KR20110018067A - Compositions for epidermal keratinocyte stem cells proliferation - Google Patents

Abstract

PURPOSE: A composition containing ortho-dihydroxyisoflavone derivative for improving skin regeneration or skin elasticity is provided to induce proliferation of epidermal keratinocyte stem cells. CONSTITUTION: A composition for improving skin regeneration or skin elasticity contains 0.001-30 weight% of ortho-dihydroxyisoflavoen derivative of 4',7,8-trihydroxyisoflavone, 4',6,7-trihydroxyisoflavone, or 3',4',7-trihydroxyisoflavone as an active ingredient. An external use skin composition, pharmaceutical composition, or cosmetic composition contains the composition.

Description

Composition for skin keratin stem cell proliferation {Compositions for Epidermal Keratinocyte Stem Cells Proliferation}

The present invention relates to an ortho-dihydroxyisoflavone derivative-containing composition having a proliferative effect on human dermal keratinocyte stem cells (KSC).

The most important role of the skin is to prevent the loss of heat and moisture from the inside while acting as a defense barrier to protect the human body from external damage and invasion of bacteria. The skin is largely structurally comprised of the epidermis and dermis, which can be divided into the stratum corneum and the living epidermis, which consist of flat hexagonal dead cells.

The living epidermis comprises four layers: Stratum Basale, Stratum Spinosum, Stratum Granulosum, and Stratum lucidum. Of these, keratinocyte stem cells (KSC) are located in the basal layer, and keratinocyte stem cells are continuously dividing to form trans-amplifying cells (TA cells). These cells divide a number of times and form keratinocytes that play a pivotal role in the skin barrier function, which is the dermal development of the skin. Keratinocytes undergo differentiation and are pushed up to the skin surface, the last step in dermatological development. In this process, keratinocyte differentiation and keratinization occur, creating the stratum corneum, which eventually leaves the stratum corneum.

As people age, their ability to proliferate these keratinocyte stem cells (KSC) and transit-amplifying cells (TA cells) decreases. Decreased ability to make keratinocytes eventually leads to a decrease in skin regeneration ability, which intensifies aging phenomena such as decreased skin elasticity (Fuchs E. Nature . 2007 Feb 22; 445 (7130): 834-42).

An object of one embodiment of the present invention is to provide a composition for improving skin regeneration or skin elasticity.

Another object of an embodiment of the present invention is to provide a cosmetic or pharmaceutical composition for improving skin regeneration or skin elasticity.

Compositions according to the present invention for achieving this object are 4 ', 7,8-trihydroxyisoflavones, 4', 6,7-trihydroxyisoflavones, and 3 ', 4', 7-trihydr It contains at least one ortho-dihydroxyisoflavone derivative selected from the group comprising oxyisoflavones as an active ingredient.

The composition according to the present invention induces the proliferation of skin keratinocytes, thereby exhibiting skin regeneration and / or skin elasticity improvement effects, and thus can be variously used in cosmetics or medicine.

The present invention relates to a composition for improving skin regeneration and / or skin elasticity improvement, characterized by containing an ortho-dihydroxyisoflavone derivative as an active ingredient. In one embodiment, the ortho-dihydroxyisoflavone derivatives are, for example, 4 ', 7,8-trihydroxyisoflavone, 4', 6,7-trihydroxyisoflavone, and 3 ', It may be one or more selected from the group containing 4 ', 7-trihydroxyisoflavone.

The ortho-dihydroxyisoflavone derivative is recognized to improve skin regeneration and improve skin elasticity through the proliferation of skin keratinocytes. Specifically, cutaneous keratinocytes divide continuously to produce Trans-Amplifying Cells (TA cells). These meta-amplified cells divide more than a dozen times and become keratinocytes, one of the skin's major cells, and rise to the skin surface through differentiation. Therefore, the proliferation of keratinocyte stem cells (KSC) ultimately plays a role in regenerating the epidermis (Fuchs E. Nature . 2007 Feb 22; 445 (7130): 834-42).

