KR101336474B1 - Cosmetic Composition Comprising Nano Capsule or Nanofiber Containing Cultured Medium of Adipose-derived Adult Stem Cells and Manufacturing Method Thereof - Google Patents

Abstract

The present invention relates to a cosmetic composition and a method for producing the same, and more particularly, to a cosmetic composition comprising a nanofiber or nanocapsules of fat-derived adult stem cell culture medium and a method for producing the same.

Description

Cosmetic composition comprising nanofibers or nanocapsules of adult stem cell cultures derived from adipose-derived cultures and a method for preparing the same (Cosmetic Composition Comprising Nano Capsule or Nanofiber Containing Cultured Medium of Adipose-derived Adult Stem Cells and Manufacturing Method Thereof)

The present invention relates to a cosmetic composition and a method for producing the same, and more particularly, to a cosmetic composition comprising a nanofiber or nanocapsules of fat-derived adult stem cell culture medium and a method for producing the same.

 Stem cells are undifferentiated cells that can divide and self-renewal for a long time, and can differentiate into various cell types given a certain condition. Stem cells are classified into embryonic stem cells and adult stem cells according to the origin of the tissue. Potential has limited disadvantages than embryonic stem cells, but many therapeutic agents have been studied for adult stem cells without ethical problems and no side effects. .

Specifically, adult stem cells isolated from adult adipocytes can be obtained through separation and purification from cells present in adipose tissue. The process of acquiring adipose tissue uses adipose tissue that has been discarded in the process of liposuction commonly performed, and its usefulness is doubled because no additional procedure is required. Specifically, the human adipose tissue is obtained by liposuction under local anesthesia, the extracellular matrix of the adipose tissue is treated with collagenase, and then centrifuged to separate monocytes, erythrocytes and various cell debris. Stem cells can be obtained. The amount of adipose stem cells obtained from aspirated adipose tissue obtained from liposuction was separated from 1 × 10 ⁷ to 6 × 10 ⁸ stem cells per 300ml, and more than 90% survival rate has been studied. Compared to the cultivation, the growth rate is fast, and the cell aging phenomenon is late. In addition, the obtained stem cells can be cultured under various conditions to obtain specific cultures.

 When the stem cells are cultured, the stem cells secrete various substances into the culture medium. This culture has been shown to have significant biochemical activity in several studies. In recent years, attempts have been made to utilize stem cells by culturing stem cells as functional cosmetic materials by maximizing effective substances.

In order to obtain a desired amount of stem cell culture medium as a material of cosmetics, a sufficient amount of stem cell culture should be made. Stem cells are normal and not immortal. As the passage continues, genetic instability increases, making it impossible to continuously produce stem cell cultures from stem cells isolated from a person's fat. Normal stem cells have a limitation in passage culture, and the stem cells used as cell therapeutics are generally considered to use only passaged cells within five times. For this reason, stem cells used in cosmetics that are released from different people, that is, adipose derived stem cells obtained in an environment of different ages and genders, may be used.

In addition, some stem cell cosmetics are difficult to commercialize because of the use of human-derived animal cells or their by-products, raising the possibility of infection and a number of unpredictable problems. In addition, in Europe, human-derived substances and related by-products have been prevented from being used in cosmetics for a long time, which prevents the use of cell culture fluids as well as direct cells or related extracts from human stem cell culture. For this reason, many cosmetic companies worldwide are preparing to develop and launch stem cell cosmetics, but the development of products is being delayed due to the difficulty of securing safe materials.

As a way to solve this problem, there is a mini pig as a stem cell source to replace the fat of the human body. Mini pigs are small in size and easy to handle, making them popular for medical and pharmaceutical research. When mini pigs are used as stem cell cosmetic raw materials, fats can be obtained from individuals of the same age and gender, so that cosmetic raw materials can be supplied constantly. Qu et al. (2007) found that stem cells isolated from pig fat were similar in form to mesenchymal stem cells isolated from fat isolated from liposuction and mesenchymal stem cells isolated from fat of other species. In addition, the differentiation capacity of various lines, such as differentiation into adipocytes, chondrocytes, bones and nerves, is also consistent with human adipose derived stem cells. In the case of mini pigs, a lot of research has already been carried out for organ transplantation and drug research, and if sterile mini pigs are grown in sterile conditions, they can be used as a source of stem cells for safe and constant stem cell cosmetics without the risk of contamination.

