Method for producing vesicle-containing liquid

Abstract

The present invention addresses the problem of providing a technique for obtaining plant-derived vesicles with higher efficiency, higher purity, or enhanced structural stability. This problem is solved by a method for producing a vesicle-containing liquid, the method being characterized by comprising obtaining the vesicle-containing liquid from a plant material, wherein the plant material is an inner layer of a fruit obtained by removing an outer layer thereof, or an outer layer of a fruit obtained by removing an inner layer thereof.

Description

Method for producing vesicle-containing liquid

[0001] The present invention relates to a method for producing a vesicle-containing liquid derived from a plant.

[0002] Vesicles derived from plants have been reported to have various physiological activities, such as anti-inflammatory, antibacterial, and angiogenic activities (Patent Document 1). Vesicles derived from plants may include extracellular vesicles secreted from plant cells and the entire endoplasmic reticulum, such as cell walls (formed of cellulose and polysaccharides) that are released when plants are crushed. Examples of extracellular vesicles include exosomes and microvesicles. Patent Document 1 describes the preparation of a vesicle-containing liquid by ultracentrifugation of juice squeezed from whole fruits.

[0003] Japanese Patent Application Publication No. 2022-525131

[0004] Although ultracentrifugation is a technique frequently used to purify vesicles such as exosomes derived from animals, it requires high energy costs, is not suitable for purifying extracellular vesicles in general, and may destroy extracellular vesicles. In the course of research, the present inventors found that when whole fruit was used as a starting material, particularly under conditions without ultracentrifugation, the vesicle structure could not be confirmed, the vesicles were buried / covered by impurities, or the vesicles were destroyed.

[0005] An objective of the present invention is to provide a technique for obtaining plant-derived vesicles with higher efficiency, higher purity, or with their structure more intact.

[0006] Another object of the present invention is to provide a technology for preserving and formulating a liquid containing plant-derived vesicles.

[0007] In view of the above-mentioned problems, the present inventors have conducted extensive research and have found that the above-mentioned problems can be solved by a method for producing a vesicle-containing liquid, which comprises obtaining a vesicle-containing liquid from a plant material, wherein the plant material is the inner layer obtained by removing the outer layer of a fruit or the outer layer obtained by removing the inner layer of a fruit. Furthermore, the present inventors have found that refrigerated storage is suitable for storing plant-derived vesicle-containing liquid, and that in order to stably formulate the plant-derived vesicle-containing liquid, it is advisable to mix the liquid with a nonionic surfactant, a polyhydric alcohol, and water to obtain a vesicle-solubilized liquid. Based on these findings, the present inventors have conducted further research and have completed the present invention. That is, the present invention encompasses the following aspects:

[0008] Item 1. A method for producing a vesicle-containing liquid, comprising obtaining a vesicle-containing liquid from a plant material, wherein the plant material is an inner layer obtained by removing an outer layer of a fruit or an outer layer obtained by removing an inner layer of a fruit.

[0009] Item 2. The method according to Item 1, wherein the particle size of the vesicles is 10 to 1,000 nm.

[0010] Item 3. The method according to Item 1 or 2, wherein the vesicles include extracellular vesicles.

[0011] Item 4. (1) The production method according to any one of Items 1 to 3, which comprises crushing and / or squeezing the plant material to obtain a crude extract.

[0012] Item 5. (2) The method according to Item 4, further comprising centrifuging the crude extract to obtain a supernatant.

[0013] Item 6. The method according to Item 5, wherein the centrifugal force of the centrifugation is 30,000 g or less.

[0014] Item 7. (3) The production method according to Item 5 or 6, further comprising ultrafiltration of the supernatant to obtain a filtrate.

[0015] Item 8. (4) The production method according to Item 7, further comprising fractionating the filtrate by size exclusion chromatography to obtain a vesicle-containing fraction.

[0016] Item 9. The production method according to any one of Items 1 to 8, wherein the fruit is a fruit of at least one plant selected from the group consisting of Cucurbitaceae plants and Rosaceae plants.

[0017] Item 10. The production method according to Item 9, wherein the fruit is at least one fruit selected from the group consisting of Cucurbitaceae plants consisting of cucumber, gourd, watermelon, pumpkin, zucchini, melon, bottle gourd, wax gourd, and bitter melon, and Rosaceae plants consisting of strawberry, apple, pear, cherry, peach, plum, apricot, Japanese plum, loquat, and prune.

[0018] Item 11. A vesicle-containing liquid obtained by the production method according to any one of Items 1 to 10.

[0019] Item 12. A plant-derived vesicle-containing liquid that has been refrigerated but has not been frozen or stored at temperatures above refrigeration.

