Patent Description:
Skin can be roughly divided into the epidermis which contacts the outer world, the dermis located beneath the epidermis and tightly adhered to the epidermis, and the subcutaneous adipose tissue further beneath the dermis and located between the dermis and muscles. The dermis located between the epidermis and the subcutaneous adipose tissue is constituted by the papillary layer and the reticular dermis layer, and the fibrous connective tissue. Collagen accounts for about <NUM>% of the dermis, and besides the dermis is constituted by extracellular matrix components such as elastic fiber (elastin) and hyaluronan. Collagen, elastin, hyaluronan, and the like constituting the dermis are produced by the dermal fibroblast present in the reticular dermis layer.

These fibers are destroyed and become old due to UV ray, dryness, age advancement, and the like thereby causing their elasticity to reduce and also causing the production function of extracellular matrix components to reduce, which are generally considered as the causes of the skin aging such as the reduction of skin firmness and elasticity, wrinkles and sagging. Thus, the decrease of extracellular matrix components in the dermis, particularly collagen and hyaluronan, is involved with the aging process such as the reduction of skin elasticity and wrinkle formation.

Consequently, it is considered that the skin aging can be prevented or improved when the function of dermal fibroblast is activated and the production of extracellular matrix components described above is increased. In particular, wrinkles formed on the face have a significant impact on the person's appearance as an aging marker. For this reason, demands on cosmetic products with a wrinkle prevention or improvement effect (anti-aging and/or anti-wrinkle cosmetic products) have been increasing as a countermeasure to keep one's youth (non-patent document <NUM>).

Here, it has been reported that deoxyribonucleic acid (DNA) used as a safe and effective functional food has an improvement effect on skin conditions (moisture level, sebum level, sulcus cutis density, wrinkle, stain, freckle, and the like) (patent documents <NUM>, <NUM>). In addition, it has been reported cosmetic products for beautifying skin (diminishing of wrinkles, roughness) comprising a hydrolysed nucleo-protein / DNA extract from salmon milt. In particular as regards the nucleic acid fraction, this hydrolysed DNA fraction makes up <NUM>,<NUM>% deoxyoligonucleotide of MW <NUM>-<NUM> (patent document <NUM>).

Additionally, it has been reported that an extract of soybean seeds, germs (embryos) or sprouts abundant with polyamines has a promoting action on the production of extracellular matrix components such as elastin, collagen, and hyaluronan which support the skin structure (patent document <NUM>). Similar report was made in patent document <NUM>.

However, there has been no report on the interaction between these deoxyribonucleic acid (DNA) and the soybean extract as far as the present inventors know.

The skin aging prevention or improvement can be expected as described above when the function of dermal fibroblast is activated and the production of extracellular matrix components, particularly collagen and hyaluronan, is increased. Accordingly, an object of the present invention is to provide a skin anti-aging agent, an agent for regulating expression of a skin anti-aging related gene, and a cosmetic product comprising the anti-aging agent or the agent for regulating expression of anti-aging related gene which have better activation effects on the dermal fibroblast and are very safe and can be used reliably for a long term.

The present inventors have conducted extensive studies on the above object and found that a special low molecular DNA and a soybean extract, which are safe even when taken for a long term, have synergistic activation actions on the dermal fibroblast, i.e., synergistic effects (synergy) on a cell proliferation action, a collagen production promoting action, and a hyaluronan production promoting action, synergistically promote hyaluronan synthesis-related gene expression, and also decrease hyaluronan breakdown-related gene expression. The present invention has been accomplished based on these findings. More specifically, the present invention is as follows.

According to the present invention, a special low molecular DNA and a soybean extract, which are the active ingredients, have synergistic activation actions on the dermal fibroblast and are hence expected to prevent or improve the skin aging such as the reduction of skin firmness and elasticity, wrinkles and Thereby, the special low molecular DNA and soybean extract are as herein defined above and in the appended claims. Both these active ingredients compose the skin anti-ageing agent as mentioned herein.

[<FIG>] A figure showing analysis results by Gel Permeation Chromatography (GPC) on the hydrolyzed DNA-Na used in Examples. The figure is plotted with retention time (min) as the horizontal axis and absorbance at a UV region (wavelength of <NUM>) as the vertical axis.

A mode of carrying out the present invention will be described below, but the following description is only an example of the modes of carrying out the present invention, and the present invention is in no way limited to the following contents described. Note that when the expression "to" is used in the present Description, such an expression should be used to include the numerical values or physical property values before and after the expression. Additionally, a content (%) of active ingredients is weight percent (wt%) unless otherwise specified.

The skin anti-aging agent of the present invention comprises: a special low molecular DNA; and a soybean extract, as active ingredients, wherein the special low molecular DNA is a hydrolysate of DNA extracted from a milt of fish, and contains <NUM> to <NUM>% of fractions having a molecular weight of <NUM>,<NUM> or less;the soybean extract is an extract of at least one selected from the group consisting of soybean seeds, germs, and sprouts; and the activation of the function is at least one action selected from the group consisting of a proliferation-promoting action, a collagen Type I production-promoting action, and a hyaluronan production-promoting action on a dermal fibroblast.

Here, the skin anti-aging means activation of a function of the dermal fibroblast, and the activation of the function means at least one action selected from the group consisting of a proliferation-promoting action, a collagen Type I production-promoting action, and a hyaluronan production-promoting action on a dermal fibroblast.

