KR20120021317A - Serum-free medium for animal cell culture comprising natural extracts or compounds isolated from them - Google Patents

Abstract

PURPOSE: A serum-free medium for culturing animal cells is provided to lower medium production cost and to basically block contamination. CONSTITUTION: A serum-free medium for culturing animal cells contains Acanthopanax sieboldianus plant extract for replacing animal serum. The extract is prepared by isolating Acanthopanax sieboldianus with water, low alcohol, hexane, chloroform, or mixture thereof. The animal cells are animal stem cells or skin cells. A cosmetic composition for improving skin condition contains the extract or culture liquid which is prepared by culturing animal cells in a serum-free medium and removing the animal cells.

Description

Serum-free medium for animal cell culture comprising natural extracts or compounds isolated from them}

The present invention relates to a serum-free medium for the cultivation of animal cells, and more particularly animal cells, in particular by adding a natural extract or a compound isolated therefrom to the basal medium has been traditionally used for in vitro animal cell culture The present invention relates to a serum-free medium for culturing animal cells, which is capable of culturing stem cells at a higher cell growth rate than conventional serum-containing medium. The present invention also relates to the use of the natural extracts added to the serum-free medium for culturing animal cells or the skin condition of the compounds isolated therefrom.

Animal cell industrialization technology, which is one of several production technologies of biopharmaceuticals, has been spotlighted as a technology for producing high value-added biopharmaceuticals by culturing large numbers of genetically engineered human and animal cells. Recently, gene therapy and cell The development of cell therapy techniques has further emphasized the importance of industrialization technology. In general, the development of animal cell-derived pharmaceutical production processes is focused on securing the safety of the products produced and reducing the high production costs by cell productivity. Animal cell culture methods currently used must supply serum to the cell culture medium for growth. At present, the most widely used serum is FBS (Fetal Bovine Serum) or FCS (Fetal Calf Serum). Although the function of these additives is not clear, they are known to play a role of promoting growth, supplying nutrients, protecting cells from oxidation and toxins, and when these components are excluded, cell culture itself is almost essential. However, the expensive serum not only increases the price of the medium, but also makes it difficult to purify the recombinant protein due to its complex components, and there is a fear that there are differences in the components of the production process, resulting in different results every time the experiment is performed. In addition, since serum is derived from an animal, there is a possibility that cells may be contaminated with a virus or infectious prion during the culturing process. Prion infected with bovine serum can be transferred directly to humans, so animal cell culture has several safeguards. Normal use of serum involves the following problems: i) large variations in the ability to promote growth to cells due to composition variations in serum; Ii) in the production of cell products for use in humans, serum has the potential for contamination by infectious agents; Iii) Bacteriophage and bovine virus are detected in commercially prepared FBS or FCS; Iii) a variety of toxins resulting from bacterial contamination also contain cell culture inhibitors; V) bacterial toxin due to contamination is present before filtration; Iii) may be a biological contaminant (mycoplasmas, bacteriophages, viruses) in cell culture; Iii) the gamma-globulin fraction contains toxins secreted by bacteria and antibodies that cross react with the culture; Iii) heat inactivation reduces the cytotoxin action of immunoglobulins but destroys unstable constituents and is not always more satisfactory than untreated serum; Iii) modification of cell surface is induced by adsorption / binding of serum proteins; Iii) disruption of cell products; xi) excessive growth of fibroblasts in the first culture is encouraged.

Since it was reported that synthetic medium could partially replace natural medium in the 1950's, attempts were made to culture cells without serum, and now, in some cases, cell culture without serum is performed for specific purposes. However, these serum-free media grow only specific cells, and proteinaceous cell growth factors used in place of serum are too expensive, and growth and product production is reported to be less stable than in serum-containing media. In addition, it is known that serum-free medium is not applicable to the culture of stem cells. The most important field in the commercial use of stem cells is a treatment technology in which stem cells obtained from the human body are cultured in a test tube and then put back into a patient as a cell therapy. Stem cell culturing is essential in these areas, and it is important to avoid animal media or serum as much as possible. Considering the therapeutic benefits of stem cell transplantation for the purpose of disease treatment, the risk of infection may be at risk.However, for cosmetic purposes, the risk of exposure to infection is greater than that of stem cell transplantation. It can be highlighted. Therefore, finding an animal serum replacement in the field of stem cell culture would be very meaningful commercially.

On the other hand, where the stem cells are located in the skin is largely as follows. The first is in the basal cell layer of epidermis, called the interfollicular epidermis. Second is the hair follicle. Hair follicles are cells before cell differentiation occurs, and there are cells that play the most important role in epidermal regeneration. Third is adipose derived stem cells present in the subcutaneous fat layer. As such, the stem cells present in the skin play an overall role directly related to the health of the skin, which is the most important cell group in relation to skin health.

Stem cells are known to inhibit the aging of cells due to harmful environment and to regenerate or repair problem areas by proliferating and releasing various cell activators through endless cell division during life. However, stem cells are also cell populations, so gradually lose their function with age. In other words, the stem cells of the skin also gradually decreases its activity as aging causes cell division to decrease and gradually becomes inactive, resulting in various problems such as wrinkles, blotch, slow wound healing, and slow recovery due to aging. Therefore, if the stem cells in the skin can be activated to prevent the aging of the stem cells will be able to restore the activity again.

In order to prevent and improve various problems caused by the reduction and loss of the function of the skin cells through the activation of the skin stem cells, the present inventors first plant a substance capable of promoting the proliferation of stem cells in serum-free medium instead of animal serum. I wanted to find from the material. As a result, the present invention has been completed, and it has also been found that the vegetable stem cell activation ability and various skin problems can be solved.

Therefore, one object of the present invention is one from the group consisting of the extract of thistle plant, the extract of the plant of the genus Ogapi, stigmasterol of the formula (1) and en-acetylgalactosamine of the formula (2) by replacing the animal-derived serum components It is to provide a serum-free medium for animal cell culture containing the selected material.

Still another object of the present invention is an extract of thistle plant, an extract of the genus Ogapi, stigmasterol of formula 1, en-acetylgalactosamine of formula 2, and animal cells in a serum-free medium for culturing the animal cells After culturing to provide a composition for improving the skin condition comprising at least one material selected from the group consisting of a culture solution from which the cultured animal cells are removed.

In one embodiment, the present invention is one species from the group consisting of the extract of thistle plant, the extract of the genus Ogapi plant, stigmasterol of formula (1) and en-acetylgalactosamine of formula (2), replacing the animal-derived serum components Provided is a serum-free medium for animal cell culture comprising the selected material.

The present inventors made an intensive effort to find a substance from the plant material that can promote the proliferation and activation of animal cells, particularly stem cells and skin cells, even in a serum-free medium in place of animal-derived serum. As a result, it was found that thistle plant extract, Ogapi plant extract, stigmasterol of Formula 1 and N-acetylgalactosamine of Formula 2 can promote the proliferation and activation of animal cells to the extent that they can replace animal-derived serum. The present invention has been completed.

