TW201922290A - Screening method of substance having firmness improving action - Google Patents

Abstract

The purpose of the present invention is to provide a substance which has a firmness improving action. The expression of adhesion molecules in at least one type of vascular endothelial cells selected from the group consisting of VE-cadherin and integrin [alpha]5 was discovered to contribute to improvement of firmness. On the basis of this discovery, a screening method was developed in which adhesion molecules in vascular endothelial cells are used as an index.

Description

Screening method for substances with tightness improving effect

The present invention relates to a technical field of beauty, makeup and food for improving firmness.

If skin firmness is lost, it will become the cause of wrinkles or sagging, and the age or appearance of the skin will have a greater impact. Therefore, maintaining and improving skin firmness is a major issue in beauty. The firmness of the skin depends mainly on the thickness of the dermis layer. As the function of dermal fibroblasts is reduced or activation of matrix metalloproteinase is caused by increasing age or exposure to ultraviolet rays, the elastic fibers represented by collagen or elastin are reduced, so the dermal layer becomes thinner and loses. Tightness. In the previous cosmetic methods, in order to achieve an improvement in firmness, efforts were made to increase elastic fibers through the activation of dermal fibroblasts. As a result, it was found that a component having dermal fibroblast activation effect or collagen production promotion effect was applied to cosmetics.

So far, isoflavone compounds, phytosterols, etc. have been reported as natural-derived components that prevent and improve the age of the skin by promoting the production of collagen, and Binhai Qianhu (Patent Document 1: WO 2013/099378), extracts of Cypress seed (made by drying the seeds of Platycladus orientalis) (Patent Document 2: WO2012 / 057123), Ryukyu Yatake, Japanese Lily, and Apricot (Patent Document 3: Japanese Patent Laid-Open) 2011-195505) plant extracts such as extracts. It is also known that fibroblasts interact with extracellular matrix through adhesion molecules such as integrins expressed in fibroblasts, and this interaction contributes to firmness (Non-Patent Document 1: J. Inv. Dermatology (2013) vol. 133, 899-906). However, the insights so far focus on the relationship between skin firmness and adhesion molecules of collagen or fibroblasts, and the relationship between skin firmness and adhesion molecules in vascular endothelial cells is completely unknown.
[Prior technical literature]
[Patent Literature]

[Patent Document 1] International Publication No. 2013/099378
[Patent Document 2] International Publication No. 2012/057123
[Patent Document 3] Japanese Patent Laid-Open No. 2011-195505
[Non-patent literature]

[Non-Patent Document 1] J. Inv. Dermatology (2013) vol. 133, 899-906

[Problems to be solved by the invention]

The object of the present invention is to provide a method for screening a substance having a tightness-improving effect by a new mechanism different from the tightness-improving agents or methods for improving tightness that have been discovered so far.
[Technical means to solve the problem]

The inventors of the present inventors made efforts to find the cause of the more rooted firmness in a living body, and surprisingly found that the firmness of the skin is related to the capillaries in the skin. As a result of further research, it was found that the expression of VE-cadherin involved in the cell adhesion of vascular endothelial cells and integrin α5 involved in the adhesion of vascular cells to the cell matrix was related to tightness, and the present invention was completed.

Specifically, the present invention relates to the following inventions:
[1] A screening method for substances with a tightness improving effect, which uses the gene expression of adhesion molecules in vascular endothelial cells as an indicator.
[2] The screening method according to item 1, wherein the adhesion molecule is VE-cadherin or integrin α5.
[3] A screening method for a substance having a tightness improving effect as described in item 1 or 2, comprising: culturing vascular endothelial cells in a medium containing a candidate agent;
The gene expression of adhesion molecules in vascular endothelial cells is measured; and when the above expression is increased in comparison with a control, a candidate agent is determined as a substance having a tightness improving effect.
[4] The screening method according to any one of items 1 to 3, wherein the control is a gene expression in a vascular endothelial cell culture without adding a candidate agent.
[5] The screening method of any one of items 1 to 4, wherein the gene expression is determined by the amount of mRNA or protein in vascular endothelial cells.
[6] A firmness improving agent comprising at least one expression enhancer of an adhesion molecule in a vascular endothelial cell selected from the group consisting of VE-cadherin and integrin α5.
[7] A method for improving the firmness, which is used for a cosmetic person, and comprises: using an adhesion molecule in a vascular endothelial cell selected from the group consisting of VE-cadherin and integrin α5 to promote the expression of adhesion molecules Agent.
[8] A method for improving firmness, which is used for a beauty person, and includes applying a heat treatment at 37 to 39 ° C to the skin.
[9] The method for improving compactness according to item 8, which utilizes the above-mentioned thermal treatment to promote the expression of adhesion molecules in vascular endothelial cells.
[10] The method for improving compactness according to item 9, wherein the adhesion molecule is VE-cadherin or integrin α5.
[Effect of the invention]