Additionally, the agent for regulating expression of a skin anti-aging related gene of the present invention comprises: a special low molecular DNA; and a soybean extract, as active ingredients, wherein: the special low molecular DNA is a hydrolysate of DNA extracted from a milt of fish, and contains <NUM> to <NUM>% of fractions having a molecular weight of <NUM>,<NUM> or less;the soybean extract is an extract of at least one selected from the group consisting of soybean seeds, germs, and sprouts; and the regulation of gene expression is promotion of gene expression of either one of or both of Human Hyaluronan Synthase <NUM> gene, and Human Hyaluronan Synthase <NUM> gene of dermal fibroblast, or decrease of gene expression of Human Hyaluronidase <NUM> gene of dermal fibroblast.

Here, in the present invention, the skin anti-aging related genes mean the genes encoding any of the enzymes involved with the synthesis or breakdown of collagen or hyaluronan in the dermal fibroblast described below.

Additionally, the regulation of gene expression means the promotion of expression (increase in expression level) of the synthase genes and/or the expression decrease (decrease in expression level) of breakdown enzyme genes described above. These regulations of skin anti-aging related gene expression preferably mean promotion of expression of at least one gene selected from the group consisting of Human Collagen Type I Alpha <NUM> gene (COL1A1), Human Hyaluronan Synthase <NUM> gene (HAS1), and Human Hyaluronan Synthase <NUM> gene (HAS2), or gene expression decrease of Human Matrix Metallopeptidase <NUM> gene (MMP1) and/or Human Hyaluronidase <NUM> gene (HYAL1).

Further, the above-described activation of a function of a dermal fibroblast may be the above-described gene expression promotion or the above-described gene expression decrease in the dermal fibroblast.

The skin anti-aging agent or the agent for regulating expression of a skin anti-aging related gene of the present invention comprises a special low molecular DNA and a soybean extract as active ingredients, as defined herein, and may further comprise other optional components as needed.

The special low molecular DNA of the present invention is a component that can be prepared by hydrolyzing DNA extracted from the testes (milt) of fish such as salmon, trout, and cod from viewpoints of abundant DNA-contained and effective utilization of wastes from processed marine products.

Extraction and purification of DNA from the fish testes can be carried out by a routine method (for example, in accordance with the description in <CIT>). For example, the fish orchis is roughly crashed, the crashed fish orchis is treated with a protein breakdown enzyme (protease) under the condition in which the DNA is not broken down, and alcohol (methanol, ethanol, isopropyl alcohol, or the like) is added to the enzymatically treated solution to precipitate the DNA in the form of a DNA salt (DNA sodium salt) and collect the precipitate. Alternatively, acid (hydrochloric acid, phosphoric acid, citric acid, or the like) is added to the enzymatically treated solution to precipitate the DNA, and the precipitate is collected, neutralized with sodium hydroxide, and dried to obtain a DNA salt (DNA sodium salt).

The obtained DNA salt is hydrolyzed using a nucleolytic enzyme such as a nuclease to obtain a special low molecular DNA. For the nuclease used for the hydrolysis treatment, for example, a nuclease derived from Penicillium can be used.

Hydrolysis can be carried out by, for example, charging the above DNA salt (DNA sodium salt) as a raw material to warm water adjusted to about <NUM>, after stirring, further heating to <NUM>, and adding a nuclease to cause the reaction. Temperature at the time of hydrolysis treatment is preferably <NUM> to <NUM>, more preferably <NUM>.

A special low molecular DNA in a powder form can be obtained by, for example, freeze-drying the obtained hydrolysate product. The special low molecular DNA by the above technique can be typically obtained in the state of a sodium salt. Note that the salt is not limited to a sodium salt, and may be, for example, a potassium salt or a calcium salt. Additionally, the special low molecular DNA may be a free form instead of a salt.

The special low molecular DNA contains <NUM> to <NUM>% of fractions having a molecular weight of <NUM>,<NUM> or less, more preferably contains <NUM> to <NUM>% of fractions having a molecular weight of <NUM>,<NUM> or less. Note that a molecular weight distribution can be measured by classifying samples based on molecular weight using GPC followed by determining quantities using a UV detector.

The soybean extract of the present invention is at least one extract selected from the group consisting of soybean seeds, germs, and sprouts. The extraction condition is not particularly limited, but a method for obtaining a plant extract comprising polyamines, for example, as described in patent document <NUM>, a method wherein soybean seeds, germs (embryos), or sprouts are crushed in a suitable medium and extracted under an acidic condition is preferred. Note that the germ used as a raw material may be those separated from seeds and collected, and the soybean sprout may be sprout parts collected from soybean seeds germinated under suitable conditions.

Here, polyamines are abundant in the soybean extract. Polyamine is a collective term for linear aliphatic hydrocarbons having two or more primary amino groups, present in various living bodies, and known as a growth factor involved with cell division and protein synthesis.

A polyamine content of the soybean extracts is not particularly limited but is preferably <NUM>% or more, more preferably <NUM>% or more, further preferably <NUM>% or more, and particularly preferably <NUM>% or more. Note that a polyamine content can be measured by high performance liquid chromatography.

Polyamines (putrescine, cadaverine, spermidine, spermine, and the like) are abundant in the soybean extracts, particularly an extract of the soybean sprout part (hereinafter, sometimes referred to as "soybean sprout extract") (patent document <NUM>). When a soybean sprout extract abundant with polyamines is used, effects provided by polyamines can be expected.

