray, suspension, emulsion, suppository, patch, or the like. It may be, but is not limited thereto.

Determination of the dosage of the active ingredient is within the level of ordinary skill in the art, the daily dosage of the drug depends on a variety of factors, such as the progress of the subject to be administered, the onset, age, health status, complications, etc. On the basis of the 1 ㎍ / kg to 200 mg / kg the composition in one aspect, 50 ㎍ / kg to 50 mg / kg in another aspect may be administered by dividing 1 to 3 times a day, the dosage Does not limit the scope of the invention in any way.

The pharmaceutical composition may be an external preparation for skin, and the external preparation for skin may be included herein as a generic term that may include anything applied outside the skin.

According to an exemplary embodiment, the composition may be a cosmetic composition.

The cosmetic composition may further include a functional additive and components included in a general cosmetic composition in addition to the extracellular vesicles. The functional additive may include a component selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, polymer peptides, polymer polysaccharides, sphingolipids and seaweed extract. In addition to the other components included, oils and fats, moisturizers, emollients, surfactants, organic and inorganic pigments, organic powders, ultraviolet absorbers, preservatives, fungicides, antioxidants, plant extracts, pH adjusters, alcohols, pigments, flavorings, blood circulation And accelerators, cooling agents, limiting agents, purified water, and the like.

The cosmetic composition is not particularly limited in formulation, and may be appropriately selected as desired. For example, skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisturizing lotion, nutrition lotion, massage cream, nutrition cream, moisturizing cream, hand cream, foundation, essence, nutrition essence, pack, soap, cleansing It may be prepared in any one or more formulations selected from the group consisting of foam, cleansing lotion, cleansing cream, body lotion and body cleanser, but is not limited thereto.

When the formulation of the present invention is a paste, cream or gel, animal carriers, vegetable fibers, waxes, paraffins, starches, tracantes, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicas, talc or zinc oxide, etc. may be used as carrier components. Can be.

When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used, and especially in the case of spray, additionally chlorofluorohydrocarbon, propane Propellant such as butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, solvating or emulsifying agent is used as the carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 Fatty acid esters of, 3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan.

When the dosage form of the present invention is a suspension, liquid carrier diluents such as water, ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline Cellulose, aluminum metahydroxy, bentonite, agar or tracant and the like can be used.

When the formulation of the present invention is a surfactant-containing cleansing, the carrier component is an aliphatic alcohol sulfate, an aliphatic alcohol ether sulfate, a sulfosuccinic acid monoester, an isethionate, an imidazolinium derivative, a methyltaurate, a sarcosinate, a fatty acid amide. Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, linolin derivatives or ethoxylated glycerol fatty acid esters and the like can be used.

According to an exemplary embodiment, the composition may be a food composition.

The food composition may be in a liquid or solid dosage form, for example, various foods, beverages, gums, teas, vitamin complexes, dietary supplements, and the like, and may be used in the form of powders, granules, tablets, capsules, or beverages. Can be. In addition to the active ingredient, the food composition of each formulation may be appropriately selected and blended by those skilled in the art according to the formulation or purpose of use, in addition to the active ingredient, and synergistic effects may occur when applied simultaneously with other raw materials.

There is no particular limitation on the liquid component that can be contained in addition to the active ingredient disclosed herein, and may include various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. Examples of the natural carbohydrates include conventional sugars such as disaccharides such as monosaccharides, glucose and fructose, polysaccharides such as maltose and sucrose, dextrins and cyclodextrins, and sugar alcohols such as xylitol, sorbitol and erythritol. Etc. As the flavoring agent, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (for example, saccharin, aspartame, etc.) can be advantageously used. The proportion of natural carbohydrates may generally be about 1-20 g, in one aspect about 5-12 g, per 100 ml of the compositions disclosed herein.

In one aspect, the food composition may contain various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and the like. Salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like. In another aspect it may include a pulp for the production of natural fruit juices and vegetable drinks. The components can be used independently or in combination. The ratio of the additive may vary, but is generally selected from 0.001 to about 20 parts by weight per 100 parts by weight of the composition disclosed herein.