Thistle plant of the present invention (Cirsium) is a plant belonging to the family Asteraceae, examples of which are not necessarily limited to this, Cirsium japonicum var. spinossimum ), thistle ( Cirsium) japonicum var. ussuriense), narrow leaves thistle (Cirsium japonicum for. nakaianum), considering thistle (Cirsium rhinoceros var. Niveoaraneum ), Thistle Cirsium schantarense ) for. albiflorum ), Cirsium toraiense , Cirsium water thistle nipponicum ), Thistle ( Cirsium) rhinoceros ), willow thistle ( Cirsium) lineare ), Thistle ( Cirsium) vulgare), Jeombongsan thistle (Cirsium zenii ), thistle ( Cirsium) chanroenicum var. lanceolata ), Jeju thistle ( Cirsium) chinense ), Canadian Thistle ( Cirsium) arvense ), Cirsium pendulum , Cirsium vlassovianum var. album , Thistle Cirsium vlassovianum for. leucanthum ) plants. In one specific embodiment of the present invention, thistle among the thistle genus plants ( Cirsium japonicum var. ussuriense ).

The plant of the genus Acanthopanax of the present invention is a plant belonging to the family Arboraceae , examples of which are not limited thereto, but are not necessarily limited to this, Acanthopanax senticosus Harms (araliaceae), Acanthopanax Senticosus Var. Koreanus ), Acanthopanax Senticosus Var. inermis ), Acanthopanax Sessmuorus ), Acanthopanax Chilsanensis ), Acanthopanax Seoulensis ), Acanthopanax Rufinerve ), Acanthopanax Koreanm ), Acanthopanax Siebololianum ) plants. In one specific embodiment of the present invention, Acanthopanax senticosus Harms).

In the present invention, the thistle genus and the genus Ogapi can use leaves, flowers, seeds, stems, roots and outposts as raw materials.

In the present invention, the thistle plant extract and the plant extract of the genus Ogapi include all of the extracts or fractions extracted by the solvent of these plants, the purified products of the extract, the concentrated or dried form of the extract or purified products, and the like.

The solvent for the extract or fraction extracts of the thistle and the organium plants, lower alcohols such as methanol, ethanol, propanol, ethylene, acetone, hexane, ether, chloroform, ethyl acetate, butyl acetate, dichloromethane, N, N Polyhydric alcohols, such as -dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1, 3- butylene glycol, and propylene glycol, and water can be used individually or as a mixture of 2 or more types.

In the present invention, the production method of the extract or fraction extract of the thistle and the genus Ogapi plants is not particularly limited, for example, cold needle extraction, ultrasonic extraction, reflux cooling extraction and the like can be used.

The purified product is a process of removing the suspended solid particles from the extract or fraction extract, it may be used to filter the particles using cotton, nylon or the like, ultrafiltration, cryofiltration, centrifugal separation, etc., but is not limited thereto. It may also further comprise a separation process by various chromatography (chromatography according to size, charge, hydrophobicity or affinity).

The extract, fraction extract or purified product may be used as it is in liquid form or may be used by concentrating and / or drying it. The concentration and / or drying methods include, but are not limited to, methods such as freeze drying, vacuum drying, hot air drying, spray drying, reduced pressure drying, foam drying, high frequency drying, infrared drying and the like.

In one specific embodiment of the present invention, the extract of thistle or scabies is prepared using water or a lower alcohol, and then fractionated extracts are sequentially extracted using organic solvents such as hexane, chloroform, butanol, and water. It was. In addition, the fraction extract was purified by silica gel chromatography, Sephadex LH-20 column and the like.

In the present invention, the stigmasterol of the formula (1) is an extract of the plant thistle, preferably thistle ( Cirsium japonicum var. ussuriense ) extract, more preferably a compound isolated from thistle chloroform soluble fraction extract. In one specific embodiment, the stigmasterol of the formula (1) is prepared by extracting thistle extract obtained by lower alcohol using a hexane to prepare a water-soluble fraction extract, and then extracted with chloroform to prepare a chloroform soluble fraction extract, ODS flash column It is prepared by eluting with 100% aqueous methanol in, followed by purification by silica gel chromatography, filtering on a Sephadex LH-20 column, dividing into a fraction collector and isolating. However, the stigmasterol of Formula 1 is a compound isolated from the extract of the plant of the thistle genus and its efficacy is found, but does not exclude the stigmasterol of Formula 1 chemically synthesized in the present invention.

En-acetylgalactosamine of the formula (2) is an extract of the genus Ogapi, preferably Ogapi ( Acanthopanax senticosus Harms) extract, more preferably a compound isolated from Ogapi chloroform soluble fraction extract. In one specific embodiment, n-acetylgalactosamine of the formula (2) is prepared by extracting the ogapi extract obtained by extraction with lower alcohol using hexane, and then extracting with chloroform to prepare a chloroform soluble fraction extract. It is prepared by eluting with 100% aqueous methanol on an ODS flash column, purifying by silica gel chromatography, filtering on a Sephadex LH-20 column, and dividing by a fraction collector. However, although N-acetylgalactosamine of Chemical Formula 2 is a compound isolated from the extract of the genus Ogapi and its efficacy has been revealed, it does not exclude the chemically synthesized N-acetylgalactosamine of Chemical Formula 2 in the present invention. .

In the present invention, "serum-free" medium means any animal cell culture medium that does not contain a predetermined amount or more of serum derived from animals including humans. Such suitable cell culture media are known to those skilled in the art, but are not limited to, for example, DMEM medium, HAM medium, IMDM medium, RPMI 1640 medium and the like. These media contain salts, vitamins, buffers, energy sources, amino acids and other substances. In addition, these mediums generally use about 5-20% of animal-derived serum in culturing cells to provide universal nutrients for growth of cells to be cultured. However, the medium of the present invention replaces the animal-derived serum necessary for the growth of the cells, and at least one substance selected from the group consisting of the extract of the thistle plant, the extract of the genus Opiaceae, stigmasterol and en-acetylgalactosamine The addition promotes the growth and proliferation of cells that are sufficiently superior to replace animal derived serum. As used herein, “replace animal-derived serum” or “contain no more than a certain amount of animal-derived serum” means at least 10%, more preferably at least 30%, even more preferably of animal-derived serum content added to the animal cell culture medium. Preferably at least 40%, even more preferably at least 50% and most preferably at least 100% at least one selected from the group consisting of the thistle plant extract, the genus Ogapi plant extract, stigmasterol and en-acetylgalactosamine Substances are added instead to grow and proliferate cells. In one specific embodiment, the cells in the medium, for example stem cells and the thistle genus plant extract, the genus Ogapi plant extract, stigmasterol and en-acetylgalactosamine of the present invention are added to the medium in place of animal-derived serum. It has been found to promote and activate the proliferation of fibroblasts and the like. Accordingly, even if the thistle plant extract of the present invention, the genus Ogapi plant extract, stigmasterol and en-acetylgalactosamine are added to animal cell culture medium instead of animal-derived serum within the above range, cells in culture medium by animal-derived serum It will be apparent to one skilled in the art that equivalent or more of proliferation and activation functions will be present.

