KR102109274B1 - Composition for skin regeneration and wound healing containing absolute of digitaria sanguinalis flower - Google Patents

Abstract

The present invention relates to a composition and cosmetic composition for skin regeneration, including absolutum extracted from barley flowers, wherein the absolute is excellent in skin regeneration, skin elasticity enhancement and wound healing through the effect of cell proliferation and migration and collagen synthesis at the skin cell level. It provides an effect.

Description

COMPOSITION FOR SKIN REGENERATION AND WOUND HEALING CONTAINING ABSOLUTE OF DIGITARIA SANGUINALIS FLOWER

The present invention relates to a composition for the treatment of skin regeneration and wounds containing absolut extracted from the dried flowers ( Digitaria sanguinalis ) as an active ingredient, and a cosmetic and skin external use thereof.

The skin is composed of three layers (epidermis, dermis, subcutaneous fat) and is an organ that performs various functions such as protecting the body from harmful external stimuli, maintaining moisture, performing sensory functions, and controlling body temperature. The epidermal layer is located on the outermost layer of the skin, and most of it consists of keratinocytes. The dermal layer is composed of several extracellular matrix, and among them, there are more than 20 types of collagen according to α-chain, and type I, III, IV, V, VI and VII type collagen is distributed in human skin. Mainly, type I collagen constitutes more than 85% of the dermal layer of the skin, and type IV collagen is the main structural protein of the basement membrane, and the synthesis of type I and IV collagen enhances skin elasticity and improves basement membrane formation. It is known that the adhesion and proliferation of skin cells are regulated, and the barrier function to selectively penetrate harmful substances and the interaction between the epidermis and the dermis are provided. For this reason, the movement and proliferation of dermal cells of the skin and the induction of collagen synthesis promote skin elasticity as well as skin regeneration and wound healing and help maintain healthy skin.

Natural fragrances are gaining attention once again, as problems such as high toxicity to the human body and hormonal disturbances have been recognized by synthetic fragrances that have been used in the modern society due to improved living standards and changes in consumer perception. In particular, research on natural flavors derived from natural products is becoming more important as natural flavors are known as well as antibacterial and anti-inflammatory and aromatherapy effects. Natural fragrances are classified in various ways according to the extract, extraction method, use and purpose, and natural fragrances derived from plants are classified into essential oils, absolute, concrete, etc. according to the extraction method. Plant-derived natural flavorings are volatile chemicals that show pharmacological effects on the human body among secondary metabolites of plants, and are mainly composed of various organic compounds such as terpene, alcohol, aldehyde, ester, and ketone. The aromatic ingredients of these natural fragrances not only show the effects of stress relief, fatigue recovery, muscle relaxation, etc. through the skin, circulatory system, or olfactory system, but also have therapeutic effects such as antibacterial action, immunity enhancement, and anti-allergic action. It has been used for prevention and prevention, and is used as a raw material for food flavoring and cosmetics. Recently, as natural materials that do not cause side effects have been attracting attention to modern people who are under threat of skin health due to changes in the environment and stress such as environmental pollution and destruction of the ozone layer, research related to the development of functional materials derived from natural products is required. .

Digitaria sanguinalis is a herbaceous perennial plant belonging to the rice family (Gramineae). Light green or purple flowers bloom from July to August. The height is 40 ~ 70 cm, the leaves are linear lanceolate, and the roots come from the nodes of the stem. It has been known since ancient times that adding water and decoction clears eyes and ears and improves digestion. A recent study on physiological activity has reported allelopathy on alfalfa plants.

Korean Patent Publication No. 10-2011-0036317

In order to solve the above problems, the present invention has been completed by completing the present invention by extracting absolut from the flowers of barley, identifying its constituents, and enhancing its skin elasticity, skin regeneration and wound healing efficacy.

In order to achieve the above object, the present invention provides a composition for skin regeneration and wound healing, comprising as an active ingredient an absolute extracted from a flower of Digitaria sanguinalis .

