BMP2 expression inhibitor

Abstract

Dermal fibroblasts constitute various subpopulations, and this research aims to improve the disruption of the properties of healthy dermal fibroblasts, particularly those caused by subpopulations that arise with age. [Solution] We screened cosmetic ingredients that can suppress BMP2 expression in dermal fibroblasts and found that Geranium thunbergii extract, yarrow extract, rose extract, and lavender extract each have BMP2 expression inhibitory effects.

Description

Technical Field

[0001] To provide an inhibitor of BMP2 expression related to property changes of dermal fibroblasts. Regarding the maintenance of skin condition such as improvement of skin elasticity, sagging, wrinkles, and firmness through the suppression of BMP2 expression.

Background Art

[0002] Histologically, the skin is classified into the epidermis, dermis, and subcutaneous tissue. Each tissue contains various cells, but the epidermis is mainly composed of keratinocytes. Keratinocytes with proliferative activity located in the basal layer stop proliferating and differentiate outward into the spinous layer, granular layer, and stratum corneum, forming the epidermis, which is the boundary with the outside world. In this way, even a single cell within a single tissue can form subpopulations with different differentiation states. Compared to cell populations like keratinocytes, which proliferate only in the basal layer and do not proliferate after differentiation, dermal cells undergo repeated differentiation and proliferation, making it difficult to identify subpopulations, and research focusing on cells belonging to specific subpopulations has lagged behind that of keratinocytes. On the other hand, with the establishment of analytical methods focusing on single cells in recent years, it has become possible to divide dermal fibroblasts into subpopulations and analyze them. For example, fibroblasts are classified into subpopulations that constitute the papillary surface layer and subpopulations that extend to the subcutaneous tissue, with differences in morphology, proliferative capacity, culture matrix production capacity, growth factor production capacity, and response to growth factors reported (Patent Document 1: JP 2024-3103). Conventional subpopulations of dermal fibroblasts are classified mainly based on their localization, but single-cell analysis using aging human skin has identified fibroblast subpopulations that characteristically appear with age. These subpopulations exhibit characteristic gene expression and release BMP2, BMP4, BMP7, TGFβ1, TGFβ2, TGFβ3, etc. to other skin cells, causing changes in the properties of other fibroblasts (Patent Document 2: JP 2024-196222, Non-Patent Document 1: 49th Annual Meeting of the Japanese Society for Investigative Dermatology). As a result, it is thought that they may promote differentiation into adipocytes or the production of chondrocyte-like matrix, for example. Therefore, these effects mediated by the aforementioned signals released by the aging fibroblast subpopulation are thought to be involved in various symptoms of skin aging, such as decreased skin elasticity, sagging, and wrinkles. [Prior art documents] [Patent Documents]

[0003] [Patent Document 1] Japanese Patent Publication No. 2024-3103 [Patent Document 2] Japanese Patent Application No. 2024-196222 [Non-patent literature]

[0004] [Non-Patent Document 1] Sawane et al., 49th Annual Meeting of the Japanese Society for Investigative Dermatology (2024) [Non-Patent Document 2] Grafe et al., Cold Spring Harb Perspect Biol 2018 [Non-Patent Document 3] Plikus et al., Science, 2017 [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] Dermal fibroblasts classified as an aging subpopulation induce adipogenic differentiation and chondrocyte-like matrix production in other fibroblasts via BMP2, BMP4, BMP7, TGFβ1, TGFβ2, and TGFβ3, leading to changes in the physical properties of the dermis. However, there has been no means to suppress skin aging induced by dermal fibroblasts classified as an aging subpopulation. [Means for solving the problem]

