KR102013164B1 - Composition for improving skin - Google Patents

Abstract

The present invention relates to a composition for improving skin. Fibroblast extract according to the present invention is a medicament for skin regeneration, atopic improvement, skin moisturizing and skin texture improvement through the effect of promoting the proliferation of skin stem cells, the excellent activity of PPAR alpha, the effect of increasing the expression of pilagrin, skin brightness It can be used to make cosmetics or food. In addition, it promotes the proliferation of skin stem cells, exhibits a stem cell maintenance effect, can be used in the production of cosmetics for promoting stem cell activity.

Description

Composition for improving skin

The present invention relates to a skin improvement composition exhibiting skin regeneration, atopic improvement, skin moisturization, skin texture improvement, stem cell activity promoting effect.

The epidermis, located at the outermost part of the skin, protects against a variety of external physical, chemical and mechanical stimuli and protects the body from excessive release of moisture through the skin. This protective function is possible by the normal formation and maintenance of the stratum corneum consisting of keratinocytes. Keratinocytes are cells formed by gradual changes in morphology and function as basal cells, which have been continuously proliferating in the stratum basale, migrate to the stratum corneum. The forming cells are detached from the skin, and the new keratinocytes from the lower epidermal layer repeat the process of epidermis differentiation or keratinization, which takes over the function. In this keratinization process, keratinocytes produce natural moisturizing factor (NMF) and intercellular lipids such as ceramides, cholesterol, and fatty acids, and the stratum corneum acts as a barrier to the outside, resulting in a skin barrier. It has a function as.

In addition, dry skin, which is considered to be one of the major diseases in modern society, is one of the symptoms caused by skin barrier dysfunction, and recently, environmental pollution, increased dry environment such as apartments and high-rise buildings, increased social stress, It is increasing due to various factors such as excessive bath culture, skin aging, etc., and it is also continuously increasing when severe symptoms require treatment.

Recently, atopic dermatitis, which occurs in 10% of children, is also known to be caused by dry skin, more fundamentally, abnormal skin barrier function. In order to treat such atopic dermatitis, a lot of researches have been conducted to supply moisture from the outside or minimize the loss of water from the body, focusing on maintaining proper moisture in the skin. Moisturizers such as ceramide) or derivatives thereof have been developed and are widely used in the pharmaceutical or cosmetic field.

However, the use of such moisturizers are mostly temporary relief of symptoms rather than fundamental treatments, and do not show sufficient effects on the treatment of dry skin and skin barrier function including atopic dermatitis. Therefore, it is urgent to develop a material that fundamentally regenerates damaged skin barriers.

Recently, when PPAR alpha (Peroxisome Proliferator Activated Receptor-alpha) present in keratinocytes is activated by binding to its ligand, it has been known to promote the differentiation of keratinocytes and reconstruct the damaged skin barrier. In the case where clofibrate [Non-Patent Document 1] or WY14643 [Non-Patent Document 2] or the like, which is an actually known PPAR alpha agonist, is applied to the skin with damaged skin barrier, the differentiation of keratinocytes is promoted. It has been confirmed that the recovery of the skin barrier is promoted.

In addition, the skin texture in the form of the skin surface is characterized by a three-dimensional microstructure formed by fine lines and significantly different by age and region. The skin texture is divided into primary (about 20-100 ㎛), secondary (about 5-40 ㎛) and tertiary (about 0.5 ㎛) lines according to the depth of the lines. It has a characteristic that the polygon formed by forming the lines and star formation (star formation) is distinct [Non-Patent Document 3].

However, as the body ages or the skin is damaged, the network structure of the fine fine lines collapses and the fine lines (secondary and tertiary, etc.) disappear and the primary lines become deeper and wrinkles are produced. Document 4,5]. In other words, wrinkles, a representative sign of skin aging, are already the accumulation of fine changes that occur over a long period of time.

