TW202042786A - APJ performance enhancer wherein the APJ performance enhancer is effective in improving skin elasticity through stabilization of blood vessels - Google Patents

Abstract

The present invention provides a substance for improving the skin elasticity and a method for screening substances with the skin elasticity improving effect. The present invention provides an APJ performance enhancing agent and an agent effective for improving the skin elasticity by enhancing the performance of APJ and stabilizing blood vessels by using the agent. The APJ performance enhancing agent, which contains one or more crude drugs selected from the group consisting of houttuynia cordata extract, cherry leaf extract, and neem leaf extract, is effective in improving the skin elasticity through stabilization of blood vessels. By the screening method of the present invention, a substance with a skin elasticity improvement effect can be selected.

Description

APJ performance enhancer

The present invention relates to an APJ performance enhancer.

Skin elasticity has a great influence on the appearance and age. Maintaining and improving skin elasticity is a major issue in beauty. There are many studies on the skin elasticity of the matrix of the dermis such as collagen, elastin, and hyaluronic acid. For example, it is widely known that the amount of collagen in the dermis has a greater influence. In the previous cosmetic methods, in order to improve the elasticity of the skin, it is focused on increasing the elastic fibers by the activation of dermal fibroblasts, and it has been discovered that the ingredients have the activation effect of dermal fibroblasts or the promotion of collagen production. , And has been applied to cosmetics.

So far, as ingredients from natural products that promote the production of collagen to prevent and improve skin aging changes, isoflavone compounds, phytosterols, etc. have been reported, and Binhai Peucedanum has been found (International Publication No. 2013/099378), Platycladus orientalis seeds (International Publication No. 2012/057123), Ryukyu Yazhu, Xueli, and Saponin (Japanese Patent Laid-Open No. 2011-195505) and other plant extracts. It is also known that fibroblasts interact with the extracellular matrix via adhesion molecules such as integrins expressed in fibroblasts, and this interaction is related to firmness (Non-Patent Document 1). The insights so far have focused on the relationship between skin elasticity and collagen or fibroblast adhesion molecules. [Prior Technical Literature] [Patent Literature]

[Patent Document 1] Japanese Patent No. 5808769 [Patent Document 2] Japanese Patent Laid-Open No. 2012-20942 [Patent Document 3] Japanese Patent Laid-Open No. 2013-209339 [Patent Document 4] International Publication No. 2012/133825 [Patent Document 5] Japanese Patent Laid-Open No. 2018-172410 [Patent Document 6] Japanese Patent Publication No. 2019-501899 [Patent Document 7] International Publication No. 2007/072980 [Patent Document 8] Japanese Patent No. 5648149 [Non-Patent Literature]

[Non-Patent Document 1] J. Inv. Dermatology (2013) vol. 133, 899-906 [Non-Patent Document 2] BLOOD, 2010, VOLUME 115, NUMBER 15, 3166-3174 [Non-Patent Document 3] DIABETES, VOL. 62, JUNE 2013, 1970-1980 [Non-Patent Document 4] J Cell Physiol. 2019;234:61-74 [Non-Patent Document 5]Kidoya et al., EMBO J. 2008 Feb 6;27(3):522-34 [Non-Patent Document 6] Kajiya et al., J Dermatol Sci. 2018 Oct;92(1):3-5 [Non-Patent Document 7] Nakatani et al., Skin Res Technol. 2013 Feb;19(1):e332-8

[The problem to be solved by the invention]

The subject of the present invention is to provide a substance for improving skin elasticity and a method for screening substances with skin elasticity improving effect by a mechanism different from the previous skin elasticity improving agents and methods. [Technical means to solve the problem]

The inventors of the present invention made intensive research to improve the elasticity of the skin. As a result, they found that by enhancing the performance of APJ, the blood vessels were stabilized and the function was improved, and/or the skin elasticity was improved by inhibiting the reduction of skin blood vessels caused by aging, and further, A novel substance that promotes the performance of APJ was also discovered, thus completing the present invention.

The present invention relates to the following inventions: (1) An APJ performance enhancer for improving skin elasticity. (2) An APJ performance enhancer comprising one or more crude drugs selected from the group consisting of houttuynia cordata extract, cherry leaf extract, and neem leaf extract. (3) A skin elasticity improving agent comprising the APJ performance promoting agent as described in (1) or (2). (4) A screening method for skin elasticity improving agents based on APJ performance. (5) As the screening method described in (4), it includes: Use a medium containing candidate agents to cultivate biological samples; Determination of APJ performance in biological samples; and When comparing APJ performance with the control group, the candidate agent is determined to be a substance with a skin elasticity improvement effect. (6) A kit for implementing the screening method described in any one of (4) or (5), which includes reagents for determining APJ performance. (7) A method for improving the skin elasticity of a subject for the purpose of beauty, which includes promoting the subject's APJ performance. (8) In the method described in (7), the promotion of the subject's APJ performance is achieved by administering the skin elasticity improving agent described in (3) to the subject. [Effects of Invention]

The APJ performance promoter of the present invention can promote the performance of APJ. If the performance of APJ is promoted, blood vessels can be stabilized and skin elasticity can be improved. APJ performance enhancer, which contains one or more crude drugs selected from the group consisting of houttuynia cordata extract, cherry leaf extract, and neem leaf extract, is effective in improving skin elasticity by stabilizing blood vessels. Furthermore, by the screening method of the present invention, a substance with a skin elasticity improvement effect can be selected. The skin elasticity improving agent selected by the screening method of the present invention improves the skin elasticity by promoting the performance of APJ and stabilizing the blood vessels. By improving skin elasticity, skin problems such as wrinkles and sagging can be improved.

