Agent for activating dermal fibroblast functions and cosmetic products including the same

Abstract

To provide an agent for activating dermal fibroblast functions that is highly safe and can be used safely over a long period, and cosmetic products including the function activating agent.SOLUTION: The present invention provides an agent for activating dermal fibroblast functions, and a cosmetic product including the function activating agent, each of which comprises DNA hydrolysates and extracellular vesicles as active ingredients. The active ingredients have the effect of activating dermal fibroblast functions, potentially preventing or improving skin aging symptoms such as reduced firmness and elasticity, wrinkles, and sagging.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an agent for enhancing the function of dermal fibroblasts, and more specifically, to an agent for enhancing the function of dermal fibroblasts that contains a DNA hydrolysate and extracellular vesicles as active ingredients, and a cosmetic product containing the same. [Background technology]

[0002] Skin can be broadly divided into the epidermis, which is in contact with the outside world, the dermis, which is located inside the epidermis and strongly adheres to the epidermis, and the subcutaneous adipose tissue, which is located further inside and between the dermis and muscle. The dermis, which is located between the epidermis and the subcutaneous adipose tissue, is composed of the papillary layer, the reticular dermis, and connective fibrous tissue. Approximately 70% of the dermis is collagen, and the rest is composed of extracellular matrix components such as elastic fibers (elastin) and hyaluronic acid. The collagen, elastin, hyaluronic acid, etc. that make up the dermis are produced by dermal fibroblasts present in the reticular dermis.

[0003] Generally, these fibers are destroyed by ultraviolet rays, dryness, aging, etc., and elasticity decreases as they age, and the production function of extracellular matrix components is reduced, which is considered to be the cause of skin aging such as skin tension and elasticity reduction, wrinkles, and sagging.Thus, the reduction of extracellular matrix components in the dermis, especially collagen and hyaluronic acid, is involved in aging phenomena such as skin elasticity reduction and wrinkle formation.Therefore, it is considered that skin aging can be prevented or improved by activating the function of dermal fibroblasts to increase the production of the above-mentioned extracellular matrix components.

[0004] Deoxyribonucleic acid (DNA), which is used here as a safe and effective functional food, has been reported to have the effect of improving skin conditions (moisture content, sebum content, skin groove density, wrinkles, age spots, freckles, etc.) (Patent Documents 1 and 2).

[0005] On the other hand, it has been reported that human stem cell culture medium containing extracellular vesicles such as exosomes has various useful properties such as whitening, wrinkle improvement, and anti-aging (Non-Patent Document 1). It has also been reported that exosomes derived from kale promote collagen production in skin fibroblasts (Patent Document 3).

[0006] However, to the best of the inventors' knowledge, there have been no reports on the interaction between these DNA (deoxyribonucleic acid) and extracellular vesicles. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] JP 2007-291062 A [Patent Document 2] JP 2008-63315 A [Patent Document 3] Patent Publication No. 2022-92330 [Non-patent literature]

[0008] [Non-Patent Document 1] "The Beauty Power of Human Stem Cell Culture Fluid" Nutrient Library Co., Ltd., published on March 26, 2020 Summary of the Invention [Problem to be solved by the invention]

[0009] As described above, if the function of dermal fibroblasts is activated to increase the production of extracellular matrix components, particularly collagen and hyaluronic acid, it is expected that skin aging can be prevented or improved. Therefore, the object of the present invention is to provide a dermal fibroblast function enhancer that has a superior dermal fibroblast activation effect, is highly safe, and can be used safely for a long period of time, and a cosmetic product containing the same. [Means for solving the problem]

[0010] As a result of intensive research into the above-mentioned problems, the inventors have found that DNA hydrolysates (hereinafter sometimes referred to as "hydrolyzed DNA"), which are safe even when taken over long periods of time, and extracellular vesicles significantly enhance the activating effect on dermal fibroblasts, i.e., synergistically enhance the collagen production-promoting effect and hyaluronic acid production-promoting effect, compared to when each is used alone. The present invention has been achieved based on these findings. That is, the present invention is as follows.

