KR102527607B1 - Plant Derived Extracellular Vesicles, Composition Comprising Thereof, and Method of Producing Thereof - Google Patents

Abstract

The present invention relates to plant-derived extracellular vesicles, a composition containing the same, and a manufacturing method thereof, and more specifically, to a method for producing stable extracellular vesicles from plants in a high yield and a composition containing the extracellular vesicles as an active ingredient.

Description

Plant-derived extracellular vesicles, compositions containing them, and methods for producing them

The present invention relates to a plant-derived extracellular vesicle, a composition containing the same, and a method for producing the same, and more particularly, to a method for producing stable extracellular vesicles from plants in high yield and containing the extracellular vesicles as an active ingredient It's about the composition.

Extracellular Vesicles (EVs) is a general term for vesicles (vesicles) having a double-phospholipid membrane structure secreted from cells of an organism. The size of extracellular vesicles is from 20 to 30 nm to 1 μm or less based on particle size, and exosomes, which are representative extracellular vesicles, are known to have a particle size of 50 to 250 nm. Extracellular vesicles can be classified into types such as exosomes, ectosomes, and microvesicles according to their size and how they are produced.

In early studies in the field of extracellular vesicle-related technology, it was recognized that extracellular vesicles only had a role in discharging unnecessary substances (excrement) from cells, but in recent studies, various types of proteins, lipids, and nucleic acids such as proteins, lipids, and nucleic acids were It contains physiologically active substances and has begun to attract attention as the roles of signal messengers, reaction mediators, and immunoreactants of extracellular vesicles have been revealed. In addition to these various roles, extracellular vesicles are emerging as next-generation drug delivery systems due to their double-phospholipid membrane structure, easy intracellular penetration, and bio-friendly characteristics. In particular, in the pharmaceutical/bio field, exosomes have been extensively studied with a focus on targeting drug delivery, and the range of application to cosmetics, health foods, and drugs is expanding.

Extracellular vesicles are secreted from animal cells, plant cells, and microbial cells. In particular, it has been reported that extracellular vesicles derived from plant cells are less cytotoxic than extracellular vesicles derived from animal cells. It is also price competitive in terms of supplying raw materials for creation/manufacturing.

However, since plant cells have a cell wall unlike animal cells, a process of crushing/crushing the cell wall is required, and a conventional method for obtaining extracellular vesicles through plant juice, etc. is known (see Patent Documents 1-2). ), because the yield of extracellular vesicles is low, a new method for improving the yield is required.

In addition, when extracting extracellular vesicles from organisms, it is difficult to separate and purify them due to their small size and low density. Typically, ultracentrifugation, biphasic aqueous solution, polymer precipitation, electrophoresis, size exclusion chromatography, and specific antibodies are used. When obtaining extracellular vesicles from organisms through, there was a problem of low efficiency in terms of process complexity and cost.

In the manufacture of cosmetics, health food, and medicines, chemical raw materials have problems such as causing side effects such as skin allergy or skin trouble. As the use of Korean food is rapidly decreasing, eco-friendly raw materials such as plants are in the spotlight.

As an example of a vegetable raw material, centella asiatica has long been used as a medicine in India, Africa, China, etc., and is a plant called tiger grass because a tiger rolled over the centella to heal wounds. Madecassic acid contained in the leaves and stems of Centella Asiatica has excellent anti-inflammatory and wound healing effects, and is applied to various product groups such as pharmaceutical ointments and cosmetic raw materials.

Under this technical background, the present inventors have made diligent efforts to produce stable plant-derived extracellular vesicles in high yield. As a result, in one embodiment of the present invention, centella asiatica is pretreated, and then cell walls of centella asiatica are disrupted in the presence of a sequestering agent. Cells obtained after filtering the centella asiatica extract obtained through isolating/obtaining crude extracellular vesicles, purifying and concentrating the crude extracellular vesicles through tangential flow filtration (TFF). It was found that a high yield of stable Centella asiatica-derived extracellular vesicles could be obtained by stabilizing the extracellular vesicles by adding sugars to the exoendoplasmic reticulum, as well as confirming the physiological synergistic effect of the active ingredients contained in the extracellular endoplasmic reticulum. , which led to the completion of the present invention.

Republic of Korea Patent No. 10-1897451 (2018.09.05) Republic of Korea Patent Registration No. 10-2207364 (2021.01.20)

An object of the present invention is to provide a method for producing a stable plant-derived extracellular vesicle in high yield, an extracellular vesicle prepared by the above method, and a composition comprising the same.

In order to achieve the above object, the present invention is a method for producing a stable plant-derived extracellular vesicle,

(a) obtaining a plant extract through cell wall disruption of a plant, preferably a pretreated plant;

(b) separating crude extracellular vesicles by filtering the plant extract;

(c) obtaining extracellular vesicles by purifying and concentrating the crude extracellular vesicles by tangential flow filtration (TFF); and

(d) provides a method for producing stable plant-derived extracellular vesicles, comprising the step of stabilizing the extracellular vesicles by adding sugars.

The present invention also provides an extracellular vesicle prepared by the above production method, and a composition comprising the same.

The method for producing plant-derived extracellular vesicles according to the present invention stably manufactures extracellular vesicles, which can be very usefully applied as active ingredients or functional materials for cosmetics, health functional foods, drugs, etc., with increased physiological activity and in high yield can do.

Figure 1 shows the result of confirming the Centella asiatica-derived extracellular vesicles according to Example 2 of the present invention with a transmission electron microscope as a photograph.
2 and 3 are graphs showing the number of extracellular vesicles per unit volume measured by nanoparticle tracking analysis (NTA) for Centella asiatica-derived extracellular vesicles according to Examples 1 and 2 of the present invention. will be.
4 and 5 are Examples 1 and 2 of the present invention, Comparative Example 1-1, Comparative Example 1-2, Comparative Example 1-3, Comparative Example 2-1, Comparative Example 2-2 and Comparative Example The average particle size of the main peak and the polydispersity index (PDI, polydispersity index).
6 to 13 are Examples 1 and 2 of the present invention, Comparative Example 1-1, Comparative Example 1-2, Comparative Example 1-3, Comparative Example 2-1, Comparative Example 2-2 and Comparative Example The distribution by particle size for each extracellular vesicle of 2-3 was measured on the day of obtaining the extracellular vesicles (day 0) and on the 10th day after obtaining them by dynamic light scattering, and is shown in a particle size graph.
14 and 15 are graphs showing the results of confirming the signal transduction enhancement effect of the extracellular vesicles according to Example 2 of the present invention as NO production inhibitory ability.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is one well known and commonly used in the art.

