A cosmetic composition containing as active ingredients a plant stem cell culture extract and osmocell containing lactic acid bacteria exosomes

Abstract

The present invention relates to a cosmetic composition containing a plant stem cell culture extract and an osmocell encapsulating lactic acid bacteria exosomes. Specifically, the present invention relates to a cosmetic composition for preventing skin aging, which is prepared by forming a lipid complex paste from cell membrane lipid components and water using a high-pressure emulsifier, and then adding the lipid complex to the aqueous solution of plant stem cell culture extract and lactic acid bacteria exosomes at room temperature to phase-invert the reverse micelle emulsion and stabilize it.

Description

[Technical field]

[0001] The present invention relates to a cosmetic composition for preventing skin aging, which contains as active ingredients a plant stem cell culture extract and osmocells encapsulating lactic acid bacteria exosomes. [Background technology]

[0002] Before skin aging, the physiological activity of skin cells is active, and in fibroblasts in the dermis, collagen, which is important for skin elasticity and wrinkle prevention, is actively synthesized and metabolized, and GAGS (Glucosaminoglycans), a glycoprotein containing hyaluronic acid, which is essential for maintaining skin moisture, is also actively synthesized, giving the skin elasticity, softness, flexibility and moisture. In addition, in the epidermis, proliferation of basal layer cells is active, and adhesion proteins such as laminin, integrin and desmosomes, which strengthen the bonds between skin cells, are produced in a balanced manner, strengthening the skin cell tissue and maintaining healthy skin. However, the skin of modern people is exposed to various harmful external environments and stresses, which causes various damages, and with age, the physiological activity of the skin decreases, the recovery speed of damaged skin slows, and various skin aging phenomena such as loss of elasticity, dryness, wrinkle formation, pigmentation, age spots, dullness, etc. Therefore, the main goal of cosmetics is to improve these skin aging symptoms, and various cosmetics have been developed for this purpose.

[0003] In general, skin aging phenomena such as loss of elasticity, skin wrinkles, and pigmentation are caused by collagen destruction, decreased collagen synthesis, loss of elastin, and melanin pigmentation due to decreased skin physiology, and can be considered as phenomena occurring in the epidermis and dermis of the skin. Recently, skin physiology research on preventing skin aging has been actively conducted, and in fact, UV blocking agents, antioxidants, cell activity promoters, etc. are used in cosmetics to prevent skin wrinkles. In addition, a method for improving skin wrinkles by collagen synthesis has been proposed. In addition, based on the development of biochemistry and molecular biology, small amounts of signal substances such as growth factors present in the human body have been discovered, and the theory of biological aging based on this has been re-established. Furthermore, this signal substance decreases in the body with age, and it has been revealed that the decrease in this growth factor is closely related to human aging. Such growth factors are produced in large quantities by stem cells present at the base of each tissue.

[0004] The use of stem cells in the field of cosmetics is to utilize growth factors as their metabolic products. Growth factors are proteins that bind to receptors on the surface of cells and stimulate cell proliferation and differentiation. The functions of growth factors are very diverse, and they can stimulate the division of various cells, but in some cases they can only act on specific cell types. It has been revealed that growth factors regulate cell differentiation, migration, and proliferation processes as essential components for stimulating cell proliferation. When a growth factor binds to a specific receptor, a signal transduction chain is activated and a signal is transmitted from the outside of the cell to the nucleus inside. As the signal transduction continues, it acts with a large number of other messengers and finally synthesizes new proteins. Thus, growth factors form a signal transduction network that is important in transmitting interactions between cells and controlling their functions. In addition, in the results of a skin wound regeneration experiment using mice, it was confirmed that the size of the wound was reduced by more than 40% when stem cell-derived proteins were applied compared to when they were not applied. Furthermore, good results were shown in the whitening effect on subjects with pigmentation, and a strong effect as a cosmetic ingredient was investigated, but ethical and safety issues remain to be resolved.

[0005] Korean Patent Publication No. 10-2007-0000005 discloses a functional cosmetic composition for wrinkle improvement and / or anti-aging, which contains allogeneic and / or autologous adipocytes, stem cells isolated from allogeneic and / or autologous adipocytes, or fibroblast growth factors isolated therefrom. Korean Patent Publication No. 10-848056 discloses a cosmetic composition for whitening, which contains a stem cell culture medium or a protein isolated from the culture medium. Korean Patent Publication No. 10-2008-0075959 discloses an additive for injections, which contains a conditioned medium obtained by culturing human adipose stem cells in a serum-free medium containing antioxidants, vitamins, amino acids, and minerals for at least one day, and is used in injections containing human stem cells for tissue regeneration.

[0006] However, the safety of such human-derived stem cells has not been ensured, and there are still limitations to their safe use as cosmetic compositions, and in particular, there are many restrictions on their future use due to remaining ethical issues. Furthermore, various active ingredients contained in human-derived stem cell culture extracts, more specifically, various cell growth factors, have difficulty in penetrating the skin, and unlike their excellent effectiveness for medical treatment purposes, their effectiveness as cosmetics is limited in healthy skin. Therefore, in the field of dermatology, in order to improve skin aging, human-derived stem cell culture extracts are currently injected directly into the skin, or fine holes are made in the skin using special equipment to induce skin penetration, but there are various skin safety issues in this process, making it difficult to use this method as a cosmetic.

[0007] Plants also have stem cells, and like human stem cells, plant stem cells have strong differentiation and regeneration capabilities. In addition, plant stem cells do not have the ethical and safety issues that human stem cells do, and interest in the use of plant stem cells has been increasing recently.

[0008] Plants have stem cells in the shoot apical meristem (SAM) and root apical meristem (RAM). Plant stem cells differentiate based on their unique properties and function to regenerate plant tissues and organs such as roots and stems. These plant stem cells are classified as pluripotent stem cells and have the ability to form various organs due to their typical functions. Stem cells in the SAM and RAM differentiate to grow plants, and the differentiation of stem cells and the maintenance of stem cell density are regulated by the relationship with the neighboring organizing center (OC) or quiescent center (QC) cells in the SAM and RAM. Unlike animals, plants were found to have totipotency in 1902 by Australian botanist Gottlieb Haberlandt. Totipotency in plants refers to the ability of plant cells to differentiate into other types of cells or the entire plant body.

[0009] Callus cells, which are undifferentiated cells, can be obtained by de-differentiating the plant body. Callus cells can be obtained by surface sterilizing the plant body, cutting and incising it, and culturing it in a solidified medium containing an appropriate concentration of plant hormones. The callus cells can be cultured in that state or in a liquid medium to form a suspension culture, which allows a large number of cells to be obtained. Callus cells can generate embryos and form roots and shoots again by adjusting the concentration of plant hormones in the medium. The embryos, shoots, and roots thus generated develop into complete plants by germination or growth. Therefore, callus cells, which are undifferentiated cells, have the ability of stem cells and are called stem cells. In terms of application as target cells for cell therapy, mesenchymal stem cells have many advantages over embryonic stem cells in that they are relatively easy and safe to collect and can be autotransplanted.

[0010] In the present invention, callus, which is a plant stem cell, is a wound-healing tissue in which cells recover their ability to divide, prevent injury, and swell when the plant body is injured. It refers to an undifferentiated tissue or cell mass that is generated when tissue excised from a plant body is cultured in a medium containing auxin, or when a certain type of plant is wounded or the wound site is treated with auxin, and can continue to divide and grow by being subcultured in a new medium.

[0011] Furthermore, there is growing interest in exosomes, which contain approximately 150 types of growth factor protein components, such as EGF (epidermal growth factor), vEGF (vascular endothelial growth factor), TGF (transforming growth factor), HGF (hepatocyte growth factor), FGF (fibroblast growth factor), and IGF (insulin-like growth factor).