In one embodiment, the 4 ', 7,8-trihydroxyisoflavone, 4', 6,7-trihydroxyisoflavone, and 3 ', 4', 7-trihydroxyisoflavone are each It may be represented as in the formula (1-3).

4 ', 6,7-trihydroxyisoflavone, 4', 7,8-trihydroxyisoflavone and 3 ', 4', 7-trihydr, which are ortho-dihydroxyisoflavone derivatives according to the invention Roxyisoflavones can be prepared by known methods known in the art. The ortho-dihydroxyisoflavone derivatives can be produced, for example, by bioconversion of daidzein, but is not limited thereto.

In one embodiment, the ortho-dihydroxyisoflavone derivatives may be contained in an amount of 0.001 to 30% by weight based on the total weight of the composition. If the content of the derivative is less than 0.001% by weight, it is not possible to sufficiently obtain the effect of skin regeneration and elasticity through the proliferation of keratinocytes of the skin keratinocytes. If the content of the derivative is more than 30% by weight, the effect is not large. Can occur.

In addition, marker markers (proteins) for keratinocyte stem cells (KSC) and trans-amplifying cells (TA cells) were identified. As a result, CD71 and alpha 6 integrin were identified. The marker was identified.

The CD71 protein (or transferrin receptor) is a protein present in the cell membrane that interacts with the transferrin protein, which binds to iron flowing through the bloodstream of the cell, thereby receptor mediated transferrin. endocytosis) to supply iron in the cell. Since iron is usually toxic, the amount of iron in the cell should be well controlled by the expression level of CD71 protein.

Another marker, alpha 6 integrin, was identified, and severe skin blisters were observed in knockout mice that do not express the protein of the marker, and are known to die at birth. . Alpha 6 integrin is mainly associated with beta 4 integrins in Keratinocyte Stem Cells (KSC), an important factor in a cellular structural complex called hemidesmosome. To achieve. Alpha6 integrins are known to affect cellular function in a variety of areas such as cellular communication, cell migration, proliferation, and differentiation.

In general, the early stages of skin formation are the keratinocyte stem cells (KSC) and transit-amplifying cells (TA cells). In keratinocyte stem cells (KSC), alpha 6 integrin expression is strong and CD71 protein (or transferrin receptor) expression is weak (Li A, Kaur P. Methods). Mol Biol . 2005; 289: 87-96). In addition, in trans-amplifying cells (TA cells), both alpha 6 integrin and CD71 protein (or Transferrin receptor) are characterized by strong expression.

In addition, FZD1 (Frizzled homolog 1) is also recognized as a marker for skin keratinocytes, but it is not known how these proteins function in these cells.

In experimental examples of the present invention, specific derivatives of ortho-dihydroxyisoflavones are determined by measuring the expression level of the above-mentioned alpha 6 integrin, CD71 protein, and FZD1 (Frizzled homolog 1) gene. It was confirmed that there is a proliferative effect of skin keratinocytes.

The present invention also provides a topical skin composition containing the ortho-dihydroxyisoflavone derivative. In one embodiment, it provides a cosmetic composition and pharmaceutical composition for improving skin regeneration and / or skin elasticity containing ortho-dihydroxyisoflavone derivatives.

The external preparation composition for skin according to the present invention may be formulated in the form of a dry powder which is in the form of a solution, a suspension or an emulsion in an oil or an aqueous medium, or dissolved in sterile, pyrogen-free water before use. Water-in-oil emulsions are oil-based vegetable oils such as olive oil or mineral oils such as liquid paraffin, derived from natural phospholipids such as soy lecithin and esters of anhydrous hecitol or fatty acids such as sorbitan monooleate, hydroxy The active ingredient is emulsified using compounds obtained by condensation of ethylene oxide with partial esters derived from anhydrous hexitol and fatty acids such as ethylene sorbitol monooleate as emulsifiers.