In one embodiment of the present invention, the absolute may be extracted by an organic solvent extraction method using a volatile solvent, and may be an absolute that removes insoluble wax by extracting the concrete extracted by the organic solvent extraction method with alcohol.

In one embodiment of the present invention, the absolute contains a volatile organic compound, for example, linolenic acid, cytostenone, cyclooctacosane, hexadecanoic acid ), Stigmasterol, Multiflora-7,9 (11) -dien-3β-ol (Multiflora-7,9 (11) -dien-3β-ol), Pentacosane, Methylenebis ( Phenol, 2,2'-methylenebis [6- (1,1-dimethylethyl) -4-methyl-), Stearic Acid, Campesterol, Heptacosane, Nonadecane , Squalene, Octacosane, 1-Docosene, 1-Nonadecene, Eicosane, Xycaine, Lanosteol It may include one or more volatile organic compounds selected from the group consisting of.

In one embodiment of the invention, the absolute may be included in the range of 0.1 ~ 100 μg / mL, for example, may be included in the range of 1 ~ 50 μg / mL.

In one embodiment of the present invention, the absolute may induce proliferation and migration of epidermal keratinocytes and dermal cells. Specifically, the absolute may induce proliferation, migration, and collagen synthesis of human skin-derived keratinocyte cell lines (HaCat) and human skin-derived dermal cells (CCD986skt).

In addition, the present invention provides a cosmetic composition comprising as an active ingredient the absolute extracted from the flowers of the barley.

In addition, the present invention provides a composition for external application for skin comprising absolute as an active ingredient extracted from dried flowers.

The present invention is to characterize the components of the absolute extracted from the flowers of barang, and demonstrate its skin elasticity enhancement, skin regeneration and wound healing efficacy, and induces cell proliferation and migration at the skin cell level, thereby providing excellent effects on skin regeneration and wound healing Have

1 is a flow chart showing the process of extracting the absolute (absolute) from the flowers of the barley in one embodiment of the present invention.
Figure 2 is a graph showing the effect of the absolute color of flowers in the basal flower on the proliferation of skin keratinocytes (A) and dermal cells (B) in one embodiment of the present invention.
Figure 3 is a graph showing the effect of the absolute color of flowers in the basal flower on the movement of skin keratinocytes (A) and dermal cells (B) in one embodiment of the present invention.
FIG. 4 is a graph quantifying the effect of collagen type I and IV synthesis on skin keratinocytes (A) and dermal cells (B) of Absolut of Abalone flowers in one embodiment of the present invention.
Figure 5 is an image showing the effect on the natural growth (out) growth of collagen sprouting (A) in the dermal cells of the Absolut (A) of the barley flower in one embodiment of the present invention and (B) collagen sprouting (sprouting) natural growth It is a graph showing quantified results on (out growth).
FIG. 6 is an image showing the effect on the phosphorylation of Absolute (A) extracellular signal regulation kinase (Erk1 / 2) and intracellular serine / threonine protein kinase (p38) in dermal cells in one embodiment, ( B) A graph showing the result of quantifying the phosphorylation of the extracellular signal regulation kinase (Erk1 / 2) in the dermal cell and (C) a graph showing the result of quantifying the effect on the phosphorylation of the serine / threonine protein kinase (p38) in the dermal cell to be.

Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings. In the following description, detailed descriptions of techniques well known to those skilled in the art can be omitted. In addition, in describing the present invention, when it is determined that detailed descriptions of related known functions or configurations may unnecessarily obscure the subject matter of the present invention, detailed descriptions thereof may be omitted. In addition, terms used in the present specification (terminology) are terms used to properly represent a preferred embodiment of the present invention, which may vary according to a user, an operator's intention, or a custom in a field to which the present invention pertains.

Therefore, the definition of these terms should be made based on the contents throughout this specification. Throughout the specification, when a part “includes” a certain component, it means that the component may further include other components, not to exclude other components, unless otherwise stated.