[0006] The present inventors screened cosmetic ingredients capable of suppressing BMP2 expression, which contributes to fibroblast degeneration, and arrived at the present invention. Therefore, the present invention relates to the following: [1-1] A BMP2 expression inhibitor comprising at least one extract selected from the group consisting of Geranium thunbergii extract, Yarrow extract, Rose extract, and Lavender extract. [1-2] A method for suppressing BMP2 expression in a subject requiring improvement of wrinkles, sagging, or firmness, comprising administering at least one extract selected from the group consisting of Geranium thunbergii extract, Yarrow extract, Rose extract, and Lavender extract. [1-3] A cosmetic method involving suppression of BMP2 expression, comprising administering at least one extract selected from the group consisting of Geranium thunbergii extract, Yarrow extract, Rose extract, and Lavender extract to a subject requiring improvement of wrinkles, sagging, or firmness. [1-4] Geranium thunbergii extract, yarrow extract, rose extract, and lavender extract for use in the treatment of wrinkles, sagging, or firmness by suppressing BMP2 expression. [1-5] Use of Geranium thunbergii extract, yarrow extract, rose extract, and lavender extract for the manufacture of BMP2 expression inhibitors. [2] A skin elasticity improving agent containing a BMP2 expression inhibitor selected from the group consisting of Geranium thunbergii extract, yarrow extract, rose extract, and lavender extract. [3] A topical skin composition containing a BMP2 expression inhibitor as described in item 1-1, or a skin elasticity improving agent as described in item 2. [4] The topical skin composition according to item 3, wherein the topical skin composition is a composition for improving wrinkles, sagging, or firmness. [Effects of the Invention]

[0007] A BMP2 expression inhibitor containing at least one component selected from the group consisting of Geranium thunbergii extract, yarrow extract, rose extract, and lavender extract can suppress BMP2 expression. BMP2 is involved in the changes in the properties of dermal fibroblasts in aged skin (promotion of adipocyte differentiation and chondrocyte-like matrix production), and suppressing BMP2 expression inhibits the alteration of fibroblasts, thereby exerting anti-aging effects, such as improved skin elasticity. [Brief explanation of the drawing]

[0008] [Figure 1] Figure 1 shows the screening results for extracts that can suppress the promotion of BMP2 expression by TNF-α addition. (A) Geranium thunbergii extract, (B) Yarrow extract, (C) Rose extract, and (D) Lavender extract each have an inhibitory effect on BMP2 expression in fibroblasts. [Modes for carrying out the invention]

[0009] One aspect of the present invention relates to a BMP2 expression inhibitor comprising at least one extract selected from the group consisting of Geranium thunbergii extract, Yarrow extract, Rose extract, and Lavender extract. BMP2 alters the properties of fibroblasts, such as increasing adipogenic differentiation of fibroblasts and the production of chondrocyte-like matrix. It has been identified as being released particularly by a subpopulation of aging fibroblasts and contributes to the degeneration of other fibroblasts with age. By suppressing BMP2 expression, dermal fibroblasts can be maintained normally, thereby maintaining the health of the dermis.

[0010] Fibroblasts possess mesenchymal cell properties and are cytologically closely related to mesoderm-derived adipocytes, bone cells, and chondrocytes. Examples of fibroblasts include myofibroblasts present during muscle and skin injury, and dermal fibroblasts present in the dermis. BMP2 contributes to the promotion of adipogenic differentiation and the production of chondrocyte-like matrix in fibroblasts. Dermal fibroblasts present in the dermis can be characterized by gene expression such as PDGFRA and DCN, but dermal fibroblasts are widely distributed in the dermis and form subpopulations with different properties depending on the region within the dermis. The dermis is the region sandwiched between the epidermis and subcutaneous tissue, and is classified into the papillary layer, subpapillary layer, reticular layer, and subcutaneous junctional layer directly below the basement membrane, with different subpopulations of dermal fibroblasts distributed in each layer. Organs such as hair follicles and sweat glands extend from the dermis to the subcutaneous tissue, and dermal papillae and hair root sheath cells also constitute a subpopulation of dermal fibroblasts. In recent years, a subpopulation of fibroblasts that appears in aging human skin has also been identified, and this subpopulation causes changes in the properties of other dermal fibroblasts via BMP2.

