JP2019194176A - R-spondin 1 production promoter - Google Patents

Abstract

To provide a composition for promoting hair growth or the like with improved efficiency.SOLUTION: We have intensively studied and searched for materials having an R-spondin 1 production promotion ability, and discovered that Nasturtium officinale can promote the production of R-spondin 1, completing this invention.SELECTED DRAWING: Figure 6

Description

  The present invention mainly relates to a composition (agent, etc.) for promoting hair growth for animals such as humans.

  Hair growth begins when resting hair follicle stem cells present in the hair follicle start cell division again and reconstitute the hair follicle. Hair follicle stem cells exist in a region called a bulge and are composed of a special environment where napped muscles and nerve endings are in contact. Factors for activating hair follicles are produced from dermal papilla cells, and dermal papilla cells (DPc) themselves start to proliferate first, after which the cells divide from within the bulge and the epithelial mesenchymal cells Hair follicles are formed by the interaction.

  The mechanism of hair follicle formation is that the outer root sheath (ORS) cells that have started to divide by the hair growth signal from the dermal papilla cells form the hair matrix region directly above the dermal papilla cells while heading to the deep dermis. To do. The cells constituting the hair matrix region are called hair matrix cells, and further proliferate and differentiate to form hair shafts and inner root sheath cells. In the bulge region and secondary germ of the outer root sheath cell, cells expressing Leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), which is a stem cell marker, are present. . In the resting phase, the secondary hair bud is in a quiescent state like a bulge, but when induced in the growth phase, LGR5-positive cells trigger the proliferation of outer root sheath cells (see the schematic diagram in FIG. 1). . LGR5-positive cells proliferate depending on the Wnt signal, and are regarded as hair follicle proliferating cells contributing to the maintenance of hair follicle homeostasis. Interestingly, LGR5-positive cells in the anagen hair follicle do not disappear after the regression phase and remain in the secondary hair buds and are composed in the next anagen phase. LGR5, which is deeply involved in the regulation of introduction and maintenance into the growth period, has long been regarded as an orphan receptor, but recently, the secreted protein R-spondin (RSPO: also referred to as RSPO in the present specification and drawings) is a ligand. (Non-Patent Documents 1 to 4).

LGR5 is a seven-transmembrane gene identified as a gene encoding a membrane receptor having a structure similar to that of the gonadotropin hormone receptor such as thyroid-stimulating hormone and follicle-stimulating hormone. It is a type of receptor and has a structural feature of having a long leucine-rich repeat outside the cell. As LGR5 was confirmed to be specifically expressed in intestinal epithelial stem cells (Non-patent Document 5), it has attracted attention as a novel stem cell-specific protein. Non-Patent Document 6 describes that as a method for stably and long-term culturing the intestine and the large intestine, cell proliferation was significantly promoted by a fusion protein of RSPO1 (R-spondin1) and immunoglobulin Fc. . Non-Patent Document 7 describes that stem cell-like cells are present in corneal limbal epithelial cells, and that LGR5 can be a phenotypic marker of human corneal limbal epithelial stem cells in which LGR5 remains. In tissues such as the outer root sheath of hair follicles (Non-patent document 8) and gastric epithelium (Non-patent document 9), LGR5 expression is specifically increased in stem cells, and stem cell niche ( stem cells maintain their properties in vivo) It has been reported that it is involved in the construction and organization of the microenvironment necessary for

  RSPOs involved in hair follicle formation are expressed in hair papilla cells, and in particular, RSPO1 has been reported to increase markedly from the end of rest period to the growth period and activate hair follicle stem cells (Non-patent Document 10). FIG. 1 shows a schematic diagram). That is, activation of hair papilla cells and promotion and / or suppression of RSPO1 expression from hair papilla cells are desirable for proliferation of LGR5-positive cells present in bulges and outer root sheath cells, and hair growth, growth phase maturation It is thought to be involved in the conversion. In addition, Dickkopf1 (DKK1) is known as an inhibitory molecule for the Wnt / β-catenin signaling pathway, and the Wnt signal is suppressed by forming a complex of the Wnt co-receptor LRP6 and the DKK1 receptor Kremen. . Since DKK1 is highly expressed at the hair loss site, Wnt signal is suppressed according to the above mechanism, but RSPO1 acts on the DKK1-LRP5 / 6-Kremen complex and regulates to promote Wnt binding ( Non-patent document 11).