APJ (alias AGTRL1: Angiotensin receptor like 1) is a 7-pass transmembrane G protein-coupled receptor that has been reported to be widely expressed in the vascular system, nervous system, and adipocytes. It has been confirmed that the performance of APJ is related to myocardial contraction in the heart, and is related to the control of vasopressin performance in the nervous system, the stability of blood vessels and lymphatic vessels, and the regulation mechanism of body fluids. It has been reported in the vascular system. The performance of vascular endothelial cells and parietal cells is considered to play an important role in the formation of blood vessels in the cardiovascular system and peripheral blood vessels. It has been reported in blood vessels that APJ endogenous ligand binds to APJ receptors and participates in the reduction of blood pressure, angiogenesis, and recovery of arteriosclerosis and ischemia. In addition, the exploration of various APJ agonists and the treatment of various diseases such as cardiovascular insufficiency, vascular diseases, and emotional diseases using APJ agonists have also been studied (Patent Documents 1-7, Non-Patent Documents 1-5) .

However, the insights so far have not yet known the relationship between skin elasticity and APJ performance. Surprisingly, the inventors of the present invention found that by promoting the performance of APJ, blood vessels are stabilized, collagen is synthesized around blood vessels, in other words, skin elasticity is improved. Furthermore, it was also found that the performance of APJ changes according to the physical properties of the surrounding environment, such as matrix elasticity, which has a greater correlation with skin elasticity. In other words, a mechanism has been discovered that APJ performance in blood vessels interacts closely with physical properties such as skin elasticity obtained by collagen synthesis, etc. APJ in vivo will enhance performance according to the physical properties of the surrounding matrix environment. As a result, Stabilize blood vessel structure, promote collagen synthesis around, skin will maintain proper elasticity.

The present invention is based on the insights of the inventors and others, and relates to an APJ performance promoter for improving skin elasticity. In one embodiment, the APJ performance enhancer of the present invention includes or consists of one or more crude drugs selected from the group consisting of houttuynia cordata extract, cherry leaf extract, and neem leaf extract. However, as long as it is a substance that can promote the expression of APJ, it is not limited to them. For example, APJ activators as described in Patent Documents 3, 4, 7, and Non-Patent Document 4 may also be used.

Houttuynia cordata (Houttuynia cordata), also known as water spinach, is a perennial herb of the water spinach family, which is widely distributed throughout Japan, China, Vietnam and other Asian regions. Plants such as leaves, stems, flowers, and roots can be used for medicinal purposes such as diuresis, hypertension, and arteriosclerosis. Houttuynia cordata extract refers to the plant body of Houttuynia cordata, especially the extract obtained by extracting the aerial parts.

Sakura leaves belong to plants of the genus Prunus, and refer to the leaves of plants such as Yoshino cherry (Prunus yedoensis Matsum.), Oshima cherry (Prunus lannesiana var. speciosa.), and Sakura (Prunus serrulata). Sakura is a deciduous broad-leaved tree of Rosaceae found in various parts of Japan, East Asia, North America and other places. It is known that cherry leaves have moisturizing effects, whitening effects, and anti-inflammatory effects. Cherry leaf extract refers to the extract obtained by extracting the leaves of the genus Cherry.

The so-called Azadirachta indica (Azadirachta indica) is a tree of the Neem family native to India, also known as Indian sandalwood. Neem is used in Ayurvedic therapy for the purpose of bactericidal and moisturizing effects in India and Sri Lanka. Neem leaf extract refers to the extract obtained by extracting the leaves of Neem.

The above-mentioned plant extracts can be used commercially as cosmetic raw materials or health food materials, or can be obtained by usual methods. The extraction method is not particularly limited, but an extraction method using a solvent is preferred. When extracting, it can be obtained by filtering and concentrating the raw plant together with the extraction solvent at room temperature or heating and immersing or refluxing. Although plants can also be used directly, they can be crushed into granular or powdered form and then used for extraction. The effective ingredients can be extracted in a short time with high extraction efficiency under stable conditions. The extraction temperature is not particularly limited, as long as it is appropriately set according to the particle size of the pulverized product and the type of solvent. It is usually set in the range from room temperature to the boiling point of the solvent. In addition, the extraction time is not particularly limited, as long as it is appropriately set according to the particle size of the pulverized product, the type of solvent, and the extraction temperature. Furthermore, during extraction, it can be stirred, can be left standing without stirring, or ultrasonic waves can be applied.