The present invention relates to a composition for skin regeneration and wound healing, comprising an absolute extracted from a barley flower as an active ingredient, a cosmetic composition comprising the same, and an external composition for skin.

The absolute is preferably obtained from dried dried flowers, and may be an absolute which removes insoluble wax by extracting concrete extracted with an organic solvent extraction method with alcohol.

Specifically, the absolute is the step of obtaining a concrete (concrete) by extracting the barley flower by an organic solvent extraction method using a volatile solvent; And extracting the obtained concrete with alcohol to remove insoluble wax and obtaining an absolute.

As an example, the absolute may be extracted as shown in the flowchart of FIG. 1. First, after drying the dried flowers, precipitated in hexane (n-hexane), the obtained extract is concentrated using a rotary evaporator to obtain concrete, and then ethyl alcohol is added to the obtained concrete. After storing and dissolving at -20 ° C for 12 hours, separation / purification using a vacuum cooling filtration and a rotary evaporator can be used to prepare a barley flower absolute according to the present invention.

The organic solvent extraction method and the method of separating absolute from the extract may include all methods that can be modified by those skilled in the art.

The absolut extracted from the dried flowers contains various physiologically active ingredients, more specifically, linolenic acid, cytostenone, cyclooctacosane, hexadecanoic acid, Stigmasterol, Multiflora-7,9 (11) -dien-3β-ol (Multiflora-7,9 (11) -dien-3β-ol), Pentacosane, Methylenebis (Phenol, 2,2'-methylenebis [6- (1,1-dimethylethyl) -4-methyl-), Stearic Acid, Campesterol, Heptacosane, Nonadecane, Squalene Group consisting of (Squalene), Octacosane, 1-Docosene, 1-Nonadecene, Eicosane, Xycaine, Lanosterol It may include one or more volatile organic compounds selected from, but is not limited thereto.

In addition, the content of these components is specifically linolenic acid (Linolenic acid) 56-57% by weight, Cytostenone (Sitostenone) 7-8% by weight, cyclooctacoic acid (Cyclooctacosane) 6-7% by weight, hexadecanoic acid (Hexadecanoic) acid) 4-5% by weight, Stigmasterol 4-5% by weight, Multiflora-7,9 (11) -dien-3β-ol (Multiflora-7,9 (11) -dien-3β-ol) 2-3% by weight, Pentacosane 2-3% by weight, Phenyl, 2,2'-methylenebis [6- (1,1-dimethylethyl) -4-methyl-) 1-2% by weight , Stearic acid (Octadecanoic acid) 1 to 2% by weight, Campesterol (1 to 2% by weight), Heptacosane (Heptacosane) to 1 to 2% by weight, Nonadecan (Nonadecane) 1 to 2% by weight, Squalene (Squalene) ) 1 ~ 2% by weight, Octacosane 1 ~ 2% by weight, 1-Docosene 1 ~ 2% by weight, 1-Nonadecene 1 ~ 2% by weight, Aikosan (Eicosane) 1 to 2% by weight, xycaine (Xycaine) 0 to 1% by weight, may be included in the range of 0 to 1% by weight of lanosterol (Lanosterol).

In the present invention, the absolute extracted from the barley contains the components exhibiting the physiological activity described above, and shows the effect of proliferation and migration of epidermal keratinocytes by these components (see FIGS. 2, 3 and 5). Clarified. In addition, the absolute extracted from the flowers of barang can induce at least one phosphorylation process or signaling process selected from the group consisting of serine / threonine kinase (p38) and extracellular signal regulation kinase (ERK1 / 2) in dermal cells.

Accordingly, it can be used as a skin regeneration and wound healing composition comprising the absolute extracted from the flowers of barang according to the present invention as an active ingredient, and contains alone or one or more pharmaceutically acceptable absolutes extracted from the pharmaceutically effective amount of barang. Carriers, excipients or diluents.

