JP7606736B2 - Epithelial or mesenchymal cell activator - Google Patents

Description

The present invention relates to an agent for activating epithelial cells or mesenchymal cells.

When cells or organs in the tissues of vertebrates (especially mammals) are damaged due to injury, disease, or aging, the regenerative system activates and attempts to repair the damage to the cells or organs. Stem cells in the tissue play a major role in this process. Stem cells have the ability to self-replicate and the pluripotency to differentiate into various cells and organs, and these properties allow them to regenerate tissues and maintain homeostasis by compensating for damaged parts of cells and organs. There is great hope for regenerative medicine, the next generation of medical care that utilizes such stem cells.

Bone marrow is the tissue in which mammalian stem cell research has progressed the most. Bone marrow contains the body's hematopoietic stem cells, which have the ability to self-replicate and produce identical cells, and the ability to differentiate into any type of blood cell. As such, transplantation of hematopoietic stem cells makes it possible to reconstruct blood cells, and so hematopoietic stem cell transplantation is being performed as a means of treating blood diseases and immunodeficiencies. However, it has been reported that the proliferation and differentiation capabilities of hematopoietic stem cells decline with age (Non-Patent Document 1), and there are cases in which the blood system cannot be reconstructed even with hematopoietic stem cell transplantation.

It is also known that some of these stem cells decrease with age, and research into techniques to prevent the decrease in stem cells in order to maintain homeostasis in each tissue is being actively conducted (Non-Patent Document 2). In recent years, in order to apply the capabilities (pluripotency) of stem cells to the regeneration of organs and tissues, techniques for isolating stem cells from living tissues and culturing and proliferating them in the fields of cell transplantation therapy and tissue engineering (regenerative medicine and regenerative cosmetics) have been developed (Non-Patent Documents 3 and 4). In other words, activating stem cells is very important in promoting tissue regeneration. Growth factors such as EGF (Epidermal Growth Factor) and FGF1 (Fibroblast Growth Factor 1) are known to activate stem cells, and contribute to the proliferation of not only stem cells but also various other cells. These growth factors activate downstream signals by binding to growth factor receptors on the cell membrane surface, activating the cells. Activated cells promote active tissue regeneration through proliferation, etc.

There have been several reports on techniques to promote the expression of growth factors. For example,
Patent Document 1 describes an insulin-like growth factor-1 (IGF-1) expression promoter that contains extracts of several kinds of plants, such as ginkgo, as active ingredients. However, a method for promoting the expression of growth factor receptors has not yet been developed. If it is possible to promote the expression of growth factor receptors rather than to control the expression of growth factors, more sustained cell activation effects and tissue regeneration effects can be expected.

Anise (scientific name: Pimpinella anisum L.) is an annual plant belonging to the Apiaceae family, and its seeds (botanically the fruit) contain essential oil components such as anethole, which are known to promote digestion. Anise is also used as a spice to flavor food and to eliminate odors. Anise extracts have been reported to be used as cholera toxin production inhibitors (Patent Document 2), skin whitening agents (Patent Document 3), and estrogen agents (Non-Patent Document 5).

JP 2009-256270 A JP 2011-105715 A JP 2005-139070 A

Age-associated characteristics of murine hematopoietic stem cells. J. Exp. Med. 2000 Nov 6;192(9):1273-80. Yasushi Hasegawa, Aesthetic Dermatology, 2013, Vol. 23, pp. 1-11 Hiroshi Mabuchi et al., Journal of the Japanese Society for Regenerative Medicine, 2007, Vol. 6, pp. 263-268 Kitagawa, J. et al., Journal of the Japanese Society for Regenerative Medicine, 2008, Vol. 7, pp. 14-18 Fennel and anise as estrogenic agents. Journal of Ethnopharmacology, 01 1980 Dec; 2(4): 337-344

In view of the above circumstances, the object of the present invention is to find a substance that promotes the expression of growth factor receptors that are activated by the binding of growth factors and play a role in controlling cell proliferation and differentiation, and to provide a cell activator that has excellent durability using said substance.

As a result of intensive research aimed at solving the above problems, the inventors discovered that anise extract has the effect of promoting the expression of growth factor receptors in epithelial cells or mesenchymal cells and the proliferation of said cells, which led to the completion of the present invention.