[0011] (1) A function-activating agent for dermal fibroblasts, comprising a DNA hydrolysate and extracellular vesicles as active ingredients, characterized in that the function activation is either one or both of a collagen production promoting effect or a hyaluronic acid production promoting effect. (2) The functional enhancer described in (1) above, wherein the DNA hydrolysate contains 10 to 80% of a fraction having a molecular weight of 12,000 or less, and the extracellular vesicles are derived from mesenchymal stem cells. (3) A cosmetic comprising the functional enhancer according to (1) or (2). Effect of the Invention

[0012] According to the present invention, the active ingredients, hydrolyzed DNA and extracellular vesicles, have the effect of significantly synergistically enhancing the activation action on dermal fibroblasts (hereinafter sometimes referred to as "synergistic effect"), and therefore anti-aging effects of the skin, i.e., prevention or improvement of skin aging such as loss of skin firmness and elasticity, wrinkles, and sagging, can be expected. [Brief description of the drawings]

[0013] [Figure 1] 1 shows the results of gel permeation chromatography (GPC) analysis of the hydrolyzed DNA-Na used in the Examples, in which the horizontal axis represents retention time (min) and the vertical axis represents absorbance in the ultraviolet region (wavelength 260 nm). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] The following describes in detail an embodiment of the present invention, but the following description is merely an example of an embodiment of the present invention, and the present invention is not limited to the following description. In this specification, the expression "to" is used to include the numerical values ​​or physical properties before and after the expression. In addition, the content (%) of the active ingredient is mass percent (wt%) unless otherwise specified.

[0015] The functional enhancer of the present invention contains hydrolyzed DNA and extracellular vesicles as active ingredients, where the hydrolyzed DNA is an ingredient that can be prepared, for example, by hydrolyzing DNA extracted from animals or plants, and the extracellular vesicles are, for example, extracellular vesicles such as exosomes and microvesicles derived from animal or plant cells.

[0016] Here, the term "activating effect on dermal fibroblasts" means either or both of the effect of promoting collagen production or the effect of promoting hyaluronic acid production by dermal fibroblasts, and "activating the function of dermal fibroblasts" means the same thing.

[0017] The agent for activating dermal fibroblast function of the present invention contains hydrolyzed DNA and extracellular vesicles as active ingredients, and may further contain other optional ingredients as necessary.

[0018] The hydrolyzed DNA in the present invention is a component that can be prepared by hydrolyzing DNA. The source of DNA may be various organisms such as animals, plants, and microorganisms, and synthetic DNA may also be used as DNA. Among these, it is particularly preferable to use DNA derived from the testes (milt) of fish such as salmon, trout, and cod, from the viewpoints of containing a large amount of DNA and being able to effectively utilize waste from fishery processing.

[0019] DNA can be extracted and purified from fish testes by a conventional method (for example, according to the description in JP 2005-245394 A). For example, fish testes are roughly crushed, and the roughly crushed fish testes are treated with a protease under conditions that do not degrade DNA. Alcohol (methanol, ethanol, isopropyl alcohol, etc.) is added to the enzyme-treated solution to precipitate DNA as a DNA salt (DNA sodium salt), and the precipitate is collected. Alternatively, an acid (hydrochloric acid, phosphoric acid, citric acid, etc.) is added to the enzyme-treated solution to precipitate DNA, and the precipitate is collected, neutralized with sodium hydroxide, and dried to obtain a DNA salt (DNA sodium salt).

[0020] The resulting DNA salt can be hydrolyzed with a nucleic acid degrading enzyme such as a nuclease to obtain a hydrolyzed DNA. For example, a nuclease derived from Penicillium can be used as the nuclease used in the hydrolysis treatment.

[0021] Hydrolysis can be carried out, for example, by adding the above DNA salt (DNA sodium salt) as a raw material to warm water adjusted to about 65°C, stirring, and then heating to 70°C, and adding nuclease to carry out the reaction. The temperature during hydrolysis is preferably 60 to 75°C, and more preferably 70°C.

[0022] The obtained hydrolysis product can be freeze-dried to obtain hydrolyzed DNA in powder form. The hydrolyzed DNA obtained by the above method can usually be obtained in the form of a sodium salt. The salt is not limited to the sodium salt, and may be, for example, a potassium salt or a calcium salt. The hydrolyzed DNA may be in a free form instead of a salt.

[0023] The hydrolyzed DNA preferably contains 10 to 80% of a fraction having a molecular weight of 12,000 or less, and more preferably contains 10 to 80% of a fraction having a molecular weight of 5,000 or less. The molecular weight distribution can be measured by fractionating a sample based on molecular weight using gel permeation chromatography (GPC) and then quantifying the sample using a UV detector.

[0024] In the present invention, extracellular vesicles refer to "nucleated (non-replicable) lipid bilayer-enclosed particles released from cells" as defined by the International Society for Extracellular Vesicles (ISEV). Extracellular vesicles are known to be produced in various cells, such as microorganisms, plants, and animals, and the properties of the extracellular vesicles they produce also vary depending on the cells of origin and the conditions at the time of production. In addition, extracellular vesicles may be those isolated from body fluids, milk such as milk, fruit juice, etc.

