Hair growth agent, hair papilla cell proliferation promoter, and method of using the same

Abstract

To provide a hair tonic, a hair papilla cell proliferation promoter and a biomolecule expression promoter.SOLUTION: Provided are a hair tonic, a hair papilla cell proliferation promoter, a WINT5A expression promoter, a vascular endothelial growth factor (VEGF) expression promoter, a fibroblast growth factor-7 (FGF-7) expression promoter, a tissue non-specific alkaline phosphatase (ALPL) expression promoter, a versican expression promoter, a tenascin C expression promoter, a chondroitin sulfate proteoglycan 4 (CSPG4) expression promoter, and a fibronectin expression promoter, which contain a fermentation product of extract from Calendula, extract from Fallopia and / or a fermentation product of extract from Fallopia as an active component. A hair restoration effect can be obtained, and symptoms such as thinning hair and hair loss can be prevented, improved, or treated.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a hair growth agent, a hair papilla cell proliferation promoter, and a biomolecule expression promoter. More specifically, the present invention relates to a hair growth agent, a hair papilla cell proliferation promoter, a WINT5A expression promoter, a vascular endothelial growth factor (VEGF) expression promoter, a fibroblast growth factor-7 (FGF-7) expression promoter, a tissue non-specific alkaline phosphatase (ALPL) expression promoter, a versican expression promoter, a tenascin-C expression promoter, a chondroitin sulfate proteoglycan 4 (CSPG4) expression promoter, and a fibronectin expression promoter, each of which contains as an active ingredient a fermented Calendula extract, a Fallopia extract, and / or a fermented Fallopia extract. [Background technology]

[0002] Hair plays an important role in maintaining life, such as maintaining body temperature and protecting the body surface, and in humans, it is distributed over the skin of the entire body except for the palms, soles of the feet, and parts of the pubic area. Hair grows out according to a hair cycle (for human scalp hair, one cycle lasts 2 to 6 years) consisting of the stages of growth, regression, and resting. An average human adult has 100,000 to 150,000 hairs on the scalp, of which 85 to 90% are in the growth phase (unless there is any symptom of thinning or hair loss), and 50 to 100 hairs in the resting phase fall out each day, but the density is kept almost constant by hair growth.

[0003] Hair is produced in an appendage of the skin called a hair follicle. The structure of a hair follicle is complex, with the hair shaft (commonly known as hair) at the center, the hair shaft surrounded by the inner root sheath (IRS), and the outermost layer wrapped in the outer root sheath (ORS). The lower part of the hair follicle in the anagen phase is spherical and bulging, and is called the hair bulb. The hair bulb contains the dermal papilla, which is a mesenchymal tissue, at its center. The dermal papilla is composed of fibroblast-like dermal papilla cells, capillaries, and intercellular matrix. The part of the epithelial system in contact with the dermal papilla where cell division is active is called the hair matrix matrix, and the cells that make up this matrix are called hair matrix cells. In the anagen phase, hair matrix cells proliferate and differentiate, causing hair to elongate, and in the catagen phase, apoptosis occurs in the lower tissue of the hair follicle, including the hair matrix, causing hair growth to stop, and the hair follicle that had extended deep into the dermis begins to retract. During the telogen phase, the hair follicle shortens close to the epidermis and takes on a rod-like shape called a club hair. During this phase, macrophages in the hair papilla phagocytose melanin pigment and cellular debris. When the growth phase begins again, hair matrix cells begin to divide and new hairs are produced, which push the club hair out. In general, the thickness of the hair is proportional to the size of the hair follicle, and the length of the hair is proportional to the duration of the growth phase. Close interaction between the hair matrix and the hair papilla cells that support it is essential for the hair cycle process, and if there is an abnormality in this regulation, problems will occur with the thickness, length, shape (wavy hair, etc.), and color of the hair.

[0004] On the other hand, in recent years, due to various factors such as increased stress and changes in diet, the number of men and women suffering from thinning hair (symptoms of hair becoming thinner and shorter) and hair loss (hair loss) is on the rise, and the demand for hair growth agents is increasing. In response to such social demands, various hair growth agents have been developed, and for example, Patent Document 1 discloses a hair growth agent containing, as an active ingredient, an extract of one or more plants selected from the group consisting of phoenix tree, white eyebrow grass, and false hawk claw. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent No. 5676837 Summary of the Invention [Problem to be solved by the invention]

[0006] However, it cannot be said that a hair growth agent that fully satisfies the demands of consumers is still being supplied. The present invention has been made to solve the above problems, and aims to provide a hair growth agent, a hair papilla cell proliferation promoter, a WINT5A expression promoter, a VEGF expression promoter, an FGF-7 expression promoter, an ALPL expression promoter, a versican expression promoter, a tenascin-C expression promoter, a CSPG4 expression promoter, and a fibronectin expression promoter, each of which contains a fermented product of a Calendula extract, a Convolvulus extract, and / or a fermented product of a Convolvulus extract as an active ingredient. [Means for solving the problem]

[0007] As a result of intensive research, the present inventors have found that the fermented product of Calendula extract, the extract of Bucca genus, and the fermented product of Bucca genus extract promote the proliferation of hair papilla cells.Furthermore, they have found that the fermented product of Calendula extract, the extract of Bucca genus, and the fermented product of Bucca genus extract promote the expression of a wide range of biomolecules involved in hair growth.Based on these findings, the present inventors have completed the following inventions.

[0008] (1) The hair growth agent of the present invention contains one or more of the following (a) to (c) as an active ingredient: (a) a fermented product of an extract of the genus Calendula, (b) an extract of the genus Convolvulus, and (c) a fermented product of an extract of the genus Convolvulus.

[0009] (2) The hair papilla cell proliferation promoter of the present invention contains one or more of the following (A) to (C) as an active ingredient: (A) a fermented product of an extract of the genus Calendula, (B) an extract of the genus Convolvulus, and (C) a fermented product of an extract of the genus Convolvulus.

[0010] (3) The WINT5A expression promoter of the present invention contains one or more of the following (a) to (c) as an active ingredient: (a) a fermented product of an extract of the genus Calendula, (b) an extract of the genus Convolvulus, and (c) a fermented product of an extract of the genus Convolvulus.

[0011] (4) The VEGF expression promoter of the present invention contains one or more of the following (a) to (c) as an active ingredient: (a) a fermented product of an extract of the genus Calendula, (b) an extract of the genus Convolvulus, and (c) a fermented product of an extract of the genus Convolvulus.

[0012] (5) The FGF-7 expression promoter of the present invention contains one or more of the following (a) to (c) as an active ingredient: (a) a fermented product of an extract of the genus Calendula, (b) an extract of the genus Convolvulus, and (c) a fermented product of an extract of the genus Convolvulus.

[0013] (6) The ALPL expression promoter of the present invention contains the following (a) or (b) as an active ingredient: (a) a fermented product of an extract of the genus Calendula, or (b) an extract of the genus Bindweed.

[0014] (7) The agent for promoting the expression of versican according to the present invention contains the following (a) or (b) as an active ingredient: (a) a fermented product of an extract of the Calendula genus, or (b) an extract of the Bindweed genus.

[0015] (8) The tenascin-C expression promoter of the present invention contains one or more of the following (a) to (c) as an active ingredient: (a) a fermented product of an extract of the genus Calendula, (b) an extract of the genus Convolvulus, and (c) a fermented product of an extract of the genus Convolvulus.

[0016] (9) The CSPG4 expression promoter according to the present invention comprises the following (a) or (b) as an active ingredient: (a) a fermented product of an extract of the Calendula genus, or (b) an extract of the Bindweed genus.

[0017] (10) The fibronectin expression promoter of the present invention comprises the following (a) or (b) as an active ingredient: (a) a fermented extract of the Calendula genus, or (b) an extract of the Bindweed genus.

[0018] (11) In the agent according to the present invention, the fermented product may be a fermented product obtained by fermentation with lactic acid bacteria or yeast.

