JP5858561B2 - Preparations or cosmetics or cosmetics for hair or scalp, comprising moth-derived VEGF production promoter and moth-derived VEGF production promoter - Google Patents

Description

  The present invention relates to a technical field related to promotion of angiogenesis. More specifically, the present invention relates to techniques such as cosmetics such as hair and scalp cosmetics, hair growth agents, hair nourishing agents, wound healing promotion and skin color improvement.

  The cocoon is a fossil that is mainly formed by the resin of a pine genus plant buried in the basement for a long time and condensed, and mainly contains resin, essential oil, succinic acid and the like. And it dissolves in a small amount in ethanol, diethyl ether or benzene (Non-patent Document 1). In addition to the use of the casket itself for decorative crafts, jewelry, insulating materials, and the use of shaving shavings as an incense, it has been used for scratches around the 19th century (Non-patent Document 2) .

  In recent years, a technique for blending wrinkle powder into cosmetics to improve skin feel (Patent Documents 1, 2, 16 and 17), a technique for blending an extract of wrinkle into a skin external preparation (Patent Documents 3 and 4), wrinkles Technology utilizing skin turnover promoting factor in extract fraction (Patent Document 5), Technology utilizing skin skin-farming effect in extract of salmon (Patent Document 6), Hyaluronic acid production in salmon extract fraction A technique using a promoting factor (Patent Document 7) has been reported. In addition, technology that blends glaze powder into cosmetics to prevent deterioration (Patent Documents 16 and 17), and technology that blends glaze powder into cosmetics to restore the youthful luster of mature human skin (Patent Documents 18 and 19) have already been introduced. Furthermore, it has been reported that the hot water extract of persimmon has a dermal papilla cell growth promoting action (Patent Document 20).

  On the other hand, angiogenesis is a phenomenon in which a new blood vessel is formed from an original blood vessel. Vascular endothelial progenitor cells first develop, assemble and adhere to form the primordial vascular plexus (vasculogenesis). From there, the process of angiogenesis such as fusion, insertion, germination, and regression of blood vessels occurs. The main factor that promotes angiogenesis is vascular endothelial growth factor (VEGF). VEGF is a group of glycoproteins involved in angiogenesis and angiogenesis, stimulating the division, migration, and differentiation of vascular endothelial cells that make up the innermost surface of blood vessels, and enhancing the vascular permeability of microvessels. Have.

  It has been reported that non-patent documents 9, 12, 13, and 14 indicate the involvement of HB-EGF in promoting VEGF production or angiogenesis.

  VEGF improves blood flow by forming new blood vessels, has a wound healing promoting effect and a skin color improving effect, and further has a hair-growth / hair-restoring effect and the like. In particular, in the hair growth / hair restoration effect, when the production of VEGF around the hair follicle increases, angiogenesis is promoted, and the nutrients necessary for the hair in the growth phase are efficiently supplied, which may lead to the effect of hair growth / hair restoration promotion. It is known (Non-patent Document 3). In addition, the paper (Non-patent Documents 4 and 5) that outer hair root sheath cells and hair papilla cells in hair follicles express VEGF, and when the amount of VEGF synthesis in the outer hair root sheath is increased, the size of hair follicles is increased. A paper (Non-Patent Document 3) has been reported that the diameter of the hair to be made increases with the increase. Therefore, VEGF production and angiogenesis are important for hair growth and hair restoration.

  Examples of VEGF production promoters that can be applied to hair growth and hair restoration effects that have been known so far include minoxidil (Non-patent document 6), eucalyptus extract (Non-patent document 7), white vanilla lupine bean extract (non- Patent Document 8), Royal Jelly (Patent Document 8), Dutch Pepper Extract (Patent Document 9), Steamed Distilled Water of Citrus Plants, and Fruit Juice (Patent Document 10), Shiitake Mushroom, Echinashi, Prunes, Moyashi, Achacharu Extracts (Patent Documents 11 and 12), Taconoki plant extract (Patent Documents 13 and 14), Chlorella extract, Yuzu fruit extract (Patent Document 15) and the like have been reported.

Patent No. 3725848 JP2004-083478 Patent No. 4034839 JP2001-131048 JP2007-314522 JP2008-189669 JP2008-266260 JP2003-192541 JP2003-313134 JP2003-313136 Patent No. 4324349 JP2004-035443 Patent No. 4050560 JP2004-043393 JP 2006-282597 Patent No. 3741429 JP2004-083477 Special table 2003-500429 JP2005-206613 JP2010-235551

Chuyaku University Dictionary Vol.2 Shanghai Science and Technology Publishing Co., Ltd. K. Kaiserling Pathloge, 22 (4), 285-286 (2001). Kiichiro Yano, et al. Journal of Clinical Investigation, 107, 409-417 (2001). Souad Lachgar, et al. The Journal of Investigative Dermatology, 106, 17-23 (1996). Urszula Kozlowska, et al. Archives of Dermatological Research, 290 (12), 661-668 (1998). S. Lachgar, British Journal of Dermatology, 138, 407-411 (1998). Shigeru Moriwaki, et al. Fragrance Journal, 35 (12), 22-27 (2007). C. Lenaers, et al. Fragrance Journal, 35 (12), 69-72 (2007). Kozo Nakai, Kozo Yoneda, Tetsuya Moriue, JunskeIgarashi, Hiroaki Kosaka and YasuoKubota, HB-EGF-induced VEGF production and eNOS activation depend on both PI3 kinase and MAP kinasein HaCaT cells, Journal of Dermatological Science, 55, 170-178 (2009) . Edited by Masafumi Shibuya, approaching the forefront of blood vessel research, p39, Yodosha (2000). Chemistry Doujin editorial edition, new edition continued, p54, Chemistry Doujin, (1987) for conducting experiments safely. Veela B. Mehta and Gail E. Besner, HB-EGF promotes angiogenesis in endothelial cells via PI3-kinase and MAPK signaling pathways, Growth Factors, 25 (4), 253-263 (2007). Veela B. Mehta, Yu Zhou, Andrei Radulescu and Gail E. Besner, HB-EGF stimulates eNOSexpression and nitric oxide production and promotes eNOSdependent angiogenesis, Growth Factors, 26 (6), 301-315 (2008). Burak M. Arkonac, Lauren C. Foster, Nicholas ES Sibinga, Cam Patterson, KaihuaLai, Jer-Chia Tsai, Mu-En Lee, Mark A. Perrella and Edgar Haber, Vascular Endothelial Growth Factor Induces Heparin-binding Epidermal Growth Factor-like Growth Factor in Vascular Endothelial Cells, The Journal of Biological Chemistry, 273, 4400-4405 (1998).

This invention makes it a subject to prepare a novel VEGF production promoter.
Furthermore, this invention makes it a subject to provide the usage method and use of the VEGF production promoter prepared by the lower alcohol extraction from straw.