[0012] Exosomes are intercellular communication substances secreted extracellularly from all cells, from microorganisms to humans, and are nano-sized extracellular vesicles with a size of 50 to 200 nm. Such nano-sized exosomes can transport membrane proteins that are insoluble in water. Membrane proteins are proteins that exist in phospholipid bilayer membranes and are hydrophobic, so intercellular transmission through blood is basically difficult. In contrast, exosomes are made of phospholipid bilayer membranes and can transport membrane proteins (Korean Patent Registration No. 10-2253418). In addition, exosomes can protect their contents from external degrading substances by surrounding them with a phospholipid bilayer membrane. A typical example is the RNA transport mechanism. RNA present outside the cell is degraded, but RNA present in exosomes is protected from these substances and can be smoothly delivered to the target cell. Technologies relating to methods for obtaining exosomes include Korean Patent Registration No. 10-2125567, entitled "Method for mass-producing plant exosomes," Korean Patent Registration No. 10-2047768, entitled "Culture medium containing highly pure and highly concentrated exosomes derived from mesenchymal stem cells and method for producing the same," Korean Patent Registration No. 10-1895916, entitled "Method for producing exosomes and / or extracellular vesicles and compositions containing the same," and Korean Patent Registration No. 10-1980482, entitled "Multiple columns for isolating exosomes and method for isolating exosomes." Summary of the Invention [Problem to be solved by the invention]

[0013] Therefore, the inventors have made extensive efforts to find a substance that improves skin aging prevention. As a result, they have overcome the ethical and safety issues associated with human-derived stem cell culture extracts, and the problem that the various cell growth factors contained in human-derived stem cell culture extracts are difficult to penetrate into the skin, resulting in limited effectiveness in healthy skin. They have discovered that a plant stem cell culture extract containing ingredients that match the active ingredients in human-derived stem cell culture extracts and lactic acid bacteria exosomes containing approximately 150 types of growth factor protein ingredients, such as EGF (epidermal growth factor), vEGF (vascular endothelial growth factor), TGF (transforming growth factor), HGF (hepatocyte growth factor), FGF (fibroblast growth factor), and IGF (insulin-like growth factor), which are active ingredients in human-derived stem cell culture extracts, are highly effective in improving skin aging phenomena such as wrinkles, loss of elasticity, loss of moisture content, and pigmentation, and have completed the present invention.

[0014] The object of the present invention is to provide a cosmetic composition which contains as active ingredients a plant stem cell culture extract and osmocell encapsulating lactic acid bacteria exosomes, and which has excellent anti-aging effects for skin and improves skin troubles. [Means for solving the problem]

[0015] The above-mentioned objectives can be achieved by a cosmetic composition for preventing skin aging, which contains as an active ingredient Osmocell, a lipid complex consisting of phospholipids, phytosphingosine, ceramide NP, cholesterol, phytosterol, β-sitosterol, and squalene, which contains a plant stem cell culture extract and lactic acid bacteria exosomes encapsulated therein.

[0016] Preferably, the plant stem cell culture extract may include two or more selected from the group consisting of wild ginseng stem cell culture extract, ginkgo stem cell culture extract, sophora japonica stem cell culture extract, green tea stem cell culture extract, edelweiss stem cell culture extract, grape stem cell culture extract, tomato stem cell culture extract, rose stem cell culture extract, aloe stem cell culture extract, and rice stem cell culture extract.

[0017] Also, preferably, the lactic acid bacteria exosomes can be isolated from a Lactobacillus plantarum culture medium.

[0018] Furthermore, preferably, the osmocell contains, relative to the total weight of the osmocell, 0.1 to 10 wt% lipid complex, 0.01 to 50.0 wt% plant stem cell culture extract, 0.00001 to 1.0 wt% lactic acid bacteria exosomes, and the remaining amount of purified water, and the lipid complex may contain, relative to the total weight of the lipid complex, 0.1 to 30.0 wt% phospholipid, 0.01 to 20.0 wt% phytosphingosine, 0.01 to 30.0 wt% ceramide NP, 0.01 to 10.0 wt% cholesterol, 0.01 to 10.0 wt% phytosterol, 0.01 to 10.0 wt% β-sitosterol, and 0.01 to 90.0 wt% squalene.

[0019] Preferably, the osmocell may be contained in an amount of 0.001 to 100.0% by weight based on the total weight of the entire cosmetic composition.

[0020] Also, preferably, the osmocell can be prepared by a method including the steps of dissolving phospholipid, phytosphingosine, ceramide NP, cholesterol, phytosterol, β-sitosterol and squalene under high pressure to form a translucent gel-like lipid complex, and mixing the lipid complex, the plant stem cell culture extract and the lactic acid bacteria exosome at room temperature.

[0021] Also, preferably, the cosmetic composition has an effect of improving skin wrinkles, a whitening effect, and an effect of improving skin elasticity. Effect of the Invention

[0022] The cosmetic composition according to the present invention contains a stabilized osmocell containing a plant stem cell culture extract and lactic acid bacteria exosomes as an active ingredient, and has a superior delivery effect to the skin and a superior effect of preventing skin aging and improving skin troubles compared to conventional cosmetics encapsulated in liposomes. Furthermore, since the active ingredient is stabilized in osmocell at room temperature, it is possible to prevent the active ingredient from being destroyed by heat and maximize the effect of the cosmetic. [Brief description of the drawings]

[0023] [Figure 1] FIG. 1 shows the particle distribution of osmocells containing a plant stem cell culture extract and lactic acid bacteria exosomes according to the present invention. [Diagram 2] FIG. 2 is an SEM image of osmocell containing plant stem cell culture extract and lactic acid bacteria exosomes produced in Production Example 1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] The present invention relates to a cosmetic composition for preventing skin aging, which contains a plant stem cell culture extract and Osmocell encapsulating lactic acid bacteria exosomes, and which improves skin aging and skin troubles. In the present invention, preventing skin aging comprehensively includes wrinkle improvement, skin whitening, and elasticity improvement effects, and improving skin troubles means alleviating skin inflammation symptoms such as erythema, edema, and itchiness that occur on the skin.

[0025] The present invention provides a cosmetic composition for preventing skin aging, which contains as active ingredients a plant stem cell culture extract and osmocell containing lactic acid bacteria exosomes.

[0026] The plant stem cell culture extract of the present invention is obtained by sterilizing the surfaces of wild ginseng roots, ginkgo stems, Chinese sophora stems, green tea stems, edelweiss flowers, grape stems, tomato fruits, rose flowers, aloe stems and rice stems, cutting and wounding them, culturing them in a solidified medium containing an appropriate concentration of plant hormone to induce callus cells, and culturing the induced callus cells in a liquid medium for suspension culture. The plant stem cell culture extracts of the present invention are wild ginseng stem cell culture extract, ginkgo stem cell culture extract, sophora japonica stem cell culture extract, green tea stem cell culture extract, edelweiss stem cell culture extract, grape stem cell culture extract, tomato stem cell culture extract, rose stem cell culture extract, aloe stem cell culture extract, and rice stem cell culture extract, which are obtained by crushing wild ginseng stem cells (callus), ginkgo stem cells (callus), sophora japonica stem cells (callus), green tea stem cell culture extract, edelweiss stem cell culture extract, grape stem cell culture extract, tomato stem cell culture extract, rose stem cell culture extract, aloe stem cell culture extract, and rice stem cell culture extract.