When the composition according to the present invention is formulated in the form of a cosmetic, it can be used for the purpose of anti-aging through improving skin regeneration and skin elasticity. The cosmetic formulation is not particularly limited, for example, supple cosmetics, astringent cosmetics, nourishing cosmetics, massage cream, eye cream, nutrition cream, cleansing cream, cleansing foam, cleansing water, powder, essence, pack, gel or skin It may be a formulation of a tacky cosmetic, and may also be a transdermal dosage form such as a lotion, ointment, gel, cream, patch or spray. In addition, in each formulation, other ingredients can be appropriately selected and blended by those skilled in the art without difficulty according to the kind or purpose of use of other cosmetic compositions.

The present invention also provides a pharmaceutical composition comprising the composition. The pharmaceutical composition according to the present invention may be a composition for improving skin regeneration and / or improving skin elasticity. Specifically, the pharmaceutical composition, through the proliferation of the skin keratinocytes, it is recognized that the effect of improving skin regeneration and skin elasticity.

When the composition according to the present invention is applied to medicines, the composition may be formulated into an oral or parenteral dosage form in the form of solid, semi-solid or liquid by adding a commercially available inorganic or organic carrier.

As the preparation for oral administration, tablets, pills, granules, soft and hard capsules, powders, fine granules, powders, emulsions, syrups, pellets, etc. Can be mentioned. In addition, preparations for parenteral administration include injections, drops, ointments, lotions, sprays, suspensions, emulsions, suppositories, and the like. In order to formulate the active ingredient of the present invention, surfactants, excipients, coloring agents, spices, preservatives, stabilizers, buffers, suspensions, and other commonly used auxiliaries can be suitably used.

The pharmaceutical composition according to the present invention may be administered orally, parenteral, rectal, topical, transdermal, intravenous, intramuscular, intraperitoneal, subcutaneous.

In addition, the dosage of the active ingredient will vary depending on the age, sex and weight of the subject to be treated, the specific disease or pathology to be treated, the severity of the disease or pathology, the route of administration and the judgment of the prescriber. Dosage determination based on these factors is within the level of skill in the art. Typical dosages are from 0.001 mg / kg / day to 2000 mg / kg / day, more specifically from 0.5 mg / kg / day to 1500 mg / kg / day.

Hereinafter, although an Example, a test example, etc. demonstrate this invention more concretely, the scope of the present invention is not limited by these.

Preparation Example Preparation of Soybean Extract

6 liters of hexane was added to soybean 2, followed by stirring extraction three times at room temperature for degreasing. Then, 4 L of 80% methanol was added to 1 kg of degreased soybean, refluxed three times, and deposited at 15 ° C. for 1 day. Subsequently, the residue and the filtrate were separated through filter cloth filtration and centrifugation, and the separated filtrate was concentrated under reduced pressure. The obtained extract was suspended in water, extracted five times with 1 L of ether to remove the pigment, and the aqueous layer was extracted three times with 500 ml of 1-butanol. The 1-butanol layer was concentrated under reduced pressure to obtain 1-butanol extract, which was dissolved in a small amount of methanol, and then a large amount of ethyl acetate was added. An additional precipitate of ethyl acetate was dried to obtain 300 g of soybean extract.

Example 1 Preparation of 4 ', 6,7-trihydroxyisoflavone (4', 6,7-trihydroxyisoflavone) using soybean extract

10 g of the soybean extract obtained in Preparation Example 1 was dissolved in 100 ml of ionized water, sterilized at 121 ° C for 30 minutes, and then cooled to 30 ° C. After cooling, pre-cultured Aspergillus niger KCCM 11885 was inoculated at 5-10% of the liquid volume. After culturing at 37 ° C. for 7 days, the scavenging rate of the substrate was confirmed by thin layer chromatography, and the reaction was terminated when the substrate was completely lost. The precipitate obtained by centrifuging the culture at 5,000 to 10,000 rpm was washed three times with distilled water and centrifuged again to obtain a precipitate. Ethanol (200 mL) was added to the precipitate and stirred (3 times). The precipitated salts were removed by filtration, and the filtrate was concentrated under reduced pressure to obtain a crude product. The obtained crude product was separated by silica gel column chromatography (chloroform: methanol = 8: 1 to 4: 1) to obtain 0.23 g of 4 ', 6,7-trihydroxyisoflavone.