  Zinc RING Finger3 (ZNRF3) and its homologue RING FINGER 43 (RNF43) also antagonize Wnt signaling in adult stem cells by ubiquitinating Frizzled receptor (FZD), which causes endocytosis of Wnt receptor, The LGR5-RSPO1 complex suppresses Frizzled ubiquitination of ZNRF3 / RNF43 and enhances Wnt signaling (Non-patent Document 12).

Carmon KS. et al. Proc Natl Acad Sci US 2011 Jul 12; 108 (28): 11452-7. de Lau W. et al. , Nature. 2011 Jul 4; 476 (7360): 293-7. Glinka A.I. et al. , EMBO Rep. 2011 Sep 30; 12 (10): 1055-61. Yoon JK, Lee JS. , Cell Signal. 2012 Feb; 24 (2): 369-77. Barker N.M. , Et al. , Nature Nature. 2007 Oct 25; 449 (7165): 1003-7. Ootani A. , Et al. Nat Med. 2009 Jun; 15 (6): 701-6. Brzesczczynska J, et al. Int J Mol Med. 2012 May; 29 (5): 871-6 Jaks V. , Et al. Nat Genet. 2008 Nov; 40 (11): 1291-9. Barker N.M. , Et al. , Cell Stem Cell. 2010 Dec 3; 7 (6): 656-70. Na Li, et al. Int J .; Mol. Soi. 2016, Apr 20; 17 (4). Binnerts ME. , Et al. Proc Natl Acad Sci US 2007 Sep; 11; 104 (37): 14700-5. Peng WC. , Et al. , PLoS One. 2013 Dec 12; 8 (12): e83110.

  The problem to be solved by the present invention is to provide a composition for promoting hair growth and the like more efficiently.

  Thus, as described above, the inventors of the present invention focused on RSPO1 that increases markedly from the end of the rest period to the growth period, and tried to search for a substance that promotes production of RSPO1. The inventor of the present invention has intensively investigated the search for a substance having the ability to promote RSPO1 production, and as a result, has found that Nasturium officinale (Netherlands mustard, Reiko Walan) promotes the production of RSPO1 and completed the present invention. .

The present invention includes the following items.
[Item 1] An R-spondin1 production promoter comprising an extract of Dutch pepper as an active ingredient.
[Item 2] The R-spondin1 of [Item 1], wherein the Dutch pepper extract is extracted with at least one solvent selected from the group consisting of water, ethanol and 1,3-butylene glycol. Production promoter.
[Item 3] A skin external preparation containing the R-spondin1 production promoter according to [Item 1] or [Item 2].
[Item 4] A skin external preparation for promoting hair growth as described in [Item 3].
[Item 5] An oral composition containing the R-spondin1 production promoter according to [Item 1] or [Item 2].
[Item 6] An oral composition for promoting hair growth as described in [Item 5].

  The present invention can provide a composition for promoting hair growth and the like more efficiently. This composition can promote the production of RSPO1 which increases significantly from the end of the rest period to the growth period.

The schematic diagram of the effect | action of RSPO1 which increases from a rest period (Telogen) to a growth period (Anagen). Results of production confirmation of RSPO1 in various plant extracts. Results of production confirmation of predetermined markers and the like in P5 hair papilla cells and P12 hair papilla cells. Results of confirmation of RSPO1 production in P5 hair papilla cells and P12 hair papilla cells. Results of ALP activity in P5 hair papilla cells and P12 hair papilla cells. Model of the mechanism of action of RSPO1. The test result which confirmed the influence which interaction with Wnt3a, RSPO1, and DKK-1 has on cell proliferation by confirming the survival rate of a human epidermal keratinocyte.