[0019] (12) In the agent according to the present invention, the lactic acid bacterium may be Lactobacillus plantarum.

[0020] (13) In the agent according to the present invention, the Calendula genus may be Calendula officinalis.

[0021] (14) In the agent according to the present invention, the genus Convolvulus may be Polygonum grandiflorum. Effect of the Invention

[0022] According to the present invention, it is possible to obtain hair growth effects such as increasing the length of hair, increasing the density of hair, or making hair thicker. Therefore, it is possible to prevent, improve, or treat symptoms of thinning hair or hair loss.

[0023] Dermal papilla cells produce many cell growth factors (insulin-like growth factor-1 (IGF-1), hepatocyte growth factor (HGF), VEGF, FGF, etc.) and are deeply involved in the division and differentiation of hair matrix cells. In addition, signals for the transition from the resting phase to the anagen phase of the hair cycle are also emitted from dermal papilla cells (Katsuoka, Norio, Is the main player in hair growth induction dermal papilla cells? Hair follicle stem cells?, [online], broadcast on August 25, 2011, Radio Nikkei, Maruho Dermatology Seminar, [searched on May 18, 2020], Internet<URL: http: / / medical.radionikkei.jp / maruho_hifuka_pdf / maruho_hifuka-110825.pdf> ) As is clear from these findings, dermal papilla cells play an important role in hair growth. According to the present invention, it is possible to promote the proliferation of such dermal papilla cells, thereby achieving a hair growth effect.

[0024] WINT5A (Wint 5A, Wnt5A) is a protein belonging to the Wnt family. The Wnt family is a secretory signal molecule involved in intercellular signal transduction, and is composed of 300 to 400 lipid-modified amino acids. WINT5A is known to be involved in morphogenesis accompanying embryonic development, as well as to promote insulin secretion (JP Patent Publication No. 2005-220022) and suppress apoptosis of hair papilla cells (JP Patent Publication No. 2008-133233). From these facts, if the expression of WINT5A can be promoted in the living body, it is considered to be effective in the prevention and treatment of diabetes, impaired glucose tolerance, insulin resistance, and the like, or in hair growth. According to the present invention, it is possible to promote the expression of such useful WINT5A. The present invention can be used in all applications in which it is meaningful to promote the production of WINT5A.

[0025] VEGF is a glycoprotein of 34-46 kDa that promotes the proliferation, migration, and differentiation of vascular endothelial cells and enhances vascular permeability of microvessels, and is also involved in the activation of monocytes and macrophages. VEGF is produced by various cells in the body (e.g., pituitary cells, smooth muscle cells, macrophages, alveolar epithelial cells, hepatic cells, ORS cells of hair follicles, and hair papilla cells, etc.). Inhibition of VEGF production in hair follicles leads to a delay in the anagen phase of the hair cycle and a decrease in hair follicle size (J. Clin. Invest., 2001, Vol. 107, p. 409-411), indicating that VEGF plays an important role in the regeneration and development of hair follicles (Patent No. 5676837). In addition, VEGF has been reported to promote autocrine proliferation of hair papilla cells in addition to angiogenesis (Lachger S, Moukadiri H, Jonca F, Charveron M, Bouhaddioui N, Vascular endothelial growth factor is an autocrine growth factor for hair dermal papilla cells. J Invest Dermatol 106, 17-23, 1996). From these findings, if the expression of VEGF can be promoted in the living body, it is considered to be effective in promoting angiogenesis and the regeneration and development of hair follicles, i.e., hair growth. According to the present invention, it is possible to promote the expression of such useful VEGF. The present invention can be used for all applications in which it is meaningful to promote the production of VEGF.

[0026] FGF-7 is a 17-30 kDa protein belonging to the fibroblast growth factor (FGF) family, and is also called keratinocyte growth factor (KGF). FGF-7 mainly exerts a growth-promoting effect specifically on epithelial cells (keratinocytes) and is involved in morphogenesis, angiogenesis, and wound healing. In addition, studies using mouse and rat homologs have reported that FGF-7 is involved in the morphogenesis of epithelial tissues, re-epithelialization of wounds, angiogenesis, hair growth, etc. (NCBI, GenBank, Homo sapiens fibroblast growth factor 7 (FGF7), mRNA, NCBI Reference Sequence: NM_002009.4, Summary, [online], [Retrieved May 18, 2020], Internet<URL: https: / / www.ncbi.nlm.nih.gov / nuccore / NM_002009> ). From these facts, if the expression of FGF-7 can be promoted in the body, it is considered to be effective for the proliferation and tissue repair of desired epithelial cells, and for promoting the proliferation of hair matrix cells, i.e., hair growth. According to the present invention, it is possible to promote the expression of such useful FGF-7. The present invention can be used for all applications in which it is meaningful to promote the production of FGF-7.

[0027] Alkaline phosphatase (ALP; EC 3.1.3.1) is an enzyme that hydrolyzes phosphate monoester bonds at alkaline pH (pH 9-11) and consists of approximately 500 amino acids. There are four known types of alkaline phosphatase family: liver / kidney / bone type (tissue-nonspecific type), small intestine type, placenta type, and germ cell type (placenta-like type). Among these, tissue-nonspecific ALP (ALPL) is expressed in a wide range of tissues and cells, including liver, bone, kidney, testis, fibroblasts, and macrophages (Yoko Ishida et al., Structure and Function of Alkaline Phosphatase, Clinical Chemistry 33, 36-44, 2004). ALPL is involved in the calcification of hard tissues (bone and teeth), and is known to be a marker of hair papilla, and is specifically expressed in hair papilla cells during the growth phase. It has also been reported that hair papilla cells with high ALPL activity have high hair follicle induction ability. From these findings, if the expression of ALPL can be promoted in a living body, it is considered to be effective for calcification of hard tissues such as bones and teeth, and activation of hair papilla cells, i.e., hair growth. According to the present invention, the expression of such useful ALPL can be promoted. The present invention can be used for all applications in which promoting the production of ALPL is meaningful.

[0028] Versican is a large chondroitin sulfate proteoglycan of 1000 kDa or more, a component of the extracellular matrix, and is distributed relatively widely in tissues such as blood vessels, brain, skin, and cartilage. Versican is involved in cell-cell adhesion, cell proliferation, migration, and angiogenesis, and also plays a major role in the morphogenesis and maintenance of tissues. In addition, versican is specifically expressed in the hair papilla (Kishimoto et al., PNAS, 96, 7336-7341), and it has been reported that the addition of its metabolic product, versikine (DPEAAE), to cultured hair papilla cells promotes their proliferation (Takada Keiko et al., Grant-in-Aid for Scientific Research (2014), Issue No. 25861705, 2015). From these facts, if the expression of versican can be promoted in the living body, it is considered to be effective in regenerative medicine, wound healing, and the promotion of hair papilla cell proliferation, i.e., hair growth. According to the present invention, the expression of such useful versican can be promoted. The present invention can be used in all applications in which it is meaningful to promote the production of versican.

[0029] Tenascin C is a glycoprotein of the extracellular matrix. The monomer has a molecular weight of 200 to 400 kKa, and is usually present in tissues as a hexamer. During fetal development (embryonic development), tenascin C is expressed in the nerves, muscles, and vascular systems, and is involved in morphogenesis. Meanwhile, in adults, tenascin C is expressed transiently in limited areas associated with wound (inflammation) healing and tissue regeneration. It is also expressed in hair follicles in the growth phase (WO 2016 / 079912). From these facts, if the expression of tenascin C can be promoted in the living body, it is considered to be effective in regenerative medicine, wound healing, and promotion of the hair follicle growth phase, i.e., hair growth. According to the present invention, it is possible to promote the expression of such useful tenascin C. The present invention can be used in all applications in which it is meaningful to promote the production of tenascin C.

