Method for improving productivity of stem cell culture solution, method for activating same, and composition comprising same

Abstract

The present invention relates to a preparation method for enhancing the productivity and efficacy of a stem cell culture solution, and a composition for wound healing, skin improvement, and the prevention or treatment of hair loss, comprising same. The method according to the present invention has excellent effects of improving the productivity of a stem cell culture solution and enhancing the efficacy of stem cells, thereby enabling efficient acquisition of a stem cell culture solution compared to conventionally known methods, and the obtained stem cell culture solution can be used as a composition or a cosmetic composition for wound healing, skin improvement, and the prevention or treatment of hair loss.

Description

Method for improving the productivity of stem cell culture solution, method for activating the same, and composition containing the same

[0001] The present invention relates to innovative new drugs and personalized healthcare, and more particularly, to a method for improving the productivity of a culture medium containing stem cells, a method for activating the culture medium, and a composition containing the same.

[0002]

[0003] Adult stem cells are exceptionally safe for medical applications. Specifically, they do not cause cancer when transplanted into the body for organ regeneration. Because they are present in an adult body, they do not trigger immune rejection, enabling autologous transplantation using the patient's own cells. Furthermore, they possess site-specific differentiation, adapting to the characteristics of surrounding tissues. Even when injected in an undifferentiated state, they do not induce cancer. This allows them to generate the cells needed immediately after transplantation, as well as self-renew, allowing them to be regenerated and stored as undifferentiated stem cells for later use. Therefore, the importance of adult stem cells has recently been recognized, and numerous studies are underway to elucidate their utility.

[0004] Meanwhile, the skin is the organ most in contact with the external environment within the human body, protecting the body's interior. It can be broadly divided into three layers: the epidermis, the dermis, and the hypodermis. Skin is a complex organ composed of cells with diverse functions and substances with corresponding physical properties. The dermis layer maintains its inherent elasticity through a combination of elastic solid substances (fibers) and viscous liquid substances. Consequently, skin exhibits a unique physical response to mechanical tension, a property known as viscoelasticity. This viscoelasticity is due to the unique three-dimensional structure of collagen and elastin fibers within a proteoglycan matrix. As we age, skin function deteriorates and atrophies due to factors such as UV rays, pollution, and stress, leading to a decrease in skin cell count and thickness. This process alters the elastic fibers, resulting in decreased skin elasticity and sagging.

[0005] Meanwhile, exosomes are a type of extracellular vesicle (ECV) released by cells, and are nanometer-sized substances secreted by eukaryotes for the exchange of information between cells. They are composed of a phospholipid bilayer membrane, which is identical in structure to the cell membrane, and contain proteins, lipids, nucleic acids, etc. similar to those inside the parent cell. In early studies, exosomes were understood to be related to the excretion of cellular waste products rather than having a specific function. However, recent studies have newly identified various roles of exosomes. For example, it has been revealed that exosomes have a similar function to cell-derived hormones or cytokines, stimulating and inhibiting cellular metabolism.

[0006]

[0007] One object of the present invention is to provide a culture medium for activated stem cells with increased productivity using a spherical leaf vesicle.

[0008] Another object of the present invention is to provide a skin improvement composition that is safe and has excellent effects such as skin whitening, wrinkle improvement, elasticity enhancement, skin regeneration, skin moisturizing, skin irritation relief, acne prevention or improvement, antioxidant, anti-inflammation, or atopy prevention or improvement, using the culture solution.

[0009] Another object of the present invention is to provide a composition for preventing or treating hair loss that is safe and has excellent hair growth and hair growth effects using the culture solution.

[0010] Another object of the present invention is to provide a wound treatment composition having an excellent effect in treating wounds, bedsores, burns, abrasions, puncture ulcer wounds, cuts, chronic skin wounds caused by active oxygen, bruises, cuts, wounds of the mucous membrane of the throat or oral cavity, lacerations, diabetic ulcers, leg ulcers, hypertensive ischemic ulcers, venous ulcers, and foot ulcers using the culture solution.

[0011] Another object of the present invention is to provide a method for producing a culture solution having the above-described excellent effect.

[0012]

[0013] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below.

[0014]

[0015] Various embodiments of the present invention are described with reference to the drawings. In the following description, various specific details, such as specific configurations, compositions, and processes, are set forth to provide a thorough understanding of the present invention. However, certain embodiments may be practiced without one or more of these specific details, or in conjunction with other known methods and configurations. In other instances, well-known processes and manufacturing techniques are not described in specific detail so as not to unnecessarily obscure the present invention. Reference throughout this specification to an embodiment means that a particular feature, configuration, composition, or characteristic described in connection with the embodiment is included in one or more embodiments of the present invention. Thus, the appearance of an embodiment in various places throughout this specification does not necessarily indicate the same embodiment of the present invention. Additionally, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments.

[0016]

[0017] The present invention is an application based on the research results of “Global material commercialization for solving skin health issues related to pigmentation, skin cell function treatment, and irritation” (Project Identification Number 2420006491 and Project Detail Number RS-2024-00509249) conducted and to be conducted by ABio materials Co., Ltd. from October 1, 2024 to September 30, 2028 as a research project of the Small and Medium Business Technology Innovation Development Project (Export-oriented) of the Korea Technology and Information Promotion Agency for SMEs of the Ministry of SMEs and Startups of the Republic of Korea.

[0018]

[0019] 1. Stem cell culture medium activated by the spore-forming plant leaves

[0020] According to one embodiment of the present invention, a stem cell culture solution activated by a bottle gourd leaf vesicle is provided.

[0021] The present invention provides a method for producing a culture solution containing stem cell-derived exosomes to enhance productivity and efficacy, and a composition containing the same for wound treatment, skin improvement, and hair loss prevention or treatment.

[0022] In the present invention, the “stem cell” refers to a cell that is maintained without differentiating into a specific cell, and has the ability to differentiate into all types of cells that make up the body, such as nerves, blood, and cartilage, when necessary. There are two main ways to obtain these stem cells: first, obtaining them from embryos developed from fertilized eggs (embryonic stem cells), and second, recovering stem cells stored in various parts of our bodies as adults (adult stem cells). Although there are differences in terms of function, both embryonic stem cells and adult stem cells have the ability to differentiate into various types of cells. Embryonic stem cells have the advantage of excellent differentiation ability and long telomeres, but they have the disadvantage of raising ethical issues and making it difficult to obtain large numbers of cells, whereas adult stem cells can be obtained in large numbers, but have the disadvantage of having a relatively low risk of infection and differentiation ability when transplanted into others.

