KR101481167B1 - Composition For Promoting Regeneration of Skin - Google Patents

Abstract

The present invention discloses a method for promoting proliferation of skin cells and a composition for accelerating skin regeneration comprising Icaricide II as an active ingredient.

The method and composition for promoting skin cell proliferation according to the present invention can promote the proliferation of metastasis-amplified cells of the skin epidermal basal layer involved in regeneration of the epidermis, thereby promoting regeneration of the skin.

Metastasis - Amplified cells, skin regeneration

Description

[Technical Field] The present invention relates to a composition for promoting skin regeneration,

The present invention relates to a method for promoting the proliferation of skin cells and a composition for promoting skin regeneration, and more particularly, to a composition for promoting proliferation of metastasis-amplifying cells.

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 body from external damage and invasion of bacteria.

This skin is composed of several layers, the outermost part of which is called the epidermis. Keratinocyte stem cells (KSC) are known to be capable of regenerating from the epidermis.

The keratinocyte stem cells continue to divide and produce two large cells: one is the same stem cell that is made by self-renewal, and the other is Transit Amplifying Cells (TA cells).

Metastasis-amplified cells are further amplified and differentiated early to become keratinocyte cells, which continue to differentiate and push them up to the surface of the skin. In this process, the keratinocytes are differentiated and keratinized, forming the stratum corneum, and after 3 ~ 4 weeks they are separated from the stratum corneum.

These keratinocyte stem cells are usually distributed in an extremely low number of about 4-7% of the basal layer cells, and increasing the number of keratinocyte cells is not as easy as increasing the number of metastasis-amplifying cells, It is not distributed in the area, so there are many difficulties in research.

Accordingly, it is an object of the present invention to solve the technical problems requested from the past. Specifically, it is an object of the present invention to provide a skin regeneration promoting composition that exhibits excellent skin regeneration ability.

In order to accomplish the above object, the present invention provides a method for promoting skin cell proliferation, which comprises treating skin cells with icarcide II.

In addition, the skin regeneration promoting composition according to the present invention is characterized by containing icariside II as an active ingredient.

Through the present invention, it is possible to promote the proliferation of metastasis-amplifying cells and keratinocyte cells of the skin epidermal basal layer involved in regeneration of the epidermis, thereby promoting skin regeneration.

The term "skin regeneration" as defined herein is a broad concept that includes the ability to generate new keratinocytes in response to overall skin aging and wounds caused by skin epidermis and the like.

Specifically, skin regeneration further amplifies the metastasis-amplifying cells generated by the division of keratinocyte stem cells existing in the basal layer of skin epidermis, and early differentiation becomes keratinocyte, which is pushed up to the surface of the skin, (Keratinization) and the formation of the stratum corneum means all the process.

Among them, the key process of skin regeneration is the proliferation of keratinocyte stem cells in the basal layer and metastasis - amplification cells which are daughter cells of the cells. In order to restore skin damage caused by external physical and chemical conditions such as a "abrasion" in which only the outer surface of the skin is lost, a "laceration" in case of scratching, a burn caused by fire, etc., And it is necessary to vigorously propagate keratinocyte stem cells and metastasis-amplifying cells which are responsible for skin regeneration. The reason for the scar remains when the skin is damaged due to age, while the scar is not left even when the skin damage occurs at a normal age. The level of proliferation of keratinocyte and metastasis-amplification cells necessary for skin regeneration is remarkable depending on the age . That is, as the aging progresses, the degree of proliferation of these cells becomes low, which leads to a decrease in skin regeneration ability, which may delay the recovery of wounds and the like and reduce skin elasticity. Recently, the importance of these two types of cells has been actively studied in wound healing studies (Roh et al., Pediatr Res. 100-103, 2006; Elaine Fuchs et al., Proc Natl. Acad. Sci., 11830-11835, 2003).

In one embodiment, a method of promoting proliferation of the skin cells comprises treating the skin cells with icarcide II, wherein the skin cells are Transient Amplifying Cells (TA cells).

The skin cell proliferation promoting method according to the present invention can be carried out in vitro, and the proliferated skin cells can be transplanted into an individual requiring transitional-amplification cells.

The above icaricide II means a compound having a structure represented by the following formula (1).

(1)

(One)

Wherein R1 is rhamnopyranose and R2 is OH.

