KR20100018278A - Pharmaceutical and cosmetic composition comprising of decursin for increasing the numbers of human keratinocyte stem cells - Google Patents

Abstract

PURPOSE: A cosmetic or pharmaceutical composition containing decursin is provided to enhance the number of epidermal keratinocyte stem cells(KSC) and ensure anti-aging. CONSTITUTION: A composition for proliferating epidermal keratinocyte stem cells contains decursin as an active ingredient. The decursin is obtained from Angelica gigas Nakai ethanol extract or Angelica gigas Nakai supercritical extract. The Angelica gigas Nakai ethanol extract is obtained by adding ethanol, stirring and removing solvent. The Angelica gigas Nakai supercritical extract is obtained by preparing micro powder, and purifying using silica gel column chromatography. A cosmetic composition and a pharmaceutical composition for anti-aging contain a composition containing decursin as an active ingredient.

Description

Pharmaceutical and cosmetic composition comprising of decursin for increasing the numbers of human keratinocyte stem cells}

The present invention relates to a cosmetic or pharmaceutical composition comprising a decursin (Decursin) to increase the number of 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 structurally divided into epidermis and dermis, and the epidermis is divided into the stratum corneum and the living epidermis, which are composed of flat hexagonal dead cells, and the living epidermis is the stratum basale, milk. It can be divided into four layers, Stratum Spinosum, Stratum Granulosum, and Stratum lucidum. Of these, keratinocyte stem cells (KSC) are known to be located in the basal layer, and the keratinocyte stem cells are continuously dividing to form trans-amplifying cells (TA cells). The cells are further amplified to form keratinocyte cells, which continue to differentiate and push up to the surface of the skin. In this process, keratinocytes are differentiated and keratinated, forming a stratum corneum, and eventually the keratinocytes are separated from the stratum corneum by about 0.5 to 1.0 g per day. In other words, Keratinocyte Stem Cells (KSC) and Transit Amplifying Cells (TA cells) play a role in overcoming epidermal loss and regeneration of the epidermis through endless cell division. (Eline Fuchs et al., Genes and Development 17: 1189-1200, 2003, Amy Li et al., J. Clin. Invest. 113: 390-400, 2004). Keratinocyte Stem Cells (KSC) and Trans-Amplifying Cells (TA cells) are used to reduce the stem cell's ability to age due to skin aging. Gradually loses the ability to make cells, which eventually leads to a decrease in skin regeneration, which intensifies skin elasticity and aging.

Therefore, researchers are actively working to develop the ability of these keratinocyte stem cells (KSC) and transit-amplifying cells (TA cells), one of which is the purpose of the study. The marker markers of these proteins were identified, and the protein known to date is CD71 protein (or Transferrin receptor) present in the cell membrane, and CD71 protein has been found to exist only in the basal layer (Stratum Basale) (Amy Li et al., Methods in Molecular Biology, 289: 87-96, 2005). The function of the CD71 protein interacts with the transferrin protein, which binds to iron flowing through the bloodstream of the cell, and supplies iron into the cell through a process called receptor mediated transferrin endocytosis, which is delivered by receptors. (Harding C et al., J. Cell. Biol. 97: 329-339, 1983).

Since iron is usually toxic, the amount of iron in cells should be well controlled by the amount of CD71 protein. In particular, activated cells and proliferating cells require a lot of iron in the cells, which is known to increase the expression of CD71 protein (K Rittenhouse-Diakun et al., Photochemistry and Photobiology, 61: 523-528). , 1995), another marker was identified alpha 6 integrin, a severe skin blister was observed in knockout mice that do not express the protein of the marker marker and dies at birth (Georges-Labouesse E et al., Nat. Genet 13: 370-373, 1996).

Alpha 6 integrins are mainly associated with beta 4 integrins in keratinocyte stem cells (KSC), an important factor in the cellular structural complex called hemidesmosome. Located on the basal surface of the basal epidermis, where alpha 6 integrins are known to affect cellular functions in various areas, such as cellular communication, cell migration, proliferation, and differentiation. (Rodius S et al., J. of Cell.Physiol. 439-449, 2006).

In addition, keratinocyte stem cells (KSC) have strong expression of alpha 6 integrin, weak expression of CD71 protein (or transferrin receptor), and low transit amplifying cells (TA). In cells, alpha 6 integrin and CD71 protein (or Transferrin receptor) are known to show strong expression (Amy Li et al., J. Clin. Invest. 113: 390-400, 2004). .