[0023] In the present invention, the "extracellular vesicles" or "extracellular vesicles" or "extracellular vesicles (EVs)" are nano-sized vesicles secreted by all cells into the external environment for intercellular information exchange. EVs contain various biologically active substances, such as proteins, lipids, nucleic acids, and metabolites. Recently, the substances constituting EVs have been identified through high-throughput analysis. Furthermore, EVs reflect the state of the cells they originate from and have been found in various body fluids. EVs interact with target cells to perform physiological functions under normal conditions, as well as pathological functions that induce diseases. Recently, research into the diagnosis and treatment of diseases using EVs has also been actively conducted.

[0024] Extracellular vesicles are called by various names such as exosomes, microvesicles, ectosomes, microparticles, membrane vesicles, nanovesicles, and outer membrane vesicles based on their origin, secretion mechanism, and size, but the extracellular vesicles in the present invention are not limited to these. Mammalian extracellular vesicles are classified into two types: extracellular vesicles, which are created when multivesicular endosomes (MVEs) mature, and intraluminal vesicles are formed when the endosomal membrane inwardly moves, and then the multivesicular endosomes fuse with the cell surface and the intraluminal vesicles are secreted out of the cell, and microvesicles, which are secreted out of the cell when the plasma membrane protrudes outward and separate. However, it is difficult to distinguish between them once they are secreted out of the cell.

[0025] In addition, extracellular vesicles (EVs) contain various biologically active substances such as proteins, lipids, nucleic acids, and metabolites, and reflect the state of the cells from which they are derived. Furthermore, EVs exist in various body fluids such as cell culture media, blood, urine, saliva, tears, semen, breast milk, and ascites, and perform various physiological and pathological functions. For example, EVs derived from cancer cells affect cancer growth, metastasis, and angiogenesis, and EVs derived from bacteria are involved in antibiotic resistance, elimination of competing bacteria, delivery of virulence factors, and host cell regulation.

[0026] Extracellular vesicles (EVs) have recently attracted attention because of their potential for disease diagnosis and treatment. Recent efforts are focused on utilizing noninvasive liquid biopsies for disease diagnosis. One approach involves using EVs, which can be isolated from various bodily fluids, to discover new diagnostic markers and use them to diagnose diseases. Furthermore, because EVs perform physiological functions and can be delivered to specific cells or tissues, they can be utilized as therapeutic agents, drug delivery systems, and vaccines.

[0027] Furthermore, the treatment using extracellular vesicles (ECVs) has gained attention due to the discovery that EVs are a crucial component of mesenchymal stem cell (MSC)-mediated cardioprotection. Subsequent studies using EVs to regenerate heart and liver tissue have demonstrated numerous advantages over living cells for tissue regeneration. Because EVs are not living cells, they can be stored and transported for long periods of time. Furthermore, because they do not divide, mammalian EVs have a lower risk of tumorigenesis, while bacterial EVs have a lower risk of infection. Furthermore, EVs offer various advantages over other nanocarriers. Because they are cell-derived, EVs have low immunogenicity. Furthermore, because they share the same membrane topology as cells, they can be genetically engineered to express various membrane proteins, enabling the delivery of drugs and vaccines to specific cells or tissues. Furthermore, because extracellular vesicles possess a membrane and lumen, they can be loaded with various drugs, allowing them to be easily delivered to target cells through the cell membrane. However, since dying cells producing extracellular vesicles can secrete apoptotic bodies or intracellular components, managing the survival rate of extracellular vesicle-producing cells is crucial.

[0028] However, because the amount of naturally secreted extracellular vesicles is limited, treatments using extracellular vesicle mimics are being attempted. To manufacture extracellular vesicle mimics, a method can be used in which cells are treated with drugs to load them, extrusion is performed, and only the drug-loaded extracellular vesicle mimics are separated using density gradient ultracentrifugation. Extracellular vesicle mimics are attracting attention as an alternative to extracellular vesicle-based treatments because they have a much higher yield and similar drug delivery efficiency to naturally secreted extracellular vesicles. Based on this, a study has been conducted in which bone marrow-derived cells were transformed into insulin-producing cells in mice using an extracellular vesicle mimic created from pancreatic beta cells.

[0029] In another embodiment of the present invention, a stem cell culture medium activated by plant-derived extracellular vesicles is provided. The plant-derived extracellular vesicles are selected from the group consisting of, but not limited to, centella asiatica, ginseng, camellia, machihyeon, green tea, lotus, lavender, houttuynia cordata, edelweiss, desert rose, hollyhock, hydrangea, ginseng (Panax ginseng), licorice, and yew (Taxus sp.).

[0030] In the present invention, the above “Centella asiatica” is a perennial herb of the Apiaceae family, Umbelliferae order, Dicotyledonous group. Centella asiatica has been used as a medicinal plant since long ago. Pentacyclic triterpenes including asiaticoside, madecasosside, asiatic acid, and madecassic acid are known as effective ingredients of Centella asiatica, and these ingredients have been reported to have excellent effects on collagen formation in skin cells, antioxidant activity, skin aging inhibition, anti-inflammatory effects, improvement of skin photoaging, and wound healing effects, and are also used as raw materials for cosmetics and wound healing ointments in Korea.

[0031] In another embodiment of the present invention, the extracellular vesicle is provided as an exosome or a microvesicle.

[0032] In the present invention, the term "exosome" refers to a small membrane-structured vesicle secreted from various cells. Exosomes have a diameter of approximately 30 to 1,000 nm, and are vesicles released into the extracellular environment through the fusion of the multivesicular body and the plasma membrane. Representative expression markers of exosomes include HSP70, CD63, and CD9.

[0033] In the present invention, the term "microvesicles," "microvesicles," or "ectosomes" refers to a type of extracellular vesicle released from the cell membrane. In multicellular organisms, microvesicles and other extracellular vesicles are found both in tissues (the interstitial space between cells) and in various types of body fluids. Microvesicles, separated by a phospholipid bilayer, can be as small as the smallest extracellular vesicle (30 nm in diameter) or as large as 1000 nm. This appears to be larger on average than intracellularly generated extracellular vesicles known as exosomes. Microvesicles play a role in intercellular communication and can transport molecules such as mRNA, miRNA, and proteins between cells.

[0034]

[0035] In another embodiment of the present invention, a culture medium is provided, wherein the stem cells are derived from at least one of bone marrow, blood, brain, skin, fat, umbilical cord blood, and umbilical cord Wharton's jelly.

[0036] In another embodiment of the present invention, the bottle leaf packets are 1 x 10 6 particle / ㎖ to 1 x 10 10 It is preferable to activate it at particle / ㎖. The above-mentioned bottle gourd leaf vesicles are 1 x 10 6If the particle / ㎖ is less than 1 x 10, the adipose-derived stem cells may not be activated, and accordingly, the culture medium extracted from the adipose-derived stem cells may not exhibit activity. In addition, if the particle / ㎖ is less than 1 x 10, the adipose-derived stem cells may not be activated, and thus, the culture medium extracted from the adipose-derived stem cells may not exhibit activity. 10 If the particle / ㎖ is higher than this, it can be economically problematic and may even hinder the growth of adipose-derived stem cells.