  The inventors of the present application were searching for a novel VEGF production promoter, and found the VEGF production promoter from a specific fraction obtained by fractionating the koji extract by column chromatography or two-phase separation, thereby completing the present invention. It has been made.

  The present invention has an extremely excellent effect of providing a novel VEGF production promoter such as a composition containing a novel VEGF production promoting component from the cocoon component. Moreover, since the novel VEGF production promoter according to the present invention is dissolved in ethanol, it can be blended in cosmetics without giving a feeling of roughness. Furthermore, the novel method for producing a VEGF production promoter of the present invention is simply immersed in ethanol at about 40 ° C. to room temperature for about 1 month, so that it can be easily prepared without preparing high energy equipment. .

Confirmation of VEGFA-mRNA expression in F2 and N fractions using HaCaT When the ratio of VEGFA-mRNA expression to GAPDH-mRNA expression in the control sample is 100, it corresponds to GAPDH-mRNA expression in the sample-added sample The ratio of VEGFA-mRNA expression level was calculated and shown. Confirmation of neutralizing activity of HB-EGF neutralizing antibody in HaCaT When the ratio of VEGFA-mRNA expression to GAPDH-mRNA expression is 100, the amount of VEGFA-mRNA expression relative to GAPDH-mRNA expression in the sample added sample The ratio was calculated and shown. ΑHB-EGF represents an anti-human HB-EGF neutralizing antibody (manufactured by R & D SYSTEMS), and IgG goat represents an nonimmune goat antibody (manufactured by Sigma). HB-EGF represents recombinant human HB-EGF (R & D SYSTEMS) dissolved in PBS to 100 μg / mL. Confirmation of VEGFA-mRNA expression in F2 and N fractions using HB-EGF neutralizing antibody in HaCaT Confirmation of promotion of angiogenesis by tube formation assay The growth after 6 hours of incubation at 37 ° C is shown in the photograph. Confirmation of promotion of angiogenesis by tube formation assay (Figure 3) When the quantification result in the control sample is 100, the total area of the blood vessels (AREA), the total length of the blood vessels (LENGTH), the blood vessels The total number of branch points (JOINT) and the average of the length of each blood vessel (PATH) were calculated and shown. Confirmation of HB-EGF-independent tube formation promotion activity of F2 and N fractions The growth after 6 hours of incubation at 37 ° C is shown in the photograph. Confirmation of HB-EGF-independent tube formation promotion activity for F2 and N fractions Using the angiogenesis assay software ver. 2.0 (manufactured by Kurabo Industries) The analysis results are shown below. The values of AREA, LENGTH, JOINT, and PATH in the sample-added sample when the quantification result in the control sample was 100 were calculated and shown. Confirmation of the ability to promote VEGF production in the epidermal layer by F2 and N fractions applied to mouse skin. Confirmation of hair growth promoting activity by F2 fraction and N fraction applied to mouse skin Photographed on the 25th day from the start of application. Confirmation of hair growth promoting activity by F2 fraction and N fraction applied to mouse skin Evaluation by hair growth score is shown.

1. Introduction 1-1. The cocoon is a fossil made mainly from a resin of the pine genus plant that has been buried underground for a long period of time, and mainly contains resin, essential oil, succinic acid and the like. It is soluble in a small amount in ethanol, diethyl ether or benzene (Non-patent Document 1). In addition to the use of the casket itself for decorative crafts, jewelry, insulating materials, and the use of shaving shavings as an incense, it was used for scratches around the 19th century (Non-patent Document 2) . Furthermore, the effect of preventing various diseases associated with aging has been handed down (Yamano Beauty Mate HP http://www.yamanobeautymate.com).

1-2. VEGF (vascular endothelial growth factor)
VEGF is a group of glycoproteins involved in angiogenesis and angiogenesis that has an action of promoting angiogenesis, and has a function of stimulating cell division, migration, and differentiation and enhancing vascular permeability of microvessels. The VEGF family includes VEGFA, VEGFB, VEGFC, VEGFD, VEGFE, and the like. Of the novel VEGF production promoter according to the present invention, the F2 fraction and the N fraction were administered to cultured human-derived immortalized epidermal keratinocytes, and a comprehensive gene expression analysis by GeneChip ^R was performed. Compared with control, the expression level of VEGFA-mRNA increased to 1355%, whereas the expression levels of VEGFB-mRNA and VEGFC-mRNA did not change. On the other hand, the N fraction increased VEGFA-mRNA expression level by 3767%, VEGFB-mRNA expression level by 281%, and VEGFR-mRNA expression level by 311%. We have not examined changes in VEGFD and VEGFE-mRNA expression levels. Among the VEGF family, VEGFA and VEGFE mainly act on angiogenesis, and VEGFB has an angiogenesis-promoting activity of 1/10 or less than VEGFA, and is involved in monocyte chemotaxis, etc. And VEGFD is involved in the proliferation of lymphatic endothelial cells (Non-patent Document 10). Therefore, in the present invention, attention was paid to VEGFA having the strongest angiogenic activity.

There are four major types of human VEGFA: alternative splicing: 121 amino acids (VEGFA ₁₂₁ ), 165 (VEGFA ₁₆₅ ), 189 (VEGFA ₁₈₉ ), 206 (VEGFA ₂₀₆ ). A splicing polymorphism exists.

  VEGF improves blood flow by forming new blood vessels, and thereby has a wound healing promotion effect, a skin color improvement effect, and a hair growth / hair restoration effect.

2. Method for preparing VEGF production promoter extracted from cocoon As a VEGF production promoter extracted from cocoon of the present invention, an extract extracted from cocoon and containing a component having an ability to promote VEGF production, the cocoon extract A crude product of the extract and a composition purified from the koji extract are included. In the present invention, in particular, a VEGF production promoter extracted from sputum and a method for preparing the same are provided.

  Specifically, the VEGF production promoter of the present invention includes the following 2-1. And 2-2. (1) Preparation of extract from cocoon containing ingredient having ability to promote VEGF production, and (2) Crude extract of extract from cocoon containing ingredient having ability to promote VEGF production And compositions obtained by purifying the extract.

2-1. Preparation of VEGF production promoter extracted from sputum As a composition that acts on and activates skin cells, it can be prepared by extracting a VEGF production promoter from sputum. The extract can also be used as a VEGF production promoter.

  The method for extracting an extract containing a component having the ability to promote VEGF production from cocoon of the present invention includes a step of extracting from the cocoon with a lower alcohol. More specifically, the present invention includes a step of extracting an extract (composition) containing at least a component having an ability to promote VEGF production as an active ingredient from straw, preferably with a lower alcohol. The treatment step includes a step of pulverizing the cocoon and, if necessary, a step of washing the pulverized cocoon with a hydrophobic organic solvent. The invention of the present application may also include a step of fractionating the extract extracted by the step of extracting from the straw with a lower alcohol using a hydrocarbon-bonded silica gel column.

