JP2010150177A - Fibroblast growth factor-7 (fgf-7) production promoter, insulin-like growth factor-1 (igf-1) production promoter, and hepatocyte growth factor (hgf) production promoter - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an FGF-7 production promoter, an IGF-1 production promoter, and an HGF production promoter having excellent action and high safety. <P>SOLUTION: There are provided an FGF-7 production promoter, an IGF-1 production promoter, and an HGF production promoter each of which contains ashitaba (Angelica keiskei) and/or luteolin-7-O-glucoside as active ingredients. It is possible to formulate them into dermatological preparations for external use including hair growing agents and hair cosmetics, to formulate them into foods and beverages and medicaments for cosmetological use, and to desirably use them as reagents for researches. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to an FGF-7 production promoter, an IGF-1 production promoter, and an HGF production promoter.

  Hair growth repeats growth and loss according to a periodic hair cycle (hair cycle) consisting of a growth period, a regression period, and a rest period. Of these hair cycles, the stage in which new hair follicles are formed from the resting phase to the growing phase is considered to be the most important for hair growth. It is thought that hair papilla cells play an important role in differentiation. The dermal papilla cell is a cell located inside the hair follicle epithelial cell composed of outer root sheath cells and matrix cells in the vicinity of the hair root, and located in the root portion of the hair root wrapped in the basement membrane. It plays an important role in differentiation into hair, such as working on epithelial cells to promote their proliferation (see, for example, Non-Patent Document 1).

  Fibroblast growth factor-7 (FGF-7) is one of a family of fibroblast growth factors (FGF) and is also called KGF (keratinocyte growth factor). FGF is known to have more than 20 families.

  FGF is a factor that promotes the proliferation of a wide range of cells generated from mesoderm and neuroectoderm. For example, FGF is a fibroblast, vascular endothelial cell, myoblast, chondrocyte, glial cell, osteoblast, etc. Induce growth. FGF is known to have an angiogenesis action, an action for inhibiting the synthesis of collagen and fibronectin, and a strong affinity for heparin.

  In addition, it was shown that FGF-7 is expressed in hair follicle dermal papilla cells, and it has been clarified that FGF-7 has a hair-growth effect through hair root activation (for example, non-patent literature). 2). Studies using knockout mice suggest that FGF-7 is involved in the direction of hair growth.

  So far, Clara has been known as a plant extract having an action of promoting FGF-7 production (see, for example, Non-Patent Document 3).

  Insulin-like growth factor-1 (IGF-1) is a peptide hormone with a molecular weight of about 7,500 that has a structure and action very similar to insulin. IGF-1 is known to actively maintain cells in a healthy state, for example, by promoting cell differentiation and helping cell proliferation (see, for example, Non-Patent Documents 4 and 5) and preventing the progression of aging. (See, for example, Non-Patent Document 6).

  In addition, it was shown that IGF-1 is expressed in the hair follicle cells of the hair follicle, and it became clear that IGF-1 has a hair-growth effect through activation of hair roots (for example, non-patent literature) 7). Currently, hair growth agents containing IGF-1 as an active ingredient are already known (see, for example, Patent Document 1). However, since IGF-1 is an animal-derived component and has a large molecular weight, there are problems such as difficulty in percutaneous absorption by external application. Moreover, the plant-derived substance which has the outstanding IGF-1 production promotion effect is calculated | required.

  Hepatocyte growth factor (HGF) was discovered in plasma of patients with fulminant hepatitis as a factor that promotes proliferation of hepatocytes. HGF is known to promote proliferation of not only the liver but also epithelial cells, endothelial cells, hematopoietic cells and the like derived from various organs. It has been reported that the blood concentration of HGF increases in various diseases such as liver disease, pneumonia, leukemia, cancer, myocardial infarction and the like. In addition, HGF has been recognized to suppress organ damage and fibrosis and promote regeneration. Furthermore, a technique using HGF as an active ingredient of a pulmonary fibrosis preventive agent has been disclosed (for example, see Patent Document 2).

  In addition, it was shown that HGF is expressed in the hair follicle cells of the hair follicle, and it has been clarified that HGF has a hair-growth effect through activation of hair roots (for example, see Non-Patent Document 2).

