New use of milk exosomes

Abstract

The present invention relates to a new use of milk exosomes, and more specifically, provides various uses of milk exosomes in, for example, functional cosmetics, functional health foods having various functions such as improvement of intestinal health and immunity, and drugs including wound healing drugs.

Description

Technical Field

[0001] This invention relates to novel uses of milk exosomes, and more specifically, to various uses of milk exosomes in functional cosmetics, health foods, pharmaceuticals, and the like. Background Technology

[0002] Exosomes are vesicles ranging in size from tens to hundreds of nanometers, composed of a phospholipid bilayer with the same structure as the plasma membrane. Exosomes contain exosomal cargo such as proteins and nucleic acids (mRNA, miRNA, etc.), and this cargo includes a wide range of signal transduction factors. These signal transduction factors are known to be cell-type specific and regulated differently depending on the environment of the secreting cell. Exosomes are known to be intercellular signal transduction mediators secreted by cells, and various cellular signals transmitted by exosomes regulate cellular behavior, including activation, growth, migration, differentiation, dedifferentiation, apoptosis, and necrosis of targeted cells. Exosomes are known to contain specific genetic material and bioactive factors depending on the nature and state of their source cells.

[0003] Since exosomes are primarily derived from cells, they differ from other nanoparticles in that they are biocompatible. Furthermore, due to their ability to carry or label drugs or bioactive components internally or on their surface, there has been ongoing effort to utilize exosomes as drug carriers or raw materials for cosmetics or pharmaceuticals.

[0004] In this regard, Korean Patent KR 2039302 discloses a cosmetic composition for enhancing and functionally improving the corneal skin barrier, comprising stem cell-derived exosomes as active ingredients; Korean Patent KR 1662405 discloses a pharmaceutical composition for treating cerebrovascular diseases, comprising stem cell-derived exosomes as active ingredients; Korean Patent KR 1894229 discloses a composition comprising exosomes derived from deer antler stem cell cultures as active ingredients for preventing or treating hair loss, or promoting hair growth; and Patent WO 2016039356A1 discloses an anti-inflammatory drug comprising milk-derived exosomes as active ingredients.

[0005] However, apart from their use as anti-inflammatory drugs as disclosed in WO2016039356A1 and as drug carriers as disclosed by Munagala et al. (Cancer Lett., 371(1):48-61, 2016), other uses of milk exosomes are little known.

[0006] The skin is composed of the following layers: the epidermis, which is composed of stratified squamous epithelium; the dermis, which is composed of dense connective tissue; and the subcutaneous layer, which is composed of loose connective tissue. The epidermis, the outermost layer of the skin, forms a waterproof protective membrane covering the body surface and consists of a layer of squamous epithelial cells and an underlying basal layer. The epidermis contains no blood vessels and only a small number of nerves associated with the central nervous system. The main cell types constituting the epidermis include keratinocytes, melanocytes, Langerhans cells, and Merkel cells. The dermis, the layer of skin beneath the epidermis, is composed of connective tissue and provides cushioning to protect the body from pressure and tension. The dermis is tightly connected to the epidermis via the basal layer, thus supporting the epidermis and supplying it with nutrients, and contains numerous nerve endings that sense contact and heat.

[0007] Skin gradually becomes damaged with age, or from ultraviolet radiation, external pollutants, and stress. As the skin's ability to protect itself from these factors declines, its protective and proliferative capacity also decreases. In this situation, our body initiates a skin regeneration process for the damaged skin. Skin regeneration is any tissue response to damage and is a complex biological process involving chemotaxis, cell differentiation and replication, matrix protein synthesis, angiogenesis, and wound reconstruction, as a series of tissue repair processes (Steed, DL et al., Clin. Plast. Surg. 25:397, 1998).

[0008] When skin is damaged, cell proliferation follows the inflammatory response, lasting 2 days to 3 weeks after the injury. During this period, fibroblasts deposit collagen to fill the damaged skin, fibroblast proliferation is induced, and the reconstruction of new cells and stromal components occurs in the wound area. This proliferative process, also known as granulation, lasts 2 days to 3 weeks after wound formation. In young skin formed from new cells, skin cell activity is high, making fibroblasts in the dermis highly active in collagen synthesis and metabolism, which is important for skin firmness and inhibiting wrinkle formation. Additionally, the synthesis of the glycoprotein GAG (glucosamine), containing hyaluronic acid, which is crucial for skin hydration, is also active, thus giving the skin firmness, moisture, and elasticity. Furthermore, in the epidermis, basal cells proliferate actively, and a balanced production of adhesion proteins that enhance connections between skin cells (such as laminin, integrins, and desmosomes) occurs, thereby strengthening cellular tissue and maintaining healthy skin.

[0009] However, because the skin's regenerative capacity declines with age, incomplete regeneration occurs when the skin is damaged by ultraviolet radiation, pollutants, or stress. As a result, the skin loses its firmness, wrinkles form, and experiences discoloration and hyperpigmentation (such as age spots, freckles, and dark spots), and loses its ability to retain moisture.

[0010] Therefore, to maintain firm, moisturized, and healthy skin, restoring the skin's regenerative capacity, which declines with age, may be of paramount importance. Prior art relating to functional cosmetic compositions for such skin regeneration purposes includes: Korean Patent KR 1888258, which relates to a composition for skin regeneration comprising an essential oil extracted from Daisy fleabane flowers as an active ingredient; Korean Patent KR 1422690, which relates to a composition for skin regeneration comprising a culture secretion of angiogenic progenitor cells derived from embryonic stem cells; and Korean Patent KR 1663912, which relates to a cosmetic composition for skin whitening, wrinkle reduction, or skin regeneration comprising exosomes derived from human adipose-derived stem cells as an active ingredient.

[0011] However, the above-mentioned existing technologies have problems such as high production costs and untested in vivo stability and safety.

[0012] Immunity is the physiological process of distinguishing between exogenous and endogenous foreign substances in the body and eliminating and metabolizing them. Immunity can be divided into innate immunity (primitive immunity) and acquired immunity (adaptive immunity). In the primary immune response, the activity of macrophages and natural killer cells (NK cells) protects the host by inhibiting foreign substances (pathogens). Macrophages produce and release the active marker TNF-α while engulfing foreign substances, while NK cells produce and release the active marker perforin to kill pathogen-infected cells. Subsequently, cytotoxic T lymphocytes, helper T lymphocytes, and B lymphocytes involved in the acquired immune response are activated to kill infected cells or produce antibodies to protect the host. Cytotoxic T lymphocytes, like NK cells, produce and release large amounts of perforin to kill pathogen-infected cells, while B lymphocytes produce antibodies, dependent on or independent of helper T lymphocytes, to protect the host. Inflammatory cytokines such as IL-6, IL-8, and TNF-α are substances that mediate immunity and are known to be particularly involved in the primary immune response. In addition, since the innate immune system plays an extremely important role as a defense mechanism against cancer cells, cell therapy products that utilize the activation of the innate immune system, such as those using NK cells and T cells as their mechanisms of action, have received more attention in recent years.

[0013] Typically, in cases of immunodeficiency, resistance to infection is impaired, and patients with antibody deficiencies are unable to defend against bacterial infections, and the phagocytic capacity of neutrophils is also impaired. Furthermore, in such cases, activation of the complement system is also impaired, failing to produce leukocyte migration factors, subsequently increasing inflammation rates and causing viremia, which can spread to the central nervous system and other sites. In addition, in the case of cancer patients, during chemotherapy or radiotherapy, not only cancer cells but also normal cells are affected, leading to adverse reactions that severely reduce the patient's immunity.

[0014] Against this backdrop, there is a need to develop immune enhancers or functional health foods that can boost immunity for the prevention or treatment of cancer or infectious diseases.

[0015] Meanwhile, according to health insurance medical expense payment data surveyed by the Health Insurance Policy Research Institute of the National Health Insurance Corporation of Korea from 2001 to 2008, the actual number of patients treated for hair loss was 103,000 in 2001, 142,000 in 2005, and 165,000 in 2008, showing a 60% increase over the past seven years. By age, 114,000 patients were aged 20-49, accounting for 69.5% of the total, while 22,000 or more patients were aged 10-19 or younger. As of 2008, among patients actually treated, 84,000 were men and 80,000 were women, indicating a slight male-to-female ratio. Furthermore, as of 2008, the specific types of "hair loss" treated by Natural Health Insurance included alopecia areata (130,000), cicatricial alopecia (2,000), androgenetic alopecia (9,000), and other non-cicatricial alopecia (8,000). Meanwhile, according to data from the International Hair and Cosmetics Research Forum in June 2003, there were 250 million people suffering from hair loss, with an incidence rate of 30-65% between the ages of 24 and 50. As of 2008, approximately 300 million people in China suffered from hair loss, with 30% of the 30-39 year old male population and 50% of the 50-59 year old male population showing signs of hair loss, and the number of hair loss patients increasing by 10-15% annually. According to data from a 2007 survey of Japanese companies selling wigs and performing hair transplant surgery, the prevalence of hair loss in the Japanese population was 26.5%, and the number of people suffering from hair loss was estimated to be approximately 12.93 million.

[0016] Currently, preparations used to treat hair loss are mainly divided into drugs, quasi-drugs, and cosmetics. Prescription drugs that can only be purchased with a doctor's prescription include Propecia, developed and marketed by Merck in the United States. Propecia's main ingredient, finasteride, was approved by the FDA in December 1997 as a treatment for hair loss. Finasteride is a drug that inhibits 5α-reductase, an enzyme that converts testosterone to dihydrotestosterone (DHT), and plays a role in promoting the growth of fine hairs into thick, long hair. Although finasteride can effectively reduce hair loss in the short term, it has accompanying side effects such as erectile dysfunction in men, decreased sexual function, and breast enlargement. Over-the-counter drugs that have been recognized for their safety and efficacy and can therefore be purchased without a doctor's prescription include minoxidil. Minoxidil was approved by the FDA in December 1997 as the first topical treatment for hair loss. Although this drug promotes hair growth by improving blood circulation and opening potassium channels, it may cause local reactions, such as itching and rashes, and may cause tachycardia.

[0017] According to an announcement by the Minister of Health and Welfare, among the products approved by the Korean Food and Drug Administration for hair loss prevention and hair growth, those that can be sold in supermarkets or convenience stores include CJ Lion's "Hair PowerCompetent," Moracle's "Hair Tonic," and LG Household & Health Care's "Mo&More," as well as shampoos or products sold as cosmetics to maintain or promote healthy skin and hair.

[0018] The human hair loss cycle is mainly divided into three phases: the anagen (growth) phase, the catagen (regression) phase, and the telogen (resting) phase. The anagen phase is characterized by active hair follicle dermal papilla cells, rapid cell division, and rapid hair growth. While the duration of the anagen phase varies depending on hair type, it typically lasts 3-6 years for scalp hair. Hair in the anagen phase accounts for 80-90% of total hair, and individuals experiencing hair loss often have a shortened anagen phase and a prolonged telogen phase, resulting in a reduction in the anagen portion of total hair. The catagen phase marks the end of the anagen phase, with hair growth gradually slowing down, and cell division and growth finally ceasing. The catagen phase lasts approximately 1-1.5 months and accounts for about 1% of total hair. The telogen phase is the final stage of growth, during which the hair follicle and dermal papilla completely separate, the follicle atrophies, and the follicle migrates further upwards, leading to hair loss. The telogen phase lasts approximately 3-4 months and accounts for 4-14% of the total hair. As the telogen phase ends and the dermal papilla becomes active again, the dermal papilla of the new hair forms, and the hair that was in the telogen phase is pushed out and completely detached from the scalp.

[0019] Because the efficacy of conventional hair loss treatments or compositions for hair growth has not been fully validated or is accompanied by various side effects, there is an urgent need to develop safer and more effective hair loss treatments or hair growth agents.

[0020] Invention disclosure

[0021] Technical issues

[0022] The present invention has been conceived to solve various problems, including those described above, and aims to provide more effective and inexpensive new uses for milk-derived exosomes in various cosmetics (such as functional cosmetic compositions for skin regeneration), foods, and pharmaceuticals. However, the scope of the invention is not limited to the above objectives.

[0023] Technical solutions

[0024] According to one aspect of the invention, a cosmetic composition for skin regeneration is provided, comprising exosomes isolated from cow's milk or goat's milk as an active ingredient.

[0025] According to another aspect of the invention, a cosmetic composition for reducing wrinkles is provided, comprising exosomes isolated from cow's milk or goat's milk as an active ingredient.

[0026] According to another aspect of the invention, a cosmetic composition for skin whitening is provided, comprising exosomes isolated from cow's milk or goat's milk as an active ingredient.

[0027] According to another aspect of the invention, a functional cosmetic for hair is provided for reducing and preventing hair loss, comprising exosomes isolated from cow's milk or goat's milk as an active ingredient.

[0028] According to another aspect of the invention, a functional health food is provided, which includes exosomes isolated from cow's milk or goat's milk as active ingredients.

[0029] According to another aspect of the invention, a pharmaceutical composition for wound treatment is provided, comprising exosomes isolated from cow's milk or goat's milk as an active ingredient.

[0030] According to another aspect of the invention, a pharmaceutical composition for treating hair loss or promoting hair growth is provided, comprising exosomes isolated from cow's milk or goat's milk as an active ingredient.