In another aspect, the technology disclosed herein is a method for producing an extracellular vesicle derived from the yeast, comprising the steps of culturing the yeast; Centrifuging the culture solution to remove residue and filtering the supernatant; It provides a method for producing extracellular vesicles comprising the step of performing ultracentrifugation of the filtrate and collecting the pellet to resuspend in a buffer solution and to separate the iodixanol density gradient ultracentrifugation.

In one exemplary embodiment, the filtration may be to filter with a 0.2 to 0.5 ㎛ filter. Alternatively, the filtration may be to filter with a 0.2 to 0.4 ㎛ filter, 0.3 to 0.5 ㎛ filter, or 0.4 to 0.5 ㎛ filter.

In an exemplary embodiment, the ultracentrifugation may be performed at 100,000 × g or more, specifically 100,000 to 200,000 × g, or 100,000 to 150,000 × g, or 150,000 to 200,000 × g.

In an exemplary embodiment, after the iodixanol density gradient ultracentrifugation, the floating density in iodixanol is 1.08 to 1.19 g / mL, more specifically 1.08 g / mL or more, 1.09 g / mL or more, 1.10 g from fractions that are at least 1.mL g, at least 1.11 g / mL, or at least 1.12 g / mL, but are at most 1.19 g / mL, at most 1.18 g / mL, at most 1.17 g / mL, at most 1.16 g / mL, or at most 1.15 g / mL. Extracellular vesicles can be obtained.

In another aspect, the technology disclosed herein is a method for producing extracellular vesicles derived from foods comprising the yeast, by centrifuging the food processed in liquid form to remove the residue, the supernatant is filtered and then filtered It provides a method for producing extracellular vesicles comprising ultracentrifuging the solution to obtain extracellular vesicles by size exclusion chromatography.

In one exemplary embodiment, the filtration may be to filter with 0.1 to 0.4 ㎛ filter. Alternatively, the filtration may be to filter with a 0.2 to 0.4 ㎛ filter, 0.3 to 0.4 ㎛ filter, 0.1 to 0.3 ㎛ filter, or 0.1 to 0.2 ㎛ filter.

In an exemplary embodiment, the ultracentrifugation may be performed at 100,000 × g or more, specifically 100,000 to 200,000 × g, or 100,000 to 150,000 × g, or 150,000 to 200,000 × g.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

Test Example  1. Cell Culture

(1) macrophages

Mouse macrophage J774A.1 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM). 10% FBS and 1% Antibiotic-Antimycotic (Invitrogen) were added to the medium. All cell lines were those that were not infected with mycoplasma identified using the e-MyCoTM Mycoplasma PCR Detection Kit (iNtRON Biotechnology. Inc., Seoul, Korea).

(2) keratinocytes

Human primary epidermal keratinocytes (ScienCell) were aliquoted onto Poly-L-lysine (PLL) coated plates and cultured in Keratinocyte medium (ScienCell).

Test Example  2. Exosomes  Separation

(1) yeast derived exosomes

Exosome, an extracellular vesicle, was isolated from yeast cells used for fermentation of bread and beer. Specifically, Saccharomyces cerevisiae BY4741 wild-type cells were cultured by shaking in Synthetic complete medium, which is a chemically defined medium at 30 ° C. for 24 hours. Thereafter, S. cerevisiae culture medium was pelleted twice at 4,000 × g for 15 minutes to remove yeast cells. The supernatant was filtered through a 0.45 μm vacuum filter and further filtered by ultrafiltration using a Minimate ^™ tangential-flow filter (TFF) capsule (Minimate TFF Capsule; Pall Corporation, East Hills, NY) with a 100K MWCO membrane. Concentrated. In addition, the concentrated supernatant was filtered through a 0.45 μm syringe filter to remove aggregates. The retained material was ultracentrifugated at 100,000 × g for 2 hours at 4 ° C. The pellet was resuspended in HEPES-buffered saline (HBS) and the samples were taken in 5, 15, 30, 40, 50% 2 mL each of Axis-Shield PoC AS, Nycomed, Oslo Norway. Placed on top of the tube. After ultracentrifugation at 200,000 × g for 15 hours at 4 ° C., 10 fractions of equivalent volume (1 mL) were collected from the top of the density gradient. Each fraction was measured for protein and particle content using a Bradford protein assay (Bio-Rad, Munich, Germany) and Nanoparticle Tracking Analysis (Malvern Instruments Ltd.).