That is, the present invention includes the following inventions.
(1) An agent for promoting growth factor receptor expression in epithelial cells or mesenchymal cells, comprising an extract of anise as an active ingredient.
(2) An epithelial cell or mesenchymal cell activator comprising an anise extract as an active ingredient.
(3) The agent according to (1) or (2), wherein the epithelial cells or mesenchymal cells are present in skin tissue.
(4) A composition for external use on the skin, comprising the agent according to any one of (1) to (3).
(5) The topical skin composition according to (4), which is a cosmetic, a pharmaceutical, or a quasi-drug.

According to the present invention, an epithelial cell or mesenchymal cell activator is provided that can promote the expression of growth factor receptors in epithelial cells or mesenchymal cells and promote the proliferation of the cells. The epithelial cell or mesenchymal cell activator of the present invention is effective in preventing, improving, or treating disorders or deterioration of the skin or hair, such as wrinkles and sagging caused by aging, and hair loss and thinning.

The present invention will be described in detail below.
1. Growth factor receptor expression promoter or cell activator The growth factor receptor expression promoter in epithelial cells or mesenchymal cells and the epithelial cell or mesenchymal cell activator according to the present invention contain anise extract as an active ingredient.

The anise (scientific name: Pimpinella anisum L.) used in the present invention is an annual plant belonging to the Apiaceae family. In the present invention, an extract of anise refers to an extract of parts of the plant such as the seeds (which are the fruit on the plant), flowers, stems, leaves, roots, buds, etc., or the whole plant (whole plant), or a mixture thereof, with the seeds being preferred as the extraction material. In addition, for extraction, these plant bodies may be used as they are, or may be processed by drying, crushing, crushing, shredding, etc.

The extraction method for obtaining an extract from anise is not particularly limited, and may be, for example, a heated extraction method, or a room temperature or cold extraction method. Examples of the solvent used for extraction include water or hot water, lower alcohols (methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, etc.), liquid polyhydric alcohols (1,3-butylene glycol, propylene glycol, glycerin, etc.), ketones (acetone, methyl ethyl ketone, etc.), acetonitrile, esters (ethyl acetate, butyl acetate, etc.), hydrocarbons (hexane, heptane, liquid paraffin, squalane, etc.), ethers (ethyl ether, tetrahydrofuran, propyl ether, etc.), etc. These solvents may be used alone or in combination of two or more. In addition, a solvent whose pH has been adjusted by adding an acid or alkali to the above extraction solvent may be used.

There is no particular limit to the amount of solvent used, and it may be, for example, 10 times or more, preferably 20 times or more, based on the amount of anise seeds (dry weight), but it is preferable that the amount is 100 times or less for convenience of operations when concentrating or isolating after extraction. The extraction temperature and time depend on the type of solvent used, but can be, for example, 10 to 100°C, preferably 30 to 90°C, and 30 minutes to 24 hours, preferably 1 to 10 hours. More specifically, an anise extract can be obtained by adding water to anise seeds and performing hot water extraction at 95 to 100°C. Alternatively, an anise extract can be obtained by adding a lower alcohol (e.g., ethanol, etc.), a liquid polyhydric alcohol (e.g., 1,3-butylene glycol, etc.), or a hydrocarbon (e.g., hexane, etc.) to anise seeds and performing extraction at room temperature (e.g., 15 to 35°C).

After the solvent extraction, the obtained solvent phase itself can be used as an extract of anise. Alternatively, if necessary, the obtained solvent phase can be subjected to processes such as concentration (vacuum concentration, membrane concentration, etc.), dilution, filtration, and decolorization and deodorization using activated carbon, etc., and the obtained product can be used as an extract of anise. In addition, the extracted solution can be subjected to drying processes such as concentration to dryness, spray drying, and freeze drying, and the obtained dried product can be used as an extract of anise.

In the present invention, epithelial cells or mesenchymal cells include not only differentiated cells but also their precursor cells and stem cells. Epithelial cells refer to cells that have the role of covering the body surface or body cavities, and are not particularly limited as long as they are present in epithelial tissues such as the skin, hair roots, cornea, nasal cavity, pharynx, respiratory tract, oral cavity, esophagus, and digestive tract (stomach, small intestine, and large intestine), but cells present in skin tissue are preferred, and specific examples include epidermal cells (mainly epidermal keratinocytes), epidermal stem cells, hair follicle cells in the bulge region, hair follicle stem cells, outer root sheath cells, inner root sheath cells, and adipocytes. In addition, mesenchymal cells include dermal cells, fibroblasts, and hair papilla cells.

