Patent Application
20070134265
The present invention relates to an anti-aging composition and a collagen production promoting composition and, more particularly, to an anti-aging composition and a collagen production promoting composition that can promote the production of collagen which is one of the components of the extracellular matrix. Further, the present invention relates to a promoting composition and a promoting method for collagen gel contraction, and to fibroblast or epidermal cell integrin production.
Wrinkle and sagging observed in aged skin are a major change in appearance caused by aging and are a serious problem for many middle-aged and elderly persons. Thinning of skin tissues due to aging is one of the causes of wrinkle and sagging of the skin. In the aged skin, the amount of collagen fibers, which are a major matrix component of the dermis, is much lower than that in the non-aged skin. This is a major cause of the reduced skin thickness. Accordingly, keeping the amount of collagen on a suitable level by promoting collagen production is considered effective for preventing and reducing wrinkle and sagging of the skin.
For example, isoflavone compounds, selected from daidzein, daidzin, genistein, and genistin, and phytosterols have hitherto been reported as natural product-derived ingredients which can promote collagen production to prevent and reduce a change in skin caused by aging.
In recent years, however, there has been a strong demand for other substances having significant collagen production promotion effect.
The present inventor has made extensive and intensive studies on substances having the effect of strongly promoting dermal fibroblast collagen biosynthesis. As a result, the present inventor has found that a hydrogen peroxide-treated yeast hydrolysate, which has been cultured in a specific nutrient medium, has significant collagen production promotion effect.
According to the present invention, there is provided an anti-aging composition characterized by comprising a collagen production promoting composition comprising, as an active ingredient, a hydrogen peroxide-treated yeast hydrolysate which has been cultured in a nutrient medium containing a non-animal-derived glycosaminoglycan. Further, there is provided a collagen production promoting composition characterized by comprising, as an active ingredient, a hydrogen peroxide-treated yeast hydrolysate which has been cultured in a nutrient medium containing a non-animal-derived glycosaminoglycan. Furthermore, there is provided a method for promoting collagen production, characterized by comprising using the above hydrogen peroxide-treated yeast hydrolysate to promote the production of collagen.
The hydrogen peroxide-treated yeast hydrolysate used in the present invention is known to have elastin production promotion effect. Up to now, however, the collagen production promotion effect of the hydrogen peroxide-treated yeast hydrolysate was not known and was found for the first time by the present inventor. In this connection, it should be noted that the elastin production promotion effect is a technical matter different from the collagen production promotion effect, and a substance having an elastin production promotion effect does not always have a collagen production promotion effect.
Further, regarding the hydrogen peroxide-treated yeast hydrolysate, the present inventor has made further studies on collagen gel contraction and epidermal cell integrin production promotion which are known to be involved in the prevention/amelioration of skin aging phenomena, for example, wrinkles and sagging. As a result, the present inventor has surprisingly found that this hydrolysate also has collagen gel contraction promotion effect, a fibroblast integrin production promotion effect, particularly for fibroblast integrin α2, integrin α3, integrin α6, integrin α2β1 and/or integrin β1 production promotion effect, as well as an epidermal cell integrin production promotion effect, particularly for epidermal cell integrin α2, integrin α3, integrin α6, integrin α2β1, integrin β1 and/or integrin β4 production promotion effect. Collagen gel contraction promotion as well as fibroblast and epidermal cell integrin production promotion are known to be effective for preventing skin aging and for ameliorating skin aging condition (for example, Japanese Unexamined Patent Publication (Kokai) Nos. 2001-39850, 10(1998)-72336, 2001-278769, and 2003-171225). Therefore, having such effects supports the anti-aging effect of the hydrogen peroxide-treated yeast hydrolysate used in the present invention.
Therefore, according to another aspect of the present invention, there is provided an anti-aging composition characterized by comprising a collagen gel contraction promoting composition comprising, as an active ingredient, a hydrogen peroxide-treated yeast hydrolysate which has been cultured in a nutrient medium containing a non-animal-derived glycosaminoglycan. Further, there is provided a collagen gel contraction promoting composition characterized by comprising, as an active ingredient, a hydrogen peroxide-treated yeast hydrolysate which has been cultured in a nutrient medium containing a non-animal-derived glycosaminoglycan. Furthermore, there is provided a method for promoting collagen gel contraction, characterized by comprising using the above hydrogen peroxide-treated yeast hydrolysate to promote the contraction of a collagen gel.