[0037] In another embodiment of the present invention, a culture solution is provided that increases the expression of any one protein or gene selected from the group consisting of procollagen, KGF (Keratinocyte Growth Factor), CD34, VEGF (vascular endothelial growth factor), LEF-1 (Lymphoid enhancer-binding factor 1), and PTC-1 (patched protein, Sonic Hedgehog (Shh) receptor).

[0038] In the present invention, the “procollagen” is a precursor of collagen, which is composed of N-terminal and C-terminal telopeptides that provide a template having a triple helix structure during synthesis.

[0039] In the present invention, the “KGF” or “Keratinocyte Growth Factor” has a molecular weight of approximately 18.9 kDa, and KGF, also known as FGF7, is a major growth factor that stimulates the differentiation, growth, and physiological activity of keratinocytes that constitute the keratin of the skin. KGF promotes the division and epidermalization of skin tissue during the wound healing process, and enables the completion of complete wound healing. In addition, it interacts with EGF, IGF, and a / bFGF to prevent skin aging and is an essential factor for the healthy recovery of wounded skin. In addition, it is an important growth factor related to cell division, differentiation, and survival, and is closely related to wound regeneration in injured body parts. In particular, in the skin, it promotes the regeneration of keratinocytes of the epidermis.

[0040] In the present invention, the “VEGF” or “Vascular endothelial growth factor” has a molecular weight of approximately 18.3 kDa, and VEGF is a growth factor closely related to PDGF that induces angiogenesis. It also induces cell migration, physiologically lowers blood pressure, and increases the capacity of microvessels. In addition, the physiological phenomenon of skin regeneration is also important for the regeneration of vascular tissue and the supply of oxygen and nutrients through it, so it is a very important factor for anti-aging, regeneration of damaged skin, and growth. In addition, the transgenic overexpression of VEGF in the outer root keratinocytes of the hair follicle strongly induces vascularization around the hair follicle, thereby accelerating hair regrowth after hair loss and increasing the size of the hair follicle and hair shaft, thereby affecting hair regeneration and growth.

[0041] In the present invention, the “LEF-1” or “Lymphoid enhancer-binding factor 1” is a protein encoded by the LEF1 gene in humans, and is a gene that promotes the morphogenesis and differentiation of hair follicles.

[0042] In the present invention, the “PTC-1” or “patched protein, Sonic Hedgehog (Shh) receptor” is a gene that forms the shape of hair follicles and regulates their growth.

[0043] In another embodiment of the present invention, a culture medium is provided, wherein the culture medium suppresses the expression of any one protein or gene selected from the group consisting of TNF-α, IL-6, and melanin.

[0044] In the present invention, the above “TNF-α” or “tumor necrosis factor-α” is widely known to play an important role in the inflammatory response and is a cytokine that is a member of the acute-phase response (acute-phase protein). TNF-α is mainly secreted by activated macrophages, but is also secreted by various cells such as helper T cells, natural killer cells, and damaged neurons.

[0045] In the present invention, the “IL-6” or “interleukin 6” is an interleukin that acts as both a pro-inflammatory cytokine and an anti-inflammatory myokine, and is a cytokine that regulates the immune and inflammatory response of our body.

[0046] In the present invention, the above-mentioned "melanin" or "melanin" refers to a general term for black or brown pigments present in tissues such as the skin and eyes of various animals. It mainly exists as a melanin protein that forms a strong bond with globulin, and although it is insoluble in water, it dissolves in alkaline solutions or concentrated sulfuric acid. Melanin has the function of blocking a certain amount of ultraviolet rays, so it maintains the skin's body temperature and protects the skin from ultraviolet rays. In addition, skin color is determined by the amount of melanin. Melanin is produced in a special structure called a melanosome, which is a small, sack-shaped structure produced within the cytoplasm of melanocytes. Melanocytes extend protrusions containing melanosomes to transfer melanin to other surrounding cells, such as keratinocytes, and if the melanosome contains a phenol type such as tyrosine and its oxidizing enzyme, it can accumulate the formed melanin within itself. The produced melanosomes are transported into the epidermal cells, causing darkening of the skin and hair.

[0047]

[0048]

[0049] 2. Cosmetic composition for skin improvement, comprising the above culture solution as an effective ingredient

[0050] In one embodiment of the present invention, a cosmetic composition for improving skin is provided, which comprises the culture solution as an effective ingredient.

[0051] The skin is composed of the epidermis, dermis, and subcutaneous tissue. The epidermis, the outer layer of the skin, is composed of stratified squamous epithelium and acts as a protective barrier against the external environment. From the outside, the epidermis is divided into the stratum corneum, stratum lucidum, stratum granulosum, stratum spinosum, and stratum basale. The dermis is the layer between the epidermis and the subcutaneous tissue, and is divided into the papillary dermis and the reticular dermis. It contains blood vessels, collagen, elastin fibers, pores, arrector pili muscles, sebaceous glands, sweat glands, various sensory nerves, fibroblasts, and macrophages, which are not present in the epidermis. It occupies the largest area of ​​the skin.

[0052] The dermis is primarily composed of collagen and elastin fibers, which support the skin. Therefore, problems with the dermis can lead to wrinkles, loss of skin elasticity, and accelerated skin aging. Collagen is known to play a crucial role in skin regeneration, moisture retention, wound healing, and wrinkle improvement, and is produced by fibroblasts. Collagen has the ability to retain a large amount of water, providing moisture to the dermis. However, as we age, collagen's ability to retain water decreases, leading to an increase in wrinkles. Collagen also plays a role in wound healing, filling in the wound with the continuous production of collagen by fibroblasts.

[0053] In addition, the cosmetic composition of the present invention can be manufactured in the form of toner, nutritional lotion, nutritional essence, massage cream, cosmetic bath additive, body lotion, body milk, bath oil, baby oil, baby powder, shower gel, shower cream, sunscreen lotion, sunscreen cream, suntan cream, skin lotion, skin cream, UV protection cosmetics, cleansing milk, hair loss agent {cosmetic}, face and body lotion, face and body cream, skin whitening cream, hand lotion, hair lotion, cosmetic cream, jasmine oil, bath soap, liquid soap, beauty soap, shampoo, hand cleanser (hand cleaner), medicated soap {non-medical}, cream soap, facial wash, body cleanser, scalp cleanser, hair rinse, cosmetic soap, teeth whitening gel, toothpaste, etc. To this end, the composition of the present invention may further include a solvent, appropriate carrier, excipient, or diluent commonly used in the manufacture of cosmetic compositions.