As the extraction solvent, any solvents generally used for extraction can be used. For example, aqueous solvents such as water, physiological saline, phosphate buffer, boric acid buffer, or organic solvents such as ethanol, propylene glycol, 1,3-Butanediol, glycerin and other alcohols, water-containing alcohols, chloroform, dichloroethane, carbon tetrachloride, acetone, ethyl acetate, hexane, etc.

Through this extraction operation, the effective ingredients are extracted and dissolved in the solvent. The solvent containing the extract can be used directly, or after the usual purification treatments such as sterilization, washing, filtration, decolorization, and deodorization are applied. In addition, it may be used after concentration by freeze-drying or the like or dilution with any solvent as needed. Furthermore, it can be used after volatilizing all the solvent and making it into a solid state (dried substance), and you may use it after redissolving this dried substance in arbitrary solvents.

In addition, the squeezed liquid obtained by squeezing the raw plant also contains the same effective ingredients as the extract, so the squeezed liquid can also be used instead of the extract.

The APJ performance enhancer of the present invention may include one or more crude drugs selected from the group consisting of houttuynia cordata extract, cherry leaf extract, and neem leaf extract, or contain them as active ingredients. In addition, the skin elasticity improving agent of the present invention contains the APJ performance promoting agent of the present invention. For example, the skin elasticity improving agent of the present invention promotes APJ performance, promotes the performance of APJ to act on blood vessels and stabilizes them, and can promote collagen synthesis around blood vessels to improve skin elasticity.

The so-called improvement of skin elasticity refers to the best state that makes the skin not too soft and not too hard. For example, when it is too soft, it may be like edema or swelling, and when it is too hard, it may become lumps, warts, and tumors. Therefore, although it is not limited, it is as described in the examples. The average contact force of the human cheek is the contact force centered at about 17.5 mN, such as the contact force in the range of about 10.0 mN～22.0 mN, about 12.0 mN～20.0 mN, or the collagen that can correspond to the contact force in this range The protein concentration is the center, and corresponds to the elastic modulus of the collagen gel concentration of about 0.5～1.00 mg/ml, about 0.6～0.9 mg/ml, which is higher in APJ, such as about 5.0～30.0 Pa, about 5.0 Storage elastic modulus in the range of ～20.0 Pa. In addition, it is known that skin loses its elasticity and becomes soft as it ages. Therefore, the so-called improvement of skin elasticity can refer to the elasticity lower than that of aging or edema, 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, 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, etc. The contact force rises, or it can be instructed to be about 10.0 The current elastic force within the appropriate value range of mN～22.0 mN, approximately 12.0 mN～20.0 mN, etc. will further increase within the above appropriate value range. The increase can be, for example, an increase in contact force or storage elastic modulus that has a statistically significant difference (such as student's t-test) with a significant level of 5%, and/or can also be, for example, 10% or more, 20% or more, An increase of over 30%, over 40%, over 50%, over 60%, over 70%, over 80%, over 90%, or over 100%. Or, it can also maintain the contact force of the skin or the storage elastic modulus within the above-mentioned appropriate value range.

The promotion of APJ performance can refer to, for example, the increase in the amount of mRNA or protein of APJ in the biological sample when the APJ performance enhancer of the present invention is added compared with the case without addition. The increase can be, for example, an increase with a statistically significant difference (such as student's t-test) with a significant level of 5%, and/or can also be, for example, 10% or more, 20% or more, 30% or more, or 40% or more , 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100% or more.

The APJ performance enhancing agent and the skin elasticity improving agent of the present invention (hereinafter sometimes collectively referred to as the "agent of the present invention") may contain any one of the above-mentioned active ingredients alone, or two or more of them may be contained in any combination and ratio.

The agent of the present invention can also be made into a composition composed of the above-mentioned effective ingredients and one or more other ingredients, such as excipients, carriers and/or diluents. The composition or form of the composition is arbitrary, as long as it is appropriately selected according to the conditions such as the active ingredient and the use. The composition can be manufactured according to a formula obtained by appropriately combining its dosage form, excipients, carriers and/or diluents, etc., and other ingredients, using a usual method.

The agent of the present invention is appropriately administered orally or parenterally (percutaneously, intravenously, intraperitoneally, etc.), and is administered by any route, preferably percutaneously in order to act on the skin. Blood vessels.

The agent of the present invention can be formulated in cosmetics, pharmaceuticals, quasi-drugs, etc. for use on humans and animals, and can also be formulated in various dietary products, such as supplements and other nutritional supplementary foods for human and animal ingestion, or administered as a pharmaceutical preparation People and animals.

When applying the present invention to external skin preparations such as cosmetics, pharmaceuticals, and quasi-drugs, the blending amount (dry mass) of the above-mentioned extracts and other active ingredients can be appropriately determined according to the type, purpose, form, use method, etc. . For example, about 0.000001% to 50.0% (in terms of dry mass) of houttuynia cordata extract, cherry leaf extract, and neem leaf extract can be blended in the total amount of cosmetics, especially about 0.0001% to 10.0% (calculated in dry mass) to add.