[0020] Item 13. A method for producing a vesicle solubilized solution, comprising mixing a plant-derived vesicle-containing solution, a nonionic surfactant, a polyhydric alcohol, and water.

[0021] Item 14. A vesicle solubilizing solution comprising a plant-derived vesicle-containing solution, a nonionic surfactant, a polyhydric alcohol, and water.

[0022] Item 15. An external or pharmaceutical composition comprising a vesicle solubilized solution obtained by the production method according to Item 13 or a vesicle solubilized solution according to Item 14.

[0023] According to the present invention, there is provided a method for producing a vesicle-containing liquid that can obtain plant-derived vesicles with higher efficiency, higher purity, or with the structure more preserved. Furthermore, according to the present invention, there is also provided a technology for preserving and formulating a plant-derived vesicle-containing liquid.

[0024] 1 shows the results of DLS analysis of the vesicle-containing liquid obtained in Example 1 when the outer layer portion was used as the starting material. Each graph line shows the results of three analyses of the same sample. 1 shows the results of DLS analysis of the vesicle-containing liquid obtained in Example 1 when the inner layer portion was used as the starting material. Each graph line shows the results of three analyses of the same sample. 1 shows the results of DLS analysis of the vesicle-containing liquid obtained in Example 1 when the whole cucumber fruit was used as the starting material. Each graph line shows the results of three analyses of the same sample. 1 shows a TEM observation image of the vesicle-containing liquid obtained in Example 1 when the outer layer portion was used as the starting material. The bar at the bottom right of each photograph indicates 200 nm. 1 shows a TEM observation image of the vesicle-containing liquid obtained in Example 1 when the inner layer portion was used as the starting material. The bar at the bottom right of each photograph indicates 200 nm. 1 shows a TEM observation image of the vesicle-containing liquid obtained in Example 1 when the whole cucumber fruit ... DLS analysis result of the vesicle-containing liquid obtained in Example 2 when the outer layer portion was used as the starting material. Each graph line shows the results of three analyses of the same sample. This shows the results of DLS analysis of the vesicle-containing liquid obtained in Example 2 when the inner layer portion was used as the starting material. Each graph line shows the results of three analyses of the same sample. This shows the results of DLS analysis of the vesicle-containing liquid obtained in Example 2 when the whole strawberry fruit was used as the starting material. Each graph line shows the results of three analyses of the same sample. This shows a TEM image of the vesicle-containing liquid obtained in Example 2 when the outer layer portion was used as the starting material. The bar at the bottom right of each photograph indicates 200 nm. This shows a TEM image of the vesicle-containing liquid obtained in Example 2 when the inner layer portion was used as the starting material. The bar at the bottom right of each photograph indicates 200 nm or 100 nm. This shows a TEM image of the vesicle-containing liquid obtained in Example 2 when the whole strawberry fruit was used as the starting material. The bar at the bottom right of each photograph indicates 200 nm or 100 nm. This shows a TEM image of the vesicle-containing liquid obtained in Example 3 when the outer layer portion was used as the starting material. The bar at the bottom right of each photograph indicates 100 nm, 200 nm, or 500 nm. This shows a TEM image of the vesicle-containing liquid obtained when the inner layer portion was used as the starting material in Example 3. The bar at the bottom right of each photograph indicates 100 nm.1 shows the results of DLS analysis of vesicle-containing liquid in Example 5. Each graph line shows the results of three analyses of the same sample. 2 shows the results of DLS analysis of lotion 1 in Example 5. Each graph line shows the results of three analyses of the same sample. 3 shows the results of DLS analysis of lotion 2 in Example 5. Each graph line shows the results of three analyses of the same sample. 4 shows the results of measurement of stratum corneum moisture content in Example 6. The vertical axis shows stratum corneum moisture content (conductivity) (unit: μs). 5 shows the results of measurement of transepidermal water loss in Example 6. The vertical axis shows transepidermal water loss (TEWL) (unit: g / h / m). 2 ) is shown.

[0025] In this specification, the expressions "contain" and "comprise" include the concepts of "contain," "comprise," "consist essentially of," and "consist only of."

[0026] 1. Method for Producing Vesicle-Containing Liquid In one aspect, the present invention relates to a method for producing a vesicle-containing liquid (sometimes referred to herein as "Production Method 1 of the Present Invention"), which comprises obtaining a vesicle-containing liquid from a plant material, wherein the plant material is an inner layer obtained by removing an outer layer of a fruit or an outer layer obtained by removing an inner layer of a fruit.