[0031] According to another aspect of the invention, a method for accelerating the regeneration of injured skin in a subject is provided, the method comprising applying a therapeutically effective amount of exosomes isolated from cow's milk or goat's milk to the injured skin of the subject.

[0032] According to one aspect of the invention, a method for enhancing the immunity of a subject is provided, the method comprising administering the subject a therapeutically effective amount of exosomes isolated from cow's milk or goat's milk.

[0033] According to one aspect of the invention, a method for preventing or treating hair loss in a subject is provided, the method comprising administering to the subject a therapeutically effective amount of exosomes isolated from cow's milk or goat's milk.

[0034] According to another aspect of the present invention, a method for reducing skin wrinkles in a subject includes administering the subject a therapeutically effective amount of exosomes isolated from cow's milk or goat's milk.

[0035] According to another aspect of the present invention, a method for whitening the skin of a subject, the method comprising administering to the subject a therapeutically effective amount of exosomes isolated from cow's milk or goat's milk.

[0036] According to another aspect of the invention, the use of exosomes isolated from cow's milk or goat's milk in the preparation of wound healing agents is provided.

[0037] According to another aspect of the invention, the use of exosomes isolated from cow's milk or goat's milk in the preparation of cosmetics for reducing skin wrinkles is provided.

[0038] According to another aspect of the invention, the use of exosomes isolated from milk or goat milk in the preparation of cosmetics for whitening is provided.

[0039] According to another aspect of the invention, the use of exosomes isolated from cow's milk or goat's milk in the preparation of medicaments for the prevention and treatment of hair loss is provided.

[0040] According to another aspect of the invention, the use of exosomes isolated from cow's milk or goat's milk in the preparation of hair growth agents is provided.

[0041] According to another aspect of the invention, the use of a composition of exosomes isolated from cow's milk or goat's milk for enhancing immunity is provided.

[0042] According to another aspect of the invention, a method for isolating exosomes from cow's milk or goat's milk is provided, the method comprising: a first centrifugation step to remove fat and cells from the cow's milk or goat's milk by centrifugation; a filtration step to filter the centrifuged cow's milk or goat's milk through a filter having a sieve aperture size of 20-60 μm to further remove fat and cells therefrom; a dilution step to dilute the filtered cow's milk or goat's milk by adding an equal volume of distilled water thereto; an isoelectric precipitation step to add acid to the diluted cow's milk or goat's milk to adjust the pH to 4-5, thereby precipitating casein in the cow's milk or goat's milk; a second centrifugation step to further centrifuge the cow's milk or goat's milk in which casein has been precipitated, and to collect the supernatant therefrom; and a filtration step to filter the collected supernatant through a 0.2 μm filter.

[0043] Beneficial effects

[0044] The cosmetic compositions for skin regeneration according to examples of the present invention not only exhibit significantly higher skin regeneration effects than ordinary exosomes isolated from cell culture media, but also possess low production costs, significantly high yields, and high material stability, thus enabling extremely economical production. However, the scope of the present invention is not limited to the aforementioned effects. Brief description of the attached diagram

[0046] Figure 1a is a flowchart schematically illustrating a known process for generating large quantities of exosomes from milk or colostrum; and Figure 1b is a flowchart schematically illustrating a process for isolating exosomes from milk or colostrum according to an example of the present invention.

[0047] Figure 2a is a graph showing the yield of exosomes isolated from commercial milk and colostrum sources according to an example of the present invention, and the yield of exosomes isolated from various cells (HaCat, B16F10 and HDF) as a control group; and Figure 2b is a graph showing the yield of exosomes isolated by an improved process without ultrafiltration according to an embodiment of the present invention, compared with a process using ultrafiltration.

[0048] Figure 3 The images are a series of photographs showing the results of Western blot analysis of exosome surface markers from commercial milk and colostrum isolated according to an example of the present invention, as well as exosomes isolated from HEK293 cells as a control group.

[0049] Figure 4 Histograms (A and C) showing the results of dynamic light scattering analysis of the particle size of milk-derived exosomes (A and B) isolated according to an example of the present invention, exosomes (C and D) isolated from HEK293 cells as a control group, and photographs (B and D) of exosomes captured by transmission electron microscopy are displayed.

[0050] Figure 5 This is a series of photographs showing the shapes of milk-derived exosomes (top) isolated according to an example of the present invention, captured by transmission electron microscopy, and HEK293-derived exosomes (bottom) as a control group, before and after two freeze / thaw cycles. The red boxes in the photographs indicate the magnified areas on the right.

[0051] Figure 6 A series of histograms show the dynamic light scattering analysis results of the particle size distribution of milk-derived exosomes (A) isolated according to an example of the present invention and HEK293-derived exosomes (B) as a control group before and after two freeze / thaw cycles.

[0052] Figure 7a shows a series of graphs displaying the results of dynamic scattering analysis of the average particle size of exosomes derived from colostrum (bottom right), exosomes derived from HaCat cells (HaCat Exo, top left), exosomes derived from B16 melanocytes (B16 Exo, top right), and exosomes derived from human dermal fibroblasts (HDF Exo, bottom left) as control exosomes before and after freezing and thawing, confirming the particle size distribution before and after freezing and thawing; Figure 7b shows a series of photographs taken by transmission electron microscopy of exosomes derived from colostrum according to an embodiment of the invention before cryopreservation (top) and after thawing and resuspending (bottom); and Figure 7c shows a series of photographs taken after immunofluorescence staining to analyze the expression level of type I collagen in human dermal fibroblasts treated with exosomes derived from colostrum according to an embodiment of the invention. The saline on the left represents the control group treated only with physiological saline without exosome treatment.

[0053] Figure 8 This is a graph showing the results of analyzing the average particle size of colostrum-derived exosomes (Col M-Exo) and various control exosomes (HaCat Exo, B16Exo, and HDF Exo) after five repeated freeze / thaw cycles.

[0054] Figure 9 The image shows a fluorescence microscopy image (right) illustrating the absorption of milk-derived exosomes isolated according to an example of the present invention into human dermal fibroblasts upon administration. The left image represents the PBS treatment group as a control. Blue indicates the DAPI fluorescence signal used for nuclear counterstaining.

[0055] Figure 10 Histograms representing the results of measuring the degree of apoptosis after treating human dermal fibroblasts (HDF) with colostrum-derived exosomes according to embodiments of the present invention at concentrations of 0.1 mg / ml and 0.3 mg / ml, respectively.

[0056] Figure 11a shows a series of fluorescence microscopy images illustrating the levels of reactive oxygen species (ROS) formed in UV-radiated human dermal fibroblasts, analyzed by DCF-DA after treatment with commercial milk-derived exosomes (Comm M-exo) and colostrum exosomes (Col M-exo), examples of the present invention, which express fluorescence through reaction with ROS. Figure 11b is a graph showing the quantified relative fluorescence intensities identified in Figure 11a.

[0057] Figure 12a shows a graph illustrating the cell proliferation rate of human keratinocytes (HaCaT) measured after treatment with exosomes isolated from commercial milk and colostrum according to an example of the present invention, respectively. Figure 12b shows a series of graphs illustrating the cell proliferation rate of human dermal fibroblasts (HDF) measured after treatment with exosomes isolated from commercial milk and colostrum according to an example of the present invention, respectively. The control group represents the degree of cell proliferation treated with PBS buffer alone, and the proliferation rate represents a relative percentage when the proliferation rate of the control group is set to 100%.

[0058] Figure 13 A series of images (A and C) showing the degree of cell migration observed after exosomes derived from commercial milk and colostrum isolated according to an example of the present invention were administered to scratch-induced human keratinocytes (HaCaT, A and B) and human dermal fibroblasts (HDF, C and D), and a series of graphs (B and D) showing the degree of cell migration quantified according to the results of A and C.

[0059] Figure 14 Presented are (a) a series of images captured by a microscope showing the extent of tube formation in mouse endothelial cells (SVEC4-10) cultured in three dimensions in Matrigel after treatment with commercial milk-derived exosomes (Comm M-exo) and colostrum exosomes (Col M-exo) as examples of the present invention, and a series of graphs showing (b) the number of branching points observed in (a) and (c) the length of the formed tubes.

[0060] Figure 15a shows a series of images captured by whole-body in vivo fluorescence imaging, illustrating the in vivo distribution of colostrum exosomes of this invention, labeled with fluorescent material (Flamma 675) and injected subcutaneously. Figure 15b is a photograph captured by an in vivo imaging system, showing the fluorescence distribution in the organs after the experimental animals were sacrificed the day after exosome injection and the major organs were harvested. Figure 15c is a graph of the quantification results of Figure 15b.

[0061] Figure 16 The diagram above shows a graph illustrating the degree of wound healing over time after applying commercially derived milk exosomes (Comm M-exo) and colostrum exosomes (Col M-exo), examples of the present invention, to wound sites in trauma model mice, along with a series of photographs of directly captured wound sites. For the control group, a group was given only saline (saline) and serum-derived exosomes (Serum Exo), and the colostrum exosome group was further divided into a group given on days 1, 4, and 7 (Col M-Exo) and a group given on days 4, 7, and 10 (Col M-Exo-D4).

[0062] Figure 17 A series of photographs showing the execution Figure 16 The results of immunohistochemical assays using anti-elastin antibodies were performed on tissue sections excised from the wound site after the experimental animals were tested.

[0063] Figure 18a is a series of fluorescence micrographs showing the expression levels of type 1 collagen in human dermal fibroblasts (HDF) analyzed by immunofluorescence assay after treatment with commercial milk-derived exosomes (Com M-exo) and colostrum exosomes (Col M-exo) according to an embodiment of the invention. Figure 18b is a graph showing the expression levels of matrix metalloproteinase-2 (MMP-2) in human dermal fibroblasts (HDF) after treatment with commercial milk-derived exosomes (Com M-exo) and colostrum exosomes (Col M-exo) according to an embodiment of the invention, analyzed by Western blot. The control groups were the saline treatment group (saline) and the HDF-derived exosome treatment group.

[0064] Figure 19The images show (a) a series of photographs of melanin extracted from melanoma cells treated with or untreated with colostrum exosomes (Col M-exo) of various concentrations of the present invention 24 hours after treatment, (b) a graph showing the measurement results of melanin amount per 10 x 6 cells, and (c) a graph showing the comparison results of melanin content between groups treated with commercial milk-derived exosomes (Comm M-exo) and colostrum exosomes (Col M-exo) of the present invention and control groups (saline, B16-derived exosome treatment (B16-exo)).

[0065] Figure 20 The illustration shows (a) a photograph of the expression levels of various cytokines in bone marrow-derived macrophages (BMDM) treated with commercial milk-derived exosomes (Comm M-exo) and colostrum exosomes (Col M-exo) according to examples of the present invention, analyzed by dot blot assay 24 hours after treatment, and (b) a graph showing the quantitative results.

[0066] Figure 21 Presents (a) dot blot assay results comparing the expression levels of various growth factors involved in tissue regeneration and angiogenesis in NIH-3T3 fibroblasts treated with commercial milk-derived exosomes (Comm M-exo) and colostrum exosomes (Col M-exo), respectively, and (b) a graph showing the quantitative results of the dot blot assay.

[0067] Figure 22a shows a series of fluorescence micrographs, which show observations of intracellular exosome uptake patterns in human dermal dermal papilla cells treated with commercial milk-derived exosomes (Comm M-exo) according to an example of the present invention for different treatment durations. Figure 22B A graph (left) shows the effect of different concentrations of colostrum exosomes on the proliferation of human dermal dermal papilla cells treated with colostrum exosomes according to an example of the present invention, and a graph (right) shows the results of the analysis of the differences in the degree of proliferation of human dermal dermal papilla cells treated with or without hair loss inducing substances. Figure 22C A series of photographs are shown, illustrating the observations of hair growth over time in experimental animals after administration of commercial milk-derived exosomes (Comm M-exo) according to an example of the present invention via transdermal administration.

[0068] Methods for implementing the present invention

[0069] Definition of terminology

[0070] The terms used in this article are defined as follows.

[0071] As used in this article, the term "exosome" refers to nanoscale cell-derived vesicles that can be present in all types of biological fluids, including blood, urine, and cell culture media. Exosomes are known to range in size from 30 nm to 100 nm and are secreted from cells either by the fusion of multiple vesicles with the plasma membrane or directly through the plasma membrane. Exosomes are known to play important roles in various processes such as coagulation, intercellular signal transduction, and metabolite management. In this context, the term "milk-derived exosomes" as used in this article refers to exosomes derived from mammalian milk (such as commercial cow's milk and colostrum). Similarly, the term "colostrum exosomes" refers to milk-derived exosomes derived from milk produced by the mother immediately after the birth of the offspring.

[0072] As used herein, the term "milk" primarily refers to cow's milk, but includes milk from other mammals such as horses, sheep, dairy goats (goats), and camels as functional equivalents. Considering availability, cost, and other factors, commercial cow's milk is the preferred choice, and commercial "goat milk" from dairy goats (goats) is an excellent alternative. In particular, goat milk has a composition similar to breast milk and is therefore easily digestible and has high nutritional value, thus being considered a superior milk source. Horse milk (mare's milk), although less common, is used as a cow's milk substitute by nomadic tribes in Central Asia. Meanwhile, "colostrum" is the form of milk produced during late pregnancy and the days following birth. Unlike regular milk, colostrum contains more nutrients and antibodies necessary for survival and growth, and contains higher levels of antioxidants such as lactoferrin and heme-binding proteins compared to regular milk, and is therefore sometimes preferred for health improvement and nutrition.