In addition to the above-mentioned ingredients, if necessary, within a range that does not impair the effects of the present invention, the ingredients used in general cosmetics, pharmaceuticals, quasi-drugs and other external skin agents, such as antioxidants, oils, and UV protection agents , Surfactants, tackifiers, alcohols, powder ingredients, colorants, water-based ingredients, water, various skin nutrients, etc.

Furthermore, disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, gluconic acid and other metal ion blocking agents, methylparaben, Preservatives such as ethyl p-hydroxybenzoate, butyl p-hydroxybenzoate, caffeine, tannin, verapamil, tranexamic acid and its derivatives, licorice extract, glycyrrhizin, hot water of papaya fruit Extracts, various crude drugs, vitamin E acetate, glycyrrhizic acid and its derivatives or salts and other agents, vitamin C, magnesium ascorbyl phosphate, ascorbyl glucoside, arbutin, kojic acid and other whitening agents, glucose, fructose, mannose , Sucrose, trehalose and other sugars.

The external preparation for skin of the present invention can be used as cosmetics, quasi-drugs, etc. applied to the skin, and is particularly preferably used as cosmetics. The dosage form is not limited as long as it can be applied to the skin. It can be applied to solution systems, solubilizers, and emulsifiers. Any dosage forms such as powder dispersion system, water-oil two-phase system, water-oil-powder three-phase system, ointment, lotion, gel, aerosol, etc.

When the agent of the present invention is used as cosmetics, for example, facial cream, massage cream, body lotion, lotion, lotion, beauty lotion, gel, facial mask, cleanser, hand cream, lotion, pressed powder, lipstick, lipstick , Hand washing powder, shower gel, bath cosmetics and other forms.

When the agent of the present invention is blended into foods and beverages, the blending amount (dry mass) of the plant body or its extract can be appropriately determined according to the type, purpose, form, use method, etc. of the plant body. For example, it can be formulated so that the daily intake of plants or their extracts for adults becomes about 0.00001 mg to 10.0 g (dry residue) and about 0.001 mg to 5.0 g (dry residue).

As the form of food and drink or feed, it can be made into any form, for example, it can be made into a granular, granular, paste, gel, solid, or liquid form. In these forms, various known substances that are allowed to be contained in foods and beverages, such as binding agents, disintegrating agents, thickening agents, dispersing agents, reabsorption promoters, flavoring agents, buffering agents, and surfactants, can be appropriately contained. , Solubilizers, preservatives, emulsifiers, isotonic agents, stabilizers or pH modifiers and other excipients.

The dosage form is also arbitrary, such as solid preparations for oral use such as tablets, granules, powders, capsules, or liquid preparations for oral use such as oral liquids and syrups, or liquid preparations for parenteral use such as injections, etc., which can be A known method is appropriately prepared in any form. If it is an external preparation, it can be used in various forms such as lotion, suspension, emulsion, liquid, ointment, and patch. In these preparations, commonly used binders, disintegrants, thickeners, dispersants, reabsorption enhancers, flavors, buffers, surfactants, solubilizers, preservatives, emulsifiers, etc. can be suitably used. Excipients such as tonicity, stabilizer or pH regulator.

However, the form that the agent of the present invention can take is not limited to the above.

In addition, the present invention relates to a method for screening skin elasticity improving agents based on APJ performance in biological samples. The screening method of the present invention includes, for example, the following steps: culturing a biological sample with a medium containing a candidate drug; measuring APJ performance in the biological sample; and when the APJ performance increases compared with a control group, determining that the candidate drug has skin A substance that improves elasticity. Here, the control group refers to APJ performance when cultured in a medium containing no candidate agent is different. The experiment of the control group can be carried out in parallel with the screening method of the present invention, or carried out in advance.

The biological sample used in the screening method of the present invention may be any of vascular endothelial cells, adipocytes, and the like, and it is not limited as long as APJ performance can be measured. For example, it can be the vascular endothelial cells and the vascular constitutive cells in the vascular model made by inducing blood vessel formation from the subcutaneous fat tissue of animals such as humans as described in the examples, and can be blood vessels obtained from living organisms. HUVEC (Human Umbilical Vein Endothelial Cell), HAEC (Human Aortic Endothelial Cell), and HAEC (Human Aortic Endothelial Cell) can also be used for endothelial cells, outer cover cells, or their descended cells or strained cells. Arterial Endothelial Cell), HMVEC (Human Microvascular Endothelial Cell, human microvascular endothelial cell), etc. Alternatively, as described in Patent Document 3 and Patent Document 4, APJ may be used to express cells.

