KR102475129B1 - Anti-aging composition comprising ginseng derived exosome like vesicles - Google Patents

Abstract

Disclosed herein is an anti-aging composition comprising ginseng-derived exosome-like vesicles as an active ingredient and a method for preparing the composition. The ginseng-derived exosome-like vesicles may have a diameter of 20 to 500 nm, and may be isolated from ginseng roots, more specifically, ginseng root juice. The composition contains ginseng-derived exosome-like vesicles as an active ingredient, thereby exhibiting anti-aging effects such as improving skin wrinkles, enhancing skin elasticity, and promoting collagen biosynthesis.

Description

Anti-aging composition comprising ginseng-derived exosome-like vesicles as an active ingredient

Disclosed herein is an anti-aging composition comprising ginseng-derived exosome-like vesicles as an active ingredient and a method for preparing the composition.

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

Extracellular vesicles are membrane-structured vesicles ranging in size from about 20 nm in diameter to about 5 μm in diameter, and are heterogeneous in size and composition, exosomes (about 30-100 nm), ectosomes It includes many different species, such as ectosomes, microvesicles (about 100-1,000 nm), microparticles, and apoptotic bodies (about 1-5 μm).

The different types of extracellular vesicles are characterized by their origin, diameter, density in sucrose, shape, sedimentation rate, lipid composition, protein markers, or mode of secretion (i.e., signaling (inducible) or spontaneous (constitutive)). ), etc. For example, a microvesicle is a membrane vesicle having an irregular shape of about 100 to 1,000 nm, which buds toward the outside of the plasma membrane (originating from the plasma membrane), and has a marker including integrin, selectin, CD40 ligand, and lipid including phosphatidylserine. It is known. On the other hand, exosomes are the smallest membranous vesicles having a cup shape of about 30 to 100 nm (<200 nm), budding from the inside of late endosomes (originating from endosomes), CD63, tetraspanin of CD9, TSG101, It is known to have markers including ESCRT, lipids including cholesterol, sphingomyelin, ceramides, and phosphatidylserine.

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

In plants, too, fusion of the plasma membrane with the polytope results in the release of small vesicles into the extracellular space, and in various plant cells of various plant species, intra-polyvesicular vesicles have been observed in the extracellular space (Marchant R, Peat A, Banbury GH. The ultrastructural basis of hyphal growth.New Phytol. R, Robards AW. Membrane systems associated with the plasmalemma of plant cells. Ann Bot. 1968;32:457-471). In addition, recent studies have reported that plant cell-derived exosome-like nanoparticles are similar in nano-sized vesicle structure and nanoparticle composition to mammalian cell-derived exosomes (An , Q, Heckelhoven, R, Kogel, KH and van Bel, AJ (2006).Multivesicular bodies participate in a cell wall-associated defense response in barley leaves attacked by the pathogenic powdery mildew fungus.Cell Microbiol 8: 1009-1019 ) (Regente, M, Pinedo, M, Elizalde, M and de la Canal, L (2012). Apoplastic exosomelike vesicles: a new way of protein secretion in plants? Plant Signal Behav 7: 544-546).

Conventionally, exosomes have been mainly used as biomarkers, and technology for using exosomes for specific purposes using the efficacy of exosomes themselves is still insufficiently developed. In particular, little is known about the specific use of exosome-like membrane-structured vesicles derived from plant cells. In the case of ginseng, the anti-aging efficacy of ginseng-derived extracts or single components has been reported, but ginseng-derived ginseng, a complex bioactive substance The anti-aging efficacy of exosome-like membrane-structured vesicles has not been reported.