A commercial product can be used as the soybean extract. Specifically, examples include soybean sprout extracts (PHYTOPOLYAMINE (registered trademark)-S (product number: SPA-<NUM>) and PHYTOPOLYAMINE-SP (product number: SPA-<NUM>)) manufactured by TOYOBO CO. and a soybean extract material (SOYPOLYA (registered trademark)) manufactured by Combi Corporation.

A content of the active ingredients in the anti-aging agent varies depending on the kind and form of a preparation, purpose of use, and frequency of use, and it is thus difficult to set a fixed content but the content and the content ratio of each component are preferably a content and a content ratio which provide synergistic effects on the activating actions on the dermal fibroblast.

For example, when the anti-aging agent is applied to skin (the anti-aging agent is a cosmetic product), the content is as follows. A content of the special low molecular DNA is preferably <NUM> to <NUM>/mL (about <NUM> to <NUM>%), more preferably <NUM> to <NUM>/mL (about <NUM> to <NUM>%), and further preferably <NUM> to <NUM>/mL (about <NUM> to <NUM>%). Additionally, a content of the soybean extract is preferably <NUM> to <NUM>/mL (<NUM> to <NUM>%), more preferably <NUM> to <NUM>/mL (<NUM> to <NUM>%), and further preferably <NUM> to <NUM>/mL (<NUM> to <NUM>%).

Additionally, the content ratio of the special low molecular DNA and the soybean extract is not particularly limited but is, in terms of weight ratio, preferably the special low molecular DNA : the soybean extract = <NUM>:<NUM> to <NUM>:<NUM>, more preferably <NUM>:<NUM> to <NUM>:<NUM>, and further preferably <NUM>:<NUM> to <NUM>:<NUM>.

Further, the polyamine concentration of the anti-aging agent is determined by the kind of a preparation and product form, purpose of use, frequency of use, and the like and not particularly limited but is typically suitable to be <NUM> to <NUM>, preferably <NUM> to <NUM>, and more preferably <NUM> to <NUM>.

When a content and a content ratio of the special low molecular DNA and the soybean extract are within preferable ranges, it is advantageous in the aspect of providing synergistic effects or significant effects on the activating actions (cell proliferation, collagen production promotion, hyaluronan production promotion, hyaluronan synthase gene expression promotion, hyaluronan breakdown enzyme gene expression decrease) on the dermal fibroblast as specifically described in Examples to be described later.

Here, the synergistic effects on the activating actions on the dermal fibroblast mean that when the special low molecular DNA and the soybean extract are used concurrently to the dermal fibroblast, either a cell proliferation rate, a collagen production rate, or a hyaluronan production rate is greater (significantly greater) than the sum of degrees when each component is used independently, or when the special low molecular DNA and the soybean extract are used concurrently to the dermal fibroblast, either an expression level of the collagen or hyaluronan synthase gene, preferably an expression level of the hyaluronan synthase gene, or an expression decrease level of the collagen or hyaluronan breakdown enzyme gene, preferably an expression decrease level of the hyaluronan breakdown enzyme, is greater (significantly greater) than the sum of degrees when each component is used independently. Additionally, the significant effect means an effect with a significant difference when compared to a negative control.

The anti-aging agent or the agent for regulating expression of anti-aging related gene of the present invention can be manufactured to an intended dosage form by mixing the special low molecular DNA and the soybean extract, and other components as needed by a routine method. Here, examples of the other components include the same components as optional components that the cosmetic product of the present invention to be described later can contain.

The cosmetic product of the present invention comprises the skin anti-aging agent or the agent for regulating expression of anti-aging related gene. Here, "comprise" means that the cosmetic product may comprise a physiologically acceptable carrier and optional components such as concurrently usable other auxiliary components according to an intended product form.

The cosmetic product of the present invention can be provided as, for example, a skin care cosmetic product or a cosmetic product for hair to be used by applying to the scalp and hair. Note that the cosmetic product in the present invention encompasses quasi-drugs in addition to the cosmetic products of the Pharmaceutical Affairs Law.

When the anti-aging agent or the agent for regulating expression of anti-aging related gene of the present invention is provided as a cosmetic product, the preparable dosage form is not particularly limited as long as applicable to skin. Specifically, the anti-aging agent or the agent for regulating expression of anti-aging related gene can be provided as a lotion, a milky lotion, a cream, a gel, a jelly, an essence, a lip balm, a pack, a mask, or the like in the dosage form of a liquid form, an emulsion form, a gel form, a cream form, an ointment form, a foam form, a mist form, an aerosol form, or the like.

The other optional components that these cosmetic products can contain are not particularly limited and additives that can be mixed in typical cosmetic products can be used. Examples of the additive include water, a fat and an oil, a wax, a hydrocarbon, a fatty acid, an alcohol, an ester, a surfactant, a flavor, an astringent, a germicidal and/or antibacterial agent, a whitening agent, a UV absorber, a moisturizer, a cell activator, an antiphlogistic and/or antiallergic agent, an antioxidant, a vitamin and a natural extract. The contents of these other optional components are not particularly limited either and can be suitably selected according to an intended dosage form, and the like.

Additionally, the anti-aging agent or the agent for regulating expression of anti-aging related gene of the present invention can be provided as food or drink to be used via oral intake. The food and drink encompass health food products (for example, a functional food product, a nutritional supplementary product, a health supplementary product, a nutritionally enriched product, a nutritionally balanced food product, and a supplement), foods with health claims (for example, a food for specified health uses, a food with nutrient function claims, and a food with functional claims), foods for special dietary uses (for example, a food for sick people, a formulated milk powder for infants, and a milk powder for pregnant or lactating mothers), and additionally products categorized as a food and a drink with a label for risk reduction, prevention or improvement of diseases or conditions (symptoms) caused by reduced function of the dermal fibroblast.