[0025] Furthermore, extracellular vesicles may be natural vesicles that are released (secreted) from cells as they are, or modified vesicles that have been modified. Modified vesicles include, for example, vesicles whose contents have been changed by gene transfer into the source cells, vesicles whose surfaces have been modified after secretion (release) from cells, vesicles whose surfaces have been modified with peptides or antibodies, and vesicles whose surfaces have been modified with specific pharmaceuticals.

[0026] Extracellular vesicles are classified as follows based on differences in their production mechanisms (Doujin News Vol. 169 Reviews: Characteristics and Functions of Extracellular Vesicles and Exosomes). (1) Exosomes: small vesicles approximately 50 to 150 nm in size derived from the endosomal membrane that are formed during endocytosis. (2) Microvesicles: Small vesicles approximately 100 to 1,000 nm in size that bud directly from the cell membrane and are secreted outside the cell. (3) Apoptotic vesicles: vesicles of about micrometer size that bud from the membrane of cells undergoing apoptosis (they are easily distinguished from other vesicles because they are separated by low-speed centrifugation at about 2,000 × g).

[0027] Extracellular vesicles may generally be nanometer to micrometer in size. In particular, (1) exosomes and (2) microvesicles have common components and sizes and are difficult to completely separate, but a size of about 50 to 1,000 nm is preferable, and a size of about 50 to 150 nm is more preferable.

[0028] The cellular vesicles of the present invention preferably do not contain (3) apoptotic vesicles, and more preferably consist essentially of (1) exosomes, or may contain (1) exosomes and (2) microvesicles.

[0029] The type of cells that can be used in the present invention is not particularly limited as long as they release (secrete) extracellular vesicles. As described above, extracellular vesicles are known to be produced in various cells such as microorganisms, plants, and animals, and the properties of the extracellular vesicles produced by them also vary depending on the cells from which they are derived and the conditions during production. Therefore, in the present invention, extracellular vesicles produced (released or secreted) from any type of cell can be used as long as they have a synergistic effect when used in combination with the hydrolyzed DNA described above.

[0030] Examples of microbial cells that can be used include lactic acid bacteria, yeast, bacteria, etc. Examples of lactic acid bacteria include Bacillus coagulans, Leuconostoc mesenteroides, and Lactobacillus paracasei.

[0031] Examples of yeast include Hanseniaspora vineae, Kloeckera apiculata, Saccharomyces cerevisiae, and Saccharomyces paradoxus.

[0032] Examples of bacteria include gram-negative bacteria such as Escherichia coli and gram-negative bacteria such as Pantoea agglomerans.

[0033] In the present invention, the plant cells may be of any species, and may be either living plant cells or cultured cells, or may be seeds or fruits (eg, acerola fruit, etc.).

[0034] In the present invention, cultured animal cells are usually used. The cultured cells may be cultured somatic cells or cultured germ cells.

[0035] Examples of somatic cells include hepatocytes, pancreatic cells, muscle cells, bone cells, osteoblasts, osteoclasts, chondrocytes, adipocytes, skin cells, fibroblasts, pancreatic cells, kidney cells, lung cells, cardiac muscle cells, synovial cells, lymphocytes, erythrocytes, leukocytes, monocytes, macrophages, and megakaryocytes, as well as diseased cells thereof. Specific examples of somatic cells include those selected from the group consisting of human osteoblasts, human chondrocytes, human cardiac muscle cells, human fibroblasts, human synovial cells, human hepatocytes, and diseased cells thereof.

[0036] Examples of reproductive cells include gametes for sexual reproduction, ie, eggs, egg cells, sperm, and sperm cells, and spores for asexual reproduction.

[0037] In the present invention, pluripotent stem cells are preferably used as cultured cells. Pluripotent stem cells are a general term for stem cells that have the ability to differentiate into cells of any tissue (differentiation pluripotency). Pluripotent stem cells include, for example, embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), and ntES cells (nuclear transfer embryonic stem cells). Among these, ES cells or iPS cells are preferred.

[0038] In the present invention, tissue stem cells are preferably used as cultured cells. Tissue stem cells refer to stem cells whose cell lineages are limited to specific tissues, but which have the ability to differentiate into various cell types (multipotency). Examples of tissue stem cells include neural stem cells, epithelial stem cells, cardiac muscle cells, hepatic stem cells, pancreatic stem cells, gastrointestinal epithelial stem cells, germline stem cells, skeletal muscle cells, hematopoietic stem cells, and mesenchymal stem cells. Among these, mesenchymal stem cells are preferred.

