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

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 osmocells encapsulating plant stem cell culture extract and lactic acid bacteria exosomes as an active ingredient.

Background Art

[0002] Before the skin ages, the physiological activities of skin cells are active. Therefore, in fibroblasts in the dermis, the synthesis and metabolism of collagen, which is important for suppressing skin elasticity and wrinkle formation, are actively carried out, and the synthesis of GAGS (Glucosaminoglycans), which are glycoproteins including hyaluronic acid essential for maintaining skin moisture, is also active. Thus, elasticity, softness, flexibility, and moisturizing properties are imparted to the skin. Also, in the epidermis, the proliferation of basal layer cells is active, and the production of adhesion proteins such as laminin, integrin, and desmosome, which strengthen the bonds between skin cells, is balanced, enabling the reinforcement of the skin cell tissue and the maintenance of healthy skin. However, the skin of modern people is exposed to various external harmful environments and stresses and suffers various damages. As people age, the physiological activities of the skin decline, the recovery rate of damaged skin slows down, and various skin aging phenomena such as loss of skin elasticity, dryness, wrinkle formation, pigmentation, senile lentigo, and dullness appear. Therefore, the biggest goal of cosmetics is to improve such skin aging symptoms, and various cosmetics have been developed for this purpose.

[0003] Generally, skin aging phenomena such as decreased skin elasticity, wrinkles, and pigmentation are caused by collagen breakdown, decreased collagen synthesis, loss of elastin, and melanin pigmentation due to a decline in skin physiology, and can be seen as phenomena occurring in the epidermis and dermis of the skin. Recently, there has been a surge in skin physiological research related to preventing skin aging, and in fact, cosmetics use UV blockers, antioxidants, and cell activity promoters to prevent wrinkles. In addition, methods for improving skin wrinkles through collagen synthesis have been proposed. Furthermore, based on advances in biochemistry and molecular biology, small amounts of signaling substances such as growth factors present in the human body have been discovered, and the theory of aging in the body based on these has been re-established. Moreover, it has become clear that these signaling substances decrease in the body with age, and that this decrease in growth factors is closely related to human aging. Such growth factors are produced in large quantities by stem cells present in the basal layer of each tissue.

[0004] The use of stem cells in cosmetics involves utilizing growth factors as their metabolites. Growth factors are proteins that bind to receptors on the surface of cells, stimulating cell proliferation and differentiation. The functions of growth factors are highly diverse; they can stimulate cell division in various cells, but in some cases, they act only on specific cell types. It has become clear that growth factors regulate cell differentiation, migration, and proliferation processes as essential components for stimulating cell proliferation. When growth factors bind to specific receptors, they activate signaling chains, transmitting signals from the outside of the cell to the nucleus inside. As signaling continues, they act with many other messengers to ultimately synthesize new proteins. Therefore, growth factors form an important signaling network in the transmission and functional regulation of intercellular interactions. Furthermore, in skin wound regeneration experiments using mice, it was confirmed that the size of wounds decreased by more than 40% when stem cell-derived proteins were applied compared to when they were not applied. In addition, good results were observed in whitening effects in subjects with hyperpigmentation, and strong effects as a cosmetic ingredient have been investigated, but ethical and safety issues remain challenges that need to be addressed.

[0005] Korean Published Patent No. 10-2007-0000005 discloses a functional cosmetic composition for wrinkle improvement and / or anti-aging containing allogeneic and / or autologous adipocytes, stem cells isolated from allogeneic and / or autologous adipocytes, or fibroblast growth factors isolated therefrom. Korean Registered Patent No. 10-848056 discloses a cosmetic composition for whitening containing stem cell culture medium or proteins isolated from that culture medium. Korean Published Patent No. 10-2008-0075959 discloses an injectable additive for use in an injectable drug containing human stem cells for tissue regeneration, which contains a conditional culture medium obtained by culturing human adipose stem cells in a serum-free medium containing antioxidants, vitamins, amino acids, and minerals for one day or more.

[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. In particular, ethical issues remain, which imposes many restrictions on their future use. Furthermore, there are difficulties in the skin penetration of the various active ingredients, more specifically, various cell growth factors, contained in human-derived stem cell culture fluid extracts. Unlike their excellent efficacy for medical therapeutic purposes, their effectiveness as cosmetics in healthy skin is limited. Therefore, currently, in the field of dermatology, to improve skin aging, human-derived stem cell culture fluid extracts are either injected directly into the skin or micro-channels (holes) are created in the skin using special devices to induce skin penetration. However, various skin safety issues arise during this process, making it difficult to use this method as a cosmetic.

[0007] Plants also have stem cells, and like human stem cells, they possess strong differentiation and regenerative capabilities. Furthermore, plant stem cells do not have the ethical or safety issues associated with human stem cells, and interest in their utilization has been steadily increasing recently.

[0008] Plants contain 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 create new plant tissues and organs such as roots and stems. Such plant stem cells are classified as pluripotent stem cells and have the ability to form various organs through their typical functions. Stem cells in the SAM and RAM contribute to plant growth through differentiation, and the differentiation of stem cells and the maintenance of stem cell density are regulated by the relationship between adjacent organizing center (OC) or quiescent center (QC) cells in the SAM and RAM. Unlike animals, plants are totipotent, a property discovered in 1902 by the Australian botanist Gottlieb Haberlandt. Plant totipotency refers to the ability of plant cells to differentiate into other cell forms or into the entire plant body.

[0009] By dedifferentiating a plant, undifferentiated cells called callus can be obtained. Callus cells can be obtained by surface sterilizing a plant, making cuts to create wounds, and culturing them in a solidified medium containing appropriate plant hormone concentrations. Callus cells can be cultured in this state or cultured in a liquid medium to form a suspension culture, which allows for the acquisition of large quantities of cells. By adjusting the concentration of plant hormones in the culture medium, callus cells can develop embryos and then form roots and buds again. These developed embryos, buds, and roots will germinate or grow into complete plants. Therefore, callus cells, being undifferentiated cells, possess stem cell capabilities and are called stem cells. From the perspective of application as target cells for cell therapy, mesenchymal stem cells have many advantages over embryonic stem cells, such as being relatively easy and safe to collect and being suitable for autologous transplantation.