The composition can also regenerate skin epidermis by promoting the proliferation of metastasis-amplified cells. The metastasis-amplifying cells, together with keratinocyte cells, overcome the epidermal loss due to the detachment of keratinocytes through continuous cell division, Can play a role of reproducing.

As evidenced by the following examples, the inventors of the present application found that when a skin regeneration-promoting composition containing icariside II according to the present invention as an active ingredient is applied to keratinocytes, The number of cells is effectively increased.

The metastasis-amplified cells occupy a large specific gravity of about 10% or more of the basal layer of skin epidermis, as compared with keratinocyte cells, which are distributed in an extremely low number of about 4-7% of the skin epidermal basal layer. And plays an important role in regeneration of the epidermis. In comparison with proliferation of keratinocyte stem cells having a small number of distributions, proliferation is relatively easy. Therefore, through the increase of the number of metastasis-amplifying cells by the composition according to the present invention, it is possible to improve the skin regeneration ability for wound healing, reduce the elasticity of the skin due to the decline of the skin regeneration ability, and prevent the aging of the skin.

The composition can increase the number of metastasis-amplifying cells expressing the CD71 protein (Cluster of Differentiation 71 Protein) and a6 integrin, particularly the number of metastasis-amplifying cells that strongly express both CD71 protein and a6 integrin And the CD71 protein and [alpha] 6 integrin are marker marker proteins specific for metastasis-amplified cells.

The CD71 protein, also referred to as the transferrin receptor, is present only in the basal layer. Specifically, the CD71 protein interacts with a transferring protein that binds to iron flowing in the cell blood stream, and acts as a receptor mediated transferrin endocytosis to supply iron in the cells do. Activated cells, or proliferating cells, require a lot of iron in the cells, which can increase the expression of the CD71 protein.

The a6 integrin mediates cellular communication and can affect cell functions in various areas such as cell migration, proliferation and differentiation, and severe skin blisters can occur when the [alpha] 6 integrin is not expressed.

In addition, the composition may also increase the expression of CD200 (Cluster of Differentiation 200), which is another marker marker of metastasis-amplified cells as a glycoprotein. In this regard, as demonstrated in the Examples below, the expression level of CD200 was increased about 2 to 3 times compared to the control without the composition according to the present invention.

In one embodiment, the composition according to the present invention promotes the proliferation of metastatic-amplified cells. As in the following Examples, it was confirmed that the composition according to the present invention can exert excellent regenerating ability in skin epidermis by increasing the number of cell colonies that act on skin regeneration.

In one embodiment, the content of the active ingredient is in vitro experiment and in When seen vivo experiment on the basis of the composition is the total of 0.001 to 10% by weight, based on the weight, and preferably may comprise from 0.01 to 5% by weight.

The skin regeneration-promoting composition may be, for example, a cosmetic composition or a pharmaceutical composition.

The cosmetic composition may be formulated in a predetermined form, and the formulation is not particularly limited. For example, it can be formulated with external ointment for skin, softening longevity, nutritional lotion, massage cream, essence, nutrition cream and pack.

The pharmaceutical composition may further contain a pharmaceutical adjuvant such as a preservative, a stabilizer, a wetting or emulsifying accelerator, a salt and / or a buffer for controlling osmotic pressure, and other therapeutically useful substances, Oral or parenteral administration.

Examples of formulations for oral administration include tablets, pills, hard and soft capsules, liquids, suspensions, emulsions, syrups, granules and the like. These formulations may contain, in addition to the active ingredient, a diluent such as lactose, dextrose, Croscarmellose, mannitol, sorbitol, cellulose and glycine), and lubricants (e.g., silica, talc, stearic acid and magnesium or calcium salts thereof and polyethylene glycols).

Tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and polyvinylpyrrolidine, optionally mixed with starch, agar, alginic acid or its sodium salt Such as disintegrants, absorbents, coloring agents, flavoring agents, and sweetening agents. Tablets can be prepared by conventional mixing, granulating or coating methods. In addition, a representative aqueous solution for parenteral administration is preferably an isotonic aqueous solution or suspension in a form of injection.

In addition, the dosage of the active ingredient will depend on the age, sex, body weight of the subject to be treated, the particular disease or condition to be treated, the severity of the disease or condition, the route of administration and the judgment of the prescriber. Dosage determinations based on these factors are within the level of ordinary skill in the art and generally the dosage may range from 0.001 mg / kg / day to approximately 2000 mg / kg / day, but limiting the scope of the invention in any way no.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are intended to illustrate the present invention, but the scope of the present invention is not limited thereto.