The screening method of the present invention can be used to select a substance having a tightness improving effect. In addition, the substance having a tightness improvement effect selected by the screening method of the present invention can promote at least one type of adhesion molecule in vascular endothelial cells selected from the group consisting of VE-cadherin and integrin α5. Gene expression, and play a role in improving firmness, thereby improving skin problems such as wrinkles, sagging.

The present invention relates to a screening method for a substance or a treatment with tightness improving effect which takes the gene expression of adhesion molecules in vascular endothelial cells as an index. In the present invention, the cell adhesion molecule is particularly preferably a cell adhesion molecule expressed in vascular endothelial cells. Among the above-mentioned cell adhesion molecules, cadherin and integrin may be listed, and in particular, VE-cadherin and integrin α5 may be listed.

The screening method of the present invention includes, for example, the following steps:
Culture vascular endothelial cells in a medium containing a candidate agent;
The gene expression of adhesion molecules in vascular endothelial cells is measured; and when the above expression is increased in comparison with a control, a candidate agent is determined as a substance having a tightness improving effect.
Here, the control refers to the gene expression of the adhesion molecule in different situations when cultured in a medium containing no candidate agent. The experiment on the control may be performed simultaneously with the screening method of the present invention, or may be performed in advance.

The screening method of the present invention may further include a pre-culturing step of culturing vascular endothelial cells before the step of culturing vascular endothelial cells in a medium containing a candidate agent. In addition, the method may include a step of culturing vascular endothelial cells in a medium containing a candidate agent, and a step of culturing after culturing in a medium containing no candidate agent. The step of culturing vascular endothelial cells in a medium containing a candidate agent may be performed by directly adding the candidate agent or a dilution thereof to the culture obtained in the pre-cultivation step, or the medium containing the candidate agent may be replaced and cultured.

The measurement of the gene expression of adhesion molecules can be determined by measuring the amount of mRNA or protein of adhesion molecules in vascular endothelial cells. The measurement of the amount of mRNA can be performed using a method known in the art such as quantitative PCR or the northern blot method. As for the protein quality, any method known in the art such as western blotting method, immunostaining, and FACS can be used. In these methods, antibodies that specifically bind to an adhesion molecule can be used.

In the present invention, the firmness means the elasticity of the skin, especially the skin of the face. The elasticity of the skin can be measured by a skin elasticity tester or a double-layer cylindrical instrument, etc., and can especially be expressed by the firmness index measured by a skin elasticity tester. Skin firmness is affected by the amount of elastic fibers such as collagen or elastin in the dermis. The present invention is based on the discovery of the correlation between blood vessels and firmness index (Ur / Uf) (Figure 1). More specifically, the average thickness of blood vessels is related to the skin firmness index. The blood vessel density is related to the firmness index. In an in vitro experiment, human fibroblasts and human umbilical cord vein endothelial cells were cultured on a collagen gel compared to the case where human fibroblasts (HF) were cultured on a collagen gel alone. The above co-cultivation situation shows a higher elasticity in skin models cultured in vitro (Figure 2). Although not wishing to be bound by theory, from these results, it is believed that the endothelial cells and collagen gels of the capillaries in the skin play a role to help firmness.

Vascular endothelial cell lines are cells that make up the inner membrane of blood vessels. The arterial or venous system includes three layers of the intima, media, and adventitia, but the capillaries only include the intima containing vascular endothelial cells. In vascular endothelial cells, endothelial cells are mainly adhered by the action of cell adhesion molecules such as VE-cadherin, and the extracellular matrix is adhered to vascular endothelial cells by the action of integrin. The vascular endothelial cells may be endothelial cells obtained from a living body and their descendant cells, or strains, or umbilical cord blood vein endothelial cells and their descendant cells. The vascular endothelial cells are not limited to the following, and HUVEC and HMEC-1 can be used.