Example 2 Preparation of 4 ', 6,7-trihydroxyisoflavone using didzin

5 g of dydazine (Sigma Cat. No. D7802) was dissolved in 100 ml of ionized water, sterilized at 121 ° C for 30 minutes and then cooled to 30 ° C. After cooling, pre-cultured Aspergillus niger KCCM 11885 was inoculated at 5-10% of the liquid volume. After culturing at 37 ° C. for 7 days, the scavenging rate of the substrate was confirmed by thin layer chromatography, and the reaction was terminated when the substrate was completely lost. The precipitate obtained by centrifuging the culture at 5,000 to 10,000 rpm was washed three times with distilled water and centrifuged again to obtain a precipitate. Ethanol (200 mL) was added to the precipitate and stirred (3 times). The precipitated salts were removed by filtration, and the filtrate was concentrated under reduced pressure to give a crude product. The obtained crude product was separated by silica gel column chromatography (chloroform: methanol = 8: 1 to 4: 1) to obtain 0.34 g of 4 ', 6,7-trihydroxyisoflavone.

Example 3 Preparation of 4 ', 6,7-trihydroxyisoflavones using daidzein

3 g of Dyzedine (Sigma Cat. No. D7802) was dissolved in 100 ml of ionized water, sterilized at 121 ° C for 30 minutes, and then cooled to 30 ° C. After cooling, the cultured Aspergillus niger KCCM 11885 was inoculated 5 to 10% of the liquid amount. The reaction was then terminated after incubation at 37 ° C. for 7 days. The precipitate obtained by centrifuging the culture at 5,000 to 10,000 rpm was washed three times with distilled water and centrifuged again to obtain a precipitate. Ethanol (200 mL) was added to the precipitate, stirred (3 times), the precipitated salts were removed by filtration, and the filtrate was concentrated under reduced pressure to give a crude product. The obtained crude product was separated by silica gel column chromatography (chloroform: methanol = 8: 1 to 4: 1) to obtain 0.45 g of 4 ', 6,7-trihydroxyisoflavone.

Example 4 Preparation of 3 ', 4', 7-trihydroxyisoflavone (3 ', 4', 7-trihydroxyisoflavone) using soybean extract

10 g of the soybean extract obtained in Preparation Example 1 was dissolved in 100 ml of ionized water, sterilized at 121 ° C for 30 minutes, and then cooled to 30 ° C. After cooling, Bacillus subtilis KCCM 11732, which was pre-cultured, was inoculated at 5-10% of the liquid amount. After incubation at 30 ° C. for 7 days, the scavenging rate of the substrate was confirmed by thin layer chromatography, and the reaction was terminated when the substrate was completely lost. The precipitate obtained by centrifuging the culture at 5,000 to 10,000 rpm was washed three times with distilled water and centrifuged again to obtain a precipitate. Ethanol (200 mL) was added to the precipitate, stirred (3 times), the precipitated salts were removed by filtration, and the filtrate was concentrated under reduced pressure to give a crude product. The obtained crude product was separated by silica gel column chromatography (chloroform: methanol = 8: 1 to 4: 1) to obtain 0.23 g of 3 ', 4', 7-trihydroxyisoflavone.

Example 5 Preparation of 3 ', 4', 7-trihydroxyisoflavone using didzin

5 g of dydazine (Sigma Cat. No. 30408) was dissolved in 100 ml of ionized water, sterilized at 121 ° C for 30 minutes and then cooled to 30 ° C. After cooling, Bacillus subtilis KCCM 11732, which was pre-cultured, was inoculated at 5-10% of the liquid amount. After incubating at 37 ° C. for 7 days, the scavenging rate of the substrate was confirmed by thin layer chromatography, and the reaction was terminated when the substrate was completely lost. The precipitate obtained by centrifuging the culture at 5,000 to 10,000 rpm was washed three times with distilled water and centrifuged again to obtain a precipitate. Ethanol (200 mL) was added to the precipitate, stirred (3 times), the precipitated salts were removed by filtration, and the filtrate was concentrated under reduced pressure to give a crude product. The obtained crude product was separated by silica gel column chromatography (chloroform: methanol = 8: 1 to 4: 1) to obtain 0.44 g of 3 ', 4', 7-trihydroxyisoflavone.