When the absolute of the present invention is used as a composition for skin regeneration and wound healing, absolute may be used at a concentration of 0.1 to 100 μg / mL. The effect of proliferation and migration of epidermal keratinocytes is insignificant when Absolute Absolute is used at a concentration of 0.1 μg / mL or less, and when Absolute is used at a concentration of 100 μg / mL or more, a marked increase in the effect of content increases. It is not desirable because it does not appear and decreases.

In the above, "a pharmacologically effective amount" refers to an amount sufficient for the physiologically active ingredient to be administered to an animal or human to exhibit a desired physiological or pharmacological activity. However, the pharmaceutically effective amount may be appropriately changed according to the severity of symptoms, the age of the patient, weight, health status, sex, administration route, and treatment period.

In addition, "pharmaceutically acceptable" in the above refers to a physiologically acceptable, when administered to a human, usually does not cause an allergic reaction such as gastrointestinal disorder, dizziness, or a similar reaction. Examples of the carrier, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In addition, fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers and preservatives may be further included.

In addition, the compositions of the present invention can be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal, and can be formulated for oral or parenteral administration. It can be formulated in form. The formulation may be in the form of a powder, granule, tablet, emulsion, syrup, aerosol, soft or hard gelatin capsule, sterile injectable solution, sterile powder, ointment, lotion, cream, plaster, paste, and the like.

The composition according to the present invention can be administered through several routes including oral, transdermal, subcutaneous, intravenous or intramuscular, and the dosage of the active ingredient is a number of factors such as the route of administration, the patient's age, gender, weight and severity of the patient. It can be appropriately selected according to. In addition, the composition of the present invention can be administered in parallel with a known compound that can enhance the desired effect.

Furthermore, the anti-obesity composition according to the present invention can be used not only as a pharmaceutical composition as described above, but also as a health functional food. For example, it can be easily utilized as a main raw material, an auxiliary raw material, a food additive, a functional food or beverage.

The "food" refers to a natural product or a processed product that contains one or more nutrients, and preferably means a state that can be directly eaten through a certain processing process, and in a general sense, food It includes food additives, functional foods and beverages.

Foods to which the food composition can be added include, for example, various foods, beverages, gums, teas, vitamin complexes, and functional foods. In addition, special nutritional foods (e.g., dairy products, English, baby food, etc.), processed meat products, fish meat products, tofu, jelly, noodles (e.g., ramen, noodles, etc.), breads, health supplements, seasoned foods (e.g. soy sauce) , Miso, red pepper paste, mixed sauce, etc.), sauces, confectionery (e.g. snacks), candy, chocolate, gum, ice cream, dairy products (e.g. fermented milk, cheese, etc.), other processed foods, kimchi, pickled foods (various kimchis) , Pickles, etc.), beverages (eg, fruit drinks, vegetable drinks, soy milk, fermented drinks, etc.), natural seasonings (eg, ramen soup, etc.). The food, beverage or food additive may be prepared by a conventional manufacturing method.

In addition, the term “functional food” or “health functional food” refers to a food group or a living body having a food composition that provides added value to act and express the function of the food for a specific purpose by using physical, biochemical, or biotechnical techniques. Refers to foods designed and processed to sufficiently express the body's regulation functions for defense rhythm control, disease prevention and recovery, etc. Specifically, it may be a health functional food. The functional food may include a food-acceptable food supplement additive, and may further include suitable carriers, excipients, and diluents commonly used in the production of functional foods.

The type of the dietary supplement is not limited thereto, but may be in the form of powder, granule, tablet, capsule or beverage.

Advantages and features of the present invention, and methods for achieving them will be clarified with reference to embodiments described below in detail. Hereinafter, the present invention will be described in detail by examples. However, these examples are intended to illustrate the present invention in detail, and the scope of the present invention is not limited to these examples.

[Example]

Example 1 Preparation of Basil Flower Absolute

According to the process shown in Figure 1 to prepare a flower Absolut.

In this example, the dried flowers were collected and used in the vicinity of the Hanaro Farm (Haenam, Korea) practice field. 3 kg of dried dried flowers were extracted using an organic solvent extraction method.

