KR102354282B1 - Cosmetic composition comprising erythorbic acid - Google Patents

Abstract

The present invention relates to a composition for inducing differentiation of preadipocytes or stem cells into adipocytes, comprising erythorbic acid. In addition, the present invention provides a cosmetic composition comprising erythorbic acid and an external preparation for skin, (a) culturing stem cells; and (b) adding a composition comprising erythorbic acid to the stem cells of step (a). The erythorbic acid of the present invention promotes the differentiation of adipose tissue-derived stem cells into mature adipocytes by promoting the expression of peroxisome proliferator-activated receptor gamma (PPAR γ), which is a key regulator of adipogenesis, thereby reducing the volume due to adipose tissue shrinkage and It can present a new possibility to improve the aesthetic and health problems of modern people caused by functional deterioration.

Description

Cosmetic composition comprising erythorbic acid

The present invention relates to a composition for inducing differentiation of preadipocytes or stem cells into adipocytes, comprising erythorbic acid. The present invention also relates to a cosmetic composition containing erythorbic acid and an external preparation for skin, and a method for inducing differentiation from stem cells into adipocytes using the composition containing erythorbic acid.

The subcutaneous tissue is located between the dermis and the fascia and is a connective tissue including adipocytes, pre-adipocytes, adipose-derived stem cells, blood vessels, and neural networks that store body fat. Subcutaneous fat basically functions as energy storage, body temperature protection from external cold and heat, and buffering action. In addition, by secreting various hormones, growth factors and cytokines to regulate immunity, body temperature control, skin tissue growth and aging, it can play a role in improving the appearance of the skin through skin regeneration and suppression of wrinkle formation. . In addition, it provides elasticity and firmness to the skin, greatly affecting the shape of the individual's external state.

It is well known that such subcutaneous fat significantly reduces the overall volume of the tissue as it ages, and its function deteriorates. However, in modern times, adipose tissue tends to shrink even at a relatively young age due to the imbalance of influence caused by excessive diet. This causes problems such as reduction in skin elasticity and strength due to reduction in adipose tissue volume, as well as structural collapse due to loss of strength, promotion of aging due to deterioration of skin tissue and function as an endocrine organ that regulates systemic metabolism, reduced immunity and regeneration do.

In order to solve this problem, a method such as autologous fat grafting using an invasive method is currently being implemented. It is a method of collecting and moving fat from the abdominal and thigh areas, which are relatively rich in fat, and can partially improve the reduction of subcutaneous fat due to aging or skin sunken due to burns, surgery, and scars. However, it has to be relatively expensive, the engraftment rate of autologous adipose tissue after surgery is not constant, and it is an invasive method that has to use a surgical method. Accordingly, there is a growing demand for a non-invasive method capable of recovering the regression of the subcutaneous fat layer while compensating for these shortcomings.

Many recent studies have demonstrated that mesenchymal-type pluripotent stem cells exist in the subcutaneous fat. These stem cells are called adipose tissue-derived stem cells (ADSC), and according to environmental signals, they differentiate into mature adipocytes. In addition, it maintains the homeostasis of fat and skin tissue by secreting various hormones and cytokines. In addition, the engraftment of adipose tissue after autologous fat grafting is regulated by the content of adipose tissue-derived stem cells. That is, by inducing adipose tissue-derived stem cells to differentiate into subcutaneous fat, a non-invasive and safe fat regeneration with the same effect as autologous fat graft surgery can be expected.

In addition, the epidermis, which is located at the outermost part of the skin, performs a protective function of protecting against various external physical, chemical and mechanical stimuli and preventing excessive dissipation of body moisture through the skin. This protective function is made possible by the normal formation and maintenance of the stratum corneum composed of keratinocytes. Keratinocytes are cells that are formed through gradual changes in shape and function as basal cells that have continuously proliferated in the stratum basale move to the stratum corneum, and after a certain period of time, old keratinocytes The forming cells are removed from the skin, and the process of epidermis differentiation or keratinization is repeated in which new keratinocytes that rise from the lowermost layer of the epidermis take over their functions. In this keratinization process, keratinocytes generate natural moisturizing factor (NMF) and intercellular lipids such as ceramide, cholesterol, and fatty acids, and the stratum corneum acts as a blocking layer from the outside, thereby creating a skin barrier. retains its function as

In addition, dry skin, considered one of the major diseases in modern society, is one of the symptoms caused by abnormal skin barrier function. Recently, environmental pollution, an increase in dry environments such as apartments and high-rise buildings, an increase in social stress, It is increasing due to various factors such as excessive bathing culture and skin aging, and the number of cases requiring treatment due to severe symptoms is also continuously increasing. Recently, atopic dermatitis, which occurs in 10% of children, is also known to be the main cause of dry skin, more fundamentally, abnormal skin barrier function. In order to treat such atopic dermatitis, conventionally, many studies have been conducted to supply moisture from the outside or to minimize moisture loss from the body by focusing on maintaining adequate moisture in the skin, and in fact, ceramides ( Moisturizers such as ceramide) or derivatives thereof have been developed and are widely used in pharmaceutical or cosmetic fields.

However, most of the use of such a moisturizer is only a temporary relief of symptoms rather than a fundamental treatment, and thus does not exhibit sufficient effect on the treatment of dry skin including atopic dermatitis and skin barrier function abnormalities. Accordingly, there is an urgent need to develop a material that fundamentally regenerates the damaged skin barrier.

