KR20100096447A - Cosmetic composition comprising matrials cultured adult stem cells derived from swine placenta tissue and proteins extracted therefrom - Google Patents

Abstract

PURPOSE: A cosmetic composition containing a porcine placenta-derived adult stem cell culture liquid or a protein thereof is provided to enhance the anti-wrinkle and anti-aging properties of cosmetics. CONSTITUTION: A functional cosmetic composition contains a porcine placenta-derived adult stem cell culture liquid as an active ingredient. The functional cosmetic composition additionally contains a protein extracted from the adult stem cells. peptides, or amino acids of the protein. The cosmetic composition is used in the form of a lotion, emulsion, cream, gel, essence, or pack. A method for manufacturing the culture liquid comprises: a step of isolating amnion from porcine placenta; a step of collecting adult stem cells from the amnion; and a step of culturing the adult stem cells in a medium containing bFGF.

Description

Cosmetic Composition Comprising Porcine Placental Tissue-Derived Stem Cells and Extracted Proteins thereof {Cosmetic Composition Comprising Matrials cultured Adult Stem Cells Derived from Swine Placenta Tissue and Proteins extracted therefrom}

The present invention provides a protein extracted from (i) porcine placental tissue-derived stem cell culture and (ii) adult porcine placental tissue-derived adult stem cells; Or it relates to a cosmetic composition containing a peptide or amino acid which is a hydrolyzate of the protein and a method for producing the same.

Stem cells are cells that have the ability to self-replicate and differentiate into two or more cells. Totipotent stem cells, pluripotent stem cells, and multipotent stem cells ( multipotent stem cells).

The placenta is an organ that develops in the womb of a pregnant mammal and nourish and sustain life through the umbilical cord. The placenta is discharged from the uterus at birth and the placenta is called postpartum. The placenta contains nucleic acids such as essential amino acids, melatonin, RNA and DNA, superoxide dismwtase (SOD), hyaluronic acid (antioxidants), antioxidants, cytokines, placental peptides and insulin-like growth promoters. (Insulin-like-grouth factor), epidermal growth factors (EGFS) and unique senescent cell activating factors (SCAFS), including cytokines and growth factors It is known to be useful.

In addition, the tissue metabolism promoting action of the placenta increases sebum secretion function to normalize the secretion of sebum as a result of reducing or removing the wrinkles. In addition, the placenta contains fibroblast growth factor (FGF), which promotes proliferation and regeneration of fibroblasts, and through its activation increases the amount of collagen, elastin and hyaluronic acid to restore skin elasticity and elasticity. It will work to improve wrinkles and sagging skin.

On the other hand, the pig placenta was found to be remarkably similar to the molecular structure of the placenta in the placenta of the mammal. The pig placenta is an important component of protein, various nutrients, DNA and RNA, and is responsible for the differentiation of cells and the development of the fetus. It has been reported as the source of active cytosin.

However, attempts to use stem cells derived from pig placenta for cosmetic or cosmetic purposes have been insufficient.

Therefore, the present inventors have made efforts to use stem cells in the fields of cosmetic and plastic surgery such as wrinkle improvement, skin sagging prevention, and cultured by obtaining adult stem cells from pig placental tissues; After obtaining the protein or amino acid extracted from the porcine placental tissue-derived stem cells, it was confirmed that the composition containing it together is effective for cosmetic and molding, to complete the present invention.

The main object of the present invention is (i) culture of adult stem cells derived from pig placental tissue; Or in addition to (ii) to provide a cosmetic composition containing a protein extracted from the adult stem cells derived from pig placental tissue, or a peptide or amino acid which is a hydrolyzate of the protein as an active ingredient.

Another object of the present invention is to provide a method for producing a culture of adult stem cells derived from pig placenta tissue containing specific components.

In order to achieve the above object, the present invention is an adult pig stem tissue-derived adult stem cell culture as an active ingredient; Or it provides a functional cosmetic composition further comprising a protein or amino acid extracted from the adult stem cells derived from the placental pig tissue.

In addition, the present invention provides a pig placental tissue-derived adult stem cell culture, which is used as a main active ingredient of the cosmetic composition, and the following method of preparing the same:

(a) separating the amnion from the pig placental tissue;

(b) obtaining adult stem cells from the separated amnion; And

(c) culturing the obtained adult stem cells in a medium containing bFGF.