Developmentally, all components of human skin are known to be derived from ectoderm or mesoderm. Epidermis, hair follicles, sebaceous glands and sweat glands originate in ectoderm, and melanocytes, nerves and special sensory receptors are derived from neuroectoderm. According to the developmental period, embryonic stem cells are differentiated to become cells of the characteristic for each tissue function, and even after adulthood, a certain number of stem cells remain in the tissue. There are mainly two stem cells in the skin. . The first is in hair follicles. It is known to play an important role in regeneration of the epidermis, and also plays an important role in hair regeneration and growth. Second is the basal layer of epidermis. Stem cells in this area are found to play an important role in protecting skin health by controlling not only the epidermis but also the fibroblasts of the dermis. Stem cells here are relatively quantitative and have the advantage of being easily available and are widely used for research on skin stem cells. The skin is continuously renewed, and stem cells existing in the epithelium of the skin, that is, epidermal stem cells of the skin, are involved in the repair of the epithelium after the wound [Non-Patent Document 6]. The epithelial stem cells of the skin are also referred to as skin stem cells, and when activated, the skin stem cells can treat skin wounds such as trauma or promote wound healing, and anti-wrinkle of the skin can also be expected. have. Integrin β1 and integrin α6 are used as indicators of skin stem cells, and maintenance of skin stem cells necessary for epithelial morphology and differentiation is reported to be regulated by p63 protein.

Skin stem cells play an important role in maintaining skin health and physiological and biochemical homeostasis. Stem cells present in the skin also cause abnormalities in function due to the effects of aging, and thus various problems occur as the homeostasis of the skin is broken. Therefore, it is possible to improve the various phenomena of skin aging through the activity of these stem cells [Non-Patent Document 7].

In addition, it is safe for the living body, the active ingredient is stable, and above all, the skin regeneration, atopy improvement, skin moisturizing and skin texture improvement activities are more effective than the substances having the effects of existing skin regeneration, atopic improvement, skin moisturizing and skin texture improvement. There is an urgent need for the development of these components.

On the other hand, the larva is said to have been eaten by poppies, and is also called the king of fruits in southern China. Asteraceae Tree is a evergreen tree belonging to the Asteraceae family, about 10 to 15m high. It is also called the English name litchi (lychee). Fruits are oval or round, 3-4 cm in diameter, 2.5-3 cm in diameter. Fruit peel is leather, and the surface has scab pattern in the center protruding. The pulp is milky and juicy, with a slightly sour and sweet taste that tastes good and has a unique aroma. Fruits inhabit, grow well in the humid acidic soils of subtropical regions, and are widely grown in the tropics as fruit trees native to southern China.

Seed fibrous nucleus is oval-oval, squashed on one side and flat, 12-22mm long, 6-16mm in diameter. The outer surface is dark reddish-purple black, smooth, glossy, sparsely vertical wrinkles, one end is dull, and the quality is relatively hard.

Nucleus fibrils have been used to drive cold energy, relieve pain, and have symptoms such as colic, testicles, and abdominal pain. Chemical components contained in the nucleus nucleus include α- (methylenecyclopropyl) glycine, α- (methylencyclopropyl) glycine, 3-acetoin, 2,3-butanediol and copan (copaene), cis-caryophyllene, allo-aromadendrene, humulene, δ-cadinene, α-curcumene , Caramenene, redol (ledol), guiazulene, xanthorrhizol, palmitic acid and the like are known (Chinese medicinal herbs).

Despite the many human benefits of fibroblasts, no studies have been made on the skin and stem cells of fibroblasts.

 Feingold et al., J. Invest. Dermatol., 110, pp 368-375, 1998.  Feingold et al., J. Invest. Dermatol., 115, pp 353-360, 2000.  Journal of the European Academy of Dermatology and Venereology. 12: 103-114, 1999  The British journal of dermatology. 110: 129-138, 1984   Skin research and technology. 5: 189-194, 1999  Epidermal Stem Cells of the Skin, Cedric Blanpain, Elaine Fuchs, Annual Review of Cell and Developmental Biology, November 2006, Vol. 22, Pages 339-373  Human skin stem cells and the ageing process, Catherin Niemann, Stem Cell Aging and Regenerative Medicine, November 2008, Pages 986-997

Accordingly, the present inventors, the fibrinomegaly extract promotes the proliferation of skin stem cells to promote the regeneration of the skin, the PPAR alpha activity is also excellent to improve the atopy, and the skin moisturizing improvement effect due to the increase in the expression of filaggrin, skin In addition to improving the skin texture due to the improvement of brightness, promoting the proliferation of skin stem cells, showing the stem cell maintenance effect has been completed the present invention by confirming the stem cell activity promoting effect.