[0030] Chondroitin sulfate proteoglycan 4 (CSPG4), also known as NG2 (Neuron-gial angigen 2), is a transmembrane proteoglycan that is a giant molecule consisting of a core protein of approximately 300 kDa bound to chondroitin sulfate. It is expressed widely in the body, including tumor cells, central nervous progenitor cells (NG2 glia), pericytes, mesenchymal stem cells, pigment cells, smooth muscle cells, and macrophages, and has been reported to be involved in cell survival, cell migration, and angiogenesis (Ampofo et al., Cellular & Molecular Biology Letters, 2017, 22, 4, DOI 10.1186 / s11658-017-0035-3). It is also expressed in the bulge of the hair follicle and the area where hair matrix cells exist at the bottom of the hair follicle. Its expression decreases during the catagen phase of the hair cycle, does not change during the telogen phase, and increases and reaches a peak during the anagen phase. That is, hair neogenesis or growth correlates with the expression intensity of CSPG4 (Yasuhisa Tamura, Kumi Takata, Asami Eguchi, Yosky Kataoka, "In vivo monitoring of hair cycle stages via bioluminescence imaging of hair follicle NG2 cells", Scientific Reports, doi: 10.1038 / s41598-017-18763-3). From these findings, if CSPG4 expression can be promoted in the living body, it is considered to be effective in regenerative medicine and hair neogenesis or growth, i.e., hair growth. According to the present invention, it is possible to promote the expression of such useful CSPG4. The present invention can be used for all applications in which it is meaningful to promote the production of CSPG4.

[0031] Fibronectin is a huge glycoprotein with a monomer length of 2146-2325 amino acids. Although there is only one gene, several dozen protein isoforms are generated by alternative splicing, which results in four types of fibronectin: plasma fibronectin (present in plasma), cellular fibronectin (present on the cell surface and in the extracellular matrix), fetal fibronectin (present in amniotic fluid), and single-chain fibronectin (present as a single chain without polymerizing). In in vitro studies, fibronectin promotes cell adhesion, growth, migration, and differentiation, and in vivo, it is thought to play many roles that support vital functions, such as adhesion of cells to the extracellular matrix, formation and maintenance of connective tissue, wound healing, and formation and maintenance of the morphology and compartments of tissues and organs during embryonic development. Fibroblasts and hepatocytes are typical synthesizing cells, but almost all cells, including undifferentiated chondrocytes, muscle cells, neutrophils, macrophages, Schwann cells, keratinocytes, and epithelial cells, synthesize fibronectin and secrete it into the cells. It is also present in body fluids and in the connective tissues and basement membranes of almost all organs, such as the kidney, brain, muscle, and cartilage. In hair follicles, it is distributed throughout the connective tissue root sheath, but fibronectin in the hair papilla disappears during the regression phase to the telogen phase, and is strongly expressed in the lower part of the hair tissue in the early anagen phase (Masaaki Ito, Histochemistry of Hair Tissue, Juntendo Medicine 37(4), 523-534, 1992). From these facts, if the expression of fibronectin can be promoted in the living body, it is considered to be effective in regenerative medicine, wound healing, and promotion of the hair follicle anagen phase, i.e., hair growth. According to the present invention, it is possible to promote the expression of such useful fibronectin. The present invention can be used in all applications in which it is meaningful to promote the production of fibronectin. [Brief description of the drawings]

[0032] [Figure 1] 1 is a bar graph showing the cell proliferation promotion rate in hair dermal papilla cells cultured in the presence of various plant extracts. [Diagram 2] 1 is a bar graph showing the cell proliferation promotion rate in hair dermal papilla cells cultured in the presence of various plant extracts. [Diagram 3]FIG. 1 shows the expression ratio of Wint5A in hair follicle papilla cells cultured in the presence of various plant extracts. [Figure 4] FIG. 1 is a diagram showing the expression ratio of Wint5A in hair follicle papilla cells cultured in the presence of a fermented product of Calendula officinalis extract or adenosine. [Diagram 5] FIG. 1 shows the expression ratio of VEGF genes in hair papilla cells cultured in the presence of various plant extracts. [Figure 6] FIG. 1 shows the expression ratio of VEGF gene in hair papilla cells cultured in the presence of a fermented calendula extract or a Panax ginseng root extract. [Figure 7] FIG. 1 shows the expression ratio of FGF-7 gene in hair papilla cells cultured in the presence of various plant extracts. [Figure 8] FIG. 1 shows the expression ratio of FGF-7 gene in hair dermal papilla cells cultured in the presence of a fermented Calendula officinalis extract or a non-fermented Calendula officinalis extract. [Figure 9] FIG. 1 shows the expression ratio of ALPL gene in hair papilla cells cultured in the presence of Knotweed extract. [Figure 10] FIG. 1 shows the expression ratio of ALPL gene in hair papilla cells cultured in the presence of fermented calendula officinalis extract, unfermented calendula officinalis extract, ginseng root extract or adenosine. [Figure 11] FIG. 1 shows the expression ratio of the versican gene in hair papilla cells cultured in the presence of various plant extracts. [Figure 12] FIG. 13 is a diagram showing the expression ratio of the versican gene in hair papilla cells cultured in the presence of a fermented product of Calendula officinalis extract or adenosine. [Figure 13] FIG. 1 shows the expression ratio of tenascin-C gene in hair papilla cells cultured in the presence of various plant extracts. [Figure 14] FIG. 1 shows the expression ratio of tenascin-C gene in hair papilla cells cultured in the presence of fermented calendula officinalis extract, unfermented calendula officinalis extract, ginseng root extract or adenosine. [Figure 15] FIG. 1 shows the expression ratio of CSPG4 genes in hair papilla cells cultured in the presence of various plant extracts. [Figure 16] FIG. 1 shows the expression ratio of CSPG4 genes in hair papilla cells cultured in the presence of fermented calendula officinalis extract, unfermented calendula officinalis extract, ginseng root extract or adenosine. [Figure 17] FIG. 1 shows the expression ratio of fibronectin gene in hair papilla cells cultured in the presence of various plant extracts. [Figure 18] FIG. 1 shows the expression ratio of fibronectin gene in hair papilla cells cultured in the presence of a fermented Calendula officinalis extract or a non-fermented Calendula officinalis extract. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] The present invention will be described in detail below. The hair growth agent, hair papilla cell proliferation promoter, WINT5A expression promoter, vascular endothelial growth factor (VEGF) expression promoter, fibroblast growth factor-7 (FGF-7) expression promoter, tissue non-specific alkaline phosphatase (ALPL) expression promoter, versican expression promoter, tenascin-C expression promoter, chondroitin sulfate proteoglycan 4 (CSPG4) expression promoter, and fibronectin expression promoter (hereinafter, these agents may be collectively referred to as "the agent") according to the present invention contain one or more of the following (a) to (c) as active ingredients; (a) Fermented extract of Calendula genus, (a) Bindweed extract, (c) Fermented extract of the genus Convolvulus.

[0034] In the present invention, "hair growth" refers to increasing the length of hair, increasing hair density (increasing the number of hairs), making hair thicker, or making hair stronger, and is used interchangeably with "hair growth" and "hair care".

[0035] In the present invention, "promoting expression" of a biological molecule such as WINT5A, VEGF, FGF-7, ALPL, versican, tenascin-C, CSPG4 and fibronectin means increasing the amount of transcription of a gene encoding all or part of the biological molecule in any cell, tissue or organ of a living organism, increasing the amount of the biological molecule, or increasing the activity of the biological molecule.

[0036] The genus Calendula refers to plants belonging to the genus Calendula in the family Asteraceae. Examples of the genus Calendula include Calendula officinalis (pot marigold, calendula) and Calendula arvensis (Japanese calendula, winter marigold, winter calendula).

[0037] The term "convolvulus" refers to plants belonging to the genus Convolvulus in the family Polygonaceae. Examples of the genus Convolvulus include Japanese knotweed (Follopia sachalinensis, Polygonum sachalinense, Reynoutria sachalinense), Japanese knotweed (Follopia japonica), Japanese knotweed (Follopia dumetora), and Japanese knotweed (Follopia dentatoalata). There is also a theory that these plants are included in the genus Convolvulus. In addition, the genera Convolvulus and Japanese knotweed were once classified as the genus Polygonum. That is, in the present invention, the genus Convolvulus is used interchangeably with the genus Polygonum and the genus Polygonum.