Since the composition for cosmetics of this invention promotes the regeneration of the skin tissue of the apply | coated site | part, it is especially excellent in wrinkle improvement and / or anti-aging effect. Therefore, it can be usefully used for wrinkle improvement or preparation of functional cosmetics for preventing aging.

Hereinafter, the present invention will be described in detail.

In one aspect, the present invention, porcine placental tissue-derived adult stem cell culture; Or it relates to a functional cosmetic composition further comprising a protein or amino acid extracted from the adult stem cells derived from the placental pig tissue.

Functional cosmetic composition of the present invention comprises an adult stem cell culture derived from pig placental tissue as an active ingredient.

Stem cells are cells that have the capacity of self-replicating and have the ability to differentiate into two or more cells. Adult stem cells are stems that appear during the development or development of each organ of the embryo. It means a cell.

The term "adult stem cell-derived adult stem cell" used in the present invention is an undifferentiated adult stem cell isolated from the placental tissue of pig, and may be abbreviated herein as "placental stem cell".

The term "adult placental tissue-derived adult stem cell culture" or "placental stem cell culture" used in the present invention, after culturing the adult stem cells derived from porcine placental tissue in a medium containing a specific component, the medium is collected After the removal of the cell debris (debris) refers to the remaining (broth). After all, the culture may be said to contain the secretion of the main components and placental stem cells contained in the medium.

Porcine placenta has been found to be easy to recover and secure a large amount, as well as surprisingly similar to the molecular structure of the placenta among mammalian placenta. Porcine placenta has been reported to be a source of bio-active cytosin that controls cell differentiation and fetal development as an important component of protein, various nutrients and DNA and RNA.

There is no particular limitation on the method for obtaining adult stem cells derived from pig placental tissue, which is the main component of the present invention, but, for example, it can be prepared from the amniotic membrane of pig placenta by the following method:

(a) separating the amnion from the pig placental tissue;

(b) obtaining adult stem cells from the separated amnion; And

(c) culturing the obtained adult stem cells in a medium containing bFGF.

Hereinafter, the method will be described in more detail.

First, the pig's placenta is washed several times in physiological saline containing antibiotics, and then the amniotic membrane, chorion and exfoliating membrane are separated from the placenta. The separated amniotic membrane is washed again two or three times with saline (or DPBS), transferred to a 60mm Petri dish, and cut using surgical scissors. After that, the tissue is placed in DMEM medium containing collagenase and subjected to histolysis process at 37 ° C. for about 1 hour and 30 minutes. After that, the degree of tissue degradation is visually checked, filtered using a 100um cell strainer, and the filtered solution is centrifuged.

Pellets derived from porcine placenta tissue were centrifuged and filtered to remove debris, and then cultured in DMEM medium containing FBS, NAC and ascorbic acid. Unattached cells are washed and cultured with DMEM containing FBS, NAC, ascorbic acid, calcium, rEGF, insulin, and hydrocortisone every 2 days.

Next, the placental tissue-derived multipotent mesenchymal stem cells of Passage 3 were incubated at 90% confluency, incubated for about 120 hours in DMEM containing NAC, ascorbic acid, calcium, insulin and hydrocortisone, and the medium was collected. Centrifugation and filtering remove cell debris to obtain pure porcine placental stem cell culture.

As a medium used for obtaining the porcine placental tissue-derived stem cell culture, a conventional medium known in the art to be suitable for culturing mammalian stem cells may be used. Preferably, DMEM (Dulbecco modified Eagle medium) or aMEM is used. .

Porcine placental tissue-derived stem cell culture medium may be supplemented with an additive that promotes proliferation of the undifferentiated phenotype of stem cells while inhibiting differentiation.

In addition, the medium generally contains neutral buffers (such as phosphates and / or high concentrations of bicarbonate) and protein nutrients (such as serum, such as FBS, serum substitutes, albumin, or essential and non-essential amino acids, such as glutamine) in isotonic solutions. can do. Furthermore, lipids (fatty acids, cholesterol, HDL or LDL extracts of serum) and other components found in most preservative media of this kind (such as insulin or transferrin, nucleosides or nucleotides, pyruvate salts, any ionized form or salt) Sugar sources such as glucose, selenium, glucocorticoids such as hydrocortisone and / or reducing agents such as β-mercaptoethanol.

The medium also contains anti-clumping agents, such as those sold by Invitrogen (Cat # 0010057AE), with the aim of preventing the cells from adhering to each other, adhering to the vessel wall, or forming too large a bundle. It can be beneficial to do so.