In the present invention, an animal cell is a functional and structural basic unit derived from an animal including a human, and a cell derived from an animal including a human is included in the scope of the present invention. Accordingly, the animal cells of the present invention include, but are not limited to, stem cells, muscle cells, germ cells, cells in the skin (eg, fibroblasts, keratinocytes).

The stem cells are cells that can divide themselves and have the ability to differentiate into a wide variety of specific cell types. In one specific embodiment, the stem cells include both embryonic stem cells and adult stem cells, preferably adult stem cells derived from animals, more preferably adult stem cells derived from humans. The adult stem cell refers to a stem cell that plays a role in regenerating damaged tissue and replacing cells that are not differentiated and proliferated by cell division, which are found throughout the adult even after an embryonic development stage. These adult stem cells have a site-specific differentiation ability to differentiate themselves according to the characteristics of the surrounding tissues. Examples of these adult stem cells are derived from various adult cells such as bone marrow, blood, brain, skin, fat (ie, adipose tissue or adipocytes), umbilical cord, umbilical cord blood, umbilical cord's Barton's jelly. In one specific embodiment, the adult stem cells are human cord blood-derived stem cells (CB-MCSs), skin-derived stem cells, or human adipose-derived stem cells, ADSCs). In another specific embodiment, the stem cells derived from the skin as adult stem cells are stem cells present in the basal layer of the interfollicular epidermis between hair follicles and hair follicles, stem cells present in hair follicles, or subcutaneous fat layers. Adipose-derived stem cells present.

The serum-free medium for culturing the animal cells of the present invention is essentially one or more selected from the group consisting of thistle plant extract, scabbard plant extract, stigmasterol and en-acetylgalactosamine, which can replace animal-derived serum. In addition, it may include antibiotics, antifungal agents, and mycoplasma inhibitors that cause contamination, if necessary. As antibiotics, antibiotics commonly used in cell culture, such as penicillin-streptomycin, can be used.Antifungal agents include amphotericin B, tylosin, gentamicin, ciprofloxacin, azithromycin, etc., as mycoplasma inhibitors. Can be used. Further, if necessary, oxidative nutrients such as glutamine and energy metabolites such as sodium pyruvate can be further added. In addition, if necessary, an attachment factor such as fibronectin and / or a growth factor for assisting the growth of cells such as interleukin, GM-CSF, G-CSF, and the like may be further added. Such additional substances and their content may be easily adjusted by those skilled in the art according to the type and characteristics of the cells.

In another embodiment, the present invention is an extract of thistle plant, the extract of the genus Ogapi, the stigmasterol of the formula (1), the en-acetylgalactosamine of the formula (2), and the animal in the serum-free medium for culturing the animal cells It provides a composition for improving the skin condition comprising at least one material selected from the group consisting of a culture solution from which the cultured cells are removed after culturing the cells.

The extract of the thistle plant, the extract of the genus Ogapi, the stigmasterol of the formula (1) and the en-acetylgalactosamine of the formula (2) are as described above, a detailed description thereof will be omitted below.

After culturing the animal cells in the animal cell culture serum-free medium of the present invention described above, the culture medium from which the animal cells are removed is cultured in the serum-free medium for animal cell culture, and then the animal cells are removed after culturing the animal cells. to be. The method for producing the culture solution can be carried out using a known method. For example, after culturing the animal cells, the cell culture solution is recovered, and the cultured medium from which the animal cells have been removed can be obtained by filtering the recovered culture solution using a micro syringe filter. The pore size of the micro filter may be appropriately selected within a size capable of removing animal cells, preferably 0.1 to 10 μm.

The "improvement of the skin condition" means to alleviate, suppress or improve the phenomenon appearing on the appearance and feel of the skin due to a skin disease or a decrease in function or loss of skin cells. The skin diseases include, for example, inflammatory skin diseases such as atopic dermatitis, skin eczema, acne and psoriasis. Wrinkles occur, skin aging such as blotch, skin elasticity decrease, skin scars, etc. may occur due to deterioration or loss of function of the skin cells.

However, thistle plant extract of the present invention, the plant of the genus Ogapi, stigmasterol of the formula (1) and en-acetylgalactosamine of the formula (2) replaces the animal-derived serum components as described above, and also animal cells, in particular It has been discovered by the inventors that the cells have an ability to promote proliferation of stem cells, preferably adult stem cells. Therefore, when the substances are applied to the skin of the human body, it is possible to improve the skin condition by promoting or activating the proliferation of skin cells, particularly adult stem cells present in the skin. Adult stem cells present in the skin are, for example, stem cells present in the basal layer of the interfollicular epidermis between hair follicles and hair follicles, stem cells present in hair follicles, or adipose derived stems present in the subcutaneous fat layer. It is a cell.

In addition, thistle plant extract of the present invention, the plant of the genus Ogapi, stigmasterol of the formula (1) and en-acetylgalactosamine of the formula (2) can promote and activate the proliferation of various animal cells other than stem cells Discovered by the inventors. Therefore, when the substances are applied to the skin of the human body, it is possible to improve the skin condition thereby promoting or activating the proliferation of fibroblasts and the like present in the skin.

In addition, the thistle plant extract of the present invention, the plant of the genus Ogapi, stigmasterol of the formula (1) and en-acetylgalactosamine of the formula (2) is added to the animal cell culture medium added to replace the serum component of the animal The culture medium from which the animal cells are removed after culturing the cells contains various physiologically active substances (for example, cytokines having stem cell proliferation or activation promoting ability) secreted from animal cells, preferably stem cells. When the culture medium from which the removed animal cells are removed is applied to the human body, an effect of improving skin condition may be obtained by promoting or activating the proliferation of various cells, particularly adult stem cells, etc. present in the human body. In addition, the culture medium from which the animal cells have been removed does not contain various biological contaminants, for example, animal bacteria and animal viruses, as well as proteins such as prions or toxins, which may be derived from animal serum components. , When the culture medium from which the animal cells are removed is applied to the human body, an infection or toxicity problem caused by the contaminant may not be generated.

The composition for improving the skin condition of the present invention may have an effect as described above, thereby improving the skin condition. Specifically, the composition for improving the skin condition of the present invention can improve the skin condition because it is effective in alleviating skin diseases, improving wrinkles, suppressing skin aging, improving skin elasticity, wound recovery such as skin scars, and skin regeneration. have.