In addition, Polistastatin (FST), Dickkopf homolog 3 (DKK3), and Frizzled homolog 1 (FZD1) are also recognized as markers for cutaneous keratinocytes and metastatic-amplified cell cells. It is not known if you do.

As mentioned above, in the early stages of skin formation, keratinocyte stem cells (KSC) and transit-amplifying cells (TA cells) play an important role. And the expression of CD71 protein (or Transferrin receptor), alpha 6 integrin, is required for signal transduction with and adjacent cells.

However, the expression of CD71 protein (or Transferrin receptor) and alpha 6 integrin are expected to play an important role in regulating skin keratin stem cell activity and epidermal regeneration. It has not been done.

It is an object of the present invention to provide a composition for proliferating keratinocyte stem cells (Keratinocyte Stem Cells, KSC) comprising a decursin (Decursin) as an active ingredient.

Another object of the present invention is to provide a cosmetic and pharmaceutical composition comprising a decursin (Decursin) as the active ingredient for the growth.

The present invention relates to a composition for proliferating keratinocyte stem cells (KSC) containing decursin as an active ingredient.

More specifically, the present invention relates to a composition for propagation, characterized in that the expression of alpha6 integrin is stronger than the expression of CD71 protein to proliferate skin keratinocytes.

After treating decursin with human neonatal keratinocyte cells, the present inventors applied CD71 protein (or Transferrin receptor), which is involved in iron transport into cells, for signal transduction and cell migration. It was confirmed that the number of keratinocyte stem cells (KSC) that simultaneously express alpha 6 integrins involved increased.

Hereinafter, the present invention will be described in more detail.

The dicursin is prepared by obtaining from the Angelica ethanol extract or Angelica supercritical extract, the Angelica ethanol extract is prepared as a fine powder of Angelica gigas and fine ethanol is added to obtain the Angelica extract after stirring and removal of solvent, Angelica supercritical extract Prepared by silver fine powder, carbon dioxide is purified by silica gel column chromatography in the Angelica extract obtained using supercritical carbon dioxide at a pressure of 300 to 500 bar, temperature of 50 to 80 ℃ to prepare a dicusin.

Human skin keratinocytes (Human neonatal keratinocyte cells) are to be secured by commercially, CO ₂ incubator (CO ₂ Incubator) at 37 ℃, 5% CO ₂ Those obtained by subcultured under 2nd to 3rd generation were used. After two days of passage, the cell density of 50% is reached. After 24 hours of serum starvation, decursin is added to a final concentration of 10 mM (Dimethyl Sulfoxide; dissolved in DMSO) for 24 hours. To be processed.

After treatment with human dermal keratinocytes, 3 ml of blocking buffer was added to the cells to suspend the cells, followed by acclimatization of the cells, followed by alpha 6 integrin antibody (BD Pharmingen, CA, US, to which Fluorescein isothiocyanate) was attached. Cat No. 555735) and CD71 antibody (BD Pharmingen, CA, US, Cat No. 551143) attached with PE-Cy (phycoerythrin-cyanine) were added, respectively, and the cells used as negative controls were FITC Rat IgG2a, k Add isotype (Cat No. 555843) and PE-Cy5 mouse IgG1 k isotype (Cat No. 555750) and react for 45 minutes at 4 ° C. When the number of skin keratin stem cells obtained after the reaction was examined, the expression of CD71 protein was weak and alpha 6 integrin was strongly expressed in the case of decursin treatment compared to the negative control group. The number of cells was increased.

Since the cell proliferation time of normal skin keratinocytes is 16 hours and the treatment time of the decusin is 24 hours, the increase in expression of CD71 protein and alpha6 integrin by decursin compared to the negative control is a skin keratin stem. Increasing the number of cells (Keratinocyte Stem Cells, KSC).

In addition, as a result of visually confirming the number and size of colonies generated during a certain time to verify the skin keratin stem cell activation and proliferation promoting effect by the decursin, more in the experimental group treated with decursin. It can be seen that a large number of cell colonies are formed.

Accordingly, the present invention provides a cosmetic or pharmaceutical composition comprising decursin to increase the number of keratinocyte stem cells (KSC).

Cosmetic or pharmaceutical composition of the present invention is a cosmetic or pharmaceutical composition containing 0.01 to 50% by weight of dicusin as an active ingredient, preferably 0.1 to 10% by weight of the active ingredient.