The process will be described below.
(i) Pretreatment step The soot is pulverized to an appropriate size so as to facilitate solvent extraction. As the pulverizing means, a file or a jet mill pulverizer can be used. Further, it is also possible to use a material obtained by pulverizing chips produced when processing a jar as a gemstone. The size to be pulverized is not particularly limited. From the viewpoint of extraction efficiency with a solvent, for example, the average particle size can be up to 100 μm, preferably about 10 to 30 μm. The ground soot is washed with a hydrophobic organic solvent as necessary. As the hydrophobic organic solvent, hexane, benzene, chloroform or a mixture thereof can be used. This washing can be omitted.

(ii) Extraction step The soot or the soot that has been crushed, ground and washed in the pretreatment step can be immersed in a lower alcohol to obtain an extract. For example, the soot or the soot that has been crushed or ground and washed in the pretreatment step is immersed in a lower alcohol for a long time at a low temperature or at room temperature to obtain an extract. Specifically, the soot or the soot that has been crushed or ground and washed in the pretreatment step is immersed in a lower alcohol at a temperature of, for example, 25 ° C. to 50 ° C. for 7 days or more, preferably 15 days or more. Obtain an extract. Although the upper limit of the immersion time is not particularly limited, it can be set to, for example, 3 months or less from the viewpoint of production efficiency. More specifically, for example, in the case of ethanol, the soot or the soot that has been crushed or ground and washed in the pretreatment step is at least 7 days at a temperature of 25 ° C. to 50 ° C., preferably about 40 ° C. Extracts are obtained by soaking for 1 month (15 to 30 days). The extract is concentrated and dried to a brownish candy-like dried product. The lower alcohol is not particularly limited as long as it is a linear, branched, or cyclic alcohol having 1 to 5 carbon atoms. For example, methanol, ethanol, propanol, butanol, or a mixture thereof can be used.

  The extract thus obtained can be used as a composition containing a component having the ability to promote VEGF production, but can be further crudely purified or purified as described below.

  In addition, this extract is a component having a heparin-binding epidermal growth factor-like factor (HB-EGF (Heparin-binding EGF-like growth factor)) and hyaluronic acid synthase 3 (HAS3 (Hyaluronic acid synthase)) production promoting activity. And can also be used as a composition having HB-EGF production promoting activity and HAS production promoting activity. The extract can also be used as an anti-drying agent, humectant, and anti-wrinkle agent for skin.

2-2. Preparation of a crudely purified product from an extract containing a component capable of promoting VEGF production or a composition purified from the extract (ii) obtained in (2-1) above, containing a component capable of promoting VEGF production The extract can be fractionated by column chromatography or two-phase separation to obtain a crude product or a purified composition.

2-2-1. Fractionation by column chromatography
(i) Column chromatography fractionation step The obtained extract (dried product) is dissolved in a lower alcohol, for example, ethanol, and column chromatography using a hydrocarbon chemically bonded silica gel, for example, octadecylsilylated silica gel as a carrier. By performing the step, the eluate can be fractionated. In particular, a fraction which is a colorless and transparent liquid which is eluted after the colorless and transparent liquid which is eluted first (for example, 1 to 5% by volume of the total amount of elution) and becomes a dark yellow candy when dried. (For example, 10 to 15% by volume of the total elution amount) is excellent in storage stability and has the ability to promote VEGF production.

  This fraction can be used as a crude product of a composition containing a component capable of promoting VEGF production from koji. In addition, the present fraction can be used after being dried or dissolved in an appropriate solvent such as a lower alcohol, specifically ethanol. Such a crudely purified product of the extract is very stable during storage. This crude product also has HB-EGF and HAS3 production promoting activity. The crude product of the extract purified by the column chromatography includes the F2 fraction in the following examples.

(ii) Purification by adsorption column chromatography Further purification can be performed by adsorption column chromatography.
Specifically, column chromatography using silica gel as a carrier can be used as the adsorption column chromatography. As an elution solvent, for example, it is possible to elute with a mixed solution of benzene and ethyl acetate as a solvent, specifically, benzene: ethyl acetate = 50: 1. What was refine | purified in this way can be used as a composition refine | purified from the koji extract containing the component which has VEGF production promotion ability.

2-2-2. Fractionation by two-phase separation (i) Removal of water-soluble components 2-1. The lower alcohol extract (dried product) of koji obtained by the above can be further fractionated into two phases, an aqueous phase and an organic solvent phase, using an appropriate organic solvent. Examples of the organic solvent include lower ethers, specifically, dimethyl ether or diethyl ether. The obtained lower alcohol extract of koji is dissolved in the organic solvent, and undissolved components are removed. Next, the organic solvent in which the soot extract is dissolved is washed with water. Specifically, pure water, for example, ultrapure water produced by an ultrapure water production apparatus is added to remove water-soluble components. If necessary, the removal of the water-soluble component using pure water is repeated 3 to 5 times.

(ii) Neutral fraction (N fraction)
Thereafter, an acidic aqueous solution and a basic aqueous solution are added several times as necessary, and mixed until the basic water-soluble component and the acidic water-soluble component are finally removed from the coloring component, respectively. ,Remove. It is desirable to wash 3 to 5 times with an acidic aqueous solution, and then wash several times with a basic aqueous solution until the organic solvent in which the koji extract is dissolved is removed. As the acidic aqueous solution, for example, dilute hydrochloric acid having a pH of 3.0, and as the basic aqueous solution, for example, a sodium hydroxide aqueous solution having a pH of 12.0 can be used. Finally, wash with pure water.

  The organic solvent is volatilized from the soot extract-dissolved organic solvent from which the water-soluble components have been removed to obtain a composition that promotes VEGF production. The composition obtained by the two-phase separation also includes a neutral fraction (N fraction) in the following examples.

  The neutral fraction can be used as a crudely purified product from a koji extract containing a component capable of promoting VEGF production or a composition purified from the extract.

2-3. Confirmation of VEGF production promoting ability The crudely purified product of the koji extract of the present invention or the composition purified from the extract has the ability to promote VEGF production. For example, these can be administered to cultured cells and VEGFA-mRNA It can be confirmed by measuring expression. Alternatively, it can be confirmed by a tube formation assay. Specifically, the crudely purified koji extract of the present invention or a composition purified from the extract is used to culture vascular endothelial cells using a medium for culturing small vascular endothelial cells, and has an angiogenesis promoting action. It can be confirmed whether or not.

  Alternatively, the chicken embryo chorioallantoic membrane method (chorioallantoic membrane assay method, CAM method), rabbit cornea method, mouse dorsal subcutaneous method (dorsal air sac method, DAS method) can also be used. Furthermore, the sample can be applied to mouse skin, and the anti-VEGF antibody can be used to confirm the ability to promote VEGF production in the epidermis layer.

  Furthermore, the crudely purified extract containing the component having the ability to promote VEGF production of the present invention or a composition purified from the extract is different from the conventionally known one having the ability to promote VEGF production. It can be shown as follows.