Among other things, it may be advantageous to use one or more additional additives as follows:

Stem cell factor (SCF, Steel factor), other ligands or antibodies that dimerize the c-kit, and other active agents of the same signal transduction pathway

Of other tyrosine kinase related receptors, such as platelet-Derived Growth Factor (PDGF), macrophage colony-stimulating factor, Flt-3 ligand and Vascular Endothelial Growth Factor (VEGF) Ligands for Receptors

Factors that increase cyclic AMP concentrations, such as forskolin

Factors inducing gp130, such as LIF or Oncostatin-M

Hematopoietic growth factor such as thrombopoietin (TPO)

Modifying growth factors such as TGFβ1

Other growth factors such as epidermal growth factor (EGF)

Neurotropins, such as CNTF

N-acetyl-L-cysteine (NAC)

Hydrocortisone

ㆍ Ascrobic Acid

In particular, the medium for culturing pig placental tissue-derived stem cells of the present invention preferably contains bFGF.

Porcine placental stem cell culture of the present invention, in addition to the medium component, may contain the following protein components secreted in high concentration by the contained pig placental stem cells:

IGF-1 (820.8 pg / ml)

fibronectin (1295.7 pg / ml)

PDGF (417.17 pg / ml)

VEGF (647.43 pg / ml)

Procollagen (598.4 ng / ml).

IGF-I (Insunlin-like Growth Factor-1) Insulin-like growth factor (IGF-I) is a 7.6-kD single-chain polypeptide hormone formed of 70 amino acids that forms bone, muscle, and nerve tissue in response to growth hormones. It stimulates the differentiation of cells, thereby regenerating damaged cells and promoting growth. VEGF (vascular endothelial growth factor) Vascular endothelial growth factor is a cytokine that increases the permeation of plasma proteins in capillaries and promotes cell division and migration and induces protease that causes cell reorganization. In addition, by suppressing cell death, the survival of newly formed blood vessels is maintained, and the antigen supply of neurons is suppressed to induce immune regulation to induce cell growth and division. It promotes the migration of vascular cells to promote the development and differentiation of new cells, thereby increasing the composition of the skin. Platelet-derived growth factor (PDGF) is a growth factor that regulates cell growth and division and plays an important role in blood vessel formation. Fibronectin is an adherent glycoprotein that aids in wound healing by promoting the migration of epithelial cells over the wound matrix. Fibronectin is one of the skin components involved in skin regeneration and elasticity. Procollagen is a protein that makes up the dermal layer of the skin and combines with moisture to maintain the skin's moisture and elasticity.

In addition, the functional cosmetic composition of the present invention as one of its active ingredients, in addition to the adult stem cell culture derived from pig placental tissue, protein extracted from porcine placental tissue derived adult stem cells; Or a peptide or amino acid which is a hydrolyzate of the protein. It can also be called a purified product of porcine placental stem cells.

In the present invention, a protein or amino acid extracted from porcine placental tissue-derived adult stem cells, for example, can be obtained by the following method.

This method relates to a method of preparing the protein constituting the porcine placental stem cells in salt precipitation and dialysis to maintain the activity. After washing the pig placental stem cells to ensure that no residual components of the pig placental stem cell cultures remain, the pig placental stem cells are crushed and Ammonium Sulfate is added to the supernatant little by little to precipitate the protein to recover the protein pellets. Then, dialysis is performed by means of removing impurities. After the dialysis, the protein is recovered by centrifugation, the supernatant is recovered, and lyophilized to extract protein components of porcine placental stem cells.

In addition, low-molecular amino acids and peptides can be prepared by hydrolyzing the proteins constituting the porcine placental stem cells. Porcine placental stem cells obtained by leaving no residual components of the pig placental stem cell culture solution are treated with a protease, stirred and subjected to enzymatic digestion. After this process, the proteins that make up the porcine placental stem cells are converted into peptide and amino acid states.

The freeze-dried porcine placental stem cell extract protein, or a low molecular peptide or amino acid obtained by the above method may be suspended in the above-described porcine placental stem cell culture solution to make a cosmetic composition of the present invention.

In addition to the above components, in order to increase the content of some of the components present in porcine placental stem cell culture (for example, procollagen), it may be used by adding a specific extract, for example licorice.

Using the said composition of this invention as an active ingredient, a functional cosmetics can be manufactured and used.