Hereinafter, the present invention will be described in more detail.

The term "about" used herein to express length, area, volume, time (duration), concentration, content, temperature, number, percent (%), multiple, etc., indicates a value or range of values up to 10%. This means that an error exists.

In one aspect, the present invention relates to a method for producing a stable plant-derived extracellular vesicle, the method comprising:

(a) obtaining a plant extract through cell wall disruption of a plant, preferably a pretreated plant;

(b) separating crude extracellular vesicles by filtering the plant extract;

(c) obtaining extracellular vesicles by purifying and concentrating the crude extracellular vesicles by tangential flow filtration (TFF); and

(d) stabilizing the extracellular vesicles by adding sugars; characterized in that they include.

In another aspect, the present invention relates to the extracellular vesicles, and a composition comprising them as an active ingredient.

In the present invention, the plant may be a natural plant or a cultivated plant (agricultural product), specifically fruits, vegetables (leaf vegetables, root vegetables), seeds, nuts, flowers, etc., preferably vegetables or flowers, more preferably centipede However, it is not limited thereto and may be any kind of industrially usable plant.

The plant used in the present invention is not limited in its acquisition method, and can be grown and used or purchased commercially. A sterilized plant may be used as the plant, and the sterilization method may be performed by a conventional method in the art.

Plant parts used in the production of the extracellular endoplasmic reticulum may be flowers, fruits, seeds, leaves, stems, roots or sheaths, but are not limited thereto.

In the present invention, the pretreatment is to improve the yield and particle size stability of extracellular endoplasmic reticulum from plants, and the pretreatment may be drying, cryogenic freezing/thawing, wrapping treatment, etc., but is not limited thereto.

The drying may be hot air drying, vacuum drying, freeze drying, etc., and the hot air drying or vacuum drying may be performed at a temperature of preferably 32 to 60 ° C, more preferably 40 to 50 ° C, and the freeze drying is - It may be made at a temperature of 60 ℃ or less, but is not limited thereto.

The moisture content of the plant during the drying is preferably 0.1% by weight or less, but is not limited thereto.

The cryogenic freezing/thawing may be repeated several times, preferably 1 to 10 times, more preferably 1 to 5 times, and most preferably 1 to 3 times at cryogenic temperatures. The freezing temperature may be cryogenic (eg, -210 to -196°C) or -40°C or lower, preferably -196 to -40°C, and most preferably -80 to -60°C; The temperature at the time of thawing may be 2 to 25 ° C, preferably refrigerated temperature (eg, 2 to 8 ° C) or room temperature (eg, 15 to 25 ° C), most preferably 4 to 7 ° C. The freezing or thawing time may be 5 seconds to 1 hour, preferably 10 seconds to 30 minutes, more preferably 30 seconds to 15 minutes, and most preferably 1 to 5 minutes.

The foaming agent treatment may be achieved by applying a liquid supplement to the plant. The liquid supplement may be any one or more of salt water, vinegar, alcohol, honey water and plant juice, but is not limited thereto.

The foam treatment is characterized in that the liquid fertilizer is brought into contact with the plant, and specifically, the foam agent treatment is performed at high or low temperature after mixing the plant and the liquid fertilizer, immersing the plant in the liquid fertilizer, or spraying the liquid fertilizer on the plant. It may be made through drying or the like. The drying may be the aforementioned hot air drying, vacuum drying, etc., but is not limited thereto.

In the present invention, the plant extract is obtained by crushing the cell wall of the plant by crushing the plant by minimizing the outflow of water contained in the plant by applying a blender, pulverizer, such as an ultrafine particle grinder, and the final product is the cell In order to more efficiently or stably obtain the exoendoplasmic reticulum, an aqueous sequestering agent solution and/or a buffer solution (PBS [phosphate buffered saline], TBS [Tris buffered saline], HBS [4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid-buffered saline], etc.), preferably an aqueous solution of a sequestering agent may be added during the crushing, but is not limited thereto.

The size of the lysate contained in the plant extract may be preferably 1 μm to 10,000 μm, more preferably 10 μm to 1,000 μm, based on the average diameter, and when the size of the lysate exceeds 10,000 μm, crushing is appropriate. On the other hand, the content of the final obtained extracellular vesicles may be lowered because it is not done, when the size of the lysate is 1,000 μm or less, the content of the finally obtained extracellular vesicles may be increased.

Minimization of the outflow of water contained in the plant when the cell wall of the pretreated plant is disrupted reduces the action of a hydrolase that requires water that may exist inside and outside the plant, or reduces contamination by microorganisms, thereby reducing cell release from plant cells. It can be characterized by maintaining the ectoplasm in a stable state.

The sequestering agent aqueous solution may enhance the effect of cell wall disruption, but is not limited to this effect, and the sequestering agent is a polyphenol contained in plants when the plant is crushed, such as iron trivalent ion (Fe ³⁺ ) to prevent discoloration.

The sequestering agent is ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (Ethylene glycol-bis (β-aminoethyl ether) -N, N, N', N'-tetraacetic acid; EGTA), di Ethylenetriaminepentaacetic acid (DTPA), Hexamethylenetetramine (HMT), Nitrilotriacetic acid (NTA), Diethylenetriaminepentaacetic acid (DTPA), Hydroxyethyl-ethylene Diaminetriacetic acid (HEDTA), 2,3-Dimercapto-1-propanesulfonic acid (DMPS), dimercaptosuccinic acid (DMSA) and phenolic acid It may be one or more selected from the group consisting of silamine (Penicillamine), but is not limited thereto. When a plant is treated with an aqueous solution using the above metal ion sequestering agent alone or in combination of two or more, it is possible to break down the cell wall more easily by blocking metal ions essential for maintaining the skeleton of the plant cell wall, thereby more easily breaking down the plant cell wall. It can be crushed by physical means (such as a blender).

