KR102486043B1 - Composition for Preventing, Improving or Treating Inflammatory Diseases Comprising Brassica spp. or Allium spp.-Derived Extracellular Vesicles as Active Ingredients - Google Patents

Abstract

The present invention relates to a composition for the prevention, improvement or treatment of inflammatory diseases comprising brassica or allium plant-derived extracellular vesicles as an active ingredient.
The extracellular vesicles derived from Brassica plants or Allium genus plants of the present invention have good intracellular penetration, and the delivered extracellular vesicles are interleukin-6 (IL-6), interleukin-1 beta (IL-1β) and COX-2 ( Since it has the effect of inhibiting the expression of cyclooxygenase-2), it can be usefully used for the treatment, improvement or prevention of inflammatory diseases.

Description

A composition for preventing, ameliorating or treating inflammatory diseases comprising brassica plants or allium plant-derived extracellular vesicles as an active ingredient {Composition for Preventing, Improving or Treating Inflammatory Diseases Comprising Brassica spp. or Allium spp.-Derived Extracellular Vesicles as Active Ingredients}

The present invention relates to a composition for the prevention, improvement or treatment of inflammatory diseases comprising brassica or allium plant-derived extracellular vesicles as an active ingredient.

Extracellular vesicles have various sizes, and in particular, exosomes having the smallest size are small membranous vesicles of 50 to 150 nm. Extracellular endoplasmic reticulum is secreted by human and animal as well as insect, plant and microbial cells. In particular, they contain specific molecules such as proteins, nucleic acids, lipids, and carbohydrates that cells have, and are surrounded by a lipid bilayer to stably protect these molecules while secreting and delivering them to other cells. Extracellular vesicles, which are found in high concentrations in bodily fluids such as blood, urine, and saliva, have potential as new biological materials due to these characteristics.

The extracellular endoplasmic reticulum is 1) a natural material and has very few side effects; 2) can reach the large intestine through oral administration; 3) It is more convenient than eating directly as food, so it can be applied to infants and children; 4) Mass production from plants is possible, leading to a new paradigm of health functional food; 5) It has the advantage that it can be applied as an efficient drug delivery system that can be loaded with pharmacological ingredients and delivered to the large intestine.

Nevertheless, studies on the biological functions and applications of Brassica or Allium-derived extracellular vesicles, as well as their extraction and purification methods, have not yet been systematically conducted. In the case of ultra-high-speed centrifugation, which is generally used for the separation of extracellular vesicles, the impurity concentration is relatively high, and physical disruption of the extracellular vesicles may be induced by strong centrifugal force. Polymer precipitation methods such as ExoQuick (SBI), which are commercialized and sold, also have a very high concentration of impurities and have contamination problems due to the use of PEG (polyethylene glycol). Therefore, for the industrial use of extracellular vesicles, the development of a new technology capable of efficiently separating them in large quantities is required.

On the other hand, extracellular endoplasmic reticulum reflects the state of the cell of origin (donor cell) that secretes it, shows various biological activities depending on which cell it is secreted from, and plays an important role in cell-to-cell interactions by transferring genetic material and proteins between cells. do

Conventionally, these extracellular vesicles have been mainly used as biomarkers, and a technology for using the extracellular vesicles for a specific purpose by using the efficacy of the extracellular vesicles themselves has not yet been developed.

Therefore, the present inventors have focused on the fact that the extracellular vesicles derived from Brassica plants or Allium genus plants can be applied to the manufacture of medicines, foods, cosmetics, etc. having an anti-inflammatory effect, and came to complete the present invention.

A number of papers and patent documents are referenced throughout this specification and their citations are indicated. The contents of the cited papers and patent documents are incorporated herein by reference in their entirety to more clearly describe the level of the technical field to which the present invention belongs and the contents of the present invention.

KR 10-2019-0028281 A

An object of the present invention is to provide a pharmaceutical composition for preventing or treating inflammatory diseases, comprising, as an active ingredient, extracellular vesicles derived from Brassica plants or Allium genus plants.

In addition, another object of the present invention is to provide a food composition for preventing or improving inflammatory diseases, comprising as an active ingredient extracellular vesicles derived from Brassica plants or Allium genus plants.

In addition, another object of the present invention is to provide a cosmetic composition for preventing or improving inflammatory diseases, comprising, as an active ingredient, extracellular vesicles derived from plants of the genus Brassica or allium genus.

In addition, another object of the present invention is to provide a method for preparing extracellular vesicles derived from Brassica plants or Allium plants.

To achieve the above object, the pharmaceutical composition for preventing or treating inflammatory diseases of the present invention may contain extracellular vesicles derived from Brassica plants or Allium genus plants as an active ingredient.

The extracellular vesicles may have an average diameter of 50 to 150 nm.

The extracellular vesicles may be separated by size exclusion chromatography.

The extracellular vesicles may suppress the expression of any one selected from interleukin-6 (IL-6), interleukin-1 beta (IL-1β), and cyclooxygenase-2 (COX-2).

The inflammatory diseases include dermatitis, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, iritis, sore throat, tonsillitis, pneumonia, gastric ulcer, pancreatitis, gastritis, Crohn's disease, inflammatory bowel disease, colitis, hemorrhoids, gout, ankylosing spondylitis, lupus, fibromyalgia ( fibromyalgia), psoriasis, rheumatoid arthritis, osteoarthritis, osteoporosis, hepatitis, cystitis, nephritis, Sjogren's syndrome, and multiple sclerosis.

The inflammatory disease may be any one inflammatory skin disease selected from acne, contact dermatitis, seborrheic dermatitis, atopic dermatitis, and psoriasis.

In addition, the food composition for preventing or ameliorating inflammatory diseases of the present invention for achieving the above other object may include extracellular vesicles derived from Brassica plants or Allium genus plants as an active ingredient.

In addition, the cosmetic composition for preventing or improving inflammatory diseases of the present invention for achieving the above another object may include extracellular vesicles derived from Brassica plants or Allium genus plants as an active ingredient.

In addition, the method for producing extracellular vesicles of the present invention for achieving another object described above includes the steps of (1) preparing a juice of a plant of the genus Brassica or Allium genus; (2) performing ultrafiltration on the juice of the Brassica or Allium plants; and (3) performing size exclusion chromatography on the filtrate.

The ultrafiltration in step (2) may have a cut-off value of 5 to 15 kDa.