According to a further aspect of the present invention, there is provided an anti-aging composition characterized by comprising an integrin production promoting composition for promoting integrin production by fibroblasts, said integrin production promoting composition comprising, as an active ingredient, a hydrogen peroxide-treated yeast hydrolysate which has been cultured in a nutrient medium containing a non-animal-derived glycosaminoglycan. Further, there is provided an integrin production promoting composition for promoting integrin production by fibroblasts, characterized in that said integrin production promoting composition comprises, as an active ingredient, a hydrogen peroxide-treated yeast hydrolysate which has been cultured in a nutrient medium containing a non-animal-derived glycosaminoglycan. Furthermore, there is provided a method for promoting fibroblast integrin production, characterized by comprising using the above hydrogen peroxide-treated yeast hydrolysate to promote fibroblast integrin production.
According to a still further aspect of the present invention, there is provided an anti-aging composition characterized by comprising an integrin production promoting composition for promoting epidermal cell integrin production, said integrin production promoting composition comprising, as an active ingredient, a hydrogen peroxide-treated yeast hydrolysate which has been cultured in a nutrient medium containing a non-animal-derived glycosaminoglycan. Further, there is provided an integrin production promoting composition for promoting epidermal cell integrin production, characterized by comprising, as an active ingredient, a hydrogen peroxide-treated yeast hydrolysate which has been cultured in a nutrient medium containing a non-animal-derived glycosaminoglycan. Furthermore, there is provided a method for promoting epidermal cell integrin production, characterized by comprising using the above hydrogen peroxide-treated yeast hydrolysate to promote epidermal cell integrin production.
The present invention will be described in detail.
The hydrogen peroxide-treated yeast hydrolysate used in the present invention is a water-soluble yeast hydrolysate produced by treating yeast in aqueous hydrogen peroxide, preferably a water-soluble yeast hydrolysate produced by subjecting yeast to the above treatment and further exposing the treated product to ultraviolet radiation. Details of the hydrogen peroxide-treated yeast hydrolysate including the production process thereof are described in U.S. Pat. No. 6,461,857. Further, the hydrogen peroxide-treated yeast hydrolysate used in the present invention is commercially available from Arch Personal Care Products, L.P. under the tradename designation “Biodynes EMPP (trademark).”
The preparation of the hydrogen peroxide-treated yeast hydrolysate used in the present invention is based on a mechanism where, when culture yeast is cultured under stress applied by hydrogen peroxide, the yeast responds to the stress to produce a cell protective component which protects cells from the stress. Specifically, the hydrogen peroxide-treated yeast hydrolysate can be prepared by adding hydrogen peroxide to cultured yeast cells and culturing the mixture in a nutrient medium containing a non-animal-derived glycosaminoglycan.
More specifically, for example, the hydrogen peroxide-treated yeast hydrolysate according to the present invention can be prepared according to the following procedure:
(a) provide a culture of yeast cells, for example, Saccharomyces cerevisae;
(b) add a sub-lethal amount, for example, about 0.1 to 2% by mass based on the total mass of the culture, of hydrogen peroxide to the culture to apply stress to the cells;
(c) optionally expose the culture to a sub-lethal amount of ultraviolet radiation (for example, UVA/UVB irradiation at an intensity of 31.5 mJ/cm2) to apply stress to the cells; and
(d) obtain a water-soluble yeast extract from the hydrogen peroxide-containing culture.
The anti-aging composition, collagen production promoting composition, collagen gel contraction composition, fibroblast integrin production promoting composition, and epidermal cell integrin production promoting composition according to the present invention can be formulated to contain, in addition to the above indispensable ingredient, optional ingredients commonly used in external preparations for skin such as cosmetics and pharmaceutical products, for example, skin-whitening agents, moisturizing agents, antioxidants, oil components, ultraviolet absorbers, surfactants, thickeners, alcohols, powder components, coloring materials, aqueous components, water, and various nutrient preparations for skin.