  So far, rice or the like has been known as a plant extract having an action of promoting HGF production (see, for example, Patent Document 3).

  As mentioned above, the demand for each agent having FGF-7 production promoting action, IGF-1 production promoting action and HGF production promoting action is extremely high.

  However, conventionally, there has been a problem that a specific active ingredient having the above action has not been proposed yet. Moreover, the thing for which the specific active ingredient was proposed also had the problem that the said effect | action and effect were not enough even if it was not derived from a plant and was derived from a plant. In addition, since it is comparatively high in plant origin, there exists an advantage that it is easy to ingest or apply | coat daily.

  Ashitaba is a plant belonging to the family Aceraceae, and its scientific name is “Angelica keiskei”. In addition, it is known that Ashitaba contains glycoside luteolin 7-O-glucoside. (See Non-Patent Document 8)

  Moreover, the technique which adds and uses an Ashitaba extract to the cosmetics for hair growth is disclosed (refer patent document 4). Moreover, the technique which uses luteolin 7-O-glucoside as an anti-inflammatory agent is disclosed (refer patent document 5). However, Patent Documents 4 and 5 do not describe what kind of action Ashitaba and / or luteolin 7-O-glucoside exerts a hair-growth effect.

Therefore, up to now, it is a natural product that is easy to obtain, inexpensive, and highly safe, and does not adversely affect the quality of the additive in terms of taste, smell, feeling of use, etc. FGF-7 production promoters, IGF-1 production promoters and HGF production promoters that can be widely used as external preparations for skin, including cosmetics, foods and drinks for cosmetics, pharmaceuticals and research reagents have not yet been provided, At present, there is a strong demand for prompt provision.
Japanese Patent Publication No. 4-60567 JP 2006-131649 A JP 2004-99503 A Japanese Examined Patent Publication No. 2-46562 JP-A-1-42427 “Trends Genet”, 1992, Vol. 8, p. 56-61 Anti-aging series (1), actual hair, hair loss and hair growth, NTS, p. 1-18, 2005 J. et al. Derm. Sci. , 30, p43-49, 2002 J. et al. Biol. Chem. 271, 28853-28860 (1996) Dermatology, 20, 325-329 (2002) Am. J. et al. Med. 115, 501-502 (2003) J. et al. Invest. Dermatol. 99, 343-349 (1992) Physother. Res. 16, S24-S27 (2002)

  An object of the present invention is to solve the conventional problems and achieve the following objects. That is, the first object of the present invention is to provide a natural FGF-7 production promoter that has an excellent FGF-7 production promoting action, has high safety, and is readily available as a raw material.

  A second object of the present invention is to provide a natural IGF-1 production promoter that has an excellent IGF-1 production promoting action, has high safety, and is easily available from raw materials.

  A third object of the present invention is to provide a natural HGF production promoter that has an excellent HGF production promoting action, has high safety, and is readily available as a raw material.

  In order to solve the above-mentioned problems, the present inventors have made extensive studies and obtained the following findings. That is, it was found that Ashitaba extract and / or luteolin 7-O-glucoside had excellent FGF-7 production promoting action, IGF-1 production promoting action and HGF production promoting action.

The present invention is based on the above findings by the present inventors, and means for solving the above problems are as follows. That is,
<1> An FGF-7 production promoter characterized by containing an Ashitaba extract and / or luteolin 7-O-glucoside as an active ingredient.
<2> An IGF-1 production promoter comprising an Ashitaba extract and / or luteolin 7-O-glucoside as an active ingredient.
<3> An HGF production promoter comprising an Ashitaba extract and / or luteolin 7-O-glucoside as an active ingredient.

  According to the FGF-7 production promoter of the present invention, natural FGF- which can solve various problems in the past, has an excellent FGF-7 production promotion action, is highly safe, and is easily available as a raw material. 7 production promoters can be provided.

  According to the IGF-1 production promoter of the present invention, natural IGF- which can solve various problems in the past, has an excellent IGF-1 production promotion action, is highly safe, and is readily available as a raw material. 1 production promoter can be provided.