[0038] In the present invention, the extract refers to an extract from all or part of the tissue of a plant (hereinafter, sometimes referred to as "plant extract"). The extract can be prepared by immersing the plant in a solvent and dissolving the components contained in the plant into the solvent. Here, the solvent or extraction method can be appropriately selected depending on the type and part of the plant, the desired functionality, etc.

[0039] A more detailed example of the method for producing the extract is to soak one or more parts of the plant body selected from the roots, stems, leaves, inflorescences, and petals in a solvent for a certain period of time to produce an extract. Here, the plant body may be used as is, or may be dried or crushed before use. Examples of the solvent include water, lower alcohols (ethanol, propanol, etc.), glycols (glycerin, 1,3-butylene glycol, propylene glycol, 1,3-propanediol, etc.), and mixtures of water, lower alcohols, and / or glycols, but there is no particular restriction as long as the solvent can extract the components contained in the plant body. Examples of the amount of the solvent include 5 to 20 parts by weight per part by weight of the plant body. Examples of the extraction temperature include 4 to 90°C, and the extraction time is 1 hour to 2 weeks.

[0040] The extract of Calendula genus may be prepared by the above-mentioned procedure, or may be a commercially available product, such as Calendula officinalis extract-J (Maruzen Pharmaceuticals), Calendula officinalis extract BG-J (Maruzen Pharmaceuticals), Falcolex calendula officinalis (Ichimaru Falcos), Marigold Herbasol extract BG (Lipoid Kosmetik AG), etc.

[0041] The extract of the genus Convolvulus may be prepared by the above-mentioned procedure, or a commercially available product such as Veraherbline (Unial) may be used.

[0042] In the present invention, the fermented product of a plant extract refers to a culture obtained by culturing a microorganism in a medium containing the plant extract or using the plant extract itself as a medium. Examples of the microorganism for obtaining the fermented product include yeast, fungi such as Aspergillus (A. niger), Acremonium, Trichoderma, Rhizopus, and Monascus, and bacteria such as lactic acid bacteria, Penicillium, Bacillus, Acetobacter, and Gluconobacter.

[0043] Yeast refers to fungi that spend most of their life cycle as single cells and grow by budding or division. For example, commonly used yeasts such as baker's yeast, sake yeast, wine yeast, and brewer's yeast can be used. More specifically, examples of yeasts include the genera Saccharomyces, Shizosaccharomyces, Pichia, Candida, Kluyveromyces, Williopsis, Debaryomyces, Galactomyces, Torulaspora, Rhodotorula, Yarrowia, and Zygosaccharomyces. Examples of the genus Saccharomyces include Saccharomyces cerevisiae, Saccharomyces sake, and Saccharomyces beticus. Examples of the genus Candida include Candida tropicalis, Candida lypolitica, Candida utilis, and Candida sake.

[0044] Yeasts such as baker's yeast, sake yeast, wine yeast, and beer yeast are commercially available, and in addition to these, yeasts that can be stored for a storage period and distributed, such as the type strain of Saccharomyces cerevisiae (NBRC 10217), can be used in the present invention. Also, yeasts may be isolated from available foods and the like according to known methods (e.g., Atsuko Kono et al., Identification and Bread-making Characteristics of Yeasts Isolated from Raisins, Seinan Jogakuin University Bulletin Vol. 14, pp. 77-82, 2010).

[0045] Lactic acid bacteria are bacteria that produce lactic acid through metabolism. Examples of lactic acid bacteria include lactic acid bacteria of the genus Lactobacillus, such as Lactobacillus plantarum (NBRC 101975), Lactobacillus delbrueckii (NBRC 13953), Lactobacillus brevis (NBRC 3345), and Lactobacillus casei (NBRC 15883); lactic acid bacteria of the genus Leuconostoc, such as Leuconostoc mesenteroides (NBRC 110676) and Leuconostoc citreum (NBRC 102476); and Streptococcus faecalis (NBRC 102476). Lactic acid bacteria of the genus Streptococcus, such as Streptococcus pyogenes (NBRC 3989) and Streptococcus pyogenes; lactic acid bacteria of the genus Enterococcus, such as Enterococcus casseliflavus (NBRC 3531) and Enterococcus sulfreus (NBRC 100680); lactic acid bacteria of the genus Lactococcus, such as Lactococcus plantarum (NBRC 100936) and Lactococcus rafinolactis (NBRC 100932); lactic acid bacteria of the genus Lactococcus, such as Pediococcus damnosus (NBRC 3889) and Pediococcus Examples of such lactic acid bacteria include lactic acid bacteria of the genus Pediococcus, such as Pediococcus pentosaceus (NBRC 3182). Among these lactic acid bacteria, the genus Lactobacillus is preferred, and Lactobacillus plantarum and Lactobacillus delbrueckii are more preferred, from the viewpoint of the effectiveness of the fermented product obtained and the fact that they are not extremely anaerobic and are easy to handle.

[0046] As the Aspergillus genus, for example, commercially available koji molds such as Aspergillus oryzae, Aspergillus sojae, Aspergillus awamori, and Aspergillus glaucus can be used. Examples of the Acremonium genus that can be used include Acremonium sp. (NBRC 30052), Penicillium genus such as Penicillium pinophilum (NBRC 33285), Trichoderma genus such as Trichoderma reesei (NBRC 31328), and Gluconobacter genus such as Gluconobacter sp., Gluconobacter frateurii, Gluconobacter cerinus, Gluconobacter oxydans (NBRC 3292), Gluconobacter albidus, and the like.

[0047] The fermented product of the plant extract can be obtained, for example, by the following method. That is, a microorganism previously cultured is inoculated into the plant extract prepared by the above method, and cultured under conditions favorable for microbial fermentation with or without the addition of a medium to prepare a culture. When a medium is added, the medium is not particularly limited as long as it can culture the microorganism.

[0048] For example, when a fermentation product is obtained using yeast, the medium used for preliminary culture or for obtaining a fermentation product can be appropriately set according to the species or strain of the yeast used. For example, in the case of Saccharomyces cerevisiae, examples of the medium include YM liquid medium (10 g glucose, 5 g peptone, 3 g yeast extract, 3 g malt extract, 1 L purified water, pH 5.4), YEP medium (20 g peptone, 10 g yeast extract, 1 L purified water, pH 6.5), and YPD medium (20 g glucose, 20 g peptone, 10 g yeast extract, 1 L purified water, pH 6.5). The culture temperature depends on the species or strain used, but examples of the culture period are about 20 to 40°C and about 12 to 14 days. The culture is performed under aerobic conditions, either stationary or with aeration and / or stirring as necessary. The amount of aeration can be, for example, about ¼ to the same amount of the fermentation liquid volume per minute of sterile air blown into the bottom of the fermentation tank. The stirring conditions can be, for example, 100 to 400 revolutions per minute (rpm).

[0049] For example, when a fermented product is obtained by lactic acid bacteria, the medium used for preliminary culture or for obtaining a fermented product can be MRS medium (10 g peptone, 10 g beef extract, 5 g yeast extract, 20 g glucose, 1 g Tween 80, 2 g K2HPO4, 5 g sodium acetate, 2 g diammonium citrate, 0.2 g MgSO4·7H2O, 0.05 g MnSO4·nH2O, 1 L of purified water), which was developed as a medium that shows good growth of all lactic acid bacteria, or a synthetic medium can be used. The composition of the synthetic medium is sufficient to contain a minimum of a carbon source, a nitrogen source, and a phosphorus source, but a vitamin source and a mineral source may be added. The culture temperature is about 25 to 40°C, and the culture period is about 3 to 50 days, for example. The culture can be performed statically, but shaking culture or aeration culture may be performed to shorten the fermentation time, etc.

