WO2020230331A1

WO2020230331A1 - Apj expression promoter

Info

Publication number: WO2020230331A1
Application number: PCT/JP2019/019573
Authority: WO
Inventors: 美加加治屋; 健太朗加治屋; 上田　有紀; 綾野松尾
Original assignee: 株式会社資生堂
Priority date: 2019-05-16
Filing date: 2019-05-16
Publication date: 2020-11-19
Also published as: CN113825492A; JPWO2020230331A1

Abstract

The present invention addresses the problem of providing a substance that improves skin elasticity and a method for screening for substances having a skin elasticity-improving action.　Provided are an APJ expression promoter and an agent that is effective in improving skin elasticity through vascular stabilization by using said promoter to enhance the expression of APJ. The APJ expression promoter, which includes one or a plurality of crude drugs selected from the group consisting of Houttuynia cordata extract, cherry leaf extract, and neem leaf extract, is effective in improving skin elasticity through vascular stabilization. A screening method according to the present invention makes it possible to select a substance having a skin elasticity-improving action.

Description

APJ expression promoter

The present invention relates to an APJ expression promoter.

Skin elasticity has a great influence on the appearance age and appearance, and maintaining and improving skin elasticity is a major cosmetic issue. For skin elasticity, many studies have been conducted on the matrix of the dermis layer such as collagen, elastin, and hyaluronic acid. For example, it is well known that the amount of collagen in the dermis layer has a large effect. Conventional cosmetological methods have focused on increasing elastic fibers by activating dermal fibroblasts in order to achieve improvement in skin elasticity, and have the effect of activating dermal fibroblasts and promoting collagen production. Ingredients have been discovered and applied to cosmetics.

So far, isoflavone compounds, phytosterols, etc. have been reported as natural product-derived components that prevent and improve age-related changes in the skin by promoting collagen production, and Button Bowfu (International Publication No. 2013/099378). , Konotegasiwa seeds (International Publication No. 2012/057123), Ryukyuchiku, Oitabi and Spinach (Japanese Patent Laid-Open No. 2011-195505) have been found. It is also known that fibroblasts interact with the extracellular matrix via adhesion molecules such as integrins expressed in fibroblasts, and such interaction is involved in firmness (Non-Patent Document 1). The findings so far focus on the relationship between skin elasticity and adhesion molecules of collagen or fibroblasts.

Japanese Patent No. 5808769 Japanese Unexamined Patent Publication No. 2012-20942 Japanese Unexamined Patent Publication No. 2013-209339 International Publication No. 2012/133825 JP-A-2018-172410 Special Table 2019-501899 International Publication No. 2007/072980 Japanese Patent No. 5648149

An object of the present invention is to provide a method for screening a substance that improves skin elasticity and a substance that has a skin elasticity improving action by a mechanism different from conventional skin elasticity improving agents and methods.

As a result of diligent research to improve skin elasticity, the present inventors have stabilized skin blood vessels by enhancing the expression of APJ, improved their functions, and / or suppressed the decrease of skin blood vessels due to aging. We have discovered that the elasticity of the skin is improved, and further discovered a novel substance that promotes the expression of APJ, leading to the present invention.

The present invention relates to the following invention:
(1) APJ expression promoter for improving skin elasticity.
(2) An APJ expression promoter consisting of one or more crude drugs selected from the group consisting of Houttuynia cordata extract, Sakura leaf extract, and neem leaf extract.
(3)
A skin elasticity improving agent containing the APJ expression promoter according to (1) or (2).
(Four)
A screening method for skin elasticity improving agents using APJ expression as an index.
(Five)
Culturing a biological sample in a medium containing a candidate drug,
To measure APJ expression in a biological sample, and to determine a candidate drug as a substance having a skin elasticity improving effect when APJ expression is increased compared to a control.
The screening method according to (4).
(6)
A kit for carrying out the screening method according to any one of (4) or (5), which comprises a reagent for determining APJ expression.
(7)
A cosmetological method for improving a subject's skin elasticity, including promoting APJ expression in the subject.
(8)
The method according to (7), wherein the promotion of APJ expression in the subject is achieved by administering the skin elasticity improving agent according to (3) to the subject.

The APJ expression promoter of the present invention can promote the expression of APJ. When APJ expression is promoted, blood vessels are stabilized and skin elasticity is improved. APJ expression promoters containing one or more crude drugs selected from the group consisting of Houttuynia cordata extract, Sakura leaf extract, and neem leaf extract are effective in improving skin elasticity through stabilization of blood vessels. Further, by the screening method of the present invention, a substance having an action of improving skin elasticity can be selected. The skin elasticity improver selected by the screening method of the present invention improves skin elasticity through the stabilization of blood vessels by promoting the expression of APJ. By improving skin elasticity, skin problems such as wrinkles and sagging can be improved.