On the other hand, skin aging can be largely classified into two types according to the factors. One of them, intrinsic aging, causes the structure and physiological functions of the skin to continuously decline with age, and the other, extrinsic aging, is caused by accumulated external stress such as sunlight. As such, the prior art is anti-aging (anti-aging) of test substances from aging indicators such as aging-related beta-galactosidase (SA-beta-Gal), aging-related factors p21, p16 or MMP-1 gene, which are increased with aging. -aging) effect has been confirmed (Journal of Biological Chemistry Vol. 279, No. 47, pp. 49439-49446, 2004, Loss of Proliferative Capacity and Induction of Senescence in Oxidatively Stressed Human Fibroblasts) (Journal of Cell Science Vol. 118, No. Pt 4, pp. 743-758, 2005, Repeated exposure of human skin fibroblasts to UVB at subcytotoxic level triggers premature senescence through the TGFB signaling pathway)(CANCER RESEARCH Vol. 66, No. 22, pp 10805-10814 , 2006, Oxidative stress induces premature senescence by stimulating caveolin-1 gene transcription through p38 mitogen-activated protein kinase/Sp1-mediated activation of two GC-rich promoter elements) (Journal of Gerontology: BIOLOGICAL SCIENCES Vol. 62A, No. 5, pp. 490-499, 2007, Age-associated declarations e in proteasome content and activities in human dermal fibroblasts: Restoration of Normal Level of Proteasome Subunits Reduces Aging Markers in Fibroblasts From Elderly Persons) (GENES & DEVELOPMENT Vol. 24, no. 22, pp 2463-2479, 2010, The essence of senescence).

All aging is accompanied by structural changes in the skin, and as a result, skin elasticity is lost and wrinkles are formed. Looking more closely at the structural changes of the skin due to aging, the thickness of the epidermis, dermis, and subcutaneous tissue, which are components of the skin, becomes thinner. In addition, the components of the extracellular matrix (ECM) of the dermal tissue responsible for elasticity and tension of the skin are changed. ECM is mainly composed of two components. One is elastic fiber, which accounts for about 2 to 4% of the entire ECM, and the other is collagen, which accounts for about 70 to 80% of the entire ECM. As aging progresses, wrinkles are formed and elasticity of the skin is greatly reduced, which is due to a decrease in collagen and elastin. Collagen and elastin are regulated by various factors. Expression of matrix metallo proteases such as collagenase and elastase degrade collagen and elastin, resulting in This results in a decrease in the amount of collagen in the skin. When collagen and elastin are reduced in the dermis, the epidermis of the skin becomes rough, and wrinkles and elasticity decrease, which are aging phenomena.

Korean Patent Publication No. 10-2008-0038811

In one aspect, an object of the present specification is to provide an anti-aging composition containing, as an active ingredient, a ginseng-derived exosome-like vesicle rather than a ginseng extract.

In another aspect, an object of the present specification is to provide a method for preparing the anti-aging composition.

In one aspect, the technology disclosed herein provides an anti-aging composition comprising ginseng-derived exosome-like vesicle as an active ingredient.

In an exemplary embodiment, the exosome-like vesicle may be isolated from ginseng root.

In an exemplary embodiment, the exosome-like vesicles may be those isolated from the juice of ginseng root.

In an exemplary embodiment, the exosome-like vesicle may have a diameter of 20 to 500 nm.

In an exemplary embodiment, the exosome-like vesicles may be precipitated by ultracentrifugation of ginseng extracellular fluid at 100,000 × g or more.

In an exemplary embodiment, the exosome-like vesicle may have a suspended density of 1.00 to 1.20 g/ml in iodixanol.

In an exemplary embodiment, the active ingredient may (a) improve skin wrinkles; (b) enhancing skin elasticity; and (c) collagen synthesis enhancing properties.

In another aspect, the technology disclosed herein is a method for preparing the anti-aging composition, comprising: (1) extracting ginseng to obtain a juice; (2) obtaining a supernatant by centrifuging the juice; and (3) ultracentrifuging the supernatant to obtain an exosome-like vesicle.

In an exemplary embodiment, the centrifugation in step (2) may be performed at 500 to 3,000 × g for 10 to 30 minutes.

In an exemplary embodiment, the ultracentrifugation in step (3) may be ultracentrifugation using a sucrose cushion density gradient and an iodixanol density gradient.

