JP5745870B2 - Collagen gel contraction promoter - Google Patents

Description

  The present invention relates to a collagen gel contraction promoter, an agent for preventing or improving skin aging, an agent for improving skin elasticity, elasticity or talmi, a skin tightening agent, and a skin wound healing agent.

  The skin is mainly divided into three layers: epidermis, dermis, and subcutaneous tissue. Among them, the dermis is extremely important for maintaining the structure of the skin, and it is made strong and flexible by fibers such as collagen and elastin, and the dermal connective tissue is Forming. Proteins constituting connective tissue fibers such as collagen and elastin are synthesized / degraded by fibroblasts. Fibroblasts control the state of connective tissue by interacting with fibers such as collagen.

  When fibroblasts are embedded and cultured in collagen gel under normal fibroblast culture conditions, the collagen gel contracts (for example, Non-Patent Document 1). Collagen gel contraction varies depending on the number of cells and the amount of serum in the medium, and becomes more noticeable as the number of fibroblasts in the gel increases and the amount of serum in the medium increases. However, such contraction does not occur at all in a collagen-only gel without fibroblasts or a gel in which floating cells such as lymphocytes are embedded.

  Regarding skin aging, it is known that fibroblasts derived from the elderly have reduced gel contraction compared to cells derived from the young, and it has become clear that aging reduces the ability of the gel to contract. (For example, Non-Patent Document 2). A decrease in the contractility of the collagen gel indicates that it is difficult to maintain the dermal connective tissue in a tight state by contracting as in the previous youth. In fact, for example, as it ages, it is a well-known fact that tarmi, which was not recognized when it was young, was formed on the skin of the cheeks, neck, arms and other parts.

  For this reason, it is considered that the dermal connective tissue of the skin loses its contractile force and further loses its strength and elasticity, resulting in tarmi. Therefore, if the contractility of the collagen gel embedded with fibroblasts can be increased, and further the gel formation strength can be increased, the dermal connective tissue can be further contracted, tightened, and increased in strength, as in a young age. It is thought that a healthy skin state can be maintained. Further, it is considered that the reduced elasticity and firmness of the skin and the talmi due to aging can be improved, the skin can be tightened, and further the healing of the wound caused by the destruction of the dermal connective tissue can be promoted. Under such circumstances, development of a collagen gel shrinkage accelerator has been desired.

  Conventionally, as a substance that promotes contraction of a fibroblast-embedded collagen gel, serum, endothelin (for example, Non-Patent Document 3), transforming growth factor β (for example, Non-Patent Document 4), platelet growth factor Although compounds such as growth promoting factors such as retinoic acid are known, there are problems of transdermal absorbability, stability, safety and price, and the effect is not sufficient.

On the other hand, bamboo rush is a plant of the poppy family and is known to have antitumor, detoxification, insecticidal, antifungal and antifungal actions. Ohirumushiro is a floating leaf perennial plant belonging to the family Amuraceae.
However, the relationship between these plants and collagen gel contraction promotion, as well as prevention or improvement of skin aging is not known.

Review: E.M. Bell et al. J. et al. Invest. Dermatol. , 81, 2s (1983) M.M. Yamato, et al. Mech. Ageing Dev. 67, 149 (1993) C. Guidry et al. J. et al. Cell Biol. 115, 873 (1991) R. Montesano et al. Pro. Natl, Acad. Sci. USA, 85, 4894 (1988)

  The present invention relates to providing a collagen gel contraction promoter.

  The present inventors have intensively studied a substance having a collagen gel contraction accelerating effect using a human skin fibroblast collagen-embedded culture system. It has been found that this is useful as a collagen gel contraction promoter, a skin aging preventive or ameliorating agent, a skin elasticity, a firmness or talmi improving agent, a skin tightening agent, and a skin wound healing agent.

That is, the present invention relates to the following 1) to 5).
1) A collagen gel contraction promoter comprising as an active ingredient at least one selected from the group consisting of bamboo rush, leeks and their extracts.
2) A skin aging preventive or ameliorating agent comprising as an active ingredient at least one selected from the group consisting of bamboo rush, cabbage, and extracts thereof.
3) A skin elasticity, elasticity or sagging improving agent comprising at least one selected from the group consisting of Bamboo cristatus, Oyrum shiro and their extracts as an active ingredient.
4) A skin tightening agent comprising as an active ingredient at least one selected from the group consisting of Bamboo cristatus, Oyrus shiro and their extracts.
5) A wound healing agent for skin comprising, as an active ingredient, at least one selected from the group consisting of bamboo rush, cabbage, and extracts thereof.