Figure 1 shows how a 3D angioplasty dermis model was created. FIG. 2 is a photomicrograph of an angiogenesis model showing the localization of blood vessels (green) stained with anti-PECAM-1 / CD31 antibody and type I collagen (red) stained with anti-type I Collagen antibody. In the left figure of FIG. 3, the sound velocity (hardness) value (m / s) of a human skin section is measured by an ultrasonic sound velocity microscope, and the obtained image and the immunostained image that visualizes the blood vessel (red) of the same section are superimposed. It is a combination. As typical examples, young people (20 years old) and middle-aged people (50 years old) are shown. The figure on the right is a graph showing the average of young people (10 to 20 years old) and middle-aged people (50 to 70 years old) by quantifying the sound velocity (hardness) value around blood vessels. In the figure, *** indicates that there is a significant difference by Student's t-test (*** p <0.001). FIG. 4 is a graph showing the relationship between the collagen concentration (mg / ml) and the storage elastic modulus G'(Pa). FIG. 5 is a graph showing the relationship between collagen concentration (mg / ml) and contact force (mN). Figure 6 shows a 3D angiogenic dermis model cultured in a collagen gel at each concentration (mg / ml) stained with anti-PECAM-1 / CD31 antibody to stain blood vessels (green) and with Cy3αSMA antibody to stain pericytes (pericytes). It is a micrograph which visualized red). The left figure of FIG. 7 is a graph showing the relationship between the collagen gel concentration and the mRNA expression level of APJ in the vascular endothelial cells cultured in the collagen gel having each concentration, and when the gel concentration is 0 mg / ml (-). It is shown as a relative value (%) with the expression level as 100. In the figure, * and ** indicate that there is a significant difference by Student's t-test (* p <0.05, ** p <0.01). The figure on the right shows blood vessels stained with anti-PECAM-1 / CD31 antibody (green), cell nuclei stained with Hoechst (blue), and APJ stained with anti-APJ antibody (red) in a 3D angiogenic dermis model formed in collagen gel. ) Is a micrograph showing the localization. The left figure of FIG. 8 shows an outline of creating a 3D angioplasty dermis model in which APJ high expression vector is transfected and APJ is highly expressed. The figure on the right is a photomicrograph showing the vascular structure (red) stained with anti-PECAM-1 / CD31 antibody in a control and a 3D angiogenic dermis model with high expression of APJ. FIG. 9 is a schematic diagram showing the concept of the relationship between skin elasticity, APJ expression, and blood vessel stabilization derived from the present application. FIG. 10 shows the expression of APJ when using Houttuynia cordata extract, Sakura leaf extract, and Neem leaf extract as a relative value (%) when the control is 100. In the figure, * and ** indicate that there is a significant difference from the control by Student's t-test (* p <0.05, ** p <0.01).

APJ (also known as AGTRL1: Angiotensin receptorlike1) is a 7-transmembrane G protein-coupled receptor whose expression has been widely reported in the vascular system, nervous system, adipocytes, etc. APJ expression has been shown to be involved in myocardial contraction in the heart, regulation of vasopressin expression in the nervous system, stability of blood vessels and lymph vessels, and regulatory mechanisms of body fluids. In the vascular system, it has been reported that it is expressed in vascular endothelial cells and parietal cells, and is considered to play an important role in angiogenesis of the cardiovascular system and peripheral blood vessels. In blood vessels, it has been reported that aperin (APJ endogenous ligand) is involved in blood pressure lowering, angiogenesis, arteriosclerosis, recovery of ischemia, etc. by binding to APJ receptors. In addition, the search for various APJ agonists and the treatment of various diseases such as cardiovascular insufficiency, angiopathy, and mood disorder using APJ agonists are also being studied (Patent Documents 1 to 7, Non-Patent Documents 1 to 5).

However, the relationship between skin elasticity and APJ expression is not known from the findings so far. Surprisingly, the present inventors have found that promoting the expression of APJ stabilizes blood vessels and synthesizes collagen around the blood vessels, that is, improves skin elasticity. Furthermore, it was also found that the expression of APJ changes according to the physical properties of the surrounding environment such as matrix elasticity, which is greatly involved in skin elasticity. In other words, the expression of APJ in blood vessels and the physical properties such as skin elasticity due to collagen synthesis are closely interacted with each other, and the expression of APJ is enhanced in vivo according to the physical properties of the surrounding matrix environment, and as a result, the vascular structure is stabilized. , Discovered a mechanism by which collagen synthesis is promoted in the surroundings and the skin maintains proper elasticity.

The present invention is based on the findings of such inventors, and relates to an APJ expression promoter for improving skin elasticity. In one embodiment, the APJ expression promoter of the present invention comprises or comprises one or more crude drugs selected from the group consisting of Houttuynia cordata extract, Sakura leaf extract, and neem leaf extract. However, the substance is not limited to these as long as it can promote the expression of APJ, and for example, APJ activators as described in Patent Documents 3, 4, 7, Non-Patent Document 4, etc. may be used. Good.