In an exemplary embodiment, the ultracentrifugation in step (3) may be performed at 100,000 to 200,000 × g for 1 to 6 hours.

In an exemplary embodiment, in step (3), the exosome-like vesicles are separated from iodixanol after ultracentrifugation of the supernatant and a fraction having a suspended density of 1.00 to 1.20 g / ml It may be obtained by including.

In one aspect, the technology disclosed herein has an effect of providing an anti-aging composition comprising ginseng-derived exosome-like vesicle as an active ingredient.

In another aspect, the technology disclosed herein has the effect of providing a method for preparing the anti-aging composition.

1 shows a step-by-step process of separating exosome-like vesicles from ginseng root juice according to an embodiment of the present specification.
2 shows transmission electron microscopy results of ginseng-derived exosome-like vesicles according to an experimental example of the present specification.
3 is a measurement of the activity of aging-related beta-galactosidase (SA-beta-Gal), which increases with aging by treating human fibroblasts with exosome-like vesicles derived from ginseng according to an experimental example of the present specification One result (*, P <0.05).
4 is a result of measuring the expression of aging-related factors p21, p16 and MMP-1 gene by treating human fibroblasts with exosome-like vesicles derived from ginseng according to an experimental example of the present specification (*, P <0.05 ).

Hereinafter, the present invention will be described in detail.

In one aspect, the present specification provides an anti-aging composition comprising ginseng-derived exosome-like vesicle as an active ingredient. The present specification provides an anti-aging composition containing exosome-like vesicles derived from ginseng, specifically, exosome-like vesicles derived from ginseng extracellular fluid, as an active ingredient, rather than a ginseng-derived extract.

In the present specification, "active ingredient" means a component that exhibits the desired activity alone or can exhibit the desired activity in combination with a carrier having no activity itself.

In an exemplary embodiment, the ginseng may be at least one selected from the group consisting of seeds, roots, stems, leaves and fruits of Panax species plants, and specific ginseng plants include Korean ginseng ( Panax ginseng ), Korean ginseng ( Panax quinquefolium ), Korean ginseng ( Panax notoginseng ), bamboo ginseng ( Panax japonicum ), trifolium ginseng ( Panax trifolium ), Himalayan ginseng ( Panax pseudoginseng ) or Vietnamese ginseng ( Panax vietnamensis ). Red ginseng, fresh ginseng, white ginseng, cultivated ginseng, camphor ginseng, wild ginseng, wild ginseng including wild ginseng cultured roots can be used without limitation.

As used herein, "exosome-like vesicle" refers to a nano-sized extracellular vesicle secreted from a cell and released into the extracellular space, that is, an extracellular vesicle. Bit-like vesicles include exosomes and include both nano-sized vesicle structures and vesicles whose composition is similar to exosomes.

The exosome-like vesicle is released into the extracellular space by fusion of the multivesicular body with the plasma membrane by exocytosis. In addition, the exosome-like vesicle is divided into inside and outside by a lipid bilayer composed of cell membrane components, and has cell membrane lipids, membrane proteins, genetic materials, and cytoplasmic components, so that the properties and conditions of the cell can be indirectly identified. allows you to In addition, the exosome-like vesicles bind to other cells and tissues to deliver membrane components, mRNAs, miRNAs, proteins (growth hormones, cytokines, etc.), etc., and deliver these transmitters to recipient cells, resulting in cell-cell It acts as an extracellular transporter that mediates liver communication.

In the present specification, "ginseng-derived exosome-like vesicles" means nano-sized exosome-like vesicles secreted from ginseng cells. The exosome-like vesicles can be isolated from the extracellular fluid of ginseng, and the separated exosome-like vesicles can be wholly or partially physically separated from the original tissues or cells.

In an exemplary embodiment, the exosome-like vesicle may be isolated from ginseng root.

In an exemplary embodiment, the exosome-like vesicles may be those isolated from the juice of ginseng root.