  In the above method for confirming VEGF production promoting ability, an antagonist such as an antibody against a composition having a known ability to promote VEGF production is applied to cultured cells together with a crudely purified product of the extract of the present invention or a composition purified from the extract. This can be confirmed by examining whether or not the ability to promote VEGF production changes.

  It was confirmed that the crudely purified product having the ability to promote VEGF production derived from the cocoon of the present invention or the composition purified from the extract contains a component having a novel ability to promote VEGF production.

2-4. Confirmation of hair-growth effect, hair-growth effect Hair-growth effect, hair-growth effect, and / or hair-growth effect are described in, for example, Daisaburo Yokoyama, Yukagaku, 44 (4), 266-273 (1995). Kuniaki Adachi, Journal of Cosmetic Association 19 (1), 20-24 (1995). Or, by the method described in Noboru Kobayashi, Pharmaceutical Journal, 113 (10), 718-724 (1993). More specifically, for example, the back of the mouse is partially shaved with an electric clipper and further shaved with a shaver. After acclimation, the sample is repeatedly applied to the shaved site. After a certain period of time, the growth of the hair can be visually observed to confirm the hair growth effect, the hair nourishing effect, and / or the hair growth effect.

  It was confirmed that the crudely purified product having the ability to promote VEGF production derived from wrinkles of the present invention or a composition purified from the extract has a hair-restoring effect, a hair-restoring effect and / or a hair-growth effect.

3. The hair nourishing agent and / or hair restoring agent extracted from the cocoon The present invention includes a hair nourishing agent and / or a hair restoring agent containing a VEGF production promoting component extracted from the cocoon using a lower alcohol. The method for extracting the hair nourishing agent and / or hair restorer of the present invention from the cocoon includes a step of extracting the cocoon from the cocoon with a lower alcohol. More specifically, the present invention includes at least a step of extracting an extract (composition) containing a component having a hair-restoring action and / or a hair-restoring action as an active ingredient from wrinkles with a lower alcohol, preferably The extraction pretreatment step includes a step of pulverizing the cocoon and, if necessary, a step of washing the pulverized cocoon with a hydrophobic organic solvent. The invention of the present application may also include a step of fractionating the extract extracted by the step of extracting from the straw with a lower alcohol using a hydrocarbon-bonded silica gel column.

  In the present invention, as the hair nourishing agent and / or hair restoring agent extracted from cocoons, the above-mentioned 2-2-1. Fractionation by column chromatography and / or 2-2-2. Includes compositions prepared by extraction from straw from fractions by two-phase separation.

When the total amount of the hair nourishing agent and / or hair restoring agent is 100, about 0.0001 to 50% by mass can be added.
For example, the above described 2-2-1. Fractionation by column chromatography and / or 2-2-2. In any case of fractionation by two-phase separation, when the total amount of the hair nourishing agent and / or hair restoring agent is 100, about 0.001 to 5.0% by mass, more preferably 0.001 to 1.0% by mass can be added. Specifically, the F2 fraction and / or the N fraction can be added in an amount of about 0.001 to 1.0% by mass when the hair nourishing agent and / or the whole hair restoring agent is taken as 100.

4). Pharmaceutical preparations or cosmetics or cosmetics for hair and scalp VEGF production promoter of the present invention, specifically, for example, an extract from salmon containing a component having an ability to promote VEGF production, a crudely purified product thereof, or the extraction The composition purified from the product can be made into, for example, a pharmaceutical preparation or a cosmetic using an appropriate excipient. For example, an extract containing a component having an ability to promote VEGF production and a crudely purified product thereof or a composition purified from the extract are absorbed into an additive such as lactose, silica gel, crystalline cellulose, or starch to obtain a powder. It can also be formulated into a preparation.

  Additives usually used in these can be added to the pharmaceutical preparation or cosmetic of the present invention, for example, proteins such as bovine serum albumin, ovalbumin, milk casein, saccharides such as maltose, sucrose, trehalose, Polysaccharides such as heparin, chitosan, alginic acid, polyacrylic acid, polymethacrylic acid and carboxymethylcellulose can also be added as stabilizers.

  Furthermore, as a thickener, water-soluble polysaccharides, such as sodium carboxymethylcellulose, sodium alginate, dextran, sodium hyaluronate, etc. are mentioned, for example. Examples of the oil phase thickener include various fatty acids and fats.

  Examples of the preservative include alkyl parabens such as methyl paraben and propyl paraben.

4-1. Cosmetics for hair and scalp VEGF production promoter extracted from the cocoon of the present invention using a lower alcohol, specifically, for example, an extract extracted from cocoon containing a component having an ability to promote VEGF production, and its crude purification The product or a composition purified from the extract can be added to cosmetics (preferably cosmetics for hair and scalp) or external preparations for skin (preferably external preparations for hair and scalp). More specifically, a cosmetic or a skin external preparation containing a VEGF production promoter extracted from cocoons of the present invention using a lower alcohol can be used as a skin external preparation. The hair-restoring agent and / or hair nourishing agent extracted from the wrinkles described in 3. above is a cosmetic (preferably, cosmetic for hair / scalp) or a skin external medicine (preferably, external medicine for hair / scalp). ).

  The koji extract of the present invention can be added to cosmetics in a dried state or a state dissolved in an appropriate solvent, for example, a state dissolved in ethanol. The content of the extract composition of the present invention can be added in an amount of about 0.0001 to 50% by mass when the total cosmetic is 100.

  For example, the above described 2-2-1. Fractionation by column chromatography and / or 2-2-2. For both fractions by two-phase separation, when the total cosmetic is 100, about 0.001 to 5.0% by mass, and more preferably 0.001 to 1.0% by mass can be added. Specifically, the F2 fraction and / or the N fraction can be added in an amount of about 0.001 to 1.0% by mass when the total cosmetic is 100.

  Cosmetics to which the VEGF production promoter extracted from the cocoon of the present invention is added are not limited to their dosage forms, but include dosage forms such as emulsions, creams, ointments, solutions, gels, packs, lotions, powders, sticks, etc. Can be mentioned. The cosmetics of the present invention can appropriately contain other additive components that are usually used as cosmetic raw materials. Further, the cosmetic of the present invention may contain other base components that are usually used as cosmetic raw materials. Base ingredients include liquid fats (olive oil, etc.), solid fats (shea fat, etc.), waxes (beeswax, etc.), hydrocarbon oils (liquid paraffin, paraffin, petrolatum, etc.), higher fatty acids (stearic acid, etc.), higher grades Oily components such as alcohol (cetanol, etc.), synthetic ester oil (octyldodecyl myristate, etc.), silicones (methylpolysiloxane, etc.), various surfactants, sequestering agents, water-soluble polymers (carboxyvinyl polymer, etc.) ), Thickeners, various powder components, fragrances, water and the like.