Recently, when PPAR α (Peroxisome Proliferator Activated Receptor-alpha) present in keratinocytes binds to its ligand and is activated, it has been reported that it has the effect of reconstructing the damaged skin barrier by promoting the differentiation of keratinocytes. In fact, it has been confirmed that, when Clofibrate or WY14643, which are known agonists of PPARα, is applied to the skin with damaged skin barrier, the differentiation of keratinocytes is promoted and the recovery of the skin barrier is promoted. .

On the other hand, erythorbic acid (erythorbic acid) is a compound having a structure of the following formula (1), also called isoascorbic acid and D-amaboascorbic acid, ascorbic acid, that is, a stereoisomer of vitamin C (stereoisomer). In addition to the above names, the compound (5R)-5-[(1R)-1,2-dihydroxyethyl]-3,4-dihydroxyfuran-2(5H)-one ((5R)-5 Also called -[(1R)-1,2-Dihydroxyethyl]-3,4-dihydroxyfuran-2(5H)-one), in addition to D-araboascorbic acid, erythorbate , isoascorbic acid (Isoascorbic acid), or E315, the chemical formula is C ₆ H ₈ O _{6 It} is a compound with a molecular weight of 176.12.

[Formula 1]

Erysorbic acid is white or yellow crystals or crystalline powder, has no odor but has a slightly sour taste, is not distributed in nature, and does not exist in food, but is made from glucose, sucrose, etc. by microorganisms. Melting point is 164~171℃, soluble in water, acetone and alcohol, slightly soluble in glycerin, insoluble in ether or benzene. It has similar chemical properties to ascorbic acid and has strong reducing power, but is weak to heat or light, and is characterized by markedly accelerated oxidation and decomposition by heavy metal ions such as copper ions. turns black

On the other hand, ascorbic acid, an isomer of erythorbic acid, is called vitamin C as one of the water-soluble vitamins. is included However, in manufacturing the cosmetic composition, most are prepared at high temperature rather than at room temperature, and even when manufactured at high temperature, it is common to be prepared through several steps in various temperature ranges. In this case, ascorbic acid is As it is easily oxidized, the stability of the formulation is lowered, and thus there is a problem in whether the efficacy of ascorbic acid is actually maintained as it is.

Under this background, the present inventors found that erythorbic acid is stable even in heat treatment compared to ascorbic acid, and in particular, by promoting the expression of Peroxisome proliferator-activated receptor gamma (PPAR γ), a key regulator of adipogenesis, the mature adipose tissue-derived stem cells The present invention was completed by discovering that erythorbic acid promotes differentiation into adipocytes and by revealing for the first time the previously unknown function of erythorbic acid to regulate adipocyte differentiation.

It is an object of the present invention to provide a composition for inducing differentiation of preadipocytes or stem cells into adipocytes, comprising an erythorbic acid compound or an acceptable salt thereof.

Another object of the present invention is to provide a cosmetic composition comprising an erythorbic acid compound or a cosmetically acceptable salt thereof.

Another object of the present invention is to provide an external preparation for skin comprising an erythorbic acid compound or an acceptable salt thereof.

Another object of the present invention comprises the steps of (a) culturing stem cells; and (b) adding the composition comprising ersorbic acid to the stem cells of step (a).

The present invention provides a composition for inducing differentiation of preadipocytes or stem cells into adipocytes, comprising an erythorbic acid compound or an acceptable salt thereof, to solve the above problems and achieve the object of the present invention to provide.

In the present invention, "erythorbic acid (Erythorbic acid)" is a compound represented by the structure of the following formula (1), molecular formula C ₆ H ₈ O ₆ , has a molecular weight of 176.12. Erythorbic acid is a stereoisomer of ascorbic acid, that is, vitamin C. In addition to the above term, erythorbic acid has various names, specifically isoascorbic acid, D-amaboascorbic acid, or (5R)-5-[(1R) )-1,2-dihydroxyethyl]-3,4-dihydroxyfuran-2(5H)-one ((5R)-5-[(1R)-1,2-Dihydroxyethyl]-3,4- It is a compound named dihydroxyfuran-2(5H)-one).

[Formula 1]

The present invention is not particularly limited to the method for obtaining erythorbic acid, and may be chemically synthesized by a method known in the art, or a commercially available material may be used.

The erythorbic acid represented by Formula 1 of the present invention may exist in a non-solvated form as well as a solvated form. The erythorbic acid of the present invention may exist in crystalline or amorphous form, and all such physical forms are included within the scope of the present invention.

In the composition according to the present invention, the content of erythorbic acid may include 0.0001 to 50% by weight of erythorbic acid, specifically 0.01 to 10% by weight, based on the total weight of the composition. There is an advantage of exhibiting an excellent lipofilling, fat filling or skin regeneration effect within the above range, and there is an advantage in that the formulation of the composition is stabilized.

In one embodiment of the present invention, as a result of comparing the degree of adipocyte differentiation after heat treatment of erythorbic acid and ascorbic acid at 40° C. for 30 minutes, ascorbic acid is easily oxidized during heat treatment and has poor stability, whereas erythorbic acid is By confirming that the stability to heat is relatively excellent, it could be confirmed that the efficacy can be stably present in the formulation of the cosmetic composition, particularly distributed through various temperatures (Table 5).