The composition for improving the skin condition of the present invention having the above-described effect is a milk thistle plant extract, a plant of the genus Ogapi, stigmasterol of the formula (1), N-acetylgalactosamine of the formula (2), and the culture medium for animal cells At least one material selected from the group consisting of a culture solution from which the cultured animal cells are removed after culturing the animal cells is contained in an amount of 0.01 to 30% by weight based on the total composition. When the content of the material is less than 0.01% by weight, one of the purposes of the present invention, the skin condition improvement effect is not seen, and when it is more than 30% by weight, it is difficult to obtain an improved improvement effect compared to the increased content and the manufacturing cost is increased. have.

The composition for improving the skin condition of the present invention is a skin condition, for example, for the purpose of alleviating skin diseases, improving wrinkles, suppressing skin aging, improving skin elasticity, wound recovery such as skin scars and skin regeneration, and the like, It is preferably provided as a formulation of cosmetics and pharmaceuticals.

As a specific embodiment, the composition for improving the skin condition of the present invention may be provided as a functional food for the purpose of improving the skin condition.

When the composition for improving the skin condition of the present invention is provided in the form of a functional food, it includes ingredients that are commonly added during food production, and include, for example, proteins, carbohydrates, fats, nutrients and seasonings. For example, when prepared as a drink, a flavoring agent in addition to at least one substance selected from the group consisting of thistle plant extract of thistle, an extract of the genus Ogapi, stigmasterol of Formula 1, and en-acetylgalactosamine of Formula 2 Or natural carbohydrates may be included as additional ingredients. For example, natural carbohydrates include monosaccharides (eg, glucose, fructose, etc.); Disaccharides (eg maltose, sucrose, etc.); oligosaccharide; Polysaccharides (eg, dextrins, cyclodextrins, etc.); And sugar alcohols (eg, xylitol, sorbitol, erythritol, and the like). As flavoring agents, natural flavoring agents (eg, taumartin, stevia extract, etc.) and synthetic flavoring agents (eg, saccharin, aspartame, etc.) can be used. Given the easy access to food, the functional food is very useful for improving skin condition.

According to a preferred embodiment of the present invention, the functional food composition comprises a formulation selected from the group consisting of solution, suspension, emulsion, paste, gel, cream, powder, bar, candy, ice cream, oil, and powder emulsion spray. Have

As another specific embodiment, the composition for improving skin condition of the present invention is provided as a cosmetic applied directly to the skin for the purpose of improving the skin condition.

The cosmetics may be prepared in any formulations commonly prepared in the art, for example, solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansing, oils, Powder foundations, emulsion foundations, wax foundations and sprays and the like can be formulated, but is not limited thereto. More specifically, it can be manufactured in the form of a soft lotion, a nutritional lotion, a nutritional cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray or a powder.

When the cosmetic formulation is a paste, cream or gel, animal oils, vegetable oils, waxes, paraffins, starches, trachants, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicas, talc or zinc oxide may be used as carrier components. have.

If the cosmetic formulation is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used, in particular, in the case of a spray, additionally chlorofluorohydrocarbon, propane / Propellant such as butane or dimethyl ether.

In addition, when the cosmetic formulation is a solution or emulsion, a solvent, a solubilizer or an emulsifier is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, Fatty acid esters of 1,3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan.

In addition, when the cosmetic formulation is a suspension, liquid carrier diluents such as water, ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Crystalline cellulose, aluminum metahydroxy, bentonite, agar or tracant and the like can be used.

In addition, when the cosmetic formulation is a surfactant-containing cleansing agent, as a carrier component, an aliphatic alcohol sulfate, an aliphatic alcohol ether sulfate, a sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid Amide ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters and the like can be used.

Ingredients included in the cosmetics of the present invention include ingredients commonly used in cosmetic compositions, in addition to the active and carrier ingredients, and include conventional auxiliaries such as antioxidants, stabilizers, solubilizers, vitamins, pigments and flavorings. can do.

According to a preferred embodiment of the present invention, the cosmetic is a solution, suspension, emulsion, paste, gel, cream, lotion, shampoo, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation and It is a form selected from the group consisting of a spray.

As another specific embodiment, the composition for improving skin condition of the present invention is provided as a medicine for the purpose of improving the skin condition.

The medicament may be prepared in unit dosage form or in multiple dosage forms by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods readily available to those of ordinary skill in the art. It may be prepared by incorporation into a container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

Pharmaceutically acceptable carriers included in the medicament are conventionally used in the preparation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, fine Crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

The medicine may be administered orally or parenterally, preferably parenterally, and in the case of parenteral administration, it may be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, topical administration, transdermal administration, or the like.

In addition, suitable dosages of the medicament may be prescribed in various ways, such as by the method of formulation, mode of administration, age, weight, sex, pathological condition, food, time of administration, route of administration, rate of excretion and response to the patient. have.

Since thistle plant extract of the present invention, the genus Ogapi plant extract, stigmasterol and en-acetylgalactosamine, the serum-free medium for animal cell culture comprising at least one selected from the group consisting of expensive animal serum is not used The manufacturing cost can be significantly lowered, and safer animal cells can be provided by fundamentally blocking the contamination of animal substances generated by using animal serum in the medium. In addition, the composition comprising the culture medium of the material of the present invention and the serum-free medium for animal cell culture as an active ingredient has a proliferation promoting and activating ability of animal cells, in particular stem cells to alleviate skin diseases, skin wrinkles It has an effect of improving skin conditions such as improvement, inhibition of skin aging, improvement of skin elasticity, wound recovery such as skin scar, and skin regeneration efficacy.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example  1: Preparation of thistle extract and its new bioactive substance, stigmasterol ( stigmasterol )

1-1. Preparation of Thistle Extract

Thistle Cirsium japonicum var. ussuriense ) 10 kg of the outpost was dried and pulverized, powdered and extracted by immersion with ethanol for 7 days. The extract was filtered and concentrated to prepare 0.5 kg of thistle extract.

1-2. Preparation of thistle nucleic acid soluble fraction extract

100 g of thistle extract of Example 1-1 was dissolved in 100 ml of 80% methanol, extracted five times with 100 ml of nucleic acid, and the supernatant was filtered and distilled to obtain 9.2 g of thistle soluble fraction extract.

1-3. Thistle Chloroform Soluble Fraction Extract

8.5 g of the water-soluble fraction obtained in Example 1-2 was dissolved in 80% ethanol, concentrated under reduced pressure, and the aqueous layer was extracted five times with 100 ml chloroform to obtain 1.75 g of a chloroform soluble fraction extract.

1-4. From thistle extract Isolated  New bioactive substance, stigmasterol ( stigmasterol )

1 g of the chloroform fraction extract obtained in Example 1-3 was added 80% aqueous ethanol in an ODS flash column to obtain a fraction extract. The fraction extract was purified by silica gel chromatography, divided into four fraction extracts, and the second fraction extract was filtered again on a Sephadex LH-20 column to separate a fraction collector to isolate new active ingredients. The structure of the novel active material was confirmed to be a compound having a molecular weight (molecular weight: 412.70, Stigmasta-5, 22-dien-3beta-ol) by using a device such as NMR, IR, or UV. Stigmasterol '.