Accordingly, it is an object of the present invention to provide a composition for skin regeneration, atopy improvement, skin moisturizing and skin texture improvement comprising a fibrinomegaly extract as an active ingredient.

In addition, another object of the present invention to provide a composition for promoting stem cell activity comprising a fibrinomegaly extract as an active ingredient.

As a means for solving the above problems, the present invention provides a composition for skin regeneration, atopy improvement, skin moisturizing and skin texture improvement, including a fibrinomegaly extract as an active ingredient for the manufacture of medicine, cosmetics or food.

As another means for solving the above problems, the present invention provides a composition for promoting stem cell activity comprising a fibrinomegaly extract as an active ingredient for the production of cosmetic formulations.

The fibrinomegaly extract according to the present invention promotes the proliferation of skin stem cells, promotes the regeneration of the skin, and has the effect of improving the atopy by the excellent activity of PPAR alpha, and improves the skin moisturizing effect due to the increase in the expression of filaggrin, skin brightness By showing the skin texture improvement effect by improvement, it can be used for manufacture of medicine, cosmetics, or food.

In addition, the fibrinomegaly extract promotes the proliferation of skin stem cells and shows a stem cell maintenance effect, which can be used for preparing a cosmetic formulation for promoting stem cell activity.

EMBODIMENT OF THE INVENTION Hereinafter, the structure of this invention is demonstrated concretely.

Skin regeneration, atopy improvement, skin moisturizing and skin texture improvement components show high activity of skin regeneration, atopy improvement, skin moisturizing and skin texture improvement at low concentration, and to penetrate skin Good absorption ability, low volatility for long time stay sufficient for skin regeneration, atopic improvement, skin moisturizing and skin texture improvement, stable active ingredient on composition or skin, medicine, cosmetic or food It is preferable that it is easy to manufacture, etc., and is safe to skin. However, among the known components, components that satisfy all of the above properties are rare. For example, some skin rejuvenation, atopy improvement, skin moisturizing and skin texture enhancing ingredients have excellent skin regeneration, atopic improvement, skin moisturizing and skin texture improving activity even at low concentrations in vitro, but have the ability to penetrate and absorb skin. It is difficult to apply to real skin apart. Other active ingredients are less hydrophilic, making them difficult to formulate in medicine, cosmetics or food. In addition, some skin rejuvenation, atopy improvement, skin moisturizing and skin texture enhancing ingredients may decompose or be transformed into other compounds when exposed to heat, light, or oxygen, and the effects are already lost before application to the skin. .

As can be seen in the following examples, the fibrinomegaly extract exhibits excellent skin stem cell proliferation promoting effect, PPAR alpha activity effect, filaggrin expression effect, and skin brightness improvement effect at low concentrations, thus regenerating skin, improving atopy, and skin. It can be used as an active ingredient in medicine, cosmetics or food compositions for moisturizing and improving skin texture.

Therefore, the present invention provides a composition for skin regeneration, atopy improvement, skin moisturizing and skin texture improvement comprising a fibrinomegaly extract as an active ingredient.

The fibrinomegaly extract can be extracted by a method known in the art, the method is not particularly limited. Alternatively, a commercially available extract can be used.

Preferably, the nucleus nucleus extract may be an extract obtained by extracting the nucleus nucleus with water and / or an organic solvent, and the organic solvent may be a polar organic solvent, a non-polar organic solvent or a mixed solvent thereof. The polar organic solvent may be lower alcohol having 1 to 5 carbon atoms, ethyl acetate or acetone, and the nonpolar organic solvent may be ether, chloroform, benzene, hexane or dichloromethane. For example, the lower alcohol having 1 to 5 carbon atoms may be methanol, ethanol, propanol, butanol or isopropanol.

In one embodiment, the fibrinomegaly extract may include a fraction obtained by fractionating the primary extract extracted using the aforementioned extraction solvent using an extraction solvent having a different polarity. For example, the fibrinomegaly extract may be a fraction extracted with an alcohol having 1 to 5 carbon atoms and then re-divided with a solvent having a different polarity such as ether, benzene, and hexane. Two or more solvents may be used in the fractionation, and each solvent extract may be prepared using sequentially or mixed according to the polarity of the solvent.