The other optional components that these foods and drinks can contain are not particularly limited and additives that can be mixed in typical foods and drinks can be used.

Hereinafter, the present invention will be described more specifically in reference to Examples.

Collagen and hyaluronan in the skin are considered to play an important role to the skin firmness, the skin softness and wetness. Accordingly, synergistic effects of test substances (a special low molecular DNA and a soybean extract) were evaluated by the proliferation action on the dermal fibroblast, and the collagen and hyaluronan production promoting actions.

Human newborn-derived dermal fibroblast cell line NB1RGB cell (RIKEN BRC, Japan) was used and cultured in a CO<NUM> incubator (CO<NUM> concentration of <NUM>%, <NUM>).

Eagle's Minimal Essential Medium (EMEM, Wako, Japan) comprising <NUM>% (v/v) Fetal Bovine Serum (FBS, Hyclone, UK) and a <NUM>% (v/v) antifungal agent (Invitrogen, USA) was used.

A hydrolyzed DNA-Na (powder) manufactured by NISSEI BIO CO. This product was prepared, by the method described earlier, by extracting a Na salt of DNA from the orchis (milt) of salmonid fish.

Note that analysis results by Gel Permeation Chromatography (GPC) on the hydrolyzed DNA-Na (manufactured by NISSEI BIO CO. ) used are shown in <FIG>, and the relation between the retention time and the molecular weight is shown in Table <NUM>. A molecular weight reference of <NUM>,<NUM> was based on the retention time of Cytochrome c (MW <NUM>,<NUM>).

According to the above analysis results, fractions by the molecular weight of the hydrolyzed DNA-Na (manufactured by NISSEI BIO CO. ) are as follows.

Fractions having a molecular weight of <NUM>,<NUM> to <NUM>,<NUM> (elution time of <NUM> to less than <NUM>): <NUM>%.

Fractions having a molecular weight of <NUM>,<NUM> or less (elution time of <NUM> and thereafter): <NUM>%.

From the above, the fractions having a molecular weight of <NUM>,<NUM> or less of the hydrolyzed DNA-Na (manufactured by NISSEI BIO CO. ) are <NUM>%.

The test substance (hydrolyzed DNA-Na) adjusted to <NUM>/mL using <NUM> L-ascorbic acid (<NPL>) and <NUM>% FBS-containing EMEM was serially diluted in a common ratio of <NUM> and prepared to the total of <NUM> concentrations (<NUM>/mL, <NUM>/mL) when used (final concentrations were <NUM>/mL, <NUM>/mL).

PHYTOPOLYAMINE (registered trademark)-SP (product number: SPA-<NUM>, mixing ratio: about <NUM>% of a soybean sprout extract, about <NUM>% of citric acid Na) manufactured by TOYOBO CO.

The test substance (SPA-<NUM>) in concentrations of <NUM>/mL, <NUM>/mL, and <NUM>/mL was prepared when used by using <NUM> L-ascorbic acid (<NPL>) and <NUM>% FBS-containing EMEM (final concentrations were <NUM>/mL, <NUM>/mL, and <NUM>/mL).

Two kinds of the test substances described above were used.

The hydrolyzed DNA-Na adjusted to <NUM>/mL using SPA-<NUM>-containing EMEM, which was adjusted to <NUM>/mL using <NUM> L-ascorbic acid (<NPL>) and <NUM>% FBS-containing EMEM, was serially diluted in a common ratio of <NUM> and prepared to the total of <NUM> concentrations (<NUM>/mL, <NUM>/mL) when used [final concentration of SPA-<NUM> was <NUM>/mL and final concentrations of the hydrolyzed DNA-Na were <NUM>/mL, <NUM>/mL].

For the calculation of the cell proliferation, collagen production, and hyaluronan production, an average value of <NUM> wells per treated group was used. Additionally, the test was carried out with one group each of a test substance administered group and an operational control group per plate. The operations relating to the test including the test substance preparations were carried out at room temperature unless otherwise specified.

A <NUM> × <NUM><NUM> cells/<NUM>µL of NB1RGB cells per well were seeded in a <NUM>-well plate (Lot. <NUM>, Corning, USA) and cultured in a CO<NUM> incubator for <NUM> hours. Additionally, <NUM>µL of Phosphate buffer saline (PBS(-), Lot. <NUM>, Nissui, Japan) was added to wells that were not used for the test to prevent drying during the culture.

<NUM> hours later, <NUM>µL of the test substance and the negative control were added to the <NUM>-well plate and cultured for <NUM> hours in the CO<NUM> incubator. For the negative control, <NUM> L-ascorbic acid and <NUM>% FBS-containing EMEM were used.

<NUM> hours later, the culture supernatant was dispensed to a new <NUM>-well plate and freeze-dried (-<NUM>). A collagen level and hyaluronan level in the culture supernatant were measured using ELISA (Enzyme-Linked Immuno Sorbent Assay) described in <NUM>-<NUM> (<NUM>) and (<NUM>) to evaluate the collagen and hyaluronan production promoting actions of the test substance. Additionally, the number of cells in the <NUM>-well plate from which the culture supernatant had been removed was counted by the method described in <NUM>-<NUM> (<NUM>) to evaluate the cell proliferation action of the test substance.