[0073] As used in this article, “raw milk” refers to milk collected from pastures that have never undergone any specific treatment (such as sterilization), while “commercial milk” refers to milk that has been packaged in a commercially available form after undergoing sterilization and homogenization processes.

[0074] As used in this article, "skin regeneration" refers to the process of the skin regeneration cycle, or in the case of a wound, the process by which cells form in the basal layer, thereby restoring the skin. Epidermal cells formed in the basal layer are gradually pushed upwards and reach the stratum corneum. This process is called the skin regeneration cycle or skin renewal process. Typically, the skin renewal process is considered to take an average of 28 days, including a 14-day period during which cells form in the basal layer and move upwards, and another 14-day period during which cells remaining in the stratum corneum die and shed. Because the regeneration and shedding processes take longer with age, the skin renewal cycle can take up to 42 days, and it is known that the skin renewal cycle generally becomes longer after age 30. As a result, the skin becomes dull and rough with age; that is, the skin begins to age. Invention Details

[0076] According to one aspect of the invention, a cosmetic composition for skin regeneration is provided, the composition comprising exosomes isolated from cow's milk or goat's milk as an active ingredient.

[0077] In cosmetic compositions used for skin regeneration, milk or goat milk may be the raw milk, commercial milk or goat milk, or colostrum.

[0078] According to another aspect of the invention, a cosmetic composition for reducing wrinkles is provided, the composition comprising exosomes isolated from cow's milk or goat's milk as an active ingredient.

[0079] In cosmetic compositions used to reduce wrinkles, milk or goat milk may be the raw milk, commercial milk or goat milk, or colostrum.

[0080] According to another aspect of the invention, a cosmetic composition for skin whitening is provided, the composition comprising exosomes isolated from cow's milk or goat's milk as an active ingredient.

[0081] In cosmetic compositions used for skin whitening, milk or goat milk may be the raw milk, commercial milk or goat milk, or colostrum.

[0082] The cosmetic composition according to one aspect of the present invention can be used in a variety of products, such as functional cosmetics, cleansers, shampoos, etc. Examples of products to which the composition can be added include various types of cosmetics, such as toners, lotions, creams, serums, essences, cosmetic solutions, and masks, and also include soaps, shampoos, hair care products, cleansers, body cleansers, facial cleansers, hair conditioners, and beauty serums.

[0083] The cosmetic compositions of the present invention may further include additional functional components selected from water-soluble vitamins, fat-soluble vitamins, polymeric peptides, polysaccharides, sphingolipids, and algal extracts.

[0084] Water-soluble vitamins can be any vitamin that can be blended into cosmetics, but are preferably vitamin B1, vitamin B2, vitamin B6, pyridoxine, pyridoxine hydrochloride, vitamin B12, pantothenic acid, niacin, niacinamide, folic acid, vitamin C, vitamin H, etc., and salts of the above components (thiamine hydrochloride, sodium ascorbate, etc.) or derivatives thereof (sodium ascorbate-2-phosphate, magnesium ascorbate-2-phosphate, etc.) are also included in the water-soluble vitamins usable in this invention. Water-soluble vitamins can be obtained by known methods such as microbial transformation, chemical synthesis, enzymatic methods, or purification from microbial cultures.

[0085] The fat-soluble vitamins can be any vitamin that can be blended into cosmetics, but are preferably vitamin A, carotene, vitamin D2, vitamin D3, vitamin E (d1-α-tocopherol, d-α-tocopherol, d-δ-tocopherol), etc., and derivatives of the above components (ascorbate palmitate, ascorbate stearate, ascorbate dipalmitate, dl-α-tocopherol acetate, dl-α-tocopherol nicotinate, vitamin E, DL-panthenol, D-panthenol, pantothenic acid ethyl ether, etc.) are also included in the oil-soluble vitamins that can be used in this invention.

[0086] Fat-soluble vitamins can be obtained from microbial cultures by known methods such as microbial transformation, chemical synthesis, enzymatic methods, or purification methods.

[0087] Polymer peptides can be any polymer peptide that can be incorporated into cosmetics, but are preferably collagen, hydrolyzed collagen, gelatin, elastin, hydrolyzed elastin, keratin, etc. Polymer peptides can be obtained from microbial culture media by known methods such as chemical synthesis, enzymatic methods, or purification methods, or can be purified and used from natural products such as the skin of pigs, cattle, etc., and silk proteins from silkworms.

[0088] The polysaccharide can be any polysaccharide that can be incorporated into cosmetics, but is preferably hydroxyethyl cellulose, xanthan gum, sodium hyaluronate, chondroitin sulfate, or its salts (sodium salts, etc.). For example, chondroitin sulfate or its salts can be purified and used from known mammals or fish.

[0089] Sphingolipids can be any sphingolipid that can be incorporated into cosmetics, but are preferably ceramides, phytosphingosine, glycosphingolipids, etc. Sphingolipids can be purified from mammals, fish, shellfish, yeast, or plants by known methods, or can be obtained by chemical synthesis.

[0090] The algae extract can be any algae extract that can be blended into cosmetics, but is preferably brown algae extract, red algae extract, green algae extract, etc., and carrageenan, alginic acid, sodium alginate, potassium alginate, etc. purified from the above-mentioned algae extracts are also included in the algae extracts used in this invention. The algae extracts can be obtained from algae by purification using known methods.

[0091] In the cosmetic materials of the present invention, other ingredients that are typically mixed in cosmetic materials may be mixed together with the above-mentioned basic ingredients.

[0092] Other ingredients that may be added include oils, moisturizers, emollients, surfactants, organic and inorganic pigments, organic particles, UV absorbers, preservatives, disinfectants, antioxidants, plant extracts, pH adjusters, alcohol, pigments, fragrances, blood circulation enhancers, coolants, antiperspirants, purified water, etc.

[0093] Oil and fat components can include ester-based oils, hydrocarbon-based oils, silicone-based oils, fluorinated oils, animal fats, and vegetable oils. Ester-based oils can include the following esters, such as: triglycerides (2-ethylhexanoate), cetyl 2-ethylhexanoate, isopropyl myristate, butyl myristate, isopropyl palmitate, ethyl stearate, octyl palmitate, isocetyl isostearate, butyl stearate, ethyl linoleate, isopropyl linoleate, ethyl oleate, isocetyl myristate, isostearyl myristate, isostearyl palmitate, octyl dodecyl myristate, isocetyl isostearate, diethyl sebacate, diisopropyl adipate, isoalkyl neopentyl ester, tri(decanoyl, decanoic acid) glycerides, trimethylolpropane tri(2-ethylhexanoate), trimethylolpropane triisostearate, pentaerythritol tetra(2-ethylhexanoate), octyl Cetyl ester, decyl laurate, hexyl laurate, decyl myristate, myristyl myristate, cetyl myristate, stearyl stearate, decyl oleate, cetyl castor oil, isostearyl laurate, isotearyl myristate, isoctyl palmitate, octyl stearate, isoctearyl stearate, isodecayl oleate, octyl dodecyl oleate, octyl dodecyl linoleate, isopropyl isostearate, cetearyl 2-ethylhexanoate, stearyl 2-ethylhexanoate, hexyl isostearate, ethylene glycol dicaprylate, ethylene glycol dioleate, propylene glycol dicaprylate, di(decanoyl decanoate)propylene glycol, propylene glycol dicaprylate, neopentyl glycol dicaprylate, neopentyl glycol dicaprylate dioctanoate), tricaprylic acid glyceride, tri-undecanoic acid glyceride, triisopalmitoyl glyceride, triisostearate glyceride, octyl dodecyl neopentanoate, isostearate, octyl isononanoate, hexyl decyl neodecanoate, octyl dodecyl neodecanoate, isocetyl isostearate, isostearate, octyl dodecyl isostearate, polyglycerol oleate, polyglycerol isostearate, triisoceryl citrate, triisoalkyl citrate, triisooctyl citrate, lauryl lactate, myristyl lactate, cetyl lactate, octyl dodecyl lactate, lemon Triethyl citrate, triethyl acetyl citrate, tributyl acetyl citrate, trioctyl citrate, diisostearyl malate, 2-ethylhexyl hydroxystearate, di(2-ethylhexyl) succinate, diisobutyl adipate, diisopropyl sebacate, dioctyl sebacate, cholesterol stearate, cholesterol isostearate, cholesterol hydroxystearate, cholesterol oleate, dihydrocholesterol oleate, phytosterol isostearate, phytosterol oleate, 12-stearoyl hydroxystearate isocetyl, 12-stearoyl hydroxystearate stearate, 12-stearoyl hydroxystearate isostearate, etc.

[0094] Hydrocarbon-based oils may include the following hydrocarbon-based oils, such as: squalene, liquid paraffin, α-olefin oligomers, isoparaffin, ceresin, paraffin wax, liquid isoparaffin, polybutene, microcrystalline wax, petrolatum, etc.

[0095] Silicone-based greases may include polymethylsiloxane, methylphenylsiloxane, methylcyclopolysiloxane, octamethylpolysiloxane, decamethylpolysiloxane, dodecylcyclosiloxane, dimethylsiloxane / methylcetoxysiloxane copolymer, dimethylsiloxane / methylstearoxysiloxane copolymer, alkyl-modified silicone oil, amino-modified silicone oil, etc.

[0096] Fluorinated greases may include perfluoropolyethers, etc.

[0097] Animal and vegetable fats and oils may include the following: avocado oil, almond oil, olive oil, sesame oil, rice bran oil, saffron oil, soybean oil, corn oil, rapeseed oil, apricot kernel oil, palm kernel oil, palm oil, castor oil, sunflower oil, grapeseed oil, cottonseed oil, palm oil, cuqueur oil, wheat germ oil, rice germ oil, shea butter, evening primrose oil, macadamia nut oil, meadowfoam seed oil, egg yolk oil, animal fat, horse oil, mink oil, sea bream oil, jojoba oil, candelabra wax, carnauba wax, liquid lanolin, and hydrogenated castor oil.

[0098] Moisturizers may include water-soluble low molecular weight moisturizers, lipid-soluble molecular moisturizers, water-soluble polymers, lipid-soluble polymers, etc.

[0099] Water-soluble low molecular weight moisturizers may include serine, glutamine, sorbitol, mannitol, sodium pyrrolidone carboxylate, glycerin, propylene glycol, 1,3-butanediol, ethylene glycol, polyethylene glycol (degree of polymerization n=2 or higher), polypropylene glycol (degree of polymerization n=2 or higher), polyglycerol (degree of polymerization n=2 or higher), lactic acid, lactate, etc.

[0100] Lipid-soluble low molecular weight moisturizers may include cholesterol, cholesterol esters, etc.

[0101] Water-soluble polymers may include carboxyvinyl polymers, polyaspartic acid, tragacanth gum, xanthan gum, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, carboxymethylcellulose, water-soluble chitin, chitosan, dextrin, etc. Fat-soluble polymers may include polyvinylpyrrolidone / eicosene copolymers, polyvinylpyrrolidone / hexadecene copolymers, nitrocellulose, dextrin fatty acid esters, high molecular weight silicones, etc.

[0102] Emollients may include long-chain acylglutamic acid cholesterol ester, hydroxystearic acid cholesterol ester, 12-hydroxystearic acid, stearic acid, rosin acid, lanolin fatty acid cholesterol ester, etc.

[0103] Surfactants can include nonionic surfactants, anionic surfactants, cationic surfactants, amphoteric surfactants, etc.

[0104] Nonionic surfactants may include self-emulsifying glyceryl monostearate, propylene glycol fatty acid esters, glyceryl fatty acid esters, polyglycerol fatty acid esters, dehydrated sorbitan fatty acid esters, POE (polyoxyethylene) dehydrated sorbitan fatty acid esters, POE sorbitan fatty acid esters, POE glyceryl fatty acid esters, POE alkyl ethers, POE fatty acid esters, POE hydrogenated castor oil, POE castor oil, POE / POP (polyoxyethylene / polyoxypropylene) copolymers, POE / POP alkyl ethers, polyether-modified silicones, lauric acid alkanolamides, alkyl amine oxides, hydrogenated soybean lecithin, etc.

[0105] Anionic surfactants may include fatty acid soaps, α-acylsulfonates, alkylsulfonates, alkylallyl sulfonates, alkylnaphthalene sulfonates, alkyl sulfates, POE alkyl ether sulfates, alkylamide sulfates, alkyl phosphates, POE alkyl phosphates, alkylamide phosphates, alkylacylalkyl taurates, N-acyl amino acid salts, POE alkyl ether carboxylates, alkyl sulfosuccinates, sodium alkyl sulfoacetate, acylated hydrolyzed collagen peptide salts, perfluoroalkyl phosphates, etc.

[0106] Cationic surfactants may include alkyltrimethylammonium chloride, stearyltrimethylammonium chloride, stearyltrimethylammonium bromide, cetearyltrimethylammonium chloride, distearate dimethylammonium chloride, stearyldimethylbenzylammonium chloride, behenyltrimethylammonium bromide, benzalkonium chloride, diethylaminoethylamide stearate, dimethylaminopropylamide stearate, lanolin derivatives, quaternary ammonium salts, etc.