[0010] In the present invention, callus, which is a plant stem cell, is a wound healing tissue that recovers its ability to divide and thickens when a plant is injured, preventing further injury. It refers to undifferentiated tissue or cell mass that is produced when tissue cut from a plant is cultured in a medium containing auxin, or when a certain type of plant is injured or the injured area is treated with auxin. By subculturing in a new medium, it can continue to divide and proliferate indefinitely.

[0011] Furthermore, there is growing interest in exosomes, which contain approximately 150 types of growth factor protein components, including 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. They are nano-sized extracellular vesicles, ranging from 50 to 200 nm in size. These nano-sized exosomes can transport membrane proteins that are insoluble in water. Membrane proteins are proteins that reside in a phospholipid bilayer and are hydrophobic, making intercellular transport via blood generally difficult. In contrast, exosomes consist of a phospholipid bilayer and can transport membrane proteins (Korean Patent No. 10-2253418). Furthermore, exosomes can protect their contents from external degradation substances by surrounding them with a phospholipid bilayer. A typical example is the RNA transport mechanism. RNA present outside the cell is degraded, but RNA present within exosomes is protected from these substances and can be smoothly transported to target cells. Examples of technologies related to obtaining exosomes include "Method for mass production of plant exosomes" in Korean Registered Patent No. 10-2125567, "Culture medium containing high-purity, high-concentration exosomes derived from mesenchymal stem cells and method for producing the same" in Korean Registered Patent No. 10-2047768, "Method for producing exosomes and / or extracellular vesicles and compositions containing the same" in Korean Registered Patent No. 10-1895916, and "Multiple columns for separating exosomes and method for separating exosomes" in Korean Registered Patent No. 10-1980482. [Overview of the project] [Problems that the invention aims to solve]

[0013] Therefore, the inventors made diligent efforts to find a substance that improves the prevention of skin aging. As a result, they overcame the ethical and safety issues associated with human-derived stem cell culture medium extracts, and overcame the problem that the various cell growth factors contained in human-derived stem cell culture medium extracts are difficult to penetrate into the skin, limiting their effectiveness in healthy skin. They discovered that plant stem cell culture extracts containing components compatible with the active ingredients of human-derived stem cell culture medium, and lactic acid bacteria exosomes containing 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), which are active ingredients in human-derived stem cell culture medium, are extremely effective in improving skin aging phenomena such as wrinkles, loss of elasticity, decreased moisture content, and pigmentation, leading to the completion of the present invention.

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

[0015] The above-mentioned problems are addressed by a cosmetic composition for preventing skin aging that contains Osmocell as an active ingredient, which is a lipid complex consisting of phospholipid, phytosphingosine, ceramide NP, cholesterol, phytosterol, β-sitosterol, and squalene, encapsulating plant stem cell culture extract and lactic acid bacteria exosomes.

[0016] Preferably, the plant stem cell culture extract may include two or more selected from the group consisting of ginseng stem cell culture extract, ginkgo stem cell culture extract, Japanese pagoda tree 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] Furthermore, preferably, the lactic acid bacteria exosomes can be isolated from the Lactobacillus plantarum culture medium.

[0018] Preferably, the osmocell contains 0.1 to 10% by weight of a lipid complex, 0.01 to 50.0% by weight of plant stem cell culture extract, 0.00001 to 1.0% by weight of lactic acid bacteria exosomes, and the remainder is purified water, relative to the total weight of the osmocell. The lipid complex may contain 0.1 to 30.0% by weight of phospholipids, 0.01 to 20.0% by weight of phytosphingosine, 0.01 to 30.0% by weight of ceramide NP, 0.01 to 10.0% by weight of cholesterol, 0.01 to 10.0% by weight of phytosterols, 0.01 to 10.0% by weight of β-sitosterol, and 0.01 to 90.0% by weight of squalene, relative to the total weight of the lipid complex.

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

[0020] Preferably, the osmocell can be produced by a method comprising 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 exosomes at room temperature.

[0021] Preferably, the cosmetic composition has the effect of improving skin wrinkles, whitening the skin, and improving skin elasticity. [Effects of the Invention]

[0022] The cosmetic composition according to the present invention contains a plant stem cell culture extract and a lactic acid bacteria exosome, includes stabilized osmocells as an active ingredient, has a better delivery effect on the skin than conventional cosmetics encapsulated in liposomes, and has a better effect of preventing skin aging and improving skin problems. Furthermore, since the active ingredient is stabilized in the osmocells at room temperature, destruction of the active ingredient by heat can be prevented and the effect of the cosmetic can be maximized.

Brief Description of the Drawings

[0023] [Figure 1] It is a diagram showing the particle distribution of osmocells containing a plant stem cell culture extract and a lactic acid bacteria exosome according to the present invention. [Figure 2] It is a SEM photograph of osmocells containing a plant stem cell culture extract and a lactic acid bacteria exosome produced in Production Example 1.

Modes for Carrying Out the Invention

[0024] The present invention relates to a cosmetic composition for preventing skin aging, which contains osmocells (OSMOCELL) encapsulating a plant stem cell culture extract and a lactic acid bacteria exosome for improving skin aging phenomena and skin problems. Prevention of skin aging in the present invention comprehensively means including improvement of wrinkles, skin whitening, and elasticity enhancement effects, and improvement of skin problems means alleviating skin inflammatory symptoms such as erythema, edema, and itching that occur on the skin.

[0025] The present invention provides a cosmetic composition for preventing skin aging, which contains osmocells encapsulating a plant stem cell culture extract and a lactic acid bacteria exosome as an active ingredient.

[0026] The plant stem cell culture extract of the present invention is obtained by sterilizing the surface of the roots of wild ginseng, the stems of ginkgo trees, the stems of Japanese pagoda tree, the stems of green tea, the flowers of edelweiss, the stems of grapes, the fruits of tomatoes, the flowers of roses, the stems of aloe, and the stems of rice, cutting and wounding them, culturing them in a solidified medium containing an appropriate concentration of plant hormones to induce callus cells, culturing the induced callus cells in a liquid medium, and performing suspension culture. The plant stem cell culture extracts of the present invention are obtained by crushing wild ginseng stem cells (callus), ginkgo stem cells (callus), Japanese pagoda tree stem cells (callus), green tea stem cells (callus), edelweiss stem cells (callus), grape stem cells (callus), tomato stem cells (callus), rose flower stem cells (callus), aloe stem cells (callus), and rice stem cells (callus), followed by pressing and filtration.

