KR101766393B1 - Screening method for materials improving dry skin using bleomycin hydrolase - Google Patents

Abstract

The present invention relates to a method for screening a dry skin improving substance, which comprises screening based on a change amount of gene expression of a bleomycin hydolase (BH) gene by a candidate substance, And provides a dry skin improving substance found by screening. Also provided is a composition for external application for skin containing as an active ingredient an LXR activating agent which increases the expression amount of BH gene. The screening method of the present invention has an effect of finding an effective dry skin improving substance, and the composition has the effect of restoring the skin barrier of the skin horny layer by improving the dry skin and preventing moisture loss of the skin.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for screening dry skin improving substances using bleomycin hydrolyzate,

The present invention relates to a method for screening dry skin improving substances using Bleomycin hydolase (BH).

The most important function of the epidermis on the outermost skin is to protect against various external stimuli such as chemicals, air pollutants, dry environment, ultraviolet rays, physical and chemical stimulants, This protective function can be maintained only when the stratum corneum composed of keratinocytes is normally formed. The outermost stratum corneum (horney layer) is formed from keratinocytes and consists of keratinocytes with complete differentiation and a surrounding lipid layer. The keratinocyte is a characteristic cell in which basal cells continuously proliferating in the lowest epidermis undergo a stepwise change in morphology and function and are elevated to the surface of the skin. After a certain period of time, And the new keratinocyte replaces its function. This repetitive sequence of changes is called "epidermal cell differentiation" or "keratinization". During keratinization, keratinocytes form the stratum corneum while producing natural moisturizing factors (NMF) and intercellular lipids (ceramides, cholesterol, fatty acids), making the stratum corneum solid and flexible, As shown in Fig.

Diseases Dry skin without skin and disease has a common feature that skin moisture is reduced and NMF is reduced, but there are differences in some features.

Skin with skin disease is characterized by reduced ceramide, decreased filaggrin and decreased W-hydroxyceramide, but in the case of dry skin without aged skin or disease, There is no change, cell proliferation is reduced, and the turnover rate of the stratum corneum is delayed.

Therefore, moisturizers applied to the skin of people with various skin diseases such as dry skin, atopy, psoriasis and the moisturizing agents applied to the skin of people with normal dry skin need to be distinguished. Therefore, it was necessary to search for marker substances involved in moisturizing in general dry skin.

In order to solve the above problems, it is an object of the present invention to provide a method for screening dry skin improving substances involved in moisturizing dry skin and to provide screened dry skin improving substances.

In order to achieve the above object, the present invention provides a method for screening a dry skin improving substance, which comprises screening based on a change amount of gene expression of a bleomycin hydolase (BH) A method for screening a substance is provided.

The present invention also provides a dry skin improving material screened by the above method.

The present invention also provides a composition for external application for skin comprising, as an active ingredient, a Liver X Receptor (hereinafter referred to as LXR) activator which increases the expression amount of BH gene.

The present invention also provides a method for providing information for diagnosing dry skin by measuring the amount of BH in the skin.

The screening method of the present invention has an effect of finding an effective dry skin improving substance. The dry skin improving substance found through the screening method improves the dry skin and prevents moisture loss of the skin, . ≪ / RTI >

Figs. 1 and 2 are diagrams illustrating a method of extracting normal skin keratin and dry skin keratin using a tape stripping method, quantifying pillar green by a dot blot method, and measuring the total amount of amino acids, i.e., (NMF) content of the sample.
Fig. 3 shows the correlation between pillared green and NMF (total amino acid).
FIG. 4 shows the results of measurement of the change in conversion enzyme that converts pilar green to NMF in the epidermis of elderly skin and young skin.
Figure 5 shows the amount of Bleomycin hydolase (BH) measured on the keratin of normal skin and dry skin.
FIG. 6 shows the amount of pillared green in the keratinocytes treated with the LXR agonist T0901317 (hereinafter referred to as T1317) and measured on days 1 and 7 according to the differentiation state.
FIG. 7 shows the amount of caspase-14 expressed in the keratinocytes treated with the LXR agonist T1317 and measured at days 1 and 7 according to the differentiation state.
FIG. 8 shows the amount of BH measured on days 1 and 7 of treatment according to the differentiation state of T1317 treated with the LXR agonist in keratinocytes.
FIG. 9 shows the amount of calpain-1 measured at days 1 and 7 according to the differentiation state by treatment with LXR agonist T1317 in keratinocytes.

Hereinafter, the present invention will be described in detail.

One embodiment of the present invention is a method for screening a dry skin improving substance, which comprises screening based on a change amount of a gene expression of a bleomycin hydolase (BH) by a candidate substance, Screening method.