[0107] Amphoteric surfactants may include carboxybetaine type, amide betaine type, sulfobetaine type, hydroxysulfobetaine type, amide sulfobetaine type, phosphate betaine type, aminocarboxylate type, imidazoline derivative type, amide-amine type, etc.

[0108] Organic and inorganic pigments can include inorganic pigments such as silicic acid, silicic anhydride, magnesium silicate, talc, sericite, mica, kaolin, ferric oxide, clay, bentonite, titanate mica, bismuth oxychloride, zirconium oxide, magnesium oxide, zinc oxide, titanium oxide, aluminum oxide, calcium sulfate, barium sulfate, magnesium sulfate, calcium carbonate, magnesium carbonate, iron oxide, ultramarine, chromium oxide, chromium hydroxide, calamine, carbon black and their complexes; and organic pigments such as polyamide, polyester, polypropylene, polystyrene, polyurethane, vinyl resin, urea resin, phenolic resin, fluororesin, silicone resin, acrylic resin, melamine resin, epoxy resin, polycarbonate resin, divinylbenzene-styrene copolymer, silk powder, cellulose, CI pigment yellow, CI pigment orange, etc.; as well as composite pigments of such inorganic and organic pigments, etc.

[0109] Organic particles may include metal soaps, such as calcium stearate; alkyl phosphate metal salts, such as sodium cetate, zinc laurate, and calcium laurate; polyvalent metal salts of acyl amino acids, such as calcium N-lauroyl-β-alanine, zinc N-lauroyl-β-alanine, and calcium N-lauroylglycine; polyvalent metal salts of amide sulfonic acids, such as calcium L-lauroyl taurate and calcium N-palmitoyl taurate; N-acyl basic amino acids, such as Nε-lauroyl-L-lysine, Nε-palmitoylizine, Nα-paritoylolnithine, Nα-lauroyl arginine, and Nα-hydrogenated beef fatty acid acyl arginine; N-acyl polypeptides, such as N-lauroylglycylglycine; α-amino fatty acids, such as α-aminooctanoic acid and α-aminolauric acid; and polyethylene, polypropylene, nylon, polymethyl methacrylate, polystyrene, divinylbenzene-styrene copolymer, tetrafluoroethylene, etc.

[0110] UV absorbers may include p-aminobenzoic acid, ethyl p-aminobenzoate, amyl p-aminobenzoate, octyl p-aminobenzoate, ethylene glycol salicylate, phenyl salicylate, octyl salicylate, benzyl salicylate, butyl phenyl salicylate, holomethyl salicylate, benzyl cinnamate, 2-ethoxyethyl p-methoxycinnamate, octyl p-methoxycinnamate, mono-2-ethylhexaneglyceryl di-p-methoxycinnamate, glyceryl, isopropyl p-methoxycinnamate, diisopropyl-diisopropylcinnamate. Mixtures of esters, uric acid, ethyl uric acid, hydroxymethoxybenzophenone, hydroxymethoxybenzophenone sulfonic acid and its salts, dihydroxymethoxybenzophenone, sodium dihydroxymethoxybenzophenone disulfonate, dihydroxybenzophenone, tetrahydroxybenzophenone, 4-tert-butyl-4'-methoxybenzoylmethane, 2,4,6-triphenylamino-p-(carbonyl-2'-ethylhexyl-1'-oxy)-1,3,5-triazine, 2-(2-hydroxy-5-methylphenyl)benzotriazole, etc.

[0111] Disinfectants may include physalisol, triclosan, trichlorohydroxydiphenyl ether, chlorhexidine gluconate, phenoxyethanol, resorcinol, isopropylcresol, azulene, salicylic acid, zinc pyrithione, benzalkonium chloride, photosensitizer No. 301, sodium mononitroguaiacol, undecenoic acid, etc.

[0112] Antioxidants may include butylated hydroxyanisole, propyl gallate, elisorbic acid, etc.

[0113] pH adjusters may include citric acid, sodium citrate, malic acid, sodium malate, fumaric acid, sodium fumarate, succinic acid, sodium succinate, sodium hydroxide, disodium hydrogen phosphate, etc.

[0114] Alcohols can include higher alcohols, such as cetyl alcohol.

[0115] Furthermore, the ingredients that can be added are not limited to those mentioned above. In addition, any of the above-mentioned ingredients may be added within a range that does not adversely affect the purpose and effect of the present invention, but preferably in an amount of 0.01-5 wt% relative to the total weight, more preferably in an amount of 0.01-3 wt%.

[0116] The cosmetic material of the present invention can be in the form of solution, emulsion, viscous mixture, etc.

[0117] Examples of cosmetic material forms are not particularly limited, but may include lotions, creams, toners, masks, foundations, serums, beauty serums, hair cosmetic materials, soaps, etc.

[0118] Specific examples of cosmetic materials of this invention may include facial cleansing cream, facial foam, cleansing cream, cleansing milk, facial cleanser, massage cream, cold cream, moisturizing cream, lotion, toner, facial pack, after-shaving cream, sunscreen, tanning oil, soap, shower gel, shampoo, hair rinse, hair treatment, hair conditioner, hair growth material, hair cream, hair liquid, hair styling liquid, hair gel, hair bridge, color rinse, color spray, permanent wave solution, pressed powder, loose powder, eyeshadow, hand cream, lipstick, etc.

[0119] The cosmetic composition of the present invention can be obtained by preparing a selection of ingredients according to known methods, such as those disclosed in the first edition of "Manual for Percutaneous Application Preparation Development" edited by Matsumoto Michio (published by Seishi Shoin in 1985): water-soluble vitamins, fat-soluble vitamins, polymeric peptides, polysaccharides, sphingolipids, and algal extracts (other ingredients may be added as needed in addition to the above-mentioned ingredients), and exosomes isolated from cow's milk or goat's milk as the active ingredient of the present invention.

[0120] According to another aspect of the invention, a functional cosmetic for hair is provided for reducing and preventing hair loss, comprising exosomes isolated from milk or goat milk as active ingredients.

[0121] According to one aspect of the invention, functional cosmetic products for reducing and preventing hair loss can be provided as shampoo formulations or as topical formulations (such as ointments, creams, and gels for scalp application).

[0122] According to another aspect of the invention, a functional health food is provided, which includes exosomes isolated from cow's milk or goat's milk as active ingredients.

[0123] The health-related functions of functional health foods may include (but are not limited to) alleviating skin allergies, improving immune function, improving gut health, improving skin health, regulating blood sugar levels, antioxidant function, or improving liver health.

[0124] The types of functional health foods according to one aspect of the present invention are not particularly limited. The types of functional health foods may be, without limitation, any of the following: dairy products, confectionery products, condiments, beverages and drinks, snacks, candy, ice cream and frozen desserts, breakfast cereals, nutrition bars, snack bar chocolate products, processed foods, cereal products and pasta, soups, sauces and seasonings, confectionery products, oil products, dairy beverages and milk beverages, soy products, frozen foods, cooked and alternative foods, meat products, cheese, yogurt, bread, rolls, yeast products, cakes, cookies and crackers. Furthermore, all items generally considered functional foods are included.

[0125] According to one aspect of the present invention, functional health foods can be formulated and used as capsules, tablets, powders, liquid suspensions, pills, granules, etc. Such formulations involve uniformly mixing the functional health food with excipients, binders, disintegrants, etc., as is or by appropriate methods, to form granules and ensure the granules are as uniform as possible. Further, flavoring agents, bittering agents, etc., may be added as needed. When the functional health food is a beverage formulation, it may contain various flavoring agents or natural carbohydrates as additional ingredients, just like ordinary beverages. Examples of natural carbohydrates include monosaccharides (such as glucose and fructose), disaccharides (such as maltose and sucrose), polysaccharides (such as dextrin and cyclodextrin), and sugar alcohols (such as xylitol, sorbitol, and erythritol). For sweeteners, natural sweeteners (such as kiwifruit protein and steviol glycoside extract) or artificial sweeteners (such as saccharin and aspartame) may be used. Functional ingredients can be added, such as deer antler extract, fructooligosaccharides that help with calcium absorption and bowel movements, acacia honey, compound scutellaria extract as a natural preservative, gellan gum as an anti-settling thickener, etc., but not limited to these. Any functional ingredients suitable for functional health foods can be used appropriately.

[0126] The exosomes isolated from cow's milk or goat's milk, which are the active ingredients in the functional health foods of this invention, are extremely safe materials because the source (i.e., cow's milk or goat's milk) is a food material that has been used by humans as a food source for a long time. Therefore, exosomes derived from cow's milk or colostrum according to examples of this invention can be used as safe and extremely effective source materials for various health functions.

[0127] According to another aspect of the invention, a pharmaceutical composition for wound treatment is provided, the composition comprising exosomes isolated from cow's milk or goat's milk as an active ingredient.

[0128] In pharmaceutical compositions, milk or goat milk may be raw milk, commercial milk or goat milk, or colostrum.

[0129] In the pharmaceutical composition of the present invention, the active ingredient may be included in an amount of 0.1-100 wt% relative to the total weight of the composition.

[0130] The compositions of the present invention may further include suitable carriers, excipients, and diluents commonly used in the preparation of pharmaceutical compositions. Additionally, solid or liquid additives for formulation may be used in the preparation of the pharmaceutical compositions. The additives used for formulation may be any type of organic or inorganic additive.

[0131] Examples of excipients may include lactose, sucrose, saccharose, glucose, corn starch, starch, talc, sorbitol, crystalline cellulose, dextrin, kaolin, calcium carbonate, and silica. Examples of binders may include polyvinyl alcohol, polyvinyl ether, ethyl cellulose, methyl cellulose, rubber arabic, gum tragali, gelatin, shellac, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, calcium citrate, dextrin, and pectin. Examples of lubricants may include magnesium stearate, talc, polyethylene glycol, silica, and hydrogenated vegetable oil. Any coloring agent generally permitted for pharmaceuticals may be used. Tablets and granules of the pharmaceutical composition may be appropriately coated as needed using sugar coating, gelatin coating, etc. Additionally, preservatives, antioxidants, etc., may be added as needed.

[0132] The pharmaceutical compositions of the present invention can be prepared in any formulation commonly produced in the corresponding industry (e.g., in the literature [Remington's Pharmaceutical Science, 15th edition; Mack Publishing Company, Easton PA]), and the dosage form is not particularly limited, but is preferably a topical formulation. The topical formulations of the present invention may include common forms of topical formulations, such as tablet formulations, liquid topical formulations, spray formulations, emulsion formulations, cream formulations, puff formulations, powder formulations, penetrating pad formulations, spray formulations, gel formulations (including hydrogels), paste formulations, liniment formulations, ointment formulations, aerosols, powder formulations, suspension formulations, transdermal formulations, etc. Such formulations are described in the literature [Remington's Pharmaceutical Science, 15th edition, 1975, Mack Publishing Company, Easton, Pennsylvania 18042 (Chapter 87: Blaug, Seymour), which is a collection of formulations commonly known in all pharmaceutical and chemical fields.

[0133] As an example of the invention, the composition can be applied directly to a wound. That is, the composition can be distributed at the wound site. When applied to a wound site, the composition in sheet form is used to properly wrap the application site to protect the wound and prevent a reduction in the therapeutic effect of the active ingredient. Any commercially available or commonly known excipients can be used. Examples of commercially available excipients include Compeel, Duoderm, Tagaderm, and Opsite.

[0134] When the pharmaceutical compositions of the present invention are provided as topical formulations, the pharmaceutically acceptable carriers, although varying depending on the formulation of the pharmaceutical composition, may include hydrocarbons such as petrolatum, liquid paraffin, and gelling hydrocarbons (referred to as Plastibase); animal and vegetable oils such as medium-chain triglycerides, lard, stearin, and cocoa butter; higher fatty acid alcohols, fatty acids, and their esters such as cetyl alcohol, stearyl alcohol, stearic acid, and isopropyl palmitate; water-soluble bases such as macrogol, 1,3-butanediol, glycerin, gelatin, sucrose, and sugar alcohols; emulsifiers such as glyceryl fatty acid esters, polyoxyltearate, and polyoxyethylene hydrogenated castor oil; adhesives such as acrylates and sodium alginate; propellants such as liquefied petroleum gas carbon dioxide; and preservatives such as para-oxybenzoic acid esters. The topical formulations of the present invention may be manufactured using any of the above components according to known methods. In addition to the above-mentioned components, stabilizers, fragrances, colorants, pH adjusters, diluents, surfactants, preservatives, antioxidants, etc., may be further blended in as needed. The topical formulation of the present invention can be used by applying it to a local wound site using known methods.

[0135] Furthermore, the topical formulation of the present invention can be adhered to a solid support, such as a common bandage with a wound dressing. As an example of the invention, the solid support is first coated with an adhesive layer to increase the adhesion of the liquid medium to the solid support. Examples of adhesives may include polyacrylates and cyanoacrylates.