When adding the sequestering agent aqueous solution to the plant, the weight ratio of the plant and the sequestering agent aqueous solution may be 1 to 20: 1 to 100, considering the volume of the plant,

When the plant is a live plant, the weight ratio of the live plant and the sequestering agent aqueous solution is 1 to 15: 1 to 15, preferably 1 to 6: 1 to 12, more preferably 1 to 3: 5 ~ 12, most preferably 1: 8 ~ 12,

When the plant is a dried plant, the weight ratio of the dried plant and the sequestering agent solution is 1-5: 10-100, preferably 1-4: 20-100, more preferably 1-4: 50-100, Most preferably, it may be 2-4: 96-98.

The sequestering agent concentration of the sequestering agent aqueous solution is 0.01 to 50 mg/ml, preferably 0.1 to 10 mg/ml, and more preferably 0.5 to 5 mg/ml, and distilled water or a buffer solution (PBS, TBS, HBS, etc.) may be used as a solvent.

In the present invention, the extracellular vesicles are the supernatant obtained by centrifuging the filtrate obtained by filtering the plant extract with a mesh having a pore size of 30 to 100 μm, preferably 30 to 50 μm. (supernatant) was obtained, and then the final filtrate (crude cells) obtained by further filtration with an absolute filter having a pore size of 0.1 to 8.0 μm, preferably 0.2 to 5.0 μm, and most preferably 0.2 μm. It can be characterized in that it is produced in the form of a concentrate by purifying and concentrating the endoplasmic reticulum) by tangential flow filtration (TFF).

The centrifugation may be characterized by centrifugation using a centrifuge at 3,800 to 10,000 rpm or 1,500 to 15,000 x g or more, preferably 8,000 rpm or 5,000 x g or more.

The absolute filter can increase the number and content of extracellular vesicles having a particle size of 20 to 300 nm, preferably 50 to 250 nm, by removing extracellular vesicles having a particle size exceeding 300 nm and other impurities in the plant extract. .

The tangential flow filtration is performed by using a filter having a molecular weight cut-off (MWCO) of 1KDa, 3KDa, 5KDa, 10KDa, 30KDa, 50KDa or 100kDa, preferably 100kDa, so that the cells contained in the final filtrate Components outside the endoplasmic reticulum, preferably 1 KDa or less, 3 KDa or less, 5 KDa or less, 10 KDa or less, 30 KDa or less, 50 KDa or less, or 100 kDa or less, more preferably 100 kDa or less, are removed (cut-off of 100 kDa or less) 20 ~ 300nm contained in the final filtrate, preferably by increasing the purity, number and content of extracellular vesicles having a particle size of 50 ~ 250nm can be obtained a concentrate with a high yield of extracellular vesicles.

In the present invention, the extracellular vesicles obtained in step (c) may be characterized in that they have 10 ⁸ or more extracellular vesicles based on volume, preferably per 1ml.

In the present invention, the extracellular vesicles are purified water, a buffer solution (PBS, TBS, HBS, etc.), preferably purified water, in order to remove low molecular weight substances and / or sequestering agents that may remain inside and outside the extracellular vesicles. It can be characterized in that it has been washed (purified) once or more times, preferably 1 to 3 times.

In the present invention, in order to improve the stability of the extracellular vesicles, saccharides, preferably aqueous saccharides, are added to the extracellular vesicles to obtain final products, or the liquid components inside and outside the extracellular vesicles are added to the same weight of the saccharide aqueous solution. substitution to obtain the final product.

The saccharide is at least one selected from the group consisting of polysaccharides, oligosaccharides, trisaccharides, disaccharides and monosaccharides, preferably disaccharides, more preferably trehalose, lactulose, cellobiose, isomaltose, turanose, sucrose, maltose or lactose, most preferred Preferably, it may be trehalose.

A disaccharide, preferably trehalose, which is mainly used as a protein stabilizer in the food and drug industry, inhibits the aggregation reaction between the extracellular vesicles so that the average particle size of the extracellular vesicles is uniformly 20 to 300 nm, preferably 50 to 300 nm. It can be maintained to have a particle size of 250 nm.

The saccharide concentration of the saccharide aqueous solution is 0.01 to 50 mg/ml, preferably 0.1 to 10 mg/ml, and more preferably 0.1 to 1 mg/ml, and distilled water or a buffer solution (PBS, TBS, HBS, etc.) is used when preparing the aqueous solution. It can be characterized in that it is used as a solvent.

In the present invention, the composition may be characterized in that it contains 10 ⁸ or more per 1ml based on the volume of the extracellular vesicles of the composition. When the composition contains less than 10 ⁸ plant-derived extracellular vesicles per 1ml of the composition, the physiological efficacy of the extracellular vesicles, such as intercellular signaling pathways, reaction intermediates, and immune response bodies, may not be sufficiently exhibited. there is.

The extracellular vesicles included as active ingredients of the composition are natural substances derived from plants and have little toxicity, so they can be continuously used in appropriate amounts as cosmetics, foods, or pharmaceuticals.

In the present invention, the composition comprises a plant-derived extract, preferably a plant-specific extract that is a plant-derived partial extract (fractional extract), more preferably a plant-quantitative extract having physiological activity (eg, centella asiatica extract, etc.) can do. The effect (activity / expression) of the plant extract included in the composition is enhanced in the target (body's biological system (circulatory system, etc.), organs, tissues, cells, etc.) /Can be promoted, but is not limited thereto.

The composition may include a cell activity regulator, and the cell activity regulator may be a substance useful for regulating cell activity, which is involved in controlling skin moisturizing power, regulating immunity, regulating inflammation, and regulating elasticity.

The extracellular vesicles included in the composition may be characterized in that they enhance signal transduction efficacy by serving to transmit the cell activity regulator between cells as an intercellular signal transducer/reactive intermediate. For example, the extracellular endoplasmic reticulum can increase/promote the activity/expression of the cell activity regulator in the recipient cell by transferring the cell activity regulator from the donor cell to the recipient cell.

The composition can be commercialized in various formulations (cosmetics, health functional foods, drugs, etc.), and can be appropriately formulated in consideration of functionality, cost, and other conditions of products to be implemented.

The composition may be a cosmetic composition, for example, basic cosmetics, makeup cosmetics, hair cosmetics, body cosmetics, etc., and the formulation is not particularly limited and may be appropriately selected according to the purpose.

The composition is a health functional food composition, for example, health supplements, and may be in the form of powder, granule, tablet, capsule, beverage, etc., but is not limited thereto.

The composition may be a pharmaceutical composition, for example, a formulation such as a tablet, ointment, lotion, gel, cream, spray, suspension, emulsion, patch, etc., but is not limited thereto.