[0027] According to one embodiment of the present invention, the plant stem cell culture extract can be prepared by the following method. First, the flowers, stems, fruits or roots of the plant are immersed in 70% (v / v) ethanol for 30 seconds, sterilized by shaking in 2% (v / v) sodium hypochlorite for 5 minutes, and then washed with sterile water to disinfect the surface. Next, the surface of the flowers, stems, fruits or roots of the plant is scratched with a sharp knife, cut, and inoculated on M&S (Murashige & Skoog's) solid medium containing growth regulators of IAA (indole-3-acetic acid) 0.5 mg / L, zeatin 0.2 mg / L, sucrose 30 g / L, and gelite 2.0 g / L, and then cultured in a dark culture room at 25±2°C for 4 weeks to induce stem cells. The induction of callus, which is a plant stem cell, was maximized by appropriately applying the plant hormones auxin and cytokinin at concentrations of 0.01 mg / L to 10 mg / L, preferably 0.1 mg / L to 1 mg / L, to M&S (Murashige & Skoog's) solid medium. In order to maximize the amount of callus, which is the induced totipotent embryogenic stem cell, a liquid culture capable of increasing the growth rate was subcultured every two weeks in dark conditions. A suspension culture vessel (gyrotory shaker) for liquid culture is inoculated with 0.1 g / L to 20 g / L, preferably 1 g / L to 5 g / L, of the callus induced using the above-mentioned M&S liquid medium, and a large amount of stem cells (callus) can be obtained by performing suspension culture. In the final subculture stage, the multiplied callus was cultured in the suspension culture vessel under dark conditions at 25 ± 2°C using MS medium without the addition of hormones. A bioreactor can also be used for mass production. The plant stem cells thus obtained are washed 2 to 3 times with purified water to remove medium components, and the plant stem cells (callus) are then crushed at high speed at 8,000 to 15,000 rpm for 2 to 3 minutes at low temperature in a frozen stock container using a homogenizer. The crushed and squeezed-extracted plant callus is then filtered under reduced pressure using filter paper to obtain the plant stem cell (callus) culture extract of the present invention.

[0028] The lactic acid bacteria exosomes according to the present invention are derived from intestinal lactic acid bacteria such as Lactobacillus acidophilus, Bifidobacterium lactis, Lactobacillus rhamnosus, Bifidobacterium longum, Bifidobacterium breve, Lactobacillus casei, Lactobacillus Salivarius, Lactobacillus reuteri, and Lactobacillus plantarum, which are also present on the skin. plantarum) was cultured in sterilized milk for 48 hours and isolated from the culture medium.

[0029] According to one embodiment of the present invention, the lactic acid bacteria exosomes can be obtained by mixing lactic acid bacteria Lactobacillus plantarum with sterilized milk and culturing for 48 hours, centrifuging the lactic acid bacteria culture solution at 1,500 to 2,000 g for 20 to 30 minutes using a centrifuge, taking only the supernatant culture solution to separate and remove the lactic acid bacteria, centrifuging the supernatant culture solution again at 10,000 to 15,000 g for 20 to 30 minutes using an ultra-high speed centrifuge, taking the supernatant to separate and remove the lactic acid bacteria and residual matter, centrifuging the supernatant again at 100,000 to 150,000 g for 2 to 4 hours using an ultra-high speed centrifuge, sedimenting the exosomes into a pellet layer, and resuspending the pellet layer in PBS using a pipette and vortex to obtain exosomes.

[0030] The present invention is characterized in that a lipid complex composed of lipid components constituting cell membranes is first produced to stabilize the heat-labile active ingredients, plant stem cell culture extract and lactic acid bacteria exosomes, and to facilitate percutaneous absorption, and then the plant stem cell culture extract, which is heat-labile at room temperature, and the active ingredients in the lactic acid bacteria exosomes are stably encapsulated and incorporated into the cosmetic.

[0031] Unlike conventional liposomes which contain lecithin or a lecithin-like emulsifier, an oily component and an aqueous component to form a bilayer membrane, the OsmoCELL of the present invention is composed only of the lipid components which make up the skin cell membrane, and by encapsulating the plant stem cell culture extract and lactic acid bacteria exosomes of the present invention, OsmoCELL, which has a bilayer membrane structure similar to that of a cell membrane, has been completed.

[0032] The osmocell of the present invention is easy to penetrate into the skin, can encapsulate an excess amount of water-soluble ingredients compared to liposomes, and is stable. The osmocell of the present invention means an osmocell that encapsulates the physiologically active ingredients of the present invention in a vesicle formed from phospholipids, phytosphingosine, ceramide NP, cholesterol, phytosterol, β-sitosterol, and squalene, which are the main lipid components constituting cell membranes. Such an osmocell has higher affinity for the skin and is more stable than conventional liposomes.

[0033] Moreover, osmocell refers to nano-sized bilayer vesicles having an average droplet size of 20 to 500 nm, preferably 50 to 250 nm, and most preferably 100 to 200 nm. When the average droplet size of the osmocell exceeds 500 nm, the skin penetration and stability of the formulation are very low. In the present invention, the term "osmocell" refers to nano-sized bilayer vesicles stabilized by encapsulating the plant stem cell culture extract and lactic acid bacteria exosomes of the present invention.

[0034] The osmocell of the present invention is produced by the following method.

[0035] Phospholipids, phytosphingosine, ceramide NP, cholesterol, phytosterol, β-sitosterol, and squalene, which are the main lipid components constituting cell membranes, are dissolved under high pressure using a high-pressure emulsifier (microfludizer) to form a translucent gel-like lipid complex. Next, an appropriate amount of this lipid complex is added to an aqueous solution in which the plant stem cell culture extract and lactic acid bacteria exosomes are dispersed at room temperature, and mixed to obtain an osmocell containing the plant stem cell culture extract and lactic acid bacteria exosomes of the present invention having a closed bilayer structure. In this case, the reaction process is performed at room temperature. The conventional liposome production process using a high-pressure emulsifier (microfludizer) has the disadvantage that liposomes are formed at high temperatures (60°C or higher), making it impossible to ensure the thermal stability of temperature-sensitive components. The present invention uses a high-pressure emulsifier to mix materials that form a lipid complex, but is different from the use of a high-pressure emulsifier to form liposomes in the prior art. In the present invention, lipid complexes that are dissolved and mixed in a high-pressure emulsifier are formed, and these form vesicles in the aqueous phase and encapsulate the active ingredients dispersed in the aqueous solution, i.e., plant stem cell culture extract and lactic acid bacteria exosomes, to form osmosels.

[0036] The osmocell contains, relative to the total weight of the osmocell, 0.1 to 10% by weight of a lipid complex, 0.01 to 50.0% by weight of a plant stem cell culture extract, 0.00001 to 1.0% by weight of lactic acid bacteria exosomes, and the remaining amount being purified water.

[0037] More preferably, the content of the plant stem cell culture extract may be 0.1 to 10.0% by weight based on the total weight of the osmocell, and the content of the lactic acid bacteria exosomes is 0.0005 to 0.1% by weight based on the total weight of the osmocell.

[0038] The lipid complex is composed of phospholipid, phytosphingosine, ceramide NP, cholesterol, phytosterol, β-sitosterol and squalene. The phospholipid is contained in an amount of 0.1 to 30.0% by weight based on the total weight of the lipid complex, and preferably in an amount of 1.0 to 10.0% by weight based on the total weight. The phytosphingosine is contained in an amount of 0.01 to 20.0% by weight based on the total weight of the lipid complex, and preferably in an amount of 0.1 to 5.0% by weight based on the total weight. The ceramide NP is contained in an amount of 0.01 to 30.0% by weight based on the total weight of the lipid complex, and preferably in an amount of 0.1 to 10.0% by weight based on the total weight. The cholesterol is contained in an amount of 0.01 to 10.0% by weight based on the total weight of the lipid complex, and preferably in an amount of 0.1 to 5.0% by weight based on the total weight. The content of phytosterol is 0.01 to 10.0% by weight based on the total weight of the lipid complex, and preferably 0.1 to 5.0% by weight based on the total weight. The content of β-sitosterol is 0.01 to 10.0% by weight based on the total weight of the lipid complex, and preferably 0.1 to 5.0% by weight based on the total weight. The content of squalene is 0.01 to 90.0% by weight based on the total weight of the lipid complex, and preferably 10.0 to 70.0% by weight based on the total weight.