[0039] In the present invention, the cultured cells may be sarcoma cells, cell lines, transformed cells, etc. Here, "sarcoma" refers to cancer occurring in connective tissue cells derived from non-epithelial cells such as bone, cartilage, fat, muscle, and blood, and sarcoma cells refer to cells derived from such sarcomas. "Cell lines" refer to cultured cells that have been maintained outside the body for a long period of time, have acquired certain stable properties, and can be subcultured semi-permanently. "Transformed cells" refer to cells whose genetic properties have been changed by introducing nucleic acid (e.g., DNA) from outside the cell.

[0040] Specifically, examples of "cell lines" include cell lines derived from various tissues of various species including humans, such as PC12 cells (derived from rat adrenal medulla), CHO cells (derived from Chinese hamster ovary), HEK293 cells (derived from human fetal kidney), HL-60 cells (derived from human white blood cells), HeLa cells (derived from human cervical cancer), Vero cells (derived from African green monkey kidney epithelial cells), MDCK cells (derived from canine kidney tubular epithelial cells), and HepG2 cells (derived from human hepatic cancer).

[0041] These cultured cells are preferably free of aging or disease. In addition, extracellular vesicles, especially exosomes, are known to be actively produced in undifferentiated cells such as stem cells and cancer cells, and it is more preferable to use stem cells, such as mesenchymal stem cells, as the cultured cells in the present invention.

[0042] Mesenchymal stem cells are a type of bone marrow stromal cell that has non-hematopoietic adhesive and self-replicating properties and is isolated as a cell population that can differentiate into fat, bone, and cartilage in a test tube (Web Dictionary of Biomedical Engineering). Mesenchymal stem cells can be derived not only from bone marrow, but also from tissues such as fat, blood, umbilical cord, umbilical cord blood, periosteum, perichondrium, and cartilage. At present, any mesenchymal stem cell that meets the following three criteria proposed by the International Society for Cell Therapy is acceptable (Drug Delivery System 29-2, 2014).

[0043] (1) Ability to adhere to plastic plates (2) Surface antigens CD105, CD73, and CD90 are positive, and CD45, CD34, CD14 or CD11b, CD79a or CD19, and HLA-DR are negative. (3) Ability to differentiate into osteoblasts, chondrocytes, and adipocytes in vitro

[0044] The cells may be isolated and cultured according to their properties, for example, by using the expression of surface antigens or the like as an indicator, using a known method that is commonly used.

[0045] Cultured cells and microorganisms are deposited at depository centers, such as the RIKEN BioResource Research Center and the American Type Culture Collection (ATCC). TM Cells listed in the catalog of the National Institute of Technology and Evaluation (NITE) Biotechnology Center (NBRC), etc., or deposited at these depository centers, may also be used. Furthermore, commercially available cells may also be purchased and used.

[0046] In the present invention, the cultured cells used for producing extracellular vesicles are preferably mesenchymal stem cells, and are preferably cells that have been cultured with appropriate removal of reactive oxygen species during culture, i.e., cells in which the intracellular glutathione concentration is appropriately maintained.

[0047] Examples of the mesenchymal stem cells that can be used include human bone marrow mesenchymal stem cells, human umbilical cord-derived mesenchymal stem cells, human embryonic stem cell derived mesenchymal stem cells, etc. Among these, human umbilical cord-derived mesenchymal stem cells are particularly preferred.

[0048] Extracellular vesicles can be obtained by recovering (separating and purifying) those released (secreted) from cells using a commonly known method. Examples of recovery methods include ultracentrifugation, ultrafiltration, density gradient centrifugation, polymer precipitation, and immunoprecipitation. These methods may be combined appropriately depending on the type of cells and the properties of the vesicles to be recovered.

[0049] Here, the ultracentrifugation method is a method of separating extracellular vesicles by ultracentrifugation at 100,000 to 200,000 × g after removing cell debris by low-speed centrifugation. The ultrafiltration method is a method of classifying extracellular vesicles using a filter according to the target size. The density gradient centrifugation method is a method of purifying the crude fraction obtained by ultracentrifugation by further applying it to a density gradient. The polymer precipitation method is a method of separating extracellular vesicles by low-speed centrifugal precipitation using a polymer. The immunoprecipitation method is a method of separating extracellular vesicles by an affinity method for marker proteins or lipids of extracellular vesicles.

[0050] More specifically, for example, extracellular vesicles of microorganisms can be recovered by the above-mentioned method after pretreating the culture supernatant, if necessary, by mixing it with an appropriate liquid.

[0051] Plant extracellular vesicles can be recovered by the above-mentioned method, for example, by pretreating the juice of a plant or fruit, if necessary, by mixing it with an appropriate liquid (for example, phosphate-buffered saline).

[0052] When producing animal extracellular vesicles, as described above, appropriate management is required to prevent contamination by mycoplasma, viruses, endotoxins, medium components, etc. during the production process, in addition to ensuring the health and safety of the cells themselves.

