JP2009149548A - Agent for promoting production of integrin and vinculin - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an agent for improving sagging skin and elasticity, which improves the sagging skin due to aging and imparts an elastic touch to the skin, and to provide a composition which has an effect for inhibiting the proliferation and metastasis of cancer cells. <P>SOLUTION: Thamnolia vermicularis having an effect for promoting the production of integrin and vinculin is contained as an active ingredient, and thereby the interaction of skin fibroblasts with an extracellular matrix is activated so that the elastic touch is imparted to the skin. Thus, the composition which improves the sagging and elasticity of the skin, and the composition having the effect for inhibiting the proliferation and metastasis of cancer cells are provided. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

The present invention relates to an integrin and vinculin production promoter comprising an extract of plant snow tea as an active ingredient, and as a field of use thereof, for example, it can be used for cosmetics, quasi drugs, pharmaceuticals, etc. The present invention relates to a composition that improves sagging and gives a feeling of elasticity, and a composition having an effect of inhibiting the growth of cancer cells and metastasis.

Conventional technology

The skin is constantly exposed to external stimuli such as dryness and ultraviolet rays, and as it ages, it loses its elasticity and elasticity, resulting in sagging. Conventionally, as a composition that improves the sagging of the skin and gives elasticity to the skin, the adhesion between the epidermis and the basement membrane is improved and strengthened, or the production of integrins in the epidermis cells is promoted to promote skin wrinkles, sagging, Proposals have been made to improve the texture structure. Moreover, what expects the effect which tightens skin with an astringent is known. Patent Document 1 exemplifies a composition for promoting the production of integrin α6β4 for keratinocytes containing plants of kizota, echinacea, pumpkin, dandelion, and angelica or extracts thereof. Patent Document 2 also includes a laminin 5 production promoter that is a skin basement membrane activation component for keratinocytes and an integrin α6β4 production promoter that is a skin basal cell activation component. Examples are compositions that prevent, prevent and improve. Furthermore, Patent Document 3 also discloses an integrin production promoting substance for keratinocytes, which is a hyssop belonging to the genus Willow genus, stevia belonging to the genus Stevia, broccoli belonging to the genus Brassica, mallows belonging to the genus Mallow, and hyacinths. Agents are illustrated.
JP 2003-171225 A JP 2003-226655 A Japanese Patent Laid-Open No. 2004-262861

However, improving and strengthening the adhesion between the epidermis and the basement membrane and promoting the production of integrins by epidermal cells is not enough to improve skin wrinkles and sagging, and it also has the effect of tightening the skin with astringents. It was only a temporary effect and could not continuously improve the sagging / elasticity of the skin. Then, this invention makes it a subject to provide the composition which improves the sagging of skin and provides a feeling of elasticity.

The present inventors paid particular attention to the interaction between dermal fibroblasts and an extracellular matrix such as collagen. In other words, the snow tea extract, which has the effect of promoting fibroblast integrin and vinculin production, acts on skin fibroblasts to enhance adhesion to the dermal extracellular matrix, thereby reducing skin sagging and elasticity. It has become possible to provide a composition having an effect of improving.

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 firmly and flexibly by fibers such as collagen, fibronectin, and elastin. Forming an organization. Human skin cells, particularly human skin fibroblasts, control the state of connective tissue by interacting with fibers such as collagen, fibronectin, and elastin. Therefore, the interaction between dermal fibroblasts and extracellular matrix such as collagen is deeply related to the maintenance of skin sagging and elasticity.

Therefore, the present inventors have focused on integrin and vinculin, which are adhesion proteins that have not attracted much attention in skin cells, and by increasing the amount of integrin and vinculin protein, skin cells, in particular skin fibroblasts, are obtained. It has been found that skin sag and elasticity can be improved by activating the interaction between cells and extracellular matrix.

On the other hand, it is known that integrin, particularly integrin β-1 subunit is strongly expressed in many cancer cells. Integrins are biomolecules that form a family of molecules that form α and β heterodimers, and are known to bind to cell surface ligands and extracellular matrix. Integrins are further classified into β-1, β-2, and β-3 subgroups by their β subunits. It has also been reported that the expression level of integrin is closely related to the malignancy such as growth rate and metastasis of cancer cells in vivo. For example, belongs alpha ₅ beta ₁ to beta-1 subgroup has been reported that not tumorigenesis was observed when the expression level is increased (Cell.
60, 849-859 (1990)). Thus, the importance of integrins has increased in recent years, and application to the treatment and prevention of leukocyte adhesion inhibition, platelet aggregation inhibition, cancer metastasis inhibition, myocardial infarction, arteriosclerosis, osteolytic disease and the like can be expected. In addition, vinculin is best known as one of the integrin-lined proteins, and it is known to enhance adhesion by enhancing expression and suppress cell movement.