The size exclusion chromatography in step (3) may be equilibrated with a phosphate buffer (PBS) of pH 6 to pH 8.

In the size exclusion chromatography in step (3), the average pore diameter of the stationary phase may be 30-75 nm.

The stationary phase may be any one gel filtration medium selected from Superdex, Sephacryl, Sephadex, Sepharose, Fractogel, Toyopearl and Bio-Gel.

The eluent of the size exclusion chromatography may be a PBS buffer solution of pH 6 to pH 8.

Particle size analysis is performed on each fraction obtained from the size exclusion chromatography, and a fraction having an average diameter of 50 to 150 nm may be selected and recovered.

The extracellular vesicles derived from Brassica plants or Allium genus plants of the present invention have good intracellular penetration, and the delivered extracellular vesicles are interleukin-6 (IL-6), interleukin-1 beta (IL-1β) and COX-2 ( Since it has the effect of inhibiting the expression of cyclooxygenase-2), it can be usefully used for the treatment, improvement or prevention of inflammatory diseases.

1 is a picture showing the state of a sample obtained by concentrating and purifying a brassica plant juice containing impurities other than brassica plant-derived extracellular vesicles.
Figure 2 is a picture showing the process of loading the juice derived from Brassica plants through size exclusion chromatography.
Figure 3 is a picture showing the fractions of cabbage-derived juice recovered through size exclusion chromatography.
Figure 4 is a picture showing the fractions of the juice derived from red cabbage recovered through size exclusion chromatography.
5A is a graph showing the concentration of extracellular vesicles (number of extracellular vesicles/mL) contained in fractions of cabbage-derived juice recovered through size exclusion chromatography, and FIG. It is a graph showing the concentration of extracellular vesicles (number of extracellular vesicle particles/mL) contained in fractions of juice derived from red cabbage.
5C is size exclusion chromatography (SEC), FIG. 5D is polymer precipitation (PEG), and FIG. 5E is a size distribution diagram of fractions of cabbage-derived juice obtained by ultracentrifugation (UC).
5F is a size distribution diagram of fractions of juice extracted from red cabbage recovered by size exclusion chromatography (SEC), FIG. 5G is polymer precipitation (PEG), and FIG. 5H is an ultracentrifugation (UC) method.
5I to 5K are size distribution diagrams (I), yield (J) and yield (J) of fractions of cabbage-derived juice recovered according to polymer precipitation (PEG), ultracentrifugation (UC), and size exclusion chromatography (SEC). It is a graph of the result of analyzing the purity (K).
5L to 5N are size distribution maps (L) and yield (M) of fractions of juice derived from red cabbage recovered according to polymer precipitation (PEG), ultracentrifugation (UC), and size exclusion chromatography (SEC) methods. And it is a graph of the result of analyzing the purity (N).
Figure 6A is a transmission electron microscope (TEM) image of cabbage-derived extracellular vesicles (Cabex) recovered through size exclusion chromatography, and Figure 6B is red cabbage-derived extracellular vesicles recovered through size exclusion chromatography ( Rabex) is an image taken with a transmission electron microscope (TEM).
6C is a graph showing zeta potential values of cabbage-derived extracellular vesicles (Cabex) and red cabbage-derived extracellular vesicles (Rabex) recovered through size exclusion chromatography.
6D to 6F show whether cabbage-derived extracellular vesicles (Cabex) and red cabbage-derived extracellular vesicles (Rabex) labeled with PKH67 were added to the cell culture medium and then photographed with a confocal microscope to determine whether the extracellular vesicles penetrated into the cells. As an image for confirming, it is an image taken of a control group (D), cabbage-derived extracellular vesicles recovered through size exclusion chromatography (E), and red cabbage-derived extracellular vesicles (F).
7 is a graph showing cell proliferation and cytotoxic effects of cabbage-derived extracellular vesicles (Cabex) and red cabbage-derived extracellular vesicles (Rabex) in HaCaT, HDF, and RAW 264.7 cells.
8 is a graph showing the effect of inhibiting the expression of IL-6, IL-1β and COX-2 by cabbage-derived extracellular vesicles (Cabex), red cabbage-derived extracellular vesicles (Rabex), and onion-derived extracellular vesicles (Onex). .
9 is an ELISA showing the inhibitory effect of IL-6, IL-1β and COX-2 expression of cabbage-derived extracellular vesicles (Cabex), red cabbage-derived extracellular vesicles (Rabex), and onion-derived extracellular vesicles (Onex); This is the result of Western blot analysis.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for the prevention or treatment of inflammatory diseases comprising, as an active ingredient, extracellular vesicles derived from plants of the genus Brassica or allium genus.

As used herein, the term "Brassica Species" refers to plants belonging to the genus Brassica, and specifically includes any one or more selected from cabbage, red cabbage, broccoli, cauliflower, kohlrabi, collards, and Brussels sprouts. It may be cabbage or red cabbage more preferably.

In the present specification, the term "Allium Species" refers to a plant belonging to the genus Allium, and may specifically include any one or more selected from onions, garlic, ginger, green onion, green onion, chives, chives, wild chives and three vegetables, Preferably it may be an onion.

As used herein, the term "extracellular vesicle" refers to a small membrane-structured vesicle secreted from various cells, having a diameter in the range of about 30-1,000 nm, and passing through the fusion of the polycystic body and the plasma membrane to the outside of the cell. released into the environment

In the present specification, "comprising as an active ingredient" means containing an amount sufficient to achieve the efficacy or activity of extracellular vesicles derived from Brassica plants or Allium plants. The present invention is an extracellular vesicle derived from a plant of the genus Brassica or Allium genus, which is a natural plant material, and since there is no side effect on the human body even when administered in excess, the upper limit of the amount of the extracellular vesicle included in the composition of the present invention is within the appropriate range for those skilled in the art. You can choose to do it within.

The extracellular vesicles of the present invention may be exosomes having an average diameter of 40 to 200 nm, preferably 50 to 150 nm, and more preferably 80 to 120 nm. The exosome is a bio-derived nanovesicle, and since MHC in its cell membrane helps to evade immune cells, it itself has less immune rejection than other polymeric or inorganic nanoparticles. Accordingly, it can be delivered to CTC (circulating tumor cells) while circulating without being decomposed for a long time even in the blood.