In the present invention, a "growth factor receptor" refers to a receptor that is expressed on the cell membrane surface of epithelial cells or mesenchymal cells, recognizes a growth factor that controls cell proliferation and differentiation, and transmits a signal. The cell that secretes the growth factor and the cell that has the receptor may be the same cell (mode of action: autocrine) or different cells (mode of action: endocrine, paracrine). Examples of growth factor receptors include epidermal growth factor receptor (EGFR), fibroblast growth factor receptor (FGFR), insulin-like growth factor receptor (IGFR), transforming growth factor receptor (Transforming Growth Factor Receptor), vascular endothelial growth factor receptor (VEGFR), and hepatocyte growth factor receptor (HGFR). Receptor) and the like. More specifically, EGFR includes ErbB1 (HER-1), ErbB2 (HER-2), and ErbB3 (HER-3), and FGFR includes FGFR-1 (Flg, N-Sam), FGFR-2 (Bek, K-Sam, KGFR), and FGFR-3.

In the present invention, "cell activation" means maintaining or enhancing cell proliferation or activity, and a cell activator is synonymous with an anti-aging agent.

The content of anise extract in the agent for promoting expression of growth factor receptors in epithelial cells or mesenchymal cells and the agent for activating epithelial cells or mesenchymal cells according to the present invention varies depending on the nature of the extract (liquid extract, concentrate, or dried product) and is not particularly limited, but is preferably 0.00001 to 10% by weight, and more preferably 0.0001 to 1% by weight, based on the total amount of the drug.

Anise extract can also be used as a culture medium additive to activate epithelial or mesenchymal cells when they are cultured for use in regenerative medicine. The epithelial or mesenchymal cells may be primary cultured cells, subcultured cells, or frozen cells.

2. Composition for external application to the skin The agent for promoting the expression of growth factor receptors in epithelial cells or mesenchymal cells and the agent for activating epithelial cells or mesenchymal cells of the present invention (hereinafter referred to as "the agent of the present invention") can be used as it is, but can also be incorporated into compositions such as cosmetics, medicines, and quasi-drugs together with appropriate additives within a range that does not impair the effects of the present invention. In particular, it is preferable to provide the agent by incorporating it into a composition for external application to the skin.

The agent of the present invention can promote the expression of growth factor receptors in epithelial cells or mesenchymal cells and activate the cells, and therefore a topical skin composition containing the agent is effective in treating, ameliorating, and preventing diseases or pathological conditions caused by a decrease in the function (such as the proliferation or differentiation ability) of the above-mentioned epithelial cells or mesenchymal cells. For example, diseases or conditions caused by a decrease in the function of epidermal keratinocytes or their stem cells include atopic dermatitis, psoriasis (accompanied by erythema, scales, and desquamation), delayed healing of burns and wounds, rough skin, dry skin, sensitive skin, keratinocyte hyperplasia, age spots, dullness, and open pores. Diseases or conditions caused by a decrease in the function of dermal fibroblasts or their stem cells include wrinkles, sagging skin, nasolabial folds (nasolabial folds), marionette lines, loss of firmness and elasticity, lack of moisture and luster, stiffness, dullness, solar elastosis, scleroderma, fibrosarcoma, xeroderma pigmentosum, cutaneous histiocytoma, linear atrophy of the skin (straea), bedsores, scars, birthmarks, and melasma.

The topical skin composition also includes a hair composition formulated in a formulation suitable for use on the scalp or hair. The hair composition can be used to prevent and/or improve alopecia. Here, "preventing and/or improving alopecia" includes preventing the occurrence of hair loss or thinning, improving the degree of hair loss or thinning (number or range), slowing down the rate of progression of hair loss or thinning, and inhibiting the loss of hair luster and elasticity associated with hair loss or thinning. The effect of preventing and/or improving alopecia includes both cases where the mechanism of action is direct on the hair and cases where the mechanism of action is transdermal on the head. In the present invention, alopecia refers to a condition in which part or all of the hair falls out due to aging, disease, various stresses such as ultraviolet rays and overwork, and part or all of the scalp is visible through the hair. Examples of alopecia include, but are not limited to, androgenetic alopecia (AGA), diffuse alopecia (FAGA), alopecia areata, senile alopecia, seborrheic alopecia, pityriasis graminea, postpartum (postpartum) alopecia, mechanical (compression or traction) alopecia, congenital alopecia, alopecia after burns or trauma, drug-induced alopecia due to anticancer drugs, cicatricial alopecia (lichen planopilaris, lupus erythematosus, folliculitis alopecia, papillary dermatitis of the head, etc.), and trichotillomania (hair pulling disorder).

