JP5732207B2 - VEGF production promoter - Google Patents

Description

  The present invention relates to a VEGF production promoter, a hair quality improving agent, and an external preparation for skin that can promote the production of vascular endothelial growth factor (VEGF).

  VEGF is known as a vascular permeability factor, but recent studies have reported that it is a major angiogenic factor in human skin, and has attracted attention in research fields such as wound healing, skin color improvement, hair restoration and hair growth. Is a molecule.

  In normal skin, VEGF is secreted in small amounts by epidermal keratinocytes and binds to specific receptors on the dermal microvascular endothelial cells, thereby ensuring the viability of the endothelial cells, thereby ensuring the upper network structure. Maintain blood vessels.

It has been reported that VEGF in the epidermis thickened during inflammation or wound healing is highly expressed, leading to an increase in blood vessels and nutrient supply in the dermis (Non-patent Document 1). In addition, the blood vessels around the hair follicle are dramatically expanded during the hair growth phase, and VEGF is expressed in the hair follicle cells. Has been reported to be suppressed (Non-patent Document 2).
Furthermore, since VEGF promotes the formation of blood vessels, it has been reported that it promotes blood circulation to supply a large amount of nutrients to the hair root and is related to the adhesion between cortex cells of hair.
In particular, in recent research, it has been reported that a decrease in VEGF expression level and a decrease in hair sharpness are correlated, and application of VEGF production promoters to hair cosmetics has attracted attention (Non-patent Document 3). .

  Therefore, promoting the production of VEGF, which is involved in the increase of blood vessels and nutrient supply in the dermis, is very effective for wound recovery and healing, as well as dullness and skin caused by decreased skin metabolism, etc. It is effective for improving skin color such as a decrease in transparency. In addition, promoting the production of VEGF, which is also involved in the growth of hair and hair follicles, is effective in preventing and improving symptoms such as hair loss, thinning hair, and reduction in sharpness.

  For this reason, various VEGF production promoters have been developed so far. For example, preparations derived from soybeans (Patent Document 1), Amihanaiguchi, Shironumeiguchi, Hanaiguchi, Utsurobenaninaichi, Amanita, Kinoboriguchi, Ezoukogi, Yellow spirit, Gentiana, Senna, Tochu, Daiou, Merirot, Yokuinin, Cucumber fruit, Home extract, ground yellow, sanshishi, licorice, carrot, red ginseng, purple root, cymbidium extract (Patent Document 2), genus Pandanus L.f. It has been reported that each extract of bean sprouts and hamachal (Patent Document 4) has a VEGF production promoting action. However, these VEGF production promoters may be blended in terms of side effects, and when an effective amount is blended, problems such as coloring and unpleasant odor may occur.

On the other hand, Aleoside A, a saponin contained in a plant belonging to the genus Ruscus belonging to the family Liliaceae such as Butcher bloom (Ruscus aculeatus L.), has an antitumor effect (Non-patent Document 4). Has been reported.
Butcher bloom has diuretic and vasoconstrictive effects, so its extract is a plant that is blended in basic cosmetics, foundations, shampoos, etc. as skin conditioning agents, astringents, etc. In addition to inhibiting the formation of hair (Patent Document 5), preventing the generation of gray hair (Patent Document 6), promoting cholesterol synthesis of skin epidermis cells and hair matrix cells, improving the amount of cholesterol in the hair and reducing hair damage It has been reported that it has an action to prevent (Patent Document 7).

  However, it is not known at all that acreoside A and butcher bloom have a VEGF production promoting action.

JP-A-11-286432 JP 2000-212059 A JP 2004-43393 A JP 2004-35443 A JP 2001-322943 A Japanese Patent Laying-Open No. 2005-068159 JP 2002-173416 A

Detmar M. The role of VEGF and thrombospondins in skin angiogenesis, J Dermatol Sci. 24 (Suppl 1), S78-84, 2000 Yano K, Brown LF, Detmar M. Control of hair growth and follicle size by VEGF-mediated angiogenesis, J Clin Invest. 107 (4), 409-17, 2001 Moriwaki, Taguchi Fragrance Journal December 2007 issue Mimaki Y. Aculeoside B, a New Bisdescosidic Spirostanol Saponin from the Underground Parts of Ruscus Aculeatus, J. Nat. Prod. 61, 1279-1282,1998

  The present invention relates to providing a VEGF production promoter, a hair quality improving agent, and a skin external preparation, which have a VEGF production promoting action and can be used as a pharmaceutical, a cosmetic or a food, or a material thereof.

  As a result of repeated studies on substances that promote VEGF production, the present inventors have found that acreoside A, a kind of saponin, has an excellent VEGF production promoting action.

That is, the present invention relates to the following 1) to 3).
1) A VEGF production promoter containing acreoside A as an active ingredient.
2) A hair quality improving agent containing acreoside A as an active ingredient.
3) A skin external preparation containing acreoside A.

  According to the present invention, there are provided pharmaceuticals, quasi-drugs, cosmetics, foods, etc. that have an excellent VEGF production promoting action and are useful for wound healing, skin color improvement, hair nourishment / hair growth, hair sharpening, etc. can do.