(2) Lab-beer (L-beer) -derived exosomes

The exosomes, which are extracellular vesicles, were isolated from beer fermented with the same yeast as above. Specifically, Saccharomyces cerevisiae BY4741 wild-type cells were fermented in Indian pale ale (IPA) for 2 weeks at room temperature without shaking. S. cerevisiae culture IPA was pelleted continuously at 4,000 × g for 15 minutes and at 10,000 × g for 30 minutes. The supernatant was filtered through a 0.22 μm vacuum filter and ultracentrifuged at 150,000 × g for 3 hours at 4 ° C. The pellet was resuspended in HEPES-buffered saline (HBS) and the sample was subjected to size-exclusion chromatography on Sephacryl S-500 column to isolate the exosomes.

(3) exosomes derived from Heineken-beer (H-beer)

Exosome, an extracellular vesicle, was isolated from commercially available Heineken beer. Specifically, Heineken beer was pelleted continuously for 15 minutes at 4,000 × g and 30 minutes at 10,000 × g. The supernatant was filtered through a 0.22 μm vacuum filter and ultracentrifuged at 150,000 × g for 3 hours at 4 ° C. The pellet was resuspended in HEPES-buffered saline (HBS) and the sample was subjected to size exclusion chromatography on a Sephacryl S-500 column to isolate the exosomes.

Test Example  3. Exosomes  analysis

(1) Nanoparticles tracking analysis (Nanoparticle Tracking Analysis; NTA)

Samples were placed in a chamber of Nanosight LM10 (Malvern Instruments Ltd.) and the number of exosomes was counted using nanoparticle tracking analysis software.

(2) Dynamic light scattering (DLS)

Size distribution of NVs (Nanovesicles) was measured with Zetasizer Nano ZS (Malvern Instrument Ltd., Malvern, U.K.). Size distribution based on relative frequency was determined by infrared light (wavelength = 633 nm) passing through the sample at scattering intensity for 10 × 30 s.

(3) Transmission Electron Microscopy ( TEM )

Purified exosomes were added to glow-discharged carbon-coated copper grids (Electron Microscopy Sciences, Fort Washington, PA). After allowing NVs to be absorbed on the grid for 1 hour, the grid was fixed for 10 minutes with 4% paraformaldehyde and washed with a drop of deionized water, then negative with 2% uranyl acetate (Ted Pella, Redding, CA). Negative staining was performed. Electron micrographs were recorded with a JEM 1011 microscope (JEOL, Tokyo, Japan) at an acceleration voltage of 100 kV.

Test Example  4. Exosomes efficacy sign  Bitwise in vitro ) analysis

(1) cell survival analysis ( WST- 1)

Mouse macrophage J774A.1 cells (2 × 10 ⁴ cells / well) were aliquoted into 96-well plates. After overnight culture, 1 ng / mL LPS and various concentrations (10-1000 ng / mL) of S. cerevisiae exosomes in DMEM containing 0.5% FBS were added to the cells and incubated for 12 hours. 2 μl of WST-1 reagent was added to each well and incubated for 1 hour. The absorbance of the converted dye was measured at a wavelength of 440 nm using background subtraction at 690 nm using a microplate reader. In this assay, values obtained from cells treated with DMEM containing 0.5% FBS were considered 100% viable.