Further, other materials, for example, disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, gluconic acid, and other sequestering agents, caffeine, tannin, verapamil, tranexamic acid and its derivatives, licorice extract, glabridin, hot-water extracts of fruits of Kakyoku (Pyracantha fortuneana), various crude drugs, tocopherol acetate, glycyrrhetinic acid and its derivatives or its salts, and other drugs, vitamin C, magnesium ascorbic acid phosphate, ascorbic acid glucoside, arbutin, kojic acid, and other skin-whitening agents, glucose, fructose, mannose, sucrose, trehalose, and other saccharides may also be optionally formulated.
The present invention can be extensively applied to cosmetics, quasidrugs and the like used for the outer skin, particularly preferably to cosmetics. The compositions and the promoting compositions according to the present invention may take a wide variety of forms such as aqueous solutions, dissolvable forms, emulsions, powders, oils, gels, ointments, aerosols, water-oil two-phase systems, water-oil-powder three-phase systems and the like. That is, in the case of basic cosmetics, they can be extensively applied in the above various forms for face cleansers, skin lotions, milky lotions, creams, gels, essences (beauty care lotions), packs, masks and the like. Further, in the case of makeup cosmetics, they can be applied in a wide range of forms such as foundations, and, in the case of toiletry products, they can be applied in a wide range of forms such as body soaps, soaps and the like. Further, in the case of quasidrugs, they can be applied in a wide range of forms such as various ointments. It should be noted that the form and type of the collagen production promoter according to the present invention are not limited to these forms and types.
The following Examples further illustrate but do not limit the present invention. Formulating amounts are in % by weight. Before the Examples, the methods for testing the effects and the results thereof will be explained.
1-1. Assay Method
(1) Culture of Human Fibroblasts
Human fibroblasts were inoculated in an amount of 1×104 cells/well in a 10% fetal bovine serum-containing DMEM medium in a 96-well plate, and, 3 to 4 hrs after the inoculation, the concentration of the serum was decreased to 0.5%. One day after that, the medium was replaced with a 0.5% serum-containing DMEM medium with Biodynes EMPP being added thereto in varied concentrations. On the 4th day from the cell inoculation, the amount of type I collagen in the supernatant of the medium was measured, and the amount of DNA was measured for the cells as a measure of the number of cells.
(2) Quantitative Determination of DNA
The amount of DNA was measured by fluorimetry with H33342 manufactured by Hoechst.
(3) Measurement of Type I Collagen
Procollagen type I carboxyterminal propeptide (PIP) produced by human fibroblasts was measured by ELISA with Kit MK-101 manufactured by Takara Shuzo Co., Ltd. The percentage of the amount of PIP per DNA of the sample relative to the amount of PIP per DNA in the control sample to be 100 was regarded as type I collagen production promotion rate.
For comparison, the collagen production promotion effect was examined in the same manner as described just above, except that “Biodynes TRF-25” (tradename: manufactured by Arch Personal Care Products L.P.), which is also a yeast culture hydrolysate, was used instead of “Biodynes EMPP” according to the present invention.
The production process of “Biodynes TRF-25” is different from the production process of “Biodynes EMPP” according to the present invention in that growth peptones with various low-molecular weights and non-animal-derived glycosaminoglycan were not provided in a satisfactory amount and, in addition, hydrogen peroxide treatment was not carried out.
1-2. Results
2-1. Assay Method
(1) Culture of Human Fibroblasts
Human skin fibroblasts were inoculated in an amount of 1×105 cells/well in a DMEM medium containing a 0.25% fetal bovine serum and 250 μM ascorbic acid glucoside in a 24-well plate. After cell adhesion, Biodynes EMPP was added. Two days after, the medium was centrifuged. The supernatant thus obtained was measured for type VII collagen and the amount of DNA for cells as a measure of the number of cells.
(2) Quantitative Determination of DNA
The amount of DNA was measured by fluorimetry with H33342 manufactured by Hoechst.
(3) Determination of Type VII Collagen by Sandwich ELISA
Type VII collagen was measured by sandwich ELISA. In this Example, the following antibody was used.