The single dose of the cosmetic or pharmaceutical composition thus prepared may be appropriately adjusted according to individual differences, dosage forms, and forms such as age and the extent of lesions, and effective for 0.01 to 1000 mg twice a day for general adults. Use in content.

The formulation of the cosmetic composition for increasing the number of epidermal keratinocytes (Keratinocyte Stem Cells, KSC) comprising the decursin of the present invention as an active ingredient is not particularly limited and may be appropriately selected as desired. For example, the cosmetic composition of the present invention may be prepared in the form of external skin ointment, softening longevity, nourishing longevity, massage cream, essence, nutrition cream, pack, and the like.

The pharmaceutical composition of the present invention may further contain pharmaceutical supplements such as preservatives, stabilizers, hydrating or emulsifying accelerators, salts or buffers for controlling osmotic pressure, and other therapeutically useful substances, and various oral methods according to conventional methods. Or in parenteral dosage forms.

Formulations for oral administration include, for example, tablets, pills, hard and soft capsules, solutions, suspensions, emulsifiers, syrups, granules, etc. These formulations may contain diluents (e.g., lactose, dextrose, water, etc.) in addition to the active ingredients. Cross, mannitol, sorbitol, cellulose and glycine), lubricants such as silica, talc, stearic acid and its magnesium or calcium salts and polyethylene glycols. Tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and polyvinylpyrrolidine, optionally starch, agar, alginic acid or its sodium salt Pharmaceutical additives such as disintegrants, absorbents, colorants, flavors, and sweeteners. Tablets may be prepared by conventional mixing, granulating or coating methods. Also representative of parenteral formulations are injectable formulations, preferably aqueous isotonic solutions or suspensions.

However, it should be understood that the actual dosage of the active ingredient should be determined in light of several relevant factors such as the severity of the symptom, the route of administration chosen, the subject's age, sex, weight and health status.

Suitable dosages for the pharmaceutical compositions of the present invention are usually those skilled in the art that can readily determine and prescribe effective dosages for the skin. More preferably, when oral administration of the pharmaceutical composition of the present invention, a suitable dosage is 1 x 10 ⁵ to 5 x 10 ⁷ TCID ₅₀ , more preferably 2 x 10 ⁶ to 2 x 10 ⁷ TCID ₅₀ , Most preferably about 1 × 10 ⁷ TCID ₅₀ , and the dosage does not limit the scope of the invention in any way.

As described above, as a composition for proliferating keratinocyte stem cells (KSC) comprising decursin as an active ingredient, skin regeneration of skin keratin stem cells is improved according to the proliferation of skin keratin stem cells, regeneration It can be usefully applied as a cosmetic or pharmaceutical composition having excellent effects on anti-aging due to improvement of elasticity and promotion of blood circulation.

Hereinafter, the present invention will be described in more detail with reference to specific examples. However, the present invention is not limited to the following examples, and it will be apparent to those skilled in the art that various modifications or changes can be made within the spirit and scope of the present invention.

[ Production Example  1] Angelica ethanol extract

1kg of Dried Angelica Angelica is used to obtain a fine powder. This was added to 5L of 95% ethanol and then extracted with gentle stirring at room temperature. Extraction time proceeds for about 6 hours, after which the extraction solution is recovered and concentrated under reduced pressure, the ethanol solvent is completely removed. 55 g of the final Angelica extract obtained in the above process was obtained. The content of dicusin contained in the extract was about 5-6 (w / w)% was confirmed by high pressure liquid chromatography.

[ Production Example  2] Angelica Supercritical  Extract manufacturer

1kg of dried fine Angelica is obtained using a grinder to obtain a fine powder. The supercritical carbon dioxide is used for the extraction process. After filling 1kg of pulverized Angelica powder into the reactor, carbon dioxide is supercritical under a pressure of 400 bar and a temperature of 60 degrees. Since supercritical carbon dioxide is introduced into the reactor at a flow rate of 100 ~ 120 ml / min. Supercritical carbon dioxide dissolves nonpolar substances contained in Angelica gigas, and then expands to normal pressure and room temperature in the separator to rapidly dissolve the solubility to separate carbon dioxide and Angelica extract. 65g of final Angelica extract obtained in the above process was obtained. The content of dicusin in the extract is about 13-15 (w / w)%.

[ Production Example  3] Angelica Supercritical After extraction column  Separated Decker  Produce

2g of the Angelica extract obtained in Preparation Example 1 was purified by silica gel column chromatography (filled with silica gel 200g). Hexane and ethyl acetate were used as a developing solvent, and fractions were obtained by increasing the concentration gradient of hexane and ethyl acetate from 10: 1 to 2: 1 to obtain 0.38 g of dicusin concentrate from these fractions. Through this process, the concentrated liquid was removed from the intrinsic odor of Angelica, it was confirmed that the content of dicusin is 65 ~ 70 (w / w)%.