(2) cytokine ELISA

Mouse macrophage J774A.1 cells (2 × 10 ⁴ cells / well) were aliquoted into 96-well plates. For co-treatment, 1 ng / mL LPS in DMEM containing 0.5% FBS and various concentrations (1-1000 ng / mL) of S. cerevisiae exosomes were added to the cells for 12 hours. Incubated. For post-treatment, 1 ng / mL LPS in DMEM containing 0.5% FBS was added to the cells. After 1 hour, S. cerevisiae exosomes at various concentrations (1-1000 ng / mL) were treated with the cells and incubated for 15 hours. In the case of Beer exosomes, Lab-beer or Heineken-beer exosomes at various concentrations (5-500 ng / mL) in DMEM containing 0.5% FBS were pre-treated to cells for 12 hours. , 1 ng / mL of LPS was further incubated for 12 hours. Each cell culture supernatant was harvested for ELISA. Quantification of human IL-8, CXCL10, IL-6 protein or mouse TNF-α, IL-6 protein was performed using the DuoSet ELISA kit (R & D Systems) according to the manufacturer's protocol.

Human keratinocytes (1 × 10 ⁵ cells / well, passage 3) were dispensed in 12-well plates and S. cerevisiae exosomes in keratinocyte media-basal (ScienCell) at different concentrations (500, 1000, 2000 ng / mL) Was added to the cells. After 12 hours, 10 ng / mL of TNF-α / IFN-α was treated in keratinocyte media-basal for further incubation for 12 hours and each cell culture supernatant was harvested for ELISA. Quantification of human IL-8, CXCL10, IL-6 protein or mouse TNF-α, IL-6 protein was performed using the DuoSet ELISA kit (R & D Systems) according to the manufacturer's protocol (see FIGS. 8A, 8B).

Test Example  5. Statistical analysis

Statistical analysis herein was analyzed using GraphPad Prism software (GraphPad Software). Data are expressed as mean ± SD. P-values were calculated using unpaired two-tailed Student's t-test, and P <0.05 was considered significant (*: P <0.05 , **: P <0.01 , ***: P <0.001 ).

Test Result 1. Exosomes  Separation and Characterization

Known and purified extracellular vesicles derived from yeast cells are mostly ultracentrifugation (Sci Rep., 14 (5): 7763, 2015, PLoS One., 5 (6): e11113, 2010). However, there was a problem in that ultracentrifugation alone could not effectively separate contaminants such as protein aggregates from extracellular vesicles.

In contrast, the extracellular vesicles were separated by density gradient ultracentrifugation or size exclusion chromatography according to the test example, and thus, pure separation and purification of the extracellular vesicles was possible. .

After culturing yeast cells in a chemical complete medium (SC medium), the exosomes derived from yeast cells were isolated and purified in high yield by ultracentrifugation and iodixanol density gradient ultracentrifugation (Fig. 1 and 2), as shown in FIGS. 3A and 3B, the shape of the exosomes and the diameter of the exosomes of about 25 to 150 nm were confirmed by transmission electron microscopy and dynamic light scattering. In addition, as a result of calculating the yield of the yeast-derived exosomes obtained through the test example, about 12 μg of protein and 1.3 × 10 ¹⁰ nanoparticles were obtained per 100 mL.

In addition, exosomes derived from beer were isolated and purified from ultra-centrifugation and size exclusion gel filtration from Lab-beer and Heineken-beer (see FIG. 4), and as shown in FIGS. 5A, 5B, and 5C, exosomes. The size and the size were observed by transmission electron microscopy, dynamic light scattering, and microparticle trace analysis. As a result, they were found to have a diameter of about 30 to 110 nm. In addition, as a result of calculating the yield of beer-derived exosomes obtained through the above test example, about 33 μg of protein and 13 × 10 ¹⁰ nanoparticles were obtained per 100 mL of Lab-Beer, and 100 mL of Heineken-Beer. About 66 μg of protein and 12 × 10 ¹⁰ nanoparticles were obtained.