At this time, cosmetics include lotion, milky lotion, cream, gel, essence (essence), pack cosmetics, and the like. Cosmetics containing an effective amount of the composition of the present invention is promoted the regeneration of the skin tissue of the applied site, in particular, it is possible to maintain a young and healthy skin by the wrinkle improvement and / or anti-aging effect.

"Cosmetic water" is generally a clear liquid cosmetic applied to the skin surface in order to keep the skin clean and healthy. The basic function of the lotion is to supply moisture or moisturizing ingredients to the stratum corneum of the skin, and the lotion also has the function of softening the skin. Dissolves and stabilizes substances that are difficult to dissolve in water to make the appearance transparent. Transparent or translucent using microemulsion or lipid nanosphere, O / W type (oil in water type), The opaque lotion emulsified by W / O type or W / S type | mold, the thing which mix | blended the water-soluble polymer, etc. are mentioned. As the treatment method of the present invention, known "cosmetics" can be appropriately used depending on the purpose thereof.

In addition to the composition of this invention, lotion contains the following components, for example. Namely, purified water for dissolving water-soluble components such as ion-exchanged water and supplying water to the stratum corneum; alcohol for dissolving and sterilizing oil-soluble components such as ethanol and propanol to give a refreshing feeling; glycerin, PEG, hyal Moisturizers for moisturizing the stratum corneum, such as lonic acid; emollients (oil components that prevent moisture from evaporating); moisturizing agents such as ester oils and vegetable oils; and raw material components such as polyoxyethylene oleyl alcohol ethers. Solubilizer for solubilizing; buffering agent for adjusting pH of products such as citric acid, lactic acid, amino acids, etc .; for adding flavors such as vanillin, orange flavor, lemon flavor, milk flavor geraniol, linarol Flavoring agent; Antiseptic for inhibiting microorganisms such as methylparaben and phenoxy ethanol to prevent decay; Colorant for coloring; Metal ion sequestrant, Ultraviolet ray Hand et al., Fading agent to prevent fading or discoloration; a drug, such as astringents and, fungicides, energetic agents, anti-inflammatory agents, or whitening agent, may contain a mixture of two or more.

When the total amount of the lotion is 100% by weight, the composition of the present invention may contain 0.00 to 20% by weight, preferably 0.01 to 10% by weight, more preferably about 0.1 to 5% by weight.

"Emulsion" is an intermediate between a lotion and a cream and is generally a fluid emulsion. Latex is a cosmetic mainly used to supply moisture, moisturizer, oil, etc. to the skin in order to maintain the skin's moisture balance, moisturizing and flexibility. The components contained in the milky milk are similar to the ingredients contained in the creams described later. However, since the milky milk is fluid, the amount of solid oil and lead is less than that of the creams. In the treatment method of this invention, a well-known "oil emulsion" can be used suitably according to the use etc.

The emulsion may contain, for example, 0.001 to 20% by weight, preferably 0.01 to 10% by weight, more preferably about 0.1 to 5% by weight of the composition of the present invention.

"Cream" is a type of emulsion in which one side of a liquid that does not mix with each other, such as water and oil, is dispersed in a stable state in the other dispersion medium. In the treatment method of this invention, well-known "cream" can be used suitably according to the use etc. These creams include emollient creams for moisturizing and softening of the skin, massage creams for promoting blood circulation, cleansing creams for cleansing the skin, hair removers for hair loss, deodorant creams for odor removal, and keratin softening. Keratin softening cream and the like.

The cream contains, for example, the following ingredients: That is, the aqueous phase components included in the cream include purified water such as ion-exchanged water; alcohols for dissolving and sterilizing oil-soluble components such as ethanol and propanol to give a refreshing feeling; for moisturizing the stratum corneum, such as glycerin, PEG and hyaluronic acid. Humectants; and mucus such as queen's seed, pectin, cellulose derivatives, and the like. Oil components included in the cream include hydrocarbons such as squalene, liquid paraffin, petrolatum and solid paraffin; oils such as olive oil, almond oil, cacao fat and castor oil; lead such as beeswax, lanolin, jojoba oil; stearic acid, oleic acid, Higher alcohols such as fatty acids such as palmitic acid, cetanol and stearyl alcohol, esters such as IPM, glycerin triester, pentaerythritol tetraester, silicone oils such as polysiloxanes, and the like. Although the quantity of each component contained in cream differs greatly by the kind and use, a well-known thing may be used suitably in the treatment method of this invention.