Example 6 Preparation of 3 ', 4', 7-trihydroxyisoflavone using daidzein

3 g of dydzein was dissolved in 100 ml of ionized water, sterilized at 121 ° C for 30 minutes, and then cooled to 30 ° C. After cooling, Bacillus subtilis KCCM 11732, which was pre-cultured, was inoculated at 5-10% of the liquid amount. After incubation at 37 ° C. for 7 days, the reaction was terminated. The precipitate obtained by centrifuging the culture at 5,000 to 10,000 rpm was washed three times with distilled water and centrifuged again to obtain a precipitate. Ethanol (200 mL) was added to the precipitate, stirred (3 times), the precipitated salts were removed by filtration, and the filtrate was concentrated under reduced pressure to give a crude product. The obtained crude product was separated by silica gel column chromatography (chloroform: methanol = 8: 1 to 4: 1) to obtain 0.45 g of 3 ', 4', 7-trihydroxyisoflavone.

Example 7 Preparation of 4 ', 7,8-trihydroxyisoflavone (4', 7,8-trihydroxyisoflavone) using soybean extract

10 g of the soybean extract obtained in Preparation Example 1 was dissolved in 100 ml of ionized water, sterilized at 121 ° C for 30 minutes, and then cooled to 30 ° C. After cooling, Bacillus subtilis KCCM 11732, which was pre-cultured, was inoculated at 5-10% of the liquid amount. After culturing at 30 ° C. for 7 days, the scavenging rate of the substrate was confirmed by thin layer chromatography to terminate the reaction when the substrate was completely lost. The precipitate obtained by centrifuging the culture at 5,000 to 10,000 rpm was washed three times with distilled water and centrifuged again to obtain a precipitate. Ethanol (200 mL) was added to the precipitate, stirred (3 times), the precipitated salts were removed by filtration, and the filtrate was concentrated under reduced pressure to give a crude product. The obtained crude product was separated by silica gel column chromatography (chloroform: methanol = 8: 1 to 4: 1) to give 0.16 g of 4 ', 7,8-trihydroxyisoflavone.

Example 8 Preparation of 4 ', 7,8-trihydroxyisoflavone using didzin

5 g of dydazine was dissolved in 100 ml of ionized water, sterilized at 121 ° C. for 30 minutes and then cooled to 30 ° C. After cooling, Bacillus subtilis KCCM 11732, which was pre-cultured, was inoculated at 5-10% of the liquid amount. After incubating at 37 ° C. for 7 days, the scavenging rate of the substrate was confirmed by thin layer chromatography, and the reaction was terminated when the substrate was completely lost. The precipitate obtained by centrifuging the culture at 5,000 to 10,000 rpm was washed three times with distilled water and centrifuged again to obtain a precipitate. Ethanol (200 mL) was added to the precipitate, stirred (3 times), the precipitated salts were removed by filtration, and the filtrate was concentrated under reduced pressure to give a crude product. The obtained crude product was separated by silica gel column chromatography (chloroform: methanol = 8: 1 to 4: 1) to give 0.55 g of 4 ', 7,8-trihydroxyisoflavone.

Example 9 Preparation of 4 ', 7,8-trihydroxyisoflavone using daidzein

3 g of dydzein was dissolved in 100 ml of ionized water, sterilized at 121 ° C for 30 minutes, and then cooled to 30 ° C. After cooling, Bacillus subtilis KCCM 11732 which had been incubated in advance was inoculated at 5-10% of the liquid amount. After incubation at 37 ° C. for 7 days, the reaction was terminated. The precipitate obtained by centrifuging the culture at 5,000 to 10,000 rpm was washed three times with distilled water and centrifuged again to obtain a precipitate. Ethanol (200 mL) was added to the precipitate, stirred (3 times), the precipitated salts were removed by filtration, and the filtrate was concentrated under reduced pressure to give a crude product. The obtained crude product was separated by silica gel column chromatography (chloroform: methanol = 8: 1 to 4: 1) to obtain 0.42 g of 4 ', 7,8-trihydroxyisoflavone.