In the present invention, the term "progenitor cell" is referred to as a committed stem cell, and when a specific differentiation-forming expression of a cell (X) corresponding to a progeny is revealed, an undifferentiated parent cell that does not express the differentiation trait is a progenitor cell of X call it In addition, "preadipocytes" refers to progenitor cells whose differentiation into adipocytes has been revealed.

In the present invention, the term "stem cell" refers to a cell that exists in all multicellular organisms as undifferentiated cells of animals, and has the ability to reproduce indefinitely, maintain normal chromosomes, and differentiate into various cells. Since it was discovered by Ernest A. McCulloch and James E. Till of Ontario Cancer Institute in Canada in the 1960s, in particular, due to the totipotent potential of being able to differentiate into any cell of the tissues constituting more than 210 organs in the body, the human body known as the master cell of These stem cells are not limited thereto, but may be embryonic stem cells, adult stem cells, umbilical cord blood stem cells, mesenchymal stem cells, or human adipose tissue-derived stem cells, depending on the origin. More specifically, it may be a mesenchymal stem cell, or more specifically, it may be an adipose tissue-derived mesenchymal stem cell.

As used herein, the term "animal" includes not only humans and primates, but also domestic animals such as cattle, pigs, sheep, horses, dogs, mice, rats or cats, specifically humans.

As used herein, the term "embryonic stem cell (ESC)" is a stem cell derived from the inner cell mass (ICM) of the blastocyst stage, which is one of the early developmental stages of the embryo. About 4-5 days later, it has 50-150 cells, and these cells are stem cells with pluripotent differentiation ability that can divide into more stem cells or differentiate into tridermal cells. .

As used herein, the term "umbilical cord blood stem cells" refers to stem cells present in blood from the placenta and fetus' umbilical cord, and has a stronger proliferation than bone marrow-derived stem cells.

As used herein, the term "mesenchymal stem cells" refers to mesenchymal stem cells that are differentiated into mesenchymal tissues such as muscle, bone, and fat, which exist in the bone marrow and are adult stem cells having the ability to differentiate into cells such as nerves and skin. It refers to one of the cells (adult stem cell, AS). These adult stem cells are found in organs that repeat continuous cell replacement, and these organs include the epidermal layer of the skin, the lining of the small intestine, bone marrow, brain, tendons, and the like.

As used herein, the term "adipose tissue-derived stem cells" refers to cells with multipotency distributed in adipose tissue. They have the same level of differentiation potential as those of bone marrow mesenchymal stem cells, and it is reported that they are superior to bone marrow mesenchymal stem cells in maintaining colony forming ability and proliferative capacity in cell culture. Adipose tissue-derived stem cells increase the growth rate and cell cycle when antioxidants such as N-acetyl-L-cystein and ascorbic acid-2-phosphate are added and the calcium concentration is low. In addition, the proliferation of adipose tissue-derived stem cells can be stimulated by FGF-2 (fibroblast growth factor), and adipocyte-derived stem cells are vascular endothelial growth factor (VEGF), hepatocyte growth factor (hepatocyte growth factor). It is known to secrete potential growth factors such as factor, HGF) and insulin like growth factor (IGF). Unlike stem cells isolated from bone marrow or umbilical cord blood, adipose tissue-derived stem cells have the advantage that they can be collected repeatedly and do not cause pain to the patient when collected like bone marrow. It has the advantage of no rejection. In addition, it is easier to culture than bone marrow-derived mesenchymal stem cells, the amount of isolated mesenchymal stem cells is 100 to 1000 times higher than that of bone marrow, and self-renewal ability and multipotent ability are also known to be excellent.

As used herein, the term “differentiation into adipocytes” refers to the differentiation of preadipocytes or stem cells of an animal, specifically, a mammal into adipocytes. As used herein, the term “composition for inducing differentiation” refers to a composition capable of inducing a process in which cells in the initial stage have characteristics as individual tissues, and for the purpose of the present invention, the preadipocytes or stem cells are transformed into adipocytes. means a composition capable of inducing differentiation into Specifically, the composition for inducing differentiation can induce the differentiation of adipocytes by promoting the expression of PPAR γ. In addition, the composition may activate PPAR α to promote differentiation of keratinocytes, thereby inducing skin regeneration to reconstruct a damaged skin barrier.

In the present invention, the term, "PPAR γ (Peroxisome Proliferator Activated Receptor-gamma)" is a peroxisome proliferator activated receptor-gamma, glitazone receptor (glitazone receptor) or NR1C3 (nuclear receptor subfamily 1, group C, member 3) ) and is a type II nuclear receptor present in humans encoded by the PPARG gene. The PPAR γ is a key regulator of adipocyte differentiation, and may be used as an adipocyte differentiation marker capable of confirming the degree of differentiation from adipose tissue-derived stem cells to adipocytes.

In the present invention, the term "PPAR α (Peroxisome Proliferator Activated Receptor-alpha)" is a peroxisome proliferator activated receptor-alpha, known as NR1C1 (nuclear receptor subfamily 1, group C, member 1), and by the PPARA gene It is a nuclear receptor protein present in humans that is encoded. It is present in keratinocytes and is known to have an effect of reconstructing a damaged skin barrier by promoting differentiation of keratinocytes when activated by binding to its ligand. In fact, it has been confirmed that, when Clofibrate or WY14643, which are known agonists of PPARα, is applied to skin with damaged skin barrier, differentiation of keratinocytes is promoted and restoration of the skin barrier is promoted. have.