In the present invention, the solvent may be removed by filtering or performing a concentration or drying process on the extract or the fraction obtained by performing the fractionation process, it is possible to perform all the filtration, concentration and drying. Specifically, the filtration may be performed using a filter paper or a reduced pressure filter, the concentration may be concentrated under reduced pressure using a reduced pressure concentrator, etc., and the drying may be performed by lyophilization.

In addition, the prepared extract or the fraction obtained by performing the fractionation process, such as silica gel column chromatography, thin layer chromatography or high performance liquid chromatography, etc. Further purified fractions can be obtained by purification using a variety of chromatography.

The compositions of the present invention can be used for the preparation of pharmaceutical formulations.

The pharmaceutical formulation may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of extracts, powders, granules, tablets or capsules.

In addition, the composition may further contain one or more active ingredients exhibiting the same or similar functions. For example, it may include known skin regeneration, atopic improvement, skin moisturizing and skin texture improving ingredients. The inclusion of additional skin regeneration, atopy improvement, skin moisturizing and skin texture improving ingredients will further enhance the skin regeneration, atopy improvement, skin moisturizing and skin texture improving effects of the compositions of the present invention. When the ingredient is added, skin safety, ease of formulation, and stability of the active ingredients may be considered. In one embodiment of the invention, the composition is a skin regeneration component known in the art, retinoic acid, TGF, protein from animal placenta, betulinic acid and chlorella extract, as antioxidant ingredients known in the art, tocopherol, selenium , Vitamin C and phenolic compounds, as whitening ingredients known in the art, substances that inhibit tyrosinase enzyme activity such as kojic acid, arbutin, hydroquinone, vitamin-C (L- Ascorbic acid) and derivatives thereof and various plant extracts may further comprise one or two or more components selected from the group consisting of. Additional ingredients may be included from 0.0001% to 10% by weight relative to the total composition weight and the content range may be adjusted according to requirements such as skin safety, ease of formulation of the fibrinomegaly extract, and the like.

In addition, the composition of the present invention may further comprise a pharmaceutically acceptable carrier.

Pharmaceutically acceptable carriers may contain various components such as buffers, sterile water for injection, general saline or phosphate buffered saline, sucrose, histidine, salts, polysorbates and the like.

The composition of the present invention may be administered orally or parenterally, and may be administered in the form of general pharmaceutical preparations, for example, in various dosage forms, orally and parenterally, in the case of clinical administration. , Diluents or excipients such as binders, wetting agents, disintegrants, surfactants and the like.

Solid form preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid form preparations may include at least one excipient such as starch, calcium carbonate, water, or the like in the pharmaceutical composition of the present invention. Can be prepared by mixing cross (Lucose) or lactose (Lactose), gelatin and the like.

In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Liquid preparations for oral use include suspensions, solutions, emulsions, syrups, and the like, and various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to water and liquid paraffin, which are commonly used simple diluents.

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

In the present invention, the 'skin regeneration effect' refers to the recovery of skin tissue against damage caused by external and internal causes. The damage caused by the external causes may include ultraviolet rays, external pollutants, wounds, traumas, and the like, and the damage caused by the internal causes may include stress.

In the present invention, 'atopic improvement effect' refers to the prevention and treatment of skin diseases such as atopy and dry skin, and refers to inhibiting or inhibiting or alleviating atopic symptoms.

In the present invention, the "moisturizing effect" refers to inhibiting or inhibiting the decrease in moisture of the skin or increasing the moisture content of the skin to smooth the surface of the skin and impart shine.

In the present invention, the 'skin improvement effect' refers to smoothing the skin surface, giving gloss and improving skin tone by suppressing or inhibiting roughening of the skin surface under the influence of aging, stress, and the like. .