[0027] According to one embodiment of the present invention, the plant stem cell culture extract can be produced by the following method. First, the flower, stem, fruit, or root of the plant is 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 flower, stem, fruit, or root of the plant is cut off after being scored with a sharp knife, and then placed 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 to induce stem cells. The induction of callus, which is a plant stem cell, was maximized by appropriately adding 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. To maximize the amount of induced totipotent embryogenic stem cells (Callus), the cells were proliferated by subculturing in liquid culture under dark conditions every two weeks to increase the growth rate. A large amount of stem cells (Callus) can be secured by inoculating a suspension culture incubator (Gyrotory Shaker) 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 performing suspension culture. The proliferated callus was cultured in the suspension incubator at 25 ± 2°C under dark conditions using MS medium without added hormones in the final subculturing stage. A bioreactor can also be used for mass production. The plant stem cells thus obtained are washed 2-3 times with purified water to remove culture medium components, and the plant stem cells (callus) are rapidly pulverized at a low temperature in a frozen stock container using a homogenizer at 8,000-15,000 rpm for 2-3 minutes. The pulverized and pressed 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 include Lactobacillus acidophilus, Bifidobacterium lactis, Lactobacillus rhamnosus, Bifidobacterium longum, Bifidobacterium breve, Lactobacillus casei, Lactobacillus Salivarius, Lactobacillus reuteri, and Lactobacillus plantarum, which are intestinal lactic acid bacteria that are also found on the skin, including Lactobacillus plantarum. This is obtained by culturing *Plantarum* in sterile milk for 48 hours and then isolating it from the culture medium.

[0029] According to one embodiment of the present invention, lactic acid bacteria exosomes can be obtained by mixing Lactobacillus plantarum, a type of lactic acid bacterium, with sterile milk and culturing it for 48 hours, centrifugating the lactic acid bacteria culture solution at 1,500-2,000 g for 20-30 minutes using a centrifuge, then separating and removing the lactic acid bacteria by taking only the supernatant culture solution, centrifugating the supernatant culture solution again at 10,000-15,000 g for 20-30 minutes using an ultra-high-speed centrifuge, then separating and removing the lactic acid bacteria and residues by taking the supernatant, centrifugating the supernatant solution again at 100,000-150,000 g for 2-4 hours using an ultra-high-speed centrifuge, then settling the exosomes into a pellet layer, and finally resuspending the pellet layer in PBS using a pipette and vortex to obtain the exosomes.

[0030] The present invention is characterized by first stabilizing heat-unstable active ingredients, plant stem cell culture extract and lactic acid bacteria exosomes, and by producing a lipid complex composed of lipid components constituting the cell membrane to facilitate transdermal absorption. Then, the heat-unstable plant stem cell culture extract and the active ingredients within the lactic acid bacteria exosomes are stably encapsulated in the complex and incorporated into a cosmetic.

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

[0032] The Osmocell of the present invention is easily absorbed into the skin, can encapsulate a larger amount of water-soluble components compared to liposomes, and is stable. In the present invention, the Osmocell refers to a vesicle formed using phospholipids, phytosphingosine, ceramide NP, cholesterol, phytosterol, β-sitosterol, and squalene, which are major lipid components constituting cell membranes, and encapsulates the physiologically active components of the present invention within this vesicle. Such an Osmocell has higher skin affinity and is more stable than conventional liposomes.

[0033] Furthermore, Osmocell refers to a nano-sized bilayer vesicle with an average droplet size of 20-500 nm, preferably 50-250 nm, and most preferably 100-200 nm. When the average droplet size of the Osmocell exceeds 500 nm, skin penetration and formulation stability are very low. In this invention, the term "Osmocell" refers to a nano-sized bilayer vesicle in which the plant stem cell culture extract and lactic acid bacteria exosomes of the present invention are encapsulated and stabilized.

[0034] The Osmocell of the present invention is manufactured by the following method.

[0035] Phospholipids, phytosphingosine, ceramide NP, cholesterol, phytosterol, β-sitosterol, and squalene, which are the main lipid components that make up cell membranes, are dissolved under high pressure using a microfludizer to form a translucent gel-like lipid complex. Next, an appropriate amount of this lipid complex is added to an aqueous solution containing dispersed plant stem cell culture extract and lactic acid bacteria exosomes at room temperature and mixed to obtain osmocells containing the plant stem cell culture extract and lactic acid bacteria exosomes of the present invention, which have a closed bilayer structure. In this case, the reaction step is carried out at room temperature. Conventional liposome manufacturing processes using microfludizers have the disadvantage that the thermal stability of temperature-sensitive components cannot be ensured because liposomes are formed at high temperatures (60°C or higher). This invention uses a high-pressure emulsifier to mix materials that form a lipid complex, but it differs from the conventional method of using a high-pressure emulsifier to form liposomes. In this invention, a lipid complex is formed by dissolving and mixing materials in a high-pressure emulsifier, and these complexes form vesicles in the aqueous solution phase, encapsulating the active ingredients dispersed in the aqueous solution, namely plant stem cell culture extract and lactic acid bacteria exosomes, to form osmocells.

[0036] The Osmocell contains, based on the total weight of the Osmocell, 0.1 to 10% by weight of lipid complex, 0.01 to 50.0% by weight of plant stem cell culture extract, 0.00001 to 1.0% by weight of lactic acid bacteria exosomes, and the remainder being purified water.

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

[0038] Furthermore, the lipid complex consists 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 of the total weight of the lipid complex, preferably 1.0 to 10.0% by weight. The phytosphingosine is contained in an amount of 0.01 to 20.0% by weight of the total weight of the lipid complex, preferably 0.1 to 5.0% by weight. The ceramide NP is contained in an amount of 0.01 to 30.0% by weight of the total weight of the lipid complex, preferably 0.1 to 10.0% by weight. The cholesterol is contained in an amount of 0.01 to 10.0% by weight of the total weight of the lipid complex, preferably 0.1 to 5.0% by weight. Phytosterol is present in an amount of 0.01 to 10.0% by weight relative to the total weight of the lipid complex, preferably 0.1 to 5.0% by weight. β-sitosterol is present in an amount of 0.01 to 10.0% by weight relative to the total weight of the lipid complex, preferably 0.1 to 5.0% by weight. Squalene is present in an amount of 0.01 to 90.0% by weight relative to the total weight of the lipid complex, preferably 10.0 to 70.0% by weight.

