JP7037162B2 - VEGF gene expression promoter and FGF7 gene expression promoter - Google Patents

Description

The present invention relates to a novel VEGF gene expression promoter and an FGF7 gene expression promoter.

Hair repeats growth and shedding according to a periodic hair cycle (hair cycle) consisting of a growth phase, a catagen phase, and a resting phase. Of this hair cycle, the stage where new hair follicles are formed from the resting phase to the growing phase is considered to be the most important for hair growth, and it is responsible for the proliferation and differentiation of hair follicle epithelial cells at this stage. It is believed that telogen cells play an important role. Hair follicle cells are cells located inside the hair follicle epithelial cells consisting of outer root sheath cells and matrix cells near the hair root, and are located in the root of the hair follicle surrounded by the basal membrane. It plays an important role in the proliferation and differentiation of hair follicle epithelial cells and the formation of hair, such as acting on epithelial cells to promote their proliferation (see Non-Patent Document 1).

As described above, the hair papilla cells play the most important role in the proliferation and differentiation of hair follicle epithelial cells and the formation of hair. Conventionally, the cultured hair papilla cells are brought into contact with the target substance to carry out the proliferative activity of the cells. A method for testing the hair-growth effect of the target substance by specifying the presence / absence and / or the strength of the substance has been proposed (see Patent Document 1).

Fibroblast growth factor-7 (FGF-7) is one of the family of fibroblast growth factor (FGF) and is also called KGF (keratinoxytegrawth factor). It is known that there are more than 20 families of FGF. FGF is a factor that promotes the proliferation of a wide range of cells generated from the mesoderm and the neuroexternal embryo. For example, division of fibroblasts, vascular endothelial cells, myoblasts, chondrocytes, glia cells, osteoblasts, etc. Induces growth. It is known that FGF has an angiogenic action, an action of suppressing the synthesis of collagen and fibronectin, and has a strong affinity for heparin.

In addition, it was shown that FGF-7 was expressed in the dermal papilla cells of hair follicles (see, for example, Non-Patent Document 2), and it was clarified that FGF-7 has a hair growth effect through activation of hair roots. Became. In addition, studies using knockout mice suggest that FGF-7 is involved in the direction of hair growth. So far, Clara and the like have been known as plant extracts having an action of promoting FGF-7 production (see Non-Patent Document 3).
Furthermore, by promoting the production of these growth factors in the periodontal tissue, the periodontal tissue can be regenerated, which is effective as a periodontal tissue regeneration promoting agent. (See Patent Document 2)

On the other hand, vascular endothelial growth factor (VEGF) is a growth factor that acts specifically on vascular endothelial cells, isolated from the culture medium of pituitary stellate follicular cells. VEGF is expressed in various cells such as dermal papilla, pituitary stellate follicular cells, macrophages, smooth muscle cells, and germ fibroblasts.

It has been reported that when a specific antibody that inhibits the movement of VEGF is injected into the abdominal cavity of a mouse, the growth phase is delayed and the size of the hair follicle is reduced, and angiogenesis is important for the development and regeneration of the hair follicle. On the contrary, it has been reported that when the amount of VEGF synthesized in the outer root sheath is increased, the size of the hair follicle is increased and the diameter of the hair produced is also increased. Further, it is known that VEGF produced from dermal papilla cells promotes hair growth and is involved in the action of minoxidil used as a hair restorer in pharmaceutical products.

From the above, it is expected to develop a hair growth-promoting agent that induces the production promotion of excellent FGF7 and VEGF for the prevention and improvement of symptoms such as hair loss and thinning hair, and there is a demand for a hair-growth agent having these actions. Is extremely expensive.
In addition, promotion of VEGF production treats or prevents peripheral arterial diseases. (See Patent Document 3)

Ishige okamurae mainly inhabits rocks in the central intertidal zone, and is widely distributed south of Ibaraki on the Pacific side of Honshu, from the coast of the Sea of Japan to the south, the Seto Inland Sea, Kyushu, the Korean Peninsula, and China.

Although dendrite elongation inhibitory action (see Patent Document 4) and skin barrier strengthening action (see Patent Document 5) have been found in Ishige, we are aware of the effective hair growth promoting action for FGF7 and VEGF. It wasn't done.

Japanese Unexamined Patent Publication No. 10-22978 Japanese Unexamined Patent Publication No. 2016-124797 Japanese Unexamined Patent Publication No. 2015-108024 Japanese Unexamined Patent Publication No. 2014-133708 Japanese Unexamined Patent Publication No. 2014-101309

"Trends Genet", 1992, Volume 8, p.56-61 Anti-Aging Series (1), Practice of White Hair / Hair Loss / Hair Growth, NTS, p.1-18, 2005 J.Derm.Sci., 30, p43-49, 2002

It is an object of the present invention to promote the expression of VEGF and FGF7 genes, to obtain a hair growth-promoting agent, and to obtain a periodontal tissue regeneration-promoting agent and a preparation effective for the treatment or prevention of peripheral arterial diseases.