[0136] This type of formulation is widely available on the market, and examples include: non-adhesive wound peeling bandages in the form of perforated plastic film (Smith & Nephew Ltd.); Johnson & Johnson's BAND-AID in the form of strips, patches, spots, and plastic strips; Colgate-Palmolive Co.'s (Kendall) Curity CURAD bandage; and American White Cross Laboratories, Inc.'s STIK-TITE elastic strip, etc.

[0137] As an example of the present invention, the pharmaceutical composition according to the present invention can be formulated as a liquid topical preparation by mixing exosomes derived from milk or goat milk according to an example of the present invention with saline at a certain volume ratio. As an example of the present invention, the pharmaceutical composition according to the present invention can be formulated as an ointment by mixing exosomes derived from milk or goat milk according to an example of the present invention with a water-soluble ointment base and adding saline thereto.

[0138] According to another aspect of the invention, a method for accelerating the regeneration of injured skin in a subject is provided, the method comprising applying a therapeutically effective amount of exosomes isolated from cow's milk or goat's milk to the injured skin of the subject.

[0139] In this method, milk or goat milk can be raw milk, commercial milk or goat milk, or colostrum.

[0140] The effective therapeutic dose can vary depending on the type of wound, application site, treatment frequency, treatment duration, dosage form, patient condition, and type of adjuvant therapy. There are no particular limitations on the dosage; however, when cell culture medium is applied to a wound, the daily effective dose of the pharmaceutical composition of this invention can be 1-50 μl / cm³. 2 Preferably, it is 5-20 μl / cm 2 .

[0141] The daily dose can be given once a day, or it can be given two or three times a day at appropriate intervals, or it can be given intermittently every few days.

[0142] The administration can be performed orally, subcutaneously, intravenously, intramuscularly, or intranasally, but intravenous or subcutaneous administration is preferred, and direct application to the wound site is also possible if formulated into a suitable skin-applied dosage form.

[0143] According to another aspect of the invention, a method for enhancing the immunity of a subject is provided, the method comprising administering the subject a therapeutically effective amount of exosomes isolated from cow's milk or goat's milk.

[0144] In this method, milk or goat milk can be raw milk, commercial milk or goat milk, or colostrum.

[0145] According to another aspect of the invention, a method for preventing or treating hair loss in a subject is provided, the method comprising administering to the subject a therapeutically effective amount of exosomes isolated from cow's milk or goat's milk.

[0146] In this method, milk or goat milk can be raw milk, commercial milk or goat milk, or colostrum.

[0147] The administration can be performed orally, subcutaneously, intravenously, intramuscularly, or intranasally, but intravenous or subcutaneous administration is preferred, and direct application to the wound site is also possible if formulated into a suitable skin-applied dosage form.

[0148] According to another aspect of the invention, a method for reducing skin wrinkles in a subject is provided, the method comprising administering to the subject a therapeutically effective amount of exosomes isolated from cow's milk or goat's milk.

[0149] In this method, milk or goat milk can be raw milk, commercial milk or goat milk, or colostrum.

[0150] The administration can be performed orally, subcutaneously, intravenously, intramuscularly, or intranasally, but intravenous or subcutaneous administration is preferred, and direct application to the wound site is also possible if formulated into a suitable skin-applied dosage form.

[0151] According to another aspect of the present invention, a method for whitening the skin of a subject is provided, the method comprising administering to the subject a therapeutically effective amount of exosomes isolated from cow's milk or goat's milk.

[0152] In this method, milk or goat milk can be raw milk, commercial milk or goat milk, or colostrum.

[0153] The administration can be performed orally, subcutaneously, intravenously, intramuscularly, or intranasally, but intravenous or subcutaneous administration is preferred, and direct application to the wound site is also possible if formulated into a suitable skin-applied dosage form.

[0154] According to another aspect of the invention, the use of exosomes isolated from cow's milk or goat's milk for the preparation of wound healing agents is provided.

[0155] According to another aspect of the invention, exosomes isolated from cow's milk or goat's milk are provided for use in the preparation of cosmetics for wrinkle reduction.

[0156] According to another aspect of the invention, exosomes isolated from cow's milk or goat's milk are provided for use in the preparation of cosmetics for whitening.

[0157] According to another aspect of the invention, there is a use for exosomes isolated from cow's milk or goat's milk in the preparation of a medicament for the prevention and treatment of hair loss.

[0158] According to another aspect of the invention, exosomes isolated from cow's milk or goat's milk are provided for use in the preparation of hair growth promoters / hair growth agents.

[0159] According to another aspect of the invention, there is a use for exosomes isolated from cow's milk or goat's milk to prepare compositions for enhancing immunity.

[0160] According to another aspect of the invention, a method for isolating exosomes from cow's milk or goat's milk is provided, the method comprising: a first centrifugation step, centrifuging the cow's milk or goat's milk to remove fat and cells therefrom; a filter filtration step, filtering the centrifuged cow's milk or goat's milk through a filter having a sieve aperture size of 20-60 μm to further remove fat and cells therefrom; a dilution step, diluting the filtered cow's milk or goat's milk by adding an equal volume of distilled water thereto; an isoelectric precipitation step, adjusting the diluted cow's milk or goat's milk to pH 4-5 by adding acid thereto to precipitate casein in the cow's milk or goat's milk; a second centrifugation step, further centrifuging the cow's milk or goat's milk in which casein has been precipitated, and collecting the supernatant therefrom; and a filtration step, filtering the collected supernatant through a 0.2 μm filter.

[0161] In the above method, the first centrifugation step can be carried out at a temperature of 4°C and a temperature of 4,000-6,000 xg for 20-40 minutes, and more preferably at a temperature of 4°C and a temperature of 4,000-5,000 xg for 30 minutes.

[0162] In the above method, the filter can be a filter with a sieve aperture size of 30-50 μm, more preferably a filter with a sieve aperture size of 35-45 μm, and most preferably a filter with a sieve aperture size of 40 μm.

[0163] In the above methods, the acid can be hydrochloric acid, nitric acid, acetic acid or sulfuric acid, more preferably hydrochloric acid, and most preferably 6N hydrochloric acid.

[0164] In the above method, the second centrifugation step can be carried out at room temperature at 4000-6000xg for 15-30 minutes, more preferably at room temperature at 4500-5500xg for 15-25 minutes, and most preferably at room temperature at 5000xg for 20 minutes.

[0165] Exosomes are known to be abundant in milk. Furthermore, since milk can serve as a platform for the mass production of exosomes at a lower cost compared to stem cells, a method for the mass production of exosomes from milk or colostrum has been established (Figure 1a). Moreover, using the method established above, it has been demonstrated that the amount of exosomes isolated from milk is significantly greater than that isolated from HEK293 cells (see Figure 2a), and in particular, it was found that when colostrum is used as a raw material, the produced exosomes have an extremely high yield, with a protein content of 0.45-1.5 mg / ml. This is 100 times or more compared to the yield from HEK293 cells (see Figure 2). Furthermore, the inventors have sought to increase the yield of exosomes from milk and have developed a simpler method that does not utilize ultrafiltration (Figure 1b), and it has been demonstrated that compared to conventional ultrafiltration techniques, this improved method can significantly increase the yield of exosomes from milk by 400-1000 times or more (Figure 2b). To verify whether the exosomes prepared by the above methods were normal exosomes, Western blot analysis was used to analyze the expression of exosome markers CD8, CD63, CD81, and TSG 101, and dynamic scattering analysis was used to analyze the particle size distribution of the exosomes. As a result, such as... Figure 3 As can be seen, exosome markers were identified as normal, although slightly lower than those of HEK293-derived exosomes used as controls, and as... Figure 4 As can be seen, the exosomes derived from milk are nanoscale particles with an average particle size of 68.05 nm and a particle size distribution of 30-100 nm. This is a similar size distribution to the control exosomes, and this result confirms that exosomes can be normally separated from milk.

[0166] The inventors conducted stability analyses to investigate the efficacy of exosomes isolated from cell culture media as a cosmetic ingredient. Specifically, exosomes isolated from HEK293 cells (a human cell line commonly used to produce recombinant exosomes) were lyophilized and thawed twice, and their morphology was examined by dynamic scattering analysis and transmission electron microscopy (see [link to product]). Figure 3 and Figure 4 As a result, it was found that exosomes derived from HEK293 cells failed to maintain their initial shape due to aggregation during the second lyophilization and thawing. Therefore, it can be confirmed that exosomes derived from common cell lines, when used as lyophilized raw materials, are unsuitable for use as ingredients in cosmetic compositions intended for later storage at room temperature.

[0167] Against this backdrop, the inventors conducted a comparative analysis of the stability of exosomes isolated from milk and exosomes derived from other cells. Specifically, particle size analysis was performed by dynamic light scattering before and after cryopreservation between colostrum-derived exosomes according to an example of the invention and exosomes derived from other cells (HaCat Exo, B16 Exo, and HDF Exo). It was found that, unlike control exosomes, the milk-derived exosomes maintained their shape intact despite freeze-drying and thawing (Figures 7a and 7b), and that the ability to induce type 1 collagen expression was maintained when the colostrum exosomes before and after freezing were administered to human dermal fibroblasts. This demonstrates that the bioactivity of the milk exosomes according to an example of the invention remains intact despite cryopreservation.

[0168] Furthermore, although milk exosomes according to the embodiments of the present invention were found to maintain their size even after repeated freezing and thawing, the particle size of exosomes from other cell sources was found to increase with increasing freezing and thawing cycles, thereby causing aggregation or fusion and exhibiting an unstable appearance (see...). Figure 8 ).

[0169] Furthermore, to confirm whether exosomes isolated from milk could be successfully delivered to skin cells, the inventors treated human dermal fibroblasts (HDF) with fluorescently labeled milk-derived exosomes and identified the location of the exosomes using fluorescence microscopy (see [link to original text]). Figure 9 The results showed that the milk-derived exosomes of the present invention were well transferred to the cytoplasm of dermal fibroblasts.

[0170] Meanwhile, to verify whether the milk exosomes of this invention could potentially cause cytotoxicity, the inventors administered milk exosomes to human dermal fibroblasts at high concentrations of 0.1 mg / ml and 0.3 mg / ml, and the results showed that no apoptosis occurred (see [link to original text]). Figure 10 ).

[0171] Furthermore, to verify whether the milk exosomes of this invention possess inhibitory activity against UV-induced oxidative cell damage, human dermal fibroblasts were treated with colostrum exosomes according to this invention after UV light-induced damage to the cells to confirm the formation of reactive oxygen species (ROS). The results showed that, unlike control exosomes derived from other cells, the colostrum exosomes according to this invention significantly inhibited UV-induced ROS formation (Figures 11a and 11b).

[0172] Furthermore, the inventors investigated whether milk-derived exosomes isolated according to examples of the present invention possess skin regeneration capabilities. To this end, human keratinocyte HaCaT cells and human dermal fibroblasts (HDF) were treated with exosomes derived from commercial milk and colostrum to examine the extent of cell proliferation (see [link to original text]). Figure 6 The results showed that, compared with the control group (PBS treatment), the milk-derived exosomes of this invention exhibited better cell proliferation effects. Specifically, exosomes derived from colostrum were found to have even higher cell proliferation capacity than exosomes derived from commercial milk. Furthermore, the inventors investigated the effect on cell migration ability, one of the measures of cell regeneration capacity (see [link to study]). Figure 13 The results showed that, compared with the control, when administered, the milk-derived exosomes of the present invention increased cell migration of human dermal fibroblasts (HDF) and human keratinocytes (HaCaT cells), and the exosomes derived from colostrum particularly exhibited significantly higher cell migration capacity.

[0173] Furthermore, in order to identify various uses of milk exosomes according to examples of the present invention, the inventors investigated the following: whether tube formation occurs when treated in vascular endothelial cells. Figure 14 Analysis of in vivo distribution patterns over time when administered in vivo (Figures 15a-15c); Comparison of wound healing capacity when artificially introduced wounds are administered to actual trauma model animals. Figure 16 Analysis of elastin expression during wound treatment ( ); Figure 17 ); When treated in human dermal fibroblasts, the skin wrinkle reduction effect after inducing type I collagen expression and inhibiting MMP-2 expression (Fig. 18a and 18b); the skin whitening effect after inhibiting melanin synthesis in melanoma cells ( Figure 19 ); and the ability to induce the expression of immune-activated cytokines upon treatment in bone marrow-derived macrophages (BMDM). Figure 20 The ability to induce the expression of inflammation-related growth factors when treated in fibroblasts. Figure 21 The study investigated the therapeutic effects on hair loss by examining the ingestion of human dermal dermal papilla cells (Fig. 22a), the proliferative capacity of human dermal dermal papilla cells under normal and hair loss-inducing conditions (Fig. 22b), and the promotion and analysis of hair growth in animal experiments (Fig. 22c). As a result, it was confirmed that milk exosomes according to embodiments of the present invention are substances exhibiting various biological activities such as wound healing, wrinkle reduction, whitening effects, immune enhancement effects, and hair loss prevention effects.

[0174] Based on this result, the inventors were able to demonstrate that the milk-derived exosomes according to the embodiments of the present invention can be used very effectively for a wide variety of purposes, including skin regeneration and wound healing, and can also be used to produce functional cosmetics, such as whitening and wrinkle reduction, hair loss treatment and immune enhancement.