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

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

[0041] Furthermore, the cosmetic composition of the present invention may 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] A specific example of the present invention is that a cosmetic composition containing Osmocell, which encapsulates plant stem cell culture extract and lactic acid bacteria exosomes as active ingredients, exhibits superior skin delivery effects compared to conventional liposome-containing cosmetics, resulting in better anti-aging and improvement effects for skin problems. Furthermore, because the active ingredients are stabilized in Osmocell at room temperature, the thermal stability of the active ingredients is ensured, maximizing the efficacy of the cosmetic composition.

[0043] The present invention will be described in detail below with reference to manufacturing examples and embodiments, but the scope of the present invention is not limited by such manufacturing examples and embodiments. For the cosmetic composition of the present invention, plant stem cell culture extract, lactic acid bacteria exosomes, and osmocells containing plant stem cell culture extract and lactic acid bacteria exosomes were manufactured as shown in the following manufacturing example.

[0044] Manufacturing Example 1-1: Production of Panax Ginseng Stem Cell Culture Extract The Panax ginseng stem cell culture extract of this invention was prepared by immersing Panax ginseng roots 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 the surface with sterile water to disinfect it. The disinfected surface of the Panax ginseng roots was scored with a sharp knife, cut off, and placed 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. After inoculation, Panax ginseng stem cells were induced by culturing in a dark culture room at 25±2℃ for 12 weeks. The induction of ginseng callus, which is ginseng stem cells, was maximized by adding the plant hormones auxin and cytokinin to 0.5 mg / L in M&S (Murashige & Skoog's) solid medium. To maximize the amount of induced ginseng callus, it was propagated by subculturing in liquid culture under dark conditions every 4 weeks, which can increase the growth rate. A large amount of ginseng stem cells (callus) could be secured by inoculating 2.5 g / L of ginseng callus induced using the above-mentioned M&S liquid medium into a suspension culture incubator and performing suspension culture. The propagated ginseng callus was cultured in the suspension incubator at 25 ± 2°C under dark conditions using MS medium without added hormones in the final subculturing stage. The ginseng stem cells thus obtained were washed twice with purified water to remove culture medium components, and the ginseng stem cells (callus) were rapidly pulverized at 10,000 rpm for 2 minutes in a frozen stock container using a homogenizer at low temperature. The pulverized and pressed ginseng callus was filtered under reduced pressure using filter paper to obtain the ginseng stem cell (callus) culture extract of the present invention.

[0045] Manufacturing Example 1-2: Production of Ginkgo Biloba Stem Cell Culture Extract The Ginkgo biloba stem cell culture extract of this invention was prepared by immersing Ginkgo biloba stems 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 the surface with sterile water to disinfect it. The disinfected surface of the Ginkgo biloba stems was scored with a sharp knife, cut off, and placed 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. The cultured medium was then incubated in a dark culture room at 25±2℃ for 12 weeks to induce Ginkgo biloba stem cells. The induction of Ginkgo biloba callus, which is Ginkgo biloba stem cells, was maximized by appropriately applying the plant hormones auxin and cytokinin at concentrations of 0.5 mg / L to M&S (Murashige & Skoog's) solid medium. To maximize the amount of induced Ginkgo biloba callus, it was propagated by subculturing in liquid culture under dark conditions every 4 weeks to increase the growth rate. A large amount of Ginkgo biloba stem cells (callus) could be secured by inoculating 2.5 g / L of the Ginkgo biloba callus induced using the aforementioned M&S liquid medium into a suspension culture in a gyrotory shaker. The propagated Ginkgo biloba callus was then cultured in the suspension incubator at 25 ± 2°C under dark conditions using MS medium without added hormones in the final subculturing stage. The Ginkgo biloba stem cells obtained in this way are washed twice with purified water to remove culture medium components, and the Ginkgo biloba stem cells (callus) are rapidly pulverized at 10,000 rpm for 2 minutes in a frozen stock container using a homogenizer at low temperature. The pulverized and pressed Ginkgo biloba callus is then filtered under reduced pressure using filter paper to obtain the Ginkgo biloba stem cell (callus) culture extract of the present invention.

[0046] Manufacturing Examples 1-3: Production of Sophora japonica stem cell culture extract The Sophora japonica stem cell culture extract of the present invention was prepared by immersing Sophora japonica stems 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 the surface with sterile water to disinfect it. The disinfected Sophora japonica stems were then cut after being scored with a sharp knife, and inoculated onto 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. The cultured medium was then incubated in a dark culture room at 25±2°C for 12 weeks to induce Sophora japonica stem cells. The induction of Sophora japonica stem cells (Sophora japonica callus) was maximized by appropriately adding the plant hormones auxin and cytokinin at concentrations of 0.5 mg / L to M&S (Murashige & Skoog's) solid medium. To maximize the amount of induced Sophora japonica callus, the cells were propagated by subculturing in liquid culture under dark conditions every four weeks to increase the growth rate. A large amount of Sophora japonica stem cells (callus) were secured by inoculating 2.5 g / L of the Sophora japonica callus induced using the aforementioned M&S liquid medium into a suspension culture in a gyrotory shaker. The propagated Sophora japonica callus was then cultured in the suspension incubator at 25 ± 2°C under dark conditions using MS medium without added hormones in the final subculturing stage. The resulting Sophora japonica stem cells are washed twice with purified water to remove culture medium components. The Sophora japonica stem cells (callus) are then rapidly pulverized at 10,000 rpm for 2 minutes in a frozen stock container using a homogenizer at low temperature. The pulverized and compressed Sophora japonica callus is then filtered under reduced pressure using filter paper to obtain the Sophora japonica stem cell (callus) culture extract of the present invention.