[0011] Bone morphogenetic proteins (BMPs) are a group of proteins identified as proteins that induce the differentiation of bone tissue and cartilage. BMPs belong to the TGF-β superfamily and induce signal transduction by binding to BMP receptors. BMPs influence the differentiation of mesenchymal cells and induce ectopic bone and cartilage formation from mesenchymal cells (Non-patent Literature 2: Grafe et al., Cold Spring Harb Perspect Biol 2018). In addition to the bone induction and formation promotion initially discovered, BMPs also contribute to neurogenesis and wound healing. In wound healing, they have also been reported to regulate the differentiation of myofibroblasts into adipocytes (Non-patent Literature 3: Pikus et al., Science, 2017). Furthermore, the dermal fibroblast subpopulation, which increases with age, causes degeneration of dermal fibroblasts via BMP2, 4, 7, TGF-β1, TGF-β2, and TGF-β3 (Patent Literature 2, Non-patent Literature 1). Since this alteration is expected to lead to abnormalities in collagen and elastin in the skin, BMP2 expression inhibitors function as preventive or corrective agents for the disruption of fibroblast properties. Furthermore, by improving the disruption of fibroblast properties, the original functions of dermal fibroblasts are enhanced. Therefore, BMP2 expression inhibitors are expected to promote the expression of elastic fibers such as collagen and elastin, and the production of extracellular matrix such as proteoglycans, thereby improving skin appearance caused by dermal fibroblast dysfunction, such as sagging, wrinkles, and loss of firmness. BMP2 expression inhibitors can also be called skin elasticity enhancers, or agents that improve sagging and wrinkles, or agents that improve firmness.

[0012] Another aspect of the present invention may relate to a method for screening agents for reducing or improving the properties of fibroblasts. By screening compounds having BMP2 expression inhibitory activity, it is possible to develop products that are effective in reducing or improving the properties of dermal fibroblasts. More specifically, the method for screening agents for reducing or improving the properties of fibroblasts is as follows: A step of culturing a culture containing fibroblasts in a culture medium containing a candidate drug; A step of measuring the BMP2 expression in the culture after culturing in a medium containing a candidate agent; and A step of comparing the measured BMP2 expression level with a control to determine the effect of reducing or improving the disorder of the properties of the candidate agent It includes. A candidate agent capable of suppressing the BMP2 expression level can be selected as an agent having an effect of reducing or improving the disorder of the properties when compared with the control. The compound having the BMP2 expression-suppressing effect thus screened can be used as an agent for reducing or improving the disorder of the properties.

[0013] In screening, fibroblasts can be used as the cells, but in particular, dermal fibroblasts are preferred. The dermal fibroblasts may have their BMP2 expression promoted by culturing in a medium containing TNF-α in advance, or may be cultured in a medium supplemented with TNF-α simultaneously with the candidate agent to promote the BMP2 expression. The dermal fibroblasts may be any subpopulation, but a subpopulation with enhanced BMP2 expression can also be selected. Such a subpopulation can also be called an aging subpopulation. The culture containing dermal fibroblasts may be a culture of isolated dermal fibroblasts or immortalized dermal fibroblasts, or a collected skin organ, or a three-dimensional skin model. The cells to be used may be cells derived from any animal, but in particular, cells derived from mammals, particularly humans, are preferred.

[0014] The candidate agent may be added to the culture at a predetermined timing, or the culture medium may be replaced with a medium containing the candidate agent. Before adding the candidate agent, it may be exposed to a starvation state. The expression of BMP2 can be determined by measuring the protein amount or the mRNA amount, and can be measured using techniques well-known in the art such as immunological techniques or quantitative PCR, respectively.

[0015] As a control, a culture containing dermal fibroblasts can be cultured in a medium that is different only in that it does not contain the candidate agent, and the measured expression of BMP2 can be used. For the control group, experiments can be performed in advance to set a threshold based on the expression of BMP2 in the control group, or the culture and measurement steps can be performed in parallel. When the expression of BMP2 is suppressed compared to the expression of BMP2 in the control, the candidate agent can be screened as an inhibitor of BMP2 expression or an agent for reducing or improving the disorder of properties. Such an agent can be used as an agent for improving skin elasticity, a remedy for sagging, wrinkles, and firmness.