The topical skin composition of the present invention is particularly preferably used as an anti-aging cosmetic. Here, "anti-aging" refers to suppressing the decline in the function and activity of skin cells due to various internal or external factors, such as aging and photoaging, as well as dryness, ultraviolet rays, excessive skin cleansing, hormonal imbalance, stress, and lifestyle habits (sleep and smoking habits), leading to a youthful and healthy skin condition with even texture, firmness, and luster. Specifically, it refers to preventing or improving dry skin, rough skin, wrinkles, sagging, dullness, loss of firmness, hardening, hair loss, etc., by suppressing the decline in the functions (proliferation ability, differentiation ability) of epidermal cells, dermal cells, hair papilla cells, and their stem cells.

When the agent of the present invention is incorporated into cosmetics or quasi-drugs, the dosage form may be any of an aqueous solution system, a solubilized system, an emulsion system, a powder system, a powder dispersion system, an oil liquid system, a gel system, an ointment system, an aerosol system, a water-oil two-layer system, or a water-oil-powder three-layer system. Furthermore, the cosmetics or quasi-drugs may be produced according to methods known in the art by appropriately blending various ingredients, additives, bases, etc. that are normally used in skin topical compositions together with the agent of the present invention, selected according to their types. The form may be any of a liquid, emulsion, cream, gel, paste, spray, etc. Examples of the ingredients include oils and fats (olive oil, coconut oil, evening primrose oil, jojoba oil, castor oil, hardened castor oil, etc.), waxes (lanolin, beeswax, carnauba wax, etc.), hydrocarbons (liquid paraffin, squalene, squalane, petrolatum, etc.), fatty acids (lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, etc.), higher alcohols (myristyl alcohol, cetanol, cetostearyl alcohol, stearyl alcohol, behenyl alcohol, etc.), esters (isopropyl myristate, isopropyl palmitate, iso ... propyl, cetyl octanoate, glycerin trioctanoate, octyldodecyl myristate, octyl stearate, stearyl stearate, etc.), organic acids (citric acid, lactic acid, α-hydroxyacetic acid, pyrrolidone carboxylic acid, etc.), sugars (maltitol, sorbitol, xylobiose, N-acetyl-D-glucosamine, etc.), proteins and protein hydrolysates, amino acids and their salts, vitamins, plant and animal extracts, various surfactants, moisturizers, UV absorbers, antioxidants, stabilizers, preservatives, disinfectants, fragrances, etc.

Types of cosmetics and quasi-drugs include, for example, lotion, milky lotion, gel, beauty serum, general cream, sunscreen cream, pack, mask, facial cleanser, cosmetic soap, foundation, face powder, bath additives, body lotion, body shampoo, hair shampoo, hair conditioner, scalp lotion, scalp cream, hair tonic, hair growth agent, etc.

When the agent of the present invention is incorporated into a pharmaceutical product, it can be mixed with pharmacologically and pharmacy-acceptable additives and formulated into various preparations in a dosage form suitable for application to the affected area. As pharmacologically and pharmacy-acceptable additives, excipients, thickeners, isotonicity agents, pH regulators, stabilizers, preservatives, dispersants, emulsifiers, gelling agents, dyes, fragrances, etc. can be used depending on the dosage form and use. The form suitable for the pharmaceutical product of the present invention is an external preparation, for example, an ointment, cream, gel, liquid, patch (poultice, plaster), etc. Ointments refer to homogeneous semi-solid external preparations, and include oleaginous ointments, emulsion ointments, and water-soluble ointments. Gels refer to external preparations in which a water-insoluble component hydrate compound is suspended in an aqueous liquid. Liquids refer to liquid external preparations, and include lotions, suspensions, emulsions, liniments, etc. When providing the pharmaceutical product of the present invention in each of the above forms, it can be manufactured by a method commonly used by those skilled in the art, such as the method shown in the General Provisions for Preparations [2] of the Japanese Pharmacopoeia for each formulation.

