WO2011021818A2

WO2011021818A2 - Composition for proliferating epidermal keratinocyte stem cells

Info

Publication number: WO2011021818A2
Application number: PCT/KR2010/005387
Authority: WO
Inventors: 신동욱; 박준성; 김동현; 노민수; 김덕희
Original assignee: (주)아모레퍼시픽
Priority date: 2009-08-17
Filing date: 2010-08-16
Publication date: 2011-02-24
Also published as: WO2011021818A3; CN102470093A; CN102470093B; KR20110018067A; HK1164747A1; JP2013502410A; KR101732594B1

Abstract

Disclosed is a composition for improving skin regeneration or skin elasticity, which contains, as an active ingredient, one or more ortho-dihydroxyisoflavone derivatives selected from a group consisting of 4',7,8-trihydroxyisoflavone, 4',6,7-trihydroxyisoflavone and 3',4',7- trihydroxyisoflavone. The composition promotes the proliferation of epidermal keratinocyte stem cells and exhibits the effects of improving skin regeneration or skin elasticity, and thus can be widely used in the field of cosmetics or pharmaceuticals.

Description

【Specification】

[Name of invention]

Skin keratin stem cell proliferation composition

Technical Field

<ι> The present invention relates to human dermal keratinocytes (Epidermal Keratinocyte Stem Cells,

KSC) ortho-dihydroxyisoflavone derivative-containing composition having a proliferative effect.

<2>.

Background Art

<3> The most important role of the skin is to act as a defensive barrier to protect the human body from external damage and invasion of bacteria, while preventing the loss of heat and moisture from the inside. The skin is largely structurally comprised of the epidermis and dermis, which can be divided into the stratum corneum and the living epidermis, which are composed of flat hexagonal dead cells.

The living epidermis comprises four layers of Stratum Basale, Stratum Spinos®, Stratum Granulosus, 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 several times and form keratinocyte eel Is, which dermatologically plays a central role in the skin barrier function of the middle and late stages. Keratinocytes undergo differentiation (di f ferentiat ions) 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.

<5> As skin ages, these skin keratinocytes

(Keratinocyte Stem Cells, KSC) and the ability to proliferate Transit Amplifying Cells (TA eel Is) are reduced. If the ability to produce keratinocytes is reduced, eventually the skin regeneration ability is reduced, and aging phenomena, such as decreased skin elasticity, are intensified (Fuchs E. Nature. 2007 Feb 22: 445 (7130): 834-42).

<6>

[Detailed Description of the Invention]

[Technical problem] 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 to provide a cosmetic or pharmaceutical composition for improving skin regeneration or skin elasticity.

<9>

Technical Solution

<ιο> The composition according to the present invention for achieving this object is 4 '， 7,8—trihydr

One or more ortho-dihydroxyisoflavone derivatives selected from the group comprising oxyisoflavones, 4 ', 6,7-trihydroxyisoflavones, and 3''4', 7-trihydroxyisoflavones It contains as an active ingredient.

<ι ι>.

Advantageous Effects

The composition according to the present invention induces the proliferation of skin keratinocytes, thereby improving skin regeneration and / or skin elasticity.

<13>

[Brief Description of Drawings]

1 to 4 are graphs showing the distribution of cells showing the difference in the amount (intensity of fluorescence) in which alpha 6 integrin (FL1−H) and CD7KFL3-H are expressed, respectively. The dotted graphs (green marks) shown in FIGS. 2-4 show the cell distribution obtained in FIG. 1 (negative control), for comparison with the results obtained from each test group used in FIGS.

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

6 is a graph showing the expression level of the FZDKFrizzled homo log 1) gene for each experimental group.

<17>

[Best form for implementation of the invention]

<18> The present invention relates to a composition for improving skin regeneration and / or improved blood ^"elastic unit, ortho-in that it contains the di-hydroxy-isoflavone derivatives as an active ingredient characterized in coming. In one embodiment, the ortho-dihydroxyisoflavone derivatives are, for example, 4 ', 7,8 ᅳ trihydroxyisoflavone, 4', 6,7-trihydroxyisoflavone, and 3 ' It may be at least one member selected from the group containing 4 ', 7-trihydroxyisoflavone. The ortho-dihydroxyisoflavone derivative is recognized to improve skin regeneration and improve skin elasticity through proliferation of skin keratinocytes. Specifically, dermal keratinocytes divide continuously to produce Transit Amplifying Cells (TA eel Is). These metastasis-amplified cells divide more than a dozen times and become keratinocytes (Keratinocyte eel Is), one of the main cells of the skin, and rise to the skin surface through differentiation. Thus, 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-trihydroxy isoflavone, and 3 ', 4', 7-trihydroxyisoflavone Each can be represented by the following formula (1) to (3).