[0027] The plant from which the plant material is derived is not particularly limited as long as it is a fruit-bearing plant. Fruit is the edible part that holds the seeds, and is a concept that includes both fruits and fruit vegetables. In addition, in this specification, fruit is a concept that includes not only typical fruits (true fruits: the mature ovary of the pistil and its associated structures, containing seeds inside), but also those in which structures other than the ovary (e.g., receptacle, perianth, etc.) have matured (pseudo fruits). When the fruit includes true fruits and false fruits, the edible part (usually the false fruits, which may also hold the true fruits) is treated as the "fruit" in the present invention.

[0028] The fruit preferably has a certain moisture content or more. The moisture content of the fruit is, for example, 70% or more, preferably 75% or more, more preferably 80% or more, even more preferably 85% or more, and even more preferably 90% or more, based on 100% by mass of the fruit. The upper limit of the moisture content is not particularly limited, and is, for example, 99%, 98%, or 97%. The moisture content of the fruit can be determined, for example, according to the Standard Tables of Food Composition in Japan (8th Edition).

[0029] More specific examples of fruits include fruits of Cucurbitaceae plants and fruits of Rosaceae plants. These are suitable for Production Method 1 of the present invention and are also preferred from the viewpoint of the skin barrier function and moisturizing properties of the vesicle-containing liquid obtained by Production Method 1 of the present invention. Examples of Cucurbitaceae plants include cucumber, gourd, watermelon, pumpkin, zucchini, melon, bottle gourd, wax gourd, and bitter melon. Examples of Rosaceae plants include strawberry, apple, pear, cherry, peach, plum, apricot, plum, loquat, and prune. Among these, cucumber, strawberry, apple, and the like are preferred from the viewpoint of being particularly suitable for Production Method 1 of the present invention. Furthermore, from the viewpoint of the skin barrier function and moisturizing properties of the vesicle-containing liquid obtained by Production Method 1 of the present invention, Cucurbitaceae plants are preferred, and cucumber is particularly preferred. Furthermore, from the viewpoint of the skin barrier function and moisturizing properties of the vesicle-containing liquid obtained by Production Method 1 of the present invention, cucumber, strawberry, apple, and the like are preferred, and cucumber is particularly preferred.

[0030] Production method 1 of the present invention is characterized in that the starting plant material is the inner layer obtained by removing the outer layer of a fruit or the outer layer obtained by removing the inner layer of a fruit, which allows plant-derived vesicles to be obtained more efficiently, with a higher degree of purification, or with their structure more preserved, compared to when the fruit itself is used as the starting material.

[0031] The outer layer is the outermost layer of the fruit, and is usually referred to as the fruit skin or exocarp. Furthermore, the outer layer often contains a large amount of pigments specific to the plant and is clearly different in color from the inner layer. When the fruit is a pseudocarp, the outer layer is the surface layer of the fruit. When the fruit is a strawberry, the true fruit (achene) containing the seeds is retained on the surface of the fruit (pseudocarp), and the outer layer can be used in a state that includes this achene. The thickness of the outer layer is, for example, 0.1 to 5 mm. The thickness can be varied as appropriate depending on the type of fruit; for example, it is preferably 0.1 to 0.9 mm for cucumbers and apples, and 2 to 4 mm for strawberries.

[0032] The inner layer is the part inside the outer layer, and can be obtained by removing part or all of the outer layer of the fruit (for example, the outer layer covering 50% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 100% of the surface area of ​​the fruit, relative to 100% of the surface area of ​​the fruit). When the inner layer contains seeds, the seeds can be used as is, or they can be used after some or all of the seeds have been removed.

[0033] The method for obtaining a vesicle-containing solution from a plant material is not particularly limited, and known purification methods such as centrifugation, ultrafiltration, and chromatography can be used alone or in combination. It is preferable that ultracentrifugation is not performed in Production Method 1 of the present invention. From this perspective, it is preferable that Production Method 1 of the present invention does not include centrifugation at a centrifugal force of 100,000 g or more, 80,000 g or more, 60,000 g or more, 50,000 g or more, 40,000 g, or more than 30,000 g.

[0034] Production method 1 of the present invention preferably includes (1) crushing and / or squeezing a plant material to obtain a crude extract. The crushing and / or squeezing method is not particularly limited and can be carried out using various devices and tools. Examples of devices include a food slicer, a food mixer, a food processor, a cutter mill, a stamp mill, a mortar, a mortar, a rice masher, a ring mill, a roller mill, a jet mill, a hammer mill, a pin mill, a rotary mill, a vibration mill, a planetary mill, an attritor, and a bead mill. After crushing and / or squeezing, the mixture is preferably filtered using gauze or the like to remove coarse solids.