VE-cadherin is cadherin 5 or a protein with a molecular weight of about 140 kDa, also known as CD144, and is a protein belonging to the cadherin superfamily. VE-cadherin is specific in endothelial cells, is expressed in lymphatic endothelial cells and vascular endothelial cells, and exists in the cell membrane. VE-cadherin molecules are known to be related to the permeability of vascular endothelium or lymphatic endothelium. As the performance of VE-cadherin increases, the adhesion of vascular endothelial cells to each other increases, which contributes to the stability of vascular endothelium. If the gene expression of VE-cadherin is inhibited, it shows that the elasticity of the skin model is weakened (FIG. 4A), and it also shows that it is difficult to form a blood vessel-like structure in the skin model (FIG. 4B-E). It is known that the firmness of the skin decreases with age (FIG. 6A), but shows that the performance of VE-cadherin also decreases with age (FIG. 6B). Although not intended to be limited by theory, technical common sense and the results of these experiments show that if the performance of VE-cadherin is inhibited, the stability of capillaries is lost, and the amount of capillaries is reduced. As a result, it is thought that it prevents the production or maturation of elastic fibers such as collagen. By promoting the performance of VE-cadherin, the capillaries are stabilized, and as a result, the production or maturation of elastic fibers such as collagen can be promoted. This can improve the firmness reduced by aging.

Integrins are cell membrane proteins that exist in the cell membrane and function as cell adhesion molecules. Integrins are present in heterodimers associated with an alpha chain and a beta chain. At least 18 species have been identified as alpha subunits and 8 species have been identified as beta subunits. It is known that integrin α5 mainly associates with integrin β1 to form integrin α5β1. Integrin α5β1 is also called VLA-5, fibronectin receptor. The integrin α5 expressed in the cell membrane of vascular endothelial cells binds to the fibronectin constituting the extracellular matrix (ECM). Collagen, which is the main component of the extracellular matrix, interacts with molecules such as fibronectin or laminin to form higher-order structures. By binding vascular endothelial cells to fibronectin via integrin α5, the collagen, which is the main component of the extracellular matrix, is contracted as if it were attracted to capillaries, thereby making collagen mature, which can help Skin firmness. If the expression of integrin α5 is inhibited, the elasticity is reduced in a skin model containing collagen (FIG. 5A). Moreover, in the skin model, although there was no change with respect to the volume of the blood vessel (FIG. 5D), the number of crossings decreased (FIG. 5E), but the length of the blood vessel increased (FIG. 5C). It is thought that the weakening of the interaction with the extracellular matrix results in easy expansion of blood vessels. On the other hand, with regard to integrin α3 or α6 expressed in the vascular endothelium, no change in elasticity caused by the suppression of the expression was observed (FIG. 5A). It is known that the firmness of the skin decreases with age (Fig. 6A), but among integrins expressed in endothelial cells, the performance of integrin α5 also decreases with age (Fig. 6D). On the one hand, the expression levels of integrin α3 and α6 change less with age (Figures 6C and E). Although not wanting to be bound by theory, technical common sense and the results of these experiments show that if the expression of integrin α5 is inhibited, the interaction between capillaries and the extracellular matrix is weakened, as a result, the production of elastic fibers such as collagen or mature. By promoting the expression of integrin α5, the capillary vessels interact with the extracellular matrix, and as a result, the production or maturation of elastic fibers such as collagen can be promoted. This can improve the firmness reduced by aging.

When the substance having a tightness improvement effect screened by the screening method of the present invention is based on the performance of the VE-cadherin gene as an index, it can also be called a VE-cadherin gene expression promoter. The VE-cadherin gene expression promoter can be any substance as long as it can promote the expression of the VE-cadherin gene in vascular endothelial cells. In one aspect of the screening method of the present invention, the gene expression of VE-cadherin in vascular endothelial cells is used as an index, and screening is performed using any material database such as cosmetic materials, food materials, and pharmaceutical materials. As the substance having the effect of improving the tightness determined in this way, a plant body extract selected from Siberian ginseng, calendula, and cistanche may be cited (FIG. 7). These substances can be called VE-cadherin gene expression promoters.

The substance having the effect of improving the tightness screened by the screening method of the present invention is also called an integrin α5 gene promoter when the expression of the integrin α5 gene is used as an index. The integrin α5 gene expression promoter can be any substance as long as it can promote the expression of the integrin α5 gene in vascular endothelial cells. One aspect of the screening method of the present invention is to use the gene expression of integrin α5 in vascular endothelial cells as an index, and use arbitrary material databases such as cosmetics materials, food materials, and pharmaceutical materials for screening. As the substance having the effect of improving the tightness determined in this manner, extracts of plant bodies and yeast extracts selected from Siberian ginseng and Cistanche deserticola can be cited (FIG. 8). These substances can be referred to as integrin α5 gene expression promoters.