Hereinafter, embodiments of the present invention will be described with reference to the drawings.
(RSPO1 production promoter)
The RSPO1 production promoter is an agent for promoting the production of RSPO1. Examples of this agent include not only liquids but also solids, for example, and are considered not only external preparations for skin but also oral compositions (for example, solid or liquid preparations).

(Hair growth promotion)
In this specification and the like, “hair growth” includes not only “hair growth” but also “hair growth”, for example. In addition, the “hair growth” includes, for example, hair growth by suppressing the expression of a hair loss factor (DKK-1, IL-6, etc.), thereby promoting hair shaft formation, hair shaft elongation, and the like.
DKK-1 is considered as one of factors that cause the hair follicle to transition to the regression phase as a factor that inhibits the Wnt / β-catenin signaling pathway, and is considered to be a factor involved in male pattern hair loss ( Journal of Investigative Dermatology (2012), Volume 132, 1554-1560).

(Form of external preparation for skin)
Skin external preparations according to the present invention are ampules, capsules, powders, granules, liquids, gels, bubbles, emulsions, sheets, mists, sprays, etc. in suitable forms for use 1) pharmaceuticals, 2) quasi drugs, 3) topical or systemic topical skin preparations (for example, basic cosmetics such as lotions, emulsions, creams, ointments, lotions, oils, packs, etc., facial cleansers such as soaps, liquid soaps and hand washes, and skin cleansings) Makeup, massage, cleansing agent, hair remover, hair remover, shaving treatment, after shave lotion, pre-show lotion, shaving cream, foundation, lipstick, blusher, eye shadow, eyeliner, mascara, etc. , Perfume, nail polish, nail enamel, nail enamel remover, poultice, plaster, tape, sheet 4) Medicinal or / and cosmetic preparations applied to the scalp / hair (eg shampoo, rinse, hair treatment, pre-hair treatment, permanent solution, hair dye, Hair conditioner, hair styling agent, hair growth / hair nourishing agent, poultice agent, plaster agent, tape agent, sheet agent, aerosol agent, etc.) 5) Bath agent used in bath water, 6) Others Examples include odorants, antiperspirants, sanitary products, sanitary cotton, and wet tissue.

(Constituents of topical skin preparation)
In addition, such agents can be produced by optionally selecting and using any of the components and additives exemplified below within a range that does not impair the effects of the present invention. The blending amount is not particularly specified, but it is generally considered that about 0.0001 to 50% is preferable.

(1) Various fats and oils Avocado oil, almond oil, fennel oil, sesame oil, olive oil, orange oil, orange raffer oil, sesame oil, cocoa butter, chamomile oil, carrot oil, cucumber oil, beef tallow fatty acid, cucumber nut oil, safflower oil , Shea fat, liquid shea fat, soybean oil, camellia oil, corn oil, rapeseed oil, persic oil, castor oil, cottonseed oil, peanut oil, turtle oil, mink oil, egg yolk oil, palm oil, palm kernel oil, owl, palm Oil, beef tallow, pork tallow, squalene, squalane, pristane or hydrogenated products of these oils (hardened oil, etc.).

(2) Wax beeswax, carnauba wax, whale wax, lanolin, liquid lanolin, reduced lanolin, hard lanolin, candelilla wax, montan wax, shellac wax, rice wax and the like.

(3) Mineral oils Liquid paraffin, petrolatum, paraffin, ozokelide, ceresin, microcrystalline wax and the like.