The effects of the test substance on the proliferation of dermal fibroblast were evaluated as follows.

Each well of the <NUM>-well plate from which the medium had been removed was gently washed twice with <NUM>µL of PBS(-) heated to <NUM>. Subsequently, <NUM>µL of a solution of <NUM>-(<NUM>,<NUM>-dimethylthiazol-<NUM>-yl)-<NUM>,<NUM>-diphenyltetrazolium bromide (MTT, <NPL>) in <NUM>/mL per well was added thereto and cultured for <NUM> hours in a CO<NUM> incubator.

After completion of the culture, the MTT solution was removed and the plate was washed with <NUM>µL of PBS(-). <NUM>µL of <NUM>-propanol (<NPL>) comprising <NUM> N hydrochloric acid (<NPL>) was added thereto and allowed to stand for <NUM> hour under a shading condition to solubilize the produced insoluble formazan.

The <NUM>-well plate was shaken for <NUM> seconds at <NUM> rpm to homogeneously disperse the pigment in the well, and then an absorbance at <NUM> (OD<NUM>) was measured using a microplate reader (SPARK(TM) <NUM>, TECAN, Switzerland).

An OD<NUM> of the test substance administered group, with the OD<NUM> of the negative control being <NUM>%, was calculated as a cell proliferation rate (%). The cell proliferation rates (%) of the negative control and the test substance administered group were subjected to a significance test by unpaired two groups test (Student's t-test). All the tests had significance levels of less than <NUM>% on two sides (p < <NUM>, p < <NUM>, p < <NUM>).

The effects of the test substance on the production of collagen Type I, which is said to impart the skin with firmness and elasticity, were evaluated by competitive ELISA as follows.

For the measurement of a collagen level in the culture supernatant, Human Collagen type I ELISA kit (Lot. EC1-E105, ACEL, Japan) was used.

A collagen standard solution was serially diluted using Dilution buffer in a common ratio of <NUM> to prepare the total of <NUM> concentrations. Additionally, the culture supernatant was diluted two-fold with Dilution buffer.

<NUM>µL of a biotin-labeled collagen antibody solution was added to <NUM>µL of a collagen standard solution and the two-fold diluted culture supernatant and mixed by tapping.

The collagen immobilized microtiter plate was washed three times with <NUM>µL of Wash buffer, <NUM>µL of the standard solution containing biotin-labeled collagen antibody and the culture supernatant were added thereto and shaken for <NUM> hour at about <NUM> rpm.

<NUM> hour later, the plate was washed three times with <NUM>µL of Wash buffer, <NUM>µL of a peroxidase-labeled avidin solution was added thereto and shaken for <NUM> hour at about <NUM> rpm. <NUM> hour later, the plate was washed three times with <NUM>µL of Wash buffer, <NUM>µL of a <NUM>,<NUM>',<NUM>,<NUM>'-tetramethylbenzidine substrate solution per well was added thereto and allowed to stand for <NUM> minutes.

Then, <NUM>µL of Stop solution per well was added thereto, the microtiter plate was shaken for <NUM> minute at <NUM> rpm to homogenize the pigment in the well, and subsequently an absorbance at <NUM> (OD<NUM>) was measured using a microplate reader.

A calibration curve was regressed from the OD<NUM> of the collagen standard solution using <NUM>-Parameter logistic model. A value obtained from the regression equation was multiplied by a dilution rate to calculate collagen production levels (pg/mL) of the negative control and the test substance.

A collagen production rate (%) of the test substance, with the collagen production level of the negative control being <NUM>%, was calculated. The collagen production rates (%) of the negative control and the test substance added group were subjected to a significance test by unpaired two groups test (Student's t-test). All the tests had significance levels of less than <NUM>% on two sides (p < <NUM>, p < <NUM>, p < <NUM>).

The effects of the test substance on the production level of hyaluronan, which is said to hold moisture in collagen and elastin, were evaluated by sandwich ELISA as follows.

For the measurement of a hyaluronan level in the culture supernatant, Hyaluronan Quantikine ELISA kit (Lot. DHYAL0, R&D Systems, USA) was used.

A hyaluronan standard solution was serially diluted using Dilution buffer in a common ratio of <NUM> to prepare the total of <NUM> concentrations. Additionally, the culture supernatant was diluted eight-fold with Dilution buffer.

To an aggrecan immobilized microtiter plate, <NUM>µL of a biotin-labeled hyaluronan antibody solution per well was added. Further, <NUM>µL of the diluted culture supernatant was added thereto and shaken for <NUM> hour at about <NUM> rpm.

<NUM> hour later, the plate was washed five times with <NUM>µL of Wash buffer, <NUM>µL of a peroxidase-labeled avidin solution was added thereto and shaken for <NUM> hour at about <NUM> rpm. <NUM> hour later, the plate was washed five times with <NUM>µL of Wash buffer, <NUM>µL of a <NUM>,<NUM>',<NUM>,<NUM>'-tetramethylbenzidine substrate solution per well was added thereto and allowed to stand for <NUM> minutes under a shading condition.

A calibration curve was regressed from the OD<NUM> of the hyaluronan standard solution using <NUM>-Parameter logistic model. A value obtained from the regression equation was multiplied by a dilution rate to calculate hyaluronan production levels (ng/mL) of the negative control and the test substance.