Experimental Example 1 Measurement of Keratinocyte Stem Cells (KSC) by Flow Cytometry

Purchased from our theory (Lonza, Cat No C2507A) of human skin keratinocytes (Human neonatal keratinocyte cells), to the subcultures in 25 cm ² T- flask, 37 ℃ in a CO ₂ incubator (CO ₂ Incubator), 5% Incubated under CO ₂ conditions. Experiments were carried out after passage of the second to third generation of cells.

The cell culture was prepared using KGM-2 Bullet kit (Bovine pituitary extract) in 500 ml KBM-2 (Clonetics CC-3103) medium according to the method of Lonza, Inc. Walkersville, MD, USA. (2 ml), human epidermal growth factor (0.5 ml), Insulin (0.5 ml), Hydrocortisone (0.5 ml), Transferrin (0.5 ml), Epinephrine (0.5 ml), Gentamycin Suflate + Amphofericin-B (GA-1000, 0.5 Ml)) was used.

Two days after cell passage, when the cell density of about 50% is seen, 24 hours after serum starvation, 4 ', 7, 8-trihydroxyisoflavones, 4', respectively prepared above Three, 6,7-trihydroxyisoflavones or 3 ', 4', 7-trihydroxyisoflavones were treated at 10 μM concentrations for 24 hours, respectively. Human skin keratinocytes were separated from a 25 cm ² T flask with 0.25% Trypsin-EDTA, then the cells were allowed to settle for 5 minutes at 1500 rpm, then the culture medium was removed and 3 ml of blocking buffer, 2 % Bovine serum protein (Bovine Serum Albumin (BSA) KGM-2) was added and the cells were suspended. The cells (approximately 10 ⁶ cells) were acclimated at 4 ° C. for 15 minutes, followed by alpha 6 integrin antibody (BD Pharmingen, CA, US, Cat No. 555735) to which Fluorescein isothiocyanate (FITC) was attached. 10 μl (1 μg / μl) of PE-Cy (phycoerythrin-cyanine) -coated CD71 antibody (BD Pharmingen, CA, US, Cat No. 551143) was added thereto and reacted at 4 ° C. for 45 minutes. On the other hand, 10 μl (1 μg / μl) of FITC Rat IgG2a, k isotype (Cat No. 555843) and PE-Cy5 mouse IgG1 k isotype (Cat No. 555750) were also added to the cells used as a negative control at 4 ° C. Reaction was carried out for 45 minutes.

After the reaction, the cells were washed after washing twice with 3 ml of Washing buffer (2% Bovine Serum Albumin (BSA) KGM-2). Then it was analyzed by flow cytometry (FACS, BD Bioscience, San Jose, CA, US). Specifically, when the cells reacted in the flow cytometry in the flow cytometry and set the quadrant on the computer image (FACS Imaging Program) (Li A, Kaur P. Methods Mol Biol . 2005; 289: 87-96), Changes in the number of cutaneous keratinocytes, characterized by weak expression of the CD71 protein and strong expression of alpha 6 integrin, result in isolation. For example, the measurement result of the flow cytometry analyzer can be shown as shown in FIG. Referring to FIG. 5, two, three, and four quadrants are provided in the counterclockwise direction based on the first quadrant of the upper right corner. Quadrant 1 is strongly expressed in CD71 and alpha6 integrin, quadrant 2 is strongly expressed in CD71, alpha6 integrin is weakly expressed, third quadrant is weakly expressed in both CD71 and alpha6 integrin, and quadrant 4 is CD71 is weakly expressed and alpha6 integrins represent strongly expressed regions.

The measurement results of the specific flow cytometry analyzer are summarized in Table 1 below.