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

[Raw materials and reagents]

Used for preparing each sample of Examples 1 to 3, Reference Example 1 to Reference Example 3, Comparative Example 1-1 to Comparative Example 1-3 and Comparative Example 2-1 to Comparative Example 2-3 of the present invention described below The raw material was Centella asiatica (outpost), which was obtained from a retailer, and the reagents used for preparing the samples and the reagents used in Experimental Examples 1 to 4 were Sigma-Aldrich's products if the manufacturer/purchaser was not indicated.

[Reference Example 1] Separation process of raw Centella asiatica-derived crude extracellular vesicles

After mixing raw Centella Asiatica with an EDTA aqueous solution (EDTA concentration: 0.5 mg/ml) at a weight ratio of 1:10, it was treated with an ultra-fine particle grinder to break the cell walls of Centella asiatica to obtain an extract. After filtering the extract through a mesh having a pore size of 30 to 50 μm, the precipitate formed when the filtrate was centrifuged at 8,000 rpm or 5,000 x g was removed, and the supernatant was filtered through a filter having a pore size of 0.2 μm. The obtained filtrate (crude extracellular vesicles) was obtained as the final product.

[Reference Example 2] Isolation process of crude extracellular vesicles derived from dried Centella asiatica pretreated at low temperature

Freeze-dried Centella asiatica dried fresh Centella asiatica at a temperature of about -70 ° C (water content of 0.1% by weight or less based on the total weight of the outpost) was 2:98 by reflecting the dry yield of EDTA aqueous solution (EDTA concentration: 0.5 mg / ml) After mixing in a ratio, the extract was obtained by treating with an ultra-fine particle grinder so that the cell walls of centella asiatica were crushed. After the extract was filtered through a mesh having a pore size of 30 to 50 μm, the precipitate generated when the filtrate was centrifuged at 8,000 rpm or 5,000 x g was removed, and the supernatant was filtered through a filter having a pore size of 0.2 μm. A filtrate (crude extracellular vesicles) was obtained as the final product.

[Reference Example 3] Separation process of crude extracellular vesicles derived from dried Centella asiatica pretreated at high temperature

The hot air-dried centella asiatica dried by hot air at a temperature of about 45 ° C (moisture content of 0.1% by weight or less based on the total weight of the outpost) was 2:98 by reflecting the dry yield with an EDTA aqueous solution (EDTA concentration: 0.5 mg / ml) After mixing in a ratio, the extract was obtained by treating with an ultra-fine particle grinder so that the cell walls of centella asiatica were crushed. After the extract was filtered through a mesh having a pore size of 30 to 50 μm, the precipitate generated when the filtrate was centrifuged at 8,000 rpm or 5,000 x g was removed, and the supernatant was filtered through a filter having a pore size of 0.2 μm. A filtrate (crude extracellular vesicles) was obtained as the final product.

[Example 1] Purification and stabilization process of fresh Centella asiatica-derived extracellular vesicles

Fresh centella asiatica and EDTA aqueous solution (EDTA concentration: 0.5 mg/ml) were mixed at a weight ratio of 1:10, and then treated with an ultra-fine particle grinder to break the cell walls of centella asiatica to obtain an extract. After filtering the extract through a mesh having a pore size of 30 to 50 μm, the precipitate formed when the filtrate was centrifuged at 8,000 rpm or 5,000 x g was removed, and the supernatant was filtered through a filter having a pore size of 0.2 μm for final filtration. A liquid (crude extracellular vesicles) was obtained. Then, when the final filtrate was tangentially filtered, a 100 kDa filter was used to remove components having a molecular weight of 100 kDa or less (cut-off of 100 kDa or less), and the concentrate obtained was washed (purified) twice with purified water. After (based on weight ratio, concentrate: purified water = 1:5), the liquid component of the concentrate is added to the washed (purified) concentrate (extracellular vesicles) so that sugars are added to an aqueous solution of trehalose (Trehalose) of the same weight ( Concentration of trehalose dissolved in purified water: 0.5 mg/ml) was used to obtain a final product stabilizing the extracellular vesicles.

[Example 2] Purification and stabilization process of extracellular vesicles derived from dried Centella asiatica pretreated at high temperature

Centella asiatica dried by hot air at a temperature of about 45 ℃ (based on the total weight of the whole plant, water content of 0.1% by weight or less) and EDTA aqueous solution (EDTA concentration: 0.5mg / ml) were mixed at a ratio of 2:98 to reflect the dry yield An extract was obtained by treating the centella asiatica with an ultra-fine particle grinder so that the cell walls were crushed. After filtering the extract through a mesh having a pore size of 30 to 50 μm, the precipitate formed when the filtrate was centrifuged at 8,000 rpm or 5,000 x g was removed, and the supernatant was filtered through a filter having a pore size of 0.2 μm for final filtration. A liquid (crude extracellular vesicles) was obtained. Then, when the final filtrate was tangentially filtered, a 100 kDa filter was used to remove components having a molecular weight of 100 kDa or less (cut-off of 100 kDa or less), and the concentrate obtained was washed (purified) twice with purified water (based on weight ratio, Concentrate: purified water = 1:5), the liquid component of the concentrate to add sugars to the washed (purified) concentrate (extracellular vesicles) in the same weight of trehalose aqueous solution (trehalose dissolved in purified water) concentration: 0.5mg/ml) to obtain a final product stabilizing the extracellular vesicles.

[Example 3] Purification and stabilization process of dried Centella asiatica-derived extracellular vesicles pretreated at low temperature

Centella asiatica dried at a temperature of about -70 ℃ (based on the total weight of the whole plant, water content of 0.1% by weight or less) and EDTA aqueous solution (EDTA concentration: 0.5mg / ml) were mixed at a ratio of 2:98 to reflect the dry yield An extract was obtained by treating the centella asiatica with an ultra-fine particle grinder so that the cell walls were crushed. After filtering the extract through a mesh having a pore size of 30 to 50 μm, the precipitate formed when the filtrate was centrifuged at 8,000 rpm or 5,000 x g was removed, and the supernatant was filtered through a filter having a pore size of 0.2 μm for final filtration. A liquid (crude extracellular vesicles) was obtained. Then, when the final filtrate was tangentially filtered, a 100 kDa filter was used to remove components having a molecular weight of 100 kDa or less (cut-off of 100 kDa or less), and the concentrate obtained was washed (purified) twice with purified water (based on weight ratio, Concentrate: purified water = 1:5), the liquid component of the concentrate to add sugars to the washed (purified) concentrate (extracellular vesicles) in the same weight of trehalose aqueous solution (trehalose dissolved in purified water) concentration: 0.5mg/ml) to obtain a final product stabilizing the extracellular vesicles.