(4) Fatty acids Lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, oleic acid, linoleic acid, linolenic acid, docosahexaenoic acid, eicosapentaenoic acid, 12-hydroxystearic acid, undecylenic acid, tall oil, lanolin fatty acid Natural fatty acids such as isononanoic acid, caproic acid, 2-ethylbutanoic acid, isopentanoic acid, 2-methylpentanoic acid, 2-ethylhexanoic acid and isopentanoic acid.

(5) Alcohols Natural alcohols such as ethanol, isopropanol, lauryl alcohol, cetanol, stearyl alcohol, oleyl alcohol, lanolin alcohol, cholesterol, phytosterol, phenoxyethanol, and synthetic alcohols such as 2-hexyldecanol, isostearyl alcohol, and 2-octyldodecanol .

(6) Polyhydric alcohols Ethylene oxide, ethylene glycol, diethylene glycol, triethylene glycol, ethylene glycol monoethyl ether, ethylene glycol monobutyl ether, diethylene glycol monomethyl ether, diethylene glycol monoethyl ether, polyethylene glycol, propylene oxide, propylene glycol, polypropylene glycol 1,3-butylene glycol, pentyl glycol, glycerin, pentaerythritol, threitol, arabitol, xylitol, ribitol, galactitol, sorbitol, mannitol, lactitol, maltitol and the like.

(7) Esters Isopropyl myristate, isopropyl palmitate, butyl stearate, hexyl laurate, myristyl myristate, oleyl oleate, decyl oleate, octyldodecyl myristate, hexyl decyl dimethyloctanoate, cetyl lactate, myristyl lactate, Diethyl phthalate, dibutyl phthalate, lanolin acetate, ethylene glycol monostearate, propylene glycol monostearate, propylene glycol dioleate, and the like.

(8) Metal soaps Aluminum stearate, magnesium stearate, zinc stearate, calcium stearate, zinc palmitate, magnesium myristate, zinc laurate, zinc undecylenate and the like.

(9) gum, saccharide or water-soluble polymer compound gum arabic, benzoin rubber, dammar gum, guaiac oil, Irish moss, karaya gum, tragacanth gum, carob gum, quinseed, agar, casein, lactose, fructose, sucrose or ester thereof, Trehalose or derivatives thereof, dextrin, gelatin, pectin, starch, carrageenan, carboxymethyl chitin or chitosan, and hydroxyalkyl (C ₂ -C ₄ ) chitin or chitosan to which alkylene (C ₂ -C ₄ ) oxide such as ethylene oxide is added Low molecular chitin or chitosan, chitosan salt, sulfated chitin or chitosan, phosphorylated chitin or chitosan, alginic acid or salt thereof, hyaluronic acid or salt thereof, chondroitin sulfate or salt thereof, heparin, ethylcellulose, Cellulose, carboxymethyl cellulose, carboxyethyl cellulose, sodium carboxyethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, nitrocellulose, crystalline cellulose, polyvinyl alcohol, polyvinyl methyl ether, polyvinyl pyrrolidone, polyvinyl methacrylate, polyacrylate, polyethylene oxide and polypropylene oxide Such as polyalkylene oxide or a crosslinked polymer thereof, carboxyvinyl polymer, polyethyleneimine and the like.

(10) Surfactant Anionic surfactant (alkyl carboxylate, alkyl sulfonate, alkyl sulfate ester salt, alkyl phosphate ester salt), cationic surfactant (alkyl amine salt, alkyl quaternary ammonium salt), amphoteric Surfactant: Carboxylic acid type amphoteric surfactant (amino type, betaine type), sulfate ester type amphoteric surfactant, sulfonic acid type amphoteric surfactant, phosphate ester type amphoteric surfactant, nonionic surfactant ( Ether type nonionic surfactant, ether ester type nonionic surfactant, ester type nonionic surfactant, block polymer type nonionic surfactant, nitrogen-containing type nonionic surfactant), other surfactants ( Natural surfactants, protein hydrolyzate derivatives, polymer surfactants, surfactants containing titanium and silicon, Carbon-based surfactants).