  Collagen gel contraction promoter and the like of the present invention include prevention or improvement of skin aging, improvement of reduced skin elasticity and elasticity, skin talmi due to aging, skin tightening and skin wound healing effect, It is useful as a quasi-drug, cosmetic, or as a material or formulation for blending into these.

Collagen gel contraction-promoting action by bamboo rush extract or cabbage extract.

  As used herein, “collagen gel” is a gel-like composition containing collagen and cellular components, gel composition containing collagen and cellular components, in vitro culture of cells, and fibroblasts in the dermis of living organisms. Includes structures comprising cells and the surrounding extracellular matrix.

  In the present specification, “non-therapeutic” is a concept that does not include a medical act, that is, a treatment act on the human body by treatment.

  As used herein, “improvement” refers to improvement of a disease, symptom or condition, prevention or delay of worsening of the disease, symptom or condition, or reversal, prevention or delay of progression of a disease or symptom.

  As used herein, “prevention” refers to preventing or delaying the onset of a disease or symptom in an individual, or reducing the risk of developing an individual's disease or symptom.

In the present specification, the bamboo rush means a poppy family (Macleya corda (Wild.) R. Br), and the scallop is a scallop ophthalmia (Potamogeton natans L). . Moreover, a bamboo rush extract or a scallop extract means an extract obtained from a bamboo squirrel or a scallop.
Such plants can be any part of them, such as whole grass, leaves, stems, buds, flowers, buds, wood parts, bark, lichens, roots, rhizomes, corms, corms, tubers, seeds, fruits, fungi A nucleus, a resin, or the like, or a combination thereof can be used, but it is preferable to use the whole plant for both the bamboo rush and the scallop.
The part may be subjected to the extraction step as it is, or may be subjected to the extraction step after being pulverized, cut or dried. The extract is derived from natural ingredients and has high safety.

Specific examples of the extraction means for obtaining the bamboo shoot or leeches extract include solid-liquid extraction, liquid-liquid extraction, immersion, decoction, leaching, reflux extraction, ultrasonic extraction, microwave extraction, stirring, and the like. it can. For example, as a suitable example of immersion, immersion at 10 to 50 ° C. for 1 hour to 14 days can be mentioned. Moreover, when shortening extraction time, solid-liquid extraction with stirring is desirable. Examples of suitable conditions for this solid-liquid extraction include stirring at 10 to 100 ° C. (preferably 70 to 90 ° C.) at 100 to 400 r / min for 1 to 30 minutes.
In order to prevent the oxidation of the extract, a means for extracting under a so-called non-oxidizing atmosphere while removing dissolved oxygen by bubbling degassing or inert gas such as nitrogen gas may be used in combination.

  As the solvent for extraction, either a polar solvent or a nonpolar solvent can be used. Specific examples of the solvent include water; alcohols such as methanol, ethanol, propanol and butanol; polyhydric alcohols such as propylene glycol and butylene glycol; ketones such as acetone and methyl ethyl ketone; methyl acetate and ethyl acetate Esters; linear and cyclic ethers such as tetrahydrofuran and diethyl ether; polyethers such as polyethylene glycol; hydrocarbons such as squalane, hexane, cyclohexane and petroleum ether; aromatic hydrocarbons such as toluene; dichloromethane and chloroform And halogenated hydrocarbons such as dichloroethane; and supercritical carbon dioxide; pyridines; organic solvents such as oils and fats, other oils such as wax; and mixtures thereof. Preferable examples include water, alcohols and aqueous solutions thereof, and ethanol is preferable as the alcohols. More preferred solvents are water and an aqueous ethanol solution.

When an aqueous ethanol solution is used as a solvent for extraction, the mixing ratio (volume ratio) of the aqueous ethanol solution and water is preferably 0.001 to 100: 99.999-0, and 5 to 95:95 to 5 is more preferable, 20 to 80:80 to 20 is more preferable, 30 to 70:70 to 30 is still more preferable, and 40 to 60:60 to 40 is still more preferable. In the case of an ethanol aqueous solution, the ethanol concentration is preferably 40 to 60% by volume.
As a usage-amount of a solvent, 1-100 mL is preferable with respect to 1 g of a bamboo rush or a cabbage (dry mass conversion), As extraction time, 1 minute-100 days are preferable, and 30 minutes-10 days are more preferable. The extraction temperature at this time is 0 ° C. to the boiling point of the solvent, more preferably 20 to 100 ° C., still more preferably 50 to 100 ° C., and still more preferably 70 to 90 ° C.