The content of the agent of the present invention in the cosmetic, pharmaceutical, or quasi-drug of the present invention is not particularly limited, but is preferably 0.001 to 30% by weight, and more preferably 0.01 to 10% by weight, calculated as the dry matter of anise extract relative to the total weight of the formulation (composition). The above amounts are merely examples, and may be set and adjusted as appropriate taking into consideration the type and form of the composition, the general amount used, efficacy, and effects. In addition, the method of adding the active ingredient in the formulation may be added in advance or during production, and may be selected as appropriate taking into consideration workability.

The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples. Note that unless otherwise specified, the percentages shown in the examples are by weight.

Example 1: Production example of anise extract The following is a production example of a solvent extract using anise.

(Production Example 1) Preparation of hot water extract of aniseed 200 mL of water was added to 10 g of aniseed, and extraction was carried out for 2 hours at 95 to 100° C. The obtained extract was filtered, and the filtrate was concentrated and freeze-dried to obtain 1.9 g of hot water extract of aniseed.

(Production Example 2) Preparation of 50% ethanol extract of aniseed 200 mL of 50% ethanol was added to 10 g of aniseed, and the mixture was soaked at room temperature for 7 days to perform extraction. The resulting extract was filtered and then concentrated to dryness using an evaporator to obtain 1.6 g of 50% ethanol extract of aniseed.

(Production Example 3) Preparation of an ethanol extract of aniseed 200 mL of ethanol was added to 10 g of aniseed, and the mixture was soaked at room temperature for 7 days to perform extraction. The resulting extract was filtered and then concentrated to dryness using an evaporator to obtain 0.4 g of an ethanol extract of aniseed.

(Production Example 4) Preparation of Hexane Extract of Anise Seeds 200 mL of hexane was added to 10 g of anise seeds, and the seeds were soaked at room temperature for 7 days to perform extraction. The resulting extract was filtered and then concentrated to dryness using an evaporator to obtain 0.3 g of a hexane extract of anise seeds.

(Production Example 5) Preparation of 1,3-butylene glycol extract of aniseed 10 g of aniseed was immersed in 200 mL of 1,3-butylene glycol at room temperature for 7 days to carry out extraction. The resulting extract was filtered to obtain 1.91 g of 1,3-butylene glycol extract of aniseed.

[Example 2] Evaluation of the skin cell activation effect of aniseed extract The skin cell activation effect of each of the aniseed seed extracts produced in Example 1 was evaluated based on the effect of promoting the expression of growth factor receptors in epithelial cells or mesenchymal cells and the effect of promoting the proliferation of the cells.

(Experimental Example 1) Evaluation of the effect of anise seed extract in promoting the expression of growth factor receptors in epidermal stem cells Commercially available normal human adult epidermal keratinocytes (Kurabo) were used as epidermis-derived cells, and epidermal stem cells were isolated using NGFR (nerve growth factor receptor: Genbank number: Nucleotide NM_002507.3; Protein NP_002498.1) as an indicator according to the method described in JP 2017-055721 A. The above-mentioned epidermal stem cells maintained in Humedia-KG2 medium (Kurabo) were seeded in a 96-well plate (Greiner) at 5 x 10 ³ cells. After 24 hours of incubation, the test substance (each anise seed extract produced in Example 1) was added to a final concentration of 100 μg/mL, and the cells were incubated for 24 hours. After incubation, the cells were fixed with 4% PFA/PBS, permeabilized with 0.2% Triton-X-100 (manufactured by Wako)/PBS, and then treated with an anti-EGFR antibody (manufactured by Gene Tex) and a fluorescently labeled secondary antibody, followed by immunostaining. The nuclei of each cell were labeled by DAPI staining. Then, the expression level of epidermal growth factor receptor (EGFR) per cell (EGFR fluorescence intensity/cell number) was calculated using a fluorescent microscope and image analysis software (ImageJ). The EGFR expression level when no test substance was added was used as a control, and the relative EGFR expression level was analyzed when the control was set to 100 (%).