In addition, because exosomes have various receptors and proteins on their membrane surface, they are better absorbed by cells than liposomes that have a similar bilayer lipid to exosomes, and are also toxic. It has already been shown to be low. Accordingly, the function of the exosome as a delivery system occurs in a different aspect from that of the liposome, and the efficiency is also different. In addition, since liposomes do not have various receptors or proteins that exist in the cell membrane of exosomes, they cannot have the above advantages.

The extracellular vesicles of the present invention may be obtained by performing size exclusion chromatography on a biological sample containing a tissue or cell of a Brassica plant or an Allium genus plant.

The present inventors have made intensive research efforts to develop a method for efficiently obtaining extracellular vesicles from plants of the genus Brassica or Allium. As a result, when separation and purification are performed using size exclusion chromatography, departing from the polymer precipitation method or ultra-high-speed centrifugation method commonly used in the prior art, the content of impurities is greatly reduced, while there is no physical disruption applied to the vesicles, and the unique pharmacological properties are obtained. And it was found that high-purity and high-quality Brassica plants or Allium plants-derived extracellular vesicles maintaining drug delivery activity as they are can be obtained.

As used herein, the term "biological sample" refers to an unprocessed or pretreated sample containing an effective amount of a component of interest derived from, isolated from, or derived from Brassica plants or Allium plants that retain biological activity. it means. Specifically, the biological sample used in the present invention is a juice of a plant of the genus Brassica or Allium.

In the present specification, the term "squeezed juice of Brassica plants or Allium plants" refers to the extraction of useful components contained inside Brassica plants or Allium plants by applying mechanical or physical force without applying heat or enzymes to the Brassica plants or Allium plants. It means a liquid fluid obtained by doing, and is not particularly limited, but specifically, the juice of the Brassica plant or Allium genus plant may be prepared by the following method.

In order to prepare the juice of the Brassica or Allium plant, after preparing the Brassica or Allium plant, the Brassica or Allium plant may be pressurized to prepare the juice. The Brassica or Allium plants may additionally undergo a washing process before pressing.

Washing the Brassica plants or Allium plants may be to wash the prepared plants using running water, but may also be performed using, for example, purified water in which PBS or sodium bicarbonate is dissolved. Since sodium bicarbonate is widely used as a food additive and is non-toxic, it can be used in the cleaning process of plants of the genus Brassica or Allium.

A step of pulverizing the Brassica genus or Allium genus to an average diameter of 1 to 50 mm may be additionally performed between the washing step and the juice extraction step so that the nutrients contained in the Brassica or Allium genus are easily extracted. there is.

By pressurizing the pulverized Brassica plants or Allium plants at a pressure of 2 to 3 kgf/cm2 to extract the juice, a Brassica plant juice from which useful components of the Brassica plants or Allium plants are sufficiently leached can be prepared.

Thereafter, a step of filtering the juice of the Brassica or Allium genus plants to remove solids may be further included. The solid material may have an average diameter of 1 mm or more.

In addition, a step of centrifuging the filtrate of the Brassica plant or Allium plant juice from which the solids are removed may be further included. The centrifugation step may be performed by first centrifuging at 7,000 to 9,000 x g and then secondarily centrifuging the supernatant at 19,000 to 21,000 x g. More specifically, the centrifugation step may be performed by first centrifuging at 7,500 to 8,500 x g and then secondarily centrifuging the supernatant at 19,500 to 20,500 x g.

According to a specific embodiment of the present invention, the step of performing ultrafiltration on the Brassica plant or Allium plant juice may be additionally included in any order.

In this specification, the term "ultrafiltration" refers to a membrane-based separation process in which each material constituting a heterogeneous mixed solution is separated along a semi-permeable membrane by a pressure or concentration gradient. Ultrafiltration membranes have a pore size with a constant cutoff value. According to a specific embodiment of the present invention, the ultrafiltration used in the present invention has a cut-off value of 3 to 100 kDa, more specifically a cut-off value of 5 to 50 kDa, Most preferably, it has a cutoff value of 5 to 15 kDa.

When the step of performing ultrafiltration is additionally included in the method of the present invention, this may be performed before size exclusion chromatography or may be performed after completion of chromatography.

More specifically, the method of the present invention sequentially includes the ultrafiltration step and the size exclusion chromatography step. In this way, when size exclusion chromatography is performed after ultrafiltration, a more efficient separation process can be achieved by applying size exclusion chromatography after the volume of the sample is reduced by concentration through ultrafiltration.

In this specification, the term "size exclusion chromatography (Size Exclusion Chromatography, SEC)" means that among the materials constituting the heterogeneous mixture, materials smaller in size than the diameter of the bead pores of the stationary phase enter the pores and elute relatively slowly , and large-sized substances are quickly eluted along the mobile phase without entering the pores, and thus means chromatography that separates substances based on the difference in migration speed according to the size of each substance caused therefrom.

According to a specific embodiment of the present invention, in the size exclusion chromatography used in the present invention, the stationary phase may have an average pore diameter of 30 to 75 nm. Preferably, the stationary phase may have an average pore diameter of 65 to 75 nm.

Specifically, the stationary phase may be any one gel filtration medium selected from the group consisting of Superdex, Sephacryl, Sephadex, Sepharose, Fractogel, Toyopearl and Bio-Gel, and more preferably Izon science qEVoriginal (Cat.No: SP1) produced by the company was used, but is not limited thereto.

The size exclusion chromatography is not particularly limited as long as it is a conventional method in the art, but may be preferably performed through the following steps.

(a) loading the vegetable juice of the genus Brassica or allium genus; and

(b) obtaining fractions as eluents.

Before loading the Brassica or Allium plant juice, the size exclusion chromatography may be equilibrated through an equilibration buffer to remove impurities and form an equilibrium with the stationary phase to achieve an efficient separation process.

The equilibration buffer and eluent may be phosphate buffered saline (PBS) of pH 6 to pH 8.

According to one embodiment of the present invention, when the Brassica plant juice is loaded on the size exclusion chromatography and the eluent is developed, large molecules such as extracellular vesicles in the Brassica plant or Allium plant juice are selectively eluted and the remaining small-sized substances may remain inside the size exclusion chromatography to separate large-sized extracellular vesicles from the sample. In general, extracellular vesicles are eluted together with the eluent preferentially during the elution process by the eluent because they belong to particles with a molecular size of 1,000 kDa or more and are larger than other impurities. The molecular weight of the eluted material is different depending on the size and pore size of the porous stationary phase, the length of the column, the flow rate of the mobile phase, etc., and the size of the eluted molecule is constant under the same conditions.