[0053] First, the cells are cultured until they are about 80% confluent, then cultured in an appropriate medium for producing extracellular vesicles, for example, a medium that does not contain components such as phenol red or serum, to release (secrete) the extracellular vesicles into the medium, and the medium is recovered. If necessary, the cells and cell debris are further removed, and the extracellular vesicles of the desired size may be separated and purified, for example, by ultracentrifugation, ultrafiltration, or a combination thereof, as described above. Furthermore, if necessary, the cells may be powdered by concentration or drying, or appropriate additives for storage may be added.

[0054] Furthermore, extracellular vesicles are commercially available as products suited to the intended purpose and application, and these may be obtained and used.

[0055] In the present invention, the content of the active ingredient in the functional enhancer varies depending on the type and form of the preparation, the purpose of use, the frequency of use, etc., and it is difficult to uniformly specify it. The content and content ratio of each ingredient are not particularly limited, but it is preferable that the content and content ratio are such that they have a synergistic effect on the activation of dermal fibroblasts.

[0056] Specifically, for example, when the functional enhancer is applied to the skin (when the functional enhancer is a cosmetic), the following applies: The lower limit of the content of hydrolyzed DNA is preferably 0.001% (0.01 mg / mL) or more, more preferably 0.01% (0.1 mg / mL) or more, and the upper limit is not particularly limited but is preferably 1% (10 mg / mL) or less, more preferably 0.5% (5 mg / mL) or less, and more preferably 0.2% (2 mg / mL) or less.

[0057] The content of extracellular vesicles is preferably 0.00001×10 as the total protein content. -3 % (0.0001 μg / mL) or more, more preferably 0.0001 × 10 -3 % (0.001 μg / mL) or more, more preferably 0.00015 × 10 -3 % (0.0015 μg / mL) or more, and although there is no particular upper limit, it is preferably 0.1 × 10 -3 % (5 μg / mL) or less, preferably 1 μg / mL or less.

[0058] In addition, the lower limit of the total number of extracellular vesicles is preferably 1 × 10 3 / mL or more, more preferably 1×10 4 / mL or more, more preferably 1 × 10 5 The upper limit is not particularly limited, but is preferably 5x10 10 / mL or less, preferably 0.5x10 8 cells / mL or less.

[0059] In addition, there is no particular limitation on the content ratio of hydrolyzed DNA to extracellular vesicles, but the mass ratio of hydrolyzed DNA to proteins contained in the extracellular vesicles is preferably hydrolyzed DNA:extracellular vesicles=1:10. -8 ~1:10 -1 is preferred, 1:10 -7 ~1:10 -2 is more preferred.

[0060] When the content and content ratio of hydrolyzed DNA and extracellular vesicles are within the preferred ranges, this is advantageous in that a remarkable synergistic effect is exerted on the activation of dermal fibroblasts (promotion of collagen production and promotion of hyaluronic acid production), as specifically shown in the examples described below.

[0061] Here, the synergistic effect in the activation of dermal fibroblasts means that when hydrolyzed DNA and extracellular vesicles are used in combination with dermal fibroblasts, either the collagen production rate or the hyaluronic acid production rate is greater (significantly greater) than the sum of the levels when each is used alone. Also, a significant effect means an effect that is significantly different when compared to a negative control.

[0062] The functional enhancer of the present invention can be produced in a desired dosage form by mixing the hydrolyzed DNA, the extracellular vesicles, and, if necessary, other components in a conventional manner. Here, the other components include the same optional components that may be contained in the cosmetic product of the present invention, which will be described later.

[0063] The cosmetic product of the present invention comprises a skin fibroblast function enhancer. Here, "comprises" means that the cosmetic product may contain optional components such as a physiologically acceptable carrier according to the desired product form and other auxiliary components that can be used in combination.

[0064] The cosmetic product of the present invention can be provided, for example, as a basic cosmetic product, a hair cosmetic product to be applied to the scalp and hair, etc. In the present invention, the cosmetic product includes not only cosmetics defined by the Pharmaceutical Affairs Law, but also quasi-drugs.

[0065] When the functional enhancer of the present invention is provided as a cosmetic product, the formulation that can be used is not particularly limited as long as it is applicable to the skin. Specifically, it can be provided in the form of a liquid, emulsion, gel, cream, ointment, foam, mist, aerosol, etc., such as a lotion, emulsion, cream, gel, jelly, essence, lip cream, pack, mask, etc.