Against this background, as a result of diligent studies, we have found an action that promotes the expression of an effective FGF7 gene in the Ishige extract, and completed the present invention.
Considering the extraction efficiency of Ishige, it is preferable to perform extraction after performing treatments such as shredding, drying and crushing. Drying may be carried out in the sun or by using a commonly used dryer. As the solvent used for the extraction, water, a hydrophilic organic solvent, or a mixed solution thereof is used.
The water that can be used as the extraction solvent includes, for example, pure water, tap water, well water, mineral spring water, mineral water, hot spring water, spring water, fresh water, and the like, as well as those subjected to various treatments. Treatments applied to water include, for example, purification, heating, sterilization, filtration, ion exchange, osmotic pressure adjustment, buffering and the like. Therefore, the water that can be used as the extraction solvent in the present invention includes purified water, hot water, ion-exchanged water, physiological saline, phosphate buffer, phosphate buffered saline and the like.
Examples of the hydrophilic organic solvent include lower alcohols having 1 to 5 carbon atoms such as methanol, ethanol, propyl alcohol and isopropyl alcohol; lower aliphatic ketones such as acetone and methyl ethyl ketone; 1,3-butylene glycol and propylene glycol. Examples thereof include polyhydric alcohols having 2 to 5 carbon atoms such as glycerin, and a mixed solvent of these hydrophilic organic solvents and water can be used.
When a mixed solvent of the water and a hydrophilic organic solvent is used, the amount of lower alcohol is 1 to 20 parts by mass with respect to 10 parts by mass of water, and the amount of lower aliphatic ketone is 10 parts by mass of water. On the other hand, it is preferable to add 1 to 15 parts by mass. In the case of polyhydric alcohol, it is preferable to add 1 to 20 parts by mass with respect to 10 parts by mass of water.
The amount of the organic solvent used for extraction is preferably about 5 to 100 times, more preferably about 10 to 50 times the amount of the plant (seaweed) as a raw material. In order to further increase the extraction efficiency, stirring or homogenization may be performed in the extraction solvent. It is appropriate that the extraction temperature is from about 5 ° C. to a temperature equal to or lower than the boiling point of the extraction solvent. The extraction time varies depending on the type of extraction solvent and the extraction temperature, but it is appropriate to set it to about 1 hour to 14 days.
The extraction operation is not limited to a one-time operation. A fresh solvent can be added again to the residue after extraction to perform an extraction operation, or the extraction solvent can be brought into contact with the extraction raw material multiple times. If necessary, further purification treatment such as deodorization and decolorization may be added within a range that does not affect the effect, and the concentration can be achieved by a vacuum concentrating device such as an evaporator or solvent removal by heating. Further, this extract can be further purified using a synthetic adsorbent (Diaion HP20, Sephadex SP825, Amberlite XAD4, MCIgelCHP20P, etc.), dextran resin (Sephadex LH-20, etc.), ultrafiltration, etc. be.

Although the pharmaceutical product of the present invention exerts a medicinal effect by oral, injectable or external use, it is preferably used as a skin external preparation. External skin preparations include skin cosmetics, quasi-drugs for external use, and external external preparations for medical use.

In addition to the above-mentioned ingredients, the formulations of the present invention include surfactants, oily ingredients, moisturizers, polymer compounds, ultraviolet absorbers, anti-inflammatory agents, and bactericidal agents used in various pharmaceutical and cosmetic formulations. Antioxidants, metal ion blockers, preservatives, vitamins, pigments, fragrances, water and the like can be blended.

As the above-mentioned surfactant, any of anionic, cationic, nonionic, natural and synthetic surfactants can be used, but it is preferable to use nonionic surfactants in consideration of skin irritation. Examples of the nonionic surfactant include glycerin fatty acid ester, propylene glycol fatty acid ester, sorbitan fatty acid ester, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene sorbit fatty acid ester, polyoxyethylene alkyl ether, and polyoxyethylene polyoxypropylene glycol. , Polyoxyethylene polyoxypropylene alkyl ether, polyethylene glycol fatty acid ester, polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil, alkyl glycoside and the like.