The average diameter of the extracellular vesicles of the present invention may be 500 nm or less, preferably 20 nm to 200 nm, and more preferably 50 to 150 nm.

Therefore, in order to maximize the separation efficiency of the extracellular vesicles, it is more preferable to analyze the particle size of the eluted fraction and select and recover the fraction having the average diameter.

In one embodiment of the present invention, a column (70 nm) filled with qEVoriginal manufactured by Izon science is loaded with Brassica or Allium plant juice on a stationary phase equilibrated by adding phosphate buffer (1 X PBS, pH 7.4) to the column (70 nm). , Phosphate buffer (1 X PBS, pH 7.4) was injected into the corresponding column as an eluent, fractions were collected by 0.5 mL, and then analyzed by a nanoparticle tracking analyzer (NTA).

In one embodiment of the present invention, the inflammatory cytokine inhibitory activity of the extracellular endoplasmic reticulum of the present invention was analyzed in vitro.

As a result, the extracellular vesicles of the present invention have an activity to effectively inhibit the expression of inflammatory cytokines, interleukin-6 (IL-6), interleukin-1 beta (IL-1β), and cyclooxygenase-2 (COX-2). confirmed to be

Therefore, the inventors of the present invention, through these experimental results, found that the extracellular vesicles of the present invention can be usefully used for the prevention, improvement, or treatment of inflammatory diseases.

As used herein, the term "inflammatory disease" may include any disease that can be caused by inflammation, and specifically, but is not limited thereto, dermatitis, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, iritis, sore throat, tonsillitis, Pneumonia, gastric ulcer, pancreatitis, gastritis, Crohn's disease, inflammatory bowel disease, colitis, hemorrhoids, gout, ankylosing spondylitis, lupus, fibromyalgia, psoriasis, rheumatoid arthritis, osteoarthritis, osteoporosis, hepatitis, cystitis, nephritis, Sjögren's syndrome and polycystic It may be any one selected from sclerosis. Preferably, the inflammatory disease may be any one inflammatory skin disease selected from acne, contact dermatitis, seborrheic dermatitis, atopic dermatitis and psoriasis.

The term "prevention" of the present invention refers to any action that suppresses symptoms or delays the progression of an inflammatory disease by administering a composition containing the extracellular vesicles of the present invention.

The term "treatment" as used herein refers to the administration of a composition comprising the extracellular vesicles of the present invention to (a) inhibit the development of a disease, disorder or symptom; (b) alleviation of the disease, condition or symptom; or (c) eliminating the disease, disorder or condition.

In the composition of the present invention, the active ingredient including the extracellular vesicles derived from Brassica plants or Allium plants inhibits, removes, or alleviates inflammatory diseases. Therefore, the composition of the present invention may be a composition for treating these diseases by itself, or may be administered together with other pharmacological ingredients to be applied as a treatment adjuvant for the above diseases. Accordingly, the terms "treatment" or "therapeutic agent" herein include the meaning of "therapeutic aid" or "therapeutic agent".

The pharmaceutical composition of the present invention can be prepared using pharmaceutically suitable and physiologically acceptable adjuvants in addition to the above active ingredients, and the adjuvants include excipients, disintegrants, sweeteners, binders, coating agents, expanding agents, lubricants, and lubricants. agents or flavoring agents may be used.

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

As used herein, the term "administering" means providing a given composition of the present invention to a subject by any suitable method. In this case, the subject refers to all animals such as humans, monkeys, dogs, goats, pigs, or rats having a disease in which the symptoms of an inflammatory disease can be improved by administering the composition of the present invention.

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

In compositions formulated as liquid solutions, acceptable pharmaceutical carriers are sterile and biocompatible, and include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added if necessary. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to prepare formulations for injections such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, or tablets.

Furthermore, using a method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA as an appropriate method in the field, it can be preferably formulated according to each disease or component.

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

The suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, administration method, patient's age, weight, sex, medical condition, food, administration time, administration route, excretion rate and reaction sensitivity, usually This allows the skilled physician to readily determine and prescribe dosages effective for the desired treatment or prophylaxis. According to a preferred embodiment of the present invention, the daily dosage of the pharmaceutical composition of the present invention is 0.001 to 10 g/kg, preferably 10 to 300 mg/kg, more preferably 100 to 200 mg/kg per day. It may be administered 1 to 4 times, but is not limited thereto.

The pharmaceutical composition of the present invention may be prepared in a unit dose form by formulation using a pharmaceutically acceptable carrier and/or excipient, or prepared by putting it into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet or capsule, and may additionally contain a dispersing agent or stabilizer.

Meanwhile, the daily dose of the extracellular vesicles of the present invention may be 10 to 1,000 mg/kg, preferably 20 to 500 mg/kg, and more preferably 50 to 500 mg/kg. And, it may be more preferably 100 to 500 mg / kg, more preferably 100 to 300 mg / kg or 200 to 500 mg / kg, more preferably 200 to 400 mg / kg, , most preferably 200 to 300 mg/kg. If the dose of the extracellular vesicles of the present invention is less than the lower limit, the effect of inhibiting inflammatory cytokine expression may not appear as desired, and if it exceeds the upper limit, the effect of inhibiting inflammatory cytokine expression does not increase as much as the dose increases. may not be Since extracellular vesicles are natural products and do not have side effects on the human body even when administered in excess, the upper limit of the amount of extracellular vesicles included in the composition of the present invention can be selected and implemented by those skilled in the art within an appropriate range.

In addition, the present invention provides a health functional food composition for preventing or improving inflammatory diseases, comprising extracellular vesicles derived from Brassica plants or Allium plants as an active ingredient.

In the present invention, the terms "brasseum plants", "allium plants", "extracellular vesicles", and "inflammatory diseases" are as described above.

The term "improvement" of the present invention means that administration of a composition comprising the extracellular vesicles of the present invention reduces at least a parameter related to the condition being treated, eg, the severity of symptoms.

When the health functional food composition of the present invention is used as a food additive, the composition may be added as it is or used together with other foods or food ingredients, and may be appropriately used according to a conventional method.

The type of food is not particularly limited, and includes all foods in a conventional sense. Non-limiting examples of foods to which the substance can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, or vitamin complexes; and the like.

The health functional food may be formulated with the extracellular vesicles in the form of capsules, tablets, powders, granules, liquids, pills, slices, pastes, syrups, gels, jellies, or bars, or beverages and teas. , Spices, chewing gum, confectionery, etc. are added to food materials and manufactured in the form of general food, which means that when ingested, it brings a specific effect on health, but unlike general drugs, long-term use of drugs using food as raw material It has the advantage that there are no side effects that may occur during use.