The present invention provides a skin fibroblast by blending a snow tea extract having an effect of promoting the production of integrin and vinculin of human fibroblasts into cosmetics, quasi drugs, pharmaceuticals, and the like, and using the composition. It has been possible to provide a composition that has an effect of acting on cells and enhancing the adhesion with the dermal extracellular matrix to improve the sagging and elasticity of the skin.

In the present invention, the integrin and vinculin are not particularly limited as long as they are expressed in human skin cells, but those expressed in human skin fibroblasts are particularly preferable. If it is expressed in human skin fibroblasts, a particularly excellent skin tightening effect can be exhibited.

Integrins are composed of α subunits and β subunits, α subunits further include α1 to α5, αL, etc., and β subunits include β1, β2, β3, etc., but collagens present in various connective tissues , Vitronectin, fibronectin, laminin, etc., and the interaction between the cells present in the connective tissue such as fibroblasts, it is preferable that the expression of α1 subunit, α2 subunit, and β1 subunit is promoted. Furthermore, it is more desirable that expression of α2 subunit is promoted and expression of β1 subunit is promoted at the same time. In particular, regarding dermal fibroblasts, expression of α2β1 integrin is preferably promoted from the viewpoint of interaction with collagen.

The integrin and vinculin production promoter of the present invention contains snow tea as an active ingredient. Yukicha is called Thamnolia vermicularis and scientific name is Musgogoke, and belongs to lichens in taxonomy, and is a symbiosis of fungi and algae. In Japan, it is a rare species found only in central Honshu and Takayama in Hokkaido and lives mainly on trees, rocks and the ground. Yukicha has long been used as a fragrance and dye in many parts of the world, but it has been very prized because it does not grow as much as 1 cm per year.

The part of snow tea in the extract used in the present invention is not particularly limited, and a useful part or the whole plant can be used. In addition to the extract obtained by directly extracting from these various extraction sites using a solvent, the extract may be extracted by adding a solvent to the squeezed liquid and / or residue obtained after the squeezing treatment.

The extraction solvent for obtaining the extract used in the present invention may be selected in consideration of the intended purpose and type of the product to be provided, or the processing performed later. For example, water; methyl alcohol, ethyl alcohol Lower monohydric alcohols such as: liquid polyhydric alcohols such as glycerin, propylene glycol, and 1,3-butylene glycol; ketones such as acetone and methyl ethyl ketone; alkyl esters such as ethyl acetate; hydrocarbons such as benzene and hexane; These can be extracted and purified using one or more of halogenated alkanes such as dichloromethane and chloroform.

The snow tea extract used in the present invention is obtained by collecting the use site, pulverizing after drying, 1 to 1000 times by weight, particularly 10 to 100 times the amount of solvent, and in the case of room temperature extraction, It is preferable to carry out at 0 ° C. or higher, particularly 20 ° C. to 40 ° C. for 1 hour or longer, particularly 3 to 7 days. Moreover, you may heat-extract at 60-100 degreeC for 1 hour.

Each of the above-mentioned extracts obtained under the above conditions may be used as an extracted solution. However, if necessary, use a product that has been refined, filtered, concentrated, and powdered as necessary. I can do it.

The form of the snow tea extract used in the present invention may be any form such as liquid, solid, powder, paste, gel, etc., and the optimum shape for configuring the final product is arbitrarily selected. can do.

The dosage form of the composition of the present invention is arbitrary, and examples of the external preparation for skin include liquids, suspensions, emulsions, creams, ointments, gels, liniments, lotions or poultices, drinks and the like. A pharmaceutical or quasi-drug is preferred. Furthermore, it can mix | blend and use for pharmaceuticals, quasi-drugs, those drinks, and cosmetics. In particular, it is applied to external preparation compositions such as pharmaceuticals, quasi drugs, and cosmetic compositions applied to the outer skin.