(11) Various vitamins Vitamin A group: retinol, retinal (vitamin A1), dehydroretinal (vitamin A2), carotene, lycopene (provitamin A), vitamin B group: thiamine hydrochloride, thiamine sulfate (vitamin B1), Riboflavin (vitamin B2), pyridoxine (vitamin B6), cyanocobalamin (vitamin B12), folic acid, nicotinic acids, pantothenic acids, biotins, choline, inositols, vitamin C group: vitamin C acid or derivatives thereof, vitamin D group: Ergocalciferol (vitamin D2), cholecalciferol (vitamin D3), dihydrotaxosterol, vitamin E group: vitamin E or derivatives thereof, ubiquinones, vitamin K group: phytonadione (vitamin K1), menaquinone (vitamin K2) , Menadione (vitamin K3), menadiol (vitamin K4), other essential fatty acids (vitamin F), carnitine, ferulic acid, γ-oryzanol, orotic acid, vitamin Ps (rutin, eriocitrin, hesperidin), vitamin U and the like.

(12) Various amino acids Valine, leucine, isoleucine, threonine, methionine, phenylalanine, tryptophan, lysine, glycine, alanine, asparagine, glutamine, serine, cysteine, cystine, tyrosine, proline, hydroxyproline, aspartic acid, glutamic acid, hydroxylysine , Arginine, ornithine, histidine and the like, and their amino acid derivatives such as sulfate, phosphate, nitrate, citrate or pyrrolidone carboxylic acid.

(13) Various additives derived from plant or animal raw materials These are processed in a conventional manner according to the product type and form to be added (for example, pulverization, milling, washing, hydrolysis, fermentation, purification) , Pressing, extraction, fractionation, filtration, drying, pulverization, granulation, dissolution, sterilization, pH adjustment, deodorization, decolorization, etc. You should provide.

  The solvent used for extraction may be selected in consideration of the intended purpose and type of the product to be provided or the processing to be performed later. Usually, water, methanol, ethanol, propyl alcohol, isopropyl alcohol, butanol , One kind selected from various organic solvents such as lower alcohols such as isobutanol or hydrous lower alcohols, polyhydric alcohols such as propylene glycol, 1,3-butylene glycol and glycerin or hydrous polyhydric alcohols, acetone and ethyl acetate Alternatively, it is desirable to use a mixture of two or more. However, when it is not preferable to contain an organic solvent depending on the use, it is sufficient to use only water or ethanol that can be easily removed after extraction, and it may be used alone or in any mixture with water. The thing extracted and extracted may be sufficient.

  In addition, when using additives derived from plant or animal-based ingredients for systemic or topical external preparations and cosmetics, it protects skin and hair, moisturizes, improves touch and texture, imparts flexibility, and stimulates Relieving stress, relieving stress due to fragrance, cell activation (preventing cell aging), suppressing inflammation, improving skin and hair quality, preventing and improving rough skin, hair growth, hair growth, preventing hair loss, imparting luster, cleansing effect, In addition to cosmetic effects such as fatigue relief, blood flow promotion, and bathing effects, effects such as flavoring, deodorization, thickening, antiseptic, and buffering can also be expected.

  In addition to this, various cosmetic and pharmaceutical effects of each of the known raw materials are expected, and by combining these, the intended effect of the present invention is promoted and multifunctional. It is also possible to make a product that is expected to have a positive effect.

  Embodiments of the present invention will be described below with reference to the accompanying drawings.

(Materials used in the experiments given in the examples)
An extract obtained by extracting Oryza sativa blanc, Prunus persica, Nasturium officinale, Calendula officinalis, Saponaria officinalis and Plantago asiatica with 50 wt% ethanol was used.

  As shown in FIG. 2, the production of RSPO1 was the highest value (ratio) in the extract of Nasturium officinale (Hollandic red pepper, Japanese orchid eggplant (also called watercress)). In addition, Cnt. Is 50% ethanol by weight of the solvent.