According to the result, the chemical structure of stigmasterol is represented by the following Chemical Formula 1.

[Formula 1]

Example  2: Separation of Ogapi Extract and N-acetylgalactosamine

2-1. Preparation of Ogapi Extract

Acanthopanax senticosus Harms) 10 kg of outpost was dried, ground and powdered and extracted by immersion with ethanol for 7 days. The extract was filtered and concentrated to prepare 0.7 kg of Ogapi extract.

2-2. Preparation of Ogapi Nucleic Acid Soluble Fraction Extract

The 100 g of the extract of Example 2-1 was dissolved in 100 ml of 80% methanol, extracted five times with 100 ml of nucleic acid, and the supernatant was filtered and distilled to obtain 10.2 g of the extract of ligated nucleic acid.

2-3. Preparation of Ogapi Chloroform Soluble Fraction Extract

8 g of the water-soluble fraction obtained in Example 2-2 was dissolved in 80% ethanol, concentrated under reduced pressure, and the aqueous layer was extracted five times with 100 ml chloroform to obtain 1.65 g of a chloroform soluble fraction extract.

2-4. N-acetylgalactosamine (N- acetylgalactosamine )

1 g of the chloroform fraction extract obtained in Example 2-3 was added to 80% aqueous ethanol in an ODS flash column to obtain 0.45 g of the fraction extract. The fraction extract was purified by silica gel chromatography, divided into four fraction extracts, and the second fraction extract was filtered again on a Sephadex LH-20 column to separate a fraction collector to isolate new active ingredients. The structure of the novel active material was confirmed to be a compound of 2-Acetamido-2-deoxy-D-galactopyranose (molecular weight: MW 221.21) using a device such as NMR, IR, UV, and the like. N-acetylgalactosamine.

According to the result, the chemical structure of N-acetylgalactosamine is represented by the following Chemical Formula 2.

[Formula 2]

Example  3: extract or from Isolated  Of stem cells in a medium where the compound replaced serum Cell viability  Measure

3-1. Cells, Medium and Culture Methods

The present inventors observed that good stem cell growth was achieved by adding the samples of Examples 1 and 2 to the medium while culturing the stem cells in serum-free medium without serum to find a serum replacement for stem cell culture.

Specifically, solvent extracts of thistle (ethanol, nucleic acid, chloroform), organo solvent extracts (ethanol, nucleic acid, chloroform), and stigmasterol and en-acetylgalactosamine isolated from these as a serum substitute in the stem cell culture medium. After selection and addition, the culture results of stem cells were confirmed.

Stem cells were derived from human umbilical cord blood-derived mesenchymal stem cells and human adipose derived stem cells.

Human cord blood-derived mesenchymal stem cells (CB-MSCs) were obtained from Cha Medical University (Seongnam, Gyeonggi-do, Korea). Cells were cultured at 37 ° C. in Dulbecco's modified eagle medium (DMEM) containing low concentrations of glucose and added 15% FBS, penicillin and streptomycin. Medium was changed every 3 days until the cells reached confluency. Human adipose-derived stem cells (ADSCs) were purchased from Invitrogen (Invitrogen, Carlsbad, Calif., USA). Cryopreserved cells were thawed at 37 ° C. and immediately incubated in MesenPRO RS ^™ medium (Gibco, Carlsbad, CA, USA) containing 2% animal serum. Cells were then grown using MesenPRO RS ^™ medium. In the serum-free condition test group, which was a negative control group, the cell culture medium, MesenPRO RS ^™ medium, was replaced with serum-free DMEM (Cat. 11885, Invitrogen) medium. As a positive control, cells were cultured in MesenPRO RS ^™ medium with DEME medium containing 5% FBS.

3-2. Cell viability ( cell viability ) Measure

Cell viability was measured by MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) analysis. MTT analysis is an analysis method based on the conversion of a substrate containing a tetrazolium ring to blue formazan by mitochondrial dehydrogenase. The level of blue formazan was spectroscopically measured and used as an indirect indicator of cell density. Briefly, cells were exposed to MTT (1 mg / ml) for 3 hours at 37 ° C., then the medium was removed and the cells were lysed with dimethyl sulfoxide (DMSO). The presence of blue formazan after complete dissolution was determined spectroscopically by measuring absorbance at wavelength 570 nm.

The MTT assay was used to evaluate the effects of thistle and agar's solvent extracts (ethanol, nucleic acid, chloroform), stigmasterol, and N-acetylgalactosamine on stem cell proliferation. Observed. Among them, the ethanol extract of thistle, the ethanol extract of Ogapi, stigmasterol and en-acetylgalactosamine were confirmed to significantly induce the proliferation of stem cells (see Table 1 below). Thistle ethanol extract, agar ethanol extract, stigmasterol and en-acetylgalactosamine significantly increased the growth rate of cord blood-derived mesenchymal stem cells (CB-MSCs) to 20.6%, 26.7%, 32.4% and 42.7%, respectively. In addition, the cell growth rate of adipose derived stem cells (ADSCs) was also significantly increased to 21.5%, 22.2%, 35.5% and 45.4%, respectively (see Table 1 below).

Example  4: extract or from Isolated  Containing compound Serum free  Of animal cells in the medium Cell viability  Measure

In this example, to find out whether thistle extract, organ extract, and stigmasterol, a new bioactive substance isolated from it, can promote the proliferation of other animal cells, human keratinocytes HaCaT and mouse fibroblasts ( NIH3T3 cells, mouse fibroblasts, were cultured in serum-free medium containing thistle extract, ooga extract and compounds isolated from these, stigmasterol and N-acetylgalactosamine The effect was observed.

The experiment of this example was carried out as follows. HaCaT and NIH3T3 cells 3 × 10 ⁵ cultured in DMEM medium containing 10% FBS, 1% antibiotics at 5% CO ₂ and 37 ° C. were inoculated into 6 well plates, and then serum After washing once with the culture solution, the serum-free condition, thistle extract, organ extract and stigmasterol and N-acetylgalactosamine isolated from them replaced 10% FBS, respectively. The experimental groups were divided and cultured by the serum-free condition added thereto. After 72 hours, the cells were washed once with serum-free medium, and then replaced with serum-free medium containing 10% MTT. After 3 hours, OD values were measured and converted into percentages.

As shown in Table 2, when the normal group (including serum) is converted to 100%, thistle extract, organ extract and stigmasterol (stigmasterol) and ene, which are isolated from them, compared to the serum-free condition (54%) -Cell growth rate of 20% or more was increased under the addition of N-acetylgalactosamine, respectively, Thistle extract, Ogai extract, and compounds isolated from these, stigmasterol and N-acetylgalactosamine (N -acetylgalactosamine) has the effect of promoting the cell proliferation of not only stem cells but also general animal cells.