[0054] The type of solvent that can be further added to the cosmetic composition of the present invention is not particularly limited, but for example, water, saline solution, DMSO, or a combination thereof can be used, and carriers, excipients, or diluents include, but are not limited to, purified water, oil, wax, fatty acid, fatty acid alcohol, fatty acid ester, surfactant, humectant, thickener, antioxidant, viscosity stabilizer, chelating agent, buffer, lower alcohol, etc. In addition, whitening agents, moisturizers, vitamins, sunscreens, perfumes, dyes, antibiotics, antibacterial agents, and antifungal agents can be included as needed.

[0055] As the above oils, hydrogenated vegetable oil, castor oil, cottonseed oil, olive oil, palm oil, jojoba oil, and avocado oil can be used, and as the waxes, beeswax, spermaceti, carnauba, candelilla, montan, ceresin, liquid paraffin, and lanolin can be used.

[0056] As fatty acids, stearic acid, linoleic acid, linolenic acid, and oleic acid can be used; as fatty acid alcohols, cetyl alcohol, octyl dodecanol, oleyl alcohol, panthenol, lanolin alcohol, stearyl alcohol, and hexadecanol can be used; and as fatty acid esters, isopropyl myristate, isopropyl palmitate, and butyl stearate can be used. As surfactants, cationic surfactants, anionic surfactants, and nonionic surfactants known in the art can be used, and surfactants derived from natural products are preferable if possible.

[0057] In addition, it may include absorbents, thickeners, antioxidants, etc., which are widely known in the cosmetics field, and the types and amounts of these are as known in the art.

[0058] In another embodiment of the present invention, a cosmetic composition is provided, wherein the skin improvement is skin whitening, wrinkle improvement, elasticity enhancement, skin regeneration, skin moisturizing, skin irritation relief, acne prevention or improvement, antioxidant, anti-inflammation, or atopy prevention or improvement.

[0059] In another embodiment of the present invention, the culture solution is preferably included in an amount of 1 to 100 μg / ml. If the culture solution is less than 1 μg / ml, the skin improvement effect may be minimal, and if it exceeds 100 μg / ml, it may have a negative effect on the physical properties of the cosmetic.

[0060] In another embodiment of the present invention, the extracellular vesicles are 1 X 10 7 ~1 X 10 12 It is preferable that the number of particles / ml is included. The extracellular vesicles are 1 X 10 7 If the number of particles is less than 100 particles / ml, the skin improvement effect may be minimal, and ~1 X 10 12 If the number of particles / ml is exceeded, it may have a negative effect on the properties of cosmetics.

[0061] In another embodiment of the present invention, the cosmetic composition is provided as a shampoo, soap, rinse, surfactant-containing cleansing, cream, lotion, ointment, tonic, treatment, conditioner, suspension, emulsion, paste, gel, oil, wax, spray, aerosol, mist, or powder.

[0062]

[0063] 3. A pharmaceutical composition for preventing or treating hair loss, comprising the above culture solution as an active ingredient.

[0064] In one embodiment of the present invention, a pharmaceutical composition for preventing or treating hair loss is provided, which comprises the culture solution as an active ingredient.

[0065] With the rise of social stress, environmental pollution, Westernized eating habits such as instant foods, and frequent perms and dyeing, the number of people suffering from hair loss is gradually increasing. The hair cycle can be divided into the growth phase (anagen), when hair grows; the catagen phase, when growth ends and the hair bulb shrinks; the telogen phase, when the hair papilla ceases activity and the hair remains on the scalp; and the development phase, when the hair papilla begins activity or new hair is produced, causing old hair to fall out.

[0066] While a normal person has a large amount of hair in the growth phase, those with alopecia have a large amount of hair in the resting phase, resulting in visible hair loss. As hair loss progresses, the growth phase shortens, causing hair to become smaller. Therefore, treating hair loss requires accelerating the transition of hair follicles from the resting phase to the growth phase and lengthening the shortened growth phase.

[0067] Male pattern baldness is a condition caused by the male hormone testosterone. Testosterone is converted into the more potent hormone dihydrotestosterone (DHT) by an enzyme called 5 alpha-reductase. This hormone then acts on hair follicles, shifting them from the growth phase to the regression phase, resulting in hair loss. Therefore, methods that inhibit the production of DHT by 5 alpha-reductase are primarily used to treat male pattern baldness.

[0068] Female pattern baldness is primarily caused by a decline in estrogen levels after menopause. Minoxidil or estrogen is the most commonly used treatment for female pattern baldness.

[0069] Alopecia areata is caused by autoimmune diseases, psychological stress, or genetic predisposition. This type of alopecia areata has fundamentally different causes than androgenetic alopecia, and its treatment methods are also different. These include administering corticosteroids, applying minoxidil to the affected area, or artificially stimulating the area.

[0070] This type of hair loss causes psychological stress, lowering one's quality of life and significantly disrupting interpersonal relationships and social life. The image one projects in social life is deeply impactful, and the importance of appearance as an invisible source of self-esteem and social vitality is driving increasing interest in the causes and treatments of hair loss.

[0071] The composition according to the present invention can be formulated in the form of a shampoo, hair treatment, scalp treatment, hair essence, hair pack, hair cream, hair tonic, general ointment, etc.

[0072] When the formulation of the present invention is a paste, cream or gel, animal fiber, plant fiber, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as a carrier component.

[0073] When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and particularly in the case of a spray, a propellant such as chlorofluorohydrocarbon, propane / butane or dimethyl ether may be additionally included.

[0074] In the case where the formulation of the present invention is a solution or emulsion, a solvent, solvating agent or emulsifying agent is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan.

[0075] When the formulation of the present invention is a suspension, liquid diluents such as water, ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tragacanth may be used as carrier components.

[0076] In addition to the ingredients described above, the composition of the present invention may further include functional additives and ingredients typically found in scalp or hair compositions. The functional additives may include ingredients selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, high molecular weight peptides, high molecular weight polysaccharides, sphingolipids, and seaweed extracts.

[0077] In another embodiment of the present invention, the culture solution is preferably included in an amount of 1 to 100 μg / ml. If the culture solution is added in an amount less than 1 μg / ml, it is difficult to expect a hair loss prevention and treatment effect, and if it is added in an amount exceeding 100 μg / ml, it may deteriorate the properties of cosmetic compositions such as shampoo.