<21> [Formula 1]

<23> [Formula 2]

<25> [Formula 3]

<27> 4 ', 6, gotriha, which are ortho-dihydroxyisoflavone derivatives according to the present invention

Idroxyisoflavones, 4 ', 7,8-trihydroxyisoflavones and 3', 4 ', 7-trihydroxy isoflavones can be prepared by known methods known in the art. The ortho-dihydroxyisoflavone derivatives, for example, by bioconversion of daidzein It can be prepared as, but is not limited thereto.

In one embodiment, the ortho-dihydroxyisoflavone derivative 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, the effect of skin regeneration and elasticity improvement through the proliferation of skin keratinocytes is not sufficiently obtained. If the content of the derivative is more than 30% by weight, the effect is not increased. Problems can arise.

In addition, keratinocyte stem cells (KSC) and metastatic-amplified cells

Labeling markers (proteins) for (Transit Amplifying Cells, TA cells) were identified. As a result, CD and alpha 6 integrins were identified as markers.

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, incorporating the receptor-mediated transferrin protein. Receptor mediated transferrin endocytosis is a process that supplies iron to cells. Since iron is usually toxic, the amount of iron in the cell should be well controlled by the amount of CD71 protein expression.

<3i> Another marker was identified, alpha 6 integrin, which showed severe skin blisters in knockout mice that did not express the marker marker protein. It is known to die as soon as possible. Alpha 6 integrin is mainly associated with beta 4 integrins in Kerat inocyte Stem Cells (KSC) and is an important component of a cell-structure complex called hemidesmosonie. It is a factor. Alpha 6 integrins are known to affect cellular function in various areas such as cellular communication, cell migration, proliferation, and differentiation.

In general, the earliest stage of skin development is the skin keratinocytes.

Keratinocyte Stem Cells (KSC) and Transit Amplifying Cells (TA cells) play an important role. In keratinocyte stem cells (KSC), alpha 6 integrin expression is strong and CD71 protein (or Transferrin receptor) expression is weak (Li A, aur 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, FZDKFrizzled homo log 1) is also a marker for skin keratin stem cells. It is still unknown how these proteins function in these cells.

In experimental examples of the present invention, ortho-dihydroxyisoflavones were measured by measuring the expression level of the above-mentioned alpha 6 integrin, CD71 protein and FZDK Frizz led homo log 1) gene. Specific derivatives of, have been confirmed to have a proliferative effect on the skin keratinocytes.

The present invention also provides a skin external preparation 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, suspension or emulsion in an oil or an aqueous medium, or dissolved in sterile, pyrogen-free water before use. Can be. Water-in-oil emulsions are derived from vegetable oils such as Eulbe oil or mineral oils such as liquid paraffin, derived from esters of fatty acid phospholipids such as soy lecithin, and anhydrous hepatites or fatty acids such as sorbitan monooleate. The active ingredient is emulsified by the compound which condensed nucleus-free and partial ester derived from fatty acid with ethylene oxide as emulsifier, such as lyoxyethylene sorbitol monooleate.

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 lotion, massage cream, eye cream, nourishing cream, cleansing cream, cleansing products, cleansing water, powder, essence, pack, gel or It may be a formulation of a skin-adhesive cosmetic, and may also be a transdermal dosage form such as 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 keratinocytes of the skin improve the skin regeneration, the effect of improving the skin elasticity is recognized.

When the composition according to the present invention is applied to a pharmaceutical product, a solid, semi-solid or liquid phase may be added by adding a commercially available inorganic or organic carrier using the composition as an active ingredient. In the form of oral or parenteral administration.

<40> The preparation for oral administration includes tablets, tablets, granules,

) Include a soft ⁱ If capsules, powders, fine granules, powders, emulsions (乳獨濟), syrups, pellets and the like. In addition, the preparations for parenteral administration include injections, drops, ointments, lotions, sprays, suspensions, emulsions, sesame seeds (坐齊 IJ) and the like. Surfactants, excipients, colorants, spices, preservatives, stabilizers, buffer crabs, suspensions, and other commonly used auxiliaries can be suitably used to formulate the active ingredients of the present invention.