  According to the HGF production promoter of the present invention, a natural HGF production promoter that can solve various problems in the past, has an excellent HGF production promoting action, is highly safe, and is readily available as a raw material. can do.

(FGF-7 production promoter, IGF-1 production promoter and HGF production promoter)
The FGF-7 production promoter, IGF-1 production promoter, and HGF production promoter of the present invention contain an Ashitaba extract and / or luteolin 7-O-glucoside as an active ingredient, and, if necessary, other It contains.

  The luteolin 7-O-glucoside can be produced by isolation and purification from a plant extract containing luteolin 7-O-glucoside, or can be produced by synthesis. In addition, when manufacturing by a synthesis | combination, the synthesis | combining method is not specifically limited, It can synthesize | combine by a well-known method.

  The plant extract containing the luteolin 7-O-glucoside can be obtained by an extraction method generally used for plant extraction. As a plant containing the said luteolin 7-O-glucoside, a plant of the family Asteraceae, Asteraceae, etc. are mentioned, for example. Among these, Ashitaba of the celery family is particularly preferable.

  The Ashitaba is a plant belonging to the family Aceraceae, and its scientific name is “Angelica keiskei”. The Ashitaba is widely grown along the coast of Honshu, such as the Kii Peninsula and Boso Peninsula, and is easily available from these areas. Examples of the part that can be used as an extraction raw material include a leaf part, a stem part, a flower (bud) part, a seed, and a root part. Among these, the leaf portion is particularly preferable in that it contains abundant luteolin 7-O-glucoside.

  The Ashitaba extract can be obtained by drying the raw material for extraction and then pulverizing it as it is or using a crusher and subjecting it to solvent extraction. Drying may be performed in the sun or using a commonly used dryer. The extraction raw material may be used as an extraction raw material after pretreatment such as degreasing with a nonpolar solvent such as hexane or benzene. In addition, the extraction process by the polar solvent of an extraction raw material can be performed efficiently by performing pretreatments, such as degreasing.

  As the solvent used for the extraction, it is preferable to use water, a hydrophilic organic solvent, or a mixed solvent thereof at a temperature from room temperature to the boiling point of the solvent.

  Examples of water that can be used as the extraction solvent include 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. Examples of the treatment applied to water include purification, heating, sterilization, filtration, ion exchange, adjustment of osmotic pressure, buffering, and the like. The water that can be used as the extraction solvent includes purified water, hot water, ion exchange 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, 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.

  In addition, when using the mixed solvent of the said water and a hydrophilic organic solvent, in the case of a lower alcohol, 1 mass part-90 mass parts with respect to 10 mass parts of water, and in the case of a lower aliphatic ketone, 10 masses of water. It is preferable to add 1 to 40 parts by mass with respect to parts. In the case of polyhydric alcohol, it is preferable to add 1 part by mass to 90 parts by mass with respect to 10 parts by mass of water.

  The extraction treatment is not particularly limited as long as the soluble component contained in the extraction raw material can be eluted in the extraction solvent, and can be performed according to a conventional method. For example, the extraction raw material is immersed in an extraction solvent 5 to 15 times (mass ratio) of the extraction raw material, the soluble components are extracted at room temperature or under reflux, and then filtered to remove the extraction residue. A liquid can be obtained. When the solvent is distilled off from the obtained extract, a paste-like concentrate is obtained, and when this concentrate is further dried, a dried product is obtained.

  The method for isolating and purifying the luteolin 7-O-glucoside from the extract obtained as described above, the concentrate of the extract or the dried product of the extract is not particularly limited and may be determined by a conventional method. It can be carried out. For example, a plant extract is concentrated, subjected to column chromatography using a porous resin, etc., and eluted in the order of water and alcohol (methanol, etc.) to obtain a fraction eluted with alcohol (methanol, etc.). . At this time, the crude product can be obtained by subjecting the fractionated product to reverse phase silica gel chromatography or recrystallization using ODS (octadecylsilylated silica gel).

  And the refined luteolin 7-O-glucoside can be obtained by isolate | separating and refine | purifying the said fraction or crudely purified product, for example using a liquid chromatography etc.