In a specific embodiment of the present invention, it was confirmed that the erythorbic acid compound promotes the differentiation of adipose tissue-derived stem cells into mature adipocytes by promoting the expression of PPAR γ, which is a key regulator of adipogenesis (Fig. 3), and also , it was confirmed that the erythorbic acid compound exhibited very strong PPAR α activity when compared to the positive control (Table 3). These results show that the composition containing the erythorbic acid compound of the present invention has a function of regulating the differentiation of adipocytes, so it is a novel possibility to improve the aesthetic and health problems of modern people caused by volume reduction and functional deterioration due to adipose tissue regression. is a result that presents

As another embodiment, the present invention provides a cosmetic composition comprising an erythorbic acid compound or a cosmetically acceptable salt thereof.

Also, specifically, the present invention may provide a cosmetic composition for lipofilling or fat filling, skin regeneration or skin moisturizing, comprising an erythorbic acid compound or a cosmetically acceptable salt thereof.

As used herein, the term "erythorbic acid" is the same as described above.

In the present invention, "lipofilling (lipofilling) or fat filling" means to relieve, improve, or fill the degree of reduction in the volume of adipose tissue due to the regression of adipose tissue in the skin. It means that the volume is improved.

As used herein, the term "skin regeneration" means to restore the original function of severely damaged skin, and may include skin elasticity or wrinkle improvement. Although not specifically limited thereto, skin regeneration relieves the degree of sagging or sagging of the skin, or reduces structural breakdown due to decreased skin elasticity and strength due to skin fat filling or adipose tissue volume reduction by filling the skin with fat. It may include maintaining elasticity. In addition, it may include inhibiting or inhibiting the generation of wrinkles on the skin, or alleviating the wrinkles already generated.

In one embodiment of the present invention, the erythorbic acid compound promotes the expression of PPAR γ, which is a key regulator of adipogenesis, thereby promoting the differentiation of adipose tissue-derived stem cells, more specifically, adipose tissue-derived mesenchymal stem cells into mature adipocytes. induction was confirmed. These results suggest that by inducing the differentiation of stem cells into mature adipocytes, it is possible to alleviate, improve, or fill the decreased volume of skin adipose tissue. This indicates that the composition can be used for lipofilling or fat filling applications.

In addition, the above results suggest that by alleviating or improving the swelling of the regressed adipose tissue, the elasticity and wrinkles of the skin are enhanced or improved, and furthermore, it is suggested that damaged skin can help to restore its original function. This suggests that the cosmetic composition comprising the erythorbic acid compound of the present invention can be used for skin regeneration purposes.

In addition, in another embodiment of the present invention, as a result of comparing the degree of adipocyte differentiation after heat treatment of erythorbic acid and ascorbic acid at 40° C. for 30 minutes, ascorbic acid is easily oxidized during heat treatment and has poor stability, whereas Since erythorbic acid has relatively good stability to heat, it was confirmed that its efficacy could be stably present in the formulation of the cosmetic composition distributed through various temperatures (Table 5).

In the present invention, "moisturizing the skin" means to increase the feeling of moisture in the skin and to maintain a moist state. The skin moisturizing effect can help to improve wrinkles and increase elasticity of the skin. The skin moisture can be confirmed by measuring the level of filaggrin expression.

The "filaggrin" is a keratin-binding protein isolated from mammalian epidermal cells, and refers to a basic protein having a molecular weight of about 260,000, and the protein binds to a keratin unit in a ratio of about 3:2, The fibers are aggregated into bundles to form giant fibrils. As a highly phosphorylated precursor with a molecular weight of about 500,000, it is accumulated in the cell and undergoes differentiation and cleavage and dephosphorylation by proteolytic enzymes to become filaggrin. is known Filaggrin is well known for its function as a precursor of the Natural Moisturizing Factor, which plays an important role in moisturizing the skin.

In one embodiment of the present invention, it was confirmed that the composition containing erythorbic acid of the present invention promotes filaggrin expression in human keratinocyte line HaCaT cells, indicating that the cosmetic composition containing erythorbic acid can be used for skin moisturizing. was confirmed (Table 4).

The cosmetic composition is a solution, external ointment, cream, foam, nutrient lotion, softening lotion, pack, soft water, emulsion, makeup base, essence, soap, liquid detergent, bath agent, sunscreen cream, sun oil, suspension, emulsion, It may be prepared in a formulation selected from the group consisting of paste, gel, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation, patch and spray, but is not limited thereto.

In addition, the cosmetic composition may further include one or more cosmetically acceptable carriers to be formulated in general skin cosmetics, and as common ingredients, for example, oil, water, surfactant, humectant, lower alcohol, thickener, A chelating agent, a colorant, a preservative, a fragrance, etc. may be appropriately mixed, but the present invention is not limited thereto.

The cosmetically acceptable carrier included in the cosmetic composition varies depending on the formulation.

When the formulation of the cosmetic composition is an ointment, paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide as a carrier component or mixtures thereof may be used.

When the formulation of the cosmetic composition is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, or a mixture thereof may be used as a carrier component, and in particular, in the case of a spray, additionally propellants such as chlorofluorohydrocarbons, propane/butane or dimethyl ether.