Houttuynia cordata, also known as Houttuynia, is a perennial plant belonging to the genus Houttuynia, which is widely distributed in various Asian regions such as Japan, China, and Vietnam. Plants such as leaves, stems, flowers and roots are used for medicinal purposes such as diuresis, hypertension and arteriosclerosis. Houttuynia cordata extract refers to an extract extracted from the plant body of Houttuynia cordata, especially the above-ground part.

Plum leaf refers to the leaves of plants belonging to the genus Plumus, such as Yoshino cherry tree (Prunus yedoensis Matsum.), Prunus lannesiana var. Speciosa., And Prunus serrulata. The genus Plum is a deciduous broad-leaved tree of the Rosaceae family found in various parts of Japan, East Asia, North America, etc. Sakura leaf is known to have a moisturizing effect, a whitening effect, an anti-inflammatory effect and the like. Plum leaf extract refers to an extract obtained by extracting leaves of the genus Plum.

Neem (Azadirachta indica) is a tree of the family Meliaceae native to India, also known as Neem tree. Neem is used in Ayurveda and the like for the purpose of bactericidal action, moisturizing action and the like in India, Sri Lanka and the like. Neem leaf extract refers to an extract obtained by extracting neem leaves.

The above-mentioned plant extract may be commercially available as a cosmetic raw material or a health food material, or may be obtained by a conventional method. The extraction method is not particularly limited, but an extraction method using a solvent is preferable. When extracting, the raw material plant can be obtained by dipping or heating at room temperature or heating with an extraction solvent, and then filtering and concentrating. Although the plant body can be used as it is, it is possible to extract the active ingredient in a short time with high extraction efficiency under mild conditions by crushing it into granules or powder and using it for extraction. The extraction temperature is not particularly limited, and may be appropriately set according to the particle size of the pulverized product, the type of solvent, and the like. Usually, it is set in the range from room temperature to the boiling point of the solvent. Further, the extraction time is not particularly limited, and may be appropriately set according to the particle size of the pulverized product, the type of solvent, the extraction temperature, and the like. Further, at the time of extraction, stirring may be performed, the mixture may be allowed to stand without stirring, or ultrasonic waves may be applied.

As the extraction solvent, any solvent can be used as long as it is usually used for extraction. For example, an aqueous solvent such as water, physiological saline, a phosphate buffer solution, a borate buffer solution, or an organic solvent such as ethanol can be used. Alcohols such as propylene glycol, 1,3-butylene glycol and glycerin, hydrous alcohols, chloroform, dichloroethane, carbon tetrachloride, acetone, ethyl acetate, hexane and the like can be used alone or in combination.

By such an extraction operation, the active ingredient is extracted and dissolved in the solvent. The solvent containing the extract may be used as it is, or may be used after being subjected to conventional purification treatments such as sterilization, washing, filtration, decolorization, and deodorization. Further, if necessary, it may be concentrated by freeze-drying or diluted with an arbitrary solvent before use. Further, the solvent may be completely volatilized to form a solid (dried product) before use, or the dried product may be redissolved in an arbitrary solvent before use.

Further, since the squeezed liquid obtained by squeezing the raw material plant also contains the same active ingredient as the extract, the squeezed liquid can be used instead of the extract.

The APJ expression promoter of the present invention may consist of one or more crude drugs selected from the group consisting of Houttuynia cordata extract, Sakura leaf extract, and neem leaf extract, or may be contained as an active ingredient. In addition, the skin elasticity improving agent of the present invention contains the APJ expression promoter of the present invention. For example, the skin elasticity improving agent of the present invention promotes APJ expression, and the promoted APJ acts on blood vessels to stabilize them, thereby promoting collagen synthesis around the blood vessels and improving skin elasticity. it can.

Improving skin elasticity means making the skin condition optimal, not too soft and not too hard. For example, if it is too soft, it may be a condition such as swelling or edema, and if it is too hard, it may be a condition such as a lump, a lump, or a tumor. Therefore, although not limited, the human cheek as described in the examples. Contact force centered on the average contact force of about 17.5 mN, for example, contact force in the range of about 10.0 mN to 22.0 mN, about 12.0 mN to 20.0 mN, or collagen corresponding to the contact force in such a range. Elastic modulus corresponding to the collagen gel concentration of about 0.5 to 1.00 mg / ml and about 0.6 to 0.9 mg / ml, which is centered on the concentration and has a high APJ expression level, for example, about 5.0 to 30.0 Pa, about 5.0 to 20.0. The storage elastic modulus in the range of Pa may be used. It is also known that the skin loses elasticity and becomes soft with aging. Therefore, improvement of skin elasticity means, for example, elasticity reduced due to aging, swelling, etc., for example, less than 5.0 mN, less than 6.0 mN, less than 7.0 mN, less than 8.0 mN, less than 9.0 mN, less than 10.0 mN, less than 11.0 mN, It may be increased from a contact force of less than 12.0 mN, less than 13.0 mN, less than 14.0 mN, less than 15.0 mN, less than 16.0 mN, less than 17.0 mN, or about 10.0 mN to 22.0 mN, about 12.0 mN to. The current elasticity within the range of the appropriate value such as 20.0 mN may be further increased within the range of the appropriate value. The increase may be, for example, a increase with a statistically significant difference in contact force or storage modulus with a significance level of 5% (eg, Student's t-test) and / or, for example, 10% or more. , 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100% or more. Alternatively, the contact force or storage elastic modulus of the skin may be maintained within the above-mentioned appropriate value range.