[0050] The plant extract or its fermentation product may be purified before use. Examples of the purification method include a method in which microbial or plant residues are removed by filtration or decantation, followed by heat sterilization. Heat sterilization may be performed by heating at 80 to 120°C, preferably 90 to 105°C, for about 30 to 60 minutes. The residues may be removed by filtration using a filter of a size capable of filtering the microbial or plant residues used, or by precipitating the residues by centrifugation, and recovering the supernatant.

[0051] The plant extract or its fermentation product may be used as it is, or may be diluted or concentrated as necessary. The extract or its fermentation product may be in the form of a liquid or a solid such as a powder, granule, or solid.

[0052] The agent may be used as it is as a cosmetic, pharmaceutical, quasi-drug, etc., or may be mixed with other ingredients to be used as a raw material for cosmetics, pharmaceuticals, quasi-drugs, etc.

[0053] The present invention will be described below based on each example. Note that the technical scope of the present invention is not limited to the characteristics shown in these examples. In the examples, "%" represents mass % ((w / w)%) unless otherwise specified. EXAMPLES

[0054] Example 1: Preparation of various plant extracts (1) Calendula officinalis extract The flower heads of Calendula officinalis were dried and powdered. This dried powder was immersed in a 50% by volume aqueous solution of 1,3-butylene glycol. After that, it was filtered to remove the residue, and the collected filtrate was named "Calendula officinalis extract." The liquid is yellow to yellowish brown in color.

[0055] (2) Fermented Calendula Extract Lactic acid bacteria (Lactobacillus plantarum: NBRC 101975) were used as a stock culture on MRS agar medium (peptone 10 g, beef extract 10 g, yeast extract 5 g, glucose 20 g, Tween 80 1 g, K2HPO4 2 g, sodium acetate 5 g, diammonium citrate 2 g, MgSO4·7H2O 0.2 g, MnSO4·nH2O 0.05 g, purified water 1 L).

[0056] The petals of Calendula officinalis were powdered. 3 g of the powdered petals were immersed in 100 g of purified water, and extracted with hot water at 85°C for 1 hour, and then cooled to 37°C. A small amount of lactic acid bacteria was inoculated into the obtained hot water extract and cultured for 3 days as a seed culture (without mixing with the components of the storage medium). The seed culture was mixed with another hot water extract and cultured for 7 days at 30°C to 37°C. After the culture was completed, the petals were removed by filtration, and the collected filtrate was heat sterilized at 85°C for 30 minutes. The residue was then removed by filtration, and 1,3-butylene glycol was added to the mixture to a concentration of 30%, and the mixture was passed through a membrane filter to obtain a "Calendula officinalis fermented extract".

[0057] (3) Japanese Knotweed Extract (BG) The underground parts (rhizomes and roots) of Polygonum sachalinense were dried and powdered. 100g of this dried powder was immersed in 900g of 30% by volume 1,3-butylene glycol aqueous solution and left to stand at room temperature (approximately 20°C) for one week. The supernatant was then transferred to a separate container and refrigerated at 4°C for one week. The residue was then removed by filtration, and the collected filtrate was named "Polygonum sachalinense extract (BG)".

[0058] (4) Polygonum orbiculatum extract (ET) The underground parts (rhizomes and roots) of Polygonum sachalinense were dried and powdered. 100g of this dried powder was immersed in 900g of 100% ethanol and left to stand at room temperature (approximately 20°C) for one week. The supernatant was then transferred to a separate container and refrigerated at 4°C for one week. The residue was then removed by filtration, and the collected filtrate was named "Polygonum sachalinense extract (ET)."

[0059] (5) Yeast fermentation product of Japanese knotweed extract Yeast (Saccharomyces cerevisiae: NBRC 10217) was cultured in YM liquid medium (glucose 10 g, peptone 5 g, yeast extract 3 g, malt extract 3 g, purified water 1 L) and then preserved and cultured on YM agar medium (glucose 10 g, peptone 5 g, yeast extract 3 g, malt extract 3 g, purified water 1 L, agar 15 g) for use.

[0060] The underground part (rhizome and root) of Polygonum sachalinense was dried and powdered. 50g of this dried powder was soaked in 950g of purified water, extracted with hot water at 85°C for 1 hour, and then cooled to 37°C. A small amount of yeast was inoculated into the obtained aqueous extract and cultured for 3 days to use as the seed culture. The seed culture was mixed with another aqueous extract and cultured for 7 days at 30°C to 37°C. After the culture was completed, the residue was removed by filtration, and the collected filtrate was heat sterilized at 85°C for 30 minutes. The residue was then removed by filtration, and 1,3-butylene glycol was added to the mixture to a concentration of 30%, and the mixture was passed through a membrane filter to use as the "Polygonum sachalinense extract yeast fermentation product".

[0061] (6) Lactic acid bacteria fermentation product of Japanese knotweed extract The underground part (rhizome and root) of Japanese knotweed was dried and powdered. 50g of this dried powder was soaked in 950g of purified water, extracted with hot water at 85°C for 1 hour, and then cooled to 37°C. A small amount of lactic acid bacteria (described in this Example 1(2)) was inoculated into the obtained water extract and cultured for 3 days as a seed culture (without mixing with the components of the storage medium). The seed culture was mixed with another water extract and cultured for 7 days at 30°C to 37°C. After the culture was completed, the residue was removed by filtration, and the collected filtrate was heat sterilized at 85°C for 30 minutes. The residue was then removed by filtration, and 1,3-butylene glycol was added to the mixture to a concentration of 30%, and the mixture was passed through a membrane filter to obtain a "Japanese knotweed extract lactic acid bacteria fermentation product".

[0062] Example 2: Evaluation of the proliferation-promoting effect on dermal papilla cells Test media were prepared by adding the plant extracts of Example 1 (1) to (6) to 0.5%, 1.0%, 1.5%, 2.0% or 3.0% to Dulbecco's modified minimal essential medium (DMEM) containing 1% fetal bovine serum (FBS). Normal human hair papilla cells (Takara Bio Inc.) were cultured at 2.0 × 10 4 The cells were seeded in a 96-well plate at a cell density of 100 cells / well and cultured for 24 hours. The medium was then replaced with the test medium or DMEM containing 1% FBS without the plant extract (control medium) and cultured for another 48 hours.

[0063] The cell proliferation promoting effect was measured using the MTT assay. After the culture was completed, the medium was replaced with 1% FBS-containing DMEM containing 0.4 mg / mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and the culture was continued for another 2 hours. The medium was removed, and 2-propanol was added to extract the formazan produced. The absorbance of the extract at 550 nm was measured, and this was taken as the amount of formazan produced.

[0064] Here, it is generally believed that the amount of formazan produced is positively correlated with the number of living cells. Therefore, the ratio of the amount of formazan produced (A) in cells cultured in a test medium to the amount of formazan produced (B) in cells cultured in a control medium can be used as an index of the cell number that has changed due to the effect of the test medium. Based on the measurement results, the cell proliferation promotion rate (%) was calculated using the following formula 1. In other words, if the cell proliferation promotion rate is significantly greater than 100%, it can be determined that the proliferation of hair papilla cells has been promoted by the effect of the plant extract added to the test medium. Formula 1: Cell proliferation promotion rate (%) = A / B x 100 A: Amount of formazan produced in cells cultured in the test medium B: Formazan production in cells cultured in control medium The cell proliferation promotion rate was calculated by calculating the average value of four similar tests. In addition, a significant difference test was performed against the sample using the control medium using the Student t-test, and a p value of <0.05 was considered to indicate a significant difference.