(Experimental Example 2) Evaluation of the effect of anise seed extract in promoting the expression of growth factor receptors in epidermal cells Commercially available normal human adult epidermal keratinocytes (Kurabo) were used as epidermis-derived cells, and epidermal stem cells were isolated using NGFR (nerve growth factor receptor: Genbank number: Nucleotide NM_002507.3; Protein NP_002498.1) as an indicator in accordance with the method described in JP 2017-055721 A. At that time, cells that were not isolated as epidermal stem cells were seeded as epidermal cells in a 96-well plate (Greiner) at 5 x 10 ³ cells in Humedia-KG2 medium (Kurabo). After 24 hours of incubation, the test substance (each anise seed extract produced in Example 1) was added to a final concentration of 100 μg/mL, and the cells were incubated for 24 hours. After incubation, the cells were fixed with 4% PFA/PBS, permeabilized with 0.2% Triton-X-100 (manufactured by Wako)/PBS, and then treated with an anti-EGFR antibody (manufactured by Gene Tex) and a fluorescently labeled secondary antibody, followed by immunostaining. The nuclei of each cell were labeled by DAPI staining. Then, the expression level of epidermal growth factor receptor (EGFR) per cell (EGFR fluorescence intensity/cell number) was calculated using a fluorescent microscope and image analysis software (ImageJ). The EGFR expression level when the test substance was not added was used as a control, and the relative EGFR expression level was analyzed when the control was set to 100 (%).

The results obtained in Experimental Examples 1 and 2 are shown in Table 1 below.

As shown in Table 1, it was found that anise seed extract (Production Examples 1 to 5) had the effect of promoting EGFR expression in epithelial cells (epidermal stem cells and epidermal cells). In particular, the effect of promoting EGFR expression in epidermal stem cells was remarkable.

(Experimental Example 3) Evaluation of the effect of anise seed extract in promoting the expression of growth factor receptors in dermal stem cells Commercially available human dermal fibroblasts (manufactured by Toyobo Co., Ltd.) were used as dermis-derived cells, and dermal stem cells were isolated using NGFR (nerve growth factor receptor: Genbank number: Nucleotide NM_002507.3; Protein NP_002498.1) as an indicator according to the method described in JP 2017-093383 A. Dermal stem cells maintained in 15% FBS-containing α-MEM medium (manufactured by Thermo Co., Ltd.) were seeded in a 96-well plate (manufactured by Greiner Co., Ltd.) to a density of 4 x 10 ³ cells. After 24 hours of incubation, the test substance (each anise seed extract produced in Example 1) was added to a final concentration of 100 μg/mL, and incubated for 24 hours. After incubation, the cells were fixed with 4% PFA/PBS, permeabilized with 0.2% Triton-X-100 (manufactured by Wako)/PBS, and then treated with anti-FGFR1 antibody (manufactured by Gene Tex) and fluorescently labeled secondary antibody, followed by immunostaining. In addition, the nuclei of individual cells were labeled by DAPI staining. Then, the expression level of fibroblast growth factor receptor 1 (FGFR1) per cell (FGFR1 fluorescence intensity/cell number) was calculated using a fluorescent microscope and image analysis software (ImageJ). The expression level of FGFR1 in the absence of any test substance was used as a control, and the relative expression level of FGFR1 was analyzed with the control set at 100(%).

(Experimental Example 4) Evaluation of the effect of anise seed extract in promoting the expression of growth factor receptors in dermal fibroblasts Commercially available human dermal fibroblasts (manufactured by Toyobo Co., Ltd.) were used as dermis-derived cells, and dermal stem cells were isolated using NGFR (nerve growth factor receptor: Genbank number: Nucleotide NM_002507.3; Protein NP_002498.1) as an indicator according to the method described in JP 2017-093383 A. At that time, the cells that were not isolated as dermal stem cells were seeded as dermal fibroblasts in a 96-well plate (manufactured by Greiner) at 4 x 10 ³ cells in 10% FBS-containing DMEM medium (manufactured by Nacalai Co., Ltd.). After 24 hours of incubation, the test substance (each anise extract produced in Example 1) was added to a final concentration of 100 μg/mL, and incubated for 24 hours. After incubation, the cells were fixed with 4% PFA/PBS, permeabilized with 0.2% Triton-X-100 (manufactured by Wako)/PBS, and then treated with anti-FGFR1 antibody (manufactured by Gene Tex) and fluorescently labeled secondary antibody, and immunostained. In addition, the nuclei of individual cells were labeled by DAPI staining. Then, the expression level of fibroblast growth factor receptor 1 (FGFR1) per cell (FGFR1 fluorescence intensity/cell number) was calculated using a fluorescent microscope and image analysis software (ImageJ). The expression level of FGFR1 in the absence of any test substance was used as a control, and the relative expression level of FGFR1 was analyzed when the control was set at 100(%).