The health functional food is very useful because it can be consumed on a daily basis. The amount of extracellular vesicles added to such a health functional food cannot be uniformly defined depending on the type of target health functional food, but it can be added within a range that does not impair the original taste of the food. It ranges from 0.01 to 50% by weight, preferably from 0.1 to 20% by weight. In addition, in the case of health functional foods in the form of capsules, tablets, powders, granules, liquids, pills, flakes, pastes, syrups, gels, jellies or bars, the amount is usually 0.1 to 100% by weight, preferably 0.5 to 80% by weight. It may be added in the range of %.

The health functional food may include not only extracellular endoplasmic reticulum as an active ingredient, but also ingredients commonly added during food preparation, and include, for example, proteins, carbohydrates, fats, nutrients, seasonings and flavors. Examples of the aforementioned carbohydrates include monosaccharides such as glucose, fructose, and the like; disaccharides such as maltose, sucrose, oligosaccharides and the like; and polysaccharides such as conventional sugars such as dextrins and cyclodextrins and sugar alcohols such as xylitol, sorbitol and erythritol. As flavoring agents, natural flavoring agents [thaumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.]) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used.

For example, when the health functional food of the present invention is made into drinks and beverages, citric acid, high fructose corn syrup, sugar, glucose, acetic acid, malic acid, fruit juice, and various plant extracts may be further included in addition to the extracellular vesicles of the present invention. there is.

In addition, the present invention provides a cosmetic composition for preventing or improving inflammatory diseases, comprising extracellular vesicles derived from Brassica plants or Allium plants as an active ingredient.

In the present invention, the terms "brasseum plants", "allium plants", "extracellular vesicles", and "inflammatory diseases" are as described above.

In a specific embodiment, after artificially overexpressing inflammatory cytokines by treating keratinocytes with LPS, as a result of treating extracellular vesicles derived from Brassica plants or Allium genus plants of the present invention, the overactivated inflammatory cytokines It was specifically confirmed that this was significantly reduced.

When the expression of such overactivated inflammatory cytokines is suppressed, inflammatory skin diseases can be alleviated.

That is, the cosmetic composition comprising the extracellular vesicles of the present invention as an active ingredient can alleviate inflammatory skin diseases.

The cosmetic composition containing extracellular vesicles derived from the Brassica plant or Allium genus plant as an active ingredient of the present invention is a basic cosmetic composition (face wash such as lotion, cream, essence, cleansing foam and cleansing water) together with a dermatologically acceptable excipient , pack, body oil), color cosmetic composition (foundation, lipstick, mascara, makeup base), hair or scalp product composition (shampoo, rinse, hair conditioner, hair gel) and soap.

The excipient is not limited thereto, but may include, for example, a skin softener, a skin penetration enhancer, a colorant, a fragrance, an emulsifier, a thickening agent, and a solvent. In addition, it may additionally include flavoring agents, pigments, bactericides, antioxidants, preservatives, moisturizing agents, and the like, and thickeners, inorganic salts, synthetic polymer materials, and the like may be included for the purpose of improving physical properties. For example, when preparing a face wash and soap with the cosmetic composition of the present invention, it can be easily prepared by adding the biarylamide derivative of the present invention to a conventional face wash and soap base. In the case of preparing a cream, it can be prepared by adding the biarylamide derivative of the present invention to a general oil-in-water (O/W) cream base. Herein, synthetic or natural materials such as flavors, chelating agents, pigments, antioxidants, preservatives, and proteins, minerals, and vitamins for the purpose of improving physical properties may be additionally added.

In addition, the present invention provides a method for preparing extracellular vesicles derived from the Brassica or Allium plants.

Hereinafter, a composition for preventing, improving or treating inflammatory diseases comprising the extracellular vesicles according to the present invention as an active ingredient will be described in detail with Examples and Comparative Examples.

<Example>

Extracted juice for separation of extracellular vesicles derived from Brassica and Allium plants

1) In order to isolate extracellular vesicles from cabbage plants such as red cabbage, cabbage, and onions, plants of the genus Brassica, pesticides were first removed from the plants with running tap water after measuring the weight. Next, tap water and pesticides were removed secondarily using flowing ultrapure distilled water. Afterwards, it was cut into pieces less than 40 mm in width and 30 mm in length using a fruit juicer, and then juiced using a juicer, and then only chunks less than 1 mm in width and 1 mm in length were filtered through cotton bags to remove large substances from the juice. .

Finally, in order to remove proteins and cell walls from the solution filtered through a cotton bag, centrifugation was performed at 8,000 x g for 1 hour at 4°C, and then only the supernatant was centrifuged again at 20,000 x g for 1 hour at 4°C. The supernatant obtained in this process was stored at -80 ℃ until further experiments.

Method for isolating and purifying extracellular vesicles derived from Brassica and Allium plants

1) In order to obtain high-purity Brassica plant-derived extracellular vesicles and Allium plant-derived extracellular vesicles, impurities including proteins contained in Brassica plants and Allium plants, respectively, should be removed. Even after centrifugation of the juice, the supernatant contains a large number of protein impurities other than extracellular vesicles, and the ultra-high-speed centrifugation method and the polymer-based sedimentation method have limitations in removing these impurity proteins. Therefore, in order to increase the purity after removing substances other than these extracellular vesicles, the size exclusion chromatography method was used for the separation and purification of extracellular vesicles, and ultrafiltration was used for efficient separation and purification to improve the separation efficiency and purity. wanted to increase

2) Amicon Ultra-15 10kDa Centrifugal filter (Merck Millipore, Cat. No: UFC901024) was loaded with 13 mL of the juice solution, centrifuged at 5,000 x g for 3 hours at 4°C, and 0.5 mL of the obtained supernatant was used in a 200nm syringe It was added to the filter and transferred to a 1.5 mL Eppendorf tube (SPL, Cat. No: 50015) (FIG. 1).