In the present invention, the 'effective amount' refers to the amount of the extract which can promote the regeneration of damaged skin, improve skin dryness such as atopy, moisturize effect or improve skin texture. When the composition of the present invention comprises an effective amount of the fibrinomegaly extract can provide a desirable skin regeneration effect, atopic improvement effect, moisturizing effect and skin texture improvement effect. The effective amount of the fibrinomegaly extract contained in the composition of the present invention will vary depending on the form in which the composition is commercialized, the method by which the compound is applied to the skin, the time it stays on the skin, and the like. For example, when the composition is commercialized in a pharmaceutical formulation, it may include the fibrinomegaly extract at a higher concentration than when it is commercialized as a cosmetic that is routinely applied to the skin. Therefore, the daily dose is 0.1 to 100 mg / kg, preferably 30 to 80 mg / kg, more preferably 50 to 60 mg / kg, 1 to 6, based on the amount of the fibrinomegaly extract May be administered once.

The compositions of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological response modifiers.

The pharmaceutical formulation of the present invention may include an external preparation for skin.

When the fibrinomegaly extract is used as an external preparation for skin, it may be further added to fatty substances, organic solvents, solubilizers, thickening and gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, Commonly used in water, ionic or nonionic emulsifiers, fillers, metal ion sequestrants and chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or external preparations for the skin It may contain adjuvants conventionally used in the field of dermatology, such as any other ingredients used. The ingredients may also be introduced in amounts generally used in the field of dermatology.

When the fibrinomegaly extract is provided in an external preparation for skin, it may have a formulation such as, but not limited to, ointment, patch, gel, cream or spray.

In addition, the composition for skin regeneration, atopic improvement, skin moisturizing and skin texture improvement of the present invention can be used for the preparation of cosmetic formulations.

The cosmetic formulation may be in the form of a general emulsion formulation and solubilization formulation. For example, lotions such as flexible lotions or nourishing lotions, emulsions such as facial lotions, body lotions, creams such as nourishing creams, moisture creams, eye creams, essences, cosmetic ointments, sprays, gels, packs, sunscreens, makeup bases, liquids Formulations such as foundations, powders, cleansing creams, cleansing lotions, makeup removers such as cleansing oils, cleansing foams, soaps, body washes and the like.

In addition, the cosmetics, in addition to the nucleus extract extracts fatty substances, organic solvents, solubilizers, thickeners and gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, ions Any commonly used in type or nonionic emulsifiers, fillers, metal ion sequestrants and chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or cosmetics It may contain adjuvants conventionally used in the cosmetic field, such as other ingredients.

The cosmetic formulation may include a relatively high concentration of the supine nucleus extract in the case of wash-off type cosmetics such as makeup remover, detergent, etc., in which the active ingredient stays on the skin within a short period of time. On the other hand, in the case of a leave-on type cosmetic such as a lotion, an emulsion, a cream, an essence, etc., in which the active ingredient stays on the skin for a long time, the extract contains a lower concentration of the supine nucleus extract than the wash-off type cosmetic. It will be okay. In one embodiment of the present invention, the composition may include 0.0001% to 10% by weight (preferably 0.0001% to 1% by weight) based on the total weight of the composition. have. When the composition of the present invention comprises less than 0.0001% by weight of the extract of the nucleus, sufficient skin regeneration, atopy improvement, skin moisturizing and skin texture improvement effect can not be expected, if it contains more than 10% by weight allergy, etc. This is to prevent unwanted reactions or problems with skin safety.

In addition, the composition for skin regeneration, atopy improvement, skin moisturizing and skin texture improvement of the present invention can be used for food formulation preparation.

The food formulation refers to a food prepared by adding the fibrinomegaly extract to food materials such as beverages, teas, spices, chewing gum, confectionary, or the like, encapsulated, powdered, or suspension.

Since the food formulation can be consumed on a daily basis, it is very useful because it can expect high skin regeneration, atopic improvement, skin moisturizing and skin texture improvement effect.

In the case of using the filtrate nucleus extract as a food additive, the filtrate nucleus extract may be added as it is or used with other food or food ingredients, and may be appropriately used according to a conventional method. The mixed amount of the active ingredient can be suitably determined according to the purpose of use (prevention, health or therapeutic treatment). Generally, in the preparation of food or beverages, the composition of the present invention is added in an amount of up to 15 parts by weight, preferably up to 10 parts by weight based on the raw materials. However, in the case of long-term intake for health and hygiene or health control, the amount may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety. .