Test Result 2. Exosomes  Anti-inflammatory efficacy analysis

(1) yeast derived exosomes

When yeast-derived exosomes were treated with mouse macrophages (J774A.1 cells) for 24 hours, representative proinflammatory cytokines, Tumor necrosis factor-α (TNF-α) and Interleukin-6 (to 1000 ng / mL) It was confirmed that no secretion of IL-6) was induced (see FIG. 6A).

In addition, in a mouse macrophage model that induced an inflammatory response by treating lipopolysaccharides (LPS), an inflammatory stimulant, the secretion of IL-6 and TNF-α, which are representative proinflammatory cytokines, were treated with yeast-derived exosomes simultaneously with LPS. It was confirmed that there is an effect to suppress. 100 ng / mL yeast-derived exosomes inhibited IL-6 secretion by 40% and TNF-α secretion by 50% (see FIG. 6B). Induction of an inflammatory response by pretreatment of LPS to mouse macrophages and treatment with yeast-derived exosomes were similarly shown to have an effect of inhibiting the secretion of representative proinflammatory cytokines IL-6 and TNF-α. 1000 ng / mL yeast-derived exosomes inhibited IL-6 secretion by 42% and TNF-α secretion by 45% (see FIG. 6C).

In conclusion, yeast-derived exosomes, by themselves, did not induce an inflammatory response in mouse macrophages, but showed a concentration-dependent anti-inflammatory effect when co-treated or post-treated with LPS in mouse macrophages. The half inhibitory concentration was estimated to be 100 ng / mL for co-treatment and <1000 ng / mL for post-treatment.

(2) exosomes derived from beer

When beer-derived exosomes were treated with mouse macrophages (J774A.1 cells) for 24 hours, it was confirmed that up to 500 ng / mL did not induce the secretion of the proinflammatory cytokine IL-6 (see FIG. 7A). ).

Pre-treatment of beer-derived exosomes in mouse macrophages and treatment of LPS, an inflammatory stimulant, induced inflammatory reactions. IL-6 secretion was inhibited by 20% in all, and the group treated with 500 ng / mL exosomes inhibited IL-6 secretion by 50% or more, respectively (see Figure 7b).

In conclusion, beer-derived exosomes by themselves do not induce an inflammatory response in mouse macrophages and have a concentration-dependent anti-inflammatory effect upon pre-treatment with mouse macrophages. It could be estimated at 500 ng / mL.

Examples of the formulation of the composition according to one aspect of the present invention will be described below, but it is also applicable to various other formulations, which are intended to explain in detail only and not intended to limit the present invention.

[ Formulation example  One] Soft capsule

Exosome 50mg, L-carnitine 80 ~ 140mg, soybean oil 180mg, palm oil 2mg, vegetable hardened oil 8mg, lead 4mg and lecithin 6mg were mixed, and filled with 400mg per capsule according to a conventional method to prepare a soft capsule.

[ Formulation example  2] tablets

Exosome 50mg, galactooligosaccharide 200mg, lactose 60mg and malt sugar 140mg was mixed and granulated using a fluidized bed dryer, and sugar ester (sugar ester) 6mg was added to the tableting machine to prepare a tablet.

[ Formulation example  3] granules

50 mg of exosomes, 250 mg of anhydrous glucose, and 550 mg of starch were mixed, molded into granules using a fluidized bed granulator, and then filled into sachets to prepare granules.

[ Formulation example  4] Drinks

Exosome 50mg, glucose 10g, citric acid 0.6g, and liquid oligosaccharide 25g was mixed and 300ml of purified water was added to each bottle 200ml each was filled. After filling the bottle sterilized for 4-5 seconds at 130 ℃ to prepare a beverage drinks.