Negative control
(daidzein) 4 ', 6,7-hydroxyisoflavones 3 ', 4', 7-hydroxyisoflavones 4 ', 7,8-hydroxy
Isoflavones 1 quadrant 158 335 1308 1650 2 quadrants 522 1756 2855 3872 3 quadrants 8954 7449 5245 3612 4 quadrants 356 448 592 856

In addition, Table 2 below shows the negative control group (Dadezine), 4 ', 7,8-trihydroxyisoflavone, 4', 6,7-trihydroxyisoflavone and 3 ', 4', 7 After 10 μM treatment of trihydroxyisoflavones each, CD71 is weakly expressed and alpha6 integrin is strongly expressed, that is, the number and negative control of keratinocyte stem cells (KSCs) corresponding to four quadrants. This is the result of displaying contrast%.

Negative control
(daidzein) 4 ', 6,7-hydroxyisoflavones 3 ', 4', 7-hydroxyisoflavones 4 ', 7,8-hydroxy
Isoflavones 4 quadrants 356 448 592 856 % Increase compared to control 25.8% 66.2% 140%

From the results in Table 2, 10 μM of 4 ', 6,7-trihydroxyisoflavones, 3', 4 ', 7-trihydroxyisoflavones or 4', 7,8-trihydroxyisoflavones were treated, respectively. In one case, the CD71 protein was weakly expressed compared to the negative control group (Daidzein), and the alpha 6 integrin was strongly expressed in 25.8% of the number of keratinocyte stem cells (KSC). 66.2%, 140% increase.

Experimental Example 2 Effect of Ortho-Dihydroxyisoflavone Derivatives on Skin Stem Cell FZD1 (Frizzled Homolog 1) Gene Expression Level

After culturing human neonatal keratinocyte cells in the same manner as described in Experiment 1, keratin stem cells were isolated and serum deficient (serum starvation) after 24 hours, 4 ', 6,7-trihydroxyisoflavones, 3 ', 4', 7-trihydroxyisoflavones and 4 ', 7,8-trihydroxyisoflavones were treated at 10 μM concentrations for 24 hours, respectively. Thereafter, trizol reagent (Trizol reagent, Invitrogen, Carlsbad, CA, USA) was used to isolate the RNA in the cells. The isolated RNA was purified cleanly with KIAGEN's RNA kit (QIAGEN RNeasy kt, QIAGEN, Valencia, CA), and then Agilent's bioanalyzer 2100 model instrument (Agilent 2100 BioAnalyzer, Agilent Technologies, Santa Clara, CA, USA) was used. Was used to confirm the quality of the RNA. CDNA was synthesized from the RNA using Invitrogen's Superscript Reverse Transcriptase (RT) kit, Invitrogen, Carlsbad, Calif., Which was then used for real time-reverse transcription polymerase chain reaction, Q-RT-PCR) for quantitative analysis. Changes in the expression pattern of each gene in cutaneous keratinocyte stem cells (TaqMan® gene expression assay kit, AppliedBiosystems, FosterCity, CA) (Frizzled homolog 1 (FZD1)-HS00268943_s1 (TagMan primer catalog name)) Expression level was confirmed using. Experimental results are shown in Figure 6 below.

6, 4 ', 6,7-trihydroxyisoflavones, 3', 4 ', 7-trihydroxyisoflavones, and 4', 7,8-trihydroxyisoflavones compared to the negative control group In the case of treatment, the expression of FZD1 (Frizzled homolog 1) was confirmed to increase by about 1.9 times, 1.4 times, 2.6 times, respectively.

Examples of the formulation of the composition are described below, but are not intended to limit the present invention but merely to be described in detail.

Formulation Example 1 Nutritional Cosmetics

Nutritional longevity was prepared according to the conventional composition described in Table 3 below.

ingredient Content (unit: weight%) Purified water Balance glycerin 8.0 Butylene glycol 4.0 Hyaluronic acid extract 5.0 Beta Glucan 7.0 Carbomer 0.1 4 ', 6,7-trihydroxyisoflavone or 3', 4 ', 7-trihydroxyisoflavone 0.05 Caprylic / Capric Triglycerides 8.0 Squalane 5.0 Cetearyl Glucoside 1.5 Sorbitan stearate 0.4 Cetearyl Alcohol 1.0 Triethanolamine 0.1

Formulation Example 2 Nutrition Cream

Nutritional cream was prepared in a conventional manner according to the composition shown in Table 4.