There is no particular limitation on the kind of food. Examples of the food to which the substance may be added include meat, sausages, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, teas, drinks, Alcoholic beverages and vitamin complexes, and the like and include all of the health foods in the conventional sense.

If the food formulation is a beverage, it may contain various flavors or natural carbohydrates, etc. as additional ingredients, as in conventional beverages. The above-mentioned natural carbohydrates are glucose, monosaccharides such as fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, sugar alcohols such as xylitol, sorbitol and erythritol. As the sweetening agent, natural sweetening agents such as tautin and stevia extract, synthetic sweetening agents such as saccharin and aspartame, and the like can be used. The proportion of such natural carbohydrates is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g, per 100 mL of the composition of the present invention.

In addition to the above, food formulations include various nutrients, vitamins, electrolytes, flavors, colorants, pectic acids and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohols, carbonated beverages. Carbonating agent used for the reaction may be contained. In addition, the food formulation may contain pulp for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components can be used independently or in combination. The proportion of such additives is not critical but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

The present invention also provides a composition for promoting stem cell activity comprising the fibrinomegaly extract as an active ingredient.

In the present invention, the term 'stem cell' is a cell capable of cell division on its own and having the ability to differentiate into a wide variety of specific cell types. The type of such stem cells is not particularly limited, and in one embodiment, the stem cells may be skin stem cells. The term “skin stem cell” refers to stem cells capable of differentiating into cells forming the skin (epidermis, dermis and subcutaneous fat layer). The skin-forming cells include keratinocytes, melanocytes and fibroblasts (mainly responsible for the biosynthesis of collagen and elastin) present in the epidermis.

The type of skin stem cells is not particularly limited. Skin stem cells used in the present invention can be used regardless of where it comes from. For example, dermal stem cells can be obtained from known dermal stem cell sources, such as the hair follicles or the basal layer of the epidermis, and the animal being harvested can be a mammal. In one embodiment, the mammal may include, but is not limited to, humans, mice, rats, guinea pigs, rabbits, monkeys, pigs, horses, cattle, sheep, antelopes, dogs, or cats. Preferably the mammal may be a human. Such methods for obtaining skin stem cells from skin stem cell sources are well known in the art.

In the present invention, the "stem cell activity promoting effect" refers to a stem cell proliferation promoting effect, and / or the specific indicator molecules of stem cells and the effect of maintaining the stem cell properties of self-dividing properties.

In addition, the compositions of the present invention can be used for preparing cosmetic formulations. Such cosmetic formulations are the same as those described in the description of the cosmetic formulations comprising the fibrinomegaly extract above.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Production Example  One: Nucleus  Preparation of Extract

After the filtrate was washed and powdered, it was cooled for 5 days in 10-fold ethanol (w / v), and then the extract was filtered and concentrated to obtain an extract. A mixed solution of purified water and ethanol was added thereto, dissolved, and filtered to prepare a final extract.

Production Example  2: Serum free Under badge conditions Skin stem cell  culture

Human epidermal stem cells purchased from Cellntec were added to 96-well plates (5 × 10 ³ cells / well) with Bovine pituitary extract (BPE) similar to fetal bovine serum. CNT-57 medium (Cellntec) was incubated for 24 hours at 5% CO ₂ and 37 ℃ conditions. Thereafter, the culture medium was removed through the suction tube, and then the medium component was removed using PBS solution (GibcoBRL Co., Ltd.), and 5 μg / ml of fibrinomycin extract was treated with CNT-57 medium without BPE to give 5% CO ₂ and Incubated for 72 hours at 37 ℃ conditions.

Experimental Example  One: CCK -8 through evaluation method Skin stem cells  Growth promoting effect

CCK-8 (Cell counting kit-8) evaluation was performed on the skin stem cells cultured in Preparation Example 2. CCK-8 is an assay that indirectly expresses the density of living cells by measuring the absorbance of formazan, which is produced by dehydrogenase in the intracellular electron transfer system, which is decomposed by tetrazolium salt.

The cells were treated with CCK-8 solution (1/10 of the volume of the medium) at 37 ° C. for 2 hours and then spectroscopically determined by measuring the absorbance at 450 nm. Proliferation promoting effect of the skin stem cells of the fibrinomegaly extract was obtained from the measured absorbance value compared to the control group (CNT-57 medium containing no extract) according to the following formula (1) to calculate the growth rate (%), the value is shown in Table 1 Indicated.