4-2. External preparation for skin The VEGF production promoter extracted from the sputum of the present invention can also be added to an external preparation for skin (preferably an external preparation for hair and scalp).

  As an external preparation for skin, it can be produced in various forms such as liquid, paste, cream, ointment, powder and patch. In addition, other externally added ingredients such as mineral oils, higher alcohols, animal and vegetable oils, waxes, silicone oils, moisturizers, wetting agents, water-soluble polymers, lower alcohols, water Antioxidants, pH adjusters, dyes, pigments, antiseptics, antifungal agents and other medicinal agents, chelating agents, and the like can also be added.

When the skin external preparation is 100 as a whole, about 0.0001 to 50% by mass can be added.
For example, the above described 2-2-1. Fractionation by column chromatography and / or 2-2-2. In any case of fractionation by two-phase separation, when the total amount of external preparation for skin is 100, about 0.001 to 5.0% by mass, more preferably 0.001 to 1.0% by mass can be added. Specifically, the F2 fraction and / or the N fraction can be added in an amount of about 0.001 to 1.0% by mass when the total amount of the external preparation for skin is taken as 100.

[Example 1]
<Preparation of 琥珀 ethanol extract>
Russian baltic sea bream was crushed to about 100 mesh, 10 g was immersed in 100 mL of hexane, allowed to stand at room temperature for one week, filtered through filter paper (No. 2), and the filtrate was discarded. The filtered powder was immersed in 100 mL of ethanol and allowed to stand in a 40 ° C. water bath or in a thermostatic chamber for one week, followed by filtration with filter paper (No. 2), and the filtrate was stored in a dark place. The filtered powder was immersed in 50 mL of ethanol, covered with aluminum foil, etc., left on a 40 ° C. water bath or in a thermostatic chamber for one week, and then filtered again with filter paper (No. 2). The filtrate was further immersed twice in 50 mL of ethanol twice, and after filtration, all the filtrates were combined. While slightly warming (about 38 ° C.), the solution was concentrated and dried by an evaporator to obtain about 1.3 g of a brownish candy-like ethanol extract.

<Preparation of F2 fraction>
Column chromatography using 0.4 g of this ethanol extract in 2 mL of ethanol, 11 g of octadecylsilylated silica gel (Wakogel 100C18 manufactured by Wako Pure Chemical Industries, Ltd.) as a carrier, and methanol (reagent special grade manufactured by Wako Pure Chemical Industries, Ltd.) as a solvent ( Column size: 2.2 cm id × 4.7 cm), and the eluate was collected. Of the total eluate volume of 132 ml, collect the dark yellow transparent fraction (collected volume: 17 mL) that was eluted after the first 5 ml of colorless and transparent liquid. 330 mg of a fraction was obtained. This fraction was designated as F2.

<Preparation of neutral (N) fraction>
Separately from F2, 0.5 g of ethanol extract was dissolved in 20 mL of diethyl ether (special grade reagent manufactured by Kokusan Kagaku Co., Ltd.), and the remaining residue was removed by filtration. Add 5 mL of ultrapure water collected from the ultrapure water production equipment (made by Nihon Millipore), plug and shake, and shake for about 1 minute until it reaches equilibrium while opening the cock and returning the internal pressure occasionally. Extracted by mixing. Thereafter, the mixture was allowed to stand for about 5 minutes until it was separated into two layers, and the lower layer was removed. Again, 5 mL of ultrapure water was added to the separatory funnel and extracted, and the work of removing the lower layer was repeated twice. Thereafter, pH 3.0 diluted hydrochloric acid prepared by adding 2 μL of hydrochloric acid (special grade reagent manufactured by Sigma) in advance to 18 mL of ultrapure water was extracted by adding to a 5 mL separating funnel, and the operation of removing the lower layer was repeated three times. Then, 5 mL of ultrapure water was added to the separating funnel for extraction, and the lower layer was removed. After that, the pH 12.0 aqueous sodium hydroxide solution prepared by adding 45 mg of sodium hydroxide (Kanto Chemical Co., Inc.) 45 mg to 62 mL of ultrapure water in advance was added to the 5 mL separatory funnel and extracted, and the lower layer was removed three times. Repeated. Usually, it is said that the extraction can be sufficiently distributed with 3-5 extractions (Non-Patent Document 11). However, when washing is performed by adding a pH 12.0 aqueous sodium hydroxide solution, the first lower layer and the third lower layer are separated. Since it had the same color (light yellow), it was judged that it was not yet sufficiently distributed, and the process was repeated about 12 times until the lower layer was no longer colored. At this time, sometimes it could not be separated into two layers by shaking. In that case, add a small amount of sodium chloride (special grade of Wako Pure Chemicals), leave it overnight, or take out the contents of the separatory funnel. They were separated by centrifugation with a centrifuge (manufactured by KUBOTA). Finally, 5 mL of ultrapure water was added to the separatory funnel and extracted, and the lower layer was removed. The ether layer remaining in the separatory funnel was collected and dried with an evaporator to obtain 105 mg of a pale yellow solid fraction. This fraction was designated N.

[Example 2]
<Verification of VEGFA gene expression by gene analysis with GeneChip ^{R for} F2 and N fractions>
Dulbecco's Modified Eagle Medium (DMEM) (Invitrogen) supplemented with 5% bovine serum (FETAL BOVINE SERUM) (EQUITECH-BIO) and 1% preservative (Penicillin-Streptomycin Glutamine) (Invitrogen) (Hereinafter referred to as DMEM medium), human-derived immortalized epidermal keratinocytes (hereinafter referred to as HaCaT) are suspended at a concentration of 2.64 × 10 ⁶ co / mL, and 13 mL of DMEM medium is placed in a 15 cm diameter. 2 mL each was seeded on a dish (Nunc) and cultured at 37 ° C. for 24 hours under 95% air-5% carbon dioxide gas. After removing the culture supernatant by aspiration, a culture solution containing 1% preservative (Penicillin-Streptomycin Glutamine) (Invitrogen) in phenol red-free Dulbecco's modified Eagle medium (Invitrogen) (hereinafter referred to as serum-free medium) Was placed in a dish at 15 mL each and further cultured for 48 hours. After removing the culture supernatant by aspiration, 0.1% bovine serum albumin (EQUITECH-BIO) and 1% preservative (Penicillin-Streptomycin Glutamine) (Invitrogen) were added to Dulbecco's modified Eagle's medium without phenol red (Invitrogen). 10 mL of a culture solution containing the above (hereinafter referred to as a treatment medium) was added. Then, the F2 fraction dissolved in ethanol to a final concentration of 30 μg / mL (origin cocoon: arrival in April 2002) and N fraction (origin cocoon: arrival in May 2006) were each treated medium. (Ethanol final concentration: 0.33%) was added dropwise to each of 5 mL. In addition, the control medium was treated with a culture medium in which ethanol was added to the treatment medium to a final ethanol concentration of 0.33%. These dishes were cultured at 37 ° C. for 24 hours under 95% air-5% carbon dioxide gas.