[0078] In another embodiment of the present invention, the extracellular vesicles are 1 X 10 7 ~1 X 10 12 It is preferable that the number of particles / ml is included. The extracellular vesicles are 1 X 10 7 If the particle count is less than 100 particles / ml, it is difficult to expect a preventive treatment effect for hair loss, and 1 X 10 12 When added in a particle count exceeding 100 particles / ml, the properties of cosmetic compositions such as shampoo may deteriorate.

[0079]

[0080] 4. A composition for wound treatment comprising the above culture solution as an effective ingredient.

[0081] In one embodiment of the present invention, a composition for wound treatment is provided, which comprises the culture solution as an active ingredient.

[0082] In another embodiment of the present invention, a composition for treating a wound is provided, wherein the wound is selected from the group consisting of a wound, a bedsore, a burn, an abrasion, a puncture ulcer, a stab wound, a chronic skin wound caused by active oxygen, a bruise, a cut, a wound of the mucous membrane of the throat or mouth, a laceration, a diabetic ulcer, a leg ulcer, a hypertensive ischemic ulcer, a venous ulcer, and a foot ulcer.

[0083] In the present invention, the “wound” is preferably selected from the group consisting of wounds, bedsores, burns, abrasions, puncture ulcer wounds, stab wounds, chronic skin wounds caused by active oxygen, bruises, cuts, wounds of the mucous membrane of the throat or mouth, lacerations, diabetic ulcers, leg ulcers, hypertensive ischemic ulcers, venous ulcers, and foot ulcers, but is not limited thereto.

[0084] In addition, the pharmaceutical composition of the present invention may further include an appropriate carrier, excipient, or diluent according to a conventional method. Carriers, excipients, and diluents that may be included in the pharmaceutical composition of the present invention include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0085] In addition, the pharmaceutical composition according to the present invention can be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, or sterile injection solutions, each according to a conventional method.

[0086] In detail, when formulating, it can be prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants that are commonly used.

[0087] Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations can be prepared by mixing the pharmaceutical composition of the present invention with at least one excipient, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used.

[0088] Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives.

[0089] Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories.

[0090] Non-aqueous solvents and suspending agents may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases may include witepsol, macrogol, Tween 61, cocoa butter, laurin butter, and glycerogelatin.

[0091] Routes of administration of the pharmaceutical composition according to the present invention include, but are not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal. Oral or parenteral administration is preferred.

[0092] The term "parenteral" as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. The pharmaceutical composition of the present invention may also be administered in the form of a suppository for rectal administration.

[0093] The pharmaceutical composition of the present invention may vary depending on various factors including the activity of the specific compound used, age, body weight, general health, sex, diet, administration time, administration route, excretion rate, drug combination, and severity of the specific disease to be prevented or treated, and the dosage of the pharmaceutical composition may vary depending on the patient's condition, body weight, degree of disease, drug form, administration route, and period, but may be appropriately selected by those skilled in the art, and may be administered at 0.0001 to 50 mg / kg or 0.001 to 50 mg / kg per day. Administration may be administered once a day or divided into several times. The dosage does not limit the scope of the present invention in any way. The pharmaceutical composition according to the present invention may be formulated as a pill, a dragee, a capsule, a liquid, a gel, a syrup, a slurry, or a suspension.

[0094]

[0095] 5. Method for preparing the above culture solution

[0096] In one embodiment of the present invention, a method for producing a culture medium is provided, comprising the steps of: (1) culturing stem cells in a medium containing serum; (2) activating stem cells in a serum-free or serum-free medium to which Centella asiatica leaf vesicles are added; (3) washing the stem cells and then culturing them in a serum-free medium; (4) removing cells and impurities from the culture medium using a 0.2 μm filter filtration system; and (5) freeze-drying the culture medium containing exosomes.

[0097] The present invention can obtain a culture solution with excellent skin regeneration, hair growth and hair growth effects with high purity and content through the above process.

[0098] In the present invention, the adult stem cell refers to an undifferentiated cell having multipotency derived from an adult cell of a mammal, including a human, preferably a human, and may be derived from various adult cells such as bone marrow, blood, brain, skin, fat (i.e., adipose tissue or fat cells), umbilical cord blood, and Wharton's jelly of the umbilical cord. In the present specification, the "adult stem cell" includes a mesenchymal stem cell derived from an adult cell.

[0099]

[0100] The stem cells or their culture solution provided in the present invention are derived from stem cells activated through the vesicles of the leaves of the purslane plant, and compared to stem cell-derived culture solutions, they have excellent wound healing or wound healing promotion activity by promoting cell migration to the wound site, and have excellent skin permeability, and when absorbed, they have excellent skin improvement effects such as skin moisturizing, skin whitening, wrinkle improvement, and anti-aging, and are safe without causing irritation or side effects to the skin.

[0101] In addition, the stem cells or their culture solution provided in the present invention are derived from stem cells activated through the spores of the leaves of the purslane plant, and compared to the stem cell culture solution, they have superior hair growth and hair restoration effects, and thus have excellent hair loss prevention and treatment effects, and are safe without side effects when consumed or applied to the skin.

[0102]

[0103] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples, in accordance with the gist of the present invention.

[0104]

[0105] Preparation example

[0106] [Preparation Example 1] Cultivation of adipose-derived stem cells under serum medium conditions

[0107] Adipose-derived stem cells were cultured in alpha-MEM medium containing L-glutamine, deoxyribonucleosides, and ribonucleosides, with 10% Fetal Bovine Serum (FBS), at a density of 3 x 10 6 After inoculation at a concentration of 10 cells / ml, subculture was performed at 37°C under 5% CO2 for approximately 18 days.

[0108]

[0109] [Preparation Example 2] Manufacturing of bottle gourd leaf packets

[0110] [Preparation Example 2-1] High-pressure treatment of bottle gourd

[0111] 30 g of dried plant material was placed in a plastic bag with distilled water, sealed well to prevent air from entering, and then ultra-high pressure treatment was performed at a temperature of 25°C and a pressure of 200 MPa for 30 seconds using an ultra-high pressure machine.

[0112] [Preparation Example 2-2] Extracting the bottle gourd juice

[0113] The ultra-high pressure-treated bottle gourd was squeezed using a general juicer with a low-speed screw of 30 rpm. The obtained bottle gourd juice was filtered through a mesh screen to remove suspended matter. The recovered bottle gourd juice was stored at -80℃ until purification.

[0114] [Preparation Example 2-3] Supernatant recovery for exosome purification

[0115] Because the extract of the plant needed to remove large contaminants for exosome purification, centrifugation was performed at 10,000 × g for 10 min at 4°C. After centrifugation, the supernatant was collected to form an aqueous two-phase system.