<41>

The pharmaceutical composition according to the present invention may be administered orally, parenterally, rectally, topically, transdermally, intravenously, intramuscularly, intraperitoneally, subcutaneously.

In addition, the dosage of the active ingredient will vary depending on the age, sex, 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 0.001 mg / kg / day to 2000 nig / kg / day, more specifically 0.5 mg / kg / day to 1500 mg / kg / day.

<44>

[Form for implementation of invention]

Hereinafter, the present invention will be described in more detail with reference to Examples and Test Examples, but the scope of the present invention is not limited thereto.

<46>

Preparation Example: Preparation of Soybean Extract

6 £ of nucleic acid was added to soybean 2, and the mixture was degreased by stirring three times at room temperature. Then, 4 kg of 80% methane was added to 1 kg of degreased soybean, refluxed three times, and soaked at 15 ^° C for 1 day. Subsequently, the residue and the filtrate were separated through filter cloth filtration and centrifugation, and the filtrate was concentrated under reduced pressure. The obtained extract was suspended in water, extracted five times with ether 1 «to remove the pigment, and the aqueous layer was extracted three times with 500 m £ of 1-butane.

The 1-butane layer was concentrated under reduced pressure to obtain an 1-butane extract, which was dissolved in a small amount of methane, and then a large amount of ethyl acetate was added. The precipitate formed by the addition of ethyl acetate was dried to obtain 300 g of soybean extract.

<49>

Example 1 Preparation of 4 ', 6,7-trihydroxyisoflavone (4', 6,7-trihydroxyisof lavone) using soybean extract <5i> 10 g of the soybean extract obtained in Preparation Example 1 was dissolved in 100 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 incubation at 37 ^° C. for 7 days, the clearance 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. Ethane (200) was added to the precipitate and stirred (three 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: 1-4: 1) to give 0.23 g of 4 ', 6,7-trihydroxyisoflavone.

<52>

Example 2 Preparation of 4 ′, 6,7-Trihydroxyisoflavone Using Daidzin 5 g of Sidma Cat. Dissolved in deionized water, 121

Sterilized for 30 minutes at ^° C and then cooled to 30 ^° C. After incubation, pre-cultured Aspergillus niger KCCM 11885 was inoculated at a liquid volume to ratio of 5-10%. After incubation 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 collected by centrifugation at 5,000 to 10,000 rpm was washed with distilled water three times and centrifuged again to obtain a precipitate. Ethanol (200) 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. Silica gel and the obtained crude product was purified by column chromatography (chloroform: methanol = 8: 1 ~ 4: 1) and separated by ^41, 6, 7-trihydroxy isoflavone 0.34 g was obtained.

<55>

Example 3 Preparation of 4 ′, 6, 7—Trihydroxy Isoflavones Using Daidzein

<57> Dyzedine 3 gCSigma Cat. No. D7802) is dissolved in 100 deionized water,

Sterilized for 30 minutes at 121 ° C and then cooled to 30 ^° C. After incubation, the cultured Aspergillus niger KCCM 11885 was inoculated at 5-10% of the liquid volume. 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 mO 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. Gained. The obtained crude product was subjected to silica gel column chromatography (chloroform: methanol

= 8: 1-4: 1) to obtain 0.45 g of 4 ', 6,7-trihydroxyisoflavone.

<58>

Example 4 Preparation of 3 ', 4', 7-trihydroxyisoflavone (3 ', 4', 7-t r hydroxy i so f 1 avone) using soy extract

10 g of soybean extract obtained in Preparation Example 1 was dissolved in ionized water of 100, sterilized at 121 ^° C. for 30 minutes, and then angled at 30 ^° C. After cooling, Bacillus subtilis KCCM 11732 which had been incubated in advance was inoculated at a ratio of 5 to 1 relative to the amount of liquid. 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 times was added to the precipitate, 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 subjected to silica gel column chromatography (chloroform: Methane was separated into = 8: 14: 1) to obtain 0.23 g of 3 ', 4', 7-trihydroxyisoflavone.