Therefore, in the present invention, by mass-producing stem cell culture solution from mini pigs through biotechnology, and fusion of nanotechnology to the obtained stem cell culture biopolymer, superior safety, absorption and mobility than conventional biomaterials We developed this good nano-stem cell culture functional material.

In this regard, Korean Patent Application No. 2005-55017 discloses a cosmetic composition containing anti-wrinkle and anti-aging containing allogeneic or self-derived adipocytes or stem cells or fibroblast growth factor isolated therefrom. Republic of Korea Patent Registration No. 848056 discloses a whitening cosmetic composition comprising a stem cell culture medium or a protein isolated from the culture medium, and Korean Patent Application No. 2007-15211 discloses human fat stem cells as antioxidants, vitamins, amino acids And an additive for injection containing a conditional medium obtained by culturing in a serum-free medium containing minerals for at least one day, and used in human stem cell-containing injections for tissue regeneration. However, there is no example provided as a cosmetic composition by the nanofibers or nano-encapsulated stem cell culture solution as the present invention.

An object of the present invention is to apply a cosmetic material to the nanofibers or nano-encapsulated bio-materials stem cells culture having excellent effects on wrinkle improvement and whitening, such as skin, lotion, which can maintain its efficacy while having a strong antioxidant effect It is to provide a cosmetic composition and a manufacturing method of various formulations such as cream essence.

As one aspect for achieving the above object, the present invention relates to a cosmetic composition comprising a nanofiber or nanocapsules of fat-derived adult stem cell culture.

Preferably, the adult stem cells are derived from the fat of mini pigs.

In addition, the fat-derived adult stem cell culture medium is vesicle endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), transforming growth factor (TGF) -β1, hepatocyte growth factor (HGF), keratinocyte growth factor (KGF) , Platelet-derived growth factor (PDGF) -AA, placenta growth factor (PGA), type I collagen, fibronectin, superoxide dismutase (SOD), interleukin-6,8,10 and RANTES.

The composition can be used for wrinkle improvement, whitening or antioxidant.

In addition, the cosmetic composition includes one formulation selected from the group consisting of softening cream, nourishing cream, nutrition cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray and powder.

As another aspect, the present invention relates to a method for producing a cosmetic composition comprising nanofibers or nanocapsules of the fat-derived adult stem cell culture.

Preferably, the manufacturing method

(1) preparing adipose derived adult stem cell culture; And

(2) nanofibers or nanoencapsulation of the adipose derived adult stem cell culture.

The present invention mass-produces stem cell cultures from mini pigs through biotechnology, and fuses nanotechnology to the obtained stem cell culture biopolymers, resulting in superior safety and better absorption and mobility than existing biomaterials. It is effective to provide a culture functional cosmetic material.

Figure 1 shows the results confirmed through the alkaline phosphate activity of adult stem cells isolated from mini-pig fat.
Figure 2 shows the results of the expression of transcription factors OCT4, SOX2 and NANOG through adipose tissue-derived stem cells through immunohistochemical staining and RT-PCR.
FIG. 3 shows the results of confirming the expression of CD29, CD44, CD90 and vimentin, which are mesenchymal stem cell-specific markers, using a flow cytometer for adipose derived adult stem cells.
Figure 4 shows the differentiation of adipocytes into bone cells by von Kossa staining (A) and the differentiation of adipocytes into adipocytes by oil red O staining (B).
5 shows the differentiation of adipose stem cells into osteoblasts through expression of bone specific markers, osteonectin (ONeonectin, ON), osteocalcin (OC), and Runx2 (A) and fat cells of adipose stem cells. The differentiation of is confirmed through the expression of fat-specific markers aP2 and PPARγ2 (B).
Figure 6 shows the manufacturing process of LA-chitosan introduced with linoleic acid (LA).
7 shows a method for synthesizing N-acetylated chitosan.
Figure 8 shows a NATO capsule containing adipose stem cell culture.
Figure 9 shows the manufacturing process of the nanofibers by electrospinning.
10 shows the nanofibers of the adipose stem cell culture prepared by electrospinning.
Figure 11 shows the anti-wrinkle effect when the control application (left) and the present application formulation (right).