[0035] The production method 1 of the present invention preferably includes (2) centrifuging the crude extract obtained in step (1) to obtain a supernatant. In production method 1 of the present invention, ultracentrifugation is preferably not performed. From this perspective, the centrifugal force of the centrifugation in step (2) is preferably 30,000 g or less. The centrifugal force is preferably 200 to 30,000 g, more preferably 500 to 25,000 g, even more preferably 1,000 to 20,000 g, and even more preferably 1,500 to 18,000 g. The centrifugation time in step (2) can be, for example, 30 minutes to 12 hours, preferably 1 hour to 8 hours. The centrifugation temperature in step (2) is, for example, 0 to 25°C, preferably 4 to 20°C, more preferably 10 to 20°C, and even more preferably 12 to 18°C.

[0036] The centrifugation in step (2) can be carried out multiple times (e.g., 2 to 3 times). The centrifugation in step (2) is preferably carried out by subjecting the supernatant obtained by low-speed centrifugation to high-speed centrifugation. The centrifugal force of the low-speed centrifugation is preferably 200 to 8,000 g, more preferably 500 to 4,000 g, even more preferably 1,000 to 3,000 g, and even more preferably 1,500 to 2,500 g. The time of the low-speed centrifugation is, for example, 1 to 12 hours, preferably 2 to 8 hours, and more preferably 3 to 6 hours. The centrifugal force of the high-speed centrifugation is preferably 4,500 to 30,000 g, more preferably 9,000 to 25,000 g, even more preferably 12,000 to 20,000 g, and even more preferably 12,000 to 18,000 g. The time of the high-speed centrifugation is, for example, 30 minutes to 4 hours, preferably 45 minutes to 2 hours.

[0037] The production method 1 of the present invention preferably includes (3) ultrafiltration of the supernatant obtained in step (2) to obtain a filtrate. The material of the ultrafiltration membrane used for ultrafiltration is not particularly limited, and examples thereof include polyethylene, polyvinylidene fluoride, polyacrylonitrile, cellulose acetate, regenerated cellulose, polyamide, polyvinyl alcohol, polysulfone, polyethersulfone, and ceramic. The structure of the ultrafiltration membrane is not particularly limited, and examples thereof include hollow fiber membranes, spiral membranes, tubular membranes, and flat membranes. The molecular weight cutoff (NMWL) of the ultrafiltration membrane is preferably 10 to 300 kDa, more preferably 30 to 200 kDa, and even more preferably 60 to 150 kDa. Ultrafiltration is preferably centrifugal. The centrifugal conditions can be the same as those for step (2) above.

[0038] The production method 1 of the present invention preferably includes (4) fractionating the filtrate obtained in step (3) by size exclusion chromatography to obtain a vesicle-containing fraction. The carrier used for size exclusion chromatography is not particularly limited, as long as it is capable of purifying extracellular vesicles such as exosomes (e.g., capable of removing or reducing substances smaller than extracellular vesicles such as exosomes (e.g., soluble proteins)). For example, a carrier column with controlled pore size can be used, which makes it difficult to trap relatively large substances such as extracellular vesicles like exosomes, while easily trapping relatively small substances such as soluble proteins. For example, a qEV column manufactured by IZON can be used as the carrier column. An aqueous solution can be used as the eluate / mobile phase in size exclusion chromatography, and a pH-adjusted buffer solution such as PBS is preferred. The pH of the aqueous solution is preferably such that the structure of plant-derived vesicles is not easily damaged, for example, 6.0 to 8.0, preferably 6.2 to 7.5, and more preferably 6.5 to 7.3.

[0039] The vesicle-containing solution obtained by Production Method 1 of the present invention contains vesicles such as extracellular vesicles (e.g., exosomes, microvesicles, etc.). The vesicles can be, for example, spherical, erythrocyte-like structures (i.e., structures with a hollow center), or structures that appear hollow.

[0040] The particle size of the vesicles is, for example, 10 to 1000 nm. The average particle size of the vesicles is, for example, 50 to 700 nm, preferably 80 to 500 nm, and more preferably 100 to 400 nm. The average particle size is the Z-average measured by DLS analysis.

[0041] In one aspect, the present invention relates to a vesicle-containing liquid obtained by Production Method 1 of the present invention.

[0042] 2. Preservation of vesicle-containing liquid In one aspect, the present invention relates to a plant-derived vesicle-containing liquid that has been refrigerated but has not been frozen or stored at temperatures above refrigeration. In another aspect, the present invention relates to a method for preserving a plant-derived vesicle-containing liquid, which comprises refrigerating the plant-derived vesicle-containing liquid but does not include frozen or stored at temperatures above refrigeration.