Example  5: extract or from Isolated  Containing compound Serum free  Of fibroblasts in the medium Cell viability  Measure

After treatment with thistle extract, organ extract and stigmasterol (N-acetylgalactosamine), which are isolated from these, in serum-free medium using human normal fibroblasts The effect on cell viability was observed. In this experiment, human normal fibroblasts 3 × 10 ⁵ cultured in DMEM medium containing 10% FBS and 1% antibiotics at 5% CO ₂ and 37 ° C. were inoculated into 6 well plates, and then serum was added the following day. After washing once with the removed broth, serum-free and this serum-free condition were added with thistle extract, organ extract and stigmasterol and N-acetylgalactosamine, respectively. The experimental groups were divided and cultured. After 72 hours, the cells were washed once with serum-free medium, and then replaced with serum-free medium containing 10% MTT. After 3 hours, OD values were measured and converted into percentages. The results are shown in Table 3 below.

As shown in the experimental results of Table 3 below, thistle extract, agaga extract and negative when treated with stigmasterol (stigmasterol) and N-acetylgalactosamine (N-acetylgalactosamine) isolated from them It showed a higher cell growth promoting effect than the control. Therefore, it was confirmed that thistle extract, organ extract and compounds isolated from these stigmasterol (stigmasterol) and N-acetylgalactosamine (N-acetylgalactosamine) promotes cell proliferation in fibroblasts.

Example  6: extract or from Isolated  Enhancement of Collagen Synthesis of Fibroblasts by Compounds

Promoting collagen biosynthesis of fibroblasts is a major indicator of preventing and alleviating wrinkles in the skin. Wrinkles are commonly known to occur as the biosynthesis of collagen decreases and the collagen produced breaks down. Therefore, if the collagen synthesis of the fibroblasts present in the dermal layer is promoted, the wrinkles as well as the generated wrinkles can be improved.

Human normal fibroblasts (Pacific) were inoculated into 6-well microplates containing DMEM medium (2 × 10 ⁵ cells / well) and incubated at 37 ° C. for 24 hours in a CO ₂ incubator at 5% concentration. . After 24 hours, the medium was removed from each well, and then treated with thistle extract, organ extract and compounds isolated from them, stigmasterol and N-acetylgalactosamine, and incubated again for 24 hours. It was. After 24 hours, cell medium was collected to prepare a sample. Collagen synthesis amount was measured by using a collagen measurement kit (Procollagen Type I C-peptide EIA kit; MK101; Takara, Kyoto, Japan) to measure the amount of Procollagen Type I C-peptide (PICP) . The experiment was performed according to the method described in the kit instructions. The measured standard values were expressed in the form of mean ㅁ standard deviation, and the significance was tested by t-test using the SPSS / PC + program. The experimental results are shown in Table 4 below.

As can be seen from the results of Table 4 below, thistle extract, organ extract and compounds isolated from these stigmasterol and N-acetylgalactosamine (N-acetylgalactosamine) are all higher collagen biosynthesis than the negative control group The rate is shown. In particular, Ogapi extract and N-acetylgalactosamine isolated therefrom showed higher collagen biosynthesis rate compared to thistle extract and stigmasterol isolated therefrom.

Example  7: extract or from Isolated  Inhibitory Effects of Compounds on Collagen Degrading Enzymes

Collagen is a structural protein constituting the dermal layer and wrinkles are produced when the amount is reduced. On the other hand, if the collagen is made well maintained wrinkles can be suppressed. Therefore, inhibiting the activity of collagenase is a major physiological indicator of anti-aging.

Human normal fibroblasts (Pacific) were inoculated into 6-well microplates containing DMEM medium (2 × 10 ⁵ cells / well) and incubated at 37 ° C. for 24 hours in a CO ₂ incubator at 5% concentration. . After 24 hours, the medium was removed from each well, and then treated with thistle extract, organ extract and compounds isolated from them, stigmasterol and N-acetylgalactosamine, respectively, and again for 24 hours. Incubated. After 24 hours, cell media were collected to prepare samples. An antibody against collagenase was used as a method of measuring the activity of collagenase, an enzyme that degrades collagen. PMA (phorbol myristate acetate) was treated with a substance that induces collagenase activity. Experiments were performed according to the method described in the manufacturer's instructions in a commercially available kit. Type I collagenase assay kit (Amersham Biosciences, RPN2629) was used and the absorbance was measured by ELISA reader. The measured standard values were expressed in the form of mean ㅁ standard deviation and tested for significance by t-test using the SPSS / PC + program. The experimental results are shown in Table 5 below.

As can be seen in Table 5, thistle extract, agai extract and the compounds isolated from these stigmasterol (stigmasterol) and N-acetylgalactosamine (N-acetylgalactosamine) all show an effect of inhibiting collagen degrading enzyme activity of 20% or more It was.

Example  8: extract or from Isolated  Activation of Skin Stem Cells Using Stem Cell Cultures Containing Compounds

Skin stem cells were treated with stem cell culture cultured in serum-free medium supplemented with thistle extract, organ extract and compounds isolated from them, stigmasterol and N-acetylgalactosamine. The degree of activation of stem cells was measured.

The skin stem cells used in this experiment were isolated according to the epidermal stem cell separation method described in the Korean Dermatological Research Society (2006; 13 (1): 1-4). That is, skin stem cells (ESCs) were obtained by separating cells having adhesion to type 4 collagen from skin tissues or cells obtained from skin. Thus obtained skin stem cells were inoculated into 6 well plates with 3 X 10 ⁵ cells, and then, washed once with a serum-free culture medium, and then serum-free (SF), thistle extract, organ extract and compounds isolated therefrom. Phosphatesterol (stigmasterol) and N-acetylgalactosamine (N-acetylgalactosamine) was added to the experimental group of serum-free (P / SF), respectively. After incubation for 72 hours, MTT analysis was performed. The results are shown in Table 6 below.

As shown in Table 6, when the normal group (CM) is converted to 100%, compared to the experimental group treated with serum-free condition, thistle stigmasterol (stigmasterol) isolated from thistle extract or thistle Survival rate of skin stem cells was significantly increased in the experimental group (P / SF) to which serum-free condition was added.

Example  9: extract or from Isolated  Preparation of external composition for improving skin condition containing compound

The test product was made from an oil in water emulsion formulation, which is used to make thistle extract or stigmasterol, a new bioactive substance isolated from thistle, generally into external skin preparations such as cosmetics and ointments. The composition of each test product is shown in Table 7 below. The aqueous phase purified water, triethanol amine and propylene glycol were dissolved by heating to 70 占 폚, and then the oily fatty acid, oily component, emulsifier and preservative were heated to 70 占 폚 and dissolved to add the emulsified solution. After emulsification was completed, the solution was cooled to 45 ° C. and 20 ppm of stigmasterol, a new bioactive substance isolated from thistle extract or thistle, was added.