Promotion of APJ expression may mean, for example, that the amount of mRNA or protein of APJ in a biological sample increases when the APJ expression promoter of the present invention is added, as compared with the case where it is not added. The increase may be, for example, an increase with a statistically significant difference (eg, Student's t-test) with a significance level of 5%, and / or, for example, 10% or more, 20% or more, 30% or more, The increase may be 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100% or more.

The APJ expression promoter and skin elasticity improving agent of the present invention (hereinafter, these may be collectively referred to as "agent of the present invention") may contain any one of the above active ingredients alone. , Two or more types may be contained in any combination and ratio.

The agent of the present invention may also be a composition in which the above-mentioned active ingredient is combined with one or more other ingredients such as excipients, carriers and / or diluents. The composition and form of the composition are arbitrary, and may be appropriately selected according to conditions such as the active ingredient and use. The composition can be produced by a conventional method with a formulation appropriately combined with an excipient, a carrier and / or a diluent and the like and other components according to the dosage form.

The agent of the present invention is appropriately used orally or parenterally (transdermal administration, intravenous administration, intraperitoneal administration, etc.), and may be administered by any route, but it acts on blood vessels of the skin. Percutaneous administration is preferred.

The agent of the present invention may be blended in cosmetics, pharmaceuticals, quasi-drugs, etc. and used for humans and animals, and may be blended in various foods and drinks, for example, dietary supplements such as supplements, and ingested by humans and animals. It may be administered to humans and animals as a pharmaceutical preparation.

When the present invention is applied to external preparations for skin such as cosmetics, pharmaceuticals, quasi-drugs, etc., the blending amount (dry mass) of the active ingredient such as the above extract depends on the type, purpose, form, usage method and the like. , Can be decided as appropriate. For example, about 0.000001% to 50.0% (dry mass conversion) of Jyuyaku extract, Sakura leaf extract, and neem leaf extract can be mixed in the total amount of cosmetics, and in particular, about 0.0001% to 10.0% (dry mass conversion). It may be added as follows.

In addition to the above ingredients, if necessary, ingredients usually used for skin external preparations such as cosmetics, pharmaceuticals, quasi-drugs, etc., such as antioxidants, oils, UV protection, within the range that does not impair the effects of the present invention. Agents, surfactants, thickeners, alcohols, powder components, coloring materials, aqueous components, water, various skin nutrients and the like can be appropriately blended as needed.

Furthermore, metal ion blockers such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid, preservatives such as methylparaben, ethylparaben, and butylparaben, caffeine, tannin, Bellapamil, tranexamic acid and its derivatives, licorice extract, glabridin, hot water extract of carin fruit, various crude drugs, tocopherol acetate, glycyrrhizinic acid and its derivatives or salts thereof, vitamin C, magnesium ascorbate phosphate, Whitening agents such as ascorbic acid glucoside, arbutin, and kodiic acid, and sugars such as glucose, fructose, mannose, sucrose, and trehalose can also be appropriately added.

The external preparation for skin of the present invention is particularly preferably applicable as a cosmetic such as a cosmetic applied to the outer skin, a non-medicinal product, etc., and the dosage form is not limited as long as it can be applied to the skin. Any dosage form such as solution system, solubilization system, emulsification system, powder dispersion system, water-oil two-layer system, water-oil-powder three-layer system, ointment, lotion, gel, aerosol, etc. is applied.

When the agent of the present invention is used as cosmetics, for example, face cream, massage cream, body cream, milky lotion, lotion, beauty essence, gel, pack, cleansing cream, hand cream, lotion, foundation, lipstick, lip balm, hand It may be used in the form of powder, body shampoo, bath cosmetics and the like.

When the agent of the present invention is blended in foods and drinks, the blending amount (dry mass) of the plant or its extract can be appropriately determined according to their types, purposes, forms, usage methods and the like. For example, it can be blended so that the daily intake of the plant or its extract for an adult is about 0.00001 mg to 10.0 g (dry residue) and about 0.001 mg to 5.0 g (dry residue).