[0043] The plant-derived vesicle-containing liquid is not particularly limited as long as it contains plant-derived vesicles. The vesicle-containing liquid obtained by Production Method 1 of the present invention is particularly suitable for the preservation method of the present invention.

[0044] The refrigerated storage is not particularly limited as long as it is stored at a refrigerated temperature. The refrigerated temperature is a temperature at which the plant-derived vesicle-containing liquid does not freeze and is lower than room temperature. The refrigerated temperature is, for example, 0 to 16°C, preferably 1 to 12°C, more preferably 1 to 10°C, even more preferably 2 to 8°C, and even more preferably 3 to 7°C. The refrigerated storage period is, for example, 6 months to 5 years, preferably 1 to 4 years, and more preferably 2 to 3 years. According to the storage method of the present invention, the vesicles can be stored while maintaining their structure more stably.

[0045] Frozen storage refers to storage at a temperature at which the plant-derived vesicle-containing liquid freezes, such as below 0°C, -1°C or lower, -2°C or lower, -3°C or lower, -4°C or lower, -5°C or lower, -6°C or lower, -7°C or lower, -8°C or lower, -9°C or lower, or -10°C or lower.

[0046] 3. Vesicle Solubilized Solution and Preparations In one aspect, the present invention relates to a method for producing a vesicle solubilized solution (Production Method 2 of the present invention), which comprises mixing a plant-derived vesicle-containing solution, a nonionic surfactant, a polyhydric alcohol, and water.

[0047] The plant-derived vesicle-containing liquid is not particularly limited as long as it contains plant-derived vesicles. The vesicle-containing liquid obtained by Production Method 1 of the present invention is particularly suitable for Production Method 2 of the present invention.

[0048] Examples of nonionic surfactants include polyoxyalkylene hydrogenated castor oil, polyoxyalkylene alkyl ether, polyoxyalkylene glycol, polyoxyalkylene fatty acid ester, polyoxyalkylene sorbite fatty acid ester, polyglycerin fatty acid ester, polyoxyalkylene glycerin fatty acid ester, polyoxyalkylene sorbitan fatty acid ester, etc. Among these, particularly preferred are PEG-7 glyceryl coconut oil fatty acid, PPG-13 decyltetradeceth-24, PEG-50 hydrogenated castor oil, glycereth-7, etc.

[0049] Examples of polyhydric alcohols include glycerin (concentrated glycerin), diglycerin, triglycerin, polyglycerin, propanediol, propylene glycol, dipropylene glycol, isoprene glycol, 1,2-butylene glycol, 1,3-butylene glycol, 1,2-pentanediol, 1,2-hexanediol, 1,2-octanediol, 1,2-decanediol, 1,2-dodecanediol, polyethylene glycol, polypropylene glycol, ethylene glycol, diethylene glycol, triethylene glycol, 1,2,4-butanetriol, sorbitol, xylitol, erythritol, trehalose, glucosyltrehalose glucose, maltose, maltitol, sucrose, and mannitol.

[0050] The ranges of the content ratios of each liquid / component in a mixed liquid containing a plant-derived vesicle-containing liquid, a nonionic surfactant, a polyhydric alcohol, and water are as follows: Plant-derived vesicle-containing liquid: For example, 0.001 to 50% by mass, preferably 0.01 to 10% by mass, and more preferably 0.05 to 5% by mass. Nonionic surfactant: For example, 0.5 to 10% by mass, preferably 1 to 5% by mass, and more preferably 2 to 4% by mass. Polyhydric alcohol: For example, 1 to 30% by mass, preferably 5 to 20% by mass, and more preferably 10 to 15% by mass. Water: For example, 10 to 98.5% by mass, preferably 65 to 94% by mass, and more preferably 75 to 85% by mass.

[0051] The temperature condition in Production Method 2 of the present invention is preferably 10 to 30°C, more preferably 15 to 25°C.

[0052] A vesicle solubilized solution can be obtained by Production Method 2 of the present invention. From this perspective, in one aspect, the present invention relates to a vesicle solubilized solution containing a plant-derived vesicle-containing solution, a nonionic surfactant, a polyhydric alcohol, and water.

[0053] In the vesicle solubilization liquid, plant-derived vesicles are stably present in a solubilized state. Therefore, by using the vesicle solubilization liquid, various preparations can be easily obtained while suppressing the occurrence of cloudiness or precipitation. From this perspective, in one aspect, the present invention relates to a preparation (e.g., a cosmetic, topical, or pharmaceutical composition) containing the vesicle solubilization liquid.