In one aspect, the exosome-like vesicles may be extracellular vesicles having a diameter of 20 to 500 nm. In another aspect, the exosome-like vesicle is 20 nm or more, 30 nm or more, 40 nm or more, 50 nm or more, 60 nm or more, 70 nm or more, 80 nm or more, 90 nm or more, or 100 nm or more, and 500 nm Hereinafter, it may be an extracellular vesicle having a diameter of 450 nm or less, 400 nm or less, 350 nm or less, 300 nm or less, 250 nm or less, 200 nm or less, 150 nm or less, or 100 nm or less.

In another aspect, the exosome-like vesicles may be sedimented in an ultracentrifugation of 100,000 × g or more, specifically, 100,000 to 200,000 × g, or 100,000 × g of the liquid outside the ginseng cells, that is, ginseng extracellular fluid.

In another aspect, the exosome-like vesicles may have a floating density in iodixanol of 1.00 to 1.20 g/ml, or 1.03 to 1.20 g/ml, or 1.06 to 1.20 g/ml. The floating density refers to the density measured by the density gradient centrifugation method.

In another aspect, the exosome-like vesicle may be, for example, a membrane component chemically or physically modified to efficiently perform a desired function in a target cell. For example, the membrane component of the exosome-like vesicle is chemically modified using a thiol group (-SH) or an amine group (-NH ₂ ), or a target-inducing substance or cell membrane fusion material is used in the exosome-like vesicle. , The membrane component may be chemically modified by chemically combining polyethylene glycol.

In an exemplary embodiment, the exosome-like vesicles are subjected to ultracentrifugation, differential centrifugation, equilibrium density centrifugation, density gradient, filtration It can be separated from ginseng using one or more methods selected from the group consisting of , dialysis and free-flow electrophoresis, but the method for separating exosome-like vesicles is not limited thereto.

Density gradient is the most commonly used method for distinguishing materials having different densities, and exosome-like vesicles according to the present specification are classified in density and can be separated through the density gradient. A specific example of this method may be carried out using a density gradient separation material such as ficoll, glycerol, sucrose, cesium chloride, or iodixanol. It is not limited. In one aspect, density gradients may be used in conjunction with ultracentrifugation or the like. In another aspect, gel filtration or ultrafiltration may be used to select exosome-like vesicles. In another aspect, dialysis can be used instead of filtration to remove small molecules. In another aspect, free flow electrophoresis can be used.

In an exemplary embodiment, the ginseng-derived exosome-like vesicle may be prepared by: (1) obtaining a juice containing extracellular fluid of ginseng from ginseng; (2) centrifuging the sap to remove residues and obtaining a supernatant; and (3) ultracentrifuging the supernatant to obtain exosome-like vesicles.

In an exemplary embodiment, the juice in step (1) is a liquid extracted from ginseng, and can be obtained by appropriately using methods such as mechanical degradation and chemical treatment. In one aspect, the juice may be obtained by squeezing ginseng, and the process for preparing the juice may be appropriately selected and performed by a person skilled in the art according to a method known in the art.

In an exemplary embodiment, the centrifugation in step (2) may be performed at 500 to 3,000 × g for 10 to 30 minutes. At this time, the centrifugation may be performed in stages by changing the speed or time.

In an exemplary embodiment, the ultracentrifugation in step (3) may be ultracentrifugation using a sucrose cushion density gradient and an iodixanol density gradient.

In an exemplary embodiment, the ultracentrifugation in step (3) may be performed at 100,000 to 200,000 × g for 1 to 6 hours. At this time, the ultracentrifugation may be performed stepwise by changing the speed or time.

In an exemplary embodiment, the ultracentrifugation in step (3) is performed at 100,000 to 200,000 × g for 1 to 6 hours by ultracentrifugation using a sucrose cushion density gradient and a second using an iodixanol density gradient. It can be carried out through a series of ultracentrifugation processes including centrifugation.