  Aculeoside A of the present invention is spirosta-5,25 (27) -diene-1β, 3β, 23α, 24α-tetrol 1-O- [2,3,4-tri-O-acetyl-α- L-rhamnopyranosyl- (1 → 4) -α-L-arabinopyranosyl] -24-O-6-deoxy-D-glycero-L-threo-4-hexopyranoside, Has a chemical structure.

  The acreoside A can be obtained by extraction and purification from a plant body. Extraction and purification from the plant can be carried out by referring to methods described in the literature (Chem. Lett. 1994, 2303) as appropriate, for example, plants of the genus Ruscus of the family Liiliaceae, specifically Column chromatography of extracts obtained by solvent extraction from rhizomes, shoots and leaves of Ruscus aculeatus L. (Butcher Bloom), Ruscus acleatusver. Angustifolius Boss., Ruscus hypoglossum L., Ruscus hypophyllum L. , And can be obtained by separation and purification using an appropriate separation and purification means such as ion exchange chromatography and high performance liquid chromatography. An example is shown below.

1) Add 30% ethanol to the rhizome of Butcher bloom (Ruscus aculeatus L.), extract at room temperature or under heating (40-80 ° C), and filter.
2) The extract obtained in 1) is passed through a column packed with an ion exchange resin to adsorb the components. After washing with 50% ethanol, elution is performed with 99.5% ethanol to obtain a crude saponin fraction.
3) Apply the crude saponin fraction obtained in 2 to a silica gel column and elute with chloroform: methanol = 9: 1 mixed solvent to obtain the saponin fraction.
4) Further, perform a gradient elution using a mixed solvent of chloroform: methanol (50: 1 → 19: 1 → 9: 1) in silica gel column chromatography to obtain a fraction containing spiracleoside A.
5) Next, the fraction containing spiracleoside A was subjected to preparative HPLC equipped with an ODS column, and fractionated by isocratic elution using a 50% acetonitrile aqueous solution as an eluent. Spirosta-5,25 (27) -diene-1β, 3β, 23α, 24α-tetrol 1-O- [2,3,4-tri-O-acetyl-α-L-rhamnopyranosyl- (1 → 4) -α -L-arabinopyranosyl] -24-O- [4,4-O- [oxy [(R) -1-[(S) -1-hydroxy-1-methylpropyl] -2-oxoethane-1 , 2-diyl] -6-deoxy-α-L-altropyranoside.
6) Spiracleoside A obtained in 5) was dissolved in methanol, stirred for 1 hour under reflux, and after the solvent was distilled off, the resulting solid matter was separated on a Sephadex LH-20 column (eluent: methanol). Purify to obtain acreoside A.

  In addition, according to such extraction / fractionation, acreoside A may be obtained not only as a single substance but also as a mixture of several kinds. Any of these may be used in the VEGF production promoter of the present invention. be able to.

  The obtained acreoside A may be used as it is, may be used as a diluted solution diluted with an appropriate solvent, or may be a concentrated extract or a dry powder, or may be prepared as a paste. Further, it can be freeze-dried and diluted with a solvent usually used for extraction at the time of use, for example, water, ethanol, water / ethanol mixed solution or the like. It can also be used by encapsulating in vesicles such as liposomes or microcapsules.

The acreoside A of the present invention has an effect of promoting the production of VEGF in human epidermal keratinocytes, as shown in the examples described later, and thus exerts effects such as improvement of endothelial cell viability and promotion of angiogenesis. (Heidemarie Rossiter, Caterina Barresi, Johannes Pammer, Michael Rendl, Jody Haigh, Erwin F. Wagner, and Erwin Tschachler. Loss of Vascular Endothelial Growth Factor A Activity in Murine Epidermal Keratinocytes Delays Wound Healing and Inhibits Tumor Formation, Cancer research 64, 3508 -3516, 2004), thought to be useful for wound healing, skin color improvement, hair nourishment / hair growth, and improvement of hairiness.
Therefore, the acreoside A of the present invention can be used as a VEGF production promoter or hair quality improving agent, and can be used to produce a VEGF production promoter or hair quality improving agent. The VEGF production promoter may be a drug for humans or animals, quasi-drugs, cosmetics or foods that exerts effects such as wound healing, skin color improvement, hair restoration / hair growth, hair sharpening improvement, etc. The raw material for mix | blending with the said pharmaceutical etc. may be sufficient. Further, in the present invention, “hair quality improvement” means an effect of hardening and / or strengthening the quality of fur and imparting firmness and stiffness, and the hair quality improving agent is used for humans or animals. It may be a pharmaceutical, a quasi-drug, a cosmetic or a food, or may be a material for blending in the pharmaceutical.
The cosmetics and foods mentioned above have the concept of promoting VEGF production, skin color improvement, hair restoration / hair growth, hair quality improvement, etc., and cosmetics, beauty foods, sick foods or specific foods with indications as necessary. Functional foods such as health foods are included.