[0047] Manufacturing Example 1-4: Production of Green Tea (Camellia Sinensis) Stem Cell Culture Extract The green tea (Camellia Sinensis) stem cell culture extract of the present invention was prepared by immersing green tea stems 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 disinfected green tea stems were then cut after being scored with a sharp knife, placed 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 inoculated. Green tea stem cells were then induced by culturing in a dark culture chamber at 25±2℃ for 12 weeks. The induction of green tea stem cells, or green tea callus, was maximized by appropriately applying the plant hormones auxin and cytokinin at concentrations of 0.5 mg / L to M&S (Murashige & Skoog's) solid medium. To maximize the amount of induced green tea callus, it was propagated by subculturing in liquid culture under dark conditions every four weeks to increase the growth rate. A large amount of green tea stem cells (callus) was secured by inoculating 2.5 g / L of the green tea callus induced using the aforementioned M&S liquid medium into a suspension culture incubator (Gyrotory Shaker) and performing suspension culture. The propagated green tea callus was then cultured in the suspension incubator at 25 ± 2°C under dark conditions using MS medium without added hormones in the final subculturing stage. The green tea stem cells thus obtained are washed twice with purified water to remove culture medium components, and the green tea stem cells (callus) are rapidly pulverized at 10,000 rpm for 2 minutes in a frozen stock container using a homogenizer at low temperature. The pulverized and pressed 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] Manufacturing Examples 1-5: Production of Edelweiss (Leontopodium Alpinum) Stem Cell Culture Extract The present invention's edelweiss (Leontopodium Alpinum) stem cell culture extract was prepared by immersing 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 the surface with sterile water to disinfect it. The disinfected surface of the edelweiss flower was then cut with a sharp knife and placed 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. Edelweiss stem cells were then induced by culturing in a dark culture room at 25±2°C for 4 weeks. The induction of edelweiss stem cells, or edelweiss callus, was maximized by appropriately adding the plant hormones auxin and cytokinin at concentrations of 0.5 mg / L to M&S (Murashige & Skoog's) solid medium. To maximize the amount of induced edelweiss callus, it was propagated by subculturing in liquid culture under dark conditions every two weeks to increase the growth rate. A large amount of edelweiss stem cells (callus) was secured by inoculating 2.5 g / L of edelweiss callus induced using the aforementioned M&S liquid medium into a suspension culture in a gyrotory shaker. The propagated edelweiss callus was then cultured in the suspension incubator at 25 ± 2°C under dark conditions using MS medium without added hormones in the final subculturing stage. The edelweiss stem cells thus obtained are washed twice with purified water to remove culture medium components. The edelweiss stem cells (callus) are then frozen in a stock container at a low temperature and homogenized at 10,000 rpm for 2 minutes using a homogenizer. The pulverized and compressed 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 Examples 1-6: Production of Grape (Vitis Vinifera) Stem Cell Culture Extract The grape (Vitis Vinifera) stem cell culture extract of this invention was prepared by immersing grape stalks in 70% ethanol for 30 seconds, sterilizing them by shaking them in 2% sodium hypochlorite for 5 minutes, and then washing the surface with sterile water to disinfect it. The disinfected surface of the grape stalks was then scored with a sharp knife and cut off. These stems were then inoculated onto 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 cultured in a dark culture chamber at 25±2°C for 6 weeks to induce grape stem cells. The induction of grape callus, which is grape stem cells, was maximized by appropriately applying the plant hormones auxin and cytokinin at concentrations of 0.5 mg / L to M&S (Murashige & Skoog's) solid medium. To maximize the amount of induced grape callus, it was propagated by subculturing in liquid culture, which can increase the growth rate, under dark conditions every two weeks. A large amount of grape stem cells (callus) could be secured by inoculating 2.5 g / L of grape callus induced using the above-mentioned M&S liquid medium into a suspension culture in a gyrotory shaker and performing suspension culture. The propagated grape callus was cultured in the suspension incubator at 25 ± 2°C under dark conditions using MS medium without added hormones in the final subculturing stage. The grape stem cells thus obtained were washed twice with purified water to remove medium components, and the grape stem cells (callus) were rapidly pulverized at 10,000 rpm for 2 minutes in a frozen stock container using a homogenizer at low temperature. The pulverized and pressed grape callus was filtered under reduced pressure using filter paper to obtain the grape stem cell (callus) culture extract of the present invention.

[0050] Manufacturing Examples 1-7: Production of Tomato (Solanum Lycopersicum) Stem Cell Culture Extract To induce stem cells (callus) from tomato fruit, the tomato (Solanum Lycopersicum) stem cell culture extract of this invention was prepared by immersing tomatoes 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 the surface with sterile water. The surface of the disinfected tomatoes was then scored with a sharp knife, cut off, and inoculated onto 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. The cultured medium was then incubated in a dark culture room at 25±2℃ for 3 weeks to induce tomato stem cells. To maximize the induction of tomato callus, which is a type of tomato stem cell, by appropriately applying the plant hormones auxin and cytokinin at concentrations of 0.5 mg / L to M&S (Murashige & Skoog's) solid medium. To maximize the amount of induced tomato callus, it was propagated by subculturing in liquid culture under dark conditions every two weeks to increase the growth rate. A large amount of tomato stem cells (callus) could be secured by inoculating 2.5 g / L of the tomato callus induced using the above-mentioned M&S liquid medium into a suspension culture in a gyrotory shaker and performing suspension culture. The propagated tomato callus was cultured in the suspension incubator at 25 ± 2°C under dark conditions using MS medium without added hormones in the final subculturing stage. The tomato stem cells thus obtained were washed twice with purified water to remove medium components, and the tomato stem cells (callus) were rapidly pulverized at 10,000 rpm for 2 minutes in a frozen stock container using a homogenizer at low temperature. The pulverized and pressed tomato callus was filtered under reduced pressure using filter paper to obtain the tomato stem cell (callus) culture extract of the present invention.

[0051] Manufacturing Example 1-8: Production of Rose (Rosa Hybrid) Stem Cell Culture Extract In this invention, to induce stem cells (callus) from rose flowers, 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 disinfected surface of the rose flower was then cut with a sharp knife and placed 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. After inoculation, rose stem cells were induced by culturing in a dark culture chamber at 25±2℃ for 4 weeks. The induction of rose callus, or rose stem cells, was maximized by appropriately adding the plant hormones auxin and cytokinin at concentrations of 0.5 mg / L to M&S (Murashige & Skoog's) solid medium. To maximize the amount of induced rose callus, the cells were propagated by subculturing in liquid culture under dark conditions every two weeks to increase the growth rate. A large amount of rose stem cells (callus) could be secured by inoculating 2.5 g / L of the rose callus induced using the aforementioned M&S liquid medium into a suspension culture in a gyrotory shaker and performing suspension culture. The propagated rose callus was then cultured in the suspension incubator at 25 ± 2°C under dark conditions using MS medium without added hormones in the final subculturing stage. The resulting rose stem cells were washed twice with purified water to remove culture medium components, and the rose stem cells (callus) were rapidly pulverized at 10,000 rpm for 2 minutes in a frozen stock container using a homogenizer at low temperature. The pulverized and pressed rose callus was then filtered under reduced pressure using filter paper to obtain the rose stem cell (callus) culture extract of the present invention.