[Comparative Example 1-1] Separation step of raw Centella asiatica-derived crude extracellular vesicles

Fresh centella asiatica and EDTA aqueous solution (EDTA concentration: 0.5 mg/ml) were mixed at a weight ratio of 1:10, and then treated with an ultra-fine particle grinder to break the cell walls of centella asiatica to obtain an extract. After the extract was filtered through a mesh having a pore size of 30 to 50 μm, the precipitate generated when the filtrate was centrifuged at 8,000 rpm or 5,000 x g was removed, and the supernatant was filtered through a filter having a pore size of 0.2 μm. A filtrate (crude extracellular vesicles) was obtained as the final product.

[Comparative Example 1-2] Isolation and Purification Process of Raw Centella asiatica-derived Crude Extracellular Vesicles

Fresh centella asiatica and EDTA aqueous solution (EDTA concentration: 0.5 mg/ml) were mixed at a weight ratio of 1:10, and then treated with an ultra-fine particle grinder to break the cell walls of centella asiatica to obtain an extract. After filtering the extract through a mesh having a pore size of 30 to 50 μm, the precipitate formed when the filtrate was centrifuged at 8,000 rpm or 5,000 x g was removed, and the supernatant was filtered through a filter having a pore size of 0.2 μm for final filtration. A liquid (crude extracellular vesicles) was obtained. Then, when the final filtrate was tangentially filtered, a 100 kDa filter was used to remove components having a molecular weight of 100 kDa or less (cut-off of 100 kDa or less), and the concentrate obtained was washed (purified) twice with purified water (weight ratio, concentrate :purified water = 1:5) to obtain the final product.

[Comparative Example 1-3] Separation process of low molecular weight components derived from fresh centella asiatica

Fresh centella asiatica and EDTA aqueous solution (EDTA concentration: 0.5 mg/ml) were mixed at a weight ratio of 1:10, and then treated with an ultra-fine particle grinder to break the cell walls of centella asiatica to obtain an extract. After filtering the extract through a mesh having a pore size of 30 to 50 μm, the precipitate formed when the filtrate was centrifuged at 8,000 rpm or 5,000 x g was removed, and the supernatant was filtered through a filter having a pore size of 0.2 μm for final filtration. A liquid (crude extracellular vesicles) was obtained. Then, when the final filtrate was filtered through a 100 kDa filter, a component having a molecular weight of 100 kDa or less was obtained as a final product.

[Comparative Example 2-1] Separation step of crude extracellular vesicles derived from dried centella asiatica

Centella asiatica dried in hot air at a temperature of about 45 ℃ (based on the total weight of the outpost, water content of 0.1% by weight or less) and EDTA aqueous solution (EDTA concentration: 0.5mg / ml) were mixed in a ratio of 2:98 to reflect the dry yield An extract was obtained by treating the centella asiatica with an ultra-fine particle grinder so that the cell walls were crushed. After the extract was filtered through a mesh having a pore size of 30 to 50 μm, the precipitate generated when the filtrate was centrifuged at 8,000 rpm or 5,000 x g was removed, and the supernatant was filtered through a filter having a pore size of 0.2 μm. A filtrate (crude extracellular vesicles) was obtained as the final product.

[Comparative Example 2-2] Separation and purification process of crude extracellular vesicles derived from dried centella asiatica

Centella asiatica dried in hot air at a temperature of about 45 ℃ (based on the total weight of the outpost, water content of 0.1% by weight or less) and EDTA aqueous solution (EDTA concentration: 0.5mg / ml) were mixed in a ratio of 2:98 to reflect the dry yield An extract was obtained by treating the centella asiatica with an ultra-fine particle grinder so that the cell walls were crushed. After filtering the extract through a mesh having a pore size of 30 to 50 μm, the precipitate formed when the filtrate was centrifuged at 8,000 rpm or 5,000 x g was removed, and the supernatant was filtered through a filter having a pore size of 0.2 μm for final filtration. A liquid (crude extracellular vesicles) was obtained. Then, when the final filtrate was tangentially filtered, a 100 kDa filter was used to remove components having a molecular weight of 100 kDa or less (cut-off of 100 kDa or less), and the concentrate obtained was washed (purified) twice with purified water (weight ratio, concentrate :purified water = 1:5) to obtain the final product.

[Comparative Example 2-3] Separation process of low molecular weight components derived from dried centella asiatica

Centella asiatica dried in hot air at a temperature of about 45 ℃ (based on the total weight of the outpost, water content of 0.1% by weight or less) and EDTA aqueous solution (EDTA concentration: 0.5mg / ml) were mixed in a ratio of 2:98 to reflect the dry yield An extract was obtained by treating the centella asiatica with an ultra-fine particle grinder so that the cell walls were crushed. After filtering the extract through a mesh having a pore size of 30 to 50 μm, the precipitate formed when the filtrate was centrifuged at 8,000 rpm or 5,000 x g was removed, and the supernatant was filtered through a filter having a pore size of 0.2 μm for final filtration. A liquid (crude extracellular vesicles) was obtained. Then, when the final filtrate was filtered through a 100 kDa filter, a component having a molecular weight of 100 kDa or less was obtained as a final product.

[Experimental Example 1] Identification of plant-derived extracellular vesicles

[1-1] Confirmation of plant-derived extracellular endoplasmic reticulum

In order to confirm the yield of plant-derived extracellular vesicles by protein content, the protein content of each sample of Reference Example 1, Reference Example 2 and Reference Example 3 was measured by Lowry protein assay (each sample 3 measurements were taken and the average value was obtained).

As a result, as shown in Table 1, the protein content of the extracellular vesicles obtained from the freeze-dried Centella asiatica (Reference Example 2) was about 21% higher than that of the extracellular vesicles obtained from the fresh Centella asiatica (Reference Example 1). , Extracellular vesicles (Reference Example 3) obtained from Centella Asiatica treated with hot air drying were also confirmed to have a high protein content of about 23%.

That is, it was confirmed that the yield of extracellular vesicles increased when the fresh centella asiatica was pre-treated by freeze-drying or hot-air drying.