[0039] The amount of the plant stem cell culture extract and osmocell encapsulating lactic acid bacteria exosomes according to the present invention is 0.001 to 100.0 wt% relative to the total weight of the cosmetic composition, and preferably 10.0 to 60.0 wt% relative to the total weight of the cosmetic composition.

[0040] The components contained in the cosmetic composition of the present invention may include, but are not limited to, various components that maintain the activity of biological components other than the plant stem cell culture extract and lactic acid bacteria exosomes as active ingredients, components commonly used in cosmetic compositions, such as conventional adjuvants such as antioxidants, stabilizers, solubilizers, vitamins, pigments and fragrances, and carriers.

[0041] The cosmetic composition of the present invention may also be in the form of a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleanser, oil, powder foundation, emulsion foundation, wax foundation, or spray.

[0042] According to a specific example of the present invention, the cosmetic composition of the present invention, which contains osmocell that encapsulates plant stem cell culture extract and lactic acid bacteria exosomes as active ingredients, has a better delivery effect to the skin than conventional cosmetics containing liposomes, and can provide better anti-aging and improved skin trouble effects. In addition, since the active ingredient is stabilized in osmocell at room temperature, the thermal stability of the active ingredient can be ensured, maximizing the efficacy of the cosmetic.

[0043] The present invention will be described in detail below with reference to Preparation Examples and Examples, but the scope of the present invention is not limited by such Preparation Examples and Examples. For the cosmetic composition of the present invention, a plant stem cell culture extract, lactic acid bacteria exosomes, and an osmocell encapsulating a plant stem cell culture extract and lactic acid bacteria exosomes were produced as in the following Preparation Examples.

[0044] Manufacturing Example 1-1: Manufacturing of Panax Ginseng stem cell culture extract In order to induce stem cells (callus) from wild ginseng roots, the wild ginseng roots were immersed in 70% (v / v) ethanol for 30 seconds, sterilized by shaking in 2% (v / v) sodium hypochlorite for 5 minutes, and then washed with sterile water to disinfect the surface. The surface of the disinfected wild ginseng roots was scratched with a sharp knife, cut, and inoculated on M&S (Murashige & Skoog's) solid medium containing growth regulators IAA (indole-3-acetic acid) 0.5 mg / L, zeatin 0.2 mg / L, sucrose 30 g / L, and gelite 2.0 g / L, and then cultured in a dark incubation room at 25 ± 2°C for 12 weeks to induce wild ginseng stem cells. The induction of wild ginseng callus, which is a wild ginseng stem cell, was maximized by adding 0.5 mg / L of plant hormones auxin and cytokinin to M&S (Murashige & Skoog's) solid medium. In order to maximize the amount of induced wild ginseng callus, liquid culture capable of increasing the growth rate was subcultured and propagated in dark conditions every 4 weeks. A large amount of wild ginseng stem cells (callus) was obtained by inoculating 2.5 g / L of wild ginseng callus induced using the above-mentioned M&S liquid medium into a suspension culture vessel for liquid culture and carrying out suspension culture. The propagated wild ginseng callus was cultured in the suspension culture vessel under dark conditions at 25 ± 2°C using MS medium without added hormones in the final subculture stage. The wild ginseng stem cells thus obtained were washed twice with purified water to remove medium components, and the wild ginseng stem cells (callus) were crushed at high speed at 10,000 rpm for 2 minutes in a frozen storage container using a homogenizer, and the crushed and squeezed wild ginseng callus was filtered under reduced pressure using filter paper to obtain the wild ginseng stem cell (callus) culture extract of the present invention.

[0045] Production Example 1-2: Production of Ginkgo Biloba Stem Cell Culture Extract In order to induce stem cells (callus) from ginkgo stems, the ginkgo stems were immersed in 70% (v / v) ethanol for 30 seconds, sterilized by shaking in 2% (v / v) sodium hypochlorite for 5 minutes, and then washed with sterile water to disinfect the surface. The surface of the disinfected ginkgo stems was scratched with a sharp knife, cut, and inoculated on M&S (Murashige & Skoog's) solid medium containing growth regulators IAA (indole-3-acetic acid) 0.5 mg / L, zeatin 0.2 mg / L, sucrose 30 g / L, and gelite 2.0 g / L, and then cultured in a dark incubation room at 25 ± 2 °C for 12 weeks to induce ginkgo stem cells. The induction of ginkgo stem cells (ginkgo callus) was maximized by appropriately applying the plant hormones auxin and cytokinin at a concentration of 0.5mg / L to M&S (Murashige & Skoog's) solid medium. In order to maximize the amount of induced ginkgo callus, liquid cultures capable of accelerating growth were grown by subculture every 4 weeks in dark conditions. A suspension culture vessel (Gyrotory Shaker) for liquid culture was inoculated with 2.5g / L of ginkgo callus induced using the above-mentioned M&S liquid medium and suspension culture was performed to secure a large amount of ginkgo stem cells (callus), and the grown ginkgo callus was cultured in the suspension culture vessel under dark conditions at 25±2℃ using MS medium without added hormones at the final subculture stage. The ginkgo stem cells thus obtained are washed twice with purified water to remove medium components, and the ginkgo stem cells (callus) are then crushed at high speed at 10,000 rpm for 2 minutes in a frozen storage container at low temperature using a homogenizer. The crushed and squeezed-extracted ginkgo callus is then filtered under reduced pressure using filter paper to obtain the ginkgo stem cell (callus) culture extract of the present invention.

[0046] Production Example 1-3: Production of Sophora Japonica stem cell culture extract To induce stem cells (callus) from the stems of Sophora japonica, the stems were immersed in 70% (v / v) ethanol for 30 seconds, sterilized by shaking in 2% (v / v) sodium hypochlorite for 5 minutes, and then washed with sterile water to disinfect the surface. The surface of the disinfected stems of Sophora japonica was scratched with a sharp knife, cut, and inoculated on M&S (Murashige & Skoog's) solid medium containing growth regulators IAA (indole-3-acetic acid) 0.5 mg / L, zeatin 0.2 mg / L, sucrose 30 g / L, and gelite 2.0 g / L, and then cultured in a dark incubation room at 25 ± 2 °C for 12 weeks to induce Sophora japonica stem cells. The induction of the soybean stem cell, soybean stem cell, soybean stem cell, soybean stem cell, soybean stem cell, soybean stem cell, was maximized by applying the plant hormones auxin and cytokinin at a concentration of 0.5mg / L to M&S (Murashige & Skoog's) solid medium. In order to maximize the amount of induced soybean stem cell, soybean stem cell, soybean stem cell, was grown by subculture every 4 weeks in a liquid culture that can increase the growth rate in a dark condition. A suspension culture vessel (gyrotory shaker) for liquid culture was inoculated with 2.5g / L of the soybean stem cell induced using the above-mentioned M&S liquid medium and cultured in suspension, thereby obtaining a large amount of soybean stem cell (callus). In the final subculture step, the grown soybean stem cell was cultured in the suspension culture vessel under dark conditions at 25±2℃ using MS medium without the addition of hormones. The Solanium stem cells thus obtained are washed twice with purified water to remove medium components, and the Solanium stem cells (callus) are then crushed at high speed at 10,000 rpm for 2 minutes at low temperature in a frozen storage container using a homogenizer. The crushed and squeezed Solanium callus is then filtered under reduced pressure using filter paper to obtain the Solanium stem cell (callus) culture extract of the present invention.