<61>

Example 5 Preparation of 3 ′, 4 ′, 7—Trihydroxyisoflavones Using Daidzin 5 g of Sidma Cat. In ionic water of 121

Sterilized at 30 ^° C for 30 minutes and then angled to 30 ^° C. After cooling, Bacillus subtilis KCCM 11732 was inoculated at 5-10% of the liquid volume. 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 solution at 5,000 to 10,000 rpm was washed three times with distilled water and centrifuged again to obtain a precipitate. Ethanol (200) 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', and 7-trihydroxyisoflavone.

<64>

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

Produce

3 g of Dyzede was dissolved in 100 ^ of ionized water, sterilized at 121 ^° C for 30 minutes, and then cooled to 30 ^° C. After culture, Bacillus subtilis subtil is) KCCM 11732 was inoculated with 5-10% of the liquid volume. The reaction was terminated after incubation at 37 ^° C for 7 days. The precipitate obtained by centrifuging the culture solution at 5,000 to 10,000 rpm was washed three times with distilled water and centrifuged again to obtain a precipitate. Ethanol (200 1) 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: methane = 8: 1-4: 1) to obtain 0.45 g of 3 ', 4', 7-trihydroxyisoflavone.

<67>

Example 7 Preparation of 4 ', 7,8-trihydroxyisoflavone (4', 7,8-t r hydroxy i sof 1 avone) ≤1 using soybean extract

10 g of the soybean extract obtained in Preparation Example 1 was dissolved in 100 of ionized water, sterilized at 121 ^° C. for 30 minutes, and cooled to 30 ^° C. After incubation, Bacillus subtilis KCCM 11732 was inoculated with 5-10% of the liquid volume. After incubation 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 ᅵ) was added to the precipitate, 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: methane = 8: 1-4: 1) to give 0.16 g of 4 ', 7,8-trihydroxyisoflavone.

<70>

<7i> [Example 8] Preparation of 4 ', 7,8—trihydroxyisoflavone using didzin

5 g of dydazine was dissolved in 100 ionized water, sterilized at 121 ^° C. for 30 minutes, and then angled at 30 ^° C. After incubation, Bacillus subtilis KCCM 11732 which had been incubated in advance was inoculated at 5-10% of the amount of liquid. After incubation at 37 ^° 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 solution at 5,000 to 10,000 rpm was washed three times with distilled water and centrifuged again to obtain a precipitate. Ethanol (200) was added to the precipitate and stirred (three 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: 1-4: 1) to give 0.55 g of 4 ', 7,8-trihydroxyisoflavone. · <73>

[Example 9] Preparation of 4 ', 7,8-trihydroxyisoflavone using daidzein 3 g of dyedzein was dissolved in 100 deionized water and 30 at 121 ^° C. Sterilized for 30 minutes and then cooled to 30 ^° C. Pre-cultivated Bacillus subtilis

subtil is) KCCM 11732 was inoculated with 5-10% of the liquid volume. The reaction was terminated after incubating for 7 days at 371 ：. The precipitate obtained by centrifuging the culture solution at 5,000 to 10,000 rpm was washed three times with distilled water and again centrifuged to obtain a precipitate. Then, ethane (200) was added to the precipitate and stirred (three times), and the precipitated salts were filtered. After removing through, the filtrate was concentrated under reduced pressure to give a crude product. The obtained crude product was separated by silica gel column chromatography (chloroform: methane = 8: 1-4: 1) to obtain 0.42 g of 4 ', 7,8-trihydroxyisoflavone.

<76>

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

<78> Human neonatal keratinocyte eel Is (Lonza ')

Cat No C2507A), subcultured to 25 ο Τ-flasks, C0 ₂ incubator

(C0 ₂ Incubator) was incubated under 37 ^° C, 5% C0 ₂ conditions. Experiments were carried out after passage of two or three generations of cells.

<79> The cell culture medium was prepared according to the method of Lonza, Inc. Walkersvi 1 le, MD, USA.

KGM— 2 Bullet kit; Bovine pituitary extract (2 ui ^), human e idermal growth factor (0.5 mi.), Insul in (0.5 μM) in 500 ι of KBM-2 (Clonetics CC-3103) medium ), Hydr ocor ti sone (0.5 in ^), Transferr in (0.5), E inephrine (0.5 mi), and Gentamycin Sufiate + Amphofer icin— B (GA_1000, 0.5 mi) were used.