3) After fixing the qEV original/70 nm (Izon science, Cat.No: SP1) to the clamp on the experimental clamp stand, remove the PBS solution in the qEV original column and equilibrate 10 mL with 1X PBS (pH7.4) solution by gravity. proceeded. Next, 0.5 mL of the Brassica plant or Allium plant juice solution filtered in the previous step was injected into the qEV original column, and when all the solution passed to the size exclusion chromatography beads, the PBS solution was added. At this time, in order to keep the gravitational force applied to the size exclusion chromatography constant, PBS was continuously injected so as not to exceed 2 mL. A total of 33 tubes were obtained as a result of pouring 500 mL of the descending solution into one Eppendorf tube of 1.5 mL (Figs. 1 to 4).

Size distribution, concentration and purity of brassica plant-derived extracellular vesicles

1) To determine the concentration and purity of Brassica plant-derived extracellular vesicles, a nanoparticle tracking analyzer (NTA) and a Bicinchoninic acid (BCA) assay were used to quantify proteins. First, each of the tubes of the isolated Brassica plant-derived extracellular vesicles was diluted 1:100 with PBS, and then, using NTA, Brassica plant-derived extracellular vesicles (cabbage-derived extracellular vesicles: 'Cabex', red cabbage-derived extracellular vesicles: The concentration and size distribution of 'Rabex') were measured.

Next, the protein concentration of each fraction was quantified through a BCA assay to confirm the protein concentration of the solution subjected to size exclusion chromatography. The concentration dilution of the protein is measured in two ways, the process of taking the solution directly without dilution for the first time (Fig. 4) and the BSA standard curve by measuring a sample diluted with PBS and Brassica plant-derived extracellular vesicles at a weight ratio of 9:1. The concentration of the protein entering the inside was confirmed (FIGS. 5A and 5B). 5A is a graph showing the concentration (number of extracellular vesicles/mL) of extracellular vesicles (hereinafter referred to as Cabax) contained in fractions of cabbage-derived juice extracted through size exclusion chromatography, and FIG. 5B is size exclusion chromatography. It is a graph showing the concentration (number of extracellular vesicle particles/mL) of extracellular vesicles (hereinafter Rabax) contained in the fractions of the extracted red cabbage-derived juice collected through the graph.

Comparison of Yield and Purity of Brassica Plant-Derived Extracellular Vesicles by Isolation and Purification Methods

1) Isolation of extracellular vesicles is currently mainly performed by ultra-high-speed centrifugation and polymer sedimentation. However, in the case of ultra-high-speed centrifugation, strong centrifugal force acts and physical force may cause disruption of extracellular vesicles, and polymer sedimentation is not preferable in terms of purity because it contains a large amount of impurities other than extracellular vesicles.

2) Size exclusion chromatography (SEC), ultra-high-speed centrifugation (UC), and polymer sedimentation method (PEG) use the same amount of cabbage or red cabbage juice (13 mL) to analyze the nanoparticle tracking of the Brassica plant-derived extracellular vesicles. A size distribution map was obtained using the device, and the protein concentration was measured through a BCA assay, and the yield, concentration, and purity were calculated as the amount of extracellular vesicles per mg of protein, and comparison was performed.

In the case of polymer precipitation (PEG) and ultra-high-speed centrifugation (UC), peaks of various sizes were observed, whereas extracellular vesicles separated by size exclusion chromatography (SEC) showed a small number of peaks, resulting in uniform particle size. It can be seen (Figs. 5C to 5H, 5I and 5L).

In addition, in terms of yield, all methods showed similar yields without statistical significance (FIGS. 5J and 5M), but in terms of purity, the case of separation using size exclusion chromatography was significantly higher than other methods (FIGS. 5K and 5M). Figure 5N).

Confirmation of intracellular penetration of Brassica plant-derived extracellular vesicles

In order to analyze the morphology of Brassica plant-derived extracellular vesicles isolated using size exclusion chromatography, they were photographed with a transmission electron microscope (TEM). As a result, the extracellular vesicles of both Cabex and Rabex exhibited a round shape with an average particle size ranging from 50 to 200 nm (FIGS. 6A and 6B).

In addition, as a result of analyzing the net charge of the extracellular vesicles with Zetasizer Nano ZS to evaluate the physical properties, the zeta potential of Cabex was -14.8 mV and the zeta potential of Rabex was -15.2 mV, indicating a significant difference between Cabex and Rabex. There was no significant difference (Fig. 6C).

On the other hand, in order for the Brassica plant-derived extracellular vesicles isolated through the above-described method to act on cells such as humans, a process of permeation into cells is required. Therefore, in order to confirm whether the Brassica plant-derived extracellular vesicles prepared by the method of the present invention also have intracellular permeability characteristics, they were analyzed as follows.

First, 0.5% (v/v) of PKH67 dye for staining Cabex and Rabex was reacted with 1 x 10 ¹¹ particles/mL of Cabex and Rabex at 22° C. for 15 minutes, respectively, and then ultrafiltration through extracellular vesicles. PKH67, which is not attached to , was filtered, and then, PKH67-attached extracellular vesicles (Cabex and Rabex) were used for experiments.

Next, human-derived keratinocytes (HaCaT) were cultured in 4-well chambered coverglass w/non-removable wells using DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin. ) was inoculated at 2.5 x 10 ⁴ cell/cm ² . After 48 hours, the medium of the cells was removed, and then washed three times with phosphate buffer (PBS). In a state where all light is blocked, PKH67 is mixed homogeneously with the extracellular vesicles in DMEM medium containing 10% FBS from which exosomes have been removed, and then the extracellular vesicles derived from Brassica plants (cabbage recovered through size exclusion chromatography) or a fraction containing red cabbage-derived extracellular cell bodies) was added and cultured for 6 hours.

When the incubation was complete, the cells were washed twice with phosphate buffer (PBS), mixed with 2 μM Hoechst 33342 and Opti-MEM, put into wells, and incubated at 37° C. for 15 minutes. After removing all the medium, washing twice with phosphate buffer (PBS), 500 μL of Opti-MEM was added, and the absorption of cabbage or red cabbage-derived extracellular vesicles was observed using a confocal microscope ( 6D to 6F).

In Figures 6D to 6F, blue represents cell nuclei and green represents cabbage-derived extracellular vesicles (Figure 6D as control; Figure 6E as Cabex; Figure 6F as Rabex). As shown in FIGS. 6D to 6F , cabbage or red cabbage-derived extracellular vesicles penetrate into cells and are located in the cytoplasm. Through this, it was confirmed that when the cabbage or red cabbage-derived extracellular vesicles of the present invention are administered, they can be easily penetrated and absorbed into cells.

variety Analysis of cell proliferation and cytotoxicity in cells (HaCaT, HDF, RAW 264.7 cell)

Human keratinocytes (HaCaT), normal fibroblasts (HDF) and macrophages (RAW264 .7) was inoculated at 1 × 10 ⁴ cell/cm ² respectively. After 24 hours, the medium was removed from each cell, washed with PBS, and an experimental group and a control group were prepared as follows.