[0047] Production Example 1-4: Production of green tea (Camellia Sinensis) stem cell culture extract In order to induce stem cells (callus) from green tea stems, the green tea stems were immersed in 70% (v / v) ethanol for 30 seconds, sterilized by shaking in 2% (v / v) sodium hypochlorite for 5 minutes, and then washed with sterile water to disinfect the surface. The surfaces of the sterilized green tea stems were scratched with a sharp knife, cut, and inoculated on M&S (Murashige & Skoog's) solid medium containing growth regulators IAA (indole-3-acetic acid) 0.5 mg / L, zeatin 0.2 mg / L, sucrose 30 g / L, and gelite 2.0 g / L, and then cultured in a dark incubation room at 25 ± 2 °C for 12 weeks to induce green tea stem cells. The induction of green tea callus, which is a green tea stem cell, was maximized by appropriately applying the plant hormones auxin and cytokinin at a concentration of 0.5mg / L to M&S (Murashige & Skoog's) solid medium. In order to maximize the amount of induced green tea callus, liquid cultures that can increase the growth rate were propagated by subculture every 4 weeks in dark conditions. A suspension culture vessel (Gyrotory Shaker) for liquid culture was inoculated with 2.5g / L of the green tea callus induced using the above-mentioned M&S liquid medium and suspension culture was performed to secure a large amount of green tea stem cells (callus), and the propagated green tea callus was cultured in the suspension culture vessel under dark conditions at 25±2℃ using MS medium without added hormones at the final subculture stage. The green tea stem cells thus obtained are washed twice with purified water to remove medium components, and the green tea stem cells (callus) are then crushed at high speed at 10,000 rpm for 2 minutes at low temperature using a homogenizer in a frozen storage container. The crushed and squeezed-extracted green tea callus is then filtered under reduced pressure using filter paper to obtain the green tea stem cell (callus) culture extract of the present invention.

[0048] Preparation Example 1-5: Preparation of Edelweiss (Leontopodium Alpinum) Stem Cell Culture Extract The edelweiss (Leontopodium Alpinum) stem cell culture extract of the present invention was used to induce stem cells (callus) from edelweiss flowers by immersing the edelweiss flowers in 70% (v / v) ethanol for 30 seconds, sterilizing them by shaking them in 2% (v / v) sodium hypochlorite for 5 minutes, and then washing them with sterile water to disinfect the surface. The surface of the disinfected edelweiss flowers was scratched with a sharp knife, cut, and inoculated on M&S (Murashige & Skoog's) solid medium containing growth regulators IAA (indole-3-acetic acid) 0.5 mg / L, zeatin 0.2 mg / L, sucrose 30 g / L, and gelite 2.0 g / L, and then cultured in a dark incubation room at 25 ± 2 °C for 4 weeks to induce edelweiss stem cells. The plant hormones auxin and cytokinin were appropriately applied to M&S (Murashige & Skoog's) solid medium at a concentration of 0.5 mg / L to maximize the induction of edelweiss stem cells, or edelweiss calluses. In order to maximize the amount of induced edelweiss calluses, they were propagated by subculturing in liquid culture every 2 weeks in dark conditions, which can increase the growth rate. A suspension culture vessel (gyrotory shaker) for liquid culture was inoculated with 2.5 g / L of the edelweiss calluses induced using the above-mentioned M&S liquid medium and suspension cultured to secure a large amount of edelweiss stem cells (callus), and the propagated edelweiss calluses were cultured in the suspension culture vessel under dark conditions at 25 ± 2 °C using MS medium without added hormones at the final subculture stage. The edelweiss stem cells thus obtained are washed twice with purified water to remove medium components, and the edelweiss stem cells (callus) are then crushed at high speed at 10,000 rpm for 2 minutes at low temperature in a frozen storage container using a homogenizer. The crushed and squeezed-extracted edelweiss callus is then filtered under reduced pressure using filter paper to obtain the edelweiss stem cell (callus) culture extract of the present invention.

[0049] Manufacturing Example 1-6: Manufacturing of grape (Vitis Vinifera) stem cell culture extract In order to induce stem cells (callus) from grape stems, the grape stems were immersed in 70% ethanol for 30 seconds, sterilized by shaking in 2% sodium hypochlorite for 5 minutes, and then washed with sterile water to disinfect the surface. The disinfected grape stems were cut after scratching the surface with a sharp knife, and inoculated on M&S (Murashige & Skoog's) solid medium containing growth regulators IAA (indole-3-acetic acid) 0.5 mg / L, zeatin 0.2 mg / L, sucrose 30 g / L, and gelite 2.0 g / L, and then cultured in a dark incubation room at 25 ± 2 °C for 6 weeks to induce grape stem cells. The plant hormones auxin and cytokinin were appropriately applied to M&S (Murashige & Skoog's) solid medium at a concentration of 0.5 mg / L to maximize the induction of grape stem cells, that is, grape callus. In order to maximize the amount of induced grape callus, liquid culture capable of increasing the growth rate was subcultured and propagated in dark conditions every 2 weeks. A large amount of grape stem cells (callus) could be obtained by inoculating 2.5 g / L of grape callus induced using the above-mentioned M&S liquid medium into a suspension culture vessel (Gyrotory Shaker) for liquid culture and carrying out suspension culture. The propagated grape callus was cultured in the suspension culture vessel under dark conditions at 25 ± 2 ° C. using MS medium without added hormones in the final subculture stage. The grape stem cells thus obtained were washed twice with purified water to remove medium components, and the grape stem cells (callus) were crushed at high speed at low temperature for 2 minutes using a homogenizer in a frozen storage container at 10,000 rpm, and the crushed and squeezed grape callus was filtered under reduced pressure using filter paper to obtain the grape stem cell (callus) culture extract of the present invention.

[0050] Production Example 1-7: Production of tomato (Solanum Lycopersicum) stem cell culture extract To induce stem cells (callus) from tomato (Solanum Lycopersicum) fruit, the tomato was immersed in 70% (v / v) ethanol for 30 seconds, sterilized by shaking in 2% (v / v) sodium hypochlorite for 5 minutes, and then washed with sterile water to disinfect the surface. The surface of the disinfected tomato was scratched with a sharp knife, cut, and inoculated on M&S (Murashige & Skoog's) solid medium containing growth regulators IAA (indole-3-acetic acid) 0.5 mg / L, zeatin 0.2 mg / L, sucrose 30 g / L, and gelite 2.0 g / L, and then cultured in a dark incubation room at 25 ± 2 °C for 3 weeks to induce tomato stem cells. The induction of tomato stem cell, tomato callus, was maximized by appropriately applying the plant hormones auxin and cytokinin at a concentration of 0.5 mg / L to M&S (Murashige & Skoog's) solid medium. In order to maximize the amount of induced tomato callus, liquid culture capable of increasing the growth rate was subcultured and propagated in dark conditions every 2 weeks. A suspension culture vessel (Gyrotory Shaker) for liquid culture was inoculated with 2.5 g / L of the tomato callus induced using the above-mentioned M&S liquid medium and suspension cultured to obtain a large amount of tomato stem cells (callus), and the propagated tomato callus was cultured in the suspension culture vessel under dark conditions at 25 ± 2 ° C. using MS medium to which no hormones were added in the final subculture stage. The tomato stem cells thus obtained were washed twice with purified water to remove medium components, and the tomato stem cells (callus) were crushed at high speed at low temperature for 2 minutes using a homogenizer in a frozen storage container at 10,000 rpm, and the crushed and squeezed tomato callus was filtered under reduced pressure using filter paper to obtain the tomato stem cell (callus) culture extract of the present invention.