That is, the dried dried flowers were precipitated in hexane (n-hexane), and the obtained precipitate was concentrated by evaporation in a rotary evaporation to obtain solid concrete. Then, after dissolving the concrete by adding ethyl alcohol, the insoluble wax was removed through a vacuum cooling filtration, and then evaporated in a rotary evaporator to separate / purify the barley flower absolute. Was prepared. And the prepared flowers of the dried flowers were stored in a low-temperature high temperature of -80 ℃.

Example 2. Component analysis of barley flower absolute

The analysis of the ingredients of the barley flower absolute was commissioned by KBSI (Korea Basic Science Institute, Seoul Center). A gas chromatography / mass spectrometer (6890N GC / 5975i MS, Agilent, USA) was used, and a column of GC / MS was DB5-MS (30 m × 50 μm, 0.25 μm), and carrier gas ( carrier gas) was analyzed by flowing at 1 mL / min using helium. The temperature of the oven was maintained at 40 ° C-2 ° C / min-230 ° C-5 ° C / min-300 ° C (5 minutes), and the injection temperature was analyzed at 280 ° C. In addition, the interface temperature was analyzed at 300 ℃, the ion source temperature at 230 ℃, and the analyzer temperature at 150 ℃, and the mass range was analyzed at 40 ~ 800 m / z.

Confirmation of the component of the flower Absolute was confirmed by comparing the retention time of volatile components (retention indices, RI) and the retention time of standard materials or by comparing the mass spectrum of volatiles with the mass spectrum of the Wiley 7Nist 05 library. RI was determined under the same conditions as the samples using C ₆ to C ₂₄ (nalkane). As a result, it was confirmed that it was composed of a total of 19 components, linolenic acid, cytostenone, cyclooctacosane, hexadecanoic acid, and stigmasterol. , Multiflora-7,9 (11) -dien-3β-ol (Multiflora-7,9 (11) -dien-3β-ol), Pentacosane, Methylenebis (Phenol, 2,2'- methylenebis [6- (1,1-dimethylethyl) -4-methyl-), stearic acid, campesterol, heptacosane, nonadecane, squalene, octane Octacosane, 1-dococene (1-Docosene), 1-nonadecene (1-Nonadecene), eicosane (Xycaine), lanosterol (Lanosterol) showed the highest content in this order. (Table 1).

Example 3. Effect of barley flower absolute on the proliferation of skin keratinocytes and dermal cells

In order to confirm the effect of the barley flower absolut obtained in Example 1 on epidermal keratinocytes and dermal cell proliferation, it was investigated as follows.

Human skin-derived epidermal keratinocytes (HaCat) were cultured using DMEM medium containing 10% fetal bovine serum and utilized in the examples of the present invention. Epidermal keratinocytes were injected into a 96 well microtiter plate so that the number of cells per well was 3 X 10 ³ cells / 100 μL / well and attached in a 37 ° C, 5% CO ₂ incubator.

Basin flower absolute was treated with 100 μL in each well at a concentration of 0.1 to 100 μg / mL to incubate for 36 hours. Ez-cytox and DMEM were mixed 1: 1 and treated with 20 μL per well, and reacted for 30 minutes in an incubator. After measuring the absorbance at 450 nm using an ELISA plate reader (Synerge2, BioTek, USA), it was converted to a percentage to secure the results for epidermal keratinocyte proliferation of barley flower absolute.

Human skin-derived dermal cells (CCD986skt) were cultured using RPMI1940 medium containing 10% fetal bovine serum and utilized in experimental examples of the present invention. Dermal cells were injected into a 96-well microtiter plate so that the number of cells per well was 3 X 10 ³ cells / 100 μL / well and attached in a 37 ° C, 5% CO ₂ incubator.