[0016] For the candidate components used in the screening method of the present invention, any library such as a cosmetic material, a food material, a pharmaceutical material, etc. can be used. As such a library, a compound library, an extract library, etc. may be used. The compounds and extracts contained in each library may be commercially available compounds and extracts, or synthesized compounds and prepared extracts may be used.

[0017] The plant extracts described herein can be obtained by conventional methods, for example, by immersing or refluxing the source plant with an extraction solvent at room temperature or under heating, followed by filtration and concentration. In another example, they can be obtained by distillation. Any solvent commonly used for extraction can be used as the extraction solvent, for example, aqueous solvents such as water, physiological saline, phosphate buffer, borate buffer, or organic solvents such as alcohols including ethanol, propylene glycol, 1,3-butylene glycol, and glycerin, aqueous alcohols, chloroform, dichloroethane, carbon tetrachloride, acetone, ethyl acetate, and hexane, each can be used alone or in combination. Preferably, a mixed solvent of water and alcohol, such as 1,3-butylene glycol, is used as the solvent. The extract obtained by extraction with the above solvent can be used as is, or the extract can be concentrated by, for example, freeze-drying. If necessary, an extract obtained by adsorption, for example, removing impurities using an ion exchange resin, or by adsorption using a porous polymer (e.g., Amberlite XAD-2) column, followed by elution with a desired solvent and further concentration can also be used. The plant extracts used can be commercially available extracts used as cosmetic ingredients, and these commercially available extracts can be incorporated at a predetermined concentration.

[0018] Geranium thunbergii extract is an extract obtained from plants belonging to the genus Geranium. In particular, it is an extract obtained from Geranium thunbergii. Geranium thunbergii is widely distributed in East Asia, especially Japan, China, and the Korean Peninsula, and has been used since ancient times. It has been used as a medicinal herb for centuries. Geranium thunbergii extract may be extracted from the plant body, particularly from the leaves, stems, flowers, seeds, fruits, or roots. Geranium thunbergii extract can be obtained by extracting the plant body with a solvent, either as is, after drying, or after fermentation. Geranium thunbergii extract is represented by the display name used in the full ingredient labeling of cosmetics as defined by the Japan Cosmetic Industry Association and by the international display name under the INCI (International Nomenclature for Cosmetic Ingredients) system as: Display Name / INCI Name: Geranium Thunbergii Flower / Leaf / Stem Extract. As an example of solvent extraction, it can be prepared by extraction using water, alcohol, or a mixture thereof. As alcohol, ethanol, propylene glycol, or butylene glycol can be used. More preferably, it can be extracted with a mixture of water and 1,3-butylene glycol in any ratio, for example, a mixture of 10:90 to 90:10, preferably 30:70 to 70:30, and even more preferably 50:50. Geranium thunbergii extract may be added to the product at a concentration of 0.005% to 10% by weight using a commercially available product, and it is more preferable to add it at a concentration of 0.01% to 5% by weight.

[0019] Yarrow extract is an extract obtained from plants belonging to the genus Achillea in the Asteraceae family. Specifically, it is an extract obtained from Achillea millefolium. Yarrow is widely distributed in Europe and has been used as a medicinal herb since ancient times. Yarrow may be extracted from the plant body, particularly the leaves, stems, flowers, fruits, seeds, or roots. Yarrow extract can be obtained by extracting the plant body with a solvent after drying or fermentation. In the labeling names used in cosmetic ingredient lists as defined by the Japan Cosmetic Industry Association and the international labeling names under the INCI (International Nomenclature for Cosmetic Ingredients) system, Yarrow extract is represented as: Labeling Name / INCI Name: Achillea Millefolium Extract / Achillea Millefolium Extract. As an example of solvent extraction, it can be prepared by extraction using water, alcohol, or a mixture thereof. Ethanol, propylene glycol, or butylene glycol can be used as alcohols. More preferably, the extract can be obtained using a mixture of water and 1,3-butylene glycol in any proportion, for example, a mixture of 10:90 to 90:10, preferably 30:70 to 70:30, and even more preferably 50:50. The yarrow extract may also be added to the product at a concentration of 0.005% to 10% by weight using a commercially available product, and more preferably at 0.01% to 5% by weight.