  The experiment (including sample preparation) shown in FIG. 2 was performed as follows. As described in “(2) Cell culture method” below, dermal papilla cells (passage 5 (P5)) were seeded on a plastic 96-well plate at a density of 5,000 cells / well. The next day, this extract was added to 1% FBS / DMEM medium so that the final concentration in the medium was 0.25%. Papilla cells were cultured for 2 days, and the culture supernatant was collected as a sample shown in FIG. Then, the amount of RSPO1 secreted from the hair papilla cells was quantified using an ELISA kit (LSbio).

(experimental method)
(1) Preparation of fraction of Nasturium officinale The crude extract of Nasturium officinale was fractionated using Diaion HP20 (Mitsubishi Chemical Corporation), 20 wt% ethanol, 40 wt% ethanol, 60 wt% ethanol, respectively. , 5 fractions (Fr.) eluted with 80 wt% ethanol and 100 wt% ethanol. As shown in FIGS. 4 and 5, the group eluted with 20 wt% ethanol is Fr. 1. The group eluted with 40 wt% ethanol is Fr. 2. The group eluted with 60 wt% ethanol is Fr. 3. The group eluted with 80 wt% ethanol is Fr. 4. The group eluted with 100 wt% ethanol is Fr. 5. Solvent administration group (group not containing Nasturium officinale extract) is Cnt. , Nasturium officinale crude extract (sample before passing through the column) shows Crude. Significant differences are observed in Cnt. It shows about the production rate of RSPO1 compared with.

(2) Cell culture method Papilla cells were seeded on a plastic 96-well plate at a density of 5,000 cells / well. The following day, each of the above-mentioned fractions was added to 1% FBS / DMEM medium so that the final concentration in the medium was 0.25%. Papilla cells were cultured for 2 days, and the supernatant of the cultured sample was collected as an ELISA assay sample shown in FIGS.

(3) Screening assay for RSPO1 An ELISA kit (LSbio) was used for screening, and the amount of RSPO1 secreted from the hair papilla cells at passage 5 (P5) was quantified. The value of RSPO1 production corrected by the number of viable cells was used (experiment shown in FIGS. 3 to 5). The number of viable cells was determined using Cell Counting Kit-8 (CCK-8, Dojindo).

(4) Bromodeoxyuridine (BrdU) Cell Proliferation Assay In order to examine the proliferation of dermal papilla cells, cell proliferation based on BrdU incorporation was quantified using the BrdU ELISA kit (Roche) (experiment shown in FIG. 2). BrdU is taken up by newly synthesized DNA in the S phase of the cell cycle. Since DNA incorporating BrdU (labeled with BrdU) can be detected with an anti-BrdU antibody, the anti-BrdU antibody can be used as a tool for detecting proliferating cells that perform DNA replication.

(5) Cell viability assay and alkaline phosphatase (ALP) activity The number of viable cells was determined using Cell Counting Kit-8 (CCK-8, Dojindo). Next, the same cells were washed with PBS, and ALP activity was measured using an ALP assay kit (Takara Bio) (experiment shown in FIG. 5). These experiments were performed using hair papilla cells at passage 5 (P5) and hair papilla cells at passage 12 (P12) (experiment shown in FIGS. 3 to 5). In addition, since hair follicle cells with repeated passages and hair follicles with reduced hair growth-inducing ability are also considered to have reduced ALP activity, ALP is a typical example that shows hair growth-inducing ability of hair papilla cells. Index (also shown in FIG. 3).

(Experimental result)
(Confirmation of predetermined markers in P5 hair papilla cells and P12 hair papilla cells)
In these cells, bromodeoxyuridine (BrdU incorporation amount), RSPO1 production amount and ALP activity were confirmed. The results are shown in FIG.