[0054] The formulation may contain other ingredients in addition to the vesicle solubilized solution, such as humectants, emollients, preservatives, surfactants, oils, higher alcohols, pH adjusters, chelating agents, plant extracts, fragrances, and colorants.

[0055] The formulation may be, for example, a water-based formulation, an emulsion formulation, an oil-based formulation, etc. Examples of the preparation methods for these are as follows.

[0056] In the case of aqueous formulations, the above-mentioned vesicle solubilizing solution is added to an aqueous formulation containing purified water and a blend of wetting agents, emollients, preservatives, surfactants, oils, pH adjusters, chelating agents, plant extracts, etc. at room temperature or below, and the mixture is stirred or solubilized to form an aqueous formulation containing the vesicle solubilizing solution.

[0057] In the case of emulsion formulations, purified water is heated to 50-90°C with an aqueous solution of wetting agents, surfactants, pH adjusters, chelating agents, etc., and a similarly heated oil mixture of oils, surfactants, higher alcohols, etc. is prepared. The aqueous solution and oil mixture are then combined at 50-90°C and emulsified... (A) (A) is then cooled, and preservatives, fragrances, plant extracts, colorants, etc. are added at around 60-35°C, followed by the vesicle solubilization solution at 30°C or below, and the resulting emulsion is prepared by stirring.

[0058] In the case of an oil-based preparation, a minute amount of the above vesicle solubilizing solution is added to make a transparent to milky white vesicle solubilizing solution-containing oil-based preparation.

[0059] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0060] Example 1. Production of vesicle-containing liquid from cucumber fruit 1 Cucumber fruits (commercially available for consumption) were separated using a blade into an outer layer (skin, approximately 0.1 to 0.9 mm thick) and an inner layer (the inside of the skin, including the seeds). Using the resulting outer layer, the resulting inner layer, and the whole cucumber fruit as starting materials, vesicle-containing liquid was produced as follows.

[0061] (Step 1) The starting material was crushed using a household slicer (handle vegetable cutter, manufactured by DAISO), and the resulting liquid crushed material was squeezed out using gauze (medical grade, manufactured by Hakujuji Co., Ltd.) to obtain a filtrate. This filtrate was then filtered again using new gauze. This process was repeated twice to obtain a crude extract.

[0062] (Step 2) The crude extract obtained in step 1 was dispensed into 15 mL centrifuge tubes and centrifuged (15°C, 2110 g, 4 h) to recover the supernatant. The obtained supernatant was filled into a syringe equipped with a filter (Minisart (registered trademark), pore size 0.45 μm, manufactured by SARTORIUS) and passed through the filter for filtration. The obtained filtrate was dispensed into 2 mL centrifuge tubes and centrifuged (15°C, 15000 g, 1 h) to recover the supernatant.

[0063] (Step 3) The supernatant finally obtained in step 2 was placed in a centrifugal ultrafiltration filter (Amicon Ultra-15, molecular weight cutoff (NMWL): 100 kDa, manufactured by Merck) and centrifuged (15°C, 2100 g, 1 hour) to obtain a concentrated filtrate.

[0064] (Step 4) The filtrate obtained in step 3 was subjected to size exclusion chromatography using PBS (pH 7.2) as the eluent. Specifically, a qEV column (qEV original, manufactured by IZON) was set in an automated chromatography system (IZON AFC(V1) automated size exclusion chromatography) and seven fractions (each with a volume of 0.5 mL) were obtained. The first two fractions were mixed and used as the vesicle-containing solution for the following analysis.

[0065] The vesicle-containing solution was analyzed by dynamic light scattering (DLS) using a ZETA SIZER Nano-ZS (Malvern) instrument. Figure 1 shows the results when the outer layer was used as the starting material, Figure 2 shows the results when the inner layer was used as the starting material, and Figure 3 shows the results when the whole cucumber fruit was used as the starting material.

[0066] The vesicle-containing solution was observed under a transmission electron microscope (TEM) to obtain images. Figure 4 shows the results when the outer layer was used as the starting material, Figure 5 shows the results when the inner layer was used as the starting material, and Figure 6 shows the results when the whole cucumber fruit was used as the starting material. When the whole cucumber fruit was used as the starting material, the number of vesicles was small and the vesicles were buried in impurities. On the other hand, when the solution was separated into the outer layer and the inner layer, vesicles (particularly spherical, red blood cell-like structures, or structures in which spherical vesicles were crushed to reveal hollow spaces, such as exosomes or microvesicles) were clearly observed.