Pretreatment method Reference example 1 Reference example 2 Reference example 3 Protein content (ppm) 862.95 1041.24 1063.53 Protein content increase rate (%) compared to Reference Example 1 - 20.6 23.2

In the experimental examples to be described later, the stability and physiological activity of the extracellular vesicles of each of the hot air-dried centella asiatica and the fresh centella asiatica were confirmed.

[1-2] Check the protein content of extracellular vesicles according to the pretreatment/stabilization process

Each sample of Example 1 and Example 2 of the present invention, Comparative Example 1-1, Comparative Example 1-2, Comparative Example 1-3, Comparative Example 2-1, Comparative Example 2-2 and Comparative Example 2-3 In order to determine whether and the quality of the extracellular vesicles were obtained, the protein content of each sample was measured using a Lowry protein assay (3 samples were measured and the average value was obtained).

As a result, as shown in Table 2, Example 1, Example 2, Comparative Example 1-1, Comparative Example 1-2, Comparative Example 1-3, Comparative Example 2-1, Comparative Example 2-2 and Comparative Example Each sample of 2-3 contained protein, confirming that extracellular vesicles were obtained. However, each sample of Comparative Example 1-1, Comparative Example 1-2, Comparative Example 2-1 and Comparative Example 2-2 was found to contain a high content of protein, which is a low molecular weight component having a size of 100 kDa or less. It was found that the purity of the extracellular vesicles was reduced by including a large amount of (eg, impurities such as organelles).

On the other hand, each sample of Comparative Example 1-2 and Comparative Example 2-2 that has undergone purification is partially removed from low molecular weight components compared to each sample of Comparative Example 1-1 and Comparative Example 2-1 that has not been washed (purified) Although the purity of the exoplasmic reticulum was slightly improved, it was confirmed that the purity of the extracellular endoplasmic reticulum was low when compared with each sample of Examples 1 and 2.

sample Example 1 Example 2 Comparative Example 1-1 Comparative Example 1-2 Comparative Example 1-3 Comparative Example 2-1 Comparative Example 2-2 Comparative Example 2-3 Protein content (ppm) 131.2965 170.575 866.0355 318.447 1674.711 1,023.587 267.616 429.351

[Experimental Example 2] Confirmation of the shape of plant-derived extracellular vesicles and the number of particles per unit volume

The structural characteristics (shape, size, etc.) of extracellular vesicles were confirmed by TEM (Transmission Electron Microscopy), and the number of extracellular vesicles per unit volume was confirmed by NTA (Nanoparticle Tracking Analysis). .

As a result, as shown in Figure 1, it was confirmed through TEM measurement that the extracellular vesicles of the sample according to Example 1 were spherical particles having a size of 200 nm or less.

In addition, as shown in Figures 2-3, it was confirmed that the extracellular vesicles of each sample of Examples 1 and 2 through NTA measurement had a concentration of 10 ⁸ particles / mL (particles / mL) or more.

[Experimental Example 3] Confirmation of particle size and stability of plant-derived extracellular vesicles

Each sample of Example 1 and Example 2 of the present invention, Comparative Example 1-1, Comparative Example 1-2, Comparative Example 1-3, Comparative Example 2-1, Comparative Example 2-2 and Comparative Example 2-3 was measured by Dynamic Light Scattering (DLS) (measuring 10 times for each sample and obtaining an average value), and confirming the particle size and particle size distribution of the extracellular vesicles for each sample.

In addition, in order to evaluate the stability over time of the extracellular vesicles for each sample at the time of the measurement, the particle size of the extracellular vesicles of each sample was measured by DLS on the day of sample acquisition (measurement on day 0), and 10 days after the sample was obtained. Measured after storage at ~7°C (measured on the 10th day), the stability of the extracellular vesicles over time was evaluated by the amount of change in particle size and polydispersity index (PDI).

As a result, as shown in FIGS. 4 to 9 and 11 to 12, Example 1, Example 2, Comparative Example 1-1, Comparative Example 1-2, Comparative Example 2-1, and Comparative Example 2-2, respectively. It was confirmed that the sample contained a large amount of extracellular vesicles (exosomes) having an average particle size of 50 to 250 nm of the main peak.

On the other hand, as shown in FIGS. 10 and 13, each sample of Comparative Example 1-3 and Comparative Example 2-3 was measured as a plurality of peaks in the particle size graph, and the average particle size of the main peak was a low molecular weight component (100 kDa) of less than 50 nm. less), that is, it was confirmed to contain impurities such as organelles other than extracellular vesicles.

As a result of looking at the particle size of the extracellular vesicles of each sample and the stability over time, as shown in Table 3 and FIGS. 2 to 7, each sample of Example 1 and Example 2 was of high purity, as confirmed in Table 1. It was confirmed that the average particle size of the main peak is 50 ~ 250 nm as well as the extracellular vesicles, and the stability is maintained high due to the low change in the particle size of the extracellular vesicles over time.

Specifically, each sample of Example 1, Example 2, Comparative Example 1-1, Comparative Example 1-2, Comparative Example 2-1 and Comparative Example 2-2 had a main peak particle size of extracellular vesicles when measured on day 0. It was 140 ~ 235 nm, and the sample of Example 1 when measured on the 10th day was 53.9 nm in the particle size change of the extracellular vesicles compared to the 0th day, and each sample of Comparative Example 1-1 and Comparative Example 1-2 was the particle size change of the extracellular vesicles It was 66.5nm and 48.2nm compared to the 0th day, and the sample of Example 2 was 29.7nm in the particle size change of the extracellular vesicles compared to the 0th day, and each sample of Comparative Example 2-1 and Comparative Example 2-2 was the amount of extracellular vesicles It was confirmed that the particle size change was 30.1 nm and 21.9 nm, and the extracellular vesicles of Example 2 to which dried Centella asiatica was applied showed a relatively lower particle size change on day 10 compared to day 0 compared to the extracellular vesicles of Example 1 to which fresh Centella asiatica was applied. confirmed that

In addition, each sample of Comparative Example 2-1 and Comparative Example 2-2 to which dry centella asiatica was applied was also better than each sample of Comparative Example 1-1 and Comparative Example 1-2 to which fresh centella asiatica was applied compared to day 0 versus day 10 of extracellular endoplasmic reticulum. It was confirmed that the change in particle size was relatively low, and it was confirmed that the stability of the extracellular vesicles was improved by the pretreatment process (drying, etc.) of centella asiatica applied to the sample.