  The ratio of BrdU positive cells in P5 was about 100%, while the ratio of BrdU positive cells in P12 was about 40% (a figure indicated as BrdU incorporation). In addition, the ratio of RS12 production of P12 significantly decreased compared to P5 (a figure indicated as RSPO1 expression). A decrease in the percentage of ALP activity at P12 compared to P5 was also significantly confirmed.

(Verification of RSPO1 production in P5 hair papilla cells and P12 hair papilla cells)
The result is shown in FIG. Differences in the RSPO1 production amount of each fraction were observed due to the difference in the number of passages of hair papilla cells, but the P5 group ((A) in FIG. 4) and the P12 group ((B) in FIG. 4). However, Fr. In 1 (group eluted with 20% ethanol), the production rate of RSPO1 was particularly high.

(Confirmation of ALP activity in P5 hair papilla cells and P12 hair papilla cells)
The result is shown in FIG. The influence on the ALP activity of each fraction was slightly different depending on the number of passages, and a slight promotion of activity was observed in the fraction eluted with a high ethanol concentration (for example, Fr. 3 to Fr. 5). However, unlike the production rate of RSPO1 shown in FIG. 4, the rate of enhancement of ALP activity due to the difference in the eluted fractions tended to be small. Therefore, it is suggested that the fraction produced by RSPO1 can be purified by utilizing the difference in fraction (difference in solvent).

(Discussion)
From the above-mentioned results, the action mechanism of RSPO1 as shown in FIG. 6 is expected. From the experimental results shown in FIG. 4 and FIG. 5, it is expected that ALP activity is not necessarily increased by the increase in production of RSPO1. This suggests the possibility that the Nasturium officinale extract is involved in increased production of RSPO1 and assists Wnt signaling. As a result, it is expected that the Nasturium officinale extract is involved in proliferation of outer root sheath cells, so-called growth phase maturation, and brings about effects such as hair growth.

(Analysis of further mechanism of hair growth)
The present inventor further analyzes the above-described expected mechanism of action of RSPO1 (FIG. 6), that is, further analyzes the mechanism of hair growth (specifically, hair growth by suppressing the hair loss factor). Analyzing whether or not is promoted).

FIG. 7 shows the test results confirming that the interaction between Wnt3a, RSPO1, and DKK-1 affects cell proliferation by confirming the survival rate of human epidermal keratinocytes. The vertical axis of FIG. 7 shows the cell survival rate when compared with the addition group (1) when the cell survival rate of the addition group (1) is 1.
Factors (Wnt3a (manufactured by R & D systems), DKK-1 (manufactured by R & D systems) and / or RSPO1 (manufactured by R & D systems))) were added to human epidermal keratinocytes (manufactured by Kurabo Industries) at 37 ° C. After culturing for 48 hours, the number of human epidermal keratinocytes was measured by CCK-8.
The addition groups (1) to (4) shown in FIG. 7 will be described. "-" In the figure indicates that no factor is added, and "+" indicates that a factor is added. For example, in Wnt3a, “+” is a group that activates the Wnt signal.
Addition group (1) is a group to which no factor is added, addition group (2) is a group to which Wnt3a is added, addition group (3) is a group to which Wnt3a and DKK-1 are added, addition group (4) Is a group to which Wnt3a, DKK-1 and RSPO1 were added.
When the cell survival rate of the addition group (1) was 1, the addition group (2) was 1.358, the addition group (3) was 1.133, and the addition group (4) was 1.411.
Comparing the cell survival rate of the addition group (2) and the addition group (3) suggests that DKK-1 suppresses cell proliferation (Wnt activity). Comparing the cell survival rate of the addition group (3) and the addition group (4), it is suggested that RSPO1 antagonizes DKK-1 and maintains (supports) Wnt activity, and RSPO1 invalidates DKK-1. It is suggested that there is an action to do.