[0051] Preparation Example 1-8: Preparation of Rose (Rosa Hybrid) Stem Cell Culture Extract In order to induce stem cells (callus) from rose flowers, the rose flowers were immersed in 70% (v / v) ethanol for 30 seconds, sterilized by shaking in 2% (v / v) sodium hypochlorite for 5 minutes, and then washed with sterile water to disinfect the surface. The surface of the disinfected rose flowers was scratched with a sharp knife, cut, and inoculated on M&S (Murashige & Skoog's) solid medium containing growth regulators IAA (indole-3-acetic acid) 0.5 mg / L, zeatin 0.2 mg / L, sucrose 30 g / L, and gelite 2.0 g / L, and then cultured in a dark incubation room at 25 ± 2 °C for 4 weeks to induce rose stem cells. The induction of rose stem cell, rose callus, was maximized by appropriately applying the plant hormones auxin and cytokinin at a concentration of 0.5 mg / L to M&S (Murashige & Skoog's) solid medium. In order to maximize the amount of induced rose callus, liquid culture capable of increasing the growth rate was subcultured and propagated in dark conditions every 2 weeks. A suspension culture vessel (gyrotory shaker) for liquid culture was inoculated with 2.5 g / L of rose callus induced using the above-mentioned M&S liquid medium and suspension cultured to obtain a large amount of rose stem cells (callus), and the propagated rose callus was cultured in the suspension culture vessel under dark conditions at 25 ± 2 ° C using MS medium to which no hormones were added in the final subculture stage. The rose stem cells thus obtained were washed twice with purified water to remove medium components, and the rose stem cells (callus) were crushed at high speed at low temperature for 2 minutes using a homogenizer in a frozen storage container at 10,000 rpm, and the crushed and squeezed rose callus was filtered under reduced pressure using filter paper to obtain the rose stem cell (callus) culture extract of the present invention.

[0052] Preparation Example 1-9: Preparation of Aloe Barbadensis stem cell culture extract In order to induce stem cells (callus) from aloe stems, the stems of aloe were immersed in 70% (v / v) ethanol for 30 seconds, sterilized by shaking in 2% (v / v) sodium hypochlorite for 5 minutes, and then washed with sterile water to disinfect the surface. The surface of the sterilized aloe stems was scratched with a sharp knife, cut, and inoculated on M&S (Murashige & Skoog's) solid medium containing growth regulators IAA (indole-3-acetic acid) 0.5 mg / L, zeatin 0.2 mg / L, sucrose 30 g / L, and gelite 2.0 g / L, and then cultured in a dark incubation room at 25 ± 2 °C for 3 weeks to induce aloe stem cells. The induction of aloe callus, which is an aloe stem cell, was maximized by appropriately applying the plant hormones auxin and cytokinin at a concentration of 0.5mg / L to M&S (Murashige & Skoog's) solid medium. In order to maximize the amount of induced aloe callus, liquid cultures that can increase the growth rate were propagated by subculture every two weeks in dark conditions. A suspension culture vessel (gyrotory shaker) for liquid culture was inoculated with 2.5g / L of aloe callus induced using the above-mentioned M&S liquid medium and suspension culture was performed to secure a large amount of aloe stem cells (callus), and the propagated aloe callus was cultured in the suspension culture vessel under dark conditions at 25±2℃ using MS medium without added hormones at the final subculture stage. The aloe stem cells thus obtained are washed twice with purified water to remove medium components, and the aloe stem cells (callus) are then crushed at high speed at 10,000 rpm for 2 minutes at low temperature in a frozen storage container using a homogenizer. The crushed and squeezed-extracted aloe callus is then filtered under reduced pressure using filter paper to obtain the aloe stem cell (callus) culture extract of the present invention.

[0053] Preparation Example 1-10: Preparation of rice (Oryza Sativa) stem cell culture extract In order to induce stem cells (callus) from rice stems, the rice stems were immersed in 70% (v / v) ethanol for 30 seconds, sterilized by shaking in 2% (v / v) sodium hypochlorite for 5 minutes, and then washed with sterile water to disinfect the surface. The disinfected rice stems were cut after scratching the surface with a sharp knife, and inoculated on M&S (Murashige & Skoog's) solid medium containing growth regulators IAA (indole-3-acetic acid) 0.5 mg / L, zeatin 0.2 mg / L, sucrose 30 g / L, and gelite 2.0 g / L, and then cultured in a dark incubation room at 25 ± 2 °C for 2 weeks to induce rice stem cells. The induction of rice callus, which is a rice stem cell, was maximized by appropriately applying the plant hormones auxin and cytokinin at a concentration of 0.5mg / L to M&S (Murashige & Skoog's) solid medium. In order to maximize the amount of induced rice callus, liquid cultures that can increase the growth rate were propagated by subculture every week under dark conditions. A suspension culture vessel (gyrotory shaker) for liquid culture was inoculated with 2.5g / L of rice callus induced using the above-mentioned M&S liquid medium and suspension cultured to secure a large amount of rice stem cells (callus), and the propagated rice callus was cultured in the suspension culture vessel under dark conditions at 25±2℃ using MS medium without added hormones at the final subculture stage. The rice stem cells thus obtained are washed twice with purified water to remove medium components, and the rice stem cells (callus) are then crushed at high speed at 10,000 rpm for 2 minutes at low temperature in a frozen storage container using a homogenizer. The crushed and squeezed-extracted rice callus is then filtered under reduced pressure using filter paper to obtain the rice stem cell (callus) culture extract of the present invention.

[0054] Production example 2: Production of lactic acid bacteria exosomes The lactobacillus exosome of the present invention is prepared by mixing lactobacillus Lactobacillus plantarum with sterilized milk and culturing for 48 hours, centrifuging the lactobacillus culture solution at 1,7000g for 25 minutes using a centrifuge, and then taking only the supernatant culture solution to separate and remove the lactobacillus. The supernatant culture solution is again centrifuged at 12,000g for 25 minutes using an ultra-high speed centrifuge, and the supernatant solution is taken to separate and remove the lactobacillus and residual substances. The supernatant solution is again centrifuged at 120,000g for 3 hours using an ultra-high speed centrifuge, and the exosomes are precipitated into the pellet layer, and the pellet layer is resuspended in PBS using a pipette and vortex to separate the exosomes from the intestinal lactobacillus culture solution, thereby obtaining the lactobacillus exosome of the present invention.

[0055] Production Example 3: Production of osmocells containing plant stem cell culture extract and lactic acid bacteria exosomes Osmocell was prepared by passing phospholipid, phytosphingosine, ceramide NP, cholesterol, phytosterol, β-sitosterol, and squalene through a microfluidizer three times at a pressure of 1,000 bar in the amounts shown in Table 1 below to produce a translucent gel-like lipid complex. A solution was prepared by dispersing the 10 plant stem cell culture extracts presented in Preparation Example 1 of the present invention and the lactic acid bacteria exosomes of Preparation Example 2 in an aqueous solution. Next, this lipid complex was added to the aqueous solution in which the plant stem cell culture extract and the lactic acid bacteria exosomes were dispersed at room temperature as shown in Table 2 to produce an osmocell containing the plant stem cell culture extract and the lactic acid bacteria exosomes. Figure 1 shows the particle distribution of the plant stem cell culture extract and osmocell encapsulating lactic acid bacteria exosomes from Production Example 3. Figure 2 shows SEM images of the plant stem cell culture extract and osmocell encapsulating lactic acid bacteria exosomes from Production Example 3.

[0056] [Table 1]

[0057] [Table 2]

[0058] In order to investigate the particle size distribution of osmocell particles encapsulating the plant stem cell culture extract and lactic acid bacteria exosomes of the present invention, Experimental Example 1 was carried out as described below.