A hyaluronan production rate (%) of the test substance, with the hyaluronan production level of the negative control being <NUM>%, was calculated. The hyaluronan production rates (%) of the negative control and the test substance added group were subjected to a significance test by unpaired two groups test (Student's t-test). All the tests had significance levels of less than <NUM>% on two sides (p < <NUM>, p < <NUM>, p < <NUM>).

The cell proliferation rates ± standard deviation of <NUM>/mL and <NUM>/mL of the test substance (hydrolyzed DNA-Na) were, with the cell proliferation level (OD<NUM>) of the negative control being <NUM>%, <NUM> ± <NUM>% and <NUM> ± <NUM>%, respectively.

The collagen production rates ± standard deviation of <NUM>/mL and <NUM>/mL of the test substance (hydrolyzed DNA-Na) were, with the collagen production level of the negative control being <NUM>%, <NUM> ± <NUM>% (p < <NUM>) and <NUM> ± <NUM>% (p < <NUM>), respectively.

The hyaluronan production rates ± standard deviation of <NUM>/mL and <NUM>/mL of the test substance (hydrolyzed DNA-Na) were, with the hyaluronan production level of the negative control being <NUM>%, <NUM> ± <NUM>% and <NUM> ± <NUM>%, respectively.

The cell proliferation rates ± standard deviation of <NUM>/mL, <NUM>/mL, and <NUM>/mL of the test substance (SPA-<NUM>) were, with the cell proliferation level (OD<NUM>) of the negative control being <NUM>%, <NUM> ± <NUM>% (p < <NUM>), <NUM> ± <NUM>% (p < <NUM>), and <NUM> ± <NUM>% (p < <NUM>), respectively.

The collagen production promotion rates ± standard deviation of <NUM>/mL, <NUM>/mL, and <NUM>/mL of the test substance (SPA-<NUM>) were, with the collagen production promoting action of the negative control being <NUM>%, <NUM> ± <NUM>% (p < <NUM>), <NUM> ± <NUM>% (p < <NUM>), and <NUM> ± <NUM>% (p < <NUM>), respectively.

The hyaluronan production rates ± standard deviation of <NUM>/mL, <NUM>/mL, and <NUM>/mL of the test substance (SPA-<NUM>) were, with the hyaluronan production level of the negative control being <NUM>%, <NUM> ± <NUM>% (p < <NUM>), <NUM> ± <NUM>% (p < <NUM>), and <NUM> ± <NUM>% (p < <NUM>), respectively.

The cell proliferation rates ± standard deviation of <NUM>/mL + <NUM>/mL and <NUM>/mL + <NUM>/mL of the test substance (hydrolyzed DNA-Na + SPA-<NUM>) were, with the cell proliferation level (OD<NUM>) of the negative control being <NUM>%, <NUM> ± <NUM>% (p < <NUM>) and <NUM> ± <NUM>% (p < <NUM>), respectively.

The collagen production rates ± standard deviation of <NUM>/mL + <NUM>/mL and <NUM>/mL + <NUM>/mL of the test substance (hydrolyzed DNA-Na + SPA-<NUM>) were, with the collagen production level of the negative control being <NUM>%, <NUM> ± <NUM>% (p < <NUM>) and <NUM> ± <NUM>% (p < <NUM>), respectively.

The hyaluronan production rates ± standard deviation of <NUM>/mL + <NUM>/mL and <NUM>/mL + <NUM>/mL of the test substance (hydrolyzed DNA-Na + SPA-<NUM>) were, with the hyaluronan production level of the negative control being <NUM>%, <NUM> ± <NUM>% (p < <NUM>) and <NUM> ± <NUM>% (p < <NUM>), respectively.

The above results are shown in Table <NUM>. In Table <NUM>, "DNA-Na" is "hydrolyzed DNA-Na". Table <NUM> reveals that the cell proliferation rates, the collagen production rates, and the hyaluronan production rates when the special low molecular DNA (hydrolyzed DNA-Na) and the soybean extract (SPA-<NUM>) were concurrently used are notably greater than the sum of degrees (rates) when each component was used independently. These results can confirm the synergistic effects of the special low molecular DNA and the soybean extract on the activating actions on the function of the dermal fibroblast (cell proliferation action, collagen production promoting action, and hyaluronan production promoting action).

Collagen and hyaluronan in the skin play an important role to the skin firmness, softness and wetness, and it is known that a decrease in a hyaluronan level in the dermis is particularly involved with the reduction of skin elasticity and wrinkle formation. Accordingly, in the present example, effects of the test substances (special low molecular DNA and soybean extract) on the expression of genes related to anti-aging such as synthesis and breakdown of hyaluronan in the dermal fibroblast were evaluated by the Real-Time PCR method.

The same cells as in <NUM>-<NUM> of Example <NUM> were cultured under the same conditions and used.

Medium (EMEM) having the same composition as in <NUM>-<NUM> of Example <NUM> was used, with the exception that a content of FBS (Fetal Bovine Serum) was changed to <NUM>% (v/v).

The same special low molecular DNA (hydrolyzed DNA-Na) as in <NUM>-<NUM> (<NUM>) of Example <NUM> was used.

The test substance (hydrolyzed DNA-Na) adjusted to <NUM>/mL using <NUM>% FBS-containing EMEM was serially diluted in a common ratio of <NUM> to prepare the total of <NUM> concentrations when used (final concentrations of <NUM>/mL, <NUM>/mL).