Production Example 1: Preparation of icaricide II utilizing beta-glucosidase

10 g of Icarin was dissolved in 500 ml of 0.1 M acetic acid buffer solution (pH 5.5), 0.5 g of beta-glucosidase (Sigma) was added thereto, and the mixture was stirred for 48 hours in a water bath at 25 ° C., After completion of the reaction by heating in hot water (80 to 100 ° C) for 10 minutes, the reaction solution was concentrated under reduced pressure to remove the solvent, and 200 ml of ethanol was added to the residue After stirring (3 times), the precipitate was 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 6.9 g of icaricide II.

Test Example 1: Measurement of transit-amplifying cells (TA cells) by flow cytometry

Human neonatal keratinocyte cells were purchased from Welskin Co, Seoul, Korea and subcultured in 25 cm ² T-flask. Cells were cultured in a CO ₂ incubator (CO ₂ Incubator) at 37 ° C and 5% And cultured under CO ₂ conditions. A passage of cells was typically performed in the second or third passages.

The cell culture medium was added to 500 ml of KBM-2 (Clonetics CC-3103) medium according to the method of Lonza, Inc. Walkersville, MD, USA using a KGM-2 bullet kit (BPE (Bovine pituitary extract: ), hEGF (human epidermal growth factor: 0.5 ml), insulin (0.5 ml), hydrocortisone (0.5 ml), transferrin (0.5 ml), epinephrine (0.5 ml), gentamicin sulfate + (Gentamycin Suflate + Amphofericin-B: GA-1000, 0.5 ml) was added thereto.

Two days after the cell passage, if serum confluency of about 50% is observed, serum is starvated and treated with icariside II at a concentration of 10 μM for 24 hours after 24 hours. Respectively. Human keratinocytes were separated from 25 cm ² Ti flasks with 0.25% trypsin (EDTA), and the cells were allowed to settle for 5 minutes at 1500 rpm. After removing the culture medium, 3 ml of blocking buffer: blocking buffer, 2% Fetal calf serum (FCS) and 2% bovine serum albumin (BSA) KGM-2 were added to suspend the cells.

Cells (about 10 ⁶ cells) were adapted for 15 minutes at 4 ° C and then mixed with α6 integrin antibody (Cat No. 555735) (BD Pharmingen, CA, US) with FITC (Fluorescein isothiocyanate) (Cat No. 551143) (BD Pharmingen, CA, US) with phycoerythrin-cyanine (Phycoerythrin-cyanine) was added at a ratio of 10: 1 and reacted at 4 ° C 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 to the cells used as a negative control, Lt; 0 > C for 45 minutes.

After the reaction was completed, the cells were washed twice with 3 ml of washing buffer (2% Bovine Serum Albumin (BSA) KGM-2), and then the cells were suspended. Subsequently, the number of cells expressing CD71 protein and a6 integrin in skin corneal stem cells was observed with FACSCalibur (BD Bioscience, San Jose, CA, US) Respectively.

Referring to FIG. 1, Table 1 and Table 2, in the case of the transient-amplified cells strongly expressing the .alpha.6 integrin when treated with 10 .mu.M of icaricide II, Which is about 3.3 times higher than that of the conventional model .

Test Example 2: Measurement of effect of icariside II on expression level of CD200 (Cluster of Differentiation 200) marker gene

Human neonatal keratinocyte cells were cultured in the same manner as described in Example 1. After 24 hours of serum starvation, icariside II was added to a concentration of 10 μM For 24 hours. Subsequently, RNA was isolated from the cells using a triazole reagent (Trizol reagent, Invitrogen, Carlsbad, Calif., USA).

(Agilent 2100 BioAnalyzer, Agilent Technologies, Santa Clara, Calif., USA) after purification of the separated RNA with an RNA kit (QIAGEN RNeasykt, QIAGEN, Valencia, CA) Were used to confirm the quality of the RNA. CDNA was synthesized from the RNA using a reverse transcription kit (Superscript Reverse Transcriptase (RT) kit, Invitrogen, Carlsbad, Calif.), And the cDNA was synthesized using real time reverse transcription polymerase chain reaction -RT-PCR).