  The Ashitaba extract and / or luteolin 7-O-glucoside has an FGF-7 production promoting action, an IGF-1 production promoting action and an HGF production promoting action. 7 production promoter, IGF-1 production promoter and HGF production promoter can be used as active ingredients. The plant extract obtained by the extraction treatment contains the luteolin 7-O-glucoside and is used as an active ingredient of the FGF-7 production promoter, IGF-1 production promoter and HGF production promoter as it is. However, the purified luteolin 7-O-glucoside may be used in a purified form. By using as an active ingredient what increased the purity of the said luteolin 7-O-glucoside, the FGF-7 production promoter, IGF-1 production promoter, and HGF production promoter which were further excellent in the use effect were obtained. Can do. The plant extract containing the luteolin 7-O-glucoside includes an extract obtained by using the plant containing the luteolin 7-O-glucoside as an extraction raw material, a diluted or concentrated solution of the extract, and the extract. A dried product obtained by drying, or a crude product or a purified product thereof is included.

  In addition, the luteolin 7-O-glucoside itself may be contained as an active ingredient of the FGF-7 production promoter, the IGF-1 production promoter, and the HGF production promoter, and its pharmacologically acceptable. Salt may be included. Moreover, the hydrate or solvate of the said luteolin 7-O-glucoside or its pharmacologically acceptable salt may be contained. The luteolin 7-O-glucoside may be modified or substituted as long as it does not impair the FGF-7 production promoting action, the IGF-1 production promoting action and the HGF production promoting action.

  The pharmacologically acceptable salt is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include hydrochloride, sulfate, hydrobromide, nitrate, hydrosulfate, and phosphoric acid. Salt, acetate, lactate, succinate, citrate, maleate, hydroxymaleate, tartrate, fumarate, methanesulfonate, p-toluenesulfonate, camphorsulfonate, sulfamine , Mandelate, propionate, glycolate, stearate, malate, ascorbate, pamonate, phenylacetate, glutamate, benzoate, salicylate, sulfanilate, 2 -Acetoxybenzoate, ethanedisulfonate, oxalate, isethionate, formate, trifluoroacetate, ethyl succinate, lactobionate, glucone Salt, glucoheptonate, 2-hydroxyethanesulfonate, benzenesulfonate, paratoluenesulfonate, lauryl sulfate, aspartate, adipate, hydroiodide, nicotinate, oxalate , Picrate, thiocyanate, undecanoate and the like.

  The FGF-7 production promoter, IGF-1 production promoter, and HGF production promoter of the present invention may be the luteolin 7-O-glucoside itself, or an acitaba extract containing the luteolin 7-O-glucoside. The thing which consists only of a thing may be sufficient and the thing which formulated the plant extract containing the said luteolin 7-O-glucoside may be used.

  The content of the Acitaba extract and / or luteolin 7-O-glucoside in the FGF-7 production promoter, the IGF-1 production promoter, and the HGF production promoter is not particularly limited and depends on the purpose. Can be selected as appropriate.

<Other ingredients>
The Ashitaba extract and / or luteolin 7-O-glucoside is in the form of powder, granules, tablets, in accordance with a conventional method using a pharmaceutically acceptable carrier such as dextrin, cyclodextrin and any other auxiliary agent. It can be provided by formulating into any dosage form such as liquid, and can be used by blending with other compositions (for example, skin cosmetics, etc.) and can be used as an ointment, liquid for external use, patch, etc. it can. In this case, as an auxiliary agent, for example, an excipient, a binder, a disintegrant, a lubricant, a stabilizer, a flavoring agent and the like can be used.

  In addition, the FGF-7 production promoter, IGF-1 production promoter and HGF production promoter of the present invention may be added to other natural extracts having FGF production promoting action, IGF-1 production promoting action and HGF promoting action as necessary. Can be blended with the plant extract containing the above-mentioned Ashitaba extract and / or luteolin 7-O-glucoside and used as an active ingredient.

  The said FGF-7 production promoter exhibits FGF-7 production promotion effect | action by the effect | action of the Ashitaba extract and / or luteolin 7-O-glucoside contained as an active ingredient.