In the present invention, the term 'stem cell culture medium' refers to a substance containing a component contained in a medium obtained by culturing stem cells, wherein the stem cells for producing the culture medium in the present invention is characterized in that the adult stem cells derived from fat do.

Preferably, the adult stem cells are derived from the fat of mini pigs.

The medium for stem cell culture can be used without limitation in a basic medium known in the art. The basic medium may be prepared by artificial synthesis, or a commercially prepared medium may be used. Examples of commercially prepared media include Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basic Medium Eagle (BME), RPMI 1640, F-10, F-12, α-MEM (α-Minimal). essential medium), G-MEM (Glasgow's Minimal Essential Medium), and Isocove's Modified Dulbecco's Medium, but are not limited thereto.

In addition, the fat-derived adult stem cell culture medium is vesicle endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), transforming growth factor (TGF) -β1, hepatocyte growth factor (HGF), keratinocyte growth factor (KGF) , Platelet-derived growth factor (PDGF) -AA, placenta growth factor (PGA), type I collagen, fibronectin, superoxide dismutase (SOD), interleukin-6,8,10 and RANTES.

The cosmetic composition of the present invention is characterized in that the fat-derived adult stem cell culture is nanofibers or nanoencapsulated. Due to the characteristics of the extract derived from the adipose stem cell culture medium, which is a polymer polysaccharide structure, when the culture medium is used as it is, the permeability of the stratum corneum is remarkably low, it is difficult to express the elastin and collagen biosynthesis effects of the raw materials in the dermal layer of the skin. In addition, due to the nature of the cosmetics in which more than 20 types of fat-soluble raw materials are mixed, interactions between raw materials may occur after mixing raw materials, and thus raw materials may be denatured or the efficacy of raw materials may be halved.

Thus, in the present invention, in order to overcome the skin absorption and raw material stability problems of the stem cell culture extract raw material itself, nano-technology (fused nanotechnology) by fusion nano-stem cell culture solution functionality is superior to the existing bio material, better safety, absorption and mobility Provide cosmetic materials.

Encapsulation technology refers to the technology of encapsulating solid, liquid, or gaseous substances or packaging inside a substance or tissue to release its contents at a controlled rate under certain conditions. It is called (nanocapsule). Nanocapsules can be defined as several nanometers (nm) to several hundred nanometers. Preferably, by introducing a functional group into chitosan can be used in nanocapsules by imparting affinity to the chitosan, it is possible to increase the absorption rate in the stratum corneum layer by using a low molecular weight of the stem cell culture medium.

In addition, nanofiber refers to a fiber made from several nanometers (nm) to several thousand nanometers by electrospinning after preparing a synthetic polymer solution by mixing the stem cell culture with a biodegradable polymer. Alternatively, the solution including the biodegradable polymer or natural polymer may be nanofiberized through electrospinning, and then, the stem cell culture solution may be dispersed therein and used as a cosmetic composition. The polymer constituting the nanofibers is selected according to the use and the purpose of use, but in the cosmetic application, a polymer that does not cause skin or human irritation is preferable, and polyamide, polyolefin, polyester, fluorine-based polymer, polyvinyl alcohol (PVA) ) Or derivatives thereof, but is not limited thereto.

The cosmetic composition of the present invention can be used for wrinkle improvement, whitening or antioxidant.

As used herein, the term 'wrinkle improvement' means to maintain or enhance the ability to be associated with wrinkles and elasticity of the skin. Collagen and elastin present in the dermal layer of the structure of the skin controls the elasticity of the skin, collagen biosynthesis is affected by the internal and external skin. Specifically, the intrinsic factor is a decrease in cellular activity due to natural aging, a decrease in collagen fiber, and an extrinsic factor such as excessive irradiation of ultraviolet rays, stress, SH) to increase the expression of collagenases such as Matrix Metalloproteinases-1 (MMP-1), an enzyme that inhibits enzymatic activity or degrades collagen and elastin, thereby increasing skin wrinkles and reducing elasticity Results.