The same soybean extract (SPA-<NUM>) as in <NUM>-<NUM> (<NUM>) of Example <NUM> was used.

The test substance (SPA-<NUM>) adjusted to <NUM>/mL using <NUM>% FBS-containing EMEM was serially diluted in a common ration of <NUM> to prepare the total of <NUM> concentrations when used (final concentrations of <NUM>/mL, <NUM>/mL).

Two of the test substances described above were used.

The hydrolyzed DNA-Na adjusted to <NUM>/mL using SPA-<NUM>-containing EMEM adjusted to <NUM>/mL using <NUM>% FBS-containing EMEM was serially diluted in a common ration of <NUM> to prepare the total of <NUM> concentrations when used (final concentration of SPA-<NUM> was <NUM>/mL, final concentrations of hydrolyzed DNA-Na were <NUM>/mL, <NUM>/mL).

The expression analysis of genes encoding the following enzymes was carried out using TaqMan Assay (Applied Biosystems, USA).

Human Glyceraldehyde-<NUM>-PhosphateDehydrogenase (GAPDH, Assay ID. Hs02786624_g1) gene was used as an internal standard gene.

For the analysis of gene expression levels, an average value of <NUM> sets of <NUM>-mm dish (Cat No. <NUM>, Thermo Scientific, USA) per treated group was used. The operations relating to the test including the test substance preparations were carried out at room temperature unless otherwise specified.

A <NUM> × <NUM><NUM> cells/<NUM> of NB1RGB cells were seeded in a <NUM>-mm dish and cultured for <NUM> hours in a CO<NUM> incubator. <NUM> hours later, the medium in the <NUM>-mm dish was removed, the test substance and negative control-containing medium were added thereto and cultured for <NUM> hours in a CO<NUM> incubator.

RNA Extraction and purification were carried out as follows using PureLink(TM) RNA Mini Kit (Cat No. 12183018A, Invitrogen, USA).

After <NUM>-hour culture, the <NUM>-mm dish from which the medium had been removed was washed twice with <NUM> of PBS(-) heated to <NUM>. To this, <NUM>µL of Dithiothreitol-containing Lysis Buffer (<NPL>) was added to dissolve the cells and the lysate was collected. Further, cells in the collected lysate were crushed using Homogenizer (Cat No. <NUM>-<NUM>, Invitrogen, USA).

<NUM>µL of a <NUM>% ethanol (<NPL>) solution was added to the cell-crushed liquid, then the liquid was moved to a silica membrane-based column, which was subsequently centrifuged for <NUM> seconds at <NUM>,<NUM> × g at room temperature, and the filtrate was discarded.

<NUM>µL of guanidine isothiocyanate-containing Wash Buffer and <NUM>µL of ethanol-containing Wash Buffer II were added to the silica membrane to wash, which was subsequently centrifuged for <NUM> seconds at <NUM>,<NUM> × g at room temperature and dried. <NUM>µL of RNase-Free Water was added to the membrane and allowed to stand for <NUM> minute at room temperature, and then the membrane was centrifuged for <NUM> seconds at <NUM>,<NUM> × g at room temperature. This procedure was repeated twice to elute RNA from the membrane.

A part of the eluted RNA was collected separately to a UV permeable <NUM>-well plate (Cat No. <NUM>, Thermo Scientific, USA) and diluted <NUM>-fold using Tris-EDTA Buffer (TE (pH <NUM>), Cat No. <NUM>-<NUM>, NIPPON GENE, Japan) to measure an absorbance at <NUM> (OD<NUM>) using a microplate reader (SPARK(TM) <NUM> TECAN, Switzerland).

RNA concentrations of the negative control and the test substance were calculated using the OD<NUM> by the following formula and diluted using TE Buffer to adjust the RNA concentrations to <NUM>µg/mL.

Reverse transcription of RNA was carried out as follows using SuperScript(TM) IV VILO(TM) Master Mix with ezDNase (Cat No. <NUM>, Invitrogen, USA).

<NUM>µL of <NUM> × ezDNase Buffer, <NUM>µL of ezDNase enzyme, <NUM>µL of Nuclease-free Water, and <NUM>µL of <NUM>µg/mL RNA per well were added to an <NUM>-tube strip (AB1182, Thermo Scientific, USA) and incubated for <NUM> minutes at <NUM>. <NUM> minutes later, <NUM>µL of SuperScript(TM) IV VILO(TM) Master Mix and <NUM>µL of Nuclease-free Water per well were added to the <NUM>-tube strip and heated for <NUM> minutes at <NUM>, <NUM> minutes at <NUM>, and <NUM> minutes at <NUM> to synthesize cDNA using a Real-Time PCR (QuantStudio(TM)<NUM>, Applied Biosystems, USA).

<NUM>µL of TaqMan(TM) Fast Advanced Master Mix (Cat No. <NUM>, Applied Biosystems, USA), <NUM>µL of TaqMan Gene Expressior, <NUM>µL of UltraPure(TM) Distilled Water (Invitrogen, Cat No. <NUM>-<NUM>, USA), and <NUM>µL of cDNA per well were added to a PCR plate (Cat No. N8010560, Thermo Scientific, USA) and the plate was hermetically sealed using a plate seal (Cat No. <NUM>, Thermo Scientific, USA).