[0052] Manufacturing Examples 1-9: Production of Aloe Barbadensis Stem Cell Culture Extract The Aloe Barbadensis stem cell culture extract of this invention was prepared by immersing aloe stems 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 the surface with sterile water to disinfect it. The disinfected surface of the aloe stem was then scored with a sharp knife and cut off. The stems were then inoculated onto 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 cultured in a dark culture chamber 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 adding the plant hormones auxin and cytokinin at concentrations of 0.5 mg / L to M&S (Murashige & Skoog's) solid medium. To maximize the amount of induced aloe callus, it was propagated by subculturing in liquid culture under dark conditions every two weeks to increase the growth rate. A large amount of aloe stem cells (callus) was secured by inoculating 2.5 g / L of the aloe callus induced using the aforementioned M&S liquid medium into a suspension culture in a gyrotory shaker. The propagated aloe callus was then cultured in the suspension incubator at 25 ± 2°C under dark conditions using MS medium without added hormones in the final subculturing stage. The resulting aloe stem cells are washed twice with purified water to remove culture medium components. The aloe stem cells (callus) are then rapidly pulverized at 10,000 rpm for 2 minutes in a frozen stock container using a homogenizer at low temperature. The pulverized and pressed 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] Manufacturing Examples 1-10: Production of Rice (Oryza Sativa) Stem Cell Culture Extract In this invention, to induce stem cells (callus) from rice stems, 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 then cut after being scored with a sharp knife, placed 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 inoculated. Rice stem cells were then induced by culturing in a dark culture chamber at 25±2℃ for 2 weeks. The induction of rice callus, which is rice stem cells, was maximized by appropriately adding the plant hormones auxin and cytokinin at concentrations of 0.5 mg / L to M&S (Murashige & Skoog's) solid medium. To maximize the amount of induced rice callus, it was propagated by subculturing in liquid culture under dark conditions every week to increase the growth rate. A large amount of rice stem cells (callus) was secured by inoculating 2.5 g / L of rice callus induced using the aforementioned M&S liquid medium into a suspension culture incubator (Gyrotory Shaker) and performing suspension culture. The propagated rice callus was then cultured in the suspension incubator at 25 ± 2°C under dark conditions using MS medium without added hormones in the final subculturing stage. The rice stem cells thus obtained are washed twice with purified water to remove culture medium components, and the rice stem cells (callus) are rapidly pulverized at 10,000 rpm for 2 minutes in a frozen stock container using a homogenizer at low temperature. The pulverized and pressed 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] Manufacturing Example 2: Production of Lactobacillus Exosomes The lactic acid bacteria exosome of the present invention is obtained by mixing Lactobacillus plantarum, a type of lactic acid bacterium, with sterile milk and culturing it for 48 hours. The lactic acid bacteria culture solution is then centrifuged at 17,000 g for 25 minutes using a centrifuge, and only the supernatant is taken to separate and remove the lactic acid bacteria. The supernatant is then centrifuged again at 12,000 g for 25 minutes using an ultra-high-speed centrifuge, and the supernatant is taken to separate and remove the lactic acid bacteria and residues. The supernatant is then centrifuged again at 120,000 g for 3 hours using an ultra-high-speed centrifuge, and the exosomes are allowed to settle into a pellet layer. The pellet layer is then resuspended in PBS using a pipette and vortex to separate the exosomes from the intestinal lactic acid bacteria culture solution and obtain the lactic acid bacteria exosome of the present invention.

[0055] Manufacturing Example 3: Production of Osmocell 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 microfludizer three times at a pressure of 1,000 bar, in the quantities shown in Table 1, to create a translucent gel-like lipid complex. A solution was prepared by dispersing the 10 plant stem cell culture extracts presented in Production Example 1 of the present invention and lactic acid bacteria exosomes from Production Example 2 in an aqueous solution. Next, as shown in Table 2, this lipid complex was added at room temperature to the aqueous solution containing the dispersed plant stem cell culture extracts and lactic acid bacteria exosomes to produce Osmocell containing the plant stem cell culture extracts and lactic acid bacteria exosomes. Figure 1 shows the particle distribution of osmocells containing plant stem cell culture extract and lactic acid bacteria exosomes according to Production Example 3. Figure 2 is an SEM image of osmocells containing plant stem cell culture extract and lactic acid bacteria exosomes according to Production Example 3.

[0056] [Table 1]

[0057] [Table 2]

[0058] To investigate the particle size distribution of the plant stem cell culture extract and osmocell particles containing lactic acid bacteria exosomes according to the present invention, Experimental Example 1 was conducted as described below.

[0059] Experimental Example 1 To analyze the particle size distribution of the osmocell particles of the present invention, the osmocells obtained in Production Example 3 were diluted to an appropriate concentration in 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 using a light scattering method with a Zetasizer NS, and the results are shown in the graph in Figure 1. The average particle size of the osmocells obtained in this way was 120 nm, which shows improved particle size homogeneity and stability compared to liposomes produced using a conventional high-pressure emulsifier (microfluidizer). Furthermore, Figure 2 is an SEM image of the osmocells containing plant stem cell culture extract and lactic acid bacteria exosomes produced in Production Example 3.

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

[0061] In Comparative Example 1, the conventional liposomes were prepared by mixing 3.0% lecithin, 10% propylene glycol, 10% ethyl alcohol, and 3% mineral oil, then heating to 6°C to dissolve. Subsequently, an aqueous solution containing 10% plant stem cell culture extract and 0.001% lactic acid bacteria exosomes was poured into the solution and stirred. The mixture was 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 (mechanical evaluation) of a cosmetic formulation containing the plant stem cell culture extract of the present invention and osmocell containing lactic acid bacteria exosomes was measured. Twenty women aged 20 years or older (average age 39.5 years) were divided into two groups (A and B). Group A was given the example formulation, and Group B was given the comparative example formulation, both according to Table 3, and the formulations were applied to a 2 x 2 cm area of ​​the upper arm. 2 While applying the solution (0.2g / time twice daily) to the area for 8 weeks, a replica of skin wrinkles was created using a transparent silicone solution, and the changes in 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) of each wrinkle was determined. m The average wrinkle roughness (R) is the value obtained by dividing the sum of (m is an integer greater than or equal to 1) by the number of wrinkles, as shown in Formula 1 below. z The wrinkle-improving effect was analyzed. The test results regarding the wrinkle-improving effect on the skin after 6 weeks are shown in Table 4 below.