  The said IGF-1 production promoter exhibits an IGF-1 production promotion effect | action by the effect | action of an Ashitaba extract and / or luteolin 7-O-glucoside contained as an active ingredient.

  The said HGF production promoter exhibits an HGF production promotion effect by the action of Ashitaba extract and / or luteolin 7-O-glucoside contained as an active ingredient.

  According to the FGF-7 production promoter of the present invention, for example, fibroblasts, vascular endothelial cells, myoblasts, chondrocytes, glial cells, osteoblasts and the like can be divided and grown through an excellent FGF-7 production promoting action. Hair growth can be promoted through hair root activation, and alopecia can be improved or treated. However, the FGF-7 production-promoting agent of the present invention can be used for all uses that are meaningful for exerting an FGF-7 production-promoting action in addition to these uses.

  According to the IGF-1 production promoter of the present invention, through excellent IGF-1 production promotion action, for example, differentiation / proliferation / growth of cells in general, suppression of aging progression, hypoglycemia, regulation of reproductive function, sulfate to cartilage It is possible to promote ion uptake and to improve or treat alopecia, skin aging, diabetes, hypopituitarism, pituitary dwarfism, and the like. However, the IGF-1 production-promoting agent of the present invention can be used for all uses that are meaningful for exerting an IGF-1 production-promoting action in addition to these uses.

  According to the HGF production-promoting agent of the present invention, through excellent HGF production-promoting action, for example, the proliferation of epithelial cells, endothelial cells, hematopoietic cells and the like derived from each organ is promoted, and organ damage and fibrosis are suppressed. , Regeneration can be promoted, and alopecia, fibrosis, liver disease, obstructive arteriosclerosis, etc. can be improved or treated. However, the HGF production-promoting agent of the present invention can be used for all uses that are meaningful for exerting an HGF production-promoting action in addition to these uses.

  Since the FGF-7 production promoter, IGF-1 production promoter and HGF production promoter of the present invention have an excellent action and are excellent in the feeling of use and safety when applied to the skin, they are external preparations for skin. It is suitable for blending. Especially as said skin external preparation, a hair restorer and hair cosmetics are preferable. Examples of the hair cosmetics include hair tonics, hair liquids, shampoos, pomades, and rinses.

  Moreover, since it has been confirmed that the FGF-7 production promoter, IGF-1 production promoter and HGF production promoter of the present invention have an excellent action and are not digested in the digestive tract. It is suitable for blending into cosmetic foods and beverages or medicines.

  In addition, since the FGF-7 production promoter, IGF-1 production promoter and HGF production promoter of the present invention have an excellent action, research on the functions of FGF-7, IGF-1 and HGF, and FGF-7 It can be suitably used as a reagent for studying diseases related to IGF-1 and HGF.

  The FGF-7 production promoter, the IGF-1 production promoter and the HGF production promoter of the present invention are preferably applied to humans. However, as long as the respective effects are exhibited, humans It can also be applied to other animals.

  Examples of the present invention will be described below, but the present invention is not limited to these examples.

Example 1
5 L of an extraction solvent (50% by mass ethanol) was added to 500 g of each dried product of Ashitaba leaves, and the mixture was heated and extracted with a reflux extractor for 3 hours and filtered while hot. Then, the obtained extract was concentrated under reduced pressure at 40 ° C. and dried with a freeze dryer to obtain an extract from Ashitaba. The yield of 50% by mass of Ashitaba ethanol extract was 22.5%.

(Example 2)
112.5 g of the extract of Ashitaba was suspended in 1 L of water, and this was applied to a column packed with a porous adsorption resin (DIAION HP-20, manufactured by Mitsubishi Chemical Corporation) and eluted in the order of 10 L of water and 10 L of methanol. It was. The obtained methanol-eluted fraction was concentrated under reduced pressure, and the obtained solid content (45.25 g) was subjected to normal phase column chromatography (silica gel 60 (0.063-0.200 mm) for column chromatography, manufactured by MERCK). Fractionated with chloroform: methanol: water = 30: 10: 1 as a solvent, and the eluate was concentrated under reduced pressure to obtain a luteolin 7-O-glucoside fraction (550 mg).