[0067] Example 2. Production of vesicle-containing liquid from strawberry fruit Strawberry fruit (commercially available for consumption, stems removed) was separated using a blade into an outer layer (a red surface layer, approximately 2 to 4 mm thick) and an inner layer (an inner white layer). The resulting outer layer, the resulting inner layer, and the whole strawberry fruit were used as starting materials to produce vesicle-containing liquid in the same manner as in Example 1, except that filtration in step 2 was not performed.

[0068] The vesicle-containing solution was subjected to DLS analysis in the same manner as in Example 1. The results when the outer layer was used as the starting material are shown in Figure 7, the results when the inner layer was used as the starting material are shown in Figure 8, and the results when the whole strawberry fruit was used as the starting material are shown in Figure 9.

[0069] The vesicle-containing solution was observed using a TEM in the same manner as in Example 1. The results when the outer layer portion was used as the starting material are shown in Figure 10, the results when the inner layer portion was used as the starting material are shown in Figure 11, and the results when the whole strawberry fruit was used as the starting material are shown in Figure 12. When the whole strawberry fruit was used as the starting material, the number of vesicles was small and the vesicles were buried in impurities. On the other hand, when the solution was separated into the outer layer portion or the inner layer portion, vesicles (particularly spherical, red blood cell-like structures or hollow structures, exosomes or microvesicles) could be clearly observed.

[0070] Example 3. Production of vesicle-containing liquid from apples Apples (commercially available for consumption, stems removed) were separated using a blade into an outer layer (the red surface layer, approximately 0.1 to 0.9 mm thick) and an inner layer (the inner white layer). Using the resulting outer layer and inner layer as starting materials, vesicle-containing liquids were produced in the same manner as in Example 1, except that filtration in step 2 was not performed.

[0071] The vesicle-containing solution was observed by TEM in the same manner as in Example 1. The results when the outer layer portion was used as the starting material are shown in Figure 13, and the results when the inner layer portion was used as the starting material are shown in Figure 14. Vesicles (particularly spherical, red blood cell-like structures or structures with visible hollow spaces, exosomes, or microvesicles) were clearly observed.

[0072] Example 4. Preparation of Vesicle-Containing Solution from Cucumber Fruit 2 The following five vesicle-containing solutions were prepared. (A) Vesicle-containing solution was prepared in the same manner as in Example 1 using the outer layer of cucumber fruit as the starting material and stored at 5°C for 3 days. Centrifugation was not performed during storage. (B) Vesicle-containing solution was prepared in the same manner as in Example 1 using the outer layer of cucumber fruit as the starting material, except that step (3) was not performed. Centrifugation was not performed during storage. (C) Vesicle-containing solution was prepared in the same manner as in Example 1 using whole cucumber fruit as the starting material, except that step (3) was not performed. Centrifugation was performed once during storage (15°C, 15,000 g, 0.5 h). (D) Vesicle-containing solution was prepared in the same manner as in Example 1 using whole cucumber fruit as the starting material and stored at -22°C for 3 days. Centrifugation was not performed during storage. (E) A vesicle-containing solution was prepared in the same manner as in Example 1 using whole cucumber fruits as the starting material and stored at 5°C for 3 days without centrifugation during storage.

[0073] Each of the five vesicle-containing solutions obtained was observed under TEM in the same manner as in Example 1 and evaluated according to the following evaluation criteria. (Evaluation criteria) ◎ The structure of exosomes or microvesicles can be clearly confirmed. △ The structure of exosomes or microvesicles can be confirmed locally. × The structure of exosomes or microvesicles cannot be confirmed, is buried / covered by impurities, or is destroyed.

[0074] The results are shown in Table 1.

[0075]

[0076] Example 5. Production of vesicle solubilized solution and lotion using same A vesicle-containing solution was produced in the same manner as in Example 1 using the outer layer of cucumber fruit as the starting material.

[0077] The resulting vesicle-containing solution was used to prepare a vesicle solubilized solution. Specifically, the procedure is as follows: At room temperature, water, coconut oil fatty acid PEG-7 glyceryl, BG, glycerin, and isopentyldiol were mixed and stirred, and the vesicle-containing solution was added dropwise to prepare a vesicle solubilized solution. The resulting vesicle solubilized solution was transparent.