[0175] Mode of implementing the present invention

[0176] The invention will be described in more detail below by way of examples and experimental embodiments. However, the invention is not limited to the examples and experimental embodiments disclosed below, but can be implemented in various different forms, and the examples and experimental embodiments are provided to make the disclosure of the invention complete and to fully inform those skilled in the art within the scope of the invention.

[0177] Comparative Example: Isolation of Exosomes from Various Cells

[0178] To isolate exosomes from HEK293 cells, HaCat cells, B16 cells, and HDF cells, cells (6 x 10⁻⁶) were prepared. 6 Cells were cultured in high-glucose medium (Dulbecco modified Eagle medium, DMEM, 4,500 mg / L glucose) supplemented with 10% FBS and 1% antibiotics, maintained at 37°C and 5% CO2. The cell culture supernatant was obtained by differential centrifugation when 80-90% confluence was observed on 15 cm culture dishes, and for the purpose of exosome isolation. The specific methods are as follows.

[0179] First, to remove cell debris and other cellular components from the exosome-containing culture medium, centrifugation was performed sequentially at 300g for 10 minutes, 2,000g for 10 minutes, and 10,000g for 30 minutes. After filtering the culture medium through a 0.22 μm filter, the medium was ultracentrifuged at 36,900 rpm for 2 hours. The resulting exosomes were then resuspended in PBS containing a protease inhibitor (Roche).

[0180] Example 1: Isolation of exosomes from commercial milk

[0181] The inventors isolated exosomes from commercial milk using the following method.

[0182] First, commercial milk was centrifuged at 3,000 g for 30 minutes, followed by sequential ultracentrifugation at 12,000 g for 1 hour, at 35,000 g for 1 hour, and then at 70,000 g for 3 hours. The resulting product was then filtered first through a 0.8 μm filter, second through a 0.45 μm filter, and third through a 0.2 μm filter. The filtrate was ultracentrifuged at 100,000 g for 1 hour, and the precipitate was collected and resuspended in PBS containing a protease inhibitor (Roche) (Figure 1). For the exosomes isolated from the milk, the protein concentration of the isolated exosomes was measured using the BCA Protein Assay Kit (Bio-Rad) as described above for the comparative examples.

[0183] Example 2: Isolation of exosomes from colostrum

[0184] Except for using colostrum (obtained from Lee Sung Young Chang-buk Ranch, located at 20, Bongsangwandong1-ro, Gyeseon-myeon, Changnyeong-gun, Gyeongsangnam-do) instead of commercial milk, the inventors isolated exosomes from the colostrum using the same method as described in Example 1 above.

[0185] Example 3: Improvement of Exosome Isolation Method

[0186] The methods used in Examples 1 and 2 require ultracentrifugation and several sequential centrifugations, which is time-consuming. Against this backdrop, the inventors sought to develop a simpler and more efficient method for exosome isolation.

[0187] Therefore, the inventors have devised a new method for exosome isolation by modifying a previously reported method for isolating exosomes from milk (Yamauchi et al., Drug Dev. Ind. Pharm. 45(3):359-364, 2019). The method reported by Yamauchi et al. utilizes the following process: centrifuging the raw milk at 2,000×g for 20 minutes at 4°C to remove fat and cells (primary centrifugation), diluting it by adding an equal volume of distilled water (distilled water dilution), titrating it to pH 4.6 by adding 6N HCl to remove casein by isoelectric precipitation (isoelectric precipitation), centrifuging it at 5,000×g for 20 minutes at room temperature (secondary centrifugation), and filtering it sequentially using 1.0, 0.45, and 0.2 μm filters (sequential filtration). The inventors have modified the above method to perform the first centrifugation process at 5,000×g for 30 minutes at 4°C, and introduce the use of a 40 μm filter before dilution with distilled water. The process of pre-filtration (in Corning, USA) further removes fat and cells, and sequential filtration is replaced by a single filtration process using a 0.2 μm filter after a second centrifugation.

[0188] Experimental Example 1: Analysis of Exosome Isolation Yield

[0189] The yields of exosomes derived from HaCat, B16, HDF, and HEK293 isolated according to the above comparative examples and exosomes isolated from commercial milk and colostrum according to Examples 1 and 2 of the present invention were analyzed using the BCA protein assay kit (Bio-Rad).

[0190] As a result, as shown in Figure 2a, exosomes isolated from HaCat, B16, and HDF cells exhibited extremely low yields, while the yield of exosomes isolated from commercial milk was 0.05 mg / ml, according to Example 1 of the present invention. Specifically, the colostrum showed a protein-based yield of at least 0.45 mg / ml, which is very high even when compared to commercial milk.

[0191] Meanwhile, as shown in Figure 2b, the results of separating exosomes from milk using the method of Example 3 demonstrate that the non-ultrafiltration exosome separation method according to embodiments of the present invention results in significantly higher yields compared to the ultrafiltration method. Specifically, the yield of colostrum exosomes exhibited was 1000 times or more that of the ultrafiltration method. This result indicates that the method for separating exosomes from milk according to embodiments of the present invention is a highly desirable method in terms of productivity.

[0192] Experimental Example 2: Identification of Exosome Markers

[0193] To verify whether exosomes isolated from commercial milk and colostrum retain the complete characteristics of exosomes, the inventors performed Western blot analysis using specific antibodies against exosome markers CD9, CD63, CD81, and TSG 101.

[0194] As a result, such Figure 3 As can be seen, in exosomes derived from commercial milk or colostrum according to embodiments of the present invention, exosome-specific markers were identified as normal.

[0195] Experimental Example 3: Characterization of Exosome Size and Shape

[0196] Subsequently, the inventors analyzed the particle size of the exosomes isolated according to the embodiments of the present invention using a dynamic light scattering (DLS) analysis device (Malvern zetasizer nanoZS, UK), and photographed the resulting exosomes using a transmission electron microscope. Figure 4 ). Result, such as Figure 4 As shown, the milk-derived exosomes prepared according to embodiments of the present invention have a particularly narrow particle size spectrum around 68.05 nm, which is similar to the control exosomes, and there is no significant difference in shape between them and the control exosomes.

[0197] Experimental Example 4: Freezing / Thawing Stability Analysis

[0198] In order to utilize the exosomes derived from commercial milk and colostrum, which were isolated in Examples 1 and 2 respectively, as cosmetic ingredients, the inventors evaluated storage stability, particularly freeze / thaw stability.

[0199] Therefore, control exosomes and commercial milk-derived exosomes isolated in Example 1 were frozen at -85°C and thawed at room temperature, and the thawed exosomes were imaged by transmission electron microscopy. Then, the thawed exosomes were frozen again at -85°C and thawed a second time at room temperature, and imaged by transmission electron microscopy. Figure 5 ) and particle size distribution analysis via dynamic light scattering ( Figure 6 ).

[0200] As a result, such Figure 5 As can be seen, although the control exosomes lost their initial exosome shape because aggregation of exosomes occurred during thawing after the first freezing, the milk-derived exosomes isolated according to embodiments of the present invention retained their initial shape even when frozen / thawed a second time.

[0201] Furthermore, to confirm whether the above differences could be attributed to the cell type of the control exosomes, the inventors conducted a comparative analysis with a broader range of controls. Specifically, the inventors used dynamic light scattering to measure the particle size of the colostrum exosomes prepared in Example 2, as well as exosomes extracted from human keratinocytes (HaCaT), mouse melanoma cells (B16), and human dermal fibroblasts (HDF), before and after freeze-drying (Fig. 7a). As a result, as shown in Fig. 7a, although the particle size of exosomes derived from the three cell types changed before and after freeze-drying, the particle size of the colostrum exosomes according to the embodiment of the present invention showed almost no change. In addition, the shape of the colostrum exosomes according to the embodiment of the present invention before and after freeze-drying was identified using transmission electron microscopy, and as shown in Fig. 7b, there was no significant change in shape before and after freeze-drying. This demonstrates that the colostrum exosomes exhibit excellent structural stability during freeze-drying. Furthermore, the inventors treated human dermal fibroblasts with colostrum exosomes according to embodiments of the present invention at a concentration of 0.1 mg / ml before and after lyophilization, and identified the expression level of type 1 collagen by immunofluorescence staining and fluorescence microscopy 24 hours later (Figure 7c). Figure 7C As can be seen, even after thawing following freeze-drying, the collagen synthesis capacity was maintained to some extent, indicating that colostrum exosomes also have functional stability during freeze-drying.

[0202] Furthermore, to verify the freeze-thaw structural stability of milk exosomes, the inventors compared particle size through freeze-thaw cycles. Figure 8Using dynamic light scattering analysis, the particle size of exosomes extracted from colostrum, as well as those extracted from human keratinocytes (HaCat), mouse melanoma cells (B16), and human dermal fibroblasts (HDF), was measured with increasing freeze-thaw cycles. It was found that the particle size of exosomes increased with increasing freeze-thaw cycles, while colostrum exosomes showed almost no change in particle size after up to 5 freeze-thaw cycles. This indicates that colostrum exosomes possess excellent structural stability during freeze-thaw cycles.

[0203] Experimental Example 5: Assessment of the ability to deliver into skin cells

[0204] The inventors investigated whether milk-derived exosomes isolated according to embodiments of the present invention were successfully delivered into skin cells.

[0205] Specifically, after staining the plasma membrane of milk-derived exosomes isolated according to embodiments of the present invention with the red fluorescent dye NHS-Cy5.5, the exosomes were administered to human dermal fibroblasts (HDF) cells, and fluorescence signals were observed by confocal fluorescence microscopy after 30 minutes. DAPI was used for nuclear counterstaining (…). Figure 9 ).

[0206] As a result, such Figure 9 As can be seen, milk-derived exosomes according to embodiments of the present invention are well absorbed into the cytoplasm of dermal fibroblasts.

[0207] Experimental Example 6: Cytotoxicity Assessment

[0208] The inventors sought to assess the cytotoxicity of colostrum exosomes by calculating the necrosis / apoptosis ratio in human dermal fibroblasts (HDFs) treated with colostrum exosomes. Twenty-four hours after administration of colostrum exosomes at concentrations of 0, 0.1, and 0.3 mg / ml, human dermal fibroblasts (HDFs) were stained with propidium iodide (PI) to stain the DNA of necrotic cells and to show fluorescence, and with Annexin V / FITC to react with phosphatidylserine in apoptotic cells and to show fluorescence. The results were analyzed by flow cytometry. As a result, such as… Figure 10 As can be seen, compared with the control, colostrum exosomes did not exhibit cytotoxicity at concentrations up to 0.3 mg / ml, given the observed ratio of necrotic or apoptotic cells. This indicates that colostrum exosomes according to embodiments of the present invention are safe substances.

[0209] Experimental Example 7: Analysis of Antioxidant Effects in UV-Damaged Cells

[0210] The inventors sought to demonstrate the antioxidant effect of milk exosomes by reducing reactive oxygen species (ROS) observed when colostrum exosomes were administered to human keratinocytes (HaCaT) damaged by UV irradiation. Twenty-four hours after treating HaCaT cells with exosomes derived from commercial milk and colostrum at a concentration of 0.05 mg / ml, cell damage was induced by UV radiation. Six hours later, the levels of ROS generated in the cells were analyzed by fluorescence microscopy using DCF-DA, which reacts with and exhibits fluorescence in response to ROS. As a result, as shown in Figures 11a and 11b, both commercial milk-derived exosomes and colostrum exosomes exhibited significant antioxidant activity in UV-damaged cells, demonstrating that this antioxidant activity was superior to that of stem cell-derived exosomes.

[0211] Experimental Example 8: Evaluation of Skin Cell Regeneration and Wound Healing Efficacy

[0212] Subsequently, the inventors investigated whether the milk-derived exosomes isolated according to embodiments of the present invention possess skin cell regeneration capabilities.

[0213] 8-1: Assessment of skin cell proliferative capacity

[0214] Specifically, HaCaT human keratinocytes (a type of skin cell) and HDF used in Example 5 of the above experiment were used at a ratio of 1 x 10⁻⁶ cells per well. 4 Cells were seeded at a concentration of [number] cells and treated with exosomes derived from commercial milk or colostrum at 0.2 mg / ml. Cells were counted after 48 hours to measure the degree of cell proliferation. The control group was treated with only an equal volume of saline (Figures 12a and 12b).

[0215] As shown in Figures 12a and 12b, the results indicate that, compared to the control, the exosomes derived from milk according to embodiments of the present invention exhibit significantly enhanced proliferative capacity in both types of skin cells. Specifically, exosomes derived from colostrum were found to show superior skin cell proliferation capacity compared to exosomes derived from commercial milk.

[0216] 8-2: Assessment of skin cell migration

[0217] In skin regeneration, skin cell migration is known to play a crucial role. Therefore, the inventors conducted a scratch assay to determine the effect of milk-derived exosomes isolated according to embodiments of the present invention on cell migration.