[0065] The results when the concentrations of the plant extracts in the test medium were 1.0% and 2.0% of calendula officinalis extract, 1.5% of fermented calendula officinalis extract, Japanese knotweed extract (BG), 1.5% of Japanese knotweed extract yeast fermented product, and 0.5% of Japanese knotweed extract (ET) are shown in Table 1 and Figure 1. The results when the concentrations in the test medium were 1.0% and 2.0% of calendula officinalis extract, 3.0% of fermented calendula officinalis extract, Japanese knotweed extract (BG), 1.5% of Japanese knotweed extract yeast fermented product, and 1.0% of Japanese knotweed extract (ET) are shown in Table 2 and Figure 2. [Table 1]

[0066] [Table 2]

[0067] As shown in Table 1 and Figure 1, no significant increase in the cell proliferation promotion rate was observed for Calendula officinalis extract at either 1.0% or 2.0% concentration in the test medium. In contrast, a significant increase in the cell proliferation promotion rate was observed for the fermented Calendula officinalis extract, Japanese knotweed extract (BG), Japanese knotweed extract yeast fermented product, and Japanese knotweed extract lactic acid bacteria fermented product at a concentration of 1.5% in the test medium. A significant increase in the cell proliferation promotion rate was also observed for Japanese knotweed extract (ET) at a concentration of 0.5% in the test medium.

[0068] Similarly, as shown in Table 2 and Figure 2, a significant increase in the cell proliferation promotion rate was observed for the fermented product of Calendula officinalis extract, Japanese Knotweed extract (BG), and Japanese Knotweed extract yeast fermented product, even at a concentration of 3.0% in the test medium. A significant increase in the cell proliferation promotion rate was observed for Japanese Knotweed extract (ET), even at a concentration of 1.0% in the test medium.

[0069] These results demonstrate that the fermented product of Calendula officinalis extract, Japanese Knotweed extract, and the fermented product of Japanese Knotweed extract are capable of promoting the proliferation of hair papilla cells.

[0070] Example 3: Evaluation of the effect of promoting WINT5A expression (1) Evaluation of various plant extracts Whether or not the plant extracts of Examples 1(2) to (6) change the expression level of Wint5A (NM_003392.4; Homo sapiens Wnt family member 5A (WINT5A), transcript variant 1, mRNA) was evaluated by real-time PCR.

[0071] First, human dermal papilla cells (Takara Bio Inc.) were pre-cultured for 18 hours in an environment of 37°C and 5% CO2 using a basal medium for dermal papilla cells (Takara Bio Inc.). Next, the plant extracts of Examples 1 (2) to (6) were added at 3.125 μg / mL, 12.5 μg / mL, 50 μg / mL, or 200 μg / mL, and the cells were cultured for 24 hours, which was used as a test group. In addition, the cells were similarly cultured for 24 hours without adding the plant extract, which was used as a control group.

[0072] After the culture was completed, the cells were thoroughly washed with PBS(-). Total RNA was extracted using NucleoSpin® RNA (Takara Bio Inc.). A portion of the obtained RNA sample was diluted 50-fold, and the RNA concentration (ng / μL) was calculated by measuring the absorbance at 260 nm. cDNA was produced by reverse transcription reaction (37°C 15 minutes, 85°C 5 seconds) by PCR using PrimeScript® RT Master Mix (Takara Bio Inc.).

[0073] Using the prepared cDNA as a template, real-time PCR was carried out under the reaction conditions described below, and the Ct value (threshold cycle value) of the target nucleic acid was measured. Reaction solution composition: 12.5 μL of SYBR Premix Ex Taq II (Tli RNaseH Plus) (2X), 1 μL each of forward and reverse primers (10 μM), 1 μL or 2 μL of cDNA, and RNase-free water to bring the total volume to 25 μL. Reaction cycle: 1 cycle of 95°C for 10 s (initial denaturation), 45 cycles of 95°C for 5 s and 60-65°C for 30 s, and 1 cycle of 60-95°C at 0.2°C / s. Primer sequences: Wint5A Forward: 5'-AGCAGCATCAGTCCACAAACACTTA-3' (SEQ ID NO: 1) Reverse; 5'-AGACCTGTGCCTTCGTGCCTA-3' (SEQ ID NO: 2) Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (internal standard) Forward: 5'-TGTGTCCGTCGTGGATCTGA-3' (SEQ ID NO: 3) Reverse; 5'-TTGCTGTTGAAGTCGCAGGAG-3' (SEQ ID NO: 4)

[0074] The measurement results were corrected by the Ct value of the internal standard, and then comparatively quantified by the ΔΔCt method to calculate the ratio of the expression level in the control group to 1.0, which was used as the expression ratio of the gene to be evaluated (Wint5A). The results are shown in Figure 3. If the expression ratio was 1.1 or higher, it was determined that the expression level of the gene to be evaluated was higher in the test group than in the control group.

[0075] As shown in Figure 3, the expression ratio of Wint5A was high in the test sections where the fermented product of Calendula officinalis extract was added at concentrations of 3.125 and 12.5 μg / mL, the test sections where the Japanese knotweed extract (ET) was added at concentrations of 3.125, 12.5 and 50 μg / mL, and the test section where the yeast fermented product of Japanese knotweed extract was added at a concentration of 200 μg / mL. In other words, the expression level of Wint5A in cells was high in the presence of the fermented product of Calendula officinalis extract, the Japanese knotweed extract, or the fermented product of Japanese knotweed extract. This result revealed that the fermented product of Calendula officinalis extract, the Japanese knotweed extract, and the fermented product of Japanese knotweed extract can promote the expression of WINT5A.

[0076] (2) Comparative evaluation of fermented calendula extracts The WINT5A expression promoting effect of the fermented Calendula officinalis extract was compared with that of adenosine, a substance known to have a hair growth effect. That is, the expression level of Wint5A was evaluated by the real-time PCR method described in Example 3(1) using the fermented Calendula officinalis extract of Example 1(2) and adenosine (FUJIFILM Wako Pure Chemical Industries) instead of the plant extract. However, the concentration of the test substance added was 3.125 and 12.5 μg / mL. The results are shown in FIG. 4.

[0077] As shown in Figure 4, the expression ratio of Wint5A was higher in the test group to which the fermented product of Calendula officinalis extract was added than in the test group to which adenosine was added at concentrations of 3.125 and 12.5μg / mL. This result revealed that the fermented product of Calendula officinalis extract is superior to adenosine, a known hair growth ingredient, in promoting the expression of WINT5A.

[0078] Example 4: Evaluation of the effect of promoting expression of vascular endothelial growth factor (VEGF) (1) Evaluation of various plant extracts Whether or not the plant extracts of Examples 1(2) to (6) change the expression level of the VEGF gene (NM_001025366.2; Homo sapiens vascular endothelial growth factor A (VEGFA), transcript variant 1, mRNA) was evaluated by the real-time PCR method described in Example 3(1). However, the primers used for amplifying the VEGF gene shown below were used instead of the primers for amplifying Wint5A. The results are shown in FIG. 5. <VEGF gene> Forward: 5'-TCACAGGTACAGGGATGAGGACAC-3' (SEQ ID NO:5) Reverse; 5'-CAAAGCACAGCAATGTCCTGAAG-3' (SEQ ID NO: 6)

[0079] As shown in FIG. 5, the expression ratio of the VEGF gene was high in the test sections where the fermented product of Calendula officinalis extract was added at concentrations of 3.125, 12.5, 50 and 200 μg / mL, the test sections where the Japanese Knotweed extract (BG) was added at concentrations of 3.125, 12.5 and 50 μg / mL, the test sections where the Japanese Knotweed extract (ET) was added at concentrations of 12.5, 50 and 200 μg / mL, and the test sections where the Japanese Knotweed extract lactic acid bacteria fermented product was added at concentrations of 3.125 and 12.5 μg / mL. In other words, in the presence of the fermented product of Calendula officinalis extract, the Japanese Knotweed extract or the fermented product of Japanese Knotweed extract, the expression level of the VEGF gene in the cells was high. This result revealed that the fermented product of Calendula officinalis extract, the Japanese Knotweed extract and the fermented product of Japanese Knotweed extract can promote the expression of VEGF.

[0080] (2) Comparative evaluation of fermented calendula extracts The VEGF expression promoting effect of the fermented calendula extract was compared with that of Panax ginseng root extract, a substance known to have hair growth effects. That is, the expression level of the VEGF gene was evaluated by the real-time PCR method described in Example 4(1) using the fermented calendula extract of Example 1(2) and Panax ginseng root extract (commercially available product) instead of the plant extract. However, the test substance was added at a concentration of 50 and 200 μg / mL. The results are shown in FIG. 6.