Furthermore, before the step of culturing the biological sample using the medium containing the candidate agent, the culturing step before culturing the biological sample as described above may be included. In the pre-culture step, blood vessel models can also be made by inducing blood vessel formation from subcutaneous fat tissue. In addition, after the step of culturing the biological sample with the medium containing the candidate agent, it may include the step of culturing with the medium without the candidate agent. The step of culturing a biological sample using a medium containing a candidate drug can be cultured by adding the candidate drug or its dilution directly to the culture obtained in the forward culturing step, or it can be replaced with a medium containing the candidate drug for cultivation.

The measurement of APJ performance can be determined by measuring the amount of mRNA or protein of APJ in a biological sample. The amount of mRNA can be measured using methods known in the art such as quantitative PCR (Polymerase Chain Reaction) or Northern blot method. For example, as described in the Examples, a probe for APJ mRNA can be used. For the amount of protein, any methods known in the art, such as Western blotting, immunostaining, and FACS (Fluorescence activated Cell Sorting), can be used. For example, an antibody that specifically binds to APJ can be used. Alternatively, for example, a screening system using APJ-expressing cells as described in Patent Document 3 and Patent Document 4 may be established and used. In addition, the present invention also provides a kit that includes the above-mentioned reagents for determining APJ performance and is used to implement the screening method of the present invention.

The substance having an APJ performance enhancer effect selected by the screening method of the present invention may be any substance as long as it can promote the performance of APJ. One aspect of the screening method of the present invention is to use APJ expression in biological samples such as vascular endothelial cells as an index, and use any gene library such as cosmetic materials, food materials, and pharmaceutical materials for screening. As the substance having APJ performance promoting effect thus determined, one or more crude drugs selected from the group consisting of houttuynia cordata extract, cherry leaf extract, and neem leaf extract can be cited.

As explained above, it has been found that the promotion of APJ performance improves skin elasticity. Therefore, the present invention also relates to a method for improving skin elasticity including the application of APJ performance enhancer. If skin elasticity can be improved, wrinkles and sagging can be expected to improve. The method of improving skin elasticity in this manual is about the cosmetic method for the purpose of beauty, which can be distinguished from the treatment performed by doctors or medical staff. This beauty method can be performed personally, or in beauty salons, cosmetics dealerships, or beauty salons.

All documents mentioned in this specification are incorporated into this specification by way of full citation.

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 only limited by the description of the scope of the patent application. It is possible to make changes to the present invention without departing from the gist of the present invention, such as addition, deletion, and replacement of constituent elements of the present invention. [Example]

Experiment 1 : Fabrication of 3D vascularization dermis model and visualization of vascular structure and collagen production . Fabrication of type I collagen gel solution using the Nitta Gelatin culture kit. Specifically, in a 15 ml test tube on ice, 2 ml of the cell matrix collagen solution of 3 mg/ml is suspended in 2 ml of 1 mM dilute hydrochloric acid, and while stirring, 10 times concentrated MEM (Minimum Essential Medium, the minimum essential medium) 500 μl and 500 μl of C buffer included with the collagen gel culture kit for neutralization. Place 100 μl of this solution on a 35 mm glass petri dish (IWAKI) and incubate at 37°C for 30 minutes to gel.

Cut human normal subcutaneous adipose tissue in PBS (Phosphate Buffered Saline) containing 10% FBS (Fatal Bovine Serum) into 1 mm squares, and place it on the gelatinized collagen gel Place 4 pieces. After that, add 100 μl of the remaining type I collagen gel solution made on ice by covering the tissue piece, and incubate at 37°C for 30 minutes to gel. This tissue slice uses EBM-2 (Endothelial cell Basal Medium-2) (Cambrex; Verviers, Belgium) added with growth factors and other additional factors (Cambrex; Verviers, Belgium) and then adds a final concentration of 50 ng/ml VEGFA ( Vascular Endothelial Growth Factor A (Vascular Endothelial Growth Factor A) is a culture medium that is cultured for 7 to 21 days. In addition, the medium is replaced every 2 to 3 days. In the visualization of type I collagen or partial analysis of APJ performance, this model was made using collagen gel with a concentration of 0.9 mg/ml.

After incubation, add 1 ml of 4% paraformaldehyde solution and fix at 4°C for 30 minutes, and then perform immunostaining. As for the primary antibody, goat anti-PECAM-1 (Platelet Endothelial Cell Adhesion Molecule-1)/CD31 (Cluster of Differentiation 31) antibody (AF806, R&D systems, MN), Cy3αSMA (Alpha Smooth Muscle Actin, α-Smooth Muscle Actin) antibody (c-6198, Sigma, MO) is used for outer cover cells, rabbit anti-APJ antibody is used for APJ, rabbit anti-type I is used for type I collagen Collagen antibody (ab34710, Abcam, UK), for secondary antibody, use Alexa Fluor488 donkey anti-goat antibody (A11015, Molecular Probes, Eugene, OR) and Alexa Fluor594 donkey anti-rabbit antibody (R37119, Molecular Probes, Eugene, OR). In addition, for nuclear staining, Hoechst 33342 (H3570, Invitrogen, CA) was used. After staining, a confocal microscope LSM880 (CarlZeiss, Germany) was used to detect the 3D blood vessel structure and image it.