The cream may contain 0.001 to 20% by weight, preferably 0.01 to 10% by weight, more preferably about 0.1 to 5% by weight of the composition of the present invention.

Gels are cosmetics with a uniform appearance, such as gels or sols, and are transparent to translucent cosmetics. As the treatment method of the present invention, a known "gel" can be appropriately used depending on the purpose thereof. And an aqueous gel for replenishing and moisturizing the skin, an oily gel for maintaining the moisture of the skin, and replenishing oil, a water-soluble gel for promoting blood circulation, and a gel for washing. The gel may be prepared by using a gelling agent containing a water-soluble polymer such as carboxyvinyl polymer and methyl cellulose. Moreover, you may use gel massage oil.

"Cosmetics (essences)" is basically a component such as the lotion, emulsion and cream described above, and includes those containing an emulsion, a solubilizer, a moisturizer, water, and other drugs. The well-known cosmetic liquid in the treatment method of this invention can be used. Emulsions included in the cosmetic liquid include natural vegetable oils such as olive oil, camellia oil, sesame oil, sunflower oil, sweet almond oil and jojoba oil; diglycerin esters or triglycerin esters of fatty acids such as caprinic acid, myristic acid, oleic acid and isostearic acid. Etc. can be mentioned. Such emulsions mainly function as emollients. Solubilizers included in the cosmetic liquid include propylene glycol fatty acid esters such as glycerin fatty acid ester, polyglycerol fatty acid ester, sorbitan fatty acid ester, and propylene glycol monostearate, glycerin, diglycerin, ethylene glycol, propylene glycol, 1,3-butyl Polyhydric alcohols, such as len glycol, etc. are mentioned. On the other hand, a solubilizer is preferable because it uses glycerin or diglycerol, and since a thermal effect is heightened in a massage. Distilled water and deionized water are mentioned as water contained in a cosmetic liquid. Other pharmaceutical agents include fungicides, preservatives, vitamins, natural extracts from plants, pigments and flavorings.

The quantity of each component which comprises a cosmetic liquid can employ | adopt a well-known component amount. For example, when the total amount of the cosmetic liquid is 100% by weight, the composition of the present invention may contain 0.001 to 20% by weight, preferably 0.01 to 10% by weight, more preferably about 0.1 to 5% by weight. Moreover, 65 to 90 weight% of an emulsion, 1 to 10 weight% of a solubilizer, 1 to 30 weight% of a moisturizer, about 0 to 10 weight% of other drugs, and the remainder are water. What is necessary is just to manufacture a cosmetic liquid according to a well-known manufacturing method.

"Pack cosmetic" is to apply to the target site for the purpose of moisturizing the skin, promoting blood circulation, and cleansing. Pack cosmetics generally include a jelly, paste, powder, creamy liquid, and the like, and may be applied to the face to form a film and then peeled off, wiped off after application, or washed off after application. . Powdered materials are used by dissolving or suspending uniformly in water or the like during use. Moreover, the pack cosmetics which impregnated cosmetics to the nonwoven fabric and collagen sheets of shapes, such as an eye mask, the pack cosmetics etc. which are immersed in cosmetics at the time of use may be sufficient.

Pack cosmetics may contain 0.001 to 20% by weight, preferably 0.01 to 10% by weight, more preferably 0.1 to 5% by weight, most preferably about 0.2 to 2% by weight of the composition of the present invention. The other composition may be a known composition used for a pack cosmetic, and may be prepared according to a known production method.

However, without being limited to the above description, the above cosmetics may be used according to the method in which each is commonly used.

Claims (5)

Functional cosmetic composition containing an adult stem cell culture derived from pig placental tissue as an active ingredient According to claim 1, Protein extracted from the adult stem cells derived from the placental pig tissue; Or a functional cosmetic composition further comprising a peptide or amino acid which is a hydrolyzate of the protein. According to claim 1, wherein the cosmetic composition is a cosmetic composition, characterized in that for wrinkle improvement or anti-aging. According to claim 1, wherein the cosmetic composition is a cosmetic composition, characterized in that selected from the group consisting of lotion, milky lotion, cream, gel, essence (essence) and pack cosmetics. Including, Method of producing adult stem cell culture medium derived from pig placenta tissue of claim 1: (a) separating the amnion from the pig placental tissue; (b) obtaining adult stem cells from the separated amnion; And (c) culturing the obtained adult stem cells in a medium containing bFGF.