[0081] As shown in Figure 6, the expression ratio of the VEGF gene was higher in the test group to which the fermented calendula extract was added than in the test group to which the ginseng root extract was added at concentrations of 50 and 200 μg / mL. This result demonstrated that the fermented calendula extract is superior in promoting the expression of VEGF to the known hair growth ingredient, ginseng root extract.

[0082] <Example 5> Evaluation of the effect of promoting the expression of fibroblast growth factor-7 (FGF-7) (1) Evaluation of various plant extracts Whether or not the plant extracts of Example 1 (2) to (6) change the expression level of the FGF-7 gene (NM_002009.4; Homo sapiens fibloblast growth factor 7 (FGF7), mRNA) was evaluated by the real-time PCR method described in Example 3. However, the primers used for amplifying the FGF-7 gene shown below were used instead of the primers for amplifying Wint5A. The results are shown in FIG. 7. <FGF-7 gene> Forward: 5'-GGATGCAGGCCACTATGACTAAC-3' (SEQ ID NO: 7) Reverse; 5'-AGAGTAGCTGACAACAATGCTGAA-3' (SEQ ID NO: 8)

[0083] As shown in Figure 7, the expression ratio of the FGF-7 gene was high in the test sections where the fermented product of Calendula officinalis extract was added at concentrations of 3.125, 12.5, 50 and 200μg / mL, the test sections where the Japanese knotweed extract (BG) was added at concentrations of 3.125, 12.5, 50 and 200μg / mL, the test sections where the Japanese knotweed extract (ET) was added at concentrations of 3.125, 12.5, 50 and 200μg / mL, the test sections where the Japanese knotweed extract yeast fermentation product was added at concentrations of 3.125 and 12.5μg / mL, and the test sections where the Japanese knotweed extract lactic acid bacteria fermentation product was added at concentrations of 3.125, 12.5, 50 and 200μg / mL. That is, in the presence of the fermented product of Calendula officinalis extract, the Japanese knotweed extract, or the fermented product of the Japanese knotweed extract, the expression level of the FGF-7 gene in the cells was high. These results demonstrated that the fermented product of Calendula officinalis extract, Japanese Knotweed extract, and the fermented product of Japanese Knotweed extract can promote the expression of FGF-7.

[0084] (2) Comparative evaluation of fermented calendula extracts The FGF-7 expression promoting effect of the fermented Calendula officinalis extract was compared with that of the unfermented Calendula officinalis extract. That is, the expression level of the FGF-7 gene was evaluated by the real-time PCR method described in Example 5(1) using the Calendula officinalis extract of Example 1(1) and the fermented Calendula officinalis extract of Example 1(2). However, the concentration of the test substance added was 12.5 and 200 μg / mL. The results are shown in FIG. 8.

[0085] As shown in Figure 8, the expression ratio of FGF-7 gene was higher in the test group to which the fermented product of Calendula officinalis extract was added than in the test group to which Calendula officinalis extract was added at concentrations of 12.5 and 200 μg / mL. This result demonstrated that the fermented product of Calendula officinalis extract is superior to the non-fermented Calendula officinalis extract in promoting the expression of FGF-7.

[0086] Example 6: Evaluation of promoting effect on the expression of tissue non-specific alkaline phosphatase (ALPL) (1) Evaluation of Japanese Knotweed Extract Whether or not the Japanese knotweed extract (ET) of Example 1(4) changes the expression level of the ALPL gene (NM_000478.6; Homo sapiens alkaline phosphatase, biomineralization associated (ALPL), transcript variant 1, mRNA) was evaluated by the real-time PCR method described in Example 3. However, the primers used for amplifying the ALPL gene were used instead of the primers for amplifying Wint5A. The results are shown in Figure 9. ALPL gene Forward; 5'-GGACCATTCCCACGTCTTCA-3' (SEQ ID NO: 9) Reverse; 5'-CAGGCCCATTGCCATACA-3' (SEQ ID NO: 10)

[0087] As shown in Figure 9, the expression ratio of the ALPL gene was high in the test groups where the Knotweed extract (ET) was added at concentrations of 3.125, 12.5, 50 and 200 μg / mL. In other words, the expression level of the ALPL gene in the cells was high in the presence of Knotweed extract. This result demonstrated that Knotweed extract can promote the expression of tissue non-specific alkaline phosphatase (ALPL).

[0088] (2) Comparative evaluation of fermented calendula extracts The ALPL expression promoting effect of the fermented calendula extract was compared with that of the non-fermented calendula extract, ginseng root extract and adenosine. That is, the expression level of the ALPL gene was evaluated by the real-time PCR method described in Example 6(1) using the calendula extract of Example 1(1) and the fermented calendula extract of Example 1(2), as well as ginseng root extract (commercial product) and adenosine (Fujifilm Wako Pure Chemical Industries) instead of the plant extract. However, the concentration of the test substance added was 50 μg / mL. The results are shown in FIG. 10.

[0089] As shown in Figure 10, the expression ratio of the ALPL gene was the highest in the test group to which the fermented calendula extract was added. This result demonstrated that the fermented calendula extract is superior in promoting the expression of ALPL to non-fermented calendula extract and known hair growth ingredients, Panax ginseng root extract and adenosine.

[0090] <Example 7> Evaluation of the effect of promoting the expression of versican (1) Evaluation of various plant extracts Whether or not the plant extracts of Examples 1 (2) to (6) change the expression level of the versican gene (NM_001126336.2; Homo sapiens versican (VCAN), transcript variant 2, mRNA) was evaluated by the real-time PCR method described in Example 3. However, instead of the primers for amplifying Wint5A, the primers for amplifying the versican gene shown below were used. The results are shown in Figure 11. <<Versican gene>> Forward: 5'-GCTGGAAATGGTTTCACTTGCTC-3' (SEQ ID NO: 11) Reverse; 5'-GATCCCATTCGCAGCCTTTAG-3' (SEQ ID NO: 12)

[0091] As shown in Figure 11, the expression ratio of the versican gene was high in the test sections where the fermented product of Calendula officinalis extract was added at concentrations of 3.125, 12.5, 50 and 200 μg / mL, the test sections where the Japanese knotweed extract (BG) was added at concentrations of 12.5, 50 and 200 μg / mL, and the test sections where the Japanese knotweed extract (ET) was added at concentrations of 12.5, 50 and 200 μg / mL. In other words, the expression level of the versican gene in cells was high in the presence of the fermented product of Calendula officinalis extract or the Japanese knotweed extract. This result revealed that the fermented product of Calendula officinalis extract and the Japanese knotweed extract can promote the expression of versican.

[0092] (2) Comparative evaluation of fermented calendula extracts The versican expression promoting effect of the fermented calendula extract was compared with that of adenosine. That is, the expression level of the versican gene was evaluated by the real-time PCR method described in Example 7(1) using the fermented calendula extract of Example 1(2) and adenosine (FUJIFILM Wako Pure Chemical Industries) instead of the plant extract. However, the concentration of the test substance added was 3.125 and 12.5 μg / mL. The results are shown in FIG. 12.

[0093] As shown in Figure 12, the expression ratio of the versican gene was higher in the test group to which the fermented product of Calendula officinalis extract was added than in the test group to which adenosine was added at concentrations of 3.125 and 12.5μg / mL. This result demonstrated that the fermented product of Calendula officinalis extract is superior to adenosine, a known hair growth ingredient, in promoting the expression of versican.