The amount of type VII collagen per DNA of the agent added sample by setting the amount of type VII collagen per DNA in the sample, to which Biodynes EMPP had not been added, (control sample) to be 100 was regarded as type VII collagen production promotion rate.
2-2. Results
As is apparent from the above results, the group to which Biodynes EMPP had been added was much superior in type I collagen production promotion effect as compared with the group to which Biodynes TRF-25 had been added. Further, it has become apparent that the group to which Biodynes EMPP had been added also had type VII collagen production promotion effect.
It is considered that, due to the difference in production process between Biodynes EMPP and Biodynes TRF-25 (various stimulation methods for yeast), in the case of Biodynes EMPP, more useful cell activation components were produced and this caused the difference in the effect on fibroblasts between Biodynes EMPP and Biodynes TRF-25.
For the yeast extract obtained by extracting yeast with an aqueous medium, no collagen production promotion effect was observed. This result is also described in Japanese Patent No. 3278138.
3-1. Assay Method
A human dermal fibroblast suspension collagen solution (using, as collagen, I-AC manufactured by KOKEN CO., LTD.) was prepared on ice and was mixed with a 0.25% FBS/DMEM medium containing Biodynes EMPP (purified water in the case of control; the concentration is expressed in mass %), and the mixture was then poured into a 3.5-cm dish. After gelation at 37° C., the gel was separated from the wall surface of the dish so as to allow collagen gel contraction. One to four days after, the diameter of the collagen gel was measured from three directions, and the measured values were averaged. The percentage contraction after the addition of the test substance was determined by setting the diameter before the contraction to be contraction 0% (for each group, n=3 or 4).
3-2. Results
As is apparent from the above results, the group to which Biodynes EMPP had been added was much superior in collagen gel contraction promotion effect to the control group, and it was also confirmed that the collagen gel contraction promotion effect depends upon the concentration of Biodynes EMPP.
4-1. Assay Method
Biodynes EMPP was allowed to act on human dermal fibroblasts, and, 24 hr after the action, the cells were separated by trypsin/EDTA. After neutralization with FCS, the cells were washed with PBS containing 0.01% FCS and 0.02% NaN3 to recover the cells. Anti-human integrin α2, anti-human integrin α3, anti-human integrin α6, anti-human integrin α2β1, and anti-human integrin β1 were each used after 100-fold dilution as a primary antibody, and FITC-labeled anti-mouse IgGl was used after 100-fold dilution as a secondary antibody. In the control, mouse IgG1 was used as the primary antibody. The amount of integrin on the cell surface was measured with FACScan.
4-2. Results
5-1. Assay Method
Biodynes EMPP was allowed to act on epidermal cells (HaCaT cells), and the amount of integrin on the surface of cells was measured with FACScan in the same manner as in Test Example 4. Anti-human integrin α2, anti-human integrin α3, anti-human integrin α6, anti-human integrin α2β1, anti-human integrin β1, and anti-human integrin β4 were each used after 100-fold dilution as a primary antibody.
5-2. Results
As is apparent from the results, the group to which Biodynes EMPP had been added could enhance integrin production in both fibroblasts and epidermal cells.
Hamamelis extract
Sophora angustifolia extract
Macadamia nut oil
Macademia nut oil
Hamamelis extract
Paeonia lactiflora extract
Macadamia nut oil
Thymus serpyllum extract
Sophora angustifolia extract
Bupleurum extract
Hypericum erectum extract
Rosa roxburghii Tratt extract
Paeonia lactiflora extract
Saxifraga stolonifera extract
The anti-aging compositions, collagen production promoting compositions and the like according to the present invention have excellent collagen production promotion effect, and are safe. Therefore, the collagen production promoting composition and the collagen production promotion methods according to the present invention can promote collagen production to maintain the collagen level and, in addition, can promote collagen gel contraction and integrin production in fibroblast or epidermal cell to effectively prevent or ameliorate wrinkle and sagging of the skin.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2004-068413 | Mar 2004 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP04/07038 | 5/18/2004 | WO | 8/8/2006 |