Examples of the oily component include fats and oils, waxes, hydrocarbons, higher fatty acids, higher alcohols, esters, essential oils, silicone oils and the like. Examples of oils and fats include soybean oil, nuka oil, jojoba oil, avocado oil, almond oil, olive oil, cacao oil, sesame oil, persic oil, castor oil, palm oil, minced oil, beef oil, pork oil and other natural oils and fats. Hardened oil obtained by hydrogenating natural fats and oils from Japan and synthetic triglycerides such as myristic acid glyceride and 2-ethylhexanoic acid triglyceride; Examples of waxes include carnauba wax, whale wax, beeswax, lanolin and the like; hydrocarbons. Examples include liquid paraffin, vaseline, paraffin microcrystalin wax, selecin, squalane, bristan, etc .; and higher fatty acids include, for example, lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, oleic acid, linoleic acid, etc. Linolenic acid, lanolinic acid, isostearic acid and the like; higher alcohols include, for example, lauryl alcohol, cetyl alcohol, stearyl alcohol, oleyl alcohol, lanolin alcohol, cholesterol, 2-hexyldecanol and the like; esters include, for example, cetyl octanate. , Octanoic acid triglyceride, myristyl lactate, cetyl lactate, isopropyl myristate, myristyl myristate, octyldodecyl myristate, isopropyl palmitate, isopropyl adipate, butyl stearate, decyl oleate, cholesterol isostearate, POE sorbit fatty acid ester, etc. The essential oils include, for example, peppermint oil, jasmine oil, show brain oil, hinoki oil, peppermint oil, ryu oil, terepine oil, calyx skin oil, bergamot oil, orange oil, shove oil, pine oil, lavender oil, and bay oil. , Clove oil, hiba oil, rose oil, eucalyptus oil, lemon oil, thyme oil, peppermint oil, rose oil, sage oil, menthol, cineole, eugenol, citral, citroneral, borneol, linalol, geraniol, camphor, timole, spirantol, Pinen, limonene, terpene-based compounds and the like; Examples of silicone oils include dimethylpolysiloxane and the like. These above-mentioned oily components can be used alone or in combination of two or more.
In the present invention, among these, myristic acid glyceride, 2-ethylhexanoic acid triglyceride, lanolin, liquid paraffin, vaseline, paraffin microcrystalin wax, squalane, lauric acid, myristic acid, palmitic acid, linoleic acid, linolenic acid, isostearic acid. , Cetyl alcohol, stearyl alcohol, oleyl alcohol, cholesterol, cetyl octanoate, triglyceride octanoate, isopropyl myristyrene, octyldodecyl myristate, cholesterol isostearate, POE sorbit fatty acid ester, peppermint oil, peppermint oil, calyx oil, rose It is preferable to use oil, menthol, cineol, eugenol, citral, citronellal, geraniol, pinen, limonene and dimethylpolysiloxane.

The following components can be further added to the pharmaceutical product of the present invention, but the components are not limited thereto. Pigments; Tar dyes specified in the Ordinance of the Ministry of Health and Welfare such as Yellow No. 4, Blue No. 1, Yellow No. 202, etc. Approved as food additives such as pigments in Appendix I and II, chlorophyll, riboflavin, crocin, safflower, anthraquinone, etc. Natural pigments, etc.
Vitamins; Vitamin A, Vitamin C, Vitamin D, Vitamin E, etc. Others; bactericides, preservatives, other ingredients necessary for formulation, etc.

The pharmaceutical product of the present invention can be produced according to a conventional method by adding the optional ingredient to the essential ingredient as necessary, and can be in the form of cream, milky lotion, lotion or the like.

Next, the present invention will be described in detail with reference to examples.

Example 1
500 g of purified water was added to 50 g of Ishige (wet weight), and homogenized using a mixer. This was centrifuged to obtain a filtrate. 4 times the amount of ethanol was added to this filtrate, and the mixture was filtered (No5C) after 24 hours with occasional stirring, and this was lyophilized.

Confirmation test (FGF7 and VEGF gene expression promotion test)
Human dermal papilla cells (PromoCell) were seeded on 6-well plates and cultured for 3 days. Three days later, the Ishige extract prepared in the example was adjusted to 1 mg / mL in DMEM medium containing no FBS, added to the above-mentioned human dermal papilla cells, and cultured for 2 hours. After culturing, RNA was extracted according to the following method, and after RT reaction, real-time PCR was performed.

Extraction of RNA Extraction of Total RNA from cells was adjusted using PureLink® RNA Mini Kit (Life Technologies Japan) according to the attached manual. RNA concentration was calculated using NanoDop1000 (Thermo Fisher scientific).

RT reaction and real-time PCR
Using the extracted Total RNA, RT reaction was performed according to the attached manual using High-Capacity RNA-to- cDNA Kit (Life Technologies Japan).
Real-time PCR was performed using the 7500 Real-Time PCR System (Life Technologies Japan).
Specifically, the above-mentioned RT products and TaqMan (registered trademark) primer (Life Technologies Japan) and TaqMan (registered trademark) Gene Expression Master Mix (Life Technologies Japan) of GAPDH as VEGF, FGF7 and endogenous control are used. Then, the gene expression levels were compared by the ΔΔCT method.

The results are shown in FIG.

It is a figure which shows the gene expression level change of VEGF and FGF7 in the confirmation test result of Example 1. FIG. The vertical axis is the gene expression level when the gene expression level when the example is not added is 1. In each case, the risk rate was 1%, which was significantly different from the control.

Claims (1)

An FGF7 gene expression promoter containing an extract of Ishige okamurae as an active ingredient (excluding uses for hair growth / growth agents).

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