[0116] [Preparation Example 2-4] Freeze-drying of the supernatant

[0117] To reduce the volume of the supernatant for mass production of exosomes, lyophilization was performed. The solution was frozen at -80°C for 20 hours and then dried in a vacuum freeze dryer for 100 hours. The vacuum typically refers to the pressure of the freeze dryer, and the freezing and drying times may vary depending on the solution volume.

[0118] [Preparation Example 2-5] Formation of a two-phase system on Mercury

[0119] Purified water was added to the freeze-dried supernatant, and an aqueous two-phase system was formed using PEG (Polyethylene glycol) / Dextran. PEG (purchased from Sigma Aldrich) with a molecular weight of 10,000–35,000 was used in an amount of 3.3 wt%, and Dextran (purchased from Sigma Aldrich) with a molecular weight of 300,000–650,000 was used in an amount of 1.7 wt% to form an aqueous two-phase system.

[0120] [Preparation Example 2-6] Recovery of exosomes derived from Centella asiatica

[0121] After mixing the supernatant and PEG / Dextran solution, centrifugation was performed at 1,000 × g for 10 minutes at 4°C. After centrifugation, the supernatant was removed to recover exosomes.

[0122] [Preparation Example 2-7] Additional Washing Process

[0123] To increase purity, the recovered supernatant was subjected to an additional washing process using the same concentration of the aqueous two-phase solution. After three repeated treatments, the final supernatant, enriched in exosomes, was recovered.

[0124]

[0125] [Preparation Example 3] Stem cell activation culture using serum medium with added spores of the spore-forming plant leaves

[0126] After washing the cells subcultured in Preparation Example 1 three times with PBS (Phosphate Buffered Saline), 1 x 10 were seeded in a medium containing 10% FBS (Fetal Bovine Serum) mixed with alpha-MEM medium containing L-glutamine, deoxyribonucleosides, and ribonucleosides. 8 The cells were inoculated at a concentration of 10 cells / ml and cultured for 48 hours.

[0127] After removing the culture medium from the subcultured cells and washing them three times with PBS (Phosphate Buffered Saline), the centella asiatica leaf vesicles prepared in Preparation Example 2 were seeded at 1 x 10 in a medium containing 10% FBS (Fetal Bovine Serum) mixed with alpha-MEM medium containing L-glutamine, deoxyribonucleosides, and ribonucleosides. 6 particle / ㎖ to 1 x 10 10 Up to particle / ㎖ can be added, 1 x 10 8 particle / ㎖ was added and cultured for 24 hours.

[0128]

[0129] [Preparation Example 4] Culturing of adipose-derived stem cells in serum-free medium conditions

[0130] In the above Preparation Example 3, the culture medium of the activated adipose-derived stem cells was removed by adding the scutellaria baicalensis leaf vesicles, and then washed three times with PBS (Phosphate Buffered Saline), and then 1 x 10 were added to a medium containing 15 mM HEPES, DMEM containing sodium bicarbonate, and Nutrient Mixture F-12 in a 1:1 mixture. 8 The cells were inoculated at a concentration of 10 cells / ㎖ and cultured for 120 hours.

[0131]

[0132] [Preparation Example 5] Culturing of adipose-derived stem cells

[0133] The cells subcultured in the above Preparation Example 3 were seeded at 1 x 10 in a medium containing 10% Fetal Bovine Serum (FBS) mixed with alpha-MEM medium containing L-glutamine, deoxyribonucleosides, and ribonucleosides. 8 The cells were inoculated at a concentration of 10 cells / ml and cultured identically for 48 hours.

[0134]

[0135] [Preparation Example 6] Culturing of stem cells without activation process

[0136] The adipose-derived stem cells in the above Preparation Example 3 were cultured in the same manner for 48 hours, but the stem cell activation culture step using the spores of the centella asiatica leaf was not performed.

[0137]

[0138] Example

[0139] [Example 1] Analysis of adipose-derived stem cells activated by the leaves of the plant

[0140] In order to compare the growth of adipose-derived stem cells activated by the spore-forming agent of the above Example 3, an MTT assay was performed. 1 x 10 adipose-derived stem cells were seeded in a 96-well plate. 5After inoculating at 10 cells / mL, the cells were cultured at 37°C for 18 hours under 5% CO2. The control group was cultured with adipose-derived stem cells for 24 or 48 hours in a medium containing 10% Fetal Bovine Serum (FBS) mixed with alpha-MEM medium containing L-glutamine, deoxyribonucleosides, and ribonucleosides, or in a medium supplemented with Centella Asiatica leaf vesicles. To measure the cell viability, MTT solution (5 mg / mL) was added, and the formazan formed over 4 hours was dissolved with Dimethyl sulfoxide (DMSO) and the absorbance at 570 nm was measured using an ELISA reader.

[0141] The results are shown in Table 1 below.

[0142] Culture time Cell growth rate (% of control) Adipose-derived stem cells Adipose-derived stem cells activated by the scutellaria baicalensis leaf vesicles 24 hours 100 115.148 hours 110.2130.25

[0143] As confirmed in Table 1 above, the growth rate of cells cultured in a medium containing adipose-derived stem cells and agar-like leaf vesicles was 115.1% at 24 hours and 130.25% at 48 hours compared to the control group, indicating that cell growth increased compared to the control group, adipose-derived stem cells.

[0144]

[0145] [Example 2-1] Effect of increasing expression of genes related to skin anti-aging and regeneration

[0146] 5 x 10 human dermal fibroblasts 4After seeding cells / well, 10 ug / ml of adipose-derived stem cell culture medium activated by adipose-derived stem cell exosomes and Centella Asiatica leaf vesicles was treated as a control group. After 24 hours, RNA was extracted from the cells of each group, cDNA was synthesized using an intron premix, and real-time PCR (PCR) was performed using this. The expression of procollagen, KGF, and CD34, which are genes related to skin aging prevention and regeneration, was confirmed in the cells of each treatment group. Procollagen, whose expression was confirmed, corresponds to a gene related to collagen synthesis, which maintains skin elasticity and is necessary for the formation of the main protein of the skin. KGF (keratinocyte growth factor) is a major growth factor that stimulates the differentiation and growth and physiological activity of skin cells, and corresponds to a gene that prevents skin aging, synthesizes collagen, regulates skin regeneration, and is involved in anti-aging. Lastly, CD34 is a factor involved in vascular regeneration and corresponds to a gene involved in skin regeneration and wound healing.

[0147] The results are shown in Table 2 below.

[0148] mRNA (% of control) Adipose-derived stem cells Adipose-derived stem cells activated by the vesicles of the plant Procollagen 100 120.2 KGF 100 123.7 CD34 100 119.7

[0149] As a result, it was confirmed that the gene expression of procollagen (120.2%), KGF (123.7%), and CD34 (119.7%) increased in the group treated with the culture medium of adipose-derived stem cells activated by Centella asiatica leaf vesicles compared to the control group. Through this, it was confirmed that the culture medium of adipose-derived stem cells activated by Centella asiatica leaf vesicles provided in the present invention prevents skin aging, promotes skin regeneration and wound healing, and has an anti-aging effect.