  The plant extract thus obtained may be used as it is as the extract or fraction, may be used as a diluted solution diluted with an appropriate solvent, or may be a concentrated extract or dry powder, or prepared in a paste form. It may be a thing. Also, freeze-dry and dilute with a solvent usually used for extraction, such as water, ethanol, propylene glycol, butylene glycol, water / ethanol mixture, water / propylene glycol mixture, water / butylene glycol mixture, etc. It can also be used. It can also be used by encapsulating in vesicles such as liposomes or microcapsules.

  In addition, the bamboo rush or euglena extract may be a crude purified product as long as it conforms to food and pharmaceutical acceptable standards and exhibits the effects of the present invention, and the obtained crude purified product is publicly known. These separation and purification methods may be combined as appropriate to increase their purity. Examples of the purification means include organic solvent precipitation, centrifugation, ultrafiltration membrane, high performance liquid chromatograph, column chromatograph and the like.

As will be described in Examples below, the plant of the present invention or an extract thereof has a contraction promoting action of a collagen gel in which fibroblasts are embedded. Therefore, the plant of this invention or its extract can be used for collagen gel contraction promotion.
Further, as described above, when fibroblasts are embedded and cultured in collagen gel under normal fibroblast culture conditions, the collagen gel contracts (for example, Non-Patent Document 1). Regarding skin aging, it is known that fibroblasts derived from elderly people have reduced gel contraction compared to cells derived from young people, and it is clear that aging reduces the ability of the gel to contract. (For example, Non-Patent Document 2). If the contractility of the collagen gel embedded with fibroblasts can be increased, and further the gel formation strength can be increased, the reduced elasticity and firmness of the skin can be improved, the skin talmi due to aging can be improved, It is believed that it can tighten the skin. It can also promote the healing of wounds resulting from the destruction of the dermal connective tissue.
Therefore, the plant of the present invention or an extract thereof can be used to improve reduced skin elasticity and firmness, to improve skin talmi due to aging, to tighten skin, and thus to prevent or improve skin aging, or to bind dermis It can be used for wound healing due to tissue destruction. The use can be in humans or non-human animals, or specimens derived therefrom, and can be therapeutic or non-therapeutic.

  Further, the plant of the present invention or an extract thereof includes a collagen gel contraction promoter, a skin aging preventive or ameliorating agent, a skin elasticity, a firmness or a talmi improving agent, a skin tightening agent, a skin wound healing agent, etc. Can be used as “collagen gel shrinkage promoters” and the like, and can also be used to produce these agents. At this time, the collagen gel contraction promoter or the like is at least one selected from the group consisting of the bamboo rush, leeches and extracts thereof alone, or in addition to this, a carrier appropriately selected as necessary Those which are acceptable in the below-mentioned objects to be blended may be used. In addition, the said formulation can be manufactured by a conventional method according to the target object which should be mix | blended.

  The collagen gel contraction promoter, etc. exerts various effects such as improvement of skin elasticity, elasticity or tarmi, tightening of the skin, and prevention or improvement of skin aging, or healing of wounds caused by destruction of dermal connective tissue. It can be used as an active ingredient in human or veterinary drugs, quasi drugs, cosmetics, foods and the like.

When the collagen gel contraction promoter of the present invention is used as an active ingredient of a pharmaceutical product, the pharmaceutical product can be administered in any dosage form. Examples of the dosage form include parenteral administration such as injections, suppositories, inhalants, transdermal absorption agents, external preparations, or oral administration such as tablets, capsules, granules, powders, syrups and the like.
In order to prepare such pharmaceutical preparations of various dosage forms, the collagen gel contraction promoter of the present invention alone or other pharmaceutically acceptable excipient, binder, extender, disintegrant , Surfactants, lubricants, dispersants, buffers, preservatives, flavoring agents, fragrances, coating agents, carriers, diluents, and the like can be used in appropriate combinations.

  Among these dosage forms, the preferred form is parenteral administration, and the content of bamboo rush, scallop or extract thereof in a preparation for parenteral administration containing a collagen gel contraction promoter, etc. (in terms of dry matter of the extract) Is generally preferably 0.00001 to 10% by mass, and more preferably 0.0001 to 1% by mass.