(Experimental Example 5) Evaluation of the effect of anise seed extract in promoting the expression of growth factor receptors in hair papilla cells Human hair was collected with tweezers, and tissue containing the hair papilla of the hair follicle tissue was collected using a scalpel or the like. After washing with PBS (-), the hair papilla wrapped in the hair matrix was extracted using tweezers or the like. The extracted hair papilla was scratched onto a culture plate, and then maintained until it became confluent using DMEM medium (manufactured by Nacalai) containing 10% FBS. The confluent cells were collected and seeded again onto the same culture plate, and the cells that subsequently engrafted and proliferated were used as hair papilla cells in the following test.

The above hair papilla cells were seeded in a 96-well plate (Greiner) at 4 x ¹⁰³ cells. After 24 hours of culture, the test substance (each anise extract produced in Example 1) was added to a final concentration of 100 μg/mL, and cultured for 24 hours. After the culture was completed, the cells were fixed with 4% PFA/PBS, permeabilized with 0.2% Triton-X-100 (Wako)/PBS, and then treated with anti-FGFR1 antibody (Gene Tex) and fluorescently labeled secondary antibody, followed by immunostaining. In addition, the nuclei of each cell were labeled by DAPI staining. Then, the expression level of fibroblast growth factor receptor 1 (FGFR1) per cell (FGFR1 fluorescence intensity/cell number) was calculated using a fluorescence microscope and image analysis software (ImageJ). The expression level of FGFR1 when no test substance was added was used as a control, and the relative expression level of FGFR1 was analyzed when the control was set to 100 (%).

The results obtained in Experimental Examples 3 to 5 are shown in Table 2 below.

As shown in Table 2, it was found that anise seed extract (Production Examples 1 to 5) had the effect of promoting FGFR1 expression in mesenchymal cells (dermal stem cells, fibroblasts, and hair papilla cells). In particular, the effect of promoting FGFR1 expression in dermal stem cells was remarkable.

(Experimental Example 6) Evaluation of the cell proliferation promoting effect of aniseed extract on epidermal stem cells The epidermal stem cells used in Experimental Example 1 were seeded in a 96-well plate (Falcon) in Humedia-KG2 medium (Kurabo) without EGF included in the kit so that the number of cells was 5 x 10 ^3. Next, the test substance (each aniseed extract produced in Example 1) was added to a final concentration of 100 μg/mL, or to a final concentration of 10 μM when EGF was added, and cultured for 24 hours. After the culture was completed, the cells were washed with PBS (-) and replaced with a medium of Cell Counting Kit-8 (Dojindo):DMEM (Nacalai) = 10:1, and cultured for 2 hours, and then the absorbance at 450 nm and 650 nm was measured using a microplate reader. The number of cells was calculated by subtracting the absorbance value at 650 nm from the absorbance value at 450 nm. The number of cells without the addition of the sample was used as a control. The increase or decrease (%) in the number of cells when the test substance was added was calculated relative to the control being 100 (%), and the effect of promoting epidermal stem cell proliferation was evaluated.

(Experimental Example 7) Evaluation of the cell proliferation promoting effect of aniseed extract on epidermal cells The epidermal cells used in Experimental Example 2 were seeded in a 96-well plate (Falcon) in Humedia-KG2 medium (Kurabo) without EGF included in the kit so that the number of cells was 5 x ^103. Next, the test substance (each aniseed extract produced in Example 1) was added to a final concentration of 100 μg/mL, or to a final concentration of 10 μM when EGF was added, and cultured for 24 hours. After the culture was completed, the cells were washed with PBS (-) and replaced with a medium of Cell Counting Kit-8 (Dojindo):DMEM (Nacalai) = 10:1, and cultured for 2 hours, and then the absorbance at 450 nm and 650 nm was measured using a microplate reader. The number of cells was calculated by subtracting the absorbance value at 650 nm from the absorbance value at 450 nm. The number of cells without the addition of the sample was used as a control. The increase or decrease (%) in the number of cells when the test substance was added was calculated relative to the control value of 100 (%) to evaluate the effect of promoting epidermal cell proliferation.

The results obtained in Experimental Examples 6 and 7 are shown in Table 3 below.

As shown in Table 3, it was found that anise seed extract (Production Examples 1 to 5) had an excellent proliferation-promoting effect on epithelial cells (epidermal stem cells, epidermal cells) in the presence of EGF (EGF(+)). Since this effect was not observed in the absence of EGF (EGF(-)), it was believed that the proliferation-promoting effect of anise seed extract on the above cells was due to the promotion of the expression of growth factor receptors, not the promotion of growth factor expression.