Examples of the dosage form of the above-mentioned pharmaceutical containing acreoside A of the present invention include oral administration or intravenous injection, eg, tablet, capsule, granule, powder, syrup, etc., intramuscular injection, suppository, inhalant, transdermal Examples include parenteral administration using an absorbent, eye drops, nasal drops and the like. In order to prepare pharmaceutical preparations of such various dosage forms, the acreoside A of the present invention is used alone or other pharmaceutically acceptable excipients, binders, extenders, disintegrants, interfaces. Activators, lubricants, dispersants, buffers, preservatives, flavoring agents, fragrances, coating agents, carriers, diluents, and the like can be used in appropriate combinations. When used as such a pharmaceutical preparation, the content of the acreoside A of the present invention in the preparation is generally preferably from 0.00001 to 50 mass%, particularly preferably from 0.0001 to 10 mass%. preferable.
The daily dose of the above pharmaceutical agent per adult is preferably 0.0003 to 3000 mg, more preferably 0.003 to 300 mg, as acreoside A, for example.

Examples of the form of the food containing the acreoside A of the present invention include the same forms (tablets, capsules, syrups, etc.) as the above-mentioned oral administration preparations.
In order to prepare various forms of food, the acreoside A of the present invention alone or other food materials, solvents, softeners, oils, emulsifiers, preservatives, fragrances, stabilizers, colorants, UV absorption An agent, an antioxidant, a humectant, a thickener and the like can be used in appropriate combination. In general, the content of acreoside A in the food is preferably 0.00001 to 100% by mass, particularly preferably 0.0001 to 70% by mass.

  The quasi-drugs and cosmetics containing acreoside A of the present invention can be skin external preparations, cleaning agents, makeup cosmetics, scalp hair cosmetics, and lotions depending on the method of use. , Emulsions, gels, creams, ointments, powders, granules and the like. Such quasi-drugs and cosmetics of various dosage forms are oil-based, which is blended in the quasi-drug of the present invention alone or in quasi-drugs, skin cosmetics, scalp hair cosmetics and detergents. Combine ingredients, moisturizers, powders, pigments, emulsifiers, solubilizers, detergents, UV absorbers, thickeners, drugs, fragrances, resins, antibacterial and antifungal agents, plant extracts, alcohols, etc. Can be prepared. In general, the content of acreoside A in the quasi-drug and cosmetic is preferably 0.00001 to 100% by mass, particularly preferably 0.0001 to 70% by mass.

Example 1 Preparation of Acreoside A (1) 30% EtOH 50 L was added to 5.0 kg of butcher bloom (Ruscus aculeatus L., Albania) rhizome, extracted at room temperature for 7 days, and filtered. The residue was extracted again with 30% EtOH, combined with the previous filtrate, and then passed through a column packed with Diaion HP-20 to adsorb the components. After washing with 50% EtOH and eluting with 99.5% EtOH, 14 g of a crude saponin fraction was obtained. This was applied to a silica gel column and eluted with chloroform: methanol = 9: 1 mixed solvent to obtain 2 g of a saponin fraction. Further, gradient elution was performed by silica gel column chromatography using a chloroform: methanol mixed solvent (50: 1 → 19: 1 → 9: 1) to obtain a fraction (0.5 g) containing spiracleoside A. This was subjected to preparative HPLC equipped with an ODS column, and fractionation was performed by isocratic elution using a 50% acetonitrile aqueous solution as an eluent to obtain spiracleoside A (89.9 mg).
(2) Spiracleoside A 38 mg obtained in (1) was dissolved in 120 mL of MeOH and stirred for 1 hour under reflux. After distilling off the solvent, the obtained solid was purified with a Sephadex LH-20 column (eluent MeOH) to obtain 28 mg of acreoside A.

Example 2 Effect on VEGF Production (1) Materials and Methods Normal human neonatal foreskin-derived epidermal keratinocytes (Kurabo: frozen NHEK (F) Lot. No. 061130-902) were used for the test. Cells were seeded on three 6-well microplates at 2 × 10 ⁴ cells / mL per well. For the culture, Defined keratinocyte-SFM (SFM medium, Gibco) was used and cultured under conditions of 2% CO ₂ and 37 ° C. After the cell density reached 50-60% confluence, the medium was replaced with Defined keratinocyte-SFM medium (SFM (−) medium) without additives. The cells were acclimated to SFM (−) medium for 24 hours, and then the medium was replaced with SFM (−) medium to which Acreoside A obtained in Examples was added at an arbitrary concentration, and the test was started.
As a negative control, a compound-free SFM (−) medium was used. 16 hours after the start of the test, the medium was collected, and the amount of VEGF secreted into the culture supernatant was quantified by ELISA kit (R & D Systems). The amount of VEGF production in the control was set to 1, and the evaluation was carried out with a relative value.

(2) Results Table 1 shows the evaluation results.

  Acreoside A was shown to have an excellent VEGF production promoting effect.

Claims (2)

  A VEGF production promoter containing acreoside A as an active ingredient.   The VEGF production promoter according to claim 1, wherein the acreoside A is derived from a plant belonging to the genus Liliumaceae.