ingredient Content (unit: weight%) Purified water Balance glycerin 3.0 Butylene glycol 3.0 Liquid paraffin 7.0 Beta Glucan 7.0 Carbomer 0.1 4 ', 6,7-trihydroxyisoflavone or 3', 4 ', 7-trihydroxyisoflavone 3.0 Caprylic / Capric Triglycerides 3.0 Squalane 5.0 Cetearyl Glucoside 1.5 Sorbitan stearate 0.4 Polysorbate 60 1.2 Triethanolamine 0.1

Formulation Example 3 Massage Cream

To prepare a massage cream in a conventional manner according to the composition described in Table 5.

ingredient Content (unit: weight%) Purified water Balance glycerin 8.0 Butylene glycol 4.0 Liquid paraffin 45.0 Beta Glucan 7.0 Carbomer 0.1 4 ', 6,7-trihydroxyisoflavone or 3', 4 ', 7-trihydroxyisoflavone 1.0 Caprylic / Capric Triglycerides 3.0 Beeswax 4.0 Cetearyl Glucoside 1.5 Sesquioleic acid sorbitan 0.9 Vaseline 3.0 paraffin 1.5

[Formulation Example 4] Pack

To prepare a pack in a conventional manner according to the composition described in Table 6.

ingredient Content (unit: weight%) Purified water Balance glycerin 4.0 Polyvinyl alcohol 15.0 Hyaluronic acid extract 5.0 Beta Glucan 7.0 Allantoin 0.1 4 ', 6,7-trihydroxyisoflavone or 3', 4 ', 7-trihydroxyisoflavone 0.5 Nonylphenyl ether 0.4 Polysorbate 60 1.2 ethanol 6.0

Formulation Example 5 Ointment

The ointment was prepared in a conventional manner according to the composition described in Table 7.

ingredient Content (unit: weight%) Purified water Balance glycerin 8.0 Butylene glycol 4.0 Liquid paraffin 15.0 Beta Glucan 7.0 Carbomer 0.1 4 ', 6,7-trihydroxyisoflavone or 3', 4 ', 7-trihydroxyisoflavone 1.0 Caprylic / Capric Triglycerides 3.0 Squalane 1.0 Cetearyl Glucoside 1.5 Sorbitan stearate 0.4 Cetearyl Alcohol 1.0 Beeswax 4.0

1 to 4 are graphs showing the distribution of cells showing the difference (fluorescence intensity) of the amount of alpha 6 integrin (FL1-H) and CD71 (FL3-H) expression, respectively. The graph of the dotted line shown in FIGS. 2 to 4 (green) shows the cell distribution obtained in FIG. 1 (negative control), for comparison with the results obtained from each test group used in FIGS. 2 to 4;

5 exemplarily shows a measurement result of a flow cytometry analyzer;

6 is a graph showing the expression level of the FZD1 (Frizzled homolog 1) gene for each experimental group.

Claims (7)

At least one selected from the group comprising 4 ', 7,8-trihydroxyisoflavones, 4', 6,7-trihydroxyisoflavones, and 3 ', 4', 7-trihydroxyisoflavones A composition for improving skin regeneration or skin elasticity, comprising an ortho-dihydroxyisoflavone derivative as an active ingredient. The method of claim 1, The ortho-dihydroxyisoflavone derivatives, through the proliferation of the skin keratinocytes, to improve skin regeneration or to improve skin elasticity for improving skin regeneration or skin elasticity. The method of claim 1, The content of the ortho-dihydroxyisoflavone derivatives, based on the total weight of the composition, 0.001 to 30% by weight composition for improving skin regeneration or skin elasticity. The method of claim 1, The ortho-dihydroxyisoflavone derivatives, the composition for improving skin regeneration or skin elasticity to increase the expression of FZD1 (Frizzled homolog 1). An external preparation composition for skin, comprising the composition according to any one of claims 1 to 4. Cosmetic composition containing a composition according to any one of claims 1 to 4. A pharmaceutical composition for improving skin regeneration or improving skin elasticity, comprising the composition according to any one of claims 1 to 4.

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