The form of food and drink and feed can be any form, for example, granules, granules, pastes, gels, solids, or liquids. These forms include various known substances known to be contained in foods and drinks, such as binders, disintegrants, thickeners, dispersants, reabsorption promoters, emulsifiers, buffers, and surfactants. Excipients such as activators, solubilizers, preservatives, emulsifiers, tonicity agents, stabilizers and pH adjusters can be appropriately contained.

The dosage form is also arbitrary, such as oral solid preparations such as tablets, granules, powders and capsules, oral liquid preparations such as oral liquids and syrups, and parenteral liquid preparations such as injections. The form of the above can be appropriately prepared by a known method. If it is an external preparation, it can be used in various forms such as lotions, suspensions / emulsions, liquids, ointments, and patches. These formulations include commonly used binders, disintegrants, thickeners, dispersants, reabsorption promoters, flavoring agents, buffers, surfactants, solubilizers, preservatives, emulsifiers, isotonic agents. , Excipients such as stabilizers and pH regulators may be used as appropriate.

However, the possible forms of the agent of the present invention are not limited to those described above.

The present invention also relates to a method for screening a skin elasticity improving agent using APJ expression in a biological sample as an index. The screening method of the present invention is, for example, the following steps: culturing a biological sample in a medium containing a candidate drug; measuring APJ expression in the biological sample; and when APJ expression is increased as compared with a control. To determine the candidate drug as a substance having a skin elasticity improving effect; Here, the control is APJ expression when different in that it is cultured in a medium containing no candidate drug. Experiments on controls may be performed in parallel with the screening method of the present invention or may be performed in advance.

The biological sample used in the screening method of the present invention is arbitrary, such as vascular endothelial cells and adipocytes, and is not limited as long as APJ expression can be measured. For example, as described in Examples, vascular endothelial cells in a vascular model created by inducing angiogenesis from the subcutaneous adipos tissue of an animal such as a human, vascular constituent cells such as pericytes may be used, and vascular endothelial cells obtained from a living body. , Pericytes, or passaged cells thereof, established cells, HUVEC, HAEC, HMVEC and the like may be used. Alternatively, APJ-expressing cells may be used as described in Patent Documents 3 and 4.

Further, a pre-culture step of culturing the biological sample as described above may be included before the step of culturing the biological sample in the medium containing the candidate drug. In the pre-culture step, angiogenesis may be induced from the subcutaneous adipose tissue to create a vascular model. Further, after the step of culturing the biological sample in the medium containing the candidate drug, a post-culturing step of further culturing in the medium containing no candidate drug may be included. In the step of culturing the biological sample in the medium containing the candidate drug, the candidate drug or a diluted solution thereof may be directly added to the culture obtained in the pre-culture step and cultured, or the medium may be replaced with a medium containing the candidate drug. The culture may be carried out.

Measurement of APJ expression can be determined by measuring the amount of APJ mRNA or protein in a biological sample. The amount of mRNA can be measured by using a method known in the present art such as quantitative PCR and Northern blotting. For example, as described in Examples, a probe for APJ mRNA may be used. The amount of protein can be determined using any technique known in the art such as Western blotting, immunostaining, FACS and the like. For example, an antibody that specifically binds to APJ may be used. Alternatively, for example, a screening system using APJ-expressing cells as described in Patent Documents 3 and 4 may be established and used. The present invention also provides a kit for carrying out the screening method of the present invention, which comprises the above-mentioned reagents for determining APJ expression.

The substance having an APJ expression-promoting agent action screened by the screening method of the present invention may be any substance as long as it can promote the expression of APJ. One aspect of the screening method of the present invention is to perform screening using an arbitrary library of cosmetic materials, food materials, pharmaceutical materials, etc., using APJ expression in a biological sample such as vascular endothelial cells as an index. Examples of the substance having the APJ expression promoting action thus determined include one or more crude drugs selected from the group consisting of Houttuynia cordata extract, Sakura leaf extract, and neem leaf extract.

As explained above, it was found that promotion of APJ expression improves skin elasticity. Therefore, the present invention also relates to a method for improving skin elasticity, including application of an APJ expression promoter. If the skin elasticity is improved, it is expected that wrinkles and sagging will be improved. The methods of improving skin elasticity herein relate to cosmetological methods for the purpose of cosmetology and can be distinguished from treatments performed by doctors and medical personnel. Such a beauty method may be performed individually, or may be performed at a beauty salon, a cosmetics store, an esthetic salon, or the like.

All documents referred to herein are incorporated herein by reference in their entirety.

The examples of the present invention described below are for illustration 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 claims. Modifications of the present invention, for example, addition, deletion and replacement of the constituent elements of the present invention may be made provided that the gist of the present invention is not deviated.