[0218] Specifically, HaCaT human keratinocytes and HDF were cultured until 80% confluence was achieved in 100 mm culture dishes at the 7th passage. The centers of the wells in 24-well plates were marked and 0.1% gelatin solution was added. The plates were incubated at 37°C for 2 hours and washed once with DPBS to prepare gelatin-coated 24-well plates. Cells were isolated using 0.25% trypsin-EDTA and cultured at 9 x 10⁶ cells per well. 4 Cells were seeded at a concentration of [number] cells per coated plate. Cells were cultured overnight at 37°C, and then scratched using a 200 μL pipette tip, with the wells rinsed twice with DPBS to remove debris. Subsequently, 1 mL of medium containing 0.5% FBS was added to each well, and images were captured using a microscope (Leica, Wetzlar, Germany) to measure the scratch time at 0 h. Next, except for the following groups [positive control (10% FBS, 1 mL DMEM), negative control (0.5% FBS, 1 mL DMEM), EGF (10 ng / mL, EGF 0.5% FBS, 1 mL DMEM)], 800 μL of medium containing 0.5% FBS was replaced in each well. Exosomes isolated in Examples 1 and 2 were placed in transwell inserts, and 200 μL of medium containing 1% FBS in DMEM was added to the inserts. After culturing the cells at 37°C for 8 hours, the scratched areas were imaged. The scratch width was measured using Image Pro Plus software (Media Cybernetics, USA) and calculated according to Equation 1 below.

[0219] [Equation 1]

[0220] Relative wound area = {(A0-At) / A0} / negative control result (in this equation, A0 is the initial wound area, and At is the wound area after 48 hours.)

[0221] As a result, as shown in Figure 11, compared with the control group, milk-derived exosomes according to embodiments of the present invention increased skin cell migration. In particular, it was found that exosomes derived from colostrum significantly increased skin cell migration compared with milk-derived exosomes. However, since commercial milk is more advantageous in terms of material supply and cost, these findings do not diminish the utility of exosomes derived from commercial milk as raw materials for functional cosmetics.

[0222] 8-3: Analysis of tube formation rate in vascular endothelial cells

[0223] The inventors treated mouse endothelial cells (SVEC4-10) with colostrum, commercial milk and serum-derived exosomes at a concentration of 200 μg / ml and compared the degree of angiogenesis among the groups.

[0224] First, to allow tube formation within endothelial cells, 50 μL of Matrigel (Corning #356237), serving as the extracellular matrix, was pipetted into 96-well plates and allowed to harden at 37°C for approximately 30 minutes. Subsequently, exosome-treated cells were attached and cultured for approximately 8 hours. The degree of tube formation was observed and compared using dissecting incandescent (DIC) microscopy, and the branching points where three or more tubes intersected, as well as the tube lengths, were digitized and displayed in graphs. Figure 14 ).

[0225] As a measurement result, such as Figure 14 As can be seen, compared with the untreated control group, the group treated with milk-derived exosomes (such as commercial milk and colostrum) showed a higher degree of tube formation, while the serum-derived exosome group showed similar results to the control group without significant difference. This demonstrates that milk-derived exosomes according to embodiments of the present invention can accelerate angiogenesis and therefore can be advantageously used for wound healing.

[0226] 8-4: In vivo imaging confirms the in vivo distribution of milk exosomes.

[0227] The inventors analyzed the in vivo distribution of administered colostrum exosomes using in vivo imaging in mice (IVIS).

[0228] The concentration of colostrum exosomes was adjusted to 1 μg / μl using PBS buffer, and 1 μl of Flamma 675NHS ester (BioActs#PWS1515) was added to every 100 μl of exosomes, and the mixture was labeled overnight at 4°C. Subsequently, unattached dye material was removed by air-fusing twice per hour. 100 μg of exosomes was subcutaneously injected into the upper left thigh of each mouse, and this was repeated at the same time daily. In vivo imaging was performed using an in vivo imaging system (PerkinElmer, USA) for 7 days starting from the day of injection (Fig. 15a). On the second day, five organs and skin tissue were extracted, and the distribution of colostrum exosomes in each organ was assessed (Figs. 15b and 15c). As a result, as shown in Fig. 15a, most colostrum exosomes left the body after 3 days, and complete departure took approximately 7 days. As a result of organ harvesting, such as... Figure 15B and 15CAs shown, the highest fluorescence intensity was detected in the skin, followed by the liver, lungs, and kidneys in that order. This result also confirms that colostrum exosomes, after being retained in the body, are subsequently excreted through the kidneys.

[0229] 8-5: Efficacy analysis using animal models of trauma

[0230] The inventors continued to conduct animal modeling of trauma to confirm the actual skin tissue regeneration effect of milk-derived exosomes.

[0231] As animals, male Balb / c 7-week-old mice were used, and wounds were induced on the back using an 8 mm diameter skin biopsy puncturer. Experimental groups were divided into a total of 5 types (control, serum, commercial milk, colostrum, and colostrum-4 days), and exosomes were administered subcutaneously at 3-day intervals based on the in vivo distribution results of Experimental Examples 8-4. Except for the colostrum-4 day group, the other four groups were administered the corresponding substances on days 1, 4, and 7, while the colostrum-4 day group was administered colostrum exosomes on days 4, 7, and 10. Here, the concentration of the administered substances was 1 μg / μl, and the total volume administered to each animal was 100 μl, with progress observed over approximately 25 days. Figure 16 ).

[0232] As a result, Figure 16 As can be seen, skin wound regeneration was significantly faster in the colostrum exosome treatment group and the colostrum-4-day group, with the colostrum-4-day group showing a particularly faster regeneration rate between days 4 and 10 compared to other groups. Skin regeneration was ranked sequentially after the colostrum treatment group: commercial milk exosome treatment group, PBS treatment control group, and serum-derived exosome treatment group, thus confirming that milk-derived exosomes have a higher skin regeneration effect than other experimental groups.

[0233] 8-6: Immunohistochemical analysis

[0234] Following animal experiments using the trauma model, the inventors employed immunohistochemistry to perform tissue analysis.

[0235] Specifically, after mice were euthanized on day 25 of the animal experiments, tissue from the wound site was excised and left in fixative overnight to fix the tissue. To allow for paraffin infiltration, dehydration processes were performed sequentially from xylene to 100%, 90%, 80%, and 70% ethanol, followed by paraffin embedding to form blocks. Using a tissue slicer, 6 μm thick paraffin tissue sections were prepared and tested according to the experimental methods provided by the DAB kit (Abcam#ab64264). First, after removing the paraffin component from the tissue on the slide and inducing a reaction with catalase blocking buffer at room temperature for 10 minutes, the slide was boiled in a retrieval solution at 95°C for 10 minutes to break the cross-links between proteins. Then, after inducing a reaction with protein blocking buffer at room temperature for 10 minutes, the slide was treated with anti-elastin primary antibody (santacruz#sc-58756) and allowed to react overnight at 4°C. The next day, biotinylated goat anti-primary antibody solution and streptavidin-peroxidase solution were added and allowed to react at room temperature for 10 minutes. DAB chromogen and DAB substrate were mixed at a ratio of 1:50 and placed on antibody-treated tissue sections, and color changes were observed at room temperature for 10 minutes. Finally, counterstaining was performed to stain cell nuclei, and slides were fixed with toluene.

[0236] As a result of tissue microscopic examination, such as Figure 17 As can be seen, compared with other groups, milk-derived exosomes, especially those treated with colostrum exosomes, exhibited a deeper brown color, which may conclusively indicate that elastin components were detected most abundantly in the tissues of this particular group. Furthermore, this enhanced elastin expression effect of milk exosomes is closely related to the wrinkle reduction effect described below.

[0237] Experimental Example 9: Analysis of Wrinkle Reduction Effect

[0238] Collagen is a major component of the extracellular matrix of the dermis. After being broken down during the skin regeneration cycle, it is typically regenerated by collagen-forming enzymes. Skin wrinkles are a symptom resulting from the inability to restore successful collagen biosynthesis in the dermal extracellular matrix. Against this backdrop, the inventors conducted a study measuring the degree of inhibition of collagenase and metalloproteinase-2 (MMP-2) expression, as well as the enhancement of collagen biosynthesis, as measures of wrinkle reduction.

[0239] Specifically, the inventors seeded human skin fibroblasts (HDF) in 35π glass-bottomed culture dishes (3 x 10⁻⁶ cells / mL). 5(10 cells) were cultured in an incubator at 37°C and 5% CO2 for 24 hours. Then, 24 hours after treatment with commercially available milk-derived exosomes and colostrum-derived exosomes isolated in Examples 1 and 2, respectively, at a concentration of 0.1 mg / ml, type 1 collagen was stained using immunofluorescence and examined by fluorescence microscopy, and the protein expression level of metalloproteinase 2 was measured by Western blotting. The results, as shown in Figures 18a and 18b, revealed increased expression levels of type 1 collagen and decreased expression levels of MMP-2 protein in HDF treated with commercially available milk-derived exosomes and colostrum exosomes. This finding, together with the results of Experiments 8-6, indicates that milk exosomes have an excellent effect on reducing skin wrinkles.

[0240] Experimental Example 10: Whitening Activity Analysis

[0241] The inventors investigated melanin production inhibition, a typical indicator of skin whitening activity, to verify whether milk-derived exosomes according to embodiments of the present invention possess skin whitening activity.

[0242] Specifically, the inventors sought to demonstrate the melanin-reducing effect by measuring the amount of melanin produced by B16F10 mouse melanoma cells after treatment with milk exosomes (commercially derived milk exosomes and colostrum exosomes). As a control group, exosomes isolated from B16F10 cells were used. B16F10 cells were treated with the corresponding exosomes at concentrations of 0, 0.002, 0.005, 0.01, and 0.02 mg / ml, and after 24 hours, the cells were stimulated by UV radiation. Then, after 24 hours, melanin was extracted from an equal volume of cells, and the extracted melanin was measured and analyzed at a wavelength of 490 nm. As a result, as shown... Figure 19 As can be seen, compared with the control group, there was no significant difference in the amount of melanin produced when the concentration of colostrum exosomes reached a maximum of 0.002 mg / ml, while a melanin-reducing effect was observed at colostrum exosome treatment concentrations of 0.005 mg / ml to 0.02 mg / ml. Furthermore, B16F10 was treated with exosomes extracted from B16F10, commercial milk-derived exosomes, and colostrum exosomes at a concentration of 0.1 mg / ml to compare the amount of melanin produced after 24 hours. Figure 19 (C in the text). As a result, the greatest reduction in melanin production was observed in cells treated with milk exosomes, both when cells were stimulated with ultraviolet light and when no stimulation was applied.

[0243] Experimental Example 11: Analysis of Immune Functional Activity

[0244] The inventors analyzed cytokines in the cell culture medium after treating BMDM (bone marrow-derived macrophages) with 50 μg / ml commercial milk-derived exosomes and colostrum exosomes.

[0245] Specifically, the inventors used 1 ml of cell culture medium to analyze cytokines in the cell culture medium and utilized the experimental methods provided in the Cytokine Array Kit (ARY006). First, a membrane bound to primary antibodies against various cytokines was blocked with blocking buffer at room temperature for 30 minutes, and then 1 ml of cell culture medium was added to the membrane, allowing it to react overnight at 4°C. Subsequently, the membrane was treated with a biotin-conjugated secondary antibody cocktail and allowed to react overnight at 4°C, followed by reaction with HRP-streptavidin at room temperature for 2 hours, and the results were visualized by chemiluminescence (Bio-Rad). As a result, as shown... Figure 19 As can be seen, compared with the untreated control group, the groups treated with commercial milk and colostrum showed relatively high expression of factors involved in T cell and dendritic cell activation and inflammatory responses, including CD54, CXCL1, CCL3, CCL5, and TNF-α. This result indicates that milk exosomes activate immunity according to embodiments of the present invention.

[0246] Experimental Example 12: Analysis of Enhanced Growth Factor Expression Ability

[0247] The inventors treated NIH-3T3 fibroblasts with commercially available oil-derived exosomes and colostrum exosomes at a concentration of 100 μg / ml, and analyzed the expression levels of various growth factors from cell culture medium and cell lysates after 24 hours. For this experiment, 500 μg of cell lysate was prepared based on an equal volume of protein, and cell culture medium was added until the total volume of the sample solution reached 1 ml. Protein expression level analysis was performed according to the experimental method provided in the Growth Factor Array Kit (RayBiotech #AAM-GF-3-4). First, the membrane bound to the primary antibody against the growth factor was treated with blocking buffer at room temperature for 30 minutes, and 1 ml of the prepared sample was added, allowing it to react overnight at 4°C. The next day, the membrane was thoroughly rinsed and treated with a biotinylated secondary antibody cocktail, allowing it to react at room temperature for 2 hours, followed by reaction with HRP-streptavidin at room temperature for 2 hours, and the results were visualized by chemiluminescence (Bio-Rad). As a result, as shown... Figure 21 As can be seen, compared with the control group, the experimental group treated with commercial milk-derived exosomes and colostrum exosomes showed higher levels of expression of HGF, IGF-1, PDGF-AA and VEGF-A, which are involved in anti-inflammatory responses and tissue regeneration or angiogenesis.

[0248] Experimental Example 13: Hepatocyte Proliferation Analysis

[0249] The inventors studied the activity of human hepatocytes to evaluate the efficacy of milk-derived exosomes according to embodiments of the present invention as a raw material for functional health foods for improving liver health.

[0250] Human liver cells were distributed at a density of 1 x 10⁻⁶ cells per well. 4 Cells were seeded at a concentration of [number] cells per well in 96-well plates and incubated at 37°C and 5% CO2 for 24 hours. (0.2) mg / ml of exosomes derived from commercial milk and colostrum, isolated in Examples 1 and 2 respectively, were added to serum-free DMEM medium and incubated for 24 hours. Cell proliferation was assessed using a CCK-8 assay (Dojindo Molecular Technologies, Japan). Controls were treated with an equal volume of saline. 10 μl of CCK-8 assay solution was added to each well, and absorbance at 450 nm was measured and compared after 4 hours of incubation.