[0059] Experimental Example 1 In order to analyze the particle size distribution of the osmocell particles of the present invention, the osmocell obtained in Preparation Example 3 was diluted to an appropriate concentration with a 10% Microplasma Hyopnemoniae OD410=0.1 buffer solution, and the particle size of the osmocell particles was measured. The particle size of the osmocell particles was analyzed by light scattering using a Zetasizer NS, and the results are shown in the graph of FIG. 1. The particle size of the osmocell thus obtained is 120 nm on average, and the particle size uniformity is improved compared to liposomes prepared using a conventional high-pressure emulsifier (microfluidizer), thereby improving stability. Furthermore, FIG. 2 is a SEM photograph of the osmocell encapsulating the plant stem cell culture extract and lactic acid bacteria exosomes prepared in Preparation Example 3.

[0060] As shown in Table 3 below, a composition containing the plant stem cell culture extract produced in Preparation Example 3 and osmocells encapsulating lactic acid bacteria exosomes (Example 1), and a composition containing a plant stem cell culture extract and a conventional liposome containing lactic acid bacteria exosomes (Comparative Example 1) were produced.

[0061] Conventional liposomes in Comparative Example 1 were prepared by mixing 3.0% lecithin, 10% propylene glycol, 10% ethyl alcohol, and 3% mineral oil, and then dissolving by heating to 6° C. Then, an aqueous solution containing 10% plant stem cell culture extract and 0.001% lactic acid bacteria exosomes was poured into the dissolving solution and stirred, and then passed through a high-pressure emulsifier (microfluidizer) twice at a pressure of 1,000 bar to obtain liposomes.

[0062] [Table 3]

[0063] Experimental Example 2 The skin wrinkle improving effect (instrumental evaluation) of cosmetics containing the plant stem cell culture extract of the present invention and osmocell encapsulating lactic acid bacteria exosomes was measured. 20 women aged 20 years or older (average age 39.5 years) were divided into two groups (A and B). Group A was administered the Example and Group B was administered the Comparative Example according to Table 3. 2 The area was applied (twice a day, 0.2 g / time) for 8 weeks, and a replica of the skin wrinkles was made using a transparent silicone solution, and changes in the skin wrinkles were measured using a skin wrinkle measuring device (SKIN VISIOMETER SV400, C+K Electronics GmbH, Germany). The replica images were analyzed in 3D using a CCD camera, and the roughness (R m The average wrinkle roughness (R z The wrinkle improvement effect was analyzed. The test results regarding the wrinkle improvement effect on the skin after 6 weeks are shown in Table 4 below.

[0064]

number

[0065] [Table 4]

[0066] Test results: average wrinkle roughness (R z ) in the case of the Example containing plant stem cell culture extract and osmocell encapsulating lactobacillus exosomes was reduced by 87.3 μm (p<0.01), and wrinkles were improved by about 106.9% more than in the Comparative Example containing plant stem cell culture extract and general liposomes encapsulating lactobacillus exosomes. Therefore, from the above results, it can be seen that Example 1 containing plant stem cell culture extract and osmocell encapsulating lactobacillus exosomes was much more effective in improving wrinkles than Comparative Example 1 containing general liposomes.

[0067] Experimental Example 3 A visual evaluation was carried out on the effect of cosmetics containing the plant stem cell culture extract of the present invention and osmocell encapsulating lactic acid bacteria exosomes on improving skin wrinkles.

[0068] 20 women aged 20 years or older (average age 39.5 years) were divided into two groups (A and B), and Example 1 was applied to Group A and Comparative Example 1 to Group B according to the formulations in Table 3 above, and the formulations were applied (twice a day) to areas around the eyes where wrinkles are present for 8 weeks. The degree of improvement in skin wrinkles (△W) was measured by classifying and evaluating the objective evaluation by an expert and the subjective evaluation by the subject into the following 6 grades. The clinical effects of improving skin wrinkles are shown in Table 5 (objective evaluation by an expert) and Table 6 (subjective evaluation by the subject) below. <Skin Wrinkle Improvement Evaluation Criteria> -3: Extremely severely worse; -2: Worsened; -1: Slightly worse; 0: No change; 1: Slightly improved; 2: Improved; 3: Extremely improved

[0069] [Table 5]

[0070] [Table 6]

[0071] As a result of the naked eye evaluation, in the case of Example 1, the expert's objective evaluation and the subjective evaluation were 2.7 and 2.7, respectively, indicating that the wrinkle improvement effect was very excellent. This shows an increase of 68.8% in the expert's objective evaluation and 50% in the subjective evaluation compared to Comparative Example 1, in which the liposomes contained a plant stem cell culture extract and lactic acid bacteria exosomes, indicating that the cosmetic composition of the present invention, in which the plant stem cell culture extract and lactic acid bacteria exosomes are encapsulated in the osmocell, improves wrinkles much more effectively than conventional cosmetic compositions in which the liposomes contain the plant stem cell culture extract and lactic acid bacteria exosomes.

[0072] Experimental Example 4 The skin whitening effect was measured by measuring the inhibitory effect on melanin production in melanocytes of osmocells (Production Example 3) containing plant stem cell culture extract and lactic acid bacteria exosomes. B-16 melanoma (ATCC CRL 6323) cell line derived from mouse was purchased and used as melanocytes. The melanoma cell line was inoculated into DMEM medium containing 4.5g / L glucose, 10% serum, and 1% antibiotic, and cultured in a 50ml T-flask at 37℃. After 24 hours of culture under 5% CO2 conditions, the cells were separated by treatment with 0.05% trypsin containing 0.02% EDTA, and then inoculated into a 50ml T-flask and cultured for 48 hours. The number of cells in this case was 5.76 x 10 6Cells / flask. Appropriate concentrations of plant stem cell culture extract and osmocells encapsulating lactobacillus exosomes (Production Example 3) and general liposomes encapsulating plant stem cell culture extract and lactobacillus exosomes are diluted in DMEM medium and treated with cultured melanoma cells and cultured at 37°C for 5 days. The amounts of 10% by weight of plant stem cell culture extract and 0.001% by weight of lactobacillus exosomes used for encapsulation in osmocells and general liposomes were the same to obtain osmocells and general liposomes to be applied in this experiment. After culturing, all of the medium was removed, and the cells were separated by treating with 1 ml of phosphate buffered saline (PBS) containing 0.02% EDTA and 0.05% trypsin, and then centrifuged for 5 minutes to collect only the cells. Treat with 5% trichloroacetic acid (TCA), stir, and then centrifuged to wash the precipitated melanin with phosphate buffered saline.

[0073] The precipitated melanin was dissolved by treatment with 1N NaOH, and the absorbance was measured at 475 nm. The melanin concentration was determined from a standard concentration curve of synthetic melanin (Sigma). The experimental results are shown in Table 7.

[0074] [Table 7]

[0075] From the above results, it can be seen that osmocell containing plant stem cell culture extract and lactobacillus exosomes has a higher melanin synthesis inhibition effect than plant stem cell culture extract and lactobacillus exosomes encapsulated in general liposomes. This is because the manufacturing process of osmocell containing plant stem cell culture extract and lactobacillus exosomes is carried out at room temperature, whereas the manufacturing process of conventional general liposomes is carried out at high temperatures (60°C or higher) when passing through a high-pressure emulsifier (microfludizer), which reduces or denatures the activity of temperature-sensitive components such as growth factors due to the heat generated. This experimental example shows that plant stem cell culture extract and lactobacillus exosomes have an excellent skin whitening effect by suppressing melanin production in melanocytes.