Although the sample of Comparative Example 2-2 had a relatively lower change in particle size than the sample of Example 2, as shown in FIG. 12, a plurality of peaks were measured on the 10th day on the particle size graph of extracellular vesicles, and as time elapsed, Accordingly, it was confirmed that the extracellular vesicles were somewhat aggregated and were in an unstable state.

As shown in FIGS. 10 and 13, in each sample of Comparative Examples 1-3 and 2-3, the amount of change in the particle size of the extracellular vesicles was 333.36 nm and 828.21 nm, and the low molecular weight components of 100 kDa or less decreased over time. It was confirmed that the particle size of the extracellular vesicles increased as a result of aggregation.

On the other hand, PDI, a numerical value representing polydispersity, can be evaluated as monodispersity when it is closer to 0 and polydispersity when it is closer to 1. As shown in Table 4, the sample of Example 1 is Comparative Example 1-1 and Comparative Example 1 -2 and Comparative Example 1-3, the PDI change was less, the sample of Example 2 was less PDI change than each sample of Comparative Example 2-1, Comparative Example 2-2 and Comparative Example 2-3 did

In summary, the crude extracellular vesicles obtained by filtering the extract obtained by crushing the cell wall of the plant through the preparation method of each sample of Examples 1, 2 and 3 according to the present invention are separated by tangential flow filtration and Upon concentration, it was possible to obtain a concentrate from which particles of less than 100 kDa were removed, and when the liquid component of the obtained concentrate was replaced with trehalose, low-molecular substances that could remain in the concentrate were removed and the aggregation reaction between extracellular endoplasmic reticulum was reduced. It was possible to obtain an extracellular vesicle having a uniform particle size distribution and improved stability by reducing the particle size.

sample Example 1 Example 2 Comparative Example 1-1 Comparative Example 1-2 Comparative Example 1-3 Comparative Example 2-1 Comparative Example 2-2 Comparative Example 2-3 Day 0 main peak particle size of extracellular vesicles (nm)
= [A] 144.6 233.1 168.4 212.5 10.24 168.6 184.3 18.59 Day 10 main peak particle size of extracellular vesicles (nm) = [B] 198.5 203.4 234.9 164.3 343.6 198.7 206.2 846.8 Change in particle size of extracellular endoplasmic reticulum (nm)
= |[B]-[A]|
53.9
29.7
66.5
48.2
333.36
30.1
21.9
828.21

sample Example 1 Example 2 Comparative Example 1-1 Comparative Example 1-2 Comparative Example 1-3 Comparative Example 2-1 Comparative Example 2-2 Comparative Example 2-3 Day 0 extracellular endoplasmic reticulum PDI
= [C] 0.309 0.208 0.161 0.298 0.355 0.169 0.201 0.422 Day 10 extracellular endoplasmic reticulum PDI = [D] 0.325 0.279 0.339 0.320 0.760 0.298 0.464 0.920 Change in PDI of extracellular endoplasmic reticulum = |[D]-[C]| 0.016 0.071 0.178 0.022 0.405 0.129 0.263 0.498

[Experimental Example 4] Confirmation of physiological efficacy of plant-derived extracellular vesicles

[4-1] The effect of enhancing the signal transduction of plant-derived extracellular vesicles was confirmed by the ability of L-NMMA to inhibit nitric oxide production

Raw 264.7 cells (Cat. No. KCLB No.40071, Korea Cell Line Bank, Korea) was prepared using DMEM (Dulbecco Modified Eagle Medium) (Welgene, Korea) containing 10% fetal bovine serum (FBS) (Welgene, Korea), and about 9x10 ⁴ cells/well (cells). /well) in a 96-well plate and cultured for 24 hours in a 37°C, 5% CO ₂ incubator, as shown in Table 5, for Raw 264.7 cells Untreated group, control group, comparative group 1, comparative group 2, and experimental group 1 were prepared with or without treatment of the treatment material (L-NMMA ( ^NG -Methyl-L-arginine acetate, Sigma-Aldrich, USA): nitric oxide ( NO) production inhibitor; Lipopolysaccharide (LPS)).

Treatment substance [treatment concentration] untreated group control group experimental group 1 comparator group 1 comparison group 2 LPS [250 ng/mL] - + + + + L-NMMA [20 μM] - - + + + Example 2 sample [50 ppm] - - + - - Comparative Example 2-1 Sample [50 ppm] - - - + -

Specifically, the untreated group is Raw 264.7 cells cultured for 24 hours without treatment, the control group is Raw 264.7 cells cultured for 24 hours and LPS is treated, and Comparative Group 2 is Raw 264.7 cells cultured for 24 hours. LPS was treated at 8 hours after L-NMMA treatment, and in Comparative Group 1, Raw 264.7 cells cultured for 24 hours were treated with L-NMMA and samples of Comparative Example 2-1 8 hours after treatment. LPS was treated at the time point, and Experimental Group 1 was prepared by treating LPS at the time point when 8 hours elapsed after treating L-NMMA and the sample of Example 2 to Raw 264.7 cells cultured for 24 hours.

After culturing the untreated group, control group, control group 1, control group 2, and experimental group 1 for 16 hours, the supernatant of the culture solution of each group was recovered, mixed with Griess reagent in a weight ratio of 1: 1, and microplate reader [ Epoch Microplate Spectrophotometer, BioTeK]) was used to measure absorbance at a wavelength of 540 nm.

As a result, as shown in FIG. 14, the experimental group 1 treated with L-NMMA and the samples of Example 2 showed a higher LPS-induced NO (nitrogen oxide) production than the samples of comparative group 2 treated with L-NMMA alone. It was confirmed that the reduction effect was high and there was a physiological synergistic effect.

In addition, it was confirmed under the above experimental conditions that each sample of Examples 1 and 3 also had a physiological synergistic effect due to an increased reduction of NO production similar to that of the sample of Example 2 (data not shown).

On the other hand, the samples of Comparative Group 1 treated with L-NMMA and the samples of Comparative Example 2-1 were somewhat more effective in reducing the amount of NO production induced by LPS than the samples of Comparative Group 2 treated with L-NMMA alone, It was confirmed that the degree of the effect was not higher than that of the experimental group treated with L-NMMA and the samples of Example 2.