[0020] Rose extract is an extract obtained from plants belonging to the genus Rosa in the family Rosaceae. While there are many species of roses, the extract is not limited to any particular species. Roses are widely distributed in the temperate regions of the Northern Hemisphere and have been cultivated as ornamental plants since ancient times. Rose extract may be obtained from the plant body, especially the leaves, stems, flowers, fruits, seeds, or roots, but the flowers, particularly the petals, can be used. Rose extract can be obtained by extracting the rose plant body with a solvent after drying or fermentation. The labeling name for rose extract used in the full ingredient list of cosmetics as defined by the Japan Cosmetic Industry Association, and the international labeling name according to the INCI (International Nomenclature for Cosmetic Ingredients) system, is expressed as: Labeling Name / INCI Name: Rosa Hybrid Flower Extract. As an example of solvent extraction, it can be prepared by extraction using water, alcohol, or a mixture thereof. Ethanol, propylene glycol, or butylene glycol can be used as alcohols. More preferably, the rose extract can be obtained using a mixture of water and 1,3-butylene glycol in any proportion, for example, a mixture of 10:90 to 90:10, preferably 30:70 to 70:30, and even more preferably 50:50. The rose extract may also be added to the product at a concentration of 0.005% to 10% by weight using a commercially available product, and more preferably at 0.01% to 5% by weight.

[0021] Lavender extract is an extract obtained from plants belonging to the genus Lavandula in the family Lamiaceae. While there are many species of lavender, representative species include common lavender (Lavandula angustifolia) and spike lavender (Lavandula latifolia). Lavender is widely distributed along the Mediterranean coast, North Africa, the Middle East, and India, and has been cultivated as a herb since ancient times. Lavender extract may be obtained from the plant body, particularly the leaves, stems, flowers, fruits, seeds, or roots, with herbaceous plants and flowers being particularly useful. Lavender extract can be obtained by extracting the plant body directly, after drying or fermentation, using a solvent, or by distillation. According to the Japan Cosmetic Industry Association's labeling system for cosmetic ingredients and the International Nomenclature for Cosmetic Ingredients (INCI), lavender extract is represented as: Lavandula Angustifolia (Lavender) Flower / Leaf / Stem Extract. For example, lavender extract can be obtained by placing lavender in a distillation still and performing steam distillation. The lavender extract may be lavender oil containing oil, or aromatic hydrosol (also called lavender water) in which aromatic components are dissolved. Commercially available lavender extract may be added to the product at a concentration of 0.005% to 10% by weight, and more preferably at 0.01% to 5% by weight.

[0022] The BMP2 expression inhibitor according to the present invention can be administered to individuals suffering from sagging, wrinkles, or loss of firmness. Transdermal administration is preferred as the method of administration from the viewpoint of direct action on the skin. Another aspect of the present invention may relate to a cosmetic method that includes applying the BMP2 expression inhibitor to the skin. By suppressing BMP2 expression, it is possible to suppress the disruption of the differentiation state of dermal fibroblasts, thereby improving skin elasticity and reducing sagging, wrinkles, or firmness. The BMP2 expression inhibitors of the present invention may be incorporated into cosmetics, pharmaceuticals, or quasi-drugs, and may also be incorporated into foods, such as nutritional supplements or functional foods. These agents may be administered orally or parenterally, for example, transdermally. When administered transdermally, they can be formulated into topical skin compositions.

[0023] The topical skin composition is not particularly limited as long as it is applicable to the skin, and any dosage form can be used, such as solution, emulsion, solid, semi-solid, powder, powder dispersion, water-oil two-layer separation, water-oil-powder three-layer separation, ointment, gel, aerosol, mousse, stick, etc. When formulated into a topical skin composition, bases and excipients commonly used in topical skin compositions, such as preservatives, emulsifiers, and pH adjusters, may be used.