[ Formulation example  5] lotion

The lotion was prepared in a conventional manner according to the composition described in Table 1 below.

ingredient	Content (% by weight)
Exosomes	2.00
L-ascorbic acid-2-magnesium phosphate	1.00
Water-soluble collagen (1% aqueous solution)	1.00
Sodium citrate	0.10
Citric acid	0.05
Licorice Extract	0.20
1,3-butylene glycol	3.00
Purified water	Remaining amount
Sum	100.00

[ Formulation example  6] cream

To prepare a cream in a conventional manner according to the composition shown in Table 2.

ingredient	Content (% by weight)
Exosomes	2.00
Polyethylene Glycol Monostearate	2.00
Self-emulsifying glycerin monostearate	5.00
Cetyl alcohol	4.00
Squalene	6.00
Tri2-ethylhexaneglyceryl	6.00
Sphingolipid	1.00
1,3-butylene glycol	7.00
Purified water	Remaining amount
Sum	100.00

[ Formulation example  7] pack

To prepare a pack in a conventional manner according to the composition described in Table 3.

ingredient	Content (% by weight)
Exosomes	2.00
Polyvinyl alcohol	13.00
L-ascorbic acid-2-magnesium phosphate	1.00
Lauroylhydroxyproline	1.00
Water-soluble collagen (1% aqueous solution)	2.00
1,3-butylene glycol	3.00
ethanol	5.00
Purified water	Remaining amount
Sum	100.00

As described above, specific portions of the present disclosure have been described in detail, and for those skilled in the art, these specific techniques are merely preferred embodiments, and the scope of the present disclosure is not limited thereto. Will be obvious. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (16)

1. Anti-inflammatory composition comprising yeast-derived extracellular vesicles (extracellular vesicles) as an active ingredient.
2. An anti-inflammatory composition comprising extracellular vesicles derived from foods containing yeast as an active ingredient.
3. The method according to claim 1 or 2,

   The yeast is Saccharomyces cerevisiae ).
4. The method according to claim 1 or 2,

   The extracellular vesicles will have a diameter of 20 to 200 nm, anti-inflammatory composition.
5. The method of claim 1,

   The extracellular vesicles are separated by density gradient ultracentrifugation, anti-inflammatory composition.
6. The method of claim 1,

   The extracellular vesicles have a floating density of 1.08 to 1.19 g / mL in iodixanol, anti-inflammatory composition.
7. The method of claim 2,

   The extracellular vesicles are separated by size exclusion chromatography (size exclusion chromatography), anti-inflammatory composition.
8. The method of claim 2,

   The extracellular vesicles derived from foods including the yeast are extracellular vesicles derived from beer, anti-inflammatory composition.
9. The method according to claim 1 or 2,

   The anti-inflammatory composition is to inhibit or inhibit the secretion of tumor necrosis factor-α (TNF-α) or Interleukin-6 (Interleukin-6, IL-6) that is an inflammation-related mediator.
10. The method according to claim 1 or 2,

    The anti-inflammatory composition is to have an anti-inflammatory activity against inflammatory skin diseases, anti-inflammatory composition.
11. The method of claim 10,

    The inflammatory skin disease is at least one selected from the group consisting of acne, contact dermatitis, seborrheic dermatitis, atopic dermatitis and psoriasis, anti-inflammatory composition.
12. The method according to claim 1 or 2,

    The anti-inflammatory composition is a pharmaceutical composition, a cosmetic composition or a food composition, anti-inflammatory composition.
13. A method for producing the extracellular vesicles according to claim 1,

    Culturing the yeast;

    Centrifuging the culture solution to remove residue and filtering the supernatant; And

    After the filtrate is ultracentrifuged, the pellet is collected and separated by iodixanol density gradient ultracentrifugation.
14. The method of claim 13,

    The filtration is to filter with a 0.2 to 0.5 ㎛ filter, a method for producing extracellular vesicles.
15. The method of claim 13,

    The ultracentrifugation is carried out at 100,000 × g or more, a method for producing extracellular vesicles.
16. The method of claim 13,

    After the iodixanol density gradient ultracentrifugation, extracellular vesicles are obtained from the fraction having a floating density of 1.08 to 1.19 g / mL in iodixanol.

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