Use: (HS00245978_m1 CD200 gene primer) in skin keratinocytes CD200 (Cluster of Differentiation 200) taekmaen gene's Applied Biosystems expression pattern changes in gene expression system (TaqMan ^ㄾ gene expression assay kit, Applied Biosystems, Foster City, CA) The results are shown in Fig. It was confirmed that the expression level of CD200 was increased by about 2.9 times in the case of treatment with icaricide II 10 μM as compared with the negative control.

Test Example 3: Identification of keratinocyte stem cell community ability by icaricide II

In this example, to verify the epidermal keratinocyte activation and proliferation promoting effects of icaricide II, the degree of keratinocyte stem cells was compared.

The growth of the epidermal layer occurs around a proliferative unit consisting of epidermal stem cells and keratin stem cells derived therefrom. Due to these characteristics, keratinocyte stem cells grow on colony on a culture dish, and the number and size of colonies formed over a period of time reflect the degree of proliferation activation of keratinocyte stem cells.

Human keratinocytes were inoculated with a small number of 100 cells per 35 mm culture dish. On the next day of inoculation, the control group was treated with DMSO and the experimental group treated with icariside II dissolved in DMSO at a concentration of 1 μM (the concentration was reduced to 1/10 by treatment with a small number of cells). After 4 days of culture, the number and size of cell clusters were visually observed. After the medium was removed, the cells were stained with a concentrated solution of 10% ethanol and 0.1% crystal violet for 5 minutes at room temperature. The results are shown in Table 3.

As shown in Table 3, it was confirmed that a larger number of cell clusters (about 20% increase) were obtained in the experimental group treated with icariside II.

[Formulation Example 1] Lotion-type preparation

Icaricid II of Preparation Example 1 3.00

L-ascorbic acid-2-phosphate magnesium salt 1.00

Water soluble collagen (1% aqueous solution) 1.00

Sodium citrate 0.10

Citric acid 0.05

Licorice extract 0.20

1,3-butylene glycol 3.00

Purified water balance

(Unit: wt%)

[Formulation Example 2]

Icaricid II of Preparation Example 1 1.00

Polyethylene glycol monostearate 2.00

Self emulsifying monostearate glycerin 5.00

Cetyl alcohol 4.00

Squalane 6.00

Tri-2-ethylhexane glyceryl 6.00

Sphingo glycolipid 1.00

1.3-butylene glycol 7.00

Purified water balance

(Unit: wt%)

[Formulation Example 3] Packed preparation

Icaricid II of Preparation Example 1 5.00

Polyvinyl alcohol 13.00

L-ascorbic acid-2-phosphate magnesium salt 1.00

Lauroylhydroxyproline 1.00

Water soluble collagen (1% aqueous solution) 2.00

1,3-butylene glycol 3.00

Ethanol 5.00

Purified water balance

(Unit: wt%)

[Formulation Example 4] Soft capsule

50 mg of Icaricid II of Production Example 1, 80 to 140 mg of L-carnitine, 180 mg of soybean oil, 2 mg of palm oil, 8 mg of vegetable hardening oil, 4 mg of yellow radish and 6 mg of lecithin were mixed and 400 mg / A capsule was prepared.

[Formulation Example 5] Tablets

50 mg of Icaricid II of Preparation Example 1, 200 mg of galactooligosaccharide, 60 mg of lactose and 140 mg of maltose were mixed, granulated using a fluid bed drier, and 6 mg of sugar ester was added thereto to prepare tablets Respectively.

[Formulation Example 6]

50 mg of Icaricid II of Preparation Example 1, 250 mg of anhydrous crystalline glucose and 550 mg of starch were mixed and granulated into granules using a fluidized bed granulator and filled in a vial.

Those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.

Figure 1 is a graph showing changes in the number of cells expressing CD71 protein and a6 integrin in the group treated with the composition according to the present invention;

2 is a graph showing changes in expression pattern of the CD200 gene in the group treated with the composition according to the present invention.

Claims (7)

delete delete A composition for accelerating skin regeneration comprising Icaricide II as an active ingredient. The method of claim 3, Wherein said composition regenerates epidermis by promoting proliferation of metastasis-amplified cells. The method of claim 3, Wherein said composition increases the number of metastasis-amplifying cells expressing CD71 protein (Cluster of Differentiation 71 Protein) and? 6 integrin. The method of claim 3, Wherein the composition enhances the expression of CD200 (Cluster of Differentiation 200). The method of claim 3, Wherein the composition promotes the proliferation of keratinocyte stem cells.

Images