These were immobilized using RNA purification kit RNeasy Mini Kit (manufactured by QIAGEN) and total RNA was extracted (the product is hereinafter referred to as total RNA). The total RNA amount was determined by absorbance at 260 nm using a spectrophotometer (Nano Drop). This total RNA is generated using GeneChip ^R Eukaryotic Poly-A RNA Control Kit (AFFYMETRIX), GeneChip ^R Exoression 3'-Amplification One-Cycle cDNA Synthesis Kit (AFFYMETRIX), GeneChipR Sample Cleanup Module (AFFYMETRIX), GeneChip ^R Using Exoression 3'-Amplification Reagents (manufactured by AFFYMETRIX), Biotin-Labeled complementary RNA (hereinafter referred to as cRNA) was prepared according to the protocol recommended by Affymetrix. After hybridization of the cRNA to the GeneChip ^R at GeneChip ^R Hybridazation Oven 640 (manufactured by Affymetrix), and washed with GeneChip ^R Fluidics Station 450 (manufactured by Affymetrix), the fluorescence intensity in staining GeneChip ^R Scanner 3000 (manufactured by Affymetrix) I read. Using the microarray data analysis software GeneSpring GX Ver. 9. 0. 5 (manufactured by Agilent Technologies), measure the ratio of the amount of luminescence based on the obtained image data, and set the VEA-mRNA expression level of the control sample to 100 The ratio of the VEGFA-mRNA expression level, the VEGFB-mRNA expression level and the VEGFC-mRNA expression level was calculated.
The results are shown in Table 1.

（表１ ヒト不死化表皮角化細胞(HaCaT)を用いたF2画分及びN画分のGeneChip^RによるVEGFA、VEGFB並びにVEGFCのmRNA発現変化）

(Table 1 Changes in mRNA expression of VEGFA, VEGFB, and VEC by GeneChip ^R of F2 and N fractions using human immortalized epidermal keratinocytes (HaCaT))

  When the F2 fraction was treated with HaCaT to a final concentration of 30 μg / mL and cultured for 24 hours, the expression level of VEGFA-mRNA increased by 1355% compared to the control sample, whereas VEGFB-mRNA and VEGFC- The mRNA expression level did not change. In addition, it was suggested that when the N fraction was treated in the same manner, the expression level of VEGFA-mRNA increased by 3767%, the expression level of VEGFB-mRNA increased by 281%, and the expression level of VEGFC-mRNA increased by 311%. Therefore, it was suggested that the F2 fraction and the N fraction have significant VEGFA-mRNA expression increasing ability.

[Example 3]
<Verification of VEGFA-mRNA expression in F2 and N fractions using HaCaT>
Suspend HaCaT at a concentration of 1.56 × 10 ⁶ co / mL in DMEM medium, and inoculate 512 μL each in a 6 cm diameter dish (CORNING) containing 4.49 mL of DMEM medium, 95% air-5% The cells were cultured at 37 ° C. for 24 hours under carbon dioxide. After removing the culture supernatant by suction, 5 mL of serum-free medium was added to the dish and further cultured for 48 hours. After removing the culture supernatant by suction, 3.75 mL of treatment medium was added. A mixture of F2 and N fractions dissolved in ethanol to a final concentration of 6, 9, 12, 25 μg / mL mixed in the treatment medium (ethanol final concentration: 0.25%) was added dropwise to each 1.25 mL. did. In addition, the control medium was treated with a culture medium in which ethanol was added to the treatment medium to a final ethanol concentration of 0.25%. These dishes were cultured for 6 hours at 37 ° C. under 95% air-5% carbon dioxide gas.

From these, total RNA was extracted by the method shown in Example 2 above, and the amount of total RNA was determined. Next, based on this total RNA, RT reaction was performed using PrimeScript ^™ RT reagent Kit (TaKaRa) according to the attached document. Further using SYBR ^R Premix Ex TaqTM II (TaKaRa Co.), the reaction solution was prepared in accordance with the instructions attached. This reaction solution was reacted with each primer (manufactured by Invitrogen) of VEGFA and GAPDH as an internal standard by a quantitative RT-PCR method. When the ratio of the VEGFA-mRNA expression level to the GAPDH-mRNA expression level in the control sample was 100, the ratio of the VEGFA-mRNA expression level to the GAPDH-mRNA expression level in the sample-added sample was calculated.

  The results are shown in FIG. When the F2 fraction was treated with HaCaT to a final concentration of 25 μg / mL and cultured for 6 hours, 577% compared to the control sample, and 43% when the N fraction was treated in the same way, significant VEGFA -It was confirmed to have the ability to increase mRNA expression. Moreover, it was found that the F2 fraction and the N fraction increase VEGFA-mRNA expression in a concentration-dependent manner.

[Example 4]
<Verification of VEGFA-mRNA expression in F2 and N fractions using HB-EGF neutralizing antibody in HaCaT>
Suspend HaCaT at a concentration of 1.46 × 10 ⁶ co / mL in DMEM medium and inoculate 240 μL each in a 3.5 cm diameter dish (Falcon) containing 760 μL of DMEM medium, 95% air -5% The cells were cultured at 37 ° C. for 24 hours under carbon dioxide. After removing the culture supernatant by aspiration, 1 mL of serum-free medium was added to the dish and further cultured for 48 hours. After removing the culture supernatant by aspiration, 710 μL of treatment medium was added. 250 μL each of F2 fraction and N fraction dissolved in ethanol so that the final concentration was 25 μg / mL was mixed with the treatment medium (ethanol final concentration: 0.25%) was added dropwise thereto. In addition, the control medium was treated with a culture medium in which ethanol was added to the treatment medium to a final ethanol concentration of 0.25%. Further, 40 μL of anti-human HB-EGF neutralizing antibody (manufactured by R & D SYSTEMS) (hereinafter referred to as αHB-EGF) dissolved in PBS so as to have a final concentration of 10 μg / mL was dropped. As a negative control, non-immune goat antibody (manufactured by Sigma) and PBS were used instead of αHB-EGF. In order to confirm that αHB-EGF has neutralizing activity, recombinant human HB-EGF (R & D SYSTEMS) (hereinafter referred to as HB-EGF protein) dissolved in PBS to a concentration of 100 μg / mL is pre-concentrated. Diluted with ethanol to 20 μg / mL and mixed with the treatment medium to a final concentration of 50 ng / mL, 250 μL each was added dropwise. These dishes were cultured for 6 hours at 37 ° C. under 95% air-5% carbon dioxide gas.

  From these, total RNA was extracted by the method shown in Example 2 above, and the amount of total RNA was determined. Subsequently, it was made to react using each primer of VEGFA and GAPDH by the quantitative RT-PCR method by the method shown in Example 3 above. When the ratio of the VEGFA-mRNA expression level to the GAPDH-mRNA expression level in the control sample was 100, the ratio of the VEGFA-mRNA expression level to the GAPDH-mRNA expression level in the sample-added sample was calculated.