[0066] There are no particular limitations on the other optional components that may be contained in these cosmetics, and additives that may be incorporated into ordinary cosmetics may be used. Examples of such additives include water, oils and fats, waxes, hydrocarbons, fatty acids, alcohols, esters, surfactants, fragrances, astringents, germicides and antibacterial agents, whitening agents, UV absorbers, moisturizers, cell activators, anti-inflammatory and anti-allergic agents, antioxidants, vitamins, natural extracts, etc. The content of these other optional components is also not particularly limited, and may be appropriately selected depending on the desired formulation, etc.

[0067] The present invention will be described in more detail below with reference to examples, but the technical scope of the present invention is not limited to these examples. EXAMPLES

[0068] [Example 1] Cell activation effect of dermal fibroblasts 1. Purpose and Overview of the Study It is believed that collagen produced by dermal fibroblasts plays an important role in skin firmness, and hyaluronic acid produced by fibroblasts plays an important role in skin flexibility and moisture. Therefore, we evaluated the collagen and hyaluronic acid production promoting effects of the test products (hydrolyzed DNA-Na and extracellular vesicles derived from mesenchymal stem cells) using dermal fibroblasts.

[0069] 2 Materials and test methods 2-1 cells This test was performed using NB1RGB cells (RIKEN BRC, Japan), a dermal fibroblast cell line derived from a human neonate, which were cultured in a CO2 incubator (CO2 concentration 5%, 37°C).

[0070] 2-2 Culture medium Eagle's Minimal Essential Medium (EMEM, Cat No. 051-07615, Wako, Japan) containing 10.0% (v / v) Fetal Bovine Serum FBS (Cat No. SH30071.03, Hyclone, UK) and 1.0% (v / v) antifungal agent (Antibiotic-Antimycotic 100X, Cat No. 15240-062, Invitrogen, USA) was used.

[0071] 2-3 Test items 2-3-1 Hydrolyzed DNA-Na (oligo DNA) The test product used was a 10% aqueous solution of hydrolyzed DNA-Na (product name) (10% aqueous solution of oligo DNA) manufactured by Nissei Bio Co., Ltd. This product was prepared by extracting the Na salt of DNA from the testes (milt) of salmonid fish using the method described above and hydrolyzing it. The results of gel permeation chromatography (GPC) analysis of the hydrolyzed DNA-Na used (manufactured by Nissei Bio Co., Ltd.) are shown in Figure 1, and the relationship between retention time and molecular weight is shown in Table 1. The molecular weight guideline of 12,000 was based on the retention time of Cytochrome C (MW 12,400).

[0072] [Table 1]

[0073] From the above analysis results, the molecular weight fractions of hydrolyzed DNA-Na manufactured by Nissei Bio Co., Ltd. are as follows: Fraction with molecular weight of 12,000 to 5,000 (elution time 20 to 25 minutes): 32.0% Fraction with molecular weight of 5,000 or less (elution time 25 minutes or later): 32.4% From the above, the fraction of hydrolyzed DNA-Na manufactured by Nissei Bio having a molecular weight of 12,000 or less is 64.4%.

[0074] The test product (10% aqueous solution of oligo DNA) was diluted with EMEM containing 0.5% FBS to prepare three concentrations (0.1%, 0.5%, 1%) at the time of use. The final concentrations of DNA-Na were 0.01% (0.1mg / ml), 0.05% (0.5mg / ml), and 0.1% (1mg / ml), respectively. EMEM containing 0.5% FBS was used as a control.

[0075] 2-3-2 Human umbilical cord mesenchymal stem cell-derived extracellular vesicles (ExoVESICLE) The test product used was ExoVESICLE (product name) manufactured by cell2in Inc. This product is made by culturing human umbilical cord-derived mesenchymal stem cells (prepared from umbilical cord samples received from the Department of Obstetrics and Gynecology at Seoul National University Hospital) until they become approximately 80% confluent, culturing them in a medium that does not contain phenol red or serum to release (secrete) extracellular vesicles into the medium, removing cells and cell debris by centrifugation, and separating them through filter filtration and ultrafiltration.

[0076] The pH of this product is 7.6, the total protein content is 30 μg / ml, and the number of vesicles (particles) is 9 × 10 8 The particle size distribution is 115.8 nm, the mean, and the median of the particle size distribution are 106.5 nm.

[0077] The test product (ExoVESICLE) was diluted with EMEM containing 0.5% FBS to prepare three concentrations (0.01%, 0.1%, 0.2%) at the time of use. The final concentrations calculated as protein amount were 0.003μg / ml, 0.03μg / ml, and 0.06μg / ml, respectively. EMEM containing 0.5% FBS was used as a control.