Figure 1 shows the production of a 3D vascularized dermal model after staining with anti-PECAM-1/CD31 antibody. It can be confirmed from Fig. 1 that vascular endothelial cells form a lumen and a 3D vascular structure is formed. Figure 2 shows an angiogenesis model stained with anti-PECAM-1/CD31 antibody and anti-type I Collagen antibody. It can be seen from Figure 2 that collagen is synthesized around blood vessels. In particular, as shown by the arrow in the right picture, collagen can be seen around the blood vessel, especially in the part of the stent where the blood vessel may continue to extend. Therefore, it is suggested that the existence of blood vessel structure contributes to collagen synthesis and even skin elasticity.

Experiment 2 : Decrease of blood vessels in aging and decrease of surrounding hardness. Although it was confirmed by the above experiment that the existence of blood vessel structure contributes to skin elasticity, it is known that blood vessels decrease in aging skin (Non-Patent Document 6). Therefore, for the purpose of suppressing the decrease of blood vessels caused by aging, the analysis focused on how to maintain the vascular structure and the interaction with the surrounding hardness and physical properties. Using the eyelid skins of human subjects from young people (10-20 years old) and middle-aged people (50-70 years old), using a supersonic wave microscope (Medical Ultrasonic Microscope, AMS-50SI, Honda Electronics Co., Ltd.) Observe the hardness of the peripheral area of the capillaries within 200 μm under the epidermis. Specifically, a frozen section of the skin is made, and the sound velocity of the skin profile is measured by the previous method using a supersonic wave microscope to obtain a sound velocity distribution image (Patent Document 8). Furthermore, using the slice after the measurement, immunostaining was performed to visualize the blood vessel structure. As for the primary antibody, goat anti-Pecam-1 antibody (AF806, R&D system, MN) was used to stain vascular endothelial cells, and rabbit anti-NG2 antibody (Milipore) was used to stain external cells. As for the secondary antibody, Alexa Fluor 594 donkey anti-goat antibody (A-11016, Molecular Probes, Eugene, OR) and Alexa Fluor 488 donkey anti-rabbit antibody (A-21206, Molecular Probes, Eugene, OR) were used. Thereafter, the sound velocity distribution image was superimposed on the immunostaining image, and the sound velocity value in the 50 μm range around the blood vessel was quantified. In the quantitative analysis, 10 specimens were used, and 15 blood vessels were quantified for each tissue. The results are shown in Figure 3. It can be seen from Fig. 3 that in aging skin, along with the reduction of blood vessels, the sound velocity in the peripheral area of the blood vessels significantly decreases. It suggests that the reduction of skin elasticity in aging is related to the reduction of blood vessels.

Experiment 3 : Determination of the hardness of collagen gel In the same way as Experiment 1, a type I collagen gel solution with various concentrations (0.24, 0.6, 0.9, 1.2, 1.8 mg/ml) was made on ice. After that, it was gelled in a 24-well plate at 37°C for 30 minutes to prepare a collagen gel. Physica MCR 300 Modular Compact Rheometer (Anton Paar, Germany) was used to measure the storage elastic modulus (Pa) of the produced collagen gel to define the hardness of the gel. The results are shown in Figure 4. It can be seen from Figure 4 that there is a corresponding relationship between the collagen gel concentration and the storage elastic modulus.

Experiment 4 : The relationship between the hardness of the collagen gel and the physical properties of the skin In order to study the relationship between the collagen concentration of the collagen gel and the actual physical properties of the skin, the skin softness sensor was used in the same way as in Non-Patent Document 7. (Non-Patent Document 7) The contact force of type I collagen gel with various concentrations (0.24, 0.6, 1.2, 1.8 mg/ml) was measured. The results are shown in Figure 5. It can be seen from Figure 5 that there is a roughly proportional relationship between the collagen concentration and the contact force up to about 2.0 mg/ml. In addition, using this device, it was confirmed that the average contact force of the cheek skin of healthy humans (about 20-50 years old, N=168) was about 17.5 mN, so the concentration was adjusted with the collagen concentration corresponding to 17.5 mN as the center. The following experiment.

Experiment 5 : Analysis of changes in the vascular structure in response to the hardness of the collagen gel. The concentration of the collagen gel was changed in the same way as in Experiment 1, thereby making type I collagen gels with various concentrations (0.24, 0.9) , 1.8 mg/ml). The gels of each concentration have storage elastic modulus of 2.6±0.8, 14.0±5.4, 48.1±17.0 (Pa), respectively. Using these gels of various concentrations, by the same method as Experiment 1, 3D blood vessel formation was induced from normal human subcutaneous adipose tissue. After culturing for 13 days, immunostaining was performed in the same manner as in Experiment 1 to visualize the vascular endothelial cells and outer covering cells.