[0064]

number

[0065] [Table 4]

[0066] Test results, average wrinkle roughness after 8 weeks (R z In the example containing Osmocell, which encapsulates plant stem cell culture extract and lactic acid bacteria exosomes, the wrinkle reduction was 87.3 μm (p<0.01), and wrinkles were further improved by approximately 106.9% compared to the comparative example containing general liposomes encapsulating plant stem cell culture extract and lactic acid bacteria exosomes. Therefore, from the above results, it can be seen that Example 1, which contains Osmocell, which encapsulates plant stem cell culture extract and lactic acid bacteria exosomes, showed a much more effective improvement in wrinkle reduction than Comparative Example 1, which contains general liposomes.

[0067] Experimental Example 3 A macroscopic evaluation was conducted on the improvement of skin wrinkles of a cosmetic product containing the plant stem cell culture extract of the present invention and osmocell containing lactic acid bacteria exosomes.

[0068] Twenty women aged 20 or older (average age 39.5 years) were divided into two groups (A and B). Group A was given Example 1, and Group B was given Comparative Example 1, both prepared according to Table 3. The mixtures were applied for 8 weeks (twice a day), mainly around the eyes where wrinkles were present. The degree of improvement in skin wrinkles (△W) was then measured by classifying the objective evaluation by experts and the subjective evaluation by the subjects into the following six grades. The clinical effects of improving skin wrinkles are shown in Table 5 (objective evaluation by experts) and Table 6 (subjective evaluation by subjects) below. <Evaluation Criteria for Improving Skin Wrinkles> -3: Severely worsened; -2: Worsened; -1: Slightly worsened; 0: No change; 1: Slightly improved; 2: Improved; 3: Greatly improved

[0069] [Table 5]

[0070] [Table 6]

[0071] The results of the visual evaluation showed that in Example 1, the objective evaluation by experts and the subjective evaluation by subjects were both 2.7, indicating that it was extremely effective in improving wrinkles. This represents a 68.8% increase in the objective evaluation by experts and a 50% increase in the subjective evaluation by subjects compared to Comparative Example 1, in which plant stem cell culture extract and lactic acid bacteria exosomes were encapsulated in liposomes. This demonstrates that the cosmetic composition containing plant stem cell culture extract and lactic acid bacteria exosomes in Osmocell of the present invention is far more effective in improving wrinkles than conventional cosmetics containing plant stem cell culture extract and lactic acid bacteria exosomes in liposomes.

[0072] Experimental Example 4 The skin whitening effect was measured by the inhibitory effect on melanocytes of Osmocell (Production Example 3), which contains plant stem cell culture extract and lactic acid bacteria exosomes. Melanocytes were purchased from a mouse-derived B-16 melanoma (ATCC CRL 6323) cell line. The melanoma cell line was inoculated into DMEM medium containing 4.5 g / L glucose, 10% serum, and 1% antibiotic, and cultured in a 50 ml T-flask at 37°C. After 24 hours of culture under 5% CO2 conditions, cells were isolated by treatment with 0.05% trypsin containing 0.02% EDTA, inoculated into a 50 ml T-flask, and cultured for 48 hours. The cell count in this case was 5.76 × 10⁶. 6This is a cell / flask. Here, melanoma cells cultured in DMEM medium are treated with osmocells (Production Example 3) containing plant stem cell culture extract and lactic acid bacteria exosomes at appropriate concentrations, and general liposomes containing plant stem cell culture extract and lactic acid bacteria exosomes, 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 lactic acid bacteria exosomes used for encapsulation in osmocells and general liposomes were the same to obtain osmocells and general liposomes applicable to this experiment. After culturing, all the medium is removed, and the cells are separated by treating with 1 ml of phosphate-buffered saline (PBS) containing 0.02% EDTA and 0.05% trypsin. Then, the cells are collected by centrifugation for 5 minutes. The cells are treated with 5% trichloroacetic acid (TCA), stirred, and then centrifuged to wash the precipitated melanin with phosphate-buffered saline.

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

[0074] [Table 7]

[0075] From the above results, it can be seen that Osmocell, which contains plant stem cell culture extract and lactic acid bacteria exosomes, exhibits a higher melanin synthesis inhibitory effect than when plant stem cell culture extract and lactic acid bacteria exosomes are encapsulated in ordinary liposomes. This is because the manufacturing process for Osmocell, which contains plant stem cell culture extract and lactic acid bacteria exosomes, is carried out at room temperature, whereas conventional liposomes undergo a high-temperature process (60°C or higher) when passing through a high-pressure emulsifier (microfluidizer) during the manufacturing process. This heat generates heat that reduces the activity or denatures temperature-sensitive components such as growth factors, resulting in a lower melanin synthesis inhibitory effect. This experimental example shows that plant stem cell culture extract and lactic acid bacteria exosomes suppress melanin production in melanocytes and exhibit a very excellent skin whitening effect.

[0076] Experimental Example 5 The skin elasticity-enhancing effect of a cosmetic formulation containing the plant stem cell culture extract of the present invention and Osmocell containing lactic acid bacteria exosomes was measured. Twenty women aged 20 years or older (average age 39.5 years) were divided into two groups (A and B) under conditions of a temperature of 24-26°C and a humidity of 75%. Group A was given Example 1 and Group B was given Comparative Example 1, both formulations according to Table 3 above. These formulations were applied mainly around the eyes for 8 weeks (twice a day), and 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 (week 0) values ​​from the Cutometer SEM 575. The R8 value indicates the viscoelasticity of the skin.

[0077] [Table 8]

[0078] From these results, it can be seen that the example containing Osmocell, which encapsulates plant stem cell culture extract and lactic acid bacteria exosomes, increased skin elasticity by 77.5% compared to the comparative example containing general liposomes encapsulating plant stem cell culture extract and lactic acid bacteria exosomes, demonstrating a high effect in enhancing skin elasticity.