Basin flower absolute was treated with 100 μL in each well at a concentration of 0.1 to 100 μg / mL to incubate for 36 hours. Ez-cytox and RPMI1640 were mixed 1: 1 and treated with 20 μL per well, and reacted for 30 minutes in an incubator. After measuring the absorbance at 450 nm using an ELISA plate reader (Synerge2, BioTek, USA), it was converted to a percentage to secure the results for skin dermal cell proliferation of barley flower absolute. 3A and 3B are graphs showing the proliferative effect of epidermal keratinocytes and dermal cells of barley flower absolute extracted by Example 1 of the present invention. Here, the negative control is not using the barley flower absolute, and the positive control is using epidermal growth factor (EGF 50 ng / mL), which is conventionally used for wound healing and / or skin regeneration.

As a result, as shown in FIGS. 2A and 2B, it was shown that the barley flower absolute according to the present invention induces the proliferation of epidermal keratinocytes and dermal cells in a concentration-dependent manner.

That is, the proliferation of epidermal keratinocytes and dermal cells by epidermal growth factor (50 ng / mL) was induced 169.87 ± 1.91%, 117.79 ± 3.58%, respectively, and compared to the negative control group, baransis flower absolute 1 μg / mL The concentrations showed 103.90 ± 5.32% and 102.49 ± 2.52%, respectively, and the concentrations of 100 μg / mL showed 185.32 ± 5.13%, 127.20 ± 4.46%, respectively, and epidermal keratinocytes. And it has been shown to effectively induce proliferation of dermal cells.

Example 4. Effect of barley flower absolut on skin keratinocytes and dermal cell migration

In order to confirm the effect of barley flower absolute obtained in Example 1 on epidermal keratinocytes and dermal cell migration, experiments were conducted as follows.

Human skin-derived epidermal keratinocytes (HaCat) were cultured using DMEM medium containing 10% fetal bovine serum and utilized in the examples of the present invention. The mobility of epidermal keratinocytes was investigated using a 48 well Boyden microchemotaxis chamber. First, the concentration of 0.1 to 100 μg / mL in each of the flowers in the lower chamber was treated in the lower chamber, and polycarbonate of 8 μm pore coated with type I collagen was applied. The membrane was loaded. Subsequently, epidermal keratinocytes were treated in the upper chamber at 5 × 10 ⁴ cells / 50 μL per well. After incubating the chamber for 3 hours and 30 minutes in a 37 ° C incubator, the membrane was stained and fixed with a Diff-Quick solution.

The number of cells that passed through the membrane was counted and converted to a percentage to secure the results of the movement of the skin epidermal keratinocytes of the flower Absolute.

Human skin-derived dermal cells (CCD986sk) were cultured using RPMI1640 medium containing 10% fetal bovine serum and utilized in the examples of the present invention. The flow rate of dermal cells was investigated using a 48 well Boyden microchemotaxis chamber. First, the concentration of 0.1 to 100 μg / mL in basal flower absolute was treated in the lower chamber, and a polycarbonate membrane of 8 μm pore coated with type I collagen was applied. I uploaded it. Subsequently, dermal cells were treated in the upper chamber at 5 × 10 ⁴ cells / 50 μL per well. After incubating the chamber in a 37 ° C incubator for 90 minutes, the membrane was stained and fixed with a Diff-Quick solution.

The number of cells that passed through the membrane was counted and converted to a percentage to obtain the results for the skin dermal cell migration of the dried flowers Absolute.

3A and 3B are graphs showing the migration effect of cutaneous keratinocytes and dermal cells of the dried flowers Absolute extracted by Example 1 of the present invention. Negative control was not used for the flower Absolute, and positive control was used for epidermal growth factor (EGF 5 ng / mL), which is conventionally used for wound healing and / or skin regeneration.

As a result, as shown in FIG. 3B, the basal flower absolut according to Example 1 of the present invention induced concentration of dermal cells in a concentration-dependent manner, and in particular, it was 104.92 ± 1.78% at a concentration of 1 μg / mL of barracus flower absolute. It showed the rate of induction of migration, and the maximum rate of migration of 156.83 ± 9.07% at a concentration of 100 μg / mL, so that it was found that barley flower absolute effectively induces the movement of dermal cells.