The results are shown in Figure 6. It can be seen from Figure 6 that in the case of a low-concentration gel (0.24 mg/ml) with a modulus of elasticity of 2.6±0.8 Pa, finer and unstable blood vessels are formed, and the higher concentration (0.9 mg/m) has In the case of a gel with a higher elastic modulus (14.0±5.4 Pa), thicker and stable blood vessels are formed. However, in the case of a gel with a higher concentration (1.8 mg/ml) and a higher elastic modulus (48.1±17.0 Pa), more thin and unstable blood vessels were observed. It can be seen from the results of Fig. 6 that the structure of blood vessels formed in response to the physical properties of the surrounding environment such as the elasticity of the matrix will change. Furthermore, it also suggests that the best physical properties of the surrounding environment contribute to the stabilization of blood vessels. Combining the results of Fig. 3, it is also found that although the blood vessels sense the hardness of the surroundings and maintain their structure stably, the hardness of the surroundings will decrease in aging skin. Therefore, it is considered that the blood vessels may become unstable and the blood vessels tend to decrease.

Experiment 6 : The analysis of vascular cell expression factor (APJ) in response to the hardness of collagen gel was made on ice in the same way as experiment 1, with various concentrations (0, 0.12, 0.6, 0.9, 1.2, 1.8 mg/ ml) Type I collagen gel solution. After culturing and fusion of human umbilical vein endothelial cells (HUVEC) seeded on a 12-well plate with 10 ⁵ cells/well, add each type I collagen gel solution to 300 μl/well and incubate at 37°C to gel. , And then cultivate. After 6 hours of culture, Trizol was added to each well to disrupt the cells. The total RNA is obtained by extracting chloroform from the obtained solution. Measure the concentration of total RNA obtained by nano drop and adjust to 35 ng/ml with RNase free water. Thereafter, the TaqMan ^TM RNA-to-C _T ^TM 1-Step Kit (Applied Biosystems, CA) and the Taqman probe for human APJ mRNA (Hs00270873, Applied Biosystems, CA) were used to compare the mRNA expression level of APJ.

The results are shown in Figure 7. The left figure is a graph showing the relationship between collagen gel concentration and APJ mRNA expression. It can be seen that the expression of APJ mRNA has the highest peak under the condition of 0.9 mg/ml collagen gel, and the decrease of expression in low and high concentration collagen gel is observed. In other words, it can be seen that the performance of APJ changes in response to the physical properties of the surrounding environment such as the elasticity of the substrate. Furthermore, this relationship between physical properties and performance is not seen in other vascular stabilizing factors such as Tie2 (data not shown), and is specific to APJ. In addition, the elasticity of the collagen gel obtained in this experiment is roughly the same as the gel concentration corresponding to the elasticity of normal human skin. Therefore, considering that the performance of APJ will change according to the elasticity of the matrix, it will contribute to the stability of the blood vessel, suggesting that its elasticity is too low or too high, and there is an appropriate range. The right panel of Figure 7 is a micrograph showing the local presence of APJ in a blood vessel model formed in a collagen gel at a concentration of 0.9 mg/ml. It can be seen that, like filopodia extending from vascular endothelial cells to the surroundings, APJ is locally present in the cell protrusions, suggesting that APJ may directly sense the surrounding matrix environment and stabilize blood vessels.

Experiment 7 : Fabrication and structural analysis of APJ high-performance 3D vascularization dermal model Insert human APJ gene sequence into pEGFP-N1 vector (Clontech, CA) to obtain APJ high-performance vector for lentivirus introduction. After that, based on this vector, APJ high-performance lentivirus (Oriental Yeast) was produced. The 3D blood vessel forming dermis model was made in the same way as experiment 1. After that, the model was transfected with the APJ expression vector obtained by lentivirus infection on the 21st day of culture, and cultured for 5 days. In the control group, the blood vessel model was cultured under the same conditions except that no transfection was performed. After incubation, 4% paraformaldehyde solution was added and fixed at 4 degrees for 30 minutes, and immunostaining was performed. For the primary antibody, goat anti-Pecam-1 antibody (AF806, R&D systems, MN) was used for vascular cells, and for the secondary antibody, Alexa Fluor 594 donkey anti-goat antibody (A-11016, Molecular Probes, Eugene, OR) was used. After staining, a confocal microscope LSM880 (CarlZeiss, Germany) was used to detect the 3D blood vessel structure and image it.

The results are shown in Figure 8. It can be seen from Fig. 8 that if the performance of APJ is enhanced, blood vessels become thicker and stabilized, and the results are consistent with the existing publication (Non-Patent Document 5).

Based on the above results, considering, for example, as shown in Fig. 9 of the schematic diagram, the skin elasticity interacts with APJ performance, and there may be a cycle in which APJ acts as a sensor of skin elasticity, thereby stabilizing blood vessels, It promotes collagen synthesis around blood vessels and feeds back skin elasticity. Therefore, it is considered that even for the skin whose elasticity is reduced due to aging, the enhancement of APJ performance can also strengthen circulation and improve skin elasticity. Therefore, the following experiments are conducted to screen for substances with APJ performance promoting effect.