Conversion enzymes that convert filaggrin to natural moisturizing factor (NMF) in skin include, but are not limited to, caspase-14, calpain-1, Bleomycin hydolase (hereinafter referred to as BH), and the present inventors have found for the first time that BH is reduced in dry skin.

In an embodiment of the present invention, the amount of change in gene expression can be measured using PCR, and PCR, RT-PCR, real-time PCR or real-time RT-PCR can be used.

When there is a substance that amplifies BH, there has been used a method of identifying proteins expressed in BH and confirming them at a protein level. The PCR method of the present invention is capable of detecting a small amount of BH increase and thus has an advantage in that it can precisely detect a substance that affects BH.

In addition, one embodiment of the present invention can increase the amount of BH mRNA by treating T1317 represented by the following formula (1) together with the PCR. T1317 can be treated before, after, or simultaneously with PCR.

[Chemical Formula 1]

To be able to use the PCR method, there must be a substance that increases the expression level of BH. The inventors have found that T1317 selectively increases the expression level of BH. Therefore, when a substance that increases BH expression level is screened, the amount of BH expression can be increased by using T1317, which makes it possible to use the PCR method. Therefore, the PCR method can be used for screening substances useful for skin moisturization, that is, dry skin improving substances, using T1317. For PCR, PCR, RT-PCR, real-time PCR or real-time RT-PCR can be used.

Also, one embodiment of the present invention can select a substance that increases the amount of BH mRNA among the candidate substances.

One embodiment of the present invention provides a dry skin enhancing material screened by the above method. The dry skin improving material selected by the screening method may be a Liver X Receptor (LXR) activator, and the LXR activator may be T1317 represented by the formula (1).

 The LXR is a nuclear hormone receptor belonging to the type II receptor superfamily. The LXR activator has the purpose of increasing the activity of BH, one of the converting enzymes that convert filaggrin into natural Moisturizing Factor (NMF) in the skin. Thus, it is possible to enhance the water retention ability of the skin by increasing the synthesis of filagreen and promoting the conversion to NMF. NMF is involved in water retention in the stratum corneum. We first found that T1316 of the above formula (1) is associated with filaggrin metabolism and increases the activity of the pilar green degrading enzyme.

An embodiment of the present invention provides an external preparation for skin comprising an LXR activator as an active ingredient which increases the expression amount of BH gene.

In one embodiment of the present invention, the LXR activator may be T0901317 (hereinafter referred to as T1317) represented by the above formula (1).

In one embodiment of the present invention, the composition is a composition for external application for skin, which is for skin dryness improvement or skin moisturization. Further, it is a composition for external application for skin barrier recovery and skin stratum corneum recovery.

In one embodiment of the present invention, the composition for external application for skin may be a cosmetic composition. The formulation of the cosmetic composition may be a formulation of a soft lotion, a nutritional lotion, a massage cream, a nutritional cream, a pack, a gel or a skin adhesive type cosmetic. The composition of the present invention can be used as a lubricant, a lubricant, a thickening agent and a gelling agent, a softener, an antioxidant, a suspending agent, a stabilizer, a foaming agent, a fragrance, a surfactant, water, In the field of cosmetics or dermatology, such as ionic sequestering and chelating agents, preservatives, vitamins, blockers, moisturizers, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or any other ingredient commonly used in cosmetics And may contain commonly used adjuvants. These adjuvants are introduced in amounts commonly used in the cosmetic or dermatological fields.

In addition, the composition for external application for skin may be a pharmaceutical composition, and may be a transdermal dosage form of lotion, ointment, gel, cream, patch or spray.

The ingredients other than essential ingredients in the composition for external application for skin of the present invention can be selected and mixed by those skilled in the art depending on the formulation and purpose of use of other external preparations.

One embodiment of the present invention provides a method for providing information for diagnosing dry skin by measuring the amount of BH in the skin. The method can measure the amount of BH mRNA and can be measured using PCR. Specifically, the BH content of the normal skin is measured to determine the normal level, and the BH content of the subject is measured. If the BH content of the subject is smaller than that of the normal skin, the skin can be evaluated as dry skin.

Hereinafter, the present invention will be described in detail with reference to examples of the present invention. It will be apparent to those skilled in the art that these embodiments are provided by way of illustration only for the purpose of more particularly illustrating the present invention and that the scope of the present invention is not limited by these embodiments .