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

[0080] Human normal fibroblast 1 x 10⁻¹⁰ 5Cells / ml were inoculated into a collagen gel structure similar to the fibrous tissue that makes up the connective tissue of the human body in a culture medium consisting of a 7:2:1 mixture of collagen aqueous solution (3 mg / ml), 5X DMEM (2.2% sodium bicarbonate), and 0.05N sodium hydroxide (200 mM Hepes buffer). The Dermal Equivalent (DE) obtained by culturing cells / ml cells in a collagen gel structure similar to the fibrous tissue that makes up the connective tissue of the human body was cultured in a medium containing 3 mg / ml collagen aqueous solution, 5X DMEM (2.2% sodium bicarbonate), and 200 mM Hepes buffer in a 0.05N sodium hydroxide mixture in a 7:2:1 ratio, and cultured at 5% CO2 and 37°C for 7 days. The Dermal Equivalent (DE) was placed inside a plate separated into inner and outer sections by a 3 μm porous polycarbonate membrane, and 1 x 10⁻¹⁶ epidermal keratinocytes were placed on the surface of the Dermal Equivalent (DE) cultured with fibroblasts. 5 Cells / ml were inoculated and cultured for 7 days with K-SFM medium containing EGF and BPE placed inside and outside the plate. Then, the medium inside the plate was discarded to allow air to come into contact with the surface of the cultured cells, and a medium containing equal amounts of K-SFM containing 10% FBS and DMEM without EGF was placed on the outside and cultured for 2 weeks to obtain artificial skin in which a multilayered epidermis and dermis were formed. The Osmocell of the present invention example and the liposome of the comparative example were then applied to the top layer of the artificial skin tissue and cultured for 4 hours. After that, the amount of EGF contained in the Osmocell of the example and the liposome of the comparative example that had permeated and passed through the epidermis and dermis of the artificial skin with DMEM medium without EGF was analyzed by HPLC. At this time, EGF was added to plant stem cell culture extract and lactic acid bacteria exosomes to prepare the Osmocell of the example and the liposome of the comparative example so that the EGF content was 50 μg / ml. The test results for skin penetration effect are shown in Table 9 below.

[0081] [Table 9]

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

[0083] Experimental Example 7 The stability of a cosmetic composition containing the plant stem cell culture extract of the present invention and osmocell containing lactic acid bacteria exosomes was measured.

[0084] To test the stability of the cosmetic formulations, the examples and comparative examples of the present invention were prepared according to Table 3, stored in opaque glass containers in a constant temperature bath maintained at 45°C for 12 weeks, and also stored in opaque glass containers in a completely light-shielded refrigerator maintained at 4°C for 12 weeks. After these tests, the degree of separation and discoloration was measured comparatively.

[0085] The results are shown in Table 10 below. At this time, the degree of product separation and discoloration was evaluated by classifying it into the following six grades. <Product discoloration evaluation criteria> 0: No change; 1: Very slight separation (discoloration); 2: Slight separation (discoloration); 3: Moderately strong separation (discoloration); 4: Strong separation (discoloration); 5: Extremely strong separation (discoloration)

[0086] [Table 10]

[0087] As can be seen from Table 10 above, the example containing Osmocell showed no discoloration or separation at both 4°C and 45°C and was stable, whereas the comparative example containing general liposomes showed no discoloration or separation at 4°C, but some separation was observed at 45°C, indicating instability.

[0088] Experimental Example 8 Skin safety tests were conducted on a cosmetic composition containing the plant stem cell culture extract of the present invention and osmocell containing lactic acid bacteria exosomes.

[0089] Twenty subjects (average age 30.5 years, age distribution 18-40 years) underwent a patch test on their upper arms using Haye's Test Chamber, after being prepared with Example 1 and Comparative Example 1 of the present invention according to Table 3. However, subjects with psoriasis, eczema, or other skin lesions, as well as pregnant or breastfeeding women, or those taking contraceptives, antihistamines, etc., were excluded from this experiment. After wiping the test site with 70% ethanol and drying it, 15 μg each of Example and Comparative Example were dropped into the chamber, and then the patch was placed on the upper arm, which was the test site, and fixed in place. The patch was left on 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, respectively. The assessment was performed 24 hours and 48 hours after removal of the patch, and skin reactions were evaluated according to the regulations of the International Contact Dermatitis Research Group (ICDRG) as 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 skin safety test results showed that both the example and the comparison example had an average irritation level of 0, indicating that they are safe cosmetics that do not irritate the skin.

[0093] Below, based on the results of the experimental examples described above, we present a cosmetic composition containing plant stem cell culture extract and osmocell encapsulating lactic acid bacteria exosomes. However, the compositions of the present invention are not limited to the formulation examples below.

[0094] Formulation Example 1: Softening Lotion (Skin Lotion)

[0095] [Table 13]

[0096] Formulation Example 2: Nourishing Lotion (Milk Lotion)

[0097] [Table 14]

[0098] Formulation Example 3: Nutritional Cream

[0099] [Table 15]

[0100] Formulation Example 4: Massage Cream

[0101] [Table 16]

[0102] Formulation example 5: Pack

[0103] [Table 17]

Claims

1. It contains Osmocell as an active ingredient, which is a lipid complex consisting of phospholipids, phytosphingosine, ceramide NP, cholesterol, phytosterols, β-sitosterol, and squalene, encapsulating plant stem cell culture extract and lactic acid bacteria exosomes. The aforementioned osmocell is a double-membrane vesicle, The Osmocell contains, based on the total weight of the Osmocell, 0.1 to 10% by weight of a lipid complex, 0.01 to 50.0% by weight of plant stem cell culture extract, 0.00001 to 1.0% by weight of lactic acid bacteria exosomes, and the remainder is purified water. The lipid complex contains, based on the total weight of the lipid complex, 0.1 to 30.0% by weight of phospholipids, 0.01 to 20.0% by weight of phytosphingosine, 0.01 to 30.0% by weight of ceramide NP, 0.01 to 10.0% by weight of cholesterol, 0.01 to 10.0% by weight of phytosterols, 0.01 to 10.0% by weight of β-sitosterol, and 0.01 to 90.0% by weight of squalene. The aforementioned plant stem cell culture extract is characterized by comprising ginseng stem cell culture extract, ginkgo stem cell culture extract, Japanese pagoda tree 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, making it a cosmetic composition for preventing skin aging.

2. The cosmetic composition according to claim 1, characterized in that the lactic acid bacteria exosomes were isolated from a Lactobacillus plantarum culture solution.

3. The cosmetic composition according to claim 1, characterized in that the osmocell is contained in an amount of 0.001 to 100.0% by weight relative to the total weight of the cosmetic composition.

4. The cosmetic composition according to claim 1, characterized in that the Osmocell is produced by a method comprising 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.

5. The cosmetic composition according to claim 1, characterized in that it has the effect of improving wrinkles on the skin, a whitening effect, and an effect of improving skin elasticity.

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