Experiment 1: Creation of 3D angiogenesis dermis model and visualization of vascular structure and produced collagen A type I collagen gel solution was prepared using a collagen gel culture kit (Nitta gelatin). Specifically, in a 15 ml tube on ice, 2 ml of a 3 mg / ml cell matrix collagen solution was suspended in 2 ml of 1 mM dilute hydrochloric acid, and 500 μl of MEM concentrated 10-fold there and a collagen gel culture kit for neutralization. The attached C buffer was added with stirring at 500 μl. 100 μl of this solution was placed on a 35 mm glass-based dish (IWAKI) and incubated at 37 ° C for 30 minutes to gel.

Human normal subcutaneous adipose tissue was shredded into 1 mm squares in PBS containing 10% FBS, and 4 pieces were placed on a gelled collagen gel. Then, 100 μl of the remaining type I collagen gel solution prepared on ice was added so as to cover the tissue pieces, and the mixture was gelled by incubating at 37 ° C. for 30 minutes. This tissue piece was cultured for 7 to 21 days using a culture solution containing EBM-2 (Cambrex; Verviers, Belgium) to which an additive factor such as a growth factor was added, and VEGFA having a final concentration of 50 ng / ml. It was. In addition, the medium was changed every 2 to 3 days. For visualization of type I collagen and analysis of APJ expression localization, this model was prepared using a collagen gel with a concentration of 0.9 mg / ml.

After culturing, 1 ml of 4% paraformaldehyde solution was added, fixed at 4 degrees for 30 minutes, and immunostained. Primary antibodies include sheep anti-PECAM-1 / CD31 antibody (AF806, R & D systems, MN) against vascular endothelial cells, Cy3αSMA antibody (c-6198, Sigma, MO) against pericite, and APJ. Rabbit anti-APJ antibody, rabbit anti-type I Collagen antibody (ab34710, Abcam, UK) is used for type I collagen, and Alexa Fluor488 donkey anti-sheep antibody (A11015, Molecular Probes, Eugene, OR) is used as the secondary antibody. And Alexa Fluor594 Donkey anti-rabbit antibody (R37119, Molecular Probes, Eugene, OR) was used. Hoechst 33342 (H3570, Invitrogen, CA) was used for nuclear staining. After staining, the 3D vascular structure was detected and imaged using a confocal microscope LSM880 (Carl Zeiss, Germany).

Figure 1 shows the creation of a 3D angioplasty dermis model stained with anti-PECAM-1 / CD31 antibody. As can be seen in FIG. 1, it was confirmed that vascular endothelial cells formed a lumen and a 3D vascular structure was formed. FIG. 2 shows an angioplasty model stained with anti-PECAM-1 / CD31 antibody and anti-type I Collagen antibody. As can be seen in Fig. 2, it was found that collagen is synthesized around the blood vessels. In particular, as shown by the arrow in the right figure, collagen was found around the blood vessel, and in particular, a large amount of collagen was formed in the part considered to be the scaffold where the blood vessel will be elongated. Therefore, it is suggested that the presence of vascular structure contributes to collagen synthesis and, by extension, skin elasticity.

Experiment 2: Decrease in blood vessels and decrease in surrounding hardness during aging The above-mentioned experiment showed that the presence of vascular structure contributes to skin elasticity, but it is known that blood vessels decrease in aged skin. (Non-Patent Document 6). Therefore, for the purpose of suppressing the decrease of blood vessels due to aging, we analyzed how the blood vessel structure is maintained, focusing on the interaction with the surrounding hardness and physical properties.
Using eyelid skin obtained from human subjects of young people (10 to 20 years old) and middle-aged people (50 to 70 years old), the hardness of the area around the capillaries existing in the epidermis 200 um range was measured with an ultrasonic sonic microscope ( Observation was performed with a medical ultrasonic microscope, AMS-50SI, Honda Electronics Co., Ltd.). Specifically, a frozen section of the skin was prepared, and the sound velocity of the skin cross section was measured by a conventional method with an ultrasonic sound velocity microscope to obtain a sound velocity distribution image (Patent Document 8). .. Further, the same section after measurement was subjected to immunostaining to visualize the vascular structure. As the primary antibody, vascular endothelial cells were stained with sheep anti-Pecam-1 antibody (AF806, R & D systems, MN), and pericytes were stained with rabbit anti-NG2 antibody (Milipore). Alexa Fluor 594 Donkey anti-sheep antibody (A-11016, Molecular Probes, Eugene, OR) and Alexa Fluor 488 Donkey anti-rabbit antibody (A-21206, Molecular Probes, Eugene, OR) were used as secondary antibodies. After that, the sound velocity distribution image and the immunostaining image were superposed, and the sound velocity value in the range of 50 μm around the blood vessel was quantified. For quantitative analysis, 10 samples were used, and the circumference of 15 blood vessels per tissue was quantified.
The results are shown in Figure 3. From FIG. 3, it can be seen that in the aged skin, the sound velocity value in the area around the blood vessels decreases significantly as the blood vessels decrease. In other words, it was suggested that the decrease in skin elasticity and the decrease in blood vessels in aging are related.