The so-called Siberian ginseng (Siberian Ginseng) refers to a plant that grows in Russia, China, Hokkaido and other regions. It is also called Eleutherococcus senticosus. Plants such as fruits, leaves, stems, flowers, and roots can be used for medicinal purposes, and the root bark of roots can be used as crude medicine. Siberian ginseng extract is an extract obtained by using Siberian ginseng plants, especially the root bark, with a solvent such as water or alcohols such as propylene glycol and ethanol. Siberian ginseng extract has been shown to improve skin elasticity by oral administration for 8 weeks (Figure 9).

Calendula extract is an extract of the flower of Calendula officinalis. Calendula is a member of the Compositae family native to southern Europe. Calendula extract refers to an extract obtained by extracting a flower of a calendula with a solvent such as water or alcohols such as propylene glycol and ethanol.

The so-called Cistanche extract refers to the plant extract of Cistanche salsa. Cistanche deserticosa is a plant of the family Cistancheaceae that is native to mainland China and Central Asia. In plants, stems, leaves, flowers, roots, etc. can be used. Especially dried succulent stems are called Cistanche deserticola for use as crude medicine. Cistanche extract refers to an extract that is extracted from Cistanche with a solvent such as water, or alcohols such as propylene glycol and ethanol.

The so-called yeast extract refers to yeast extracts such as Saccharomyces cerevisiae. Yeast extract is obtained by destroying the cell wall of yeast with acid treatment, alkali treatment, or enzyme treatment.

The substances with tightness improving effect selected by the screening method of the present invention can also be called tightness improving agents, which can be formulated in cosmetics, pharmaceuticals, or quasi drugs, and can also be formulated in foods such as supplements, etc. In nutritional supplements. The VE-cadherin gene expression enhancer, or integrin gene expression enhancer, and tightness improver of the present invention, which has a tightness improving effect, can also be administered through any route, but the In terms of the function of the cells, administration routes such as oral administration, transdermal administration, and transmucosal administration are preferred.

As explained above, it was found that the promotion of VE-cadherin gene expression and / or integrin α5 gene expression in vascular endothelial cells contributes to improvement of skin firmness. Therefore, another aspect of the present invention relates to the improvement of skin firmness caused by the promotion of VE-cadherin gene expression and / or integrin α5 gene expression. More specifically, the present invention relates to a firmness improving agent containing a performance-enhancing agent for adhesion molecules in vascular endothelial cells. Here, the adhesion molecules in vascular endothelial cells are VE-cadherin and / or integrin α5. Furthermore, in another aspect, it relates to a method for improving the firmness of skin including applying a VE-cadherin gene expression promoter and / or an integrin α5 gene expression promoter to the skin.

In addition, even in the case where the VE-cadherin gene expression promoter or the integrin α5 gene expression promoter is not dependent, as long as the gene expression can be promoted, compactness improvement can be achieved. Therefore, in a further aspect of the present invention, it is also related to a method for improving compactness by promoting the expression of VE-cadherin gene and / or promoting the expression of integrin α5 gene.

Examples of the method for improving the tightness include a thermal treatment. The surface temperature of the skin is about 31 ° C to 33 ° C in a healthy state. Therefore, the term "thermal treatment" refers to the application of a temperature above the surface temperature to the skin. The lower limit of the thermal treatment is 35 ° C, more preferably 37 ° C from the viewpoint of applying a temperature higher than the skin surface temperature. The upper limit temperature of the thermal treatment is preferably less than 45 ° C, more preferably 42 ° C, and still more preferably 39 ° C from the viewpoint of not causing burns to the skin. The time of the heat treatment can be arbitrarily selected within the range exhibiting the effect of improving the tightness. As an example, the time range is from 1 minute to 60 minutes. As the lower limit of the heat treatment time, from the viewpoint of exerting the effect of improving the tightness, 1 minute, more preferably 2 minutes, and still more preferably 3 minutes are selected. As the upper limit of the heat treatment time, from the viewpoint of ease of treatment, 60 minutes, more preferably 30 minutes, and still more preferably 10 minutes are selected. It was shown that by giving the skin a warming of 37 ° C. to 39 ° C. for 5 minutes, the firmness was improved after the treatment (FIG. 10). In addition, it was shown that changes in the gene expression of VE-cadherin and the gene expression level of integrin α5 caused by temperature changes in the cell culture system of vascular endothelial cells (Figure 11). Based on these results, it is shown that by increasing the performance of VE-cadherin or integrin α5 in vascular endothelial cells by thermally treating the skin, firmness can be improved. In order to confirm the effect of the tightness improving method of the present invention, the gene expression of VE-cadherin or integrin α5 can also be confirmed.