[0150]

[0151] [Example 2-2] Effect of increasing expression of genes related to skin elasticity

[0152] 5 x 10 human dermal fibroblasts 4 After inoculating cells / well, 10 ug / ml of adipose-derived stem cell culture medium activated by adipose-derived stem cell exosomes and Centella Asiatica leaf vesicles was treated as a control group. After 24 hours, RNA was extracted from the cells of each group, cDNA was synthesized using an intron premix, and real-time PCR (PCR) was performed using this. The expression of elastin, a gene related to skin elasticity, was confirmed in the cells of each treatment group, and the results are shown in Table 3 below.

[0153] mRNA (% of control) Adipose-derived stem cells activated by the vesicles of the plant's leaves Elastin 100121.7

[0154] As a result, it was confirmed that the gene expression of elastin (121.7%) increased in the group treated with the culture medium of adipose-derived stem cells activated by the Centella asiatica leaf vesicle compared to the control group. Through this, it was confirmed that the culture medium of adipose-derived stem cells activated by the Centella asiatica leaf vesicle provided by the present invention can maintain skin elasticity.

[0155]

[0156] [Example 2-3] Inhibition and activation of melanin production in B16F10 cells (whitening effect)

[0157] B16F10 cells were cultured in DMEM (GIBCO, Cat No. 11995-065) supplemented with 100 IU / mL penicillin (GIBCO, Cat No. 15140122), 100 μg / mL streptomycin (GIBCO, Cat No. 15140122) and 10% FBS (GIBCO, Cat No. 16000-044) at 37°C in a 5% CO atmosphere for 18 h. 2were cultured under. After culture, the medium was removed, and melanin production was induced with α-melanocyte stimulating hormone (α-MSH, Sigma, Cat No. M4135), and 10 ug / ml of each sample (negative control, positive control (Arbutin 0.01%), adipose-derived stem cell culture medium, and adipose-derived stem cell culture medium activated by Centella asiatica leaf vesicle) was treated. After 48 hours of culture, the cells were collected. After treating the collected cells with 1 N NaOH and completely dissolving the melanin at high temperature, the absorbance was measured at 490 nm using an ELISA reader (Molecular device, USA), and a standard curve was obtained with synthetic melanin (Sigma, Cat No. M0418) to quantify the amount of melanin, which is shown in Table 4 below.

[0158] (% of Negative control) Negative control Positive control (Arbutin 0.01%) Adipose-derived stem cells Adipose-derived stem cells activated by the vesicles of the leaves of the plant α-MSH (100 μg / mL) Melanin amount 1005257.443.9

[0159] As a result, it was confirmed that melanin production was reduced by 48% in the positive control group (Arbutin 0.01%) compared to the negative control group. In addition, it was confirmed that melanin production was inhibited by 56.1% in the group treated with the culture medium of adipose-derived stem cells activated by the Centella asiatica leaf vesicle provided by the present invention compared to the group treated with adipose-derived stem cells. Through this, it was confirmed that the culture medium of adipose-derived stem cells activated by the Centella asiatica leaf vesicle provided by the present invention has a whitening effect by inhibiting melanin production.

[0160]

[0161] [Example 2-4] Effect of increasing expression of genes related to skin regeneration

[0162] 5 x 10 human dermal fibroblasts 4 After inoculating cells / well, 10ug / ml of adipose-derived stem cell culture medium and adipose-derived stem cell culture medium activated by the Centella Asiatica leaf vesicle provided by the present invention were treated as a control group. After 24 hours, RNA was extracted from the cells of each group, cDNA was synthesized using an intron premix, and real-time PCR (PCR) was performed using this. The gene expression of VEGF, a gene related to skin regeneration, was confirmed in the cells of each treatment group, and the results are shown in Table 5 below.

[0163] mRNA (% of control) Adipose-derived stem cells VEGF100130.5 activated by adipose-derived stem cells VEGF100130.5

[0164] As a result, it was confirmed that the gene expression of KGF (124.2%), CD34 (118.2%), and VEGF (130.5%) increased in the group treated with the adipose-derived stem cell culture solution activated by the Centella asiatica leaf vesicle provided by the present invention compared to the control group. Through this, it was confirmed that the adipose-derived stem cell culture solution activated by the Centella asiatica leaf vesicle provided by the present invention has skin regeneration and wound healing effects.

[0165]

[0166] [Example 2-5] Migration effect of human skin fibroblasts at the wound site

[0167] 5 x 10 human dermal fibroblasts 4 After inoculating cells / well, scratches were made at regular intervals, and 10ug / ml of adipose-derived stem cell culture solution and adipose-derived stem cell culture solution activated by the scutellaria baicalensis leaf vesicle were treated as a control group. After 48 hours, the degree of cell migration was checked, and the results are shown in Table 6.

[0168] Adipose-derived stem cells activated by the leaf vesicles of the adipose-derived stem cells (% of control) 100113.7

[0169] As shown in Table 7 above, it can be confirmed that the degree of cell migration increased by 113.7% in the group treated with the culture medium of adipose-derived stem cells activated by the scutellaria baicalensis leaf vesicle compared to the control culture medium.

[0170]

[0171] [Example 3] Effect of increasing the expression of genes related to hair follicle growth and development in hair papilla cells

[0172] 5 x 10 human breast papilla cells 5 After inoculating cells / well, 10ug / ml of adipose-derived stem cell culture solution activated by the adipose-derived stem cell culture solution Centella asiatica leaf vesicle was treated as a control group, and after 24 hours, RNA was extracted from the cells of each group, cDNA was synthesized using an intron premix, and real-time PCR (Real-time PCR) was performed using this. In the cells of each treatment group, the gene expression of LEF-1, PTC-1, and KGF, which are genes related to the growth and development of hair follicles, was confirmed, and the results are shown in Table 7 below. The LEF-1 (lymphoid enhancer-binding factor-1), whose expression was confirmed, is a gene that promotes the morphogenesis and differentiation of hair follicles, PTC-1 (patched protein, Sonic Hedgehog (Shh) receptor) is a gene that forms the morphology of hair follicles and regulates their growth, and KGF (keratinocyte growth factor) is a gene that has an important influence on the growth, development, and differentiation of hair follicles.