EXAMPLES Example 1 Extraction of keratin and analysis of pillared green content

Normal skin and dry skin were measured by conoid meter, and if it was over 30, normal skin and 20 were classified as dry skin. Normal skin keratin and dry skin keratin were extracted by tape stripping method, quantified by dot blot method and quantified by high performance liquid chromatography (HPLC) to determine the total amount of amino acid, that is, Natural Moisturizing Factor: NMF) were analyzed. The results are shown in Fig. 1 and Fig. There was no difference in the amount of pillared green in normal skin and dry skin, but the total amino acid content was decreased. Total amino acid content of normal keratin was about 167 ug / mg. In addition, the correlation between pillared green and NMF (total amino acid) is shown in Fig. 3 and Table 1. [ Correlation analysis between the pillared green and total amino acid contents in the dry skin showed a negative value. This result indicates that there may be a problem with the degradation of the fila green in the dry skin.

≪ Example 2 >

Changes in the conversion enzymes that convert pilar green to NMF in the epidermis of aged skin and young skin were measured. Experimental method was to separate the epidermis from the skin of the younger buttocks and the buttocks of the elderly. The epidermis was then broken up using liquid nitrogen, and then 2mm glass beads and cell lysis solution were added, vortexed to obtain supernatant Proteins were quantified. Proteins of young and aged epidermis were loaded on SDS-Gel and then analyzed for the expression of caspase-14, calpain-1, Bleomycin hydrolase (BH) Were blotted using antibodies. Relative quantification corrected actin. As a result, bleomycin hydolase (BH) was selected as a priority. The results are shown in Fig. Thereafter, BH was decreased in the dry skin keratin compared to normal skin keratin. The results are shown in FIG.

≪ Example 3 >

In the case of LXR agonist T1317, 10 uM treatment of keratinocytes resulted in an increase in the number of pillared green algae, which was differentiated by normal keratinocytes. (T1317) was selected because the increase in the expression level of pilar green was very high. In order to investigate the effect of T1317 on the pilar green degrading enzymes in addition to the amount of pilar green, expression of pilar green degrading enzymes at the gene level in keratinocytes The amounts were compared between day 1 and day 7.

Since the stratum corneum of the human skin is differentiated, it is compared at the early stage (1 day) and at the completion stage (7 days) of keratinocyte experiment. Specific experimental methods are as follows. Real-time and semi-quantitative polymerase chain reaction (RT-PCR) was used. Total RNA was extracted from isolated keratinocytes using the Trizol method (Life Technologies), and 1 μg of total RNA was extracted from the cDNA using a first strand cDNA synthesis kit (MBI Fermentas) according to the manufacturer's instructions (Applied Biosystems: ABI) To quantitatively measure the expression level of each gene mRNA, a 7500 real-time PCR system (Applied Biosciences) was performed using a TaqMan Universal PCR Master Mix (Takara Bio Inc.) according to the manufacturer's instructions Biosystems). Each primer was purchased from Applied Biosystems and the information was caspase-14 (Hs00201637_m1), calpain (Hs00559804_m1), BH (Hs00166071_m1), gapdh (4333764F) filaggrin (Hs00856927_g1).

FIG. 6 shows the increase in the amount of pillared green in the keratinocytes treated with T1317 and in the untreated control (con) (days 1 and 7). In the case of T1317, the amount of pillared green increased on the 7th day. Pilar green is also a moisturizing factor, but it is a differentiation marker, so it can be seen that keratinocytes are normally differentiated.

FIG. 7 shows the expression levels of caspase-14, a papaverolytic enzyme, in the differentiated state (1 day and 7 days) between keratinocyte T1317 treated and untreated control (con) Did not act on caspase-14.

FIG. 8 is a graph comparing the expression levels of BH, a pilar green degrading enzyme, in the differentiated state (1 day and 7 days) between the treatment with keratinocyte T1317 and the untreated control (con). In the case of T1317, . This suggests that T1317 selectively increases the expression of BH, which is the most important factor in degrading fila green.

FIG. 9 shows calpain-1. In the state of treatment with keratinocyte T1317 and the untreated control (con) (1 day and 7 days), calpain-1 The expression level of T1317 did not affect Klepain-1.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (13)

A method for screening a dry skin enhancing material,
Characterized in that screening is carried out based on the degree of liver X receptor (LXR) activation by a candidate substance. The method according to claim 1,
Wherein the level of activation of the liver X receptor is determined by measuring the gene expression level of bleomycin hydolase (BH). 3. The method of claim 2,
Wherein the gene expression level of BH is measured by PCR, and the expression level of BH gene expressed by treating T1317 expressed by the following formula (1) is compared with that measured by PCR.
[Chemical Formula 1]

The method according to claim 2 or 3,
Wherein the amount of expression of BH gene is mRNA expression level. delete delete delete delete A skin external composition for improving skin dryness or skin moisturizing, comprising T1317 represented by the following formula (1) as an LXR agonist as an active ingredient.
[Chemical Formula 1]

delete delete delete delete

Images