Example  One: Flow cell  Keratin stem cells through analysis ( Keratinocyte Stem Cells , KSC ) Measurement

To the human skin keratinocytes (Human neonatal keratinocyte cells) purchased from welseukin (Welskin Co, Seoul, Korea) , 25 ㎠ to the passages in T-flask, CO ₂ Incubator (CO ₂ Incubator) 37 ℃ from, 5% CO _The culture was carried out under ₂ conditions. Typically, experiments were carried out after passage of two to three generations of cells. Cell cultures were prepared in KGM-2 Bullet kit (Bovine pituitary extract (2 ml), human in 500 ml KBM-2 (Clonetics CC-3103) medium according to the method of Lonza, Inc. Walkersville, MD, USA. Add epidermal growth factor (0.5ml), Insulin (0.5ml), Hydrocortisone (0.5ml), Transferrin (0.5ml), Epinephrine (0.5ml), Gentamycin Suflate + Amphofericin-B (GA-1000, 0.5ml) It was. If cell density of 50% is observed about two days after cell passage, serum deprivation (serum starvation) 24 hours after decursin (Cel No. CEB-008 standard) and stomach Each of three different amounts of dikersin prepared was treated at 10 mM concentrations for 24 hours. Human skin keratinocytes were separated from 25 cm2 Tiflask with 0.25% Trypsin -EDTA, and then the cells were allowed to settle for 5 minutes at 1500rpm, then the culture medium was removed, followed by 3 ml of blocking buffer and 2% Bovine Serum Albumin. (BSA) KGM-2) is added to suspend the cells. After adapting the cells (approximately 106 cells) at 4 ° C. for 15 minutes, alpha 6 integrin antibody (BD Pharmingen, CA, US, Cat No. 555735) and PE with Fluorescein isothiocyanate (FITC) were attached. -Cy (phycoerythrin-cyanine) -attached CD71 antibody (BD Pharmingen, CA, US, Cat No. 551143) was added to each 10 ml (1 mg / ml), and reacted at 4 ℃ for 45 minutes. Meanwhile, FITC Rat IgG2a, k isotype (Cat No. 555843) and PE-Cy5 mouse IgG1 k isotype (Cat No. 555750) were also added 10 ml (1 mg / ml) to the cells used as a negative control at 4 ° C. Reaction for 45 minutes at.

After the reaction, the cells are washed twice with 3 ml of Washing buffer (2% Bovine Serum Albumin (BSA) KGM-2) and the cells are suspended. Since FACSCalibur (BD Bioscience, San Jose, CA, U.S.) observed changes in the number of cells expressing CD71 protein and alpha 6 integrin in the skin keratin stem cells are shown in Table 1 below.

Table 2 below shows the result of expressing the number of% and the negative control group of keratinocyte Stem Cells (KSC), which expresses CD71 weakly and alpha6 integrins strongly, after 10 mM treatment of the negative control and the dicusin. As a result, when treated with 10 mM standard decursin, the number of keratinocyte stem cells (KSC), which expresses CD71 protein weakly and strongly expresses alpha 6 integrin, was higher than that of the negative control group. 38.3% increase. Table 3 below shows that as the dickerin content increases, the rate of change of the number of skin keratin stem cells present in the fourth quadrant increases.

Quadrant 1-Strong expression of CD71 and alpha 6 integrins

Quadrant 2-CD71 is strongly expressed and Alpha6 integrin is weakly expressed

Quadrant 3-weak expression of both CD71 and alpha6 integrin expression

Quadrant 4-CD71 weakly expressed and alpha6 integrin strongly expressed

TABLE 1

TABLE 2

[Table 3]

Example  2: Marker Marker Follistatin  ( FST ), Dickkopf homolog  3 ( DKK3 ), Frizzled  homolog 1 ( FZD1 The level of expression of the gene Of decursin  Effect measurement