Specific use forms of the present invention include aqueous components, oily components, plant extracts, animal extracts, powders, excipients, surfactants, oils, alcohols, pH adjusters, preservatives, antioxidants, A lotion, emulsion, cream, pack, powder, spray, ointment, dispersion, cleaning agent for external preparation composition by mixing thickeners, sweeteners, pigments, fragrances and the like as necessary. In addition, various dosage forms such as liquid, paste, capsule, powder, and tablet can be used.

The blending amount of the extract according to the present invention into the skin composition is slightly different depending on the degree of expected action and is not particularly limited, but is usually 0.0001% by mass or more, preferably 0.01- A concentration range of 20.0 mass% is effective.

Hereinafter, test examples relating to the effects of the integrin and vinculin production promoter according to the present invention will be shown and application prescription examples to external preparations using the materials will be described, but the examples are not limited thereto. Needless to say.

Example 1 Preparation of Extract
50 ml of purified water was added to 1 g of dried and crushed snow tea, and extracted by heating at 80 ° C. for 1 hour. The extract was filtered and the residue dried under reduced pressure at 40 ° C. was dried. The dried product was prepared to be a 1% aqueous solution and used as a sample solution.

(Example 2) [Cell culture]
・ Cell: HFSKF-II (RIKEN)
・ Medium: D-MEM + FBS15%
Normal human fibroblast HFSKF-II cells were cultured in D-MEM medium supplemented with 15% FBS. The cells were seeded and cultured on a 50 cm ² plate at about 50% confluence. The next day, the extract prepared in Example 1 was added. After the addition, the cells were collected 48 hours later and used as Western blotting samples.

[Western blotting]
After electrophoresis on 7.6% acrylamide gel, transfer to PVDF membrane, Integrinβ-1 (M-106) sc-8978 (SANTA CRUZ BIOTECHNOLOGY, INC) Human (Rabbit polyclonal) as integrin antibody, Vinculin (H -10) sc-25336 (SANTA CRUZ BIOTECHNOLOGY, INC) Human (mouse monoclonal), ECL ^TM Anti-rabbit IgG as secondary antibody, Horseradish Peroxidase linked whole
antibody (from donkey) NA934V (Amersham
Bioscience UK Limited), ECL ^TM Anti-mouse IgG, Horseradish Peroxidase linked whole antibody (from sheep) NA931V (Amersham Bioscience UK Limited), Western blotting confirmed 130KDa integrin protein and 117KDa vinculin protein did.

[Measurement of integrin and vinculin protein levels]
The band intensity obtained by Western blotting was analyzed by Dolphin-DOC (WEALTEC). The area of the integrin protein band intensity in the non-sample-added group was defined as 100, and the integrin band intensity area in each extract-added group was proportionally calculated to obtain the integrin protein amount. Vinculin protein was also treated in the same manner as integrin.

The experimental results are shown in Table 1. As a result of analysis of the band intensity area, when the control group is set to 100, the integrin β1 production amount is 125 in the group with 340 ppm of snow tea extract and 136 in the group with 680 ppm addition, and the integrin β1 production promoting effect by the snow tea extract Was confirmed. As for vinculin protein, the production amount of 121 was obtained in the 340 ppm addition group of snow tea extract and 134 was produced in the 680 ppm addition group, and the effect of promoting the production of vinculin by the snow tea extract was confirmed.

(Example 3) [Manufacture of various compositions]
Various compositions according to the present invention were prepared. Although the formulation example is shown below, this invention is not necessarily limited to these.

(1) Cream composition
(weight%)
a) Beeswax
2.0
b) Stearyl alcohol
5.0
c) Stearic acid
8.0
d) Squalane
10.0
e) Self-emulsifying glyceryl monostearate
3.0
f) Polyoxyethylene cetyl ether (20E.O.) 1.0
g) Preservatives and antioxidants
Appropriate amount
h) Purified water
The rest
i) 1,3-butylene glycol
5.0
j) Snow tea extract

0.01
k) Potassium hydroxide
0.3
Manufacturing method: Heat up to a) to g) and keep at 80 ° C Heat up to h) to j), keep at 80 ° C., add to a) to f) and emulsify. Thereafter, k), which has been dissolved in part of h) in advance, is added and cooled to 40 ° C. with stirring.