As used herein, the term 'whitening' is an element associated with darkening of the color of the skin by inhibiting the ability to synthesize melanin pigment in melanocytes (melanocytes) that determine the color of the skin.

As used herein, the term "antioxidative ability" means a function of inhibiting oxidation of skin components by highly reactive free radicals and free radicals under the influence of ultraviolet rays. Active oxygen and free radicals oxidize the constituents of the skin to produce peroxides, resulting in structurally and functionally damaging the skin to promote aging. The antioxidant function of the present invention functions to protect the skin from this . The composition comprising a culture of stem cells of the present invention can perform the function of inactivating the free radicals.

As used herein, the term 'cosmetic composition' is a composition comprising the stem cell culture, the formulation may be in any form. Examples of such formulations are cosmetics prepared using the cosmetic composition, such as creams, packs, lotions, essences, lotions, foundations and makeup bases, and any form of these formulations to achieve the object of the present invention. Furnace can also be produced and commercialized, but is not limited to the above examples. Preferably, the cosmetic composition may include one formulation selected from the group consisting of softening cream, nourishing cream, nutrition cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray and powder have.

In addition, the cosmetic composition according to the present invention may optionally add purified water, humectant, higher alcohol, higher fatty acid, emollient, chelating agent, thickener, permanent ingredient, pH adjusting agent, flavoring agent, etc. according to the formulation or purpose of use of the cosmetic. have.

Method for producing a cosmetic composition comprising a nanofiber or nanocapsules of the fat-derived adult stem cell culture of the present invention,

(1) preparing adipose derived adult stem cell culture; And

(2) nanofibers or nanoencapsulation of the adipose derived adult stem cell culture.

The preparing of the adipose-derived adult stem cell culture solution may be achieved by separating and culturing the adult stem cells from the fat of the mini pigs to obtain a culture solution. Isolation and culturing of stem cells can employ conventional methods known in the art.

For example, adipose tissue can be separated from the abdomen and thigh around the mini pig, treated with extracellular matrix of adipose tissue with collagenase, and then centrifuged to obtain dense stem cells, which are then removed through a cell filter. After separating mononuclear cells only, the non-induced culture medium and non-adhesive cells can be removed to obtain adipose stem cells.

The isolated adipose stem cells are basal medium known in the art, for example, DMEM, MEM, BME, RPMI 1640, F-10, F-12, α-MEM, G-MEM and Isocove's Modified Dulbecco's Medium, and the like. For example, it can be cultured using α-MEM medium, and after incubation, the medium can be collected and then the cell debris is removed and the remaining broth can be obtained as a stem cell culture. Eventually, the culture may be said to contain the secretion of the main components and fat stem cells contained in the medium.

Next, a step of nanofibers or nanoencapsulation of the fat-derived adult stem cell culture may be performed.

As a specific preferred embodiment of nanoencapsulation, a solvent evaporation method, a solvent displacement method or an emulsification solvent diffusion method, a supercritical fluid extraction process and the like can be used.

The solvent evaporation method dissolves a substance encapsulated in a volatile organic solvent and an oil-soluble substance in water, disperses the solution in an aqueous solution containing an anti-agglomerating agent to form particles, and distills the volatile organic solvent to prepare particles. will be. At this time, the size of the particles largely depends on the stirring speed at the time of dispersing, and when using a typical agitator, particles of several microns are formed, and when using a high speed stirring homogenizer, hundreds of nanometers are used. Particles may be formed. In general, in order to obtain particles of several tens of nanometers, particles having a size of 100 nanometers or less may be obtained by using a high pressure homogenizer. When the solvent evaporation method is used, the organic solvent uses a highly volatile solvent, and specific examples thereof include methylene chloride and ethyl acetate.