[0076] Experimental Example 5 The skin elasticity enhancing effect of cosmetics containing the plant stem cell culture extract of the present invention and osmocell containing lactic acid bacteria exosomes was measured. 20 women over 20 years old (average age 39.5 years old) were divided into two groups (A and B) under conditions of temperature 24 to 26 ° C and humidity 75%, and Example 1 was formulated for Group A and Comparative Example 1 was formulated for Group B according to Table 3 above, and applied for 8 weeks (2 times / day) mainly around the eyes, and then skin elasticity was measured using a skin elasticity measuring device (Cutometer SEM 575, C + K Electronic Co., Germany). The test results are shown in Table 8 below as β week-R8 (0 week)) values ​​of Cutometer SEM 575. The R8 value indicates the nature of skin viscoelasticity.

[0077] [Table 8]

[0078] From the above results, it can be seen that the embodiment containing osmocell encapsulating plant stem cell culture extract and lactic acid bacteria exosomes has a high effect of promoting skin elasticity, increasing skin elasticity by 77.5% compared to the comparative embodiment containing general liposomes encapsulating plant stem cell culture extract and lactic acid bacteria exosomes.

[0079] Experimental Example 6 The skin penetration effect of the plant stem cell culture extract of the present invention and osmocells encapsulating lactic acid bacteria exosomes was measured.

[0080] Human normal fibroblast 1x10 5The cells were inoculated into a collagen gel structure similar to the fibrous tissue that constitutes the connective tissue of the human body, in a collagen solution of 3mg / ml: 5X DMEM: a medium containing 2.2% sodium bicarbonate and 200mM Hepes buffer and 0.05N sodium hydroxide mixed in a ratio of 7:2:1, and cultured for 7 days at 5% CO2 and 37℃ to obtain Dermal Equivalents (DE). The DE was placed inside plates separated into inside and outside by a membrane made of 3μm porous polycarbonate material, and 1x10 epidermal keratinocytes were cultured on the surface of the Dermal Equivalents (DE) in which fibroblasts were cultured. 5 The plates were inoculated with 10 ...

[0081] [Table 9]

[0082] From the above results, it can be seen that Osmocell containing plant stem cell culture extract and lactic acid bacteria exosome has a very high skin penetration effect, more than twice as high as general liposome containing plant stem cell culture extract and lactic acid bacteria exosome. This is thought to be because the Osmocell of the present invention has a higher affinity to the skin, and because the manufacturing process is carried out at room temperature, Osmocell maintains a higher activity in the stability of heat-sensitive EGF than general liposomes, resulting in a higher skin penetration amount.

[0083] Experimental Example 7 The stability of the plant stem cell culture extract of the present invention in cosmetics containing osmocell encapsulating lactic acid bacteria exosomes was measured.

[0084] To test the stability of the cosmetics, the examples of the present invention and the comparative examples were formulated according to Table 3, placed in opaque glass containers, and stored in a thermostatic chamber maintained at a constant temperature of 45° C. for 12 weeks. Also, the examples were placed in opaque glass containers and stored in a completely light-shielded refrigerator maintained at a constant temperature of 4° C. for 12 weeks, after which the degree of separation and discoloration were comparatively measured.

[0085] The results are shown in Table 10. The degree of product separation and discoloration was evaluated according to the following six grades. <Product discoloration evaluation criteria> 0: no change; 1: very slight separation (discoloration); 2: slight separation (discoloration); 3: moderately severe separation (discoloration); 4: severe separation (discoloration); 5: extremely severe separation (discoloration).

[0086] [Table 10]

[0087] As can be seen from Table 10, the examples containing Osmocell were stable at both 4°C and 45°C without any discoloration or separation, whereas the comparative examples containing general liposomes showed no discoloration or separation at 4°C, but showed some separation at 45°C, indicating that they were unstable.

[0088] Experimental Example 8 A skin safety test was conducted on cosmetics containing the plant stem cell culture extract of the present invention and osmocell encapsulating lactic acid bacteria exosomes.

[0089] Twenty subjects (average age 30.5 years, age distribution 18-40 years) were given a patch test on the upper arm using Haye's Test Chamber with Example 1 of the present invention and Comparative Example 1 formulated according to Table 3. However, subjects with psoriasis, eczema, or other skin lesions, pregnant women, breastfeeding women, or those taking contraceptives or antihistamines were excluded from this experiment. The test site was wiped with 70% ethanol and then dried, and 15 μg of each of the Example and Comparative Example were dropped into the chamber, which was then placed and fixed on the upper arm, which was the test site. The patch was applied for 24 hours, and after removing the patch, the test site was marked with a marking pen, and the test site was observed after 24 hours and 48 hours. The evaluation was performed 24 hours and 48 hours after removing the patch, and the skin reaction was evaluated according to the International Contact Dermatitis Research Group (ICDRG) regulations shown in Table 11 below. The results of the skin stability test are shown in Table 12 below.

[0090] [Table 11]

[0091] [Table 12]

[0092] As shown in Table 12, the results of the skin safety test showed that the average irritation level for both the Examples and the Comparative Examples was 0, indicating that they are safe cosmetics that do not irritate the skin.

[0093] Based on the results of the above experimental examples, a cosmetic composition containing a plant stem cell culture extract and an osmocell encapsulating lactic acid bacteria exosomes is presented below. However, the composition of the present invention is not limited to the following formulation examples.

[0094] Formulation example 1: Softening lotion (skin lotion)

[0095] [Table 13]

[0096] Formulation example 2: Nutritious lotion (milk lotion)

[0097] [Table 14]

[0098] Formulation example 3: Nutritious cream

[0099] [Table 15]

[0100] Formulation example 4: Massage cream

[0101] [Table 16]

[0102] Formulation example 5: Pack

[0103] [Table 17]

Claims

1. A cosmetic composition for preventing skin aging, comprising as an active ingredient Osmocell, a lipid complex consisting of phospholipid, phytosphingosine, ceramide NP, cholesterol, phytosterol, β-sitosterol and squalene, which contains a plant stem cell culture extract and lactic acid bacteria exosomes.

2. 2. The cosmetic composition according to claim 1, wherein the plant stem cell culture extract comprises two or more selected from the group consisting of wild ginseng stem cell culture extract, ginkgo stem cell culture extract, sophora japonica stem cell culture extract, green tea stem cell culture extract, edelweiss stem cell culture extract, grape stem cell culture extract, tomato stem cell culture extract, rose stem cell culture extract, aloe stem cell culture extract, and rice stem cell culture extract.

3. The cosmetic composition according to claim 1, wherein the lactic acid bacteria exosomes are isolated from a Lactobacillus plantarum culture solution.

4. The cosmetic composition according to claim 1, characterized in that the osmocell contains, relative to the total weight of the osmocell, 0.1 to 10 wt% of a lipid complex, 0.01 to 50.0 wt% of a plant stem cell culture extract, 0.00001 to 1.0 wt% of lactic acid bacteria exosomes, and the remaining amount of purified water, and the lipid complex contains, relative to the total weight of the lipid complex, 0.1 to 30.0 wt% of phospholipid, 0.01 to 20.0 wt% of phytosphingosine, 0.01 to 30.0 wt% of ceramide NP, 0.01 to 10.0 wt% of cholesterol, 0.01 to 10.0 wt% of phytosterol, 0.01 to 10.0 wt% of β-sitosterol, and 0.01 to 90.0 wt% of squalene.

5. The cosmetic composition according to claim 1, wherein the osmocell is contained in an amount of 0.001 to 100.0% by weight based on the total weight of the cosmetic composition.

6. The cosmetic composition according to claim 1, wherein the osmocell is prepared by a method including the steps of dissolving phospholipid, phytosphingosine, ceramide NP, cholesterol, phytosterol, β-sitosterol and squalene under high pressure to form a translucent gel-like lipid complex, and mixing the lipid complex, the plant stem cell culture extract and the lactic acid bacteria exosome at room temperature.

7. The cosmetic composition according to claim 1, which has an effect of improving wrinkles on the skin, a effect of whitening the skin, and an effect of improving skin elasticity.

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