[0251] Experimental Example 14: Analysis of the effects of hair loss prevention, hair growth promotion, and hair growth enhancement.

[0252] 14-1: Analysis of milk exosome uptake by dermal dermal papilla cells

[0253] In order for milk exosomes according to embodiments of the present invention to exhibit preventive or therapeutic effects on hair loss, the milk exosomes need to exhibit dermal papilla cell proliferative activity that promotes the absorption and proliferation of dermal papilla cells, which are cells that contribute to hair growth. In this context, the inventors investigated whether milk exosomes according to embodiments of the present invention are readily absorbed by cultured dermal papilla cells.

[0254] Specifically, the inventors treated cultured human dermal papilla cells with commercially available milk-derived exosomes labeled with Cy5.5 fluorescent dye at 100 μg / ml for 1 hour, 4 hours, 12 hours, and 24 hours, respectively, and observed the extent of cellular uptake using a fluorescence microscope (Figure 22a). As a result, as shown in Figure 22a, it was confirmed that milk exosomes according to embodiments of the present invention were readily absorbed by human dermal papilla cells in proportion to the treatment duration.

[0255] 14-2: Dermal dermal papilla cell proliferation analysis

[0256] The inventors investigated the effects of milk-derived exosomes according to embodiments of the present invention on the proliferation of human dermal papilla cells to evaluate their effectiveness in improving hair health and reducing hair loss.

[0257] Specifically, the inventors first treated human dermal dermal papilla cells with commercially available milk-derived exosomes prepared in Example 1 at concentrations of 100, 250, and 500 μg / ml for 48 hours, and counted the cells using the experimental methods provided in the Cell Counting Kit-8 (CK04-20). 20 μl of the reaction solution from the cell counting kit was added to 200 μl of cell culture medium, and the cells were allowed to react at 37°C and 5% CO2 for 30 minutes. Measurements were then taken at 450 nm and visualized. Alternatively, human dermal dermal papilla cells were treated with 100 μM of alopecia-inducing substance (dihydrotestosterone, DHT) for 24 hours, and then treated with 500 μg / ml of commercially available milk-derived exosomes for 48 hours. 20 μl of the reaction solution from the cell counting kit was then added to 200 μl of cell culture medium, and the cells were allowed to react at 37°C and 5% CO2 for 30 minutes. Measurements were then taken at 450 nm and visualized (Figure 22b). As a result, as shown in Figure 22b, it was found that the group treated with 500 μg / ml commercial milk-derived exosomes actively induced cell proliferation compared to the untreated control group. Simultaneously, it was found that the group treated only with the hair loss inducing substance showed decreased cell proliferation activity compared to the control group, but in the group treated with 500 μg / ml commercial milk-derived exosomes after treatment with the hair loss inducing substance, cell proliferation activity recovered to the level before DHT treatment. This indicates that milk exosomes according to embodiments of the present invention are also effective in preventing hair loss.

[0258] 14-3: Analysis of Hair Growth Promotion and Hair Growth Effect

[0259] Based on the results of the above experimental examples 14-2, the inventors used experimental animals to study the hair growth promotion and hair growth effect, in order to verify whether milk exosomes according to the embodiments of the present invention are effective in promoting hair growth and hair regrowth in addition to preventing hair loss.

[0260] Specifically, the inventors shaved the entire back region of male C57BL / 67-week-old mice, applied Veet cream, and wiped away any remaining hair with Kimtech (day 0). Starting from day 1, 20 μl of commercially available milk-derived exosomes were administered intradermally to five sites along the mid-back (along the spine) every other day, for a total of 100 μl (200 μg). Hair growth was then monitored every 3–4 days. As a result, as shown in Figure 22c, hair grew at a faster rate in the group treated with commercially available milk-derived exosomes according to embodiments of the present invention.

[0261] Therefore, it can be confirmed that the milk exosomes of the present invention are also highly effective in promoting hair growth and preventing hair loss.

[0262] Preparation Example 1: Skin Lotion

[0263] A skin care liquid containing exosomes derived from commercial milk or colostrum obtained according to embodiments of the present invention is prepared by mixing according to the composition ratios shown in Table 1 below.

[0264] Table 1

[0265] Mixing ratio of skin care lotion

[0266] Element Content (unit: Wt%) Milk-derived exosomes 0.5 Glyceryl stearate SE 1.5 Cetearyl alcohol 1.5 Lanolin 1.5 Polysorbate 60 1.3 Sorbitol stearate 0.5 Hydrogenated palm oil 4.0 mineral oil 5.0 Trioctyl glyceride 2.0 Dimethyl silicone oil 0.8 Tocopheryl acetate 0.5 Carboxyvinyl polymers 0.12 glycerin 5.0 1,3-Butanediol 3.0 Sodium hyaluronate 5.0 Triethanolamine 0.12 Uniside-U13 0.02 distilled water The rest Total 100

[0267] Preparation Example 2: Nourishing Cream

[0268] A nutritional cream containing exosomes derived from commercial milk or colostrum obtained according to embodiments of the present invention is prepared by mixing the ingredients in the proportions shown in Table 2 below.

[0269] Table 2

[0270] Mixing ratio of nourishing cream

[0271]

[0272]

[0273] Preparation Example 3: Preparation of a Shampoo for Reducing Hair Loss

[0274] A shampoo for reducing hair loss containing exosomes derived from colostrum or commercial milk according to an embodiment of the present invention is prepared by mixing the ingredients according to the mixing ratios shown in Table 3 below.

[0275] Table 3

[0276] Shampoo mixing ratio for reducing hair loss

[0277] Element Content (unit: Wt%) Milk-derived exosomes 1.0 Sodium lauryl ether sulfate 0.7 Sodium lauryl sulfate 0.7 Polyquaternium-7 1 Hydrolyzed silk 0.5 Panthenol 1.0 glycerin 2.5 Cocamidopropyl Betaine 0.7 Polyquaternium-10 0.5 Ethylhexylglycerin 3.5 Disodium EDTA 1.0 Citric acid 0.7 Butylene glycol 0.7 Phenoxyethanol 0.5 spices 0.2 distilled water The rest Total 100

[0278] Preparation Example 4: Preparation of Functional Health Food (Soft Capsule Formulation)

[0279] A soft capsule formulation containing exosomes derived from colostrum or commercial milk according to an embodiment of the present invention is prepared by mixing according to the mixing ratios shown in Table 4 below. The following amounts of ingredients are mixed and homogenized, and then filled into soft capsules of a predetermined weight according to known methods.

[0280] Table 4

[0281] Preparation of soft capsule formulations

[0282] Element Content (unit: mg) Exosomes derived from freeze-dried milk 1000 Hyaluronic acid 75 Collagen peptides 75 Vitamin C 75 Vitamin B2 4 Vitamin B6 3 Tocopherol 50 Dietary fiber 100 Wheat germ oil 3,500 beeswax 500 wax 500

[0283] Exosomes derived from commercial milk and colostrum according to examples of the present invention have been found to exhibit not only extremely high yields compared to exosomes derived from conventionally cultured cells, but also storage stability and extremely high skin cell regeneration capacity not possessed by conventionally cultured cells. Therefore, exosomes derived from milk or colostrum according to examples of the present invention can be used highly effectively as raw materials for functional cosmetics for skin regeneration. Although the invention has been described with reference to the above embodiments, these embodiments are merely exemplary, and those skilled in the art should understand that various modifications and equivalent embodiments are possible. Therefore, the true scope of protection of the invention should be determined based on the technical concept of the appended claims.

[0284] Industrial availability

[0285] The cosmetic compositions for skin regeneration according to examples of the present invention not only exhibit significantly higher skin regeneration effects than conventional exosomes isolated from cell culture media, but also possess low production costs, significantly high yields, and high material stability, and are therefore extremely economical to produce. Consequently, milk exosomes according to examples of the present invention can be used for wound healing, immune function enhancement, hair loss prevention, and the preparation of pharmaceutical compositions for hair growth and hair growth promotion, as well as for the preparation of various functional cosmetics for reducing or whitening skin wrinkles.

Claims

1. Use of milk-derived exosomes in the preparation of cosmetic compositions for skin regeneration. The exosomes are prepared by the following method, the method comprising: The first centrifugation step removes fat and cells from the milk by centrifugation, wherein the first centrifugation step is carried out at a temperature of 4°C for 20-40 minutes at 4,000-6,000 x g. The filter step involves filtering the centrifuged milk using a filter with a sieve aperture size of 20-60 μm to further remove fat and cells. The dilution step involves diluting the filtered milk by adding an equal volume of distilled water. The isoelectric precipitation step involves adjusting the pH to 4-5 by adding acid to the diluted milk, thereby precipitating casein in the milk. A second centrifugation step is performed to further centrifuge the milk in which casein has precipitated, and the supernatant is collected. The second centrifugation step is carried out at 4000-6000 x g for 15-30 minutes at room temperature. and The filtration step involves filtering the collected supernatant through a 0.2 μm filter.

2. The use of claim 1, wherein the milk is raw milk, commercial milk, or colostrum.

3. Use of milk-derived exosomes in the preparation of cosmetic compositions for wrinkle reduction. The exosomes are prepared by the following method, the method comprising: The first centrifugation step removes fat and cells from the milk by centrifugation, wherein the first centrifugation step is carried out at a temperature of 4°C for 20-40 minutes at 4,000-6,000 x g. The filter step involves filtering the centrifuged milk using a filter with a sieve aperture size of 20-60 μm to further remove fat and cells. The dilution step involves diluting the filtered milk by adding an equal volume of distilled water. The isoelectric precipitation step involves adjusting the pH to 4-5 by adding acid to the diluted milk, thereby precipitating casein in the milk. A second centrifugation step is performed to further centrifuge the milk in which casein has precipitated, and the supernatant is collected. The second centrifugation step is carried out at 4000-6000 x g for 15-30 minutes at room temperature. and The filtration step involves filtering the collected supernatant through a 0.2 μm filter.

4. The use of claim 3, wherein the milk is raw milk, commercial milk, or colostrum.

5. Use of milk-derived exosomes in the preparation of cosmetic compositions for skin whitening. The exosomes are prepared by the following method, the method comprising: The first centrifugation step removes fat and cells from the milk by centrifugation, wherein the first centrifugation step is carried out at a temperature of 4°C for 20-40 minutes at 4,000-6,000 x g. The filter step involves filtering the centrifuged milk using a filter with a sieve aperture size of 20-60 μm to further remove fat and cells. The dilution step involves diluting the filtered milk by adding an equal volume of distilled water. The isoelectric precipitation step involves adjusting the pH to 4-5 by adding acid to the diluted milk, thereby precipitating casein in the milk. A second centrifugation step is performed to further centrifuge the milk in which casein has precipitated, and the supernatant is collected. The second centrifugation step is carried out at 4000-6000 x g for 15-30 minutes at room temperature. and The filtration step involves filtering the collected supernatant through a 0.2 μm filter.

6. The use of claim 5, wherein the milk is raw milk, commercial milk, or colostrum.

7. The use of milk-derived exosomes in the preparation of functional shampoos for reducing hair loss. The exosomes are prepared by the following method, the method comprising: The first centrifugation step removes fat and cells from the milk by centrifugation, wherein the first centrifugation step is carried out at a temperature of 4°C for 20-40 minutes at 4,000-6,000 x g. The filter step involves filtering the centrifuged milk using a filter with a sieve aperture size of 20-60 μm to further remove fat and cells. The dilution step involves diluting the filtered milk by adding an equal volume of distilled water. The isoelectric precipitation step involves adjusting the pH to 4-5 by adding acid to the diluted milk, thereby precipitating casein in the milk. A second centrifugation step is performed to further centrifuge the milk in which casein has precipitated, and the supernatant is collected. The second centrifugation step is carried out at 4000-6000 x g for 15-30 minutes at room temperature. and The filtration step involves filtering the collected supernatant through a 0.2 μm filter.

8. The use of claim 7, wherein the milk is raw milk, commercial milk, or colostrum.

9. Use of milk-derived exosomes in the preparation of pharmaceutical compositions for treating wounds. The exosomes are prepared by the following method, the method comprising: The first centrifugation step removes fat and cells from the milk by centrifugation, wherein the first centrifugation step is carried out at a temperature of 4°C for 20-40 minutes at 4,000-6,000 x g. The filter step involves filtering the centrifuged milk using a filter with a sieve aperture size of 20-60 μm to further remove fat and cells. The dilution step involves diluting the filtered milk by adding an equal volume of distilled water. The isoelectric precipitation step involves adjusting the pH to 4-5 by adding acid to the diluted milk, thereby precipitating casein in the milk. In the second centrifugation step, the milk in which casein has precipitated is further centrifuged, and the supernatant is collected. The secondary centrifugation step is carried out at room temperature at 4000-6000 x g for 15-30 minutes; and The filtration step involves filtering the collected supernatant through a 0.2 μm filter.

10. The use of claim 9, wherein the milk is raw milk, commercial milk, or colostrum.

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