In the experimental group, cabbage plant-derived extracellular vesicles (Cabex) or red cabbage plant-derived extracellular vesicles (Rabex) were added and cultured at a concentration of 1 x 10 ¹¹ particles/mL. cultured using it.

Cell proliferation was analyzed using the WST-1 assay (EZ-Cytox kit (Dogen Bio, Korea)), and cytotoxicity assay was performed using the trypan blue exclusion assay. All values were expressed as mean±standard deviation (*p<0.1, **p<0.01 and ***p<0.001; n = 4) (FIG. 7).

As shown in Figure 7, when cabbage-derived extracellular vesicles (Cabex) or red cabbage-derived extracellular vesicles (Rabex) were used as culture medium additives for HaCaT, HDF, and RAW264.7 cells, respectively, the addition of Cabex or Rabex Inhibition of cell proliferation and cytotoxicity were not observed.

Anti-inflammatory effect of brassica or allium-derived extracellular vesicles

1) qRT-PCR analysis

The anti-inflammatory activity of Cabex, Rabex or Onex (onion-derived extracellular vesicles) was evaluated by their effect on the expression of pro-inflammatory mediator COX-2 and pro-inflammatory cytokines (IL-1β and IL-6).

To this end, RAW264.7 cells were seeded at 5×10 ⁴ cell/cm ² in a 6-well cell culture plate using DMEM supplemented with 10% FBS, 1% penicillin and streptomycin. After 24 hours, the medium was removed from each cell, washed with PBS, and an experimental group and a control group were prepared as follows.

In the experimental group, Cabex, Rabex, or Onex (onion-derived extracellular vesicles) was added and cultured at a concentration of 1 x 10 ¹¹ particles/mL, and in the control group (CTRL), a general culture medium was used without addition of extracellular vesicles. Subsequently, inflammation was induced in the cells by treatment with 0.1 μg/ml of LPS and cultured for 24 hours. Then, the cells were washed twice with nuclease-free PBS, total RNA was isolated using RNeasy Mini Kit (Qiagen, Germany) according to the manufacturer's manual, cDNA was synthesized from the isolated total RNA, and the synthesized cDNA was used as a template Expression of pro-inflammatory mediators and cytokines was measured by qRT-PCR using SYBR green master mixture (THUNDERBIRD SYBR qPCR mix; Toyobo, Japan) and the primers shown in Table 1 below. GAPDH was used as a loading control.

primer base sequence Murine GAPDH Forward 5'-CGGGGACCTGACTGACTACC-3' SEQ ID NO: 1 Murine GAPDH Reverse 5' - AGGAAGGCTGGAAGAGTGC - 3' SEQ ID NO: 2 Murine IL-6 Forward 5'-GACAAAGCCAGAGTCCTTCAGAGA-3' SEQ ID NO: 3 Murine IL-6 Reverse 5'-CACTAGGTTTGCCGAGTAGATCTC-3' SEQ ID NO: 4 Murine IL-1β Forward 5'-CCAGCTTCAAATCTCACAGCAG-3' SEQ ID NO: 5 Murine IL-1β Reverse 5'-CTTCTTTGGGTATTGCTTGGGATC-3' SEQ ID NO: 6 Murine COX-2 Forward 5'-GAAGTCTTTGGTCTGGTGCCTG-3' SEQ ID NO: 7 Murine COX-2 Reverse 5′-GTCTGCTGGTTTGGAATAGTTGC-3′ SEQ ID NO: 8

8 is a graph showing the results. Cabbage-derived extracellular vesicles (Cabex), red cabbage-derived extracellular vesicles (Rabex), and onion-derived extracellular vesicles (Onex) are pro-inflammatory mediators increased by LPS stimulation. It showed a remarkable inhibitory effect on COX-2 and pro-inflammatory cytokines IL-1β and IL-6.

2) ELISA - Evaluation of IL-1β and IL-6 expression inhibition efficacy

ELISA kits were used to determine the effect of Cabex, Rabex or Onex on the expression of pro-inflammatory cytokines in cells.

To this end, RAW264.7 cells were seeded at 5×10 ⁴ cell/cm ² in a 6-well cell culture plate using DMEM supplemented with 10% FBS, 1% penicillin and streptomycin. After 24 hours, the medium was removed from each cell, washed with PBS, and an experimental group and a control group were prepared as follows.

The experimental group was cultured with Cabex, Rabex or Onex at a concentration of 1 x 10 ¹¹ particles/mL, and the control group (CTRL) was cultured using a general culture medium without addition of extracellular vesicles. Subsequently, inflammation was induced in the cells by treatment with 0.1 μg/ml of LPS and cultured for 24 hours. Then, the expression level of pro-inflammatory cytokines in the supernatant of each well was quantified using the mouse IL-6 DuoSet ELISA kit (R&D) (FIG. 9).

9A and 9B are graphs showing the results. Cabex, Rabex, and Onex showed significant inhibitory effects on IL-1β and IL-6, which are pro-inflammatory cytokines, which were increased by LPS stimulation.

3) Western blot analysis - Evaluation of COX-2 expression inhibition efficacy

Western blot analysis was performed to confirm the effect of Cabex and Rabex on the expression of COX-2, a proinflammatory mediator of cells.

To this end, RAW264.7 cells were inoculated at 5×10 ⁴ cell/cm ² and cultured using DMEM supplemented with 10% FBS, 1% penicillin and streptomycin in a 6-well cell culture plate. After 24 hours, the medium was removed from each cell, washed with PBS, and an experimental group and a control group were prepared as follows.

The experimental group was cultured with Cabex and Rabex at a concentration of 1 x 10 ¹¹ particles/mL, and the control group (CTRL) was cultured using a general culture medium without the addition of extracellular vesicles. Subsequently, inflammation was induced in the cells by treatment with 0.1 μg/ml of LPS and cultured for 24 hours. Then, the expression level of COX-2, a pro-inflammatory mediator, in the supernatant of each well was confirmed by Western blot analysis, which was quantified using the ChemiDoc XRS + System (Bio-Rad, USA) (FIG. 9).