[0078] 2-3-3 Mixture of Oligo DNA and ExoVESICLE The test product (oligo DNA + ExoVESICLE) was diluted with EMEM containing 0.5% FBS to prepare three concentrations (1 + 0.01%, 1 + 0.1%, 1 + 0.2%) at the time of use. The final concentration of DNA-Na was 0.1% (1mg / ml), and the final concentrations of ExoVESICLE (protein equivalent) were 0.003μg / ml, 0.03μg / ml, and 0.06μg / ml, respectively. EMEM containing 0.5% FBS was used as a control.

[0079] 2-4 Test configuration For the calculation of cell proliferation, collagen production, and hyaluronic acid production, three wells of a 96-well plate (cell proliferation: Cat No. 3860-096, Iwaki, Japan; collagen production and hyaluronic acid production: Cat No. 3855, Thermo Scientific, USA) were used per treatment group. In addition, one test group and one control group were set up per plate for the test. All operations related to the test, including the preparation of the test products, were carried out at room temperature unless otherwise specified.

[0080] 2-5 Test operation 2-5-1 Cell culture 1.0 x 10 in a 96-well plate 4 NB1RGB cells were seeded at a density of 1000 cells / well and cultured for 24 hours in a CO2 incubator. To prevent drying during culture, 200 μL of phosphate buffer saline (PBS(-), Cat No. 198601, Nissui, Japan) was added to wells not used for the test.

[0081] After 24 hours, the medium was removed, and 100 μL of the test product and control were added to the 96-well plate, followed by incubation in a CO2 incubator for 48 hours.

[0082] After 48 hours, the culture supernatant was dispensed into a new 96-well plate and frozen (-80℃). The collagen and hyaluronic acid amounts in the culture supernatant were measured by Enzyme-Linked Immunosorbent Assay (ELISA) using the methods shown in 2-5-3 and 2-5-4, and the collagen and hyaluronic acid production promoting effect of the test product was evaluated. In addition, the number of cells in the 96-well plate from which the culture supernatant was removed was evaluated using the method shown in 2-5-2, and the cell proliferation effect of the test product was evaluated.

[0083] 2-5-2 Cell proliferation evaluation The effect of the test product on the proliferation of dermal fibroblasts was evaluated as follows. The 96-well plate from which the medium had been removed was washed with 100 μL of PBS(-). Next, 100 μL of 0.5 mg / mL 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, CAS No. 298-93-1, Sigma-Aldrich, USA) solution was added and incubated in a CO2 incubator for 2 hours.

[0084] After removing the MTT solution and washing with 100 μL of PBS(-), 200 μL of 2-propanol (CAS No. 67-63-0, Wako, Japan) was added to dissolve the insoluble formazan. After uniformly dispersing the dye in the 96-well plate, the plate was read using a microplate reader (SPARK TM The absorbance (OD) at 570 nm was measured using a 10M filter (TECAN, Switzerland). 570 ) was measured.

[0085] OD of the control group 570 The OD of the test sample-added group is taken as 100%. 570 That is, the effect of cell proliferation was calculated as the cell proliferation effect (%) of the test product.

[0086] 2-5-3 Evaluation of collagen production promotion effect The effect of the test product on the production of type I collagen was evaluated by direct ELISA as follows: 100 μL of the culture supernatant diluted 4-fold with PBS(-) was added to a high-adsorption 96-well plate and incubated overnight at 4°C. Type I collagen solution (Cat No. 009-001-103, RCK, USA) was used as a standard substance.

[0087] The solidified culture supernatant was removed, and the microplate was washed with 200 μL of PBS(-) containing 0.05% Tween 20 (PBS-T, Tween 20: CAS No. 9005-64-5, Sigma-Aldrich, USA). 150 μL of 1% Bovine Serum Albumin (BSA, Cat No. PRL68700-50G, Proliant, USA) solution was added, and the plate was incubated at 37°C for 1 hour.

[0088] The BSA solution was removed, and the plate was washed with 200 μL of PBS-T. Then, 100 μL of a 100 ng / mL biotin-labeled Collagen Type I Antibody (Cat No. 600-406-103, ROCKLAND, USA) solution prepared in PBS(-) containing 0.5% BSA was added, and the plate was incubated at 37°C for 1 hour.

[0089] After removing the antibody solution and washing with 200 μL of PBS-T, 100 μL of Streptavidin-HRP (Cat No. CJ30H-1, Agilent Technologies, USA, 1:10000) solution prepared in 0.5% BSA-containing PBS(-) was added and incubated at room temperature for 30 minutes. Next, the Streptavidin-HRP solution was removed, washed with 200 μL of PBS-T, and 100 μL of 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonicacid) diammonium salt (ABTS, Cat No. 5110-0010, KPL, USA) solution was added to confirm color development. After homogenizing the dye in the 96-well plate, the absorbance (OD) at 405 nm was measured using a microplate reader. 405) was measured.