  When the collagen gel shrinkage accelerator of the present invention is used as an active ingredient of a quasi-drug or cosmetic, the form can be a skin external preparation, a cleaning agent, a makeup cosmetic, etc. Accordingly, it can be provided in various dosage forms such as lotion, emulsion, gel, cream, ointment, powder, granule and the like. Such quasi-drugs and cosmetics in various dosage forms are blended in one or more selected from the bamboo rush, Ohirumushiro and their extracts of the present invention, quasi-drugs, skin cosmetics and cleansing agents. Oil components, moisturizers, powders, pigments, emulsifiers, solubilizers, detergents, UV absorbers, thickeners, drugs (eg anti-inflammatory agents, bactericides, antioxidants, vitamins, fat metabolism It can be prepared by appropriately combining a promoting agent or a drug or natural product known to have an uncoupling protein expression promoting effect), a fragrance, a resin, an antifungal agent, a plant extract, an alcohol and the like.

  In general, the content of the quasi-drug, the bamboo rush, the silkworm or the extract thereof (in terms of a dried product of the extract) is preferably 0.00001 to 10% by mass, 0.0001 It is more preferable to set it to 1 mass%.

When using the collagen gel shrinkage accelerator of the present invention as an active ingredient of food, the form of the food is bread, cakes, noodles, confectionery, jelly, frozen food, ice cream, dairy product, beverage, etc. In addition to these various foods, the same forms (tablets, capsules, syrups, etc.) as the above-mentioned oral preparations can be mentioned.
To prepare various forms of food, the collagen gel shrinkage promoter of the present invention alone or other food materials, solvents, softeners, oils, emulsifiers, preservatives, fragrances, stabilizers, colorings An agent, an antioxidant, a humectant, a thickener and the like can be used in appropriate combination. In general, the content of the bamboo shoots, cabbage or extract thereof (in terms of dry matter of the extract) in the food is preferably 0.01 to 100% by mass, and preferably 0.1 to 100% by mass. Is more preferable, and more preferably 1 to 100% by mass.

Production Example 1
(1) Preparation of bamboo rush extract Add 2250 mL of 50 (v / v)% ethanol to 150 gram of bamboo rush, extract at 85 ° C for 3 hours, filter to obtain a crude extract, concentrate to dryness and extract 22.0 g of solid content was obtained.
This extracted solid was dissolved in 50 (v / v)% ethanol so that the evaporation residue was 1.0 (w / v)% to prepare a bamboo extract 50 (v / v)% ethanol extract.

(2) Preparation of Oyorumushiro Extract 2200 mL of 50 (v / v)% ethanol was added to 200 g of Oyorumushiro, extracted for 3 hours at 85 ° C., filtered to obtain a crude extract, concentrated to dryness and extracted. 12.0 g of solid content was obtained.
This extracted solid was dissolved in 50 (v / v)% ethanol so as to have an evaporation residue of 1.0 (w / v)% to prepare an extract of Ohirumushiro 50 (v / v)%.

Test Example 1 Measurement of Collagen Gel Contraction Promoting Ability Collagen gel was prepared by a method referring to literature “J. Cell Science, 102, 315 (1992)” or “J. Invest. Dermatol, 93, 792 (1989)”. . That is, HEPES (containing 0.01 M sodium hydroxide), DMEM (GIBCO DMEM, manufactured by Nitta Gelatin Co., Ltd., tape IA (3.0 mg / mL, pH = 3)) under ice-cooling. low glucose) 5-fold concentrated solution, FBS (5%, Fatal Bovine Serum) and purified water, and after sufficiently neutralizing with stirring, final concentrations of 0.001, 0.0005, 0.0001 mass% (in terms of dry solids) Mass%)), 0.05 (v / v)% ethanol was added as a test substance (each plant extract obtained in Production Example 1) or as a control, and finally human skin fibroblasts (derived from human foreskin, Kurabo Industries Co., Ltd.) suspension was added to 1 × 10 ⁵ cells / well. After sufficiently stirring and removing bubbles, 0.6 mL of each hole was poured into a 24-well plate and immediately gelled at 37 ° C. The collagen concentration at this time was adjusted to 1.5 mg / mL.
After 24 hours, liquid medium was added to each hole of the plate, the periphery of the gel was peeled off, and then further cultured at 37 ° C. for 48 hours.

After culturing for 48 hours, the weight of the gel was measured with a balance.
The collagen gel contraction promoting effect of each plant extract was evaluated as a contraction promotion rate (%) by correcting the weight of the gel added with each plant extract with the weight of the control.

  As shown in FIG. 1, the weight of the collagen gel, that is, the volume, was reduced by the bamboo rush and the silkworm, and the collagen gel contraction promoting effect by these two types of extracts was recognized.

Claims (1)

Ohirumushi b及 beauty collagen gel contraction accelerator comprising as an active ingredient, at least one selected from the group consisting of the extract.