The solution was spun down using a plate centrifuge, foams were removed, and then Real-Time qPCR was carried out using a Real-Time PCR system to calculate a Threshold Cycle (Ct) value, which is the number of cycles at which a fluorescence signal of each gene of the negative control and the test substance crosses any threshold. A Ct value was corrected to be a ΔCt value using the internal standard gene. The ΔCt value was corrected to be a ΔΔCt value using an average of the ΔCt values of the negative controls. Assuming that a difference of the detection per cycle by the ΔΔCt method is two times the volume in difference, a gene expression level of the test substance was analyzed by assigning to <NUM>-ΔΔCt when the gene expression level of the negative control is <NUM>. Gene expression levels of the negative control and the test substance added group were subjected to a significance test by paired two groups test (paired t-test). All the tests had significance levels of less than <NUM>% on two sides (p < <NUM>, p < <NUM>, p < <NUM>).

The HAS1 expression levels ± standard deviation of <NUM>/mL and <NUM>/mL of the test substance (hydrolyzed DNA-Na) were, with the HAS1 expression level of the negative control being <NUM>, <NUM> ± <NUM> and <NUM> ± <NUM>, respectively.

The HAS2 expression levels ± standard deviation of <NUM>/mL and <NUM>/mL of the test substance (hydrolyzed DNA-Na) were, with the HAS2 expression level of the negative control being <NUM>, <NUM> ± <NUM> (p < <NUM>) and <NUM> ± <NUM> (p < <NUM>), respectively.

The HYAL1 expression levels ± standard deviation of <NUM>/mL and <NUM>/mL of the test substance (hydrolyzed DNA-Na) were, with the HYAL1 expression level of the negative control being <NUM>, <NUM> ± <NUM> and <NUM> ± <NUM>, respectively.

The HAS1 expression levels ± standard deviation of <NUM>/mL and <NUM>/mL of the test substance (SPA-<NUM>) were, with the HAS1 expression level of the negative control being <NUM>, <NUM> ± <NUM> and <NUM> ± <NUM>, respectively.

The HAS2 expression levels ± standard deviation of <NUM>/mL and <NUM>/mL of the test substance (SPA-<NUM>) were, with the HAS2 expression level of the negative control being <NUM>, <NUM> ± <NUM> (p < <NUM>) and <NUM> ± <NUM> (p < <NUM>), respectively.

The HYAL1 expression levels ± standard deviation of <NUM>/mL and <NUM>/mL of the test substance (SPA-<NUM>) were, with the HYAL1 expression level of the negative control being <NUM>, <NUM> ± <NUM> and <NUM> ± <NUM>, respectively.

The HAS1 expression levels ± standard deviation of <NUM>/mL + <NUM>/mL and <NUM>/mL + <NUM>/mL of the test substance (hydrolyzed DNA-Na + SPA-<NUM>) were, with the HAS1 expression level of the negative control being <NUM>, <NUM> ± <NUM> and <NUM> ± <NUM> (p < <NUM>), respectively.

The (HAS2) expression levels ± standard deviation of <NUM>/mL + <NUM>/mL and <NUM>/mL + <NUM>/mL of the test substance (hydrolyzed DNA-Na + SPA-<NUM>) were, with the (HAS2) expression level of the negative control being <NUM>, <NUM> ± <NUM> (p < <NUM>) and <NUM> ± <NUM> (p < <NUM>), respectively.

The (HYAL1) expression levels ± standard deviation of <NUM>/mL + <NUM>/mL and <NUM>/mL + <NUM>/mL of the test substance (hydrolyzed DNA-Na + SPA-<NUM>) were, with the (HYAL1) expression level of the negative control being <NUM>, <NUM> ± <NUM> (p < <NUM>) and <NUM> ± <NUM>, respectively.

The results described above are shown in Table <NUM>. In Table <NUM>, "DNA-Na" is "hydrolyzed DNA-Na". Table <NUM> reveals that when the special low molecular DNA (hydrolyzed DNA-Na) and the soybean extract (SPA-<NUM>) were used concurrently, the expression level of Human Hyaluronan Synthase-<NUM> gene (HAS1) synergistically increases and the expression level of Human Hyaluronan Synthase-<NUM> gene (HAS2) significantly increases compared to those of the negative control. Further, it is revealed that the expression level of Human Hyaluronidase <NUM> gene (HYAL1) synergistically decreases. These results can confirm that when the special low molecular DNA (hydrolyzed DNA-Na) and the soybean extract (SPA-<NUM>) are used concurrently, the promotion of the expression of genes related to hyaluronan synthesis and the decrease in the expression of genes related to hyaluronan breakdown occur simultaneously.

The above results suggest that the synergistic production increases of collagen and hyaluronan when the special low molecular DNA (hydrolyzed DNA-Na) and the soybean extract (SPA-<NUM>) were used concurrently shown in Example <NUM> is caused by the simultaneous occurrence of the promotion of the expression of the genes related to synthesis of collagen and hyaluronan and the decrease in the expression of the gene related to breakdown thereof.

Claim 1:
Use of a special low molecular DNA and a soybean extract as active ingredients for a cosmetic product in the activation of a function of a dermal fibroblast, wherein
the special low molecular DNA is a hydrolysate of DNA extracted from a milt of fish, and contains <NUM> to <NUM>% of fractions having a molecular weight of <NUM>,<NUM> or less;
the soybean extract is an extract of at least one selected from the group consisting of soybean seeds, germs, and sprouts; and
the activation of the function is at least one action selected from the group consisting of a proliferation-promoting action, a collagen Type I production-promoting action, and a hyaluronan production-promoting action on a dermal fibroblast.