  The luteolin 7-O-glucoside fraction (550 mg) thus obtained was purified by recycle HPLC under the following conditions to obtain luteolin 7-O-glucoside (122 mg).

<HPLC conditions>
Stationary phase: JAIGEL GS-310 (manufactured by Nippon Analytical Industrial Co., Ltd.)
Column length: 500mm
Column diameter: 21.5mm
Temperature: Room temperature Mobile phase: Water Mobile phase flow rate: 5 mL / min
Detector: Differential refractometer

The luteolin 7-O-glucoside was analyzed by ¹³ C-NMR (nuclear magnetic resonance apparatus).
The results are shown below.
-Luteolin 7-O-glucoside-
< ¹³ C-NMR chemical shift δ (assigned carbon)>
60.6 (Glc-6), 69.5 (Glc-4), 73.0 (Glc-2), 76.3 (Glc-3), 77.1 (Glc-5), 94.6 (C8 ), 99.4 (C6), 99.9 (Glc-1), 103.0 (C3), 105.2 (C10), 113.4 (C2 ′), 115.8 (C5 ′), 119. 0 (C6 ′) 121.2 (C1 ′), 145.6 (C3 ′), 149.7 (C4 ′) 156.7 (C9), 160.9 (C5), 162.7 (C7), 164 .2 (C2), 181.6 (C4)

(Example 3)
<Production promotion test of FGF-7, IGF-1 and HGF>
Using the acitaba extract of Examples 1 and 2 and luteolin 7-O-glucoside as samples, the production promoting effects of FGF-7, IGF-1 and HGF were tested by the following test method.

First, normal human hair hair papilla cells (TOYOBO, CA60205) were cultured using a hair papilla cell growth medium (TOYOBO, TPGM-250), and then cells were collected by trypsin treatment. The collected cells were diluted with 10% FBS-containing DMEM medium to a concentration of 2 × 10 ⁵ cells / mL, then seeded in 5 mL portions in a 60 mm diameter petri dish, and cultured overnight. The next day, after changing to serum-free DMEM medium with samples added and culturing for 4 hours, total RNA was extracted with ISOGEN (Nippon Gene, Cat. No. 311-02501), and the amount of each RNA was stored in a spectrophotometer. And total RNA was prepared to 200 μg / mL.

Using this total RNA as a template, the expression levels of FGF-7, IGF-1, HGF and the internal standard GAPDH mRNA were measured. The detection was performed by real-time 2 Step RT-PCR reaction using TaKaRa SYBR PrimeScript ™ RT-PCT Kit (Perfect Real Time) (code No. RR063A) using a real-time PCR apparatus Smart Cycler (Cepheid). After correcting the expression levels of FGF-7, IGF-1 and HGF mRNA with the value of the expression level of GAPDH in the same sample, mRNA expression of FGF-7, IGF-1 and HGF according to the following formula (1) The acceleration rate (%) was calculated. The results are shown in Tables 1 and 2.
FGF-7, IGF-1 and HGF mRNA expression promotion rate (%)
= B / A x 100 (1)
[However, in the formula (1),
A: Correction value when no sample is added,
B: represents a correction value at the time of sample addition. ]

  From the results of Tables 1 and 2, it was confirmed that the extract of Ashitaba and luteolin 7-O-glucoside had a high FGF-7 production promoting effect, an IGF-1 production promoting effect, and an HGF production promoting effect.

  Since the FGF-7 production promoter, IGF-1 production promoter and HGF production promoter of the present invention have excellent FGF-7 production promotion action, IGF-1 production promotion action and HGF production promotion action, It can be suitably used as a reagent for research, blended in skin external preparations such as hair cosmetics, blended in beauty foods and beverages and pharmaceuticals.

Claims (3)

FGF-7 production promoter characterized by containing an Ashitaba extract and / or luteolin 7-O-glucoside as an active ingredient. A promoter for IGF-1 production comprising an extract of Ashitaba and / or luteolin 7-O-glucoside as an active ingredient. A HGF production promoter comprising an Ashitaba extract and / or luteolin 7-O-glucoside as an active ingredient.