Solvent replacement or emulsification solvent diffusion method dissolves the encapsulating material and oil-soluble material in the volatile organic solvent mixed with water, dispersing the solution in an aqueous solution containing an anti-flocculation agent to form particles, and distilling the volatile organic solvent to distill the particles. To prepare. Since this method uses a volatile organic solvent mixed with water, particles are formed by the difference in the solubility of the encapsulating material and the useful material in the cosolvent of water and the organic solvent. Therefore, water-soluble surfactants and other surfactants for preventing aggregation It is preferable to include. In addition, hydrophobic surfactants may be used to stabilize the encapsulated oil soluble actives. Factors affecting the size of the particles produced by the solvent replacement method is the ratio of the organic solvent and water, the concentration of the encapsulating agent and sparingly soluble active ingredient used in the organic solvent used, the amount of hydrophobic surfactant, The amount of hydrophilic surfactant, and the stirring speed, and in general, the size of particles obtained through these components and methods is between 100 and 300 nm.

Supercritical fluid extraction process can be widely used in medicine, food, cosmetics, etc., because it has no toxic solvent or surfactant in the particles, compared to other extraction processes, RESS (Rapid Expansion of Supercritical Solutions), GAS (Gas Anti-Solvent), Supercritical fluid Anti-Solvent (SAS), Aerosol Solvent Extraction System (ASES), Solution Enhanced Dispersion by Supercritical Fluids (SEDS), Particles from Gas Saturated Solutions (RPGSS), and Reactive Precipitaion in Supercritical Solution (RPSS) methods can be used.

The biodegradable polymer used as the encapsulating agent should have excellent biocompatibility and biodegradability in consideration of the safety of the body when the prepared nanoparticles penetrate the skin. For example, as a natural polymer, starch, chitosan, alginic acid, lecithin, glucomannan, Polysaccharides, acacia gum, Irish moss, karaya gum, gum tragacanth, gum guaiac, xanthan gum, locust bean gum and derivatives thereof, and proteins such as casein, gelatin, collagen, albumin, globulin, fibrin and derivatives thereof, Naturally derived carbohydrates and derivatives thereof, such as cellulose, dextrin, pectin, starch, agar and mannan, are synthetic polymers and polyvinyl polymers and derivatives thereof (e.g., polyvinylpyrrolidone, polyvinyl alcohol, polyvinyl methyl ether, Polyvinyl ethers, etc.), polycarboxylic acids and derivatives thereof (e.g. polyacrylic acid, polymethacrylic acid, polymethylmethacrylate) Etc.), hydrocarbone such as polyethylene and polypropylene, isomers thereof, polysaccharides and derivatives thereof (polysucrose, polyglucose, polylactose and salts thereof), and the like. Is possible. Naturally-derived polymers are known to have excellent biocompatibility, and the polymers described above may be applied to the nanoparticles of the present invention as polymers having excellent biocompatibility.

Also, for example, fatty acid polymers (e.g., polylactic acid, polyglycolic acid, polycitric acid, polymaline acid, etc.) and derivatives thereof, poly-, alpha-, cyanoacrylic acid and derivatives thereof, poly-, beta-, Hydroxybutyric acid, polyalkylene oxalate (e.g. polymethylene oxalate, polytetramethylene oxalate, etc.), polyorthoesters, polyorthocarbonates, polycarbonates (e.g. polyethylene carbonate, polyethylene-propylene carbonate Etc.), and polyamino acids (e.g., polygammabenzylglutamic acid, polyalanine, polygammamethylglutamic acid, etc.) can be used, and polystyrene, polyacrylic acid and its derivatives, polymethacrylic acid and derivatives thereof having excellent biocompatibility. , Copolymers of acrylic acid-methacrylic acid, polyamides (eg nylon), polyethylene terephthalate, polyamino acids, silicone high Monopolymers such as porcelain, dextranstearate, ethylcellulose, acetylcellulose, nitrocellulose, polyurethane, maleic anhydride copolymer, ethylene-vinylacetate copolymer, polyvinylacetate, polyvinyl alcohol, polyacrylamide Polymers, two or more copolymers, and mixtures of these polymers are available, and derivatives and salts thereof may be used.

In addition, the nanocapsules in the present invention may be composed of amphiphilic biopolymers having hydrophobicity and hydrophilicity. Preferably, N-acylated chitosan may be formed by combining linoleic acid or by combining alkyl anhydrides or halides with C-2 of chitosan, which is a biopolymer. Synthesis can give the chitosan affinity (amphipathicity) properties that have both hydrophilicity and hydrophobicity, and then nanocapsules can be prepared by ultrasonication method by putting in an O / W emulsion method or a buffer solution. Chitosan nanocapsules prepared as described above can be usefully used for cosmetic delivery including stem cell cultures with excellent biocompatibility, degradability, and stability.