Example  10: extract or from there Isolated  Acne improvement effect of the composition containing the compound

Inflammatory skin improvement effects of thistle extract, organ extract and compounds isolated from them, stigmasterol and N-acetylgalactosamine itself, were measured through clinical trials. Specifically, 20 ppm of thistle extracts, organ extracts and stigmasterol (N-acetylgalactosamine), which are compounds isolated from them, were included in patients with acne (40 patients each). Creams (test groups 1 to 8) were applied to the left side of the face of acne skin subjects, and on the other side thistle extracts, organ extracts and stigmasterol and N-acetylgalactosamine (N-acetylgalactosamine) compounds isolated from them. ) Was not applied as a control (controls 1 and 2), respectively. The application number of times was 1 day / 2 times for 1 month, and after 1 month of use, acne improvement was measured. The measurement was carried out by drawing an imaginary three-square centimeter square below two centimeters below the eyes under the condition that the temperature was kept constant at 24-26 ° C. and 38-40% of humidity, and then counting the number of acne entering therein. The results are shown in Table 8 below.

As can be seen in Table 8, it was confirmed that the cream according to the present invention has an acne improvement effect compared to the cream of the control group 1. As a result, it was confirmed that the symptoms improved only in the left face of the thistle extract, organ extract, and the cream containing stigmasterol and N-acetylgalactosamine, respectively, isolated from them. .

Example  11: extract or from Isolated  Skin inflammation improvement effect of the composition containing the compound

Chronic inflammatory skin improvement effects such as atopy or psoriasis of thistle extract, organ extract, and stigmasterol and N-acetylgalactosamine isolated from them were measured through clinical trials. Specifically, this study contained 20 ppm each of thistle extracts, organ extracts, and stigmasterol and N-acetylgalactosamine, which are isolated from these, for patients with atopic dermatitis (60 patients per test). One cream (test groups 1 to 8) and a cream containing no extracts and compounds (controls 1 and 2) were allowed to be used throughout the body for 1 month, and then the atopic improvement effect was measured. In the measurement, the temperature was maintained at 24-26 ℃ and the humidity was 38-40%, and 6 spots were randomly selected around the fold of the arm, and the erythema was calmed using Minolta's CR-300 colorimeter. After measuring the itch through a questionnaire, two values were corrected to quantify the final skin improvement. As for the criterion of comparison, the relative value was set by setting 0 as the case where no change occurred and 1 to 5 degree of improvement. The higher the value, the better the erythema and itching. The worsening cases are indicated from -1 to -5. The experimental results for this are shown in Table 9 below.

Example  12: extract or therefrom Isolated  Anti-aging Effect of External Composition for Skin Improvement Containing Compound

Anti-aging effects on the human body can be determined by various criteria, but wrinkles on the skin can be easily measured. In this experiment, the skin wrinkle improvement effects of thistle extract, organ extract and compounds isolated from them, stigmasterol (stigmasterol) and N-acetylgalactosamine (N-acetylgalactosamine) were measured. Specifically, the creams of the eight types and the control group prepared in Table 7 were applied to the face of 20 healthy women (25 to 35 years old) for 1 day / 2 times for 3 months, and then the temperature was 24-26. Wrinkle improvement effect was measured using Cutometer SEM 474 (Courage + Khazaka, Cologne, Germany) under the condition of maintaining a constant humidity of 38-40% ℃. As a criterion of comparison, the relative elasticity was compared with the case where skin elasticity was not 0 and many cases were 5. The results are shown in Table 10 below.

As can be seen in Table 10, thistle cream according to the invention, Ogai extract and a cream containing the compounds isolated from them stigmasterol (stigmasterol) and N-acetylgalactosamine (N-acetylgalactosamine), respectively (test group 1 8 to 8) it was found that the wrinkle improvement effect is much better than the cream (control) not containing them at all. That is, it was found that thistle extract, organ extract and compounds isolated from these stigmasterol (stigmasterol) and N-acetylgalactosamine (N-acetylgalactosamine) all have the effect of improving skin wrinkles.

Example  13: extract or from Isolated  Anti-aging Effect by Oral Administration of Compounds

Aging is a phenomenon that occurs with time in all living organisms and appears in all tissues and organs. In the case of animals, the skin is the largest organ that constitutes the outermost part of the individual. Wrinkles on the skin are the most easily distinguishable indicators of aging. In particular, when irradiated with UV light, the aging process can be artificially promoted. Therefore, after the UV light is removed from the animal test, the test substance is administered orally or transdermally and the effect is measured to determine whether the substance has an anti-aging effect. .

Therefore, in this experiment, to investigate the effects of thistle extracts, organ extracts, compositions containing stigmasterol and N-acetylgalactosamine isolated from them on the photoaging symptoms The mice were selected as an animal model and tested. Female hairless mice (Skh: HR-1) of 6-7 weeks old (Skh: HR-1) per group, UV control group, UV / thistle extract group, UV / Organic extract group, UV / stigmasterol group And UV / N-acetylgalactosamine (N-acetylgalactosamine) administration group was bred during the experimental period. The normal group and the UV control group were orally administered with 0.5 ml of saline solution, UV / thistle extract group, UV / Organic extract group, UV / stigmasterol group, and UV / En-acetylgalactosamine (N-acetylgalactosamine). The administration group was orally administered with a liquid administration syringe by mixing 500 mg of thistle extract, organ extract, stigmasterol or en-acetylgalactosamine in 0.5 ml saline on a solids basis.

The administration period was 5 weeks in total and administered at the same time 5 days a week. From 2 to 5 weeks after oral administration, Note 3 to the UV control group, UV / thistle extract group, UV / organic extract group, UV / stigmasterol group and UV / N-acetylgalactosamine group. UV was irradiated similarly to gray sunlight. In this case, the total UV radiation amount was 600 mJ / cm ² during the experiment. For the objective determination of the anti-aging effect as the main phenomenon of anti-aging effect, the skin template was taken from the back of hairless mouse using silicon polymer before necropsy. The molds were sampled to compare the anti-aging effect, that is, the anti-wrinkle effect. The results are shown in Table 11 below.

As can be seen in Table 11, R1, R2, R3, R4, and R5 values indicating wrinkle improvement are all thistle extracts, organ extracts, and new bioactive substances isolated from these, stigmasterol and en-acetylgal. Hairless mice each administered lactosamine were significantly reduced compared to the UV / control group without the extract and compound. Through this result, even if this oral administration of a composition containing thistle extract, organ extract of the present invention, stigmasterol (stigmasterol) and N-acetylgalactosamine (N-acetylgalactosamine) isolated from them exhibits anti-wrinkle effect It was confirmed that there is an aging effect.