[Equation 1]

% Growth = (absorbance of sample treatment / absorbance of control) × 100

Proliferation promoting effect of skin stem cells (repeated number = 3) Sample added density % Cell Proliferation vs Control Fermented core extract 5 µg / ml 152% Control - 100%

As shown in Table 1, the skin stem cells in the medium condition treated with the fibrinomegaly extract showed an excellent growth promoting effect.

Experimental Example  2: Skin stem cell  Stem Cellularity stemness ) Check the maintenance effect

In order to confirm the stem cell maintenance effect of the skin stem cells cultured in Preparation Example 2 was evaluated the degree of p63 expression. p63 is an intracellular transcription factor and is well known as a maintenance marker of skin stem cells. RNA was isolated from the cells to synthesize cDNA, and then real-time PCR was performed with Taqman staining to measure the expression level of p63. The concentration of RNA in this experiment was normalized to S16 ribosomal RNA.

The experimental results are shown in Table 2 below. In Table 2 below, the value of p63 increase fold means a fold compared to the control (BPE-added CNT-57 medium).

Stemness retention effect (repeat count = 3) Sample added density p63 increase multiple (times) Fermented core extract 5 μg / mL 1.85 Control - 1.0

As shown in Table 2, the medium condition treated with the fibrinomegaly extract promotes p63 expression of skin stem cells, and has excellent stem cell maintenance effect.

Example  One: PPAR  Alpha activation promoting effect verification

In order to confirm the effect of promoting PPAR alpha activation of the fibrinomegaly extract, the following experiment was performed using the fibrinomegaly extract. C2C12 cells (ATCC CRL-1772), a myoblast cell line derived from the mouse, were passaged in DMEM (Dulbeco's Modified Eagle's Medium) medium containing 10% fetal bovine serum. The vector used here is

1) DNA fragments encoding Ser ₁₆₇ to Tyr ₄₆₈ in the DNA binding domain (GAL4-DBD) and yeast transcription factor GAL4-DBD (Ligand Binding Domain) in the SV40 promoter of pZEO vector (Invitrogen) (gene bank information, NM 005036);

2) a vector inserted in the GAL4 response sequence into multiple restriction enzyme recognition sites of pGL3 vector (Promega) expressing luciferase, and

3) A vector expressing β-galactosidase (β-galactodisase) was used as a transfection internal control.

The cultured cells were plated into 4 well plates in 4 × 10 ⁴ cells and incubated for 12 hours, and the three kinds of plasmid genes were transfected using transient lipopectamine. After 8 hours, the fibrinomegaly extract was diluted and treated, incubated for 12 hours, washed with 1 × PBS and lysed with 1 × reporter lysis buffer, and then luciferase assay kit (Promega). And the activity of luciferase using the β-galactosidase assay kit (Promega). That is, PPAR alpha activity was measured by 'luciferase activity / β-galactose activity'.

In this experiment, Wy-14,643 (Calbiochem), which is known to be the strongest of PPAR alpha ligands, was used as a positive control, and 0.05% DMSO used to dissolve the sample was used as a negative control.

According to the experimental method, the fibrinomegaly extract was treated with C2C12 cells to measure the activity of PPAR alpha according to the concentration change, and the results are shown in Table 3 below. In the following Table 3, the value of PPAR alpha activity means a percentage compared to a negative control (DMSO 0.05%).

Activity of PPAR Alpha sample PPAR alpha activity (%) DMSO (0.05%) 100 Wy-14,643 (0.5 μM) 324 Wy-14,643 (1 μM) 928 Lymphocytic extract (0.05%) 502 Lymphocytic extract (0.5%) 920

As shown in Table 3, compared with the positive control, it was confirmed that the fibrils extract shows a very strong PPAR alpha activity.

Example  2: Of Filaggrin  Expression promoting effect

The fibroblast extract was added to 5 ㎍ / mL and 10 ㎍ / mL, respectively, and added to the culture medium of HaCaT cells of human keratinocytes, and then cultured for 1 day. -time PCR was performed. RNA concentrations were normalized to S16 ribosomal RNA.