In addition, liquid crystal technology (Liquid Crysta), which shows the intermediate properties of solid and liquid, promotes the delivery of active ingredients into the skin, or double-layered liquid crystal using hydrogenated lecithin. Forming techniques can be used. In addition, the surface of the powder particles may be coated with another raw material such as silicon to modify the powder surface properties.

Meanwhile, in the case of nanofibers, a stem cell culture solution may be mixed with a biodegradable polymer to prepare a synthetic polymer solution, and then may be prepared by electrospinning. Alternatively, the solution including the biodegradable polymer or natural polymer may be nanofiberized through electrospinning, and then, the stem cell culture solution may be dispersed therein and used as a cosmetic composition. Electrospinning process is a technology that forms fibers using electrical attraction and repulsive force generated when the polymer solution or molten polymer is charged to a predetermined voltage. The electrospinning process has various diameters ranging from several nm to several thousand nm. Fibers can be produced. The polymer constituting the nanofibers is selected according to the use and the purpose of use, but in the cosmetic application, a polymer that does not cause skin or human irritation is preferable, and polyamide, polyolefin, polyester, fluorine-based polymer, polyvinyl alcohol (PVA) ) Or derivatives thereof, but is not limited thereto. As an example of a method for producing nanoparticles using electrospinning, reference may be made to Korean Patent Registration No. 10-0743502.

Hereinafter, the present invention will be described in more detail. However, the following examples are illustrative of the present invention, and the present invention is not limited by the following examples.

Example  1. Local origin Mesenchymal stem cells  Isolation, Culture, and Assay

1-1. Isolation and Culture of Adipose Stem Cells

Fats from liposuction and fats from the abdomen and thighs of mini pigs were collected and separated into single cells through collagenase treatment. 39 ℃, 5% CO ₂ in stem cell culture medium (ADMEM) containing various growth factors (basic fibroblast growth factor, stem cell factor, leukemia inhibitory factor, etc.) Incubated in the. Adipose-derived mesenchymal stem cells were cultured by removing all cells except the adhered cells because they have adhesion to the culture dish.

1-2. Verification and Characterization of Adipose Stem Cells

Cells isolated and cultured in Example 1-1 were stained using AP chromogen kit (BCIP / NBT) to confirm alkaline phosphate activity (FIG. 1), and immunohistochemical staining (Immunocytochemistry) and RT Expression of the transcription factors OCT4, SOX2 and NANOG was examined via -PCR (FIG. 2). In addition, in order to identify mesenchymal stem cell specific markers, expression levels of CD29, CD44, CD90 and vimentin were confirmed using flow cytometry (FIG. 3).

1-3. In vitro differentiation of fat stem cells

When the cells isolated and cultured in Example 1-1 were 70-80% confluence, some were in 10% FBS, 0.1 uM dexamethasone, 50 uM ascorbate-2-phosphate, 10 mM β- in DMEM. Differentiation was induced in the culture medium containing glycerol phosphate for 3 weeks to induce differentiation into bone cells, and some contained 10% FBS, 1uM dexamethasone, 10uM insulin, 200uM indomethacin and 500uM 3-isobutyl-1-methylxanthine (IBMX) in DMEM. Differentiation was induced into adipocytes by culturing for 3 weeks in the culture.

The differentiation of adipose stem cells into osteoblasts was confirmed by von Kossa staining (FIG. 4A) and the expression of osteonectin (osteonectin, ON), osteocalcin (OC), and Runx2, which are bone specific markers (FIG. 5A). Differentiation of adipocytes into adipocytes was confirmed by oil red O staining (Fig. 4B) and expression of adipose specific markers aP2 and PPARγ2 (Fig. 5B).

Example  2. Identification of Stem Cell Culture Solution

Stem cells isolated in Example 1-1 were added to various growth factors (basic fibroblast growth factor, stem cell factor, leukemia inhibitory factor, etc.) in a stem cell culture medium (ADMEM) at 39 ° C. and 5% CO _2. After culturing in the culture medium was recovered at regular intervals. In order to identify the components of the culture, the assay was performed by ELISA (Enzyme-linked immunosorbent assay), Western blot assay and RT-PCR.