Human neonatal keratinocyte cells were cultured in the same manner as described in Example 1, after serum deprivation (serum starvation) for 24 hours, and at 24 mM decursin 10 mM concentration for 24 hours. Treated during. 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, Calif.), Followed by Agilent's Bioair Analyzer 2100 model instrument (Agilent 2100 BioAnalyzer, Agilent Technologies, Santa Clara, CA, USA). Was used to confirm the quality of RNA. CDNA was synthesized from the RNA using Invitrogen's Superscript Reverse Transcriptase (RT) kit, Invitrogen, Carlsbad, Calif., Which was used for real time-reverse transcription polymerase chain reaction (Q). Quantitatively via -RT-PCR). Changes in the expression pattern of each gene in cutaneous keratinocyte stem cells were carried out by TaqMan® gene expression assay kit, AppliedBiosystems, FosterCity, CA (Follistatin (FST)-HS00246260_m1; Dickkopf homolog 3 (DKK3)-HS00247426_m1) Expression level was determined using Frizzled homolog 1 (FZD1)-HS00268943_s1 (TagMan primer catalog name), Follistatin (FST), Dickkopf homolog 3 (DKK3), It was confirmed that the expression of Frizzled homolog 1 (FZD1) increased about 1.8-fold, 4.6-fold, and 2.2-fold, respectively, and the results are shown in Fig. 2 (representative results after three repeated experiments).

Example  3: On dicker  Of keratin stem cell colony

In this example, the degree of colonization of epidermal keratinocytes was compared in order to verify the effects of epidermal keratinocytes activation and proliferation promoting effect by dicusin.

Epidermal growth occurs around the proliferative unit, which consists of epidermal stem cells and keratin stem cells derived therefrom. Due to these characteristics, keratin stem cells proliferate in colonies on a culture dish, and the number and size of colonies generated over a period of time reflect the extent of proliferation activation of keratin stem cells.

Human skin keratinocytes were inoculated with as few as 100 cells per 35 mm dish. The day after the inoculation, serum starvation was performed for 24 hours, followed by treatment with DMSO in the control group and DMSO dissolved in the DMSO group for 48 hours at 0.5 mM and 1 mM concentrations, respectively. Therefore, to reduce toxicity, reduce the concentration to 1/10 or less). After incubation for 3 more days, the medium was removed to visually check the number and size of the stem cell colonies (size less than 200 mm), and the cells were concentrated at room temperature in a solution of 10% ethanol and 0.1% crystal violet at room temperature. After staining for 4 minutes, it was observed by washing four times with PBS, the results are shown in Table 4.

TABLE 4

As can be seen in Table 4, it was confirmed that the experimental group treated with dimercin 0.5 mM and 1 mM, respectively, had a larger number of cell populations (approximately 65%, 82% increase).

Figure 1 shows the results of observing 10,000 cells, respectively, using FACSCalibur in the negative control group, the standard dickerin (10 mM) treated cells. Keratinocyte Stem Cells (KSC) sections expressing CD71 weakly and strongly expressing alpha6 integrin, respectively, are indicated by arrows (corresponding to quadrant 4) (FIG. 1A). For this keratinous stem cell section (quadrant 4), the negative control group (green line) and the standard dickerin (purple section) were compared for the fluorescence and cell numbers of CD71 and alpha6 integrins.

FIG. 2 is a reverse transcription polymerase chain reaction using RNA isolated from cells treated with a negative control group, standard decosin (10 mM), Follistatin (FST), Dickkopf homolog 3 (DKK3), and Frizzled homolog 1 (FZD1). This is the result of measuring the change of expression level of genes.

Claims (8)

A composition for proliferating keratinocyte stem cells (KSC) containing decursin as an active ingredient. The method of claim 1, The dickerin is a proliferation composition, characterized in that the proliferation of keratin stem cells expressing alpha 6 integrin more strongly than the expression of CD71 protein. The method of claim 1, The dicusin is a composition for propagation, characterized in that to improve the skin elasticity by improving the skin regeneration according to the growth of skin keratinocytes. The method of claim 1, The dicusin is a composition for proliferation, characterized in that the anti-aging of the skin by the promotion of blood circulation according to the proliferation of skin keratinocytes. The method of claim 1, The dikerin is a composition for propagation, characterized in that it increases the amount of expression of the skin keratin stem cell marker Follistatin (FST), Dickkopf homolog 3 (DKK3), Frizzled homolog 1 (FZD1). In claim 1, The dicusin is a composition for propagation, characterized in that obtained from Angelica ethanol extract or Angelica supercritical extract. A skin anti-aging cosmetic composition comprising the proliferation composition according to any one of claims 1 to 6 as an active ingredient. A pharmaceutical composition for preventing skin aging comprising the composition for propagation according to any one of claims 1 to 6 as an active ingredient.

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