Example 5. Investigation of the effects of barley flower absolute on collagen Type I or IV synthesis and secretion of skin keratinocytes and dermal cells

In order to confirm the effect on the synthesis of collagen Type I, IV, which is an ECM component required for the proliferation and migration of epidermal keratinocytes and dermal cells, the barley flower absolute obtained in Example 1 was tested as follows.

Monoclonal anti-collagen I or IV Ab, (2 μg / well / PBS) was injected into a 96 well microtiter plate and coated at RT for 12 hours. After blocking with blocking buffer for 1 hour, the sample was processed at 100 μL per well and reacted at RT for 90 min. Samples were cultured (CM) of epidermal keratinocytes that were not treated with basal absolut and cultured (CM) of epidermal keratinocytes treated with basal absolute 10 μg / mL and epidermal keratin treated with basal absolute 100 μg / mL. Culture medium (CM) of forming cells was treated. Thereafter, polyclonal anti-collagen I or IV Ab was treated in each well to react for 90 minutes at RT, and then avidin-HRP was treated for each well to react for 60 minutes at RT. Supersignal ELISA pico chemiluminescent substrate was injected for 100 μL per well and reacted for 10 minutes, and then the luminescence degree was measured using an ELISA platereader. Results for type I or IV collagen synthesis and secretion of epidermal keratinocytes of BABY Absolute Was secured (FIG. 4A).

Monoclonal anti-collagen I or IV Ab, (2 μg / well / PBS) was injected into a 96 well microtiter plate and coated at RT for 12 hours. After blocking with blocking buffer for 1 hour, the sample was processed at 100 μL per well and reacted at RT for 90 min. Samples are cultures of dermal cells (CM) that have not been treated with Absolute Absolute and cultures of dermal cells (CM) treated with concentration of 10 μg / mL Absolut and cultures of dermal cells treated with concentration of 100 μg / mL Absolut (CM) ). Thereafter, polyclonal anti-collagen I or IV Ab was treated in each well to react for 90 minutes at RT, and then avidin-HRP was treated for each well to react for 60 minutes at RT. After injecting 100 μL of each supersignal ELISA pico chemiluminescent substrate for 10 minutes and reacting for 10 minutes, the degree of luminescence was measured using an ELISA platereader, and the results for type I or IV collagen synthesis and secretion of dermal cells of BABY Absolute were obtained. Did.

As a result, as shown in FIG. 4B, it was shown that barley absolut significantly promotes the synthesis and secretion of type I collagen and type IV collagen in dermal cells. In particular, the synthesis of type I collagen in dermal cell culture (CM) was induced by 169.39 ± 11.96% at a concentration of 100 μg / mL, and the synthesis of type IV collagen was induced by 196.33 ± 17.36% at a concentration of 100 μg / mL, so that Absolute Absolute Was found to effectively induce type I and IV collagen synthesis and secretion of dermal cells.

Example 6. Effect of barley flower absolute on out growth of dermal cell collagen sprouting

In order to measure the effect on the outgrowth of collagen sprouting dermal cell collagen obtained by Example 1, the flowers of barley flowers were investigated as follows.

After mixing Type I collagen and dermal cells to 2.5 × 10 ⁷ cells / mL, neutralize by treating with NaOH (1 N) solution, spotting 10 μL in a 24 well plate, 37 ° C for 5 minutes, 5% After drying in a CO ₂ incubator, it was dried at room temperature for about 10 minutes to form artificial skin tissue. Samples were processed and incubated for 48 hours at 37 ° C. in a 5% CO ₂ incubator. The culture solution was removed, and cells and tissues were fixed and stained using Diff-Quik solution, and the stained cells and tissues were photographed to obtain results.

Figure 5 is a result showing the effect on the outgrowth of dermal cell collagen sprouting of the dried flowers Absolut extracted by Example 1 of the present invention, the negative control is not using the dried flowers Absolute, the positive control is a conventional wound It uses epidermal growth factor (EGF 50 ng / mL), which is used for healing and / or skin regeneration.