In this specification, the method for improving firmness refers to a cosmetic method for cosmetic purposes, and can be distinguished from a treatment performed by a physician or a medical related person. The above-mentioned cosmetic method may be performed individually, or may be performed in a beauty salon, a cosmetics sales shop, a beauty salon, or the like.

All documents mentioned in this specification are incorporated by reference in their entirety into this specification.

The embodiments of the present invention described below are for illustrative purposes only and do not limit the technical scope of the present invention. The technical scope of the present invention is limited only by the description of the scope of patent applications. Changes to the present invention such as additions, deletions, and replacements of the constituent elements of the present invention can be made without departing from the gist of the present invention.
[Example]

Example 1: Correlation between the state of the capillaries in the skin and the firmness index Using a skin elasticity tester (400 mbar), the firmness index Ur / Uf of the skin on the cheek area was measured (females aged 26 to 39 years old 15 Bit). In addition, (ILS-Bio) purchased skin from Asian men and women (aged 20 to 40) as the remainder of the operation. Skin samples were taken from the cheek area and the skin of human skin was made transparent using the technology of ImPACT. As the primary antibody, an anti-CD31 goat antibody (: R & Dsystems) diluted 100-fold with PBS was used, and as the secondary antibody, an AlexaFluoro594 labeled anti-sheep IgG antibody (Invitrogen) diluted 200-fold with PBS was used. The stained skin samples were observed in a light microscope (Carl Zeiss). Using image analysis software (Imaris), the average thickness (μm) and density (%) of blood vessels were measured to obtain a histogram of the skin tightness index Ur / Uf measured in advance (Figure 1). The tightness index is related to the average thickness of blood vessels and the density of blood vessels, respectively.

Example 2: Relationship between skin model and elasticity A skin model was made by using three holes in a 6-well plate and using the following method. A collagen gel was prepared by putting 16.7 mL of 0.3% collagen in a 50 mL tube on ice, and slowly adding 10.6 mL of DMEM (-) medium while stirring. Next, human fibroblasts (HF) were prepared in DMEM (-) medium at 10 × 10 ⁵ cells / mL. Human umbilical cord blood vein endothelial cells (HUVEC) were prepared at 10 × 10 ⁵ cells / mL in EBM2 medium supplemented with 0.5% FBS. In the HF group, 2 ml of the prepared HF culture was divided, and in the HF-HUVEC group, 2 ml of the prepared HF culture and HUVEC culture were separated. 10 mL of cell fluid was prepared by mixing with 6 mL of EBM2 medium supplemented with 0.5% FBS. 10 ml of the prepared cell solution and 10 mL of the collagen gel solution were thoroughly stirred on ice, and 6 ml was added to each well of the 6-well plate. In a 5% humidified environment, shake at 37 ° C for a while, and then stand still at 37 ° C for 5 days to make HF skin models and HF-HUVEC skin models. In the HF-HUVEC skin model, the shape of HUVEC cells into blood vessels was observed (data not shown). For these skin models, a rheometer (PHYSICA MCR300) was used to measure the elastic force using a PP12 jig (Figure 2). There is a significant difference between the elasticity of the HF skin model and the elasticity of the HF-HUVEC skin model (p <0.05).

Example 3: Correlation between blood vessels and firmness in the skin model. In the preparation of the HF-HUVEC skin model in Example 2, angiopoietin 1 (Ang1) was added at 500 ng / ml (every day before the shaking culture). The concentration was added to the medium. In a skin model, vascular endothelial cells were visualized using an anti-CD31 goat antibody (R & D systems) that recognized vascular endothelial cells, and AlexaFluor594 labeled anti-goat antibody (Invitrogen) as a secondary antibody, and observed using a fluorescence microscope. Before making the skin model, PKH67 Green Fluorescent Cell Linker Kit (Sigma) was used to visualize fibroblasts and observed with a fluorescence microscope. It was observed that angiopoietin 1 was added to increase the shape of blood vessels (Fig. 3A). The image analysis software (Imaris) was used to measure the length (μm) (Figure 3B), volume (μm ³ ) (Figure 3C), and cross region (μm ² ) (Figure 3D) of the blood vessels. When added, the volume of the vascular structure increased (Fig. 3C) ( ^* : p <0.05). The elasticity was measured using a rheometer (PHYSICA MCR300) and a PP12 jig. As a result, the skin firmness was increased by the addition of angiopoietin 1 (Fig. 3E) ( ^* : p <0.05). Since angiopoietin 1 has an angiogenesis effect, it is thought that this effect can be used to increase the blood vessel volume in a skin model.