[0090] OD of the control group 405 The collagen production rate of the test product was calculated based on the OD of the control and the test product. 405 was measured using OD 2-5-2 (cell proliferation evaluation). 570 The collagen production rate per cell of the test product was calculated by dividing the value by 1. The collagen production rate per cell of the control group was set to 100%.

[0091] 2-5-4 Evaluation of hyaluronic acid production promotion effect The effect of the test product on the production amount of hyaluronic acid, which is said to hold water between collagen and elastin, was evaluated by sandwich ELISA as follows.

[0092] 100 μL of hyaluronan binding protein (HABP, Cat No. BC40, Hokudo, Japan, 1:5500) solution prepared with PBS was added to a high-adsorption 96-well plate and incubated overnight at 4°C.

[0093] The immobilized HABP solution was removed, and the plate was washed with 200 μL of PBS-T solution. Then, 150 μL of 1% BSA solution was added, and the plate was incubated at room temperature for 1 hour. After removing the BSA solution and washing with 200 μL of PBS-T, 100 μL of culture supernatant diluted 100-fold with PBS(-) was added and incubated at room temperature for 1 hour. Sodium hyaluronate (Cat No. 087-04511, Wako, Japan) was used as a standard substance.

[0094] The culture supernatant was removed, and the cells were washed with 200 μL of PBS-T. Then, 100 μL of a solution of biotin-labeled HABP (Cat No. BC41, Hokudo, Japan, 1:2000) prepared in PBS(-) containing 0.5% BSA was added, and the cells were allowed to stand overnight at 4°C.

[0095] After removing the biotin-labeled HABP solution and washing with 200 μL of PBS-T, 100 μL of Streptavidin-HRP solution (1:10000) prepared in PBS(-) containing 0.5% BSA was added and left to stand at room temperature for 30 minutes. After removing the Streptavidin-HRP solution and washing with 200 μL of PBS-T, 100 μL of ABTS solution was added and color development was confirmed.

[0096] After homogenizing the dye in the 96-well plate, the optical density at 405 nm (OD405) was measured using a microplate reader.

[0097] OD of the control group 405 The hyaluronic acid production rate of the test product was calculated based on the OD405 of the control and test products measured in 2-5-2 (evaluation of cell proliferation). 570 The value divided by was calculated as the hyaluronic acid production rate per cell. The hyaluronic acid production rate per cell of the test product was calculated by taking the hyaluronic acid production rate per cell of the control group as 100%.

[0098] 3. Significance test For each study, significance was tested between the control and test-product-added groups using unpaired t-tests, with the significance level set at less than 5% for both sides.

[0099] 4. Test results 4-1 Oligo DNA There was no significant change in cell proliferation at any concentration. On the other hand, the collagen production rate and hyaluronic acid production rate per cell were significantly increased compared to the control (Table 2). This result suggests that oligo-DNA enhances collagen production and hyaluronic acid production in fibroblasts.

[0100] 4-2 ExoVESICLE There was no significant change in cell proliferation at any concentration. On the other hand, the hyaluronic acid production rate per cell was significantly increased compared to the control (Table 2). These results suggest that ExoVESICLE enhances hyaluronic acid production in fibroblasts.

[0101] 4-3 Combination of Oligo DNA and ExoVESICLE There was no significant change in cell proliferation at any concentration. On the other hand, the collagen production rate and hyaluronic acid production rate per cell were significantly increased compared to the control (Table 2). These results suggest that the combined use of oligo DNA and ExoVESICLE significantly increases collagen production and hyaluronic acid production in fibroblasts.

[0102] [Table 2]

[0103] From Table 2, it can be seen that the collagen production rate and hyaluronic acid production rate per cell when oligo-DNA (hydrolyzed DNA-Na) and ExoVESICLE (extracellular vesicles derived from human umbilical cord mesenchymal stem cells) were used in combination was significantly greater than the sum of the rates when each was used alone. This confirms the synergistic effect of oligo-DNA and ExoVESICLE on the functional activation of dermal fibroblasts (promoting collagen production and hyaluronic acid production).

Claims

1. A function activating agent for dermal fibroblasts, comprising a DNA hydrolysate and extracellular vesicles as active ingredients, characterized in that the function activation is either one or both of a collagen production promoting effect or a hyaluronic acid production promoting effect.

2. The function enhancer according to claim 1, wherein the DNA hydrolysate contains 10 to 80% of a fraction having a molecular weight of 12,000 or less, and the extracellular vesicles are derived from mesenchymal stem cells.

3. A cosmetic comprising the functional enhancer according to claim 1 or 2.

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