When the formulation of the cosmetic composition is a solution or emulsion, a solvent, solubilizer or emulsifier is used as a carrier component, for example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol , 1,3-butylglycol oil can be used, and in particular, cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol fatty ester, fatty acid ester of polyethylene glycol or sorbitan may be used. can

When the formulation of the cosmetic composition is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester; Microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tracanth may be used.

When the formulation of the cosmetic composition is soap, alkali metal salts of fatty acids, fatty acid hemiester salts, fatty acid protein hydrolsate, isethionate, lanolin derivatives, aliphatic alcohols, vegetable oil, glycerol, sugar, etc. are used as carrier components can be

As another embodiment, the composition comprising the erythorbic acid compound of the present invention may be used as a food composition.

The food composition may be for lipofilling or fat filling containing an erythorbic acid compound or a pharmaceutically acceptable salt thereof, for skin regeneration, or for skin moisturizing.

The food composition may be used in the form of a health functional food, but is not limited thereto.

The erythorbic acid or a pharmaceutically acceptable salt thereof contained in the food composition may be included in the form of animals and plants containing erythorbic acid, an extract thereof, a fraction thereof, or a processed product thereof. In addition, the composition may include a food pharmaceutically acceptable food supplement additive in addition to the active ingredient.

In the present invention, "food supplement additive" refers to a component that can be added to food as an auxiliary, added to the manufacture of health functional food of each formulation, and can be appropriately selected and used by those skilled in the art. Examples of food supplement additives include various nutrients, vitamins, minerals (electrolytes), synthetic flavoring agents and flavoring agents such as natural flavoring agents, coloring agents and fillers, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners , pH adjuster, stabilizer, preservative, glycerin, alcohol, carbonation agent used in carbonated beverages, etc., but the above examples are not limited to the type of food supplement additive of the present invention.

The food composition of the present invention may include a health functional food. In the present invention, "health functional food" refers to food manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. using raw materials or ingredients useful for the human body. Here, "functional" means to obtain useful effects for health purposes, such as regulating nutrients or physiological actions with respect to the structure and function of the human body. The health functional food can be prepared by a method commonly used in the art, and at the time of manufacture, it can be prepared by adding raw materials and components commonly added in the art. In addition, the dosage form of the health functional food may be manufactured without limitation as long as it is a dosage form recognized as a health functional food. The food composition of the present invention can be prepared in various forms, and unlike general drugs, it has the advantage that there are no side effects that may occur during long-term administration of the drug using food as a raw material, and excellent portability, the present invention health functional food can be taken as a supplement to enhance the effects of lipofilling or fat filling, skin regeneration, and skin moisturizing.

There is no limitation in the form that the health functional food of the present invention can take, and it may include any food in a conventional sense, and may be used interchangeably with terms known in the art, such as functional food. In addition, the health functional food of the present invention can be prepared by mixing known additives with other suitable auxiliary ingredients that may be included in food according to the selection of those skilled in the art. Examples of foods that can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages and There are vitamin complexes, and the like, and it can be prepared by adding the compound represented by Formula 1 according to the present invention as a main component to juice, tea, jelly, juice, and the like. It also includes food used as feed for animals.

As another embodiment, the composition comprising erythorbic acid of the present invention can be used as a quasi-drug composition.

In addition, specifically, the quasi-drug composition may provide a quasi-drug composition for lipofilling or fat filling, skin regeneration, or skin moisturizing including the erythorbic acid or a pharmaceutically acceptable salt thereof.

The quasi-drug composition may further include a pharmaceutically acceptable carrier, excipient or diluent, if necessary, in addition to the above ingredients. The pharmaceutically acceptable carrier, excipient or diluent is not limited as long as it does not impair the effects of the present invention, and includes, for example, fillers, extenders, binders, wetting agents, disintegrants, surfactants, lubricants, sweeteners, fragrances, preservatives, etc. may include

Representative examples of the pharmaceutically acceptable carrier, excipient or diluent of the quasi-drug composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, maltitol, starch, gelatin, glycerin, acacia gum, alginate, calcium phosphate, Calcium carbonate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, propylene glycol, polyethylene glycol, vegetable oils, injectable esters, witepsol, macrogol, tween 61, cacao butter, laurage, and the like.

In addition, when erythorbic acid of the quasi-drug composition is used as a quasi-drug, it may further contain one or more active ingredients exhibiting the same or similar function. For example, it may include known lipofilling or fat filling, skin regeneration, or skin moisturizing ingredients. When additional lipofilling or fat filling, skin regeneration, or skin moisturizing ingredients are included, the lipofilling or fat filling, skin regeneration, or skin moisturizing improvement effect of the quasi-drug composition of the present invention can be further increased. will be. When adding the above ingredients, skin safety according to combined use, ease of formulation, and stability of active ingredients can be considered.

The quasi-drug composition of the present invention may exemplify, but is not limited to, disinfectant cleaner, shower foam, ointment, wet tissue, coating agent, etc. It may be appropriately selected from conventional techniques.

In addition, the erythorbic acid of the present invention can be used in a method for lipofilling or fat filling, skin regeneration, or skin moisturizing, including applying to the skin of an individual. The subject includes, without limitation, mammals including rats, livestock, humans, and the like.

As another embodiment, the present invention provides an external preparation for skin comprising an erythorbic acid compound or an acceptable salt thereof.