[0024] When incorporated into cosmetics, it can be used in facial or body cosmetics such as lotions, emulsions, serums, creams, packs, essences, and gels, as well as makeup cosmetics such as foundations, makeup bases, and concealers, and even bath additives. When incorporated into food products, it can be used in capsules, tablets, beverages, and the like. By using cosmetics, foods, pharmaceuticals, and quasi-drugs containing the components of the present invention, an inhibitory effect on BMP2 expression is achieved. The BMP2 expression inhibitor can be administered over a long period of time as a food or cosmetic product of the present invention. From the viewpoint of improving the disorder of differentiation in the dermis, it may be administered for several days or more, one week or more, two weeks or more, one month or more, three months or more, or six months or more. There is no particular upper limit, but it may be several years or less, for example, one year or less.

[0025] The BMP2 expression inhibitor of the present invention can be expressed at any concentration from the viewpoint of achieving a desired effect, such as improving the disorder of the differentiation state of dermal fibroblasts. The BMP2 expression inhibitors, Geranium thunbergii extract, Yarrow extract, rose extract, and lavender extract, can be formulated individually or in combination. From the viewpoint of formulation as a topical skin composition, the extracts according to the present invention can be formulated at a concentration of 0.005% to 10% by weight. From the viewpoint of fully exhibiting the effect, it is preferable to formulate it at a concentration of 0.01% or more, and more preferably at a concentration of 0.1% or more. From the viewpoint of avoiding a strong odor, it is preferable to formulate it at a concentration of 5% by weight or less, and more preferably at a concentration of 1% or less.

[0026] All references made herein are incorporated herein by citation in their entirety.

[0027] The embodiments of the present invention described below are for illustrative purposes only and do not limit the technical scope of the invention. The technical scope of the invention is limited solely by the claims. Modifications to the invention, such as additions, deletions, and substitutions of constituent elements of the invention, can be made without departing from the spirit of the invention. [Examples]

[0028] Example 1: Screening of BMP2 expression inhibitors We screened cosmetic ingredients that can suppress BMP2 expression, which is involved in the disruption of fibroblast properties. Dermal fibroblasts were raised in 10% FBS-supplemented DMEM medium in 8 × 10⁶ cells. 4Cells were seeded in wells and cultured for 24 hours at 37°C under a 5% CO2 atmosphere. The culture medium was replaced with DMEM medium supplemented with 1% FBS, and cultured for another 24 hours. Next, the culture medium was replaced with DMEM medium supplemented with 10 ng / mLTNF-α (R&D Systems, Inc.) and 31 candidate cosmetic ingredients, and cultured for 4 hours. As a control, a medium supplemented with only TNF-α and no candidate ingredients was used, and as a negative control, a medium without TNF-α or candidate ingredients was used. The experiment was conducted under the same conditions except for these differences. When 0.1% Geranium thunbergii extract (Maruzen Pharmaceutical Co., Ltd.), 0.1% Sawtooth Grass extract (Koei Kogyo Co., Ltd.), 0.1% Rose extract (Nichirei), and 0.1% Lavender extract (Ikeda Bussan) were used as candidate drugs, it was shown that the BMP2 expression promoting effect by TNF-α could be significantly suppressed (Figure 1).

Claims

1. A BMP2 expression inhibitor comprising at least one extract selected from the group consisting of Geranium thunbergii extract, yarrow extract, rose extract, and lavender extract.

2. A skin elasticity improving agent containing a BMP2 expression inhibitor selected from the group consisting of Geranium thunbergii extract, yarrow extract, rose extract, and lavender extract.

3. A topical skin composition containing the BMP2 expression inhibitor described in claim 1, or the skin elasticity improving agent described in claim 2.

4. The topical skin composition according to claim 3, wherein the topical skin composition is a composition for improving wrinkles, sagging, or firmness.

Images