Example  14: extract or therefrom Isolated  Of hairless mice by oral administration of compounds Percutaneously  Evaluation of Efficacy on Activation of Existing Skin Stem Cells

As in Example 13, this week for 5 weeks a total of five weeks this week, thistle extract, ooga extract, stigmasterol and N-acetylgalactosamine (N-acetylgalactosamine) isolated from these hairless mice, respectively After oral administration, dermal stem cells were isolated from the percutaneous skin. Separation method of skin stem cells was carried out according to the method described in Example 8. The isolated skin stem cells were inoculated into 6 well plates with 3 × 10 5 cells and then placed therein, and then cultured in a medium containing serum for 48 hours, and then cell activity was measured by MTT assay (cell viability measurement method). The results are shown in Table 12 below.

As shown in Table 12, it was observed that the stem cell proliferation rate increased compared to the UV / control group. These results indicate that thistle extracts, organ extracts, and stigmasterol and N-acetylgalactosamine, compounds isolated therefrom, have an effect on the activation of adult stem cells. .

Example  15: Acute Oral Toxicity Test

Acute oral toxicity tests are conducted to determine whether thistle extract, organ extract, and the compounds isolated from these, stigmasterol and N-acetylgalactosamine, can be safely applied to medicines, foods and cosmetics. It was. Specifically, acute toxicity test was conducted under the following conditions using 20 SPF SD rats 5 to 6 weeks old as experimental animals.

Temperature and humidity conditions: temperature 22 ± 2 ℃. Relative Humidity 50 ± 10%

ㆍ Contrast cycle: Fluorescent lighting (08 o'clock, 20 o'clock off)

Roughness: 200 to 300 Lux

ㆍ Free drinking water treated with ultraviolet rays

ㆍ Dilute the test substance in sterile distilled water to prepare a sample for administration.

On the day of administration, the general condition was observed every hour up to 4 hours, and once a day from the day after the administration of the administration, the general condition change, intoxication symptoms, motility, appearance, autonomic nerves and the presence of dead animals were carefully observed. Pathological anatomical verification was performed to determine the toxicity. The results are shown in Table 13 below. As can be seen in Table 13, thistle extract of the present invention, Ogai extract, the compounds isolated from these stigmasterol (stigmasterol) and N-acetylgalactosamine (N-acetylgalactosamine) are all non-toxic, drug, It can be used safely in food and cosmetics.

Example  16: Manufacture of health food composition

Thistle extract of the present invention, the organ extract, the stigmasterol (stigmasterol) and N-acetylgalactosamine (N-acetylgalactosamine) isolated from these in Example 15 showed no toxicity, as seen in Example 13, etc. Since the above substances of the present invention had an anti-aging effect, they tried to develop health foods based on them. Specifically, it was prepared by the conventional liquid preparation method in the same composition as the preparation example shown in Table 14. The final amount of each liquid was 100 ml. The following composition example is one example, and may be prepared in various forms used in general food forms as well as liquids, tablets, powders, granules and the like.

Example  17. Energy synergy

Thistle ethanol extract (test group 1), thistle nucleic acid extract (test group 2), thistle chloroform extract (test group 3), stigmasterol (test group 4), organo ethanol extract (test group 5), used in Example 14, The energy synergistic effect was observed in hairless mice using the organo nucleic acid extract (test group 6), the organo chloroform extract (test group 7), and en-acetylgalactosamine (test group 8). Specifically, the composition of the test group was orally inserted into the hairless mice at the same time for a total of four weeks, five days a week, and sacrificed five weeks later, and then splenocytes (SP), peripheral blood mononuclear cells (PBMC), and lymph node (LN) from the organs. After separation of immune cells, the effect of the test group on ATP synthesis was observed in comparison with the control group. Each cell was washed twice with PBS to obtain a lysate, and then centrifuged by protein removal using 0.6 M perchloric acid. The supernatant was obtained and neutralized with KOH so that protein pellets were not incorporated, and then centrifuged lightly to remove potassium perchlorate. The resulting supernatant was used for ATP synthesis. ATP was measured by reduction of NADP + in the presence of glucose, hexokinase, and glucose-6-phosphate dehydrogenase. Oligomycin was used as an inhibitor of ATPase activity. The results are shown in Table 15 below.

As can be seen in Table 15, thistle ethanol extract (test group 1), thistle nucleic acid extract (test group 2), thistle chloroform extract (test group 3), steig masterol (test group 4), Ogapi ethanol extract (test group 5) ), Organo nucleic acid extract (test group 6), organo chloroform extract (test group 7), and en-acetylgalactosamine (test group 8) were all confirmed to have an energy synergistic effect on immune cells.

Claims (12)

Serum-free medium for animal cell culture comprising an extract of the genus Ogapi by replacing animal-derived serum components. The serum-free medium for animal cell culture according to claim 1, wherein the extract of the genus Ogapi is extracted with a solvent in which water, lower alcohol, nucleic acid, and chloroform are used alone or in combination of two or more thereof. The serum-free medium for animal cell culture according to claim 1, wherein the replacement of the animal-derived serum is that at least 50% of the animal-derived serum content added to the animal cell culture medium is instead added as an extract of the genus Ogapi. The serum-free medium for animal cell culture according to claim 1, wherein the animal cell is an animal-derived stem cell or skin cell. Cosmetics for skin condition improvement comprising a culture solution from which cultured cells are removed after culturing animal cells in a serum-free medium for cultivating animal cells, which comprises extracts of plants of the genus Ogapi or serum components of animal origin. Composition. The cosmetic composition for improving skin condition according to claim 5, wherein the extract of the genus Ogapi is extracted by a solvent in which water, lower alcohol, nucleic acid, and chloroform are used alone or in combination of two or more thereof. The skin condition according to claim 5, wherein the skin condition improvement is at least one selected from the group consisting of alleviation of inflammatory skin disease, improvement of skin wrinkles, inhibition of skin aging, improvement of skin elasticity, wound recovery and skin regeneration. Improvement cosmetic composition. According to claim 7, wherein the inflammatory skin disease is atopic dermatitis, skin eczema (eczema), acne or psoriasis cosmetic composition for improving skin conditions, characterized in that psoriasis. The cosmetic composition for improving skin condition according to claim 5, wherein the animal cells are stem cells or skin cells. The culture medium according to claim 5, wherein the culture medium is removed from the cultured animal cells after culturing the animal cells in a serum-free medium for culturing the animal cells, including the extracts of the plants of the genus Ogapi by replacing the extract of the genus Ogapi or animal-derived serum components. Cosmetic composition for improving skin conditions, characterized in that to promote or activate the proliferation of stem cells or skin cells. Pharmaceutical for skin condition improvement comprising a culture medium in which animal cells are cultured in a serum-free medium for animal cell culture containing an extract of the genus Ogapi by replacing an extract of the genus Ogapi or an animal-derived serum component and then removing the cultured animal cells Composition. Food for skin condition improvement comprising a culture solution from which cultured cells are removed after culturing animal cells in a serum-free medium for cultivating animal cells, which comprises extracts of plants of the genus Ogapi or serum components of animal origin Composition.