Experiment 8 : Screening method for substances with skin elasticity-improving effects that promote APJ performance. A total of 49 raw materials containing plant extracts that are said to have anti-aging effects were used as candidate agents for screening. Use Houttuynia cordata extract (Houttuynia cordata ET-50) manufactured by Xiangrong Industrial Co., Ltd., cherry leaf extract (Falco Rex Sakura leaf B) manufactured by ICHIMARU PHARCOS, and Neem leaf extract manufactured by ICHIMARU PHARCOS (Neem Liquid B). The extracts were dissolved in dimethyl sulfide (DMSO) to a final concentration of 0.1% (w/w) to prepare samples. As a control, a DMSO solution without these extracts was used. After HUVEC seeded with 10 ⁵ cells/well on a 12-well plate is cultured to confluence, the above sample is added so that the final concentration becomes 0.1%, and incubated at 37°C for 4 hours. Compare APJ by the same method as Experiment 6. The expression level of mRNA.

The results are shown in Figure 10. It can be seen from FIG. 10 that when Houttuynia cordata extract, cherry leaf extract, and neem leaf extract are used as candidate agents, the performance of APJ is significantly increased compared with the control group. These extracts are selected as substances with APJ performance promoting effect.

Figure 1 shows the production of a 3D vascularization dermal model. Figure 2 shows blood vessels (green) stained with anti-PECAM-1 (Platelet Endothelial Cell Adhesion Molecule-1)/CD31 (Cluster of Differentiation 31) antibody and anti-type I Collagen Microscopic picture of the blood vessel formation model of type I collagen (red) after antibody staining. The left image of Figure 3 is an image obtained by measuring the speed of sound (hardness) value (m/s) of a human skin section by a supersonic wave microscope and an immunostained image obtained by visualizing the blood vessel (red) of the section. By. As a representative example, a young person (20 years old) and a middle-aged person (50 years old) are shown. The graph on the right quantifies the speed of sound (hardness) around the blood vessel and shows the average value of the young people (10-20 years old) and the middle-aged people (50-70 years old). 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 collagen concentration (mg/ml) and storage elastic modulus G'(Pa). Figure 5 is a graph showing the relationship between collagen concentration (mg/ml) and contact force (mN). Figure 6 is the 3D blood vessel formation dermis model cultured in collagen gel with various concentrations (mg/ml) stained with anti-PECAM-1/CD31 antibody and blood vessels (green) are visualized, and Cy3αSMA (Alpha Smooth Muscle) Actin, α-smooth muscle actin) antibody staining and visualization of the outer cover cell (red) micrograph. The left graph of Fig. 7 is a graph showing the relationship between the collagen gel concentration and the expression level of APJ mRNA (messenger ribonucleic acid) in vascular endothelial cells cultured in collagen gels with various concentrations. When the gel concentration is 0 mg/ml(-), the expression level is set to 100 as a relative value (%). In the figure, * and ** indicate that there is a significant difference by Student's t-test (*p<0.05, **p<0.01). The right picture shows the blood vessel stained with anti-PECAM-1/CD31 antibody (green), the cell nucleus stained with Hoechst (blue), and anti-APJ in the 3D vascularization dermis model formed in collagen gel A micrograph of the partial presence of APJ (red) after antibody staining. The left panel of Fig. 8 shows the outline of the production of a dermal model of 3D blood vessel formation with APJ high expression vector by transfection of APJ high expression vector. The picture on the right shows the microscopic photograph of the blood vessel structure (red) stained with anti-PECAM-1/CD31 antibody in the 3D blood vessel formation dermis model with high performance of APJ in the control group. Fig. 9 is a schematic diagram showing the concept of the relationship between skin elasticity and APJ performance obtained from this application, and the associated blood vessel stabilization. Fig. 10 shows the performance of APJ when the Houttuynia cordata extract, the cherry leaf extract, and the neem leaf extract are used as the relative value (%) when the control is set to 100. In the figure, * and ** indicate that there is a significant difference from the control group by Student's t test (*p<0.05, **p<0.01).

Claims (8)

An APJ performance enhancer used to improve skin elasticity. An APJ performance enhancer comprising one or more crude drugs selected from the group consisting of houttuynia cordata extract, cherry leaf extract, and neem leaf extract. A skin elasticity improving agent comprising the APJ performance promoting agent as claimed in claim 1 or 2. A method for screening skin elasticity improving agents, which uses APJ performance as an indicator. Such as the screening method of claim 4, which includes: Use a medium containing candidate agents to cultivate biological samples; Determination of APJ performance in biological samples; and When comparing APJ performance with the control group, the candidate agent is determined to be a substance with a skin elasticity improvement effect. A kit for implementing the screening method according to any one of Claims 4 or 5, which contains reagents for determining APJ performance. A method for improving the skin elasticity of a subject for the purpose of beauty, which includes promoting the subject's APJ performance. The method of claim 7, wherein the promotion of APJ performance of the subject is achieved by administering the skin elasticity improving agent of claim 3 to the subject.

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