Example 4: Gene expression of vascular endothelial cells is inhibited in HUVEC cells, and VE-cadherin (cad), integrin α3 (inta3), integrin α5 (inta5), and integrin α6 (inta6) are inhibited using siRNA Gene expression. As the siRNA, Ambion Silencer Select SiRNA, CDH5 (ID: s2780, s2781, s2782), ITGA3 (ID: s7541, s7542, s7543), ITGA5 (ID: s7547, s7548, s7549), ITGA6 (ID: s7492, s7493, s7494). HUVEC cells were prepared at 10 × 10 ⁵ cells / mL. Each cell was pelleted, and 100 μL of a set solution was added after aspiration of the culture medium and mixed. Add 1 μL each of the above siRNA (Ambion) to the mixed cells, and transfer the total amount to the 4D-Nucleofecter (Lonza) colorimetric tube. Install CELL TYPE to HUVEC and start 4D-Nucleofecter for gene introduction. Add 500 μL of EBM2 (+) medium to each colorimetric tube at the end of gene introduction, and add the total content of the colorimetric tube to a 10 cm Petri dish with 10 mL of EBM2 (+) medium previously added. After standing still at 37 ° C for a while, a skin model was made in the same manner as in Example 2 using the cells the next day. Quantitative PCR was performed on the cells into which each siRNA was introduced, and the gene expression of the target of inhibition was confirmed (data not published).

In skin models in which siRNA was introduced to suppress the expression of cadherin, integrin α3, integrin α5, and integrin α6, the elasticity was measured using a rheometer (PHYSICA MCR300) and a PP12 jig. Compared with the control using siCont, in the case of using siRNA for VE-cadherin and the case of using siRNA for integrin α5, the elasticity was significantly reduced (Figs. 4A and 5A).

In these skin models, anti-CD31 sheep antibodies (R & D systems) were used as primary antibodies, and AlexaFluor594 labeled anti-sheep antibodies (Invitrogen) were used as secondary antibodies to visualize vascular endothelial cells (Figures 4B and 5B). In skin models that inhibit the expression of VE-cadherin and integrin α5, changes in the structure of blood vessels can be seen. The image analysis software (Imaris) was used to measure the length (μm), volume (μm ³ ), and intersecting area (μm ² ) of the blood vessels. As a result, it was found that in the case of inhibiting the expression of VE-cadherin, all aspects were compared with the control. The ratios were significantly reduced (Fig. 4C-E) ( ^* : p <0.05, ^** : p <0.01). In the case of inhibiting the expression of integrin α5, the blood vessel length (μm) was significantly increased compared to the control, and on the other hand, the cross-sectional area (μm ² ) was significantly reduced compared to the control (Figure 5C, E) ( ^* : p <0.05).

Example 5: Effect of age in skin model HUVEC cells were replaced with vascular endothelial cells from young subjects (0 years old) and vascular endothelial cells from older subjects (50 years old). Make skin models in the same way. In the skin model produced, the elasticity was measured using a rheometer (PHYSICA MCR300) and a PP12 jig (FIG. 6A). There is a significant difference between the elasticity of the skin model of young subjects and the elasticity of the skin model of old subjects ( ^* : p <0.05).

The vascular endothelial cells of young subjects (0 years old) and the vascular endothelial cells of old subjects (50 years old) were seeded in 6-well plates at 2 × 10 ⁵ cells / well, and left standing for a while. RNA was extracted using RNeasy mini kit (QIAGEN). The concentration of the extracted RNA was measured by nano drop, and was prepared to 100 ng / ml by RNace-free water. The prepared RNA was quantified using a TaqMan RNA-to-C 1-Step Kit (Applied Biosystems) and primers for the following genes (Applied Biosystems) using Real Time PCR (Roche Light Cycler480II). Using β-actin (b-actin: Cat # Hs01060665_g1) as an internal standard, it was found that in cells derived from young subjects and cells derived from older subjects, VE-cadherin ( VE-Cadherin: Cat # Hs00170986_m1) and integrin α5 (Integrin alpha5: Cat # Hs01547673_m1) showed significant differences (Figure 6B and D: *: P <0.05). On the other hand, integrin α3 ( No significant difference was seen in the expressions of Integrin alpha3: Cat # Hs01076879_m1) and integrin α6 (Integrin alpha6: Cat # Hs01041011_m1) (Figures 6C and E).