[0078] A nanoemulsion lotion was prepared using the resulting vesicle solubilized solution or jojoba seed oil. Specifically, the process is as follows: Various ingredients were blended into the water phase at room temperature while stirring with a disperser. Tamarind gum dispersed in BG or glycerin was added and stirred to dissolve. Next, a mixture of cetyl ethylhexanoate, PEG-50 hydrogenated castor oil, sorbitan oleate, DPG, phenoxyethanol, citric acid, sodium citrate, and water (Nicofine CIO (Nikko Chemicals)) was stirred and dissolved. A mixture of PPG-13 decyltetradeceth-24 and phenoxyethanol was stirred and dissolved, and dissolution was confirmed to produce a nanoemulsion lotion. The composition of the lotion is shown in Table 2. The values ​​in the table indicate the percentage (by mass) of each ingredient in the lotion.

[0079]

[0080] The vesicle-containing solution, lotion 1, and lotion 2 were subjected to DLS analysis in the same manner as in Example 1. The results for the vesicle-containing solution are shown in Figure 15, the results for lotion 1 in Figure 16, and the results for lotion 2 in Figure 17. Figures 16 and 17 confirm that lotions 1 and 2 are nanoemulsions. Furthermore, rather than emulsion uptake, it was predicted that vesicles of approximately 100 nm in the vesicle-containing solution, which was a bicontinuous nanoemulsion (BCME bicontinuous microemulsion), were incorporated into nanoemulsions of approximately 20 nm.

[0081] Example 6 Evaluation of Moisturizing Effect Three different lotions (lotions 1-1, 1-2, and 1-3) were prepared using the lotion 1 of Example 5, each using a vesicle-containing liquid from a different production batch.

[0082] The inner forearm of a human was washed with water and left for 20 minutes. One drop of Lotion 1-1, Lotion 1-2, Lotion 1-3, a commercially available lotion containing exosomes (EVs), or a commercially available moisturizing lotion was applied to the inner forearm. Stratum corneum moisture content (measured with a Corneometer, Integral) and transepidermal water loss (measured with a Tewameter, Integral) were measured immediately after application, and at 10, 20, 30, 60, and 120 minutes after application. Measurements were also performed before and after washing (before application).

[0083] The measurement results for stratum corneum moisture content are shown in Figure 18, and the measurement results for transepidermal water loss are shown in Figure 19. When using lotion 1 (lotion 1-1, lotion 1-2, lotion 1-3), the stratum corneum moisture content was significantly higher and transepidermal water loss was comparable compared to when using a commercially available lotion containing EVs or a commercially available moisturizing lotion, suggesting that the skin's barrier function and moisturizing ability were improved.

Claims

1. A method for producing a vesicle-containing liquid, which comprises obtaining a vesicle-containing liquid from a plant material, characterized in that the plant material is the inner layer obtained by removing the outer layer of a fruit or the outer layer obtained by removing the inner layer of a fruit.

2. The method of claim 1, wherein the particle size of the vesicles is 10 to 1000 nm.

3. The method of claim 1, wherein the vesicles comprise extracellular vesicles.

4. The method of claim 1, comprising: (1) crushing and / or pressing the plant material to obtain a crude extract.

5. The method of claim 4, which comprises (2) centrifuging the crude extract to obtain a supernatant.

6. The method of claim 5, wherein the centrifugal force of the centrifugation is 30,000 g or less.

7. The method of claim 5, further comprising: (3) ultrafiltering the supernatant to obtain a filtrate.

8. The method of claim 7, further comprising: (4) fractionating the filtrate by size exclusion chromatography to obtain a vesicle-containing fraction.

9. The method of claim 1, wherein the fruit is the fruit of at least one plant selected from the group consisting of Cucurbitaceae plants and Rosaceae plants.

10. The method according to claim 9, wherein the fruit is at least one fruit selected from the group consisting of Cucurbitaceae plants consisting of cucumber, gourd, watermelon, pumpkin, zucchini, melon, bottle gourd, wax gourd, and bitter melon, and Rosaceae plants consisting of strawberry, apple, pear, cherry, peach, plum, apricot, plum, loquat, and prune.

11. A vesicle-containing liquid obtained by the manufacturing method according to any one of claims 1 to 10.

12. A liquid containing plant-derived vesicles that has been stored under refrigeration but has not been frozen or stored above refrigeration temperatures.

13. A method for producing a vesicle solubilized solution, comprising mixing a plant-derived vesicle-containing solution, a nonionic surfactant, a polyhydric alcohol, and water.

14. A vesicle solubilizing solution comprising a plant-derived vesicle-containing solution, a nonionic surfactant, a polyhydric alcohol, and water.

15. A cosmetic, topical or pharmaceutical composition containing the vesicle solubilized solution obtained by the manufacturing method according to claim 13 or the vesicle solubilized solution according to claim 14.

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