(Experimental Example 8) Evaluation of the cell proliferation promoting effect of aniseed extract on dermal stem cells The dermal stem cells used in Experimental Example 3 were seeded in a 96-well plate (Falcon) in 1% FBS-containing α-MEM medium (Thermo) so that the number of cells was 3×10 ^3. Next, the test substance (each aniseed extract produced in Example 1) was added to a final concentration of 100 μg/mL, and when FGF was added, the final concentration was 10 μM, and the cells were cultured for 24 hours. After the culture was completed, the cells were washed with PBS (-) and replaced with a medium of Cell Counting Kit-8 (Dojindo):DMEM (Nacalai) = 10:1, and after 2 hours of culture, the absorbance at 450 nm and 650 nm was measured using a microplate reader. The number of cells was calculated by subtracting the absorbance value at 650 nm from the absorbance value at 450 nm. The number of cells without the addition of the sample was used as a control. The increase or decrease (%) in the number of cells when the test substance was added was calculated relative to the control being 100 (%), and the effect of promoting dermal stem cell proliferation was evaluated.

(Experimental Example 9) Evaluation of the cell proliferation promoting effect of aniseed extract on dermal fibroblasts The dermal fibroblasts used in Experimental Example 4 were seeded in a 96-well plate (Falcon) in 1% FBS-containing DMEM medium (Nacalai) to a cell number of 3 x 10 ³ . Next, the test substance (each aniseed extract produced in Example 1) was added to a final concentration of 100 μg/mL, and when FGF was added, the final concentration was 10 μM, and the cells were cultured for 24 hours. After the culture was completed, the cells were washed with PBS (-), and replaced with a medium of Cell Counting Kit-8 (Dojindo):DMEM (Nacalai) = 10:1, and after 2 hours of culture, the absorbance at 450 nm and 650 nm was measured using a microplate reader. The number of cells was calculated by subtracting the absorbance value at 650 nm from the absorbance value at 450 nm. The number of cells without the addition of the sample was used as a control. The increase or decrease (%) in the number of cells when the test substance was added was calculated relative to the control of 100 (%) to evaluate the effect of promoting proliferation of dermal fibroblasts.

(Experimental Example 10) Evaluation of the cell proliferation promoting effect of aniseed extract on hair papilla cells The hair papilla cells used in Experimental Example 5 were seeded in a 96-well plate (Falcon) in 1% FBS-containing DMEM medium (Nacalai) to a cell count of ³ x 103. Next, the test substance (each aniseed extract produced in Example 1) was added to a final concentration of 100 μg/mL, and when FGF was added, the final concentration was 10 μM, and the cells were cultured for 24 hours. After the culture was completed, the cells were washed with PBS (-) and replaced with a medium of Cell Counting Kit-8 (Dojindo):DMEM (Nacalai) = 10:1, and after 2 hours of culture, the absorbance at 450 nm and 650 nm was measured using a microplate reader. The number of cells was calculated by subtracting the absorbance value at 650 nm from the absorbance value at 450 nm. The number of cells without the addition of the sample was used as a control. The increase or decrease (%) in the number of cells when the test substance was added was calculated relative to the control being 100 (%), and the effect of promoting proliferation of hair papilla cells was evaluated.

The results obtained in Experimental Examples 8 to 10 are shown in Table 4 below.

As shown in Table 4, it was confirmed that anise seed extract (Production Examples 1 to 5) has a proliferation-promoting effect on mesenchymal cells (dermal stem cells, fibroblasts, and hair papilla cells) in the presence of FGF (FGF(+)). Since this effect was not observed in the absence of FGF (FGF(-)), it was believed that the proliferation-promoting effect of anise seed extract on the above cells was due to the activation of growth factor receptors, not the promotion of growth factor expression.

INDUSTRIAL APPLICABILITY The present invention is useful in the fields of manufacturing anti-aging cosmetics, quasi-drugs, and pharmaceuticals, and in the fields of tissue regeneration and homeostasis maintenance, such as regenerative medicine, regenerative cosmetics, and anti-aging.

Claims (2)

A growth factor receptor expression promoter in epithelial cells or mesenchymal cells, containing anise extract as an active ingredient. The agent according to claim 1 , wherein the epithelial cells or mesenchymal cells are present in skin tissue.