In addition, each of the samples of Comparative Example 1-1, Comparative Example 1-2, Comparative Example 1-3, Comparative Example 2-2, and Comparative Example 2-3 also reduced the amount of NO produced to a degree similar to that of the sample of Comparative Example 2-1. The effect was confirmed under the same experimental conditions as above (data not shown).

[4-2] The effect of enhancing signal transduction of plant-derived extracellular endoplasmic reticulum was confirmed by the nitric oxide production inhibitory ability of centella asiatica extract.

In order to confirm the physiological synergistic effect exhibited when the extracellular vesicles prepared by the pretreatment / purification / stabilization process according to Example 2 are used in combination with titrated extract of Centella asiatica (TECA), the above [4-1] Experiments were performed in the same manner as in Table 6, but as shown in Table 6, TECA was used instead of L-NMMA, and raw 264.7 cells were treated with or without treatment materials, untreated group, control group, control group 3, control group 4 and Experimental group 2 was prepared.

(Here, the TECA is a partial extract (fractional extract) containing 3 to 3.5% by weight based on the total weight of the crude extract in the crude extract in the form of a powder obtained by extracting hot air-dried centella asiatica with ethanol and concentrating under reduced pressure. , The TECA contains Madecassic acid, Asiaticoside and Asiatic acid as main components (see Korean Patent No. 10-0379067 and Korean Patent No. 10-0228873 ), the TECA has the ability to inhibit production of nitric oxide (refer to Korean Patent Registration No. 10-2223534), and the TECA is based on the total weight of TECA according to the results of LC analysis (Liquid Chromatography Analysis) using an LC analyzer (manufacturer: Waters) It was confirmed that it contained about 30% by weight of madecassic acid, about 40% by weight of asiaticoside and about 30% by weight of asiatic acid.)

Specifically, the untreated group is Raw 264.7 cells cultured for 24 hours without treatment, the control group is Raw 264.7 cells cultured for 24 hours and LPS is treated, and Comparative Group 4 is Raw 264.7 cells cultured for 24 hours. LPS was treated at 8 hours after TECA treatment, and in Comparative Group 3, Raw 264.7 cells cultured for 24 hours were treated with TECA and the sample of Comparative Example 2-1, and LPS was treated at 8 hours after treatment. Experimental group 2 was prepared by treating Raw 264.7 cells cultured for 24 hours with LPS at 8 hours after treating TECA and the sample of Example 2.

After culturing the untreated group, the control group, the control group 3, the control group 4, and the experimental group 2 for 16 hours, the supernatant of the culture medium of each group was collected, mixed with Griess reagent at a weight ratio of 1: 1, and mixed with a microplate reader (Epoch Microplate Spectrophotometer). , BioTeK) was used to measure absorbance at a wavelength of 540 nm.

Treatment substance [treatment concentration] untreated group control group experimental group 2 comparator group 3 comparator group 4 LPS [250 ng/mL] - + + + + TECA [1.5 ppm] - - + + + Example 2 sample [50 ppm] - - + - - Comparative Example 2-1 Sample [50 ppm] - - - + -

As a result, as shown in FIG. 15, the experimental group 2 treated with TECA and the sample of Example 2 had a higher effect of reducing the amount of NO production induced by LPS than the sample of comparative group 4 treated with TECA alone, resulting in a physiological increase. It was confirmed that it worked.

In addition, it was confirmed under the above experimental conditions that each sample of Examples 1 and 3 also had a physiological synergistic effect due to an increased reduction of NO production similar to that of the sample of Example 2 (data not shown).

In summary, unlike Comparative Examples, Examples 1, 2 and 3 have high yields that exhibit increased physiological efficacy through the purification (separation and purification) and stabilization process of extracellular vesicles unique to the present invention. It was confirmed that stable extracellular vesicles could be obtained.

statistical processing

Statistical analysis used a t-test test as a significance test between the two groups, and compared to the control group, * P <0.05, ** P <0.01, and *** P <0.001 were considered valid differences.

Having described specific parts of the present invention in detail above, it is clear to those skilled in the art that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (7)

As a method for producing plant-derived extracellular vesicles that increase the activity of physiologically active substances,
(a) obtaining a plant extract by pre-treating the plant, adding an aqueous sequestering agent, and breaking the cell walls of the plant;
(b) the plant extract was filtered through a mesh having a pore size of 30 to 50 μm, centrifuged to remove the precipitate, and the supernatant was filtered through an absolute filter having a pore size of 0.2 to 5.0 μm to form crude extracellular vesicles ( Crude Extracellular Vesicles) Separating the filtrate;
(c) Purifying and concentrating the crude extracellular vesicle filtrate by tangential flow filtration (TFF) using a filter having a molecular weight cut-off (MWCO) of 100 kDa to obtain purified extracellular fluid. obtaining an endoplasmic reticulum concentrate; and
(d) stabilizing the extracellular vesicles by adding an aqueous saccharide solution to the purified extracellular vesicle concentrate;
The physiologically active substance is contained in extracellular vesicles or used in combination with extracellular vesicles,
The size of the lysate contained in the plant extract is 1 μm to 10,000 μm based on the average diameter,
The saccharide is trehalose, lactulose, cellobiose, isomaltose, turanose, sucrose, maltose or lactose;
The pretreatment is characterized in that at least one selected from the group consisting of plant drying treatment, wrapping treatment, freezing and thawing treatment, manufacturing method. According to claim 1,
In step (a), the concentration of the sequestering agent in the aqueous sequestering agent solution is 0.5 to 5 mg / ml, and the weight ratio of the pretreated plant and the sequestering agent aqueous solution is 1 to 4: 50 to 100,
In step (b), centrifugation is performed at 1,500 to 15,000 xg,
In the step (d), the aqueous saccharide solution contains trehalose at a concentration of 0.5 to 10 mg/ml. According to claim 1, wherein the manufacturing method is characterized in that, reducing the amount of change in the particle size of the extracellular vesicles, inhibiting the aggregation reaction, manufacturing method. delete delete A composition comprising an extracellular vesicle that increases the activity of a physiologically active substance prepared by the method of any one of claims 1 to 3,
The extracellular vesicles are characterized in that 10 ⁸ or more per 1ml based on the volume of the composition, the composition. The composition according to claim 6, wherein the composition further comprises at least one of a cell activity regulator and a plant-derived extract as a physiologically active substance used in combination with the extracellular endoplasmic reticulum.

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