In addition, specifically, the present invention can provide an external preparation for lipofilling or fat filling, skin regeneration or skin moisturizing, comprising an erythorbic acid compound or an acceptable salt thereof.

In the present invention, the terms "erythorbic acid", "for lipofilling or fat filling", "for skin regeneration", and "for skin moisturizing" are the same as described above.

As used herein, the term "external preparation" is a preparation provided for external use, and includes external acid preparations, external tablets, external solutions, ointments, warning preparations, suppositories, and the like. External preparation means dispersing zincized starch, etc., erosions and ulcers of the skin mucous membrane, external tablet means dissolving or vaginal tablet, and external preparation is a liquid containing water, ethanol, oil, etc. as a solvent It is a preparation, and is used for brushing teeth, compresses, washing, eye drops, and nasal drops. In addition, an ointment is a semi-solid formulation of suitable roughness applied to the skin, a warning is a solid at room temperature, a skin application that softens at body temperature, and a suppository is a formulation applied to the anus, vagina, and urethra.

The "external preparation for skin" refers to a preparation that acts externally on the skin among external preparations, and in the present invention, it refers to an external preparation for skin containing the erythorbic acid compound or an acceptable salt thereof as an active ingredient. Specifically, for external skin preparations, creams, gels, patches, sprays, ointments, warning agents, lotions, liniments, pasta agents, or cataplasmas can be prepared and used as pharmaceuticals or quasi-drugs for external use, but is not limited thereto. does not

As another one embodiment, the present invention comprises the steps of (a) culturing stem cells; and (b) adding a composition comprising an erythorbic acid compound to the stem cells of step (a).

In the present invention, the terms "erythorbic acid", "stem cells" and "differentiation induction" are the same as described above.

The step (a) is a step of culturing undifferentiated cells, which are stem cells, and can be cultured according to a conventional culture method known in the art. , more specifically, may be adipose tissue-derived mesenchymal stem cells.

The step (b) is a step of inducing differentiation into adipocytes by treating the stem cells cultured in step (a) with the composition containing the erythorbic acid of the present invention. Cytokines added to the culture medium to induce differentiation can be easily selected by those skilled in the art from cytokines for adipocyte differentiation known in the art, and can be used by adding an effective concentration for inducing differentiation to the medium, It is not limited thereto.

Specifically, as described above, the method for inducing differentiation of adipocytes of the present invention can promote the expression of PPAR γ, and the adipocytes differentiated from the stem cells produced in the present invention are used as cosmetics, food, quasi-drugs, or external preparations for skin. In particular, the cosmetic, food, quasi-drug, or skin external preparation may be used for lipofilling or fat filling, skin regeneration, or skin moisturizing improvement.

In addition, it can be used as a cell composition, such as a cell therapeutic agent for the treatment of rare incurable diseases and diseases related to adipocyte transplantation.

As described above, the erythorbic acid of the present invention promotes the differentiation of adipose tissue-derived stem cells into mature adipocytes by promoting the expression of Peroxisome proliferator-activated receptor gamma (PPAR γ), which is a key regulator of adipogenic differentiation, thereby promoting the differentiation of adipose tissue-derived stem cells into mature adipocytes. It can present a new possibility to improve the aesthetic and health problems of modern people caused by volume reduction and functional deterioration due to regression.

Figure 1 is indomethacin (Indomethacin), dexamethason (Dexamethason), isobutyl methyl xanthine (IBX), insulin (Insulin) containing adipocyte differentiation conditions, adipose tissue-derived mesenchymal stem cells fat After inducing cell differentiation, fat was stained with Oil Red O staining reagent and observed under a microscope. The non-differentiation group was a control group under culture conditions that did not induce differentiation, the untreated control group was a control group added with only adipocyte differentiation medium, a positive control group was performed by adding Rosiglitazone to the adipocyte differentiation medium, and the experimental group was an adipocyte differentiation medium. It was carried out by adding erythorbic acid.
Figure 2 is a graph showing the relative adipogenic differentiation level compared to the untreated control group after the adipose tissue-derived mesenchymal stem cells were treated with an adipocyte differentiation medium containing erythorbic acid.
3 is a graph showing the expression level of PPAR γ, a genetic marker for adipocyte differentiation, compared to a control group after adipose tissue-derived mesenchymal stem cells were treated with an adipocyte differentiation medium containing erythorbic acid.

Hereinafter, the configuration and effects of the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Reference example One: erythorbic acid ( erythorbic acid ) substance information

[Formula 1]

Substance name: erythorbic acid

CAS No. : 89-65-6

Molecular Formula: C ₆ H ₈ O ₆

Molecular Weight: 176.12

Where to buy: Sigma-Aldrich

Example 1: Confirmation of the efficacy of promoting differentiation of human adipose tissue-derived stem cells into adipocytes

Human adipose-derived stem cells (hADSCs) were purchased from Lonza (Lonza, Inc. Walkersville, MD, USA) and cultured according to Lonza's instructions. Adipocyte differentiation was achieved using the method recommended by Lonza. Specifically, 0.5 µM of insulin, 0.5 µM of dexamethasone (DEXA), 0.25 µM of isobutyl methyl xanthine (IBMX), and 50 µM of indomethacin were added to the adipose tissue-derived stem cell culture medium and cultured Thus, stem cells were differentiated into adipocytes, and 10 ppm of erythorbic acid was added to the medium under the same conditions to induce adipocyte differentiation. As a positive control, 0.5 μM of troglitazone (ROSI) was used and treated together with IDX medium. 14 days after the initiation of adipocyte differentiation of adipose tissue-derived stem cells, the cell culture medium was removed, the cells were washed with 10 ml of Phosphate Buffered Saline (PBS), and then the cells were cultured using 10% formalin solution. was fixed for a minute. The fixed cells were washed once with a 60% isopropanol solution, and the lipids in the adipocytes were stained with a staining solution in which Oil Red O was dissolved in 60% isopropanol for 10 minutes. Then, it was observed under a microscope, and the results are shown in FIG. 1 .