As a result, the adipose stem cell culture medium contained VEGF (Vascular endothelial growth factor), bFGF (basic fibroblast growth factor), TGF (transforming growth factor) -β1, HGF (hepatocyte growth factor), KGF (keratinocyte growth factor), and PDGF (platelet). -derived growth factor (AA) -AA, placenta growth factor (PGA), type I collagen, fibronectin (fibronectin), superoxide dismutase (SOD), interleukin-6,8,10 and RANTES were included. Among them, proteins contained at high concentrations are summarized in Table 1.

protein density PDGF 42.31 ± 2.50 pg / mL bFGF 130.23 ± 30.30 pg / mL KGF 86.30 ± 20.40 pg / mL TGF-β1 105.40 ± 1.60 pg / mL HGF 674.80 ± 85.12 pg / mL VEGF 802.54 ± 93.23 pg / mL Type I collagen 923.42 ± 48.60 ng / mL Fibronectin 1460.15 ± 461.23 ng / mL

Example  3. Nanoencapsulation and Nanofiberization of Stem Cell Cultures

3-1. Preparation of Amphiphilic Chitosan Nanocapsules

N-acylated chitosan (N-acylated) by inducing EDC reaction to C-2 of biopolymer chitosan to bind linoleic acid or alkyl anhydrides or halides Chitosan) was synthesized to give the chitosan amphipathicity having both hydrophilicity and hydrophobicity.

First, chitosan (1 g) was dissolved in 1% (w / v) acetic acid aqueous solution (100 ml) and 85 ml of methanol. Linoleic acid at room temperature was added at a rate of 0.54 mol / mol per glucosamine residue of chitosan, and 15 ml of EDC-methanol solution (0.07 g / L) was added dropwise. After 24 hours, the reaction solution was added to 200 ml of methanol / ammonia solution (7/3, v / v) and neutralized with stirring. The precipitate formed was filtered off, washed with distilled water, methanol and ether, and vacuum-dried at room temperature for 24 hours to synthesize LA-chitosan (FIG. 6).

Chitosan was also dissolved in 200 ml of 0.6% acetic acid aqueous solution and methanol mixed solution. The same amount of anhydride or halide at room temperature was added dropwise to the chitosan solution and stirred for 5 hours. The reaction solution was poured into 200 ml of methanol / ammonia solution (7/3, v / v) and neutralized with stirring. The precipitate formed was filtered, washed with distilled water, methanol and ether, and vacuum dried at room temperature for 24 hours to synthesize N-acylated chitosan (FIG. 7).

3-2. Manufacture of nanocapsules

Using the amphiphilic chitosan prepared in Example 3-1, a nano-carrier having a size of 100-200 nm was prepared by a solvent diffusion method, and an emulsifier (emulsifier of polyoxyethylene type, saturated and unsaturated alkyl sulfate, ammonium of fatty acid, alkansulfo) Nate, fatty alcohol phosphorate, fatty acid partial glycerides, polyglycerol esters, propylene glycol esters of fatty acids, lactates of fatty acids), followed by stirring in a high speed agitation homogenizer, whereby the adipose stem cell culture is It was prepared as a cosmetic formulation including a solvent cream containing (Fig. 8)

3-3. Preparation of Nanofibers

Nanofibers were prepared using the stem cell culture solution obtained in Example 2 using an electrospinning device Nano NC NC-30K. The manufacturing process is shown in FIG. 8, and the obtained adipose stem cell-containing nanofibers is shown in FIG. 9.

Example  4. Evaluation of efficacy of cosmetic preparations

4-1. Antioxidant efficacy

In order to observe the scavenging effect of free radicals, the stem cell culture solution prepared in Example 2 was added to 0.5 ml of 0.2 mM 1,1-diphenyl-2-picrylhydrazyl methanol solution at various concentrations (wt%) and 10% ethanol solution 1 After the addition of ㎖ and mixed for 10 minutes at 37 ℃ absorbance (%) was measured at 517nm. Table 2 below shows the free radical inhibition rate for each sample according to the concentration, and vitamin C was used as a control.