After about 80% of cell passage, if the cell density of about 50% is seen, serum starvation is performed 24 hours later, and 4 ', 7, 8-trihydroxyiso is prepared above, respectively. The flavones, 4 ', 6,7-trihydroxyisoflavones or 3', 4 ', 7-trihydroxyisoflavones were treated for 24 hours at 10 μΜ concentrations, respectively. Human skin keratinocytes were separated from a 25 en / T flask with 0.25% Trypsin-EDTA, allowed to settle for 5 minutes at 1500 rpm, then the culture medium was removed, followed by 3 blocking buffers, 2%. Bovine Serum Protein (BSA)

KGM-2) was added and the cells were suspended. Cells (approximately 10 ⁶ cells) for 15 min at 4 ^° C After detoxification, alpha 6 integrin antibody (BD Pharmingen, CA, US, Cat No. 555735) and F—C—phycoerythrin-cyanine (7) attached to FITCCFluorescein isothiocyanate (CD) antibody (BD Pharmingen) , CA, US, Cat No. 551143) were added 10 (1 / ίΛΥΑ), respectively, and reacted at 4Τ for 45 min. Meanwhile, cells used as negative controls were FITC Rat IgG2a, k isotype (Cat No. 555843) and PE- Cy5 mouse IgGl k isotype (Cat No. 555750) was also added 10! Ig / and reacted at 4 ^° C for 45 minutes.

<8i> After the reaction, 3 ml of Washing buffer: 2% Bovine Serum

The cells were suspended after washing twice with Albumin (BSA) KGM-2). After flow cytometry (FACS, BD Bioscience, San Jose, CA, U.S.) was analyzed. Specifically, when the reaction cells are added to the flow cytometry, and the quadrant is set on the computer image (FACS Imaging Program) (Li A, Kaur P. Methods Mol Biol. 2005; 289: 87-96), Changes in the number of dermal keratinocytes characterized by weak expression of the CD71 protein and strong expression of alpha 6 integrins result in isolation. For example, the measurement result of the flow cytometry may be shown in FIG. 5. Referring to FIG. 5, the quadrants 2, 3, and 4 become counterclockwise with respect to the first quadrant of the upper right corner. Quadrant 1 is strongly expressed in CD and alpha 6 integrins, quadrant 2 is strongly expressed in CD, alpha 6 integrin is weakly expressed, and quadrant 3 is weakly expressed in both CD and alpha 6 integrin, and 4 Quadrants show weakly expressed CD groups and alpha 6 integrins.

<82>^"measurement results of the specific flow cytometry fluorescence analyzers, are summarized in Table 1 below.

<83> [Table 1]

In addition, Table 2 shows the negative control group (Dadezane; Daidzein) and 4 ', 7,8-trihydroxyisoflavone, 4', 6,7-trihydroxyisoflavone and 3 ', 4 ', 7—Trihydroxyisoflavones treated with 10 μΜ each, CD group is weakly expressed and alpha 6 integrin is strongly expressed, that is, keratinocyte stems in four quadrants Cells, KSC) and the percentage of the negative control.

Table 2

From the results in Table 2 10 μΜ 4 ', 6,7-trihydroxyisoflavone, 3', 4 ', 7-trihydroxyisoflavone or 4', 7,8-trihydroxyisoflavone , The CD71 protein is weakly expressed compared to the negative control group (Daidzein), and alpha 6 integrin is strongly expressed in the number of keratinocyte stem cells (KSC). 25.8%, 66.2%, and 140% increase respectively.

<87>

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

<89> After culturing human neonatal keratinocyte cells in the same manner as described in Experiment 1, keratin stem cells were isolated, and serum deprivation (serum starvation) after 24 hours. , 4 ', 6,7-trihydroxyisoflavones, 3 ¹ , 4', 7-trihydroxyisoflavones and 4 ', 7,8-trihydroxyisoflavones, respectively, at 10 μΜ concentrations for 24 hours Treated during. Subsequently, RNA in the cells was isolated using a trizol reagent (Trizol reagent, Invitrogen, Carlsbad, CA, USA). 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 reverse transcriptase kit (Superscript Reverse Transcriptase (RT) kit, Invitrogen, Carlsbad, Calif.), Which was then used for real time-reverse transcript ion polymerase chain reaction. quantitatively by ion, Q 一 RT 一 PCR). Changes in the expression pattern of each gene in cutaneous keratinocyte stem cells were reported by TaqMan® gene expression assay kit (AqMan® gene expression assay kit, Ap Ied Biosys terns, Foster Ci ty, CA) (Frizz led homo log l (FZDl)- Expression level was confirmed using HS00268943_sl (TagMan primer catalog name)). Experimental results are shown in Figure 6 below.