Experiment 3: Collagen gel hardness measurement A type I collagen gel solution having each concentration (0.24, 0.6, 0.9, 1.2, 1.8 mg / ml) was prepared on ice by the same method as in Experiment 1. Then, it was gelled in a 24-well plate at 37 degrees for 30 minutes to prepare a collagen gel. The stored elastic modulus (Pa) of the prepared collagen gel was measured using a Physica MCR 300 Modular Compact Rheometer (Anton Paar, Germany) to determine the hardness of the gel.
The results are shown in Fig. 4. From FIG. 4, it can be seen that the collagen gel concentration and the storage elastic modulus have a corresponding relationship.

Experiment 4: Relationship between collagen gel hardness and skin physical properties In order to investigate the relationship between collagen gel collagen concentration and actual skin physical properties, I have each concentration (0.24, 0.6, 1.2, 1.8 mg / ml). The contact force of the type collagen gel was measured using a skin softness sensor (Non-Patent Document 7) in the same manner as in Non-Patent Document 7.
The results are shown in Fig. 5. As shown in FIG. 5, it can be seen that the collagen concentration and the contact force are almost proportional to each other up to about 2.0 mg / ml. In addition, since it was confirmed using this device that the average contact force of the buccal skin of healthy humans (20-50s, N = 168) was about 17.5 mN, the collagen concentration corresponding to 17.5 mN. The following experiment was carried out by varying the concentration centering on.

Experiment 5: Analysis of changes in vascular structure in response to collagen gel hardness By changing the collagen gel concentration in the same manner as in Experiment 1, type I collagen gel (0.24, 0.9, 1.8 mg / ml) at each concentration Was produced. The gels at each of these concentrations had storage elastic moduli of 2.6 ± 0.8, 14.0 ± 5.4, and 48.1 ± 17.0 (Pa), respectively. Using gels of each of these concentrations, 3D angiogenesis was induced from normal human subcutaneous adipose tissue by the same method as in Experiment 1. After culturing for 13 days, immunostaining was performed in the same manner as in Experiment 1 to visualize vascular endothelial cells and pericytes.

The results are shown in Fig. 6. From Fig. 6, in the case of a low-concentration gel (0.24 mg / ml) with an elastic modulus of 2.6 ± 0.8 Pa, thin and unstable blood vessels were formed, but at a higher concentration (0.9 mg / m), a high elastic modulus (14.0) was formed. In the case of the gel having ± 5.4 Pa), thick and stable blood vessels were formed. However, in the case of a gel having a higher elastic modulus (48.1 ± 17.0 Pa) at a higher concentration (1.8 mg / ml), on the contrary, many thin and unstable blood vessels were observed. From the results shown in FIG. 6, it can be seen that the structure of blood vessels formed in response to the physical properties of the surrounding environment, such as the elasticity of the matrix, changes. Furthermore, it is suggested that there are optimal physical properties of the surrounding environment for stabilizing blood vessels. Considering together with the results in Fig. 3, blood vessels sense the hardness of the surroundings and maintain their structure in a stable manner, but in aged skin, the hardness of the surroundings decreases, so the blood vessels become unstable. It is thought that the blood vessels tend to decrease.

Experiment 6: Analysis of vascular cell expression factor (APJ) in response to collagen gel hardness Type I having each concentration (0, 0.12, 0.6, 0.9, 1.2, 1.8 mg / ml) in the same manner as in Experiment 1. A collagen gel solution was prepared on ice. Human umbilical cord blood venous endothelial cells (HUVEC) seeded at 10 ⁵ cells / well on a 12-well plate were cultured to confluence, 300 μl / well of each type I collagen gel solution was added, and gelation was performed by incubating at 37 ° C. The cells were further cultured. After 6 hours of culturing, Trizol was added to each well to disrupt the cells. Chloroform extraction was performed from the obtained solution to obtain total RNA. The concentration of the obtained total RNA was measured by nano drop and adjusted to 35 ng / ml with RNase free water. ^{Thereafter, TaqMan TM RNA-to-C} T TM 1-Step Kit (Applied Biosystems, CA) Taqman probe for human APJ mRNA (Hs00270873, Applied Biosystems, CA) was used to compare the mRNA expression level of APJ.