[0173] mRNA (% of control) Adipose-derived stem cells activated by the leaf vesicle of the plant LEF-1100115.7 PTC-1100130.1 KGF100122.7

[0174] As a result, it was confirmed that the gene expression of LEF-1, PTC-1, and KGF increased in the group treated with the adipose-derived stem cell culture solution activated by the Centella asiatica leaf vesicle provided by the present invention compared to the control group. Through this, it was confirmed that the adipose-derived stem cell culture solution activated by the Centella asiatica leaf vesicle provided by the present invention has the effect of promoting the growth, development, and differentiation of hair follicles.

[0175]

[0176] [Example 4] Regulation of anti-inflammatory gene expression in RAW 264.7 cells

[0177] RAW 264.7 cells were cultured in DMEM (GIBCO, Cat No. 11995-065) supplemented with 100 IU / mL penicillin (GIBCO, Cat No. 15140122), 100 μg / mL streptomycin (GIBCO, Cat No. 15140122) and 10% FBS (GIBCO, Cat No. 16000-044) at 37°C in a 5% CO atmosphere for 18 h. 2 After culturing, the medium was removed, and 10 ug / mL of the negative control, 10 ug / mL of LPS (Sigma, Cat No. L263) alone, adipose-derived stem cell culture medium treated with LPS, and adipose-derived stem cell culture medium activated by the Centella Asiatica leaf vesicle provided by the present invention were treated, and then cultured for 24 hours. After culturing, RNA was extracted using an RNA extraction kit (Bioneer, Cat No. K-3140), and qRT-PCR was performed using a qPCR master mix (Bioneer, Cat No. K6251). The Real-Time PCR System was performed using Applied Biosystems, USA, and the results are shown in Table 8 below.

[0178] Relative mRNA expression level (normalized to GAPDH mRNA) Negative control LPS Adipose-derived stem cells Adipose-derived stem cells activated by the vesicles of the scutellaria baicalensis-LPS (10 μg / mL) Tumor necrosis factor-α (TNF-α) 1 2.5 7.16.12 Interleukin-6 (IL-6) 1 7 0 0 4 12 3 20

[0179] As a result, it was confirmed that the mRNA expression of inflammatory genes, TNF-α and IL-6, was reduced in the group treated with the culture medium of adipose-derived stem cells activated by Centella asiatica leaf vesicles compared to the group treated with adipose-derived stem cells. Through this, it was confirmed that the culture medium of adipose-derived stem cells activated by Centella asiatica leaf vesicles provided by the present invention has an anti-inflammatory effect by suppressing the expression of anti-inflammatory genes.

[0180]

[0181] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0182]

Claims

1. Stem cell culture medium activated by the spores of the leaves of the plant.

2. In paragraph 1, A stem cell culture medium, wherein the stem cells are derived from at least one of bone marrow, blood, brain, skin, fat, umbilical cord blood, and umbilical cord Wharton's jelly.

3. In paragraph 2, The above stem cell culture medium is a stem cell culture medium that increases the growth rate of stem cells.

4. In paragraph 3, In the above stem cell culture medium, the stem cell culture medium contains at least one growth factor selected from the group consisting of transforming growth factor beta1 (TGF-beta1), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), and vascular endothelial growth factor (VEGF).

5. In paragraph 4, The above bottle leaf packet is 1 x 10 6 particle / ㎖ to 1 x 10 10 A stem cell culture medium having particles / ㎖.

6. In paragraph 5, The above stem cell culture medium increases the expression of any one protein or gene selected from the group consisting of procollagen, KGF (Keratinocyte Growth Factor), CD34, VEGF (vascular endothelial growth factor), LEF-1 (Lymphoid enhancer-binding factor 1), and PTC-1 (patched protein, Sonic Hedgehog (Shh) receptor).

7. In paragraph 5, The above stem cell culture medium is a stem cell culture medium that suppresses the expression of any one protein or gene selected from the group consisting of TNF-α, IL-6, and melanin.

8. Stem cells activated by the vesicles of the leaves of the plant.

9. In paragraph 8, The above stem cells are stem cells derived from at least one of bone marrow, blood, brain, skin, fat, umbilical cord blood, and umbilical cord Wharton's jelly.

10. In paragraph 9, The above stem cells are stem cells that increase the production of the above stem cell-derived extracellular vesicles.

11. A cosmetic composition for skin improvement, comprising a stem cell culture solution of any one of claims 1 to 7 as an active ingredient.

12. In paragraph 11, A cosmetic composition wherein the above skin improvement is skin whitening, wrinkle improvement, elasticity enhancement, skin regeneration, skin moisturizing, skin irritation relief, acne prevention or improvement, antioxidant, anti-inflammation, or atopy prevention or improvement.

13. In paragraph 12, The cosmetic composition is a cosmetic composition that is a shampoo, soap, rinse, surfactant-containing cleansing, cream, lotion, ointment, tonic, treatment, conditioner, suspension, emulsion, paste, gel, oil, wax, spray, aerosol, mist, or powder.

14. A pharmaceutical composition for preventing or treating hair loss, comprising a stem cell culture solution of any one of claims 1 to 7 as an active ingredient.

15. A composition for wound treatment, comprising a stem cell culture solution of any one of claims 1 to 7 as an active ingredient.

16. In paragraph 15, A composition for treating a wound, wherein the above wound is selected from the group consisting of wounds, bedsores, burns, abrasions, puncture ulcer wounds, stab wounds, chronic skin wounds caused by active oxygen, bruises, cuts, wounds of the mucous membrane of the throat or mouth, lacerations, diabetic ulcers, leg ulcers, hypertensive ischemic ulcers, venous ulcers, and foot ulcers. 17.(1) A step of culturing stem cells in a medium containing serum; (2) a step of activating stem cells with a serum medium containing a spore-filled bottle gourd leaf; A method for producing a stem cell culture solution activated by a spore-containing herbicide.

18. In paragraph 17, (3) A method for producing a stem cell culture solution, further comprising a step of washing the stem cells and then culturing them under serum-free or serum-free medium conditions.

19. In paragraph 18, (4) A method for producing a stem cell culture solution, further comprising a step of removing cells and impurities from the culture solution using a 0.2 ㎛ filter filtration system.

20. In paragraph 19, (5) A method for producing a stem cell culture solution, further comprising a step of freeze-drying the culture solution containing exosomes.

21. In paragraph 20, (6) A step of forming an aqueous two-phase system using PEG (Polyethylene glycol) / dextran in the above freeze-dried product; and (7) A method for producing a stem cell culture solution, further comprising the step of obtaining a lower layer containing concentrated extracellular vesicles among the above two-phase aqueous solutions.

22. A method for preventing or treating hair loss, comprising a step of administering to a subject a stem cell culture solution according to any one of claims 1 to 7.

23. A wound treatment method comprising a step of administering a stem cell culture solution of any one of claims 1 to 7 to a subject.