  The results are shown in FIGS. 2-1 and 2-2. When the HB-EGF protein is treated to a final concentration of 50 ng / mL in HaCaT and cultured for 6 hours, the result of the αHB-EGF-treated sample is compared with the PBS-treated sample and the non-immune goat antibody-treated sample. As a result, the αHB-EGF used was found to have neutralizing activity. In contrast, when the F2 fraction and N fraction were treated with HaCaT to a final concentration of 25 μg / mL and cultured for 6 hours, compared to the PBS-treated sample and the nonimmune goat antibody-treated sample, αHB There was no significant change in the results of the -EGF-treated sample, and it was confirmed that it had the ability to enhance VEGFA-mRNA expression, and that the enhancement of VEGFA-mRNA expression by the F2 and N fractions was not mediated by HB-EGF . In Example 3, when the F2 fraction was treated to a final concentration of 25 μg / mL and cultured for 6 hours, it was increased by 577% compared to the control sample, and when the N fraction was treated in the same manner. An increase of 433% was observed, but in this sample where PBS was added under the same conditions, the F2 fraction was 127% and the N fraction was 181%, indicating a difference in the enhancement rate. This is thought to be due to differences in cell conditions.

[Example 5]
<Confirmation of angiogenesis promotion by tube formation assay>
5% bovine serum (FETAL BOVINE SERUM) (manufactured by EQUITECH-BIO) and 0.01 μg / mL Fibroblast Growth Factor (manufactured by PEPROTEC) in low serum medium for human microvascular endothelial cell culture (hereinafter referred to as MCDB131) (manufactured by Sigma) ) Was added to a human skin microvascular endothelial cell (hereinafter referred to as HMVEC) at 37 ° C. in 95% air-5% carbon dioxide gas. For the experiment, BD MatrigelTM Basement Membrane Matrix Growth Factor Reduced, Phenol Red Free (BD) (hereinafter referred to as Matrigel) was dispensed at 50 μL / well into a 96-well plate (Falcon) at 37 ° C for 1 hour. The coated one was used. Suspend cultured HMVEC at a concentration of 1.0 × 10 ⁵ co / mL in MCDB131 supplemented with 1% bovine serum and 0.01 μg / mL Fibroblast Growth Factor (hereinafter referred to as MCDB131 starvation medium). 50 μL each was seeded on a 96-well plate coated with Matrigel. A culture solution in which the F2 fraction and N fraction dissolved in ethanol so as to have final concentrations of 15 and 25 μg / mL were mixed with MCDB131 starvation medium (ethanol final concentration: 0.25%) was added dropwise thereto in an amount of 50 μL. In addition, a control solution obtained by treating an MCDB131 starvation medium with ethanol added to a final ethanol concentration of 0.25% was used as control. The plate was incubated at 37 ° C. for 6 hours under 95% air-5% carbon dioxide. For the analysis, angiogenesis quantification software ver. 2.0 (manufactured by Kurabo Industries) was used. When the quantification result in the control sample is 100, the total area of the blood vessel in the sample added sample (hereinafter referred to as AREA), the total length of the blood vessel (hereinafter referred to as LENGTH), the total number of branch points of the blood vessels (hereinafter referred to as JOINT), and the average length of each blood vessel (hereinafter referred to as PATH) was calculated.

  The results are shown in FIGS. When the F2 fraction was treated at HMVEC to a final concentration of 15 μg / mL and cultured for 6 hours, compared to the control sample, AREA was 198%, LENGTH was 233%, JOINT was 533%, PATH was 319%, and When the N fraction was treated in the same manner, AREA was 198%, LENGTH was 220%, JOINT was 495%, and PATH was 296%.

[Example 6]
<Confirmation of HB-EGF-independent tube formation promotion activity in F2 and N fractions>
HMVEC was cultured in MCDB131 medium at 37 ° C under 95% air-5% carbon dioxide. In the experiment, Matrigel was dispensed into a 48-well plate (manufactured by Falcon) at 150 μL / hole and coated at 37 ° C. for 1 hour. HMVEC was suspended in MCDB131 starvation medium at a concentration of 1.0 × 10 ⁵ cells / mL, and 150 μL was seeded in 48-well plates that had been coated with Matrigel in advance. 150 μL each of a culture solution (ethanol final concentration: 0.25%) in which the F2 fraction and N fraction dissolved in ethanol were mixed with MCDB131 starvation medium to a final concentration of 15 μg / mL was added thereto. In addition, a control solution obtained by treating an MCDB131 starvation medium with ethanol added to a final ethanol concentration of 0.25% was used as control. Furthermore, 6 μL of αHB-EGF dissolved in PBS was added to a final concentration of 10 μg / mL. As a negative control, an immune-free goat antibody (manufactured by Sigma) and PBS were used instead of αHB-EGF. In order to confirm that αHB-EGF has neutralizing activity, HB-EGF protein dissolved in PBS to a concentration of 100 μg / mL was diluted with PBS to a concentration of 2.0 μg / mL in advance, to a final concentration of 5 ng / mL. 150 μL each of the mixture in the processing medium was added. The plate was incubated at 37 ° C. for 6 hours under 95% air-5% carbon dioxide. For the analysis, angiogenesis quantification software ver. 2.0 (manufactured by Kurabo Industries) was used. The values of AREA, LENGTH, JOINT, and PATH in the sample added sample when the quantification result in the control sample was 100 were calculated.

  The results are shown in FIGS. When HMVEC is treated with HB-EGF protein to a final concentration of 5 ng / mL and cultured for 6 hours, compared to the PBS-treated sample (A-2) and the non-immune goat antibody-treated sample (C-2), αHB -EGF-treated sample (B-2) was used because the promotion of angiogenesis was suppressed in A-1 vs A-2 and C-1 vs C-2 (B-1 vs B-2) ΑHB-EGF was confirmed to have neutralizing activity. In contrast, when F2 fraction and N fraction were treated at HMVEC to a final concentration of 15 μg / mL and cultured for 6 hours, PBS treated samples (A-3 and A-4, respectively) Compared with goat antibody-treated samples (C-3 and C-4, respectively), there was no significant inhibition by αHB-EGF treatment (B-3 and B-4, respectively), depending on the F2 and N fractions. The ability to promote angiogenesis was confirmed even when HB-EGF activity was neutralized, and it was found that the promotion of angiogenesis by the F2 and N fractions was not mediated by HB-EGF.

[Example 7]
<Confirmation of ability to promote VEGF production in the epidermal layer by F2 and N fractions applied to mouse skin>
Six-week-old C3H mice (Japan SLC) were purchased and bred for one week, and then the back was depilated with a hair remover (Kanebo) (about 2.5 cm × 4.0 cm). After acclimatization overnight, remove 200μL of each sample (25mg / mL or 50mg / mL) in which F2 and N fractions were dissolved in ethanol to a volume of 5mg / day or 10mg / day, respectively. Applied to the site. As a control, 200 μL of ethanol was similarly applied. This was applied 5 times per week (5 times every 24 hours from Monday to Friday and closed on Saturdays and Sundays) for 3 weeks, for a total of 21 days. Two mice were used per group.