Example 6: Screening method for substances with tightness improvement effect using VE-cadherin gene expression as an index Human umbilical cord blood vein endothelial cells (HUVEC) were prepared in EBM2 medium supplemented with 0.5% FBS to 10 × 10 ⁵ cells / mL, cultured at 37 ° C in a 5% CO ₂ humidified environment. As a candidate drug, a cosmetic material library is used. Candidate agents were added and cultured for 6 hours. After the culture, the medium was removed, and RNA was extracted from the cells using an RNeasy mini kit (QIAGEN). The concentration of the extracted RNA was measured by nano drop, and was prepared to 100 ng / ml by RNace-free water. The prepared RNA was quantified using a TaqMan RNA-to-C 1-Step Kit (Applied Biosystems) and primers for the VE-cadherin gene using Real Time PCR (Roche Light Cycler480II).

In the case of using Siberian ginseng extract, calendula extract, and Cistanche extract as candidate agents, the performance of the VE-cadherin gene was significantly increased compared with the control (p <0.05: FIG. 7). These extracts were selected as a substance having a tightening effect.

Example 7: Screening method for substances with tightness improvement effect using integrin α5 gene expression as an index Human umbilical cord blood vein endothelial cells (HUVEC) were prepared in EBM2 medium supplemented with 0.5% FBS to 10 × 10 ⁵ cells / mL, cultured at 37 ° C in a 5% CO ₂ humidified environment. Candidate agents were added and cultured for 6 hours. After the culture, the medium was removed, and RNA was extracted from the cells using an RNeasy mini kit (QIAGEN). The concentration of the extracted RNA was measured by nano drop, and was prepared to 100 ng / ml by RNace-free water. The prepared RNA was quantified using a TaqMan RNA-to-C 1-Step Kit (Applied Biosystems) and primers for the integrin α5 gene using Real Time PCR (Roche Light Cycler480II).

When Siberian ginseng extract, yeast extract, and Cistanche extract were used as candidate drugs, the expression of the integrin α5 gene was significantly increased compared with the control (p <0.05: FIG. 8). These extracts were selected as a substance having a tightening effect.

Example 8: Tightness improvement effect by ingestion of a substance having a tightness improving effect In order to target vascular endothelial cells, investigate the effects of promoting the expression of the VE-cadherin gene and the integrin α5 gene in Siberia The ginseng firmness improvement effect was performed in 20 30-year-old women for an 8-week intake test of Siberian ginseng extract. At the beginning of the test, after 4 weeks and 8 weeks, the skin elasticity tester MPA580 (Integral) was used to measure the elasticity of the cheeks. As a result, the elasticity of the skin was significantly increased after 8 weeks (p <0.05: Fig. 9).

Example 9: Firmness improvement effect by warm stimulation was applied to cheeks of healthy women (14 persons), and Eyener & Lipron was applied at 39 ° C for 5 minutes. Before and after application, the skin elasticity of the cheek was measured using a skin elasticity tester MPA580 (Integral) (Figure 10). The elasticity of the skin was significantly improved before and after application (p <0.05).

Example 10: Human umbilical cord blood vein endothelial cells (HUVEC) were prepared into 10 × 10 ⁵ cells / mL in an EBM2 medium supplemented with 0.5% FBS by a thermal stimulation. After sowing the cells, they were cultured at a temperature of 29 ° C, 33 ° C, and 37 ° C in a 5% CO ₂ humidified environment. After 1 day of culture, the cells were recovered, and RNA was extracted from the cells using an RNeasy mini kit (QIAGEN). The concentration of the extracted RNA was measured by nano drop, and was prepared to 100 ng / ml by RNace-free water. The prepared RNA was quantified using TaqMan RNA-to-C 1-Step Kit (Applied Biosystems) and primers for integrin α5 gene and VE-cadherin using Real Time PCR (Roche Light Cycler480II) (Figure 11) ). The gene expression of VE-cadherin increased as the temperature increased from 29 ° C to 37 ° C. Although the gene expression level of integrin α5 did not change between 29 ° C and 33 ° C, the gene expression level was higher at 37 ° C.

FIG. 1 is a graph obtained by plotting the relationship between the tightness index and the average thickness of the blood vessel (A), the tightness index and the blood vessel density (B).

Figure 2 shows the skin model made from human fibroblast (HF) and collagen gel, and the skin model made from human fibroblast (HF) and human umbilical cord blood endothelial cells (HUVEC) and collagen gel. A graph comparing skin elasticity in skin models.

FIG. 3A is a fluorescence microscope photograph of a labeled blood vessel structure in a skin model made using human umbilical cord blood venous endothelial cells (HUVEC), and also shows the addition of angiopoietin 1 as an angiogenesis promoter.