The results are shown in Fig. 7. The figure on the left is a graph showing the relationship between collagen gel concentration and APJ mRNA expression level. It was clarified that the APJ mRNA expression level reached its peak under the condition of 0.9 mg / ml collagen gel, and it was observed that the expression decreased in the low concentration or high concentration collagen gel. In other words, it can be seen that the expression of APJ changes in response to the physical properties of the surrounding environment such as the elasticity of the matrix. Such a relationship between physical properties and expression was not found in other vascular stabilizing factors such as Tie2 (data not shown), and was APJ-specific. In addition, the elasticity of the collagen gel derived in this experiment was almost the same as the gel concentration corresponding to normal human skin elasticity. Therefore, it is considered that the expression of APJ changes according to the elasticity of the matrix and contributes to the stability of blood vessels, and the elasticity is inappropriate if it is too low or too high, and there is an appropriate range. Is suggested. The right figure of FIG. 7 is a photomicrograph showing the localization of APJ in a vascular model formed in a collagen gel having a concentration of 0.9 mg / ml. It was found that APJ was localized in cell process sites such as filopodia extending from vascular endothelial cells toward the periphery, suggesting that APJ may directly sense the surrounding matrix environment and stabilize blood vessels. It was.

Experiment 7: Preparation of APJ high expression 3D angioplasty dermis model and structural analysis A human APJ gene sequence was inserted into a pEGFP-N1 vector (Clontech, CA) to obtain an APJ high expression vector for lentivirus introduction. Then, APJ highly expressed lentivirus was prepared based on this vector (Oriental yeast). A 3D angioplasty dermis model was created by the same method as in Experiment 1. Then, on the 21st day after the start of culturing, this model was transfected with APJ expression vector by lentivirus infection and cultured for 5 days. As a control, a blood vessel model prepared under the same conditions except that transfection was not performed was cultured. After culturing, a 4% paraformaldehyde solution was added, and the cells were fixed at 4 degrees for 30 minutes and immunostained. Sheep anti-Pecam-1 antibody (AF806, R & D systems, MN) was used as the primary antibody against vascular cells, and Alexa Fluor 594 Donkey anti-sheep antibody (A-11016, Molecular Probes, Eugene,) was used as the secondary antibody. OR) was used. After staining, the 3D vascular structure was detected and imaged using a confocal microscope LSM880 (Carl Zeiss, Germany).

The results are shown in Fig. 8. From FIG. 8, it was shown that when the expression of APJ was enhanced, the blood vessels became thicker and more stable, which was consistent with the previous report (Non-Patent Document 5).

From the above results, for example, as shown in FIG. 9 as a schematic diagram, skin elasticity and APJ expression interact with each other, and APJ acts as a sensor for skin elasticity to stabilize blood vessels and blood vessels. It is possible that there is a cycle in which collagen synthesis is promoted in the surroundings and fed back to skin elasticity. Therefore, it is considered that even if the skin has decreased elasticity due to aging or the like, it is possible to strengthen the cycle and improve the skin elasticity by enhancing APJ expression. Therefore, in the following experiments, substances having an APJ expression promoting action were screened.

Experiment 8: Screening method for substances having a skin elasticity improving effect that promotes APJ expression Screening was performed using a total of 49 kinds of raw materials including a plant extract that is said to have an anti-aging effect as a candidate drug. Uses Juyaku extract (Juyaku extract ET-50) manufactured by Koei Kogyo Co., Ltd., Sakura Leaf extract (Falcolex Sakura Leaf B) manufactured by Ichimaru Falcos, and Neem Leaf Extract (Neem Leaf Liquid B) manufactured by Ichimaru Falcos did. These extracts were dissolved in dimethyl sulfoxide (DMSO) to prepare samples at a final concentration of 0.1% (w / w). As a control, a DMSO solution free of these extracts was used. HUVEC seeded at 10 ⁵ cells / well on a 12-well plate was cultured to confluence, the above sample was added to a final concentration of 0.1%, incubated at 37 ° C, and cultured for 4 hours, as in Experiment 6. The mRNA expression level of APJ was compared by the method of.

The results are shown in Fig. 10. From FIG. 10, when the Houttuynia cordata extract, Sakura leaf extract, and neem leaf extract were used as candidate drugs, the expression of APJ was significantly increased as compared with the control. These extracts were selected as substances having an APJ expression promoting action.

Claims

APJ expression promoter to improve skin elasticity.
APJ expression promoter consisting of one or more crude drugs selected from the group consisting of Houttuynia cordata extract, Sakura leaf extract, and neem leaf extract.
A skin elasticity improving agent containing the APJ expression promoter according to claim 1 or 2.
A screening method for skin elasticity improving agents using APJ expression as an index.
Culturing a biological sample in a medium containing a candidate drug,
To measure APJ expression in a biological sample, and to determine a candidate drug as a substance having a skin elasticity improving effect when APJ expression is increased compared to a control.
4. The screening method according to claim 4.
A kit for carrying out the screening method according to any one of claims 4 or 5, which comprises a reagent for determining APJ expression.
A cosmetological method for improving the subject's skin elasticity, including promoting the subject's APJ expression.
The method according to claim 7, wherein the promotion of APJ expression of the subject is achieved by administering the skin elasticity improving agent according to claim 3 to the subject.