In an exemplary embodiment, in step (3), the exosome-like vesicles are separated into fractions having a floating density of 1.00 to 1.20 g/ml obtained by fractionation after ultracentrifugation of the supernatant. It can be obtained including. At this time, the separated fraction may further include diluting in a buffer solution and ultracentrifuging, specifically ultracentrifuging at 100,000 × g for 2 hours to obtain pelleted exosomes having a diameter of 20 to 500 nm Pseudo-vesicles can be separated.

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

The present specification also relates to a composition comprising a ginseng-derived exosome-like vesicle as an active ingredient and having a lyophilized formulation; And sterilized water or purified water; it provides an anti-aging kit containing. The kit may be in a ready-to-use sealed package or packaging container.

Aging is meant to include all skin changes that occur with age, and in this specification, “anti-aging” refers to aging caused by internal factors including genetic factors and external factors including ultraviolet rays and/or the like. or to prevent, delay or ameliorate the symptoms of aging. For example, it refers to preventing, delaying, improving, or alleviating symptoms such as skin changes caused by aging, such as reduction in skin elasticity, wrinkle formation, increase in the depth or number of wrinkles, collagen reduction, and dryness.

In an exemplary embodiment, the active ingredient may (a) improve skin wrinkles; (b) enhancing skin elasticity; and (c) enhancing collagen synthesis.

In another aspect, the technology disclosed herein provides an amount of ginseng-derived exosome-like vesicles effective for preventing and/or delaying aging and/or preventing, delaying, and/or improving aging symptoms to a subject in need thereof. Provided are methods for preventing and/or delaying aging, and/or preventing, delaying, and/or improving aging phenomena, including administering. In another aspect, the technology disclosed herein provides an amount of ginseng-derived exosome-like vesicles effective for preventing and/or delaying aging, and/or preventing, delaying, and/or improving aging phenomena, to a subject in need thereof Provided is a method for preventing, delaying, and/or improving skin wrinkle formation, skin elasticity reduction, and/or collagen reduction, comprising administering to a subject. In another aspect, the technology disclosed herein provides an amount of ginseng-derived exosome-like vesicles effective for preventing, delaying, and/or improving skin wrinkles, reduction in skin elasticity, and/or reduction in collagen to a subject in need thereof. Provided is a method for preventing, retarding and/or ameliorating skin wrinkling, reducing skin elasticity and/or reducing collagen, comprising administering.

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

In addition to ginseng-derived exosome-like vesicles, the pharmaceutical composition may further contain pharmaceutical adjuvants and other therapeutically useful substances such as preservatives, stabilizers, hydration or emulsification accelerators, salts and/or buffers for osmotic pressure control, and , It can be formulated into various oral or parenteral dosage forms according to conventional methods.

The oral administration agent includes, for example, tablets, pills, hard and soft capsules, solutions, suspensions, emulsifiers, syrups, powders, powders, granules, granules, pellets, etc., and these formulations contain surfactants in addition to active ingredients , diluents (eg lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and glycine), lubricants (eg silica, talc, stearic acid and its magnesium or calcium salts and polyethylene glycol). . Tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and polyvinylpyrrolidine, optionally starch, agar, alginic acid or its sodium salt. and pharmaceutical additives such as disintegrants, absorbents, colorants, flavoring agents, and sweeteners. The tablets may be prepared by conventional mixing, granulating or coating methods.

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

Determination of the dose of the active ingredient is within the level of a skilled person, and the daily dose of the drug varies depending on various factors such as the degree of progression of the subject to be administered, the time of onset, age, health condition, and complications, but adult Based on this, in one aspect, 1 μg/kg to 200 mg/kg of the composition, and in another aspect, 50 μg/kg to 50 mg/kg may be divided and administered 1 to 3 times a day, and the dosage does not limit the scope of the present invention in any way.

The pharmaceutical composition may be an external skin preparation, and the external skin preparation is a general term that may include anything that is applied outside the skin, and may include pharmaceuticals of various formulations.