(2) Emulsion composition
(weight%)
a) Beeswax
0.5
b) Petrolatum
2.0
c) Squalane
8.0
d) Sorbitan sesquioleate
0.8
e) Polyoxyethylene oleyl ether (20E.O.)
1.2
f) Snow tea extract
0.0001
g) 1,3-butylene glycol
7.0
h) Carboxyvinyl polymer
0.2
i) Potassium hydroxide
0.1
j) Purified water
The rest
k) Preservatives and antioxidants
Appropriate amount
l) Ethanol
7.0
Preparation method: Heat up to a) to e) and keep at 80 ° C. F) to k) are dissolved by heating, kept at 80 ° C., added to a) to e), emulsified, and cooled to 50 ° C. with stirring. Add l) at 50 ° C and cool to 40 ° C.

(3) Lotion-like composition
(weight%)
a) Snow tea extract

10.0
b) α-Linolenic acid

0.1
c) Glycerin
5.0
d) Polyoxyethylene sorbitan monolaurate (20E.O.)
1.0
e) ethanol
6.0
f) Fragrance
Appropriate amount
g) Preservatives and antioxidants
Appropriate amount
h) Purified water
The remainder of the manufacturing method: a) to h) are mixed and dissolved uniformly.

(4) Packing agent
(weight%)
a) Snow tea extract
5.0
b) Vinyl acetate resin emulsion
15.0
c) Polyvinyl alcohol
10.0
d) Cuui seed oil
3.0
e) Glycerin
5.0
f) Titanium oxide
8.0
g) Kaolin
7.0
h) ethanol
8.0
i) Fragrance
Appropriate amount
j) Preservatives and antioxidants
Appropriate amount
k) Purified water
The remainder of the manufacturing method: a) to k) are mixed, stirred and dispersed well to make uniform.

(5) Ointment
a) Liquid paraffin 20.0
b) White petrolatum 50.0
c) Cetanol 10.0
d) Polyoxyethylene (60) hydrogenated castor oil 18.0
e) Snow tea extract 1.0
Production method: a) to e) are added and mixed while heating to prepare an ointment.

(6) Gel agent
a) Polyoxyethylene (60) hydrogenated castor oil 9.0
b) Carbopol 941 0.5
c) Ethanol 22.0
d) Triethanolamine 1.5
e) Snow tea extract 0.001
f) Purified water 63.0
Production method: While heating, add a), b) and e) to f) and dissolve, then add d) and c) and cool with stirring to prepare a gel.

(7) Liquid agent
a) Polyoxyethylene (60) hydrogenated castor oil 10.0
b) Ethanol 20.0
c) Snow tea extract 20.0
d) Purified water 65.0
Production method: a) to d) are added and dissolved to prepare a solution.

(8) Tablet (wt%)
a) Starch 72.0
b) Sucrose fatty acid ester 8.0
c) Snow tea extract 20.0
Manufacturing method: The raw materials of a) to c) are mixed and molded with a tableting machine.
An uncoated tablet with a diameter of mm was manufactured. This uncoated tablet was film-coated by a conventional method to complete a tablet.

(9) Capsule a) Lactose 190mg
b) Magnesium stearate 10mg
c) Snow tea extract

10mg
210mg total
Production method: a) to c) were mixed uniformly, passed through a 60 mesh sieve, and the whole powder was filled into a No. 2 gelatin capsule.

(10) Drink a) Lemon juice
20mL
b) Propolis 0.3g
c) Vitamin C 0.2g
d) Honey 13.0g
e) Snow tea extract
1.0g
f) Water proper amount total 100mL
Production method: After sufficiently stirring a) to e), f) was added to make the total amount 100 mL.

Applicability of invention

The integrin and vinculin production promoter comprising the snow tea extract of the present invention as an active ingredient is a composition that improves skin sagging and gives a feeling of elasticity, and has a cancer cell growth inhibitory and metastasis inhibitory effect. Applications can be expected for cosmetics, quasi-drugs, and pharmaceuticals.

Claims (4)

An integrin production promoter characterized by comprising an extract of snow tea as an active ingredient. The integrin production promoter according to claim 1, wherein the integrin is integrin β1. The integrin production promoter according to claim 1, wherein the integrin is integrin α2β1. A vinculin production promoter characterized by comprising an extract of snow tea as an active ingredient.