[0094] <Example 8> Evaluation of the effect of promoting expression of tenascin C (1) Evaluation of various plant extracts Whether or not the plant extracts of Example 1 (2) to (6) change the expression level of the tenascin C gene (NM_002160.4; Homo sapiens tenascin C (TNC), mRNA) was evaluated by the real-time PCR method described in Example 3. However, the primers used for amplifying the tenascin C gene shown below were used instead of the primers for amplifying Wint5A. The results are shown in FIG. 13. Tenascin-C gene Forward: 5'-CTCCCAGTGACAACATCGCAATA-3' (SEQ ID NO: 13) Reverse; 5'-GGATGGCTTCCAATGACACATTTA-3' (SEQ ID NO: 14)

[0095] As shown in Figure 13, the expression ratio of the tenascin-C gene was high in the test section where the fermented product of Calendula officinalis extract was added at a concentration of 200μg / mL, the test section where the Japanese knotweed extract (BG) was added at a concentration of 200μg / mL, the test section where the Japanese knotweed extract (ET) was added at a concentration of 200μg / mL, and the test section where the Japanese knotweed extract lactic acid bacteria fermented product was added at a concentration of 200μg / mL. In other words, in the presence of the fermented product of Calendula officinalis extract, the Japanese knotweed extract, or the fermented product of Japanese knotweed extract, the expression level of the tenascin-C gene in the cells was high. From this result, it was revealed that the fermented product of Calendula officinalis extract, the Japanese knotweed extract, and the fermented product of Japanese knotweed extract can promote the expression of tenascin-C.

[0096] (2) Comparative evaluation of fermented calendula extracts The expression promoting effect of the fermented calendula extract was compared with that of the unfermented calendula extract, ginseng root extract, and adenosine. That is, the expression level of the tenascin-C gene was evaluated by the real-time PCR method described in Example 8(1) using the calendula extract of Example 1(1) and the fermented calendula extract of Example 1(2), as well as ginseng root extract (commercial product) and adenosine (FUJIFILM Wako Pure Chemical Industries) instead of the plant extract. However, the concentration of the test substance added was 3.125 μg / mL. The results are shown in FIG. 14.

[0097] As shown in Figure 14, the expression ratio of the tenascin-C gene was highest in the test group to which the fermented calendula extract was added. This result demonstrated that the fermented calendula extract is superior in promoting the expression of tenascin-C to the non-fermented calendula extract and to the known hair growth ingredients, ginseng root extract and adenosine.

[0098] <Example 9> Evaluation of the expression-promoting effect of chondroitin sulfate proteoglycan 4 (CSPG4) (1) Evaluation of various plant extracts Whether or not the plant extracts of Example 1 (2) to (6) change the expression level of the CSPG4 gene (NM_001897.5; Homo sapiens chondroitin sulfate proteoglycan 4 (CSPG4), mRNA) was evaluated by the real-time PCR method described in Example 3. However, the primers used for amplifying the CSPG4 gene shown below were used instead of the primers for amplifying Wint5A. The results are shown in FIG. 15. CSPG4 gene Forward: 5'-TGCTTTGGAGGCTCTGGACA-3' (SEQ ID NO: 15) Reverse; 5'-GTATGCAAGCCGACGCAGAC-3' (SEQ ID NO: 16)

[0099] As shown in Figure 15, the expression ratio of the CSPG4 gene was high in the test group where the fermented product of Calendula officinalis extract was added at a concentration of 200μg / mL and the test group where the Japanese knotweed extract (BG) was added at a concentration of 200μg / mL. In other words, the expression level of the CSPG4 gene in the cells was high in the presence of the fermented product of Calendula officinalis extract or the Japanese knotweed extract. This result revealed that the fermented product of Calendula officinalis extract and the Japanese knotweed extract can promote the expression of CSPG4.

[0100] (2) Comparative evaluation of fermented calendula extracts The CSPG4 expression promoting effect of the fermented calendula extract was compared with that of the non-fermented calendula extract, ginseng root extract, and adenosine. That is, the expression level of the CSPG4 gene was evaluated by the real-time PCR method described in Example 9(1) using the calendula extract of Example 1(1) and the fermented calendula extract of Example 1(2), as well as ginseng root extract (commercial product) and adenosine (FUJIFILM Wako Pure Chemical Industries) instead of the plant extract. However, the concentration of the test substance added was 12.5 μg / mL. The results are shown in FIG. 16.

[0101] As shown in Figure 16, the expression ratio of the CSPG4 gene was the highest in the test group to which the fermented calendula extract was added. This result demonstrated that the fermented calendula extract is superior in promoting the expression of CSPG4 to the non-fermented calendula extract and the known hair growth ingredients Panax ginseng root extract and adenosine.

[0102] Example 10: Evaluation of the effect of promoting expression of fibronectin (1) Evaluation of various plant extracts Whether or not the plant extracts of Example 1 (2) to (6) change the expression level of the fibronectin gene (NM_001306129.1; Homo sapiens fibronectin 1 (FN1), transcript variant 8, mRNA) was evaluated by the real-time PCR method described in Example 3. However, the primers used for amplifying the fibronectin gene shown below were used instead of the primers for amplifying Wint5A. The results are shown in FIG. 17. <Fibronectin gene> Forward: 5'-GCCTTGCACGATGATATGGAGA-3' (SEQ ID NO: 17) Reverse; 5'-CTTGTGGGTGTGACCTGAGTGAA-3' (SEQ ID NO: 18)

[0103] As shown in Figure 17, the expression ratio of the fibronectin gene was high in the test sections where the fermented product of Calendula officinalis extract was added at concentrations of 3.125, 12.5, 50 and 200 μg / mL, and in the test section where the Japanese knotweed extract (BG) was added at a concentration of 200 μg / mL. In other words, in the presence of the fermented product of Calendula officinalis extract or the Japanese knotweed extract, the expression level of the fibronectin gene in the cells was high. From this result, it was revealed that the fermented product of Calendula officinalis extract and the Japanese knotweed extract can promote the expression of fibronectin.

[0104] (2) Comparative evaluation of fermented calendula extracts The fibronectin expression promoting effect of the fermented Calendula officinalis extract was compared with that of the unfermented Calendula officinalis extract. That is, the expression level of the fibronectin gene was evaluated by the real-time PCR method described in Example 10(1) using the Calendula officinalis extract of Example 1(1) and the fermented Calendula officinalis extract of Example 1(2). However, the concentration of the test substance added was 12.5 and 200 μg / mL. The results are shown in FIG. 18.

[0105] As shown in Figure 18, the expression ratio of the fibronectin gene was higher in the test group to which the fermented product of Calendula officinalis extract was added than in the test group to which Calendula officinalis extract was added at concentrations of 12.5 and 200μg / mL. This result revealed that the fermented product of Calendula officinalis extract is superior in promoting the expression of fibronectin to the non-fermented Calendula officinalis extract.

Claims

1. A hair growth agent whose active ingredient is a lactic acid fermentation product of calendula extract.

2. A hair papilla cell proliferation promoter having as its active ingredient a lactic acid fermentation product of calendula extract.

3. The agent according to claim 1 or 2, which is an agent for promoting the expression of WINT5A.

4. The agent according to claim 1 or 2, which is an agent for promoting expression of vascular endothelial growth factor (VEGF).

5. The agent according to claim 1 or 2, which is an agent for promoting the expression of fibroblast growth factor-7 (FGF-7).

6. The agent according to claim 1 or 2, which is an agent for promoting the expression of tissue-nonspecific alkaline phosphatase (ALPL).

7. The agent according to claim 1 or 2, which is an agent for promoting the expression of versican.

8. The agent according to claim 1 or 2, which is an agent for promoting the expression of tenascin-C.

9. The agent according to claim 1 or 2, which is an agent for promoting the expression of chondroitin sulfate proteoglycan 4 (CSPG4).

10. The agent according to claim 1 or 2, which is an agent for promoting the expression of fibronectin.

11. The agent according to any one of claims 1 to 10, wherein the lactic acid bacterium is Lactobacillus plantarum.

12. A hair growth method (excluding medical procedures) using a lactic acid bacteria fermentation product of calendula extract.

13. A method for promoting the proliferation of hair papilla cells (excluding medical procedures) using a lactic acid bacteria fermentation product of calendula extract.

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