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

In addition to the ginseng-derived exosome-like vesicles, the cosmetic composition may further include functional additives and components included in general cosmetic compositions. The functional additive may include a component selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, high-molecular peptides, high-molecular polysaccharides, sphingolipids, and seaweed extracts. Ingredients other than those included include fats and oils, moisturizers, emollients, surfactants, organic and inorganic pigments, organic powders, ultraviolet absorbers, preservatives, bactericides, antioxidants, plant extracts, pH adjusters, alcohols, pigments, fragrances, blood circulation accelerators, cooling agents, antiperspirants, purified water and the like.

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

When the formulation of the present invention is a paste, cream or gel, animal fiber, vegetable fiber, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as a carrier component. can

When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and in particular, in the case of a spray, additionally chlorofluorohydrocarbon, propane / May contain a propellant such as butane or dimethyl ether.

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

When the formulation of the present invention is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, an ethoxylated isostearyl alcohol, a suspending agent such as polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline Cellulose, aluminum metahydroxide, bentonite, agar or tracanth and the like may be used.

When the formulation of the present invention is a surfactant-containing cleanser, as carrier components, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, linolin derivatives, or ethoxylated glycerol fatty acid esters may be used.

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

The food composition may be a liquid or solid formulation, for example, various foods, beverages, gum, tea, vitamin complexes, health supplements, etc., and may be used in the form of powders, granules, tablets, capsules or beverages. can The food composition of each formulation can be selected and blended without difficulty by a person skilled in the art according to the formulation or purpose of use other than the active ingredient, and synergistic effects can occur when applied simultaneously with other ingredients.

There is no particular limitation on liquid ingredients that can be contained in addition to the active ingredients disclosed in this specification, and various flavoring agents or natural carbohydrates may be included as additional ingredients, like conventional beverages. Examples of the natural carbohydrate include monosaccharides, disaccharides such as glucose and fructose, polysaccharides such as maltose and sucrose, common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. etc. As the flavoring agent, natural flavoring agents (thaumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (eg, saccharin, aspartame, etc.) can be advantageously used. The proportion of the natural carbohydrates may be generally about 1 to 20 g, and in one aspect about 5 to 12 g per 100 ml of the composition disclosed herein.

In one aspect, the food composition includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and enhancers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, and the like may be included. In another aspect, it may include fruit pulp for the preparation of natural fruit juices and vegetable beverages. These components may be used independently or in combination. The proportions of the additives may vary, but are generally selected from the range of 0.001 to about 20 parts by weight per 100 parts by weight of the composition disclosed herein.

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.

Example 1.

(Step 1) First, 4-year-old ginseng roots were prepared and juiced to prepare a juice containing extracellular fluid of ginseng roots.

(Step 2) The juice was centrifuged at 4° C. and 500×g for 10 minutes to obtain a supernatant, which was centrifuged again at 4° C. and 3,000×g for 20 minutes to obtain a supernatant.

(Step 3) 0.5 ml of 2.5 M sucrose, 1 ml of 0.8 M sucrose, and 32 ml of the supernatant from which foreign substances were removed were sequentially put into a 35 ml ultracentrifugation tube, and 4°C, 100,000 × g was ultracentrifuged for 2 hours. After the ultracentrifugation, the ginseng root exosome-like vesicles were located between the 2.5 M sucrose layer and the 0.8 M sucrose layer by their density. Accordingly, the layer containing ginseng root exosome-like vesicles was separated by removing the solution from the top of the tube.

(Step 4) Density gradient ultracentrifugation was additionally performed on the layer containing the separated ginseng root exosome-like vesicles. Specifically, 0.42 ml of the layer containing the separated ginseng root exosome-like vesicles and 2.08 ml of 60% iodixanol were mixed to finally make 2.5 ml of 50% iodixanol, put in a 12 ml ultra-high-speed centrifugal tube, and then 35 2.5 ml of % iodixanol, 2.5 ml of 20% iodixanol, and 2.5 ml of 150 mM NaCl/20 mM HEPES were stacked sequentially and ultracentrifuged at 4 °C and 200,000 ×g for 4 hours. Thereafter, a fraction was obtained by 1 ml from the top of the tube and the density was measured to obtain fraction 3 (1.08 g/ml) corresponding to the exosome-like vesicle density (see FIG. 1).