As a result, as can be seen in FIG. 1 , when erythorbic acid was added and then adipogenic differentiation of adipose tissue-derived stem cells was induced, compared to the case of induced adipocyte differentiation of adipose tissue-derived stem cells under the conditions of no addition. It was confirmed that the differentiation of adipocytes occurred more effectively (FIG. 1).

These results indicate that the composition containing erythorbic acid has a lipolytic application by promoting the differentiation of adipose tissue-derived stem cells into adipocytes, and furthermore, the composition containing erythorbic acid is used for lipofilling or fat filling, or skin This suggests that it can be used for purposes of regeneration.

Example 2: Quantitative confirmation of adipocyte differentiation promoting effect of human adipose tissue-derived stem cells

After inducing differentiation of adipocytes from adipose tissue-derived stem cells in the same manner as in Example 1, adipocytes were stained with Nile Red reagent (AdipoRed™, Lonza). The staining method followed the method recommended by Lonza. Fluorescence of the stained adipocytes was measured at 485 nm/575 nm of 500 nm, and the results are shown in Table 1 and FIG. 2 .

non-clad group 0.114±0.035 untreated control 1±0.058 positive control 4.998±0.616 erythorbic acid 1.7758±0.013

As a result, as can be seen in Tables 1 and 2 above, as a result of treating adipose tissue-derived mesenchymal stem cells with erythorbic acid, differentiation of adipose tissue-derived stem cells into adipocytes was induced under conditions in which erythorbic acid was not added. It was confirmed that the differentiation of adipocytes occurred more effectively than in one case (Table 1 and FIG. 2).

These results also indicate that the composition containing erythorbic acid has a lipolysis application by promoting the differentiation of adipose tissue-derived stem cells into adipocytes, and furthermore, the composition containing erythorbic acid is used for lipofilling or fat filling, or This suggests that it can be used for skin regeneration.

Example 3: Confirmation of adipocyte-specific gene expression level in human adipose tissue-derived stem cells

After inducing the differentiation of adipocytes from adipose tissue-derived stem cells in the same manner as in Example 1, the expression level of PPAR γ, a marker of adipocyte differentiation, was evaluated to confirm the adipocyte differentiation degree of adipose tissue-derived stem cells. did Cell RNA was isolated to synthesize cDNA, and then, real-time-PCR was performed with Taqman staining solution to measure the expression level of PPAR γ. In this experiment, the RNA concentration was normalized to S16 ribosomal RNA, and the results are shown in Table 2 and FIG. 3 .

non-clad group 0.240±0.027 untreated control 1±0.005 positive control 4.1421±0.101 erythorbic acid 1.6374±0.096

As a result, as can be seen in Table 2 and FIG. 3, the medium condition treated with erythorbic acid promotes the expression of PPAR γ, and has the effect of promoting differentiation of adipose tissue-derived stem cells into adipocytes compared to the case where erythorbic acid is not added. was confirmed to be excellent (Table 2 and FIG. 3).

These results indicate that the composition containing erythorbic acid has a lipolytic application by promoting the differentiation of adipose tissue-derived stem cells into adipocytes, and furthermore, the composition containing erythorbic acid is used for lipofilling or fat filling, or skin This suggests that it can be used for purposes of regeneration.

Example 4: PPAR Verification of α activation promoting effect

In order to confirm the PPAR α activation promoting effect of erythorbic acid, the following experiment was performed using erythorbic acid. C2C12 cells (ATCC CRL-1772), a mouse-derived myblast cell line, were subcultured in DMEM (Dulbeco's Modified Eagle's Medium) medium containing 10% fetal bovine serum. The vectors used here include 1) DNA encoding Ser167 to Tyr468 in the yeast transcription factor GAL4-DBD (DNA binding domain) and human PPAR alpha LBD (Ligand Binding Domain) in the SV40 promoter of the pZEO vector (Invitrogen). vector containing the fragment (gene bank information, NM 005036); 2) the GAL4 response sequence was inserted into the multiple restriction enzyme recognition site of the pGL3 vector (Promega) expressing luciferase, and 3) β-galactosidase as a transfection internal control (transfection internal control) ( A vector expressing β-galactosidase) was used.

The cultured cells ^{were plated on a 24-well plate at a concentration of 4×10 4} , cultured for 12 hours, and the three types of plasmid genes were transiently transfected using Lipopectamine (Lipopectamine). did After 8 hours, 50 ppm of erythorbic acid was treated and incubated for 12 hours, washed with 1×PBS and lysed with 1× reporter lysis buffer, a luciferase assay kit (Promega) and The activity of luciferase was measured using a β-galactosidase assay kit (Promega). That is, the PPARα activity was measured as 'luciferase activity/β-galactosidase activity'.