9C and 9D are graphs showing the results. Cabex and Rabex showed significant inhibitory effects on COX-2, a pro-inflammatory mediator increased by LPS stimulation.

conclusion

Inflammation is the body's immune response system and plays an essential role in eliminating pathogens from the outside and maintaining tissue homeostasis. However, persistent chronic inflammation, such as fever and headaches, may occur and may progress to pneumonia. Therefore, although a technology for suppressing chronic inflammation is required in various inflammatory diseases, safety and efficiency problems of additive substances are emerging.

The extracellular vesicles derived from plants of the genus Brassica or Allium genus of the present invention are purified with high purity and high efficiency through size exclusion chromatography so that they can be treated in the human body, and penetrate well into cells. In addition, the extracellular vesicles delivered into cells have been shown to effectively suppress both the expression of COX-2, a proinflammatory mediator, and IL-1β and IL-6, which are proinflammatory cytokines, which are the main cause of inflammation, improving inflammatory diseases, It was found that it can be usefully used as a material for treatment or prevention.

Therefore, since the extracellular vesicles derived from plants of the genus Brassica or Allium genus of the present invention can be administered orally and can be mass-produced from plants, they can be applied to the production of health functional foods or cosmetics. In addition, it can be loaded with pharmacological components, so it can be used as a drug delivery system in the treatment of the above-mentioned diseases.

Although the present invention has been described in terms of the preferred embodiments mentioned above, various modifications and variations are possible without departing from the spirit and scope of the invention. The appended claims also cover such modifications and variations as fall within the subject matter of this invention.

<110> INCHEON UNIVERSITY INDUSTRY ACADEMIC COOPERATION FOUNDATION <120> Composition for Preventing, Improving or Treating Inflammatory Diseases Comprising Brassica spp. or Allium spp.-Derived Extracellular Vesicles as Active Ingredients <130> HP9548 <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> artificial sequence <220> <223> Murine GAPDH Forward <400> 1 cggggacctg actgactacc 20 <210> 2 <211> 19 <212> DNA <213> artificial sequence <220> <223> Murine GAPDH Reverse <400> 2 agggaaggctg gaagagtgc 19 <210> 3 <211> 24 <212> DNA <213> artificial sequence <220> <223> Murine IL-6 Forward <400> 3 gacaaagcca gagccttca gaga 24 <210> 4 <211> 24 <212> DNA <213> artificial sequence <220> <223> Murine IL-6 Reverse <400> 4 cactaggttt gccgagtaga tctc 24 <210> 5 <211> 22 <212> DNA <213> artificial sequence <220> <223> Murine IL-1B Forward <400> 5 ccagcttcaa atctcacagc ag 22 <210> 6 <211> 24 <212> DNA <213> artificial sequence <220> <223> Murine IL-1B Reverse <400> 6 cttctttggg tattgcttgg gatc 24 <210> 7 <211> 22 <212> DNA <213> artificial sequence <220> <223> Murine COX-2 Forward <400> 7 gaagtctttg gtctggtgcc tg 22 <210> 8 <211> 23 <212> DNA <213> artificial sequence <220> <223> Murine COX-2 Reverse <400> 8 gtctgctggt ttggaatagt tgc 23

Claims (15)

A pharmaceutical composition for the prevention or treatment of inflammatory diseases comprising, as an active ingredient, extracellular vesicles derived from cabbage, red cabbage or onion. According to claim 1,
The extracellular vesicles are characterized in that the average diameter of 50 to 150 nm, a pharmaceutical composition for the prevention or treatment of inflammatory diseases. According to claim 1,
The extracellular vesicles are characterized in that separated by size exclusion chromatography (size exclusion chromatography), a pharmaceutical composition for preventing or treating inflammatory diseases. According to claim 1,
The extracellular vesicles are characterized by inhibiting the expression of any one selected from interleukin-6 (IL-6), interleukin-1 beta (IL-1β) and cyclooxygenase-2 (COX-2) of inflammatory diseases. A pharmaceutical composition for prevention or treatment. According to claim 1,
The inflammatory diseases include dermatitis, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, iritis, sore throat, tonsillitis, pneumonia, gastric ulcer, pancreatitis, gastritis, Crohn's disease, inflammatory bowel disease, colitis, hemorrhoids, gout, ankylosing spondylitis, lupus, fibromyalgia ( fibromyalgia), psoriasis, rheumatoid arthritis, osteoarthritis, osteoporosis, hepatitis, cystitis, nephritis, Sjögren's syndrome, and multiple sclerosis, characterized in that any one selected from, a pharmaceutical composition for preventing or treating inflammatory diseases. According to claim 1,
The inflammatory disease is characterized in that any one inflammatory skin disease selected from acne, contact dermatitis, seborrheic dermatitis, atopic dermatitis and psoriasis, a pharmaceutical composition for preventing or treating inflammatory diseases. A food composition for preventing or improving inflammatory diseases, comprising, as an active ingredient, extracellular vesicles derived from cabbage, red cabbage or onion. Cabbage, red cabbage or onion-derived extracellular vesicles (extracellular vesicles) as an active ingredient, a cosmetic composition for preventing or improving inflammatory diseases. A method for producing the extracellular vesicles according to claim 1,
(1) preparing a juice solution of cabbage, red cabbage or onion;
(2) performing ultrafiltration on the juice; and
(3) performing size exclusion chromatography on the filtrate; method for producing extracellular vesicles including. According to claim 9,
The ultrafiltration in step (2) is characterized in that it has a cut-off value of 5 to 15 kDa, a method for producing extracellular vesicles. According to claim 9,
The size exclusion chromatography in step (3) is characterized by equilibrating with a phosphate buffer (PBS) of pH 6 to pH 8, a method for producing extracellular vesicles. According to claim 9,
The method for producing extracellular vesicles, characterized in that the size exclusion chromatography in step (3) has an average pore diameter of 30-75 nm in the stationary phase. According to claim 12,
The method of producing extracellular vesicles, characterized in that the stationary phase is any one gel filtration medium selected from Superdex, Sephacryl, Sephadex, Sepharose, Fractogel, Toyopearl and Bio-Gel. According to claim 9,
The eluent of the size exclusion chromatography is a PBS buffer solution of pH 6 to pH 8, characterized in that, the method for producing extracellular vesicles. According to claim 9,
Particle size analysis is performed on each fraction obtained from the size exclusion chromatography, and a fraction having an average diameter of 50 to 150 nm is selected and recovered.

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