(Step 5) The obtained fraction 3 was diluted in 60 ml of HBS (HEPES-buffered saline), placed in a 70 ml ultracentrifuge tube, and pelletized by ultracentrifugation at 4°C and 100,000 × g for 2 hours. ginseng-derived exosome-like vesicles were obtained.

Experimental example 1.

The size and shape of the ginseng-derived exosome-like vesicles obtained in Example 1 were analyzed using a transmission electron microscope (TEM).

2 is a transmission electron microscope image of ginseng-derived exosome-like vesicles, and it was confirmed that the ginseng-derived exosome-like vesicles had a diameter of 50 to 100 nm and were generally spherical.

Experimental example 2.

Aging-related beta-galactosidase (SA) increased with aging by treating dermal fibroblasts with ginseng-derived exosome-like vesicles (1-10 μg/ml) obtained in Example 1 -beta-Gal) activity was measured.

Specifically, human fibroblasts (adult skin fibroblasts, purchased from Lonza) were placed in a 60-mm plate culture medium at 2 × 10 ⁴ cells/well and cultured in DMEM medium supplemented with 10% FBS. After 24 hours, the medium was replaced with ginseng-derived exosome-like vesicles 1 μg/ml, 10 μg/ml, or compound K 1 ppm, respectively, and a new medium (ginseng-derived exosome-like vesicles or compound K 1 ppm) was replaced every 4 days. containing K). Cells were harvested on day 18 of culture, and beta-galactosidase activity was measured using a kit (purchased from ThermoFisher Scientific) for measuring the activity of senescence-associated beta-galactosidase (SA-beta-Gal). Specifically, cells were harvested using M-PER provided in the kit, and β-Galactosidase Assay Reagent was added to the cells, followed by reaction at 37 °C for 6 hours, and then absorbance was measured at a wavelength of 405 nm.

As a result, as shown in FIG. 3, it was confirmed that the ginseng-derived exosome-like vesicles had an anti-aging effect by reducing the activity of beta-galactosidase.

Experimental example 3.

Human dermal fibroblasts were treated with ginseng-derived exosome-like vesicles (1-10 μg/ml) obtained in Example 1 to measure the expression of aging-related factors p21, p16 and MMP-1 genes did

Specifically, human fibroblasts (adult skin fibroblasts, purchased from Lonza) were placed in a 60-mm plate culture medium at 2 × 10 ⁴ cells/well and cultured in DMEM medium supplemented with 10% FBS. After 24 hours, the medium was replaced with a medium containing 1 μg/ml and 10 μg/ml of ginseng-derived exosome-like vesicles, respectively, and was replaced with a new medium (containing ginseng-derived exosome-like vesicles) every 4 days. Cells were harvested on day 31 of culture, RNA was isolated, and cDNA was synthesized through RT-PCR (reverse transcriptional polymerase chain reaction). Thereafter, Taqman real-time PCR was performed using the synthesized cDNA to measure the expression of the aging-related factors p21, p16, and MMP-1 relative to the expression of a house-keeping gene (GAPDH).

As a result, as shown in FIG. 4 , it was confirmed that the ginseng-derived exosome-like vesicles had an anti-aging effect by reducing the expression of the aging-related factors p21 and p16 and the MMP-1 gene.

Formulation examples of the composition according to one aspect of the present specification will be described below, but can also be applied to various other formulations, which are only intended to be specifically described and not intended to limit the present invention.

[Formulation Example 1] Soft capsule preparation

Ginseng-derived exosome-like vesicles 50mg, L-carnitine 80-140mg, soybean oil 180mg, palm oil 2mg, vegetable hydrogenated oil 8mg, yellow wax 4mg, and lecithin 6mg were mixed, and 400mg per capsule was filled according to the usual method to obtain a soft capsule. manufactured.