In this experiment, as a positive control, Wy-14,643 (Calbiochem), which is known to be the strongest of the ligands of PPAR α, was used, and as a negative control, 0.05% of DMSO used for dissolving the sample was used. According to the above experimental method, C2C12 cells were treated with erythorbic acid to measure the activity of PPARα according to the concentration change, and the results are shown in Table 3 below. In Table 3 below, the values of PPAR α activity mean the percentage compared to the negative control (DMSO 0.05%).

As a result, as can be seen in Table 3, compared to the positive control, erythorbic acid was confirmed to exhibit very strong PPAR α activity (Table 3). These results indicate that erythorbic acid promotes the activity of PPARα present in keratinocytes, thereby promoting the differentiation of keratinocytes when applied to damaged skin, thereby promoting skin barrier recovery. Furthermore, it suggests that erythorbic acid has an effect of skin regeneration.

Example 5: of Filaggrin Expression promoting effect

After adding erythorbic acid to the culture medium of human keratinocyte line HaCaT cells at 5 μg/mL and 10 μg/mL, respectively, and culturing for 1 day, RNA of the cells was isolated, cDNA was synthesized, and Real- with Taqman staining solution time PCR was performed. The RNA concentration was normalized to S16 ribosomal RNA, and the results are shown in Table 4 below.

As a result, as can be seen in Table 4, it was confirmed that erythorbic acid has the ability to promote filaggrin expression in the human keratinocyte line HaCaT cells (Table 4). These results include erythorbic acid of the present invention as an active ingredient. This indicates that the composition can be used as a cosmetic composition for skin moisturizing.

Example 6: erythorbic acid and Comparison of adipocyte differentiation degree after heat treatment of ascorbic acid

Considering that the cosmetic composition is distributed through various temperatures, erythorbic acid and ascorbic acid were heat-treated at 40 ° C. for 30 minutes to confirm that erythorbic acid has better heat stability than its isomer, ascorbic acid Then, the degree of adipocyte differentiation was compared. The specific adipocyte differentiation and quantification process was evaluated in the same manner as in Example 1, and the results are shown in Table 5 below. For reference, in Table 5 below, AA is ascorbic acid, and EA is erythorbic acid.

experimental group unprocessed
control positivity
control AA 10ppm AA 20 ppm AA 50ppm EA 10 ppm EA 20 ppm EA 50 ppm Fluorescence intensity 1.0 10.5 7.4 8.1 9.6 11.2 12.5 16.3

As a result, as can be seen in Table 5, in the case of ascorbic acid, it was confirmed that the degree of differentiation increased at a marginal rate from 7.4 to 9.6 even when the content was increased, whereas in the case of erythorbic acid, the degree of differentiation increased as the content increased. From 11.2 to 16.3, it was confirmed that the increase was significantly increased compared to ascorbic acid (Table 5).

This indicates that ascorbic acid is easily oxidized when heat is treated and the stability is lowered, but erythorbic acid has relatively excellent heat stability and thus promotes fat differentiation even at various temperatures. It was found that the efficacy of erythorbic acid rather than ascorbic acid may be stably present in the formulation of the composition.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention, rather than the above detailed description, all changes or modifications derived from the meaning and scope of the following claims and their equivalents.

Claims (19)

A composition for inducing differentiation into adipocytes from preadipocytes or stem cells, comprising an erythorbic acid compound or an acceptable salt thereof.
The composition of claim 1, wherein the stem cells are mesenchymal stem cells (MSCs).
The composition of claim 1, wherein the stem cells are human adipose tissue-derived stem cells.
The composition of claim 1, wherein the composition promotes the expression of PPAR γ.
A cosmetic composition for lipofilling or fat filling, or skin regeneration comprising an erythorbic acid compound or a cosmetically acceptable salt thereof.
delete delete delete The cosmetic composition according to claim 5, wherein the lipofilling, fat filling, or skin regeneration is by differentiation of preadipocytes or stem cells into adipocytes.
The cosmetic composition according to claim 5, wherein the skin regeneration is by promoting differentiation of keratinocytes.
The cosmetic composition of claim 9, wherein the stem cells are mesenchymal stem cells (MSCs).
The cosmetic composition of claim 9, wherein the stem cells are human adipose tissue-derived stem cells.
The cosmetic composition according to claim 5, wherein the cosmetic composition is a solution, external ointment, cream, foam, nourishing lotion, softening lotion, pack, softening water, emulsion, makeup base, essence, soap, liquid detergent, bathing agent, sunscreen cream, sun oil. , suspension, emulsion, paste, gel, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation, patch and spray. , cosmetic composition.
A cosmetic composition for external application for lipofilling or fat filling, or for skin regeneration, comprising an erythorbic acid compound or an acceptable salt thereof.
delete The cosmetic composition for external application for skin according to claim 14, wherein the lipofilling, fat filling, or skin regeneration is by differentiation from preadipocytes or stem cells to adipocytes.
(a) culturing stem cells; and
(b) A method for inducing differentiation from stem cells into adipocytes, comprising adding the composition of claim 1 to the stem cells of step (a).
The method of claim 17, wherein the stem cells are mesenchymal stem cells.
The method of claim 17, wherein the stem cells are derived from human adipose tissue.

Images