FIGS. 3B to D are obtained by measuring the length (μm), volume (μm ³ ), and intersecting area (μm ² ) of the blood vessel in the fluorescence microscope photograph of FIG. 3A, and comparing the control with the angiopoietin 1 addition group. chart. FIG. 3E is a graph showing the elasticity of an angiopoietin 1 addition group in a skin model made using human umbilical cord blood vein endothelial cells (HUVEC).

FIG. 4A is a graph showing a decrease in elasticity in a skin model when VE-cadherin gene expression is inhibited by siRNA compared with a control. FIG. 4B is a skin model made using a control human umbilical cord blood vein endothelial cell (HUVEC) and a skin model made using a human umbilical cord blood vein endothelial cell (HUVEC) which inhibits gene expression of VE-cadherin, Fluorescence micrograph of labeled vessel structure.

FIG 4C ~ E based on a fluorescence microscope photograph of FIG. 4B, the measurement of the length of the blood vessels ([mu] m) (FIG. 4C), a volume ⁽³ μm) (FIG. 4D), and cross-area (μm ²⁾ (FIG. 4E), and Graphs compared to controls.

FIG. 5A shows the changes in the elasticity of human umbilical cord blood endothelial cells (HUVEC) in the case of inhibiting the gene expression of integrin α3, 5, and 6 by siRNA, respectively. Fig. 5B is a fluorescence microscope photograph of a labeled blood vessel structure in a skin model made using a control human umbilical cord blood vein endothelial cell (HUVEC) and a human umbilical cord blood vein endothelial cell (HUVEC) that inhibits gene expression of integrin α5 .

5C to E are measured in the fluorescent microscope photograph of FIG. 5B, and the length (μm) (FIG. 5C), volume (μm ³ ) (FIG. 5D), and cross region (μm ² ) (FIG. 5E) of the blood vessel were measured, and Graphs compared to controls.

FIG. 6A is a comparison of elasticity between a skin model made using vascular endothelial cells obtained from young subjects and a skin model made using vascular endothelial cells obtained from older subjects chart. Figures 6B-E show VE-cadherin, integrin α3, integrin α5, and integration in vascular endothelial cells obtained from young subjects and vascular endothelial cells obtained from older subjects A graph obtained by comparing the gene expression levels of α6.

FIG. 7 is a graph showing the effects of siberian ginseng, calendula, and cistanche extract on VE-cadherin candidate candidates with tightness improvement effect screened using VE-cadherin gene performance as an index.

FIG. 8 is a graph showing the effects of Siberian ginseng, yeast extract, and cistanche extract on integrin α5, which are candidates for compactness improvement screened using the integrin α5 gene performance as an index.

FIG. 9 is a graph showing changes in the elasticity of the skin in the Siberian ginseng intake group.

FIG. 10 is a graph showing changes in skin firmness caused by thermal treatment of the skin.

11 (A) and 11 (B) are graphs showing changes in the expression levels of VE-cadherin and integrin α5 due to different culture temperatures of vascular endothelial cells.

Claims (10)

A screening method for substances with a tightness improving effect, which uses the gene expression of adhesion molecules in vascular endothelial cells as an indicator. The screening method according to claim 1, wherein the adhesion molecule is VE-cadherin or integrin α5. The screening method for substances having a tightness improving effect as claimed in item 1 or 2 includes: Culture vascular endothelial cells in a medium containing a candidate agent; Measuring the genetic expression of adhesion molecules in vascular endothelial cells; and In the case where the above-mentioned performance is increased in comparison with the control, a candidate drug is decided as a substance having a tightness improving effect. The screening method according to any one of claims 1 to 3, wherein the control is a gene expression in a vascular endothelial cell culture without adding a candidate agent. The screening method according to any one of claims 1 to 4, wherein the gene expression is determined by the amount of mRNA or protein in vascular endothelial cells. A firmness-improving agent comprising at least one expression enhancer of an adhesion molecule in a vascular endothelial cell selected from the group consisting of VE-cadherin and integrin α5. A method for improving the firmness, which is used for cosmetic purposes, and includes: applying at least one expression promoting agent of an adhesion molecule in a vascular endothelial cell selected from the group consisting of VE-cadherin and integrin α5. A method for improving firmness, which is used for cosmetic purposes, and includes: applying a heat treatment at 37 to 39 ° C to the skin. If the method for improving the tightness of item 8 is used, the above-mentioned warm heat treatment is used to promote the expression of adhesion molecules in vascular endothelial cells. The tightness improving method according to claim 9, wherein the adhesion molecule is VE-cadherin or integrin α5.