[Formulation Example 2] Tablets

50 mg of ginseng-derived exosome-like vesicles, 200 mg of galacto-oligosaccharide, 60 mg of lactose, and 140 mg of maltose were mixed and granulated using a fluidized bed dryer, and then 6 mg of sugar ester was added and tableted with a tableting machine to prepare tablets.

[Formulation Example 3] Granules

50 mg of ginseng-derived exosome-like vesicles, 250 mg of anhydrous crystalline glucose, and 550 mg of starch were mixed, molded into granules using a fluid bed granulator, and filled into bags to prepare granules.

[Formulation Example 4] Drinking agent

After mixing 50 mg of ginseng-derived exosome-like vesicles, 10 g of glucose, 0.6 g of citric acid, and 25 g of liquid oligosaccharide, 300 ml of purified water was added and each bottle was filled with 200 ml. After filling the bottle, it was sterilized at 130 ° C. for 4 to 5 seconds to prepare a drink beverage.

[Formulation Example 5] Lotion

A lotion was prepared in a conventional manner according to the composition shown in Table 1 below.

[ formulation example 6] Cream

Cream was prepared in a conventional manner according to the composition shown in Table 2 below.

[ formulation example 7] Pack

A pack was prepared in a conventional manner according to the composition shown in Table 3 below.

In the above, specific parts of the present invention have been described in detail, and for those skilled in the art, these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. It will be clear. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (14)

A cosmetic composition for improving skin wrinkles, enhancing skin elasticity, and/or enhancing collagen synthesis, comprising exosome-like vesicles derived from ginseng as an active ingredient.
According to claim 1,
The exosome-like vesicle is a cosmetic composition for improving skin wrinkles, enhancing skin elasticity and / or enhancing collagen synthesis, which is isolated from ginseng root.
According to claim 1,
The exosome-like vesicle is a cosmetic composition for improving skin wrinkles, enhancing skin elasticity and / or enhancing collagen synthesis, which is isolated from the juice of ginseng root.
According to claim 1,
The exosome-like vesicles having a diameter of 20 to 500 nm, improving skin wrinkles, improving skin elasticity and / or enhancing collagen synthesis cosmetic composition.
According to claim 1,
The exosome-like vesicles are precipitated in ultracentrifugation of 100,000 × g or more of ginseng extracellular fluid, a cosmetic composition for improving skin wrinkles, enhancing skin elasticity and / or enhancing collagen synthesis.
According to claim 1,
The exosome-like vesicles have a floating density of 1.00 to 1.20 g / ml in iodixanol, a cosmetic composition for improving skin wrinkles, enhancing skin elasticity and / or enhancing collagen synthesis.
A food composition for improving skin wrinkles, enhancing skin elasticity, and/or enhancing collagen synthesis, comprising exosome-like vesicles derived from ginseng as an active ingredient.
According to claim 7,
The exosome-like vesicle is a food composition for improving skin wrinkles, enhancing skin elasticity and / or enhancing collagen synthesis, which is isolated from ginseng root.
According to claim 7,
The exosome-like vesicle is a food composition for improving skin wrinkles, enhancing skin elasticity and / or enhancing collagen synthesis, which is isolated from the juice of ginseng root.
According to claim 7,
The exosome-like vesicles having a diameter of 20 to 500 nm, improving skin wrinkles, improving skin elasticity and / or food composition for enhancing collagen synthesis.
According to claim 7,
The exosome-like vesicles are precipitated in ultracentrifugation of 100,000 × g or more of ginseng extracellular fluid, a food composition for improving skin wrinkles, improving skin elasticity and / or enhancing collagen synthesis.
According to claim 7,
The exosome-like vesicles have a suspended density of 1.00 to 1.20 g / ml in iodixanol, a food composition for improving skin wrinkles, enhancing skin elasticity and / or enhancing collagen synthesis. delete delete

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