As a result, as shown in FIG. 5A, it was shown that barley flower absolut induces concentration-dependent out growth of dermal cell collagen sprouting. In other words, compared to the positive control group, it was found that the inflorescence of inflorescence was effectively induced out of dermal cell collagen sprouting at a concentration of 100 μg / mL. In particular, as shown in FIG. 5B, 179.60 ± 11.06% was induced at a concentration of 1 μg / mL Absolut, and 572.82 ± 2.51% was induced at a concentration of 100 μg / mL.

Example 7. Investigation of the effect of barley flower absolut on the signaling process in dermal cells

The effect of the basal flower absolute obtained in Example 1 on the phosphorylation process of the extracellular signal-regulating kinase (Erk1 / 2) and serine / threonine kinase (p38), which are the main signaling processes that regulate the proliferation and migration of dermal cells. In order to measure, the concentration of barley flower absolute presented in Example 3 was processed, and the immunoblot method was performed using each specific antibody (P-Erk1 / 2, Erk1 / 2, P-p38, p38). Was utilized.

Figure 6A is a result showing the effect on the phosphorylation process of the intracellular dermal extracellular signal regulation kinase (Erk1 / 2) and intracellular serine / threonine kinase (p38) of the dried flowers Absolute extracted by Example 1 of the present invention, Negative control was not used for the flower Absolute, and positive control was used for epidermal growth factor (EGF 50 ng / mL), which is used for conventional wound healing and / or skin regeneration.

As a result, as shown in Figs. 6B and 6C, barley flower absolut induced phosphorylation of intradermal extracellular signal regulatory kinase (Erk1 / 2) and intracellular serine / threonine kinase (p38) in a concentration-dependent manner. Phosphorylation of extracellular signal regulatory kinase (ERK1 / 2) was induced by 205.82 ± 28.43% at a concentration of 100 μg / mL flower absolute, and phosphorylation of intracellular serine / threonine kinase (p38) was found to be induced by 212.34 ± 9.90%.

So far, the present invention has been focused on the preferred embodiments. Those skilled in the art to which the present invention pertains will appreciate that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered in terms of explanation, not limitation. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the equivalent range should be interpreted as being included in the present invention.

Claims (8)

A composition for skin regeneration and wound healing that includes absolute extracted from flowers of Digitaria sanguinalis as an active ingredient. According to claim 1,
The absolute is a composition for skin regeneration and wound treatment that is extracted by an organic solvent extraction method using a volatile solvent. According to claim 2,
The absolute is a composition for skin regeneration and wound treatment that is an absolute that removes insoluble wax by extracting the concrete extracted with the organic solvent extraction method with alcohol. According to claim 1,
The absolute is linolenic acid, cytostenone, cyclooctacosane, hexadecanoic acid, stigmasterol, multiflora-7,9 (11) -dien- 3β-ol (Multiflora-7,9 (11) -dien-3β-ol), Pentacosane, Methylenebis (Phenol, 2,2'-methylenebis [6- (1,1-dimethylethyl) -4 -methyl-), stearic acid, campesterol, heptacosane, nonadecane, squalene, octacoane, 1-docosene ), 1-Nonadecene (1-Nonadecene), Icosan (Eicosane), Xycaine (Xycaine), and contains one or more volatile organic compounds selected from the group consisting of Lanosteol (Lanosterol) for skin regeneration and wound treatment Composition. The method according to any one of claims 1 to 4,
The absolute is a composition for skin regeneration and wound treatment that is included in the range of 0.1 to 100 μg / mL. The method according to any one of claims 1 to 4,
The absolute is a composition for skin regeneration and wound treatment that induces proliferation and migration of epidermal keratinocytes and dermal cells. Cosmetic composition comprising as an absolute active ingredient extracted from flowers of Digitaria sanguinalis . A composition for external application for skin regeneration and wound healing, comprising absolute extracted from flowers of Digitaria sanguinalis as an active ingredient.

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