1.0 1.4 1.8 2.2 2.6 3.0 Stem Cell Culture
(Example 2) 35 39 50 57 68 71 Vitamin C 22 29 40 48 60 66

4-2. Automatic Oxidation Inhibitory Effect

The degree of inhibition of automatic oxidation was measured using linoleic acid, which is a well-automated material. 3 ml of a 10 mM linoleic acid solution and 0.075 ml of a sample adjusted to 4 mg / ml were added to an 8 ml cap tube, and the mixture was left at 4 ° C. for 24 hours. 0.1 ml of the mixed solution was added to 4.7 ml of 75% ethanol, followed by stirring. Then, 0.1 ml of 30% ammonium thiocyanate solution and 0.1 ml of 20 mM ferrous chloride were added for 3 minutes to prevent color development, and the absorption wavelength was measured at 500 nm. Antioxidant effect was measured in comparison with the control without the sample. The results are shown in Table 3 below, and vitamin C was used as a control.

Stem Cell Culture Solution (Example 2) Vitamin C Antioxidative effect 94% 89%

4-3. Wrinkle Improvement

20 healthy women were asked to apply a control product (physiological saline 1cc) and nanocapsules and nanofiber products (1cc) prepared in Example 3 around the eyes twice a day (morning, before bedtime). After 4 weeks and 8 weeks, the wrinkle relief effect was visually observed and the results are shown in Table 3 and FIG. 11 (unit: persons).

A noticeable effect Mitigating effect no effect Control group 0 0 20 Nanocapsules of Example 3 7 13 0 Nanofiber of Example 3 9 11 0

4-4. Whitening effect

As a whitening experiment, the nanocapsules and nanofiber products prepared in Example 3 of the present invention and a competitor product A were applied to the faces of 30 testers (10 testers and 1 group). That is, the samples of the present invention were applied to the faces of each group group 10 times twice a day for one month. In the same manner, competitor A's product A was applied to the tester face of one group of the group group. After completion of the experiment, the skin color of the applied areas on both sides of the face was analyzed by image analysis, and the dark color was set to 5, the middle color to 3, and the light color to 1 and evaluated. Looking at the following Table 5, the sample of the present invention was shown to be lower than the competitor's product A, it can be seen that the whitening effect is excellent.

One 2 3 4 5 6 7 8 9 10 Competitor A Products 3.5 3.0 3.0 3.5 4.0 3.5 3.5 3.0 3.0 3.0 Nanocapsules of Example 3 2.0 2.0 2.5 2.0 2.5 2.0 2.0 1.5 1.5 1.5 Nanofiber of Example 3 1.5 2.0 2.0 2.0 2.0 1.5 2.0 1.5 2.5 2.0

Although the preferred embodiments of the present invention have been described, the present invention is not limited to the specific embodiments described above. It will be apparent to those skilled in the art that numerous modifications and variations can be made in the present invention without departing from the spirit or scope of the appended claims. And equivalents should also be considered to be within the scope of the present invention.

Claims (7)

Cosmetic composition comprising the nanofibers of the fat-derived adult stem cell culture.
The cosmetic composition according to claim 1, wherein the adult stem cells are derived from fat of mini pigs.
delete The method of claim 1, wherein the adipose-derived adult stem cell culture medium is vesicular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), transforming growth factor (TGF) -β1, hepatocyte growth factor (HGF), keratinocyte growth factor, platelet-derived growth factor (PDGF) -AA, placenta growth factor (PGA), type I collagen, fibronectin, superoxide dismutase (SOD), interleukin-6,8,10, and regulated on activation (RANTES) Cosmetic composition comprising a normal T cell expressed and secreted).
The cosmetic composition according to claim 1, wherein the composition is for wrinkle improvement, whitening or antioxidant.
According to claim 1, wherein the cosmetic composition is a flexible cosmetics, nourishing cosmetics, nourishing cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray and powder one formulation selected from the group Cosmetic composition to have.
(1) preparing adipose derived adult stem cell culture; And
(2) nanofibrillating the adipose derived adult stem cell culture solution,
The manufacturing method of the cosmetic composition of any one of Claims 1, 2, and 4-6.

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