Furthermore, the present inventor uses human hair papilla cells to administer an extract of Nasturium officinale and dihydrotestosterone (DHT), thereby increasing the production amount of DKK-1, IL-6 and androgen receptor (AR). The change was confirmed. The confirmation results are shown in Tables 1 to 3.
Fr. shown in Tables 1 to 3. 1 corresponds to Fr. 1. This Fr. 1 is presumed to have the ability to produce RSPO1 from the experimental results shown in FIGS.
Human hair papilla cells (P5) were seeded on plates on 1% FBS / DMEM medium at a density of 5000 cells / well in each addition group. After preparation, the Fr. 1 and / or DHT was added. Thereafter, dermal papilla cells were cultured at 37 ° C. for 2 days, and the supernatant of the cultured sample was collected as an ELISA assay sample shown in Tables 1 and 2. Tables 1 and 2 show the results of measuring the protein production using ELISA (Millipore). When DHT was added, it was added so that the final concentration in the medium was 250 nM.
In addition, the crude protein solution extracted from the cells cultured in the same manner was quantified by Bradford method, subjected to SDS-PAGE, and analyzed by Western blot. For blocking, PVDF Blocking Reagent (TOYOBO) was used, and detection was performed by chemiluminescence using an anti-AR antibody (manufactured by CST) and a horseradish peroxidase-conjugated secondary antibody. The detected results are shown in Table 3.
In Tables 1 to 3, the following are indicated. The non-added group is Fr. This is a group containing neither 1 nor DHT. Regarding DHT, “None” is a group to which DHT is not added, and “Yes” is a group to which DHT is added. Fr. For “1”, “None” indicates Fr. The group in which 1 is not added and the group in which the numerical values are described (addition group 2, addition group 3, addition group 5, addition group 6, addition group 8 and addition group 9) have a final concentration in the medium, It is the group added so that it might become the density | concentration each described in the table | surface.
The measurement results of ELISA are shown in Tables 1 and 2, and the results of Western blot are shown in Table 3. In Table 1 and Table 2, the protein production amount of the addition group 1 to the addition group 6 is described with the protein production amount of the non-addition group being 1. In Table 3, the amount of protein in the addition group 7, the amount of protein in the addition group 8, the addition group 8, and the addition group 9 is described as 1.
As shown in Tables 1 and 2, from the results of the addition group 2 and the addition group 3 and the results of the addition group 5 and the addition group 6, Fr. A concentration-dependent administration of 1 (fraction estimated to have RSPO1 production ability) showed a tendency to suppress the production of hair loss factors (DKK-1 and IL-6). Further, as shown in Table 3, from the results of the addition group 8 and the addition group 9, Fr. The concentration-dependent administration of 1 (the fraction presumed to have RSPO1 production ability) showed a tendency to suppress the expression level of AR (relaxing effect of male hormone receptor stimulation).
Based on the results shown in FIG. 1 to FIG. 7, the experimental results shown in Tables 1 to 3 are considered. It may be possible to increase the LRP level on the cell membrane by inhibiting the complex formation of Kremen and LRP, and to suppress the expression of the hair loss factor itself, and to activate the Wnt / β-catenin signal.

  As mentioned above, although embodiment (including an Example) of this invention has been demonstrated with reference to drawings, the concrete structure of this invention is not restricted to this, The range which does not deviate from the summary of this invention However, even if there is a design change or the like, it is included in the present invention.

  The present invention is used, for example, as a composition (agent etc.) for promoting hair growth for animals such as humans.

Claims (6)

  A production promoter for R-spondin1, comprising an extract of Dutch pepper as an active ingredient.   The production promoter of R-spondin1 according to claim 1, wherein the Dutch pepper extract is extracted with at least one solvent selected from the group consisting of water, ethanol and 1,3-butylene glycol.   A skin external preparation containing the R-spondin1 production promoter according to claim 1 or 2.   The skin external preparation for hair growth promotion of Claim 3.   The oral composition containing the production promoter of R-spondin1 of Claim 1 or 2.   The oral composition for promoting hair growth according to claim 5.