  Mouse back skin tissue was collected and fixed by immersion in 4% paraformaldehyde aqueous solution at 4 ° C. overnight. After dehydration with ethanol in accordance with a conventional method, it was replaced with xylene and finally processed into a paraffin section. The paraffin of this section was washed with xylene, then immersed in 0.1 M citrate buffer (pH 6.0) that had been heated to 95 ° C., and incubated for 10 minutes. After cooling, it was washed with PBS buffer, immersed in 3% hydrogen peroxide solution, and incubated at room temperature for 10 minutes. After washing with PBS buffer solution, 1.5% goat normal serum (VECTOR, attached to VECTASTAIN Elite ABC Rabbit IgG kit) -blocking by incubation with PBS buffer at room temperature for 1 hour, this solution was removed, and then 1.5% Incubate overnight at 4 ° C with anti-human VEGF capture antibody (Peprotech, attached to Mini Elisa Development Kit, hereinafter referred to as αVEGF) diluted to a final concentration of 0.2 μg / ml in% goat normal serum-PBS buffer did. As a negative control, an immunoimmune rabbit antibody having the same concentration (manufactured by Jackson ImmunoResearch Laboratories, hereinafter referred to as IgG rabbit) was used. After washing with PBS buffer, it was incubated with a biotin-labeled anti-rabbit antibody for 30 minutes at room temperature according to the protocol of VECTASTAIN Elite ABC Rabbit IgG kit, and then incubated with HRP-labeled streptavidin for 30 minutes at room temperature. After washing with PBS buffer, it was incubated with a diaminobenzidine solution at room temperature for 9 minutes according to the protocol of DAB PEROXIDASE SUBSTRATE KIT (manufactured by VECOR). After washing with ultrapure water, soaked in Mayer's hematoxylin solution (Muto Chemical Co., Ltd.) for 20 seconds, washed with purified water, then soaked in 0.1M sodium tetraborate buffer (pH 8.5) for 1 minute, and purified water Washed with. Thereafter, it was washed with ethanol and xylene according to a conventional method, dried, sealed with Histological Mounting Medium (manufactured by national diagnostics), observed under a microscope, and photographed.

  The results are shown in FIG. When αVEGF was used to stain human VEGF in brown, the control mouse skin tissue treated with ethanol (A-1) showed almost no brown staining image, whereas the F2 and N fractions showed 5 In mouse skin tissues treated with a mg / day volume (A-2 and A-3, respectively), a brown stained image was observed in the epidermis layer. Similar results were observed in mouse skin tissue treated with a dose of 10 mg / day. Further, in the groups using IgG rabbit as a negative control (B-1, B-2, B-3), no brown stained image was observed. From the above results, it was found that the F2 fraction and the N fraction contain an activity to increase the amount of VEGF produced from epidermal keratinocytes in vivo.

[Example 8]
<Confirmation of hair growth promoting activity by F2 fraction and N fraction applied to mouse skin>
After purchasing a 5-week-old C3H mouse (Japan SLC) and acclimatized for 2 weeks, the back was shaved (about 2.5 cm x 4.0 cm) with an electric clipper (Slive), and a shaver (Daiei) And then shaved further. After acclimatization overnight, 200 μL of each sample (10 mg / mL or 25 mg / mL) dissolved in ethanol so that the F2 fraction and N fraction were 2 mg / day or 5 mg / day respectively. It was applied to the shaved site. As a control, 200 μL of ethanol was similarly applied. Further, as a positive control, a 1% solution (10 mg / mL) in which minoxidil (manufactured by Sigma-Aldrich Japan) was dissolved in ethanol to 2 mg / day was applied in the same manner as 200 μL. This was applied 5 times per week (5 times every 24 hours from Monday to Friday and closed on Saturdays and Sundays) for 3.5 weeks and applied for a total of 24 days. Three mice were used per group. Hair growth was evaluated on the 25th day from the start of application by visual observation / photographing and hair growth score. The hair growth score is visually 0: almost no hair growth, 1: hair growth, 2: normal hair appearance restored to 0-10%, 3: normal hair growth Appearance 10-20% recovered, 4: Normal hair appearance 20-30% recovered, 5: Normal hair appearance 30-40% recovered, 6: Normal hair appearance 40-50% 7: Normal hair appearance 50-60% recovered, 8: Normal hair appearance 60-70% recovered, 9: Normal hair appearance 70-80% recovered, 10 : Normal hair appearance was recovered from 80 to 100%, and the hair growth score of each mouse was determined.

  The results are shown in FIGS. 8-1 and 8-2. In the control group to which ethanol was applied as a control, hair growth was hardly observed, but a sample (10 mg / mL) in which the F2 fraction was dissolved in ethanol to a volume of 2 mg / day was applied. Obvious hair growth was observed in the group. Similarly, hair growth was also observed in the group to which a sample (10 mg / mL) in which the N fraction was dissolved in ethanol so as to have a volume of 2 mg / day was applied. Furthermore, it was found that the F2 fraction and the N fraction promote hair growth in a concentration-dependent manner.

  The invention of the present application is the manufacture of cosmetics such as cosmetics for hair and scalp containing VEGF production promoting ingredients, and further the production of hair restoration and hair nourishing agents containing the same ingredients, or the manufacture of pharmaceuticals such as wound healing promoters and skin color improving agents. Can be used.

Claims (6)

VEGF production comprising a step of pulverizing a cocoon and washing with a hydrophobic organic solvent, a step of immersing the cocoon washed in the washing step in a lower alcohol to obtain an extract, and a step of fractionating the extract by two-phase separation A method for producing an accelerator , comprising:
The step of fractionation by the two-phase separation is a step of fractionation by two-phase separation with a lower ether phase and an aqueous phase, and an aqueous basic solution and an aqueous basic solution are used. The manufacturing method including the process of removing a neutral component and an acidic water-soluble component, and obtaining a neutral fraction . The method according to claim 1 , wherein the lower alcohol is immersed for 7 days or longer at a temperature of 25 ° C. to 50 ° C. or at room temperature . The method for producing a VEGF production promoter according to claim 1 or 2 , wherein the hydrophobic organic solvent is one or more organic solvents selected from the group consisting of benzene, hexane and chloroform. The method according to any one of claims 1 to 3 , wherein the lower alcohol is ethanol, methanol, propanol or butanol, or a mixture of two or more alcohols selected from the group consisting of these alcohols. The method of any one of Claims 1-4 in which the process of fractionating by the said two-phase separation includes the process of removing a water-soluble component using water for an aqueous phase. The VEGF production promoter obtained by the method of any one of Claims 1-5 .

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