6, 4 ', 6, 7-trihydroxy isoflavone, compared to the negative control group,

When 3 ', 4', gtrihydroxyisoflavones, and 4 ', 7, and 8-trihydroxyisoflavones were respectively treated, the expression of FZDKFrizzled homo log 1) was about 1.9, 1.4, and 2.6 fold, respectively. It was confirmed that the degree increased.

<91>

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

<93>

<94> [Formulation Example 1] Nutritional Cosmetics

According to the composition shown in Table 3 below, nutrient lotion was prepared by a conventional method. .

<96> [Table 3]

<97>

<98> Formulation Example 2 Nutritional Cream

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

<ιοο> [Table 4] Ingredient Content (Dee: Weight>)

Purified water level

Glycerine ^ 3.0

Butylene glycol 3.0

Liquid Paraffin 7.0

Beta Glucan 7.0

Carbomer 0.1

4 ', 6,7—trihydroxyisoflavones or 3.0

3 '.4', 7-trihydroxyisopolar

Capririck / Capric Triglycerides 3.0

Squalam 5.0

Cetearim Glucoside 1.5

Sorbitan Stearate 0.4

Polysomate 60 1.2

Triethanol? 1 0.1

I 3] Massage Cream

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

[Table 5]

[Formulation Example 4] Pack

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

Table 6 Component Content (Unit: Weight%)

Purified water

Glycerin 4.0

Polyvinyl ammo 15.0

Hyaluronic Acid Extract 5.0

Beta Glucan 7.0

Allantoin 0.1

4 ', 6,7-trihydroxyisoflavones or 0.5

3 '.4' .7-trihydroxyisopolar

Old pene ether 0.4

Foamysomate 60 1.2

Ethane 6.0

<109>

<110> [Formulation Example 5] Ointment

<111> ^c by a conventional method according to the composition shown in Table 7

<112> [Table 7]

Component Content (Unit: Weight%)

Purified water level

Glycerin 8.0

Bugim Recall Recall 4.0

Liquid Paraffin 15.0

Beta Glucan 7.0

Carbomer 0.1

4 ', 6,7-trihydroxyisoflavones or 1.0

3 '.4', 7-trihydroxyisopolar

Capririck / Capric Triglycerides 3.0

Scuamran 1.0

Cetearim Glucoside 1.5

Sorbitan ste o> rate 0.4

Cetearim Alcohol 1.0

2-4.0

<113>

Those skilled in the art to which the present invention pertains will be able to perform various applications and modifications within the scope of the present invention based on the above contents.

<115>

Industrial Applicability

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

Claims

[Range of request]

[Claim 11

At least one selected from the group consisting of 4 ', 7,8-trihydroxyisoflavone, 4', 6,7-trihydroxyisoflavone, and 3 ', 4', 7-trihydroxyisoflavone A composition for improving skin regeneration or skin elasticity, comprising an ortho-dihydroxyisoflavone derivative as an active ingredient.

[Claim 2]

The method of claim 1,

The ortho-dihydroxy isoflavone derivative, through the proliferation of the skin keratinocytes, to improve the skin regeneration or improve the skin elasticity composition for improving skin regeneration or skin elasticity.

[Claim 3]

The method of claim 1,

The content of the ortho-dihydroxyisoflavone derivative, 0.001 to 30% by weight based on the total weight of the composition for improving skin regeneration or improving skin elasticity.

[Claim 4]

The method of claim 1,

The ortho-dihydroxy isoflavone derivative, the composition for improving skin regeneration or skin elasticity to increase the expression of FZDKFrizzled homo log 1).

[Claim 5]

An external preparation composition for skin, comprising the composition according to any one of claims 1 to 4.

[Claim 6]

Cosmetic composition containing a composition according to any one of claims 1 to 4.

A pharmaceutical composition for improving skin regeneration or skin elasticity, comprising the composition according to any one of claims 1 to 4.