Effects of Filaggrin Expression According to Concentration (Repeat Number = 3) Sample added Increased pilargreen compared to the control Control group (no addition) 1.00 Lymphocytic extract (5 ㎍ / ml) 26.7 Lymphocytic extract (10 ㎍ / ml) 53.5

As shown in Table 4, the fibrinomegaly extract has the ability to promote filaggrin expression of human keratinocyte line HaCaT cells.

Example  3: To improve turn-over  About Efficacy

The nutritional cream of Formulation Example 2 was tested for the improvement of keratin exfoliation in healthy 20s to 50s women as follows.

THA was applied to 20 test subjects aged 20 to 50 with DHA (Dihydroxyacetone, Sigma Aldrich, USA) 1.5% solution closed closed for 8 hours and then applied to the test site twice daily. . After 4 days of application, 8 days after application, and 12 days after application, Chromameter CR-400 (Minolta, Japan) was used to measure the skin brightness of the deposition site and photographed using DSLR. The measured value was evaluated by calculating the average value of three times except the maximum value and the minimum value, indicating that the higher the skin brightness improvement of the deposition site, the better the exfoliation effect. The results are shown in Table 5 below.

Skin brightness improvement effect division 4 days after application 8 days after application 12 days after application % Improvement 1.95 6.23 13.59

As shown in Table 5, it was found that the use of the nourishing cream according to the present invention has an improvement in the exfoliation of keratin.

Formulation example  1: Preparation of Pharmaceutical Formulations

1. Preparation of Tablets

Lymphocytic extract 0.2mg

Corn Starch 100mg

Lactose 100mg

2 mg magnesium stearate

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

Formulation example  2: manufacture of cosmetics

1. Preparation of nutrition cream

As in the following composition, a nourishing cream containing fibrinomegaly extract as an active ingredient was prepared according to a conventional method.

0.2 wt% fibroblast extract

Beta-1,3-Glucan 5.0 wt%

Beeswax 10.0 wt%

Polysorbate60 1.5 wt%

Sebum 60 Cured Castor Oil 2.0 wt%

0.5 wt% sorbitan sesquioleate

10.0% by weight of liquid paraffin

Squalane 5.0 wt%

Caprylic / Capric Triglycerides 5.0 wt%

Glycerin 5.0 wt%

Butylene Glycol 3.0 wt%

Propylene Glycol 3.0 wt%

0.2% by weight of triethanolamine

0.05 wt% preservative

0.05% by weight of pigment

0.05% by weight fragrance

Purified water to 100% by weight

Formulation example  3: Preparation of external preparation for skin

1. Manufacture of ointments

As shown in the following composition, an ointment containing fibrinomegaly extract as an active ingredient was prepared according to a conventional method.

0.5 wt% fibroblast extract

Beta-1,3-Glucan 10.0 wt%

Beeswax 10.0 wt%

Polysorbate 60 5.0 wt%

Sebum 60 Cured Castor Oil 2.0 wt%

Sorbanthesquioleate 0.5 wt%

Vaseline 5.0 wt%

10.0% by weight of liquid paraffin

Squalane 5.0 wt%

Shea Butter 3.0 wt%

Caprylic / Capric Triglycerides 5.0 wt%

Glycerin 10.0 wt%

Propylene Glycol 10.2 wt%

0.2% by weight of triethanolamine

0.05 wt% preservative

0.05% by weight of pigment

0.05% by weight fragrance

Purified water to 100% by weight

Claims (8)

Composition for improving atopic dermatitis, including the nucleus extract as an active ingredient.
The method of claim 1,
Filtration nucleus extract is an extract obtained by extracting filtrate nuclei with one or more selected from the group consisting of water and organic solvents, atopic composition for improving.
The method of claim 2,
The organic solvent may be a polar solvent including a lower alcohol having 1 to 5 carbon atoms, ethyl acetate, or acetone; Nonpolar solvents including ether, chloroform, benzene, hexane or dichlorohexane; Or a mixed solvent thereof, a composition for improving atopy.
The method of claim 1,
The composition is for atopy improvement medicine, cosmetics or food manufacturing, composition for improving atopy.
delete delete delete delete