JP5123092B2 - Topical skin preparation - Google Patents

Description

The present invention relates to an external preparation for the growth promotion of stem cells present in the skin, which is characterized by containing an extract of seeds of Prunus avium L. belonging to the genus Rosaceae. An external preparation for promoting differentiation of stem cells existing in the skin into fibroblasts, comprising a seed extract of purple wheat (scientific name: Hordeum vulgare L.) belonging to the genus (Scientific name: Starcaria scaphigera WALL) seed extract characterized by containing an extract for promoting differentiation induction of stem cells existing in the skin into keratinocytes, and topical use for skin containing these It relates to the agent.

In the case of vertebrate, particularly mammalian tissue, when cell or organ damage occurs due to injury or disease, or aging, the regeneration system works to try to recover the damage of the cell or organ. Stem cells provided in the tissue play a major role in this effect. Stem cells have the pluripotency to differentiate into all cells and organs, and this property is thought to lead to recovery by compensating for damaged parts of cells and tissues. Expectations are gathered for regenerative medicine, which is the next generation of medicine using such stem cells.

The most advanced tissue in stem cell research in mammals is the bone marrow. Bone marrow contains living hematopoietic stem cells, which have been shown to be the source of all blood cell regeneration. Furthermore, it has been reported that the bone marrow contains stem cells that can be differentiated into other organs (eg, bone, cartilage, muscle, fat, etc.) in addition to hematopoietic stem cells (see Non-Patent Document 1).
Furthermore, in recent years, it has been clarified that stem cells exist in all organs / tissues such as liver, pancreas, fat, skin, etc. in addition to bone marrow, and are responsible for the regeneration and homeostasis of each organ / tissue. It has been understood (see Non-Patent Documents 2 to 5).
Pittenger M.M. F. , Et al. , Science, 1999, 284, 143-147. Goodell M.M. F. , Et al. , Nat. Med. , 1997, 3, 1337-1345. Zulewski H. , Et al. , Diabetes, 2001, 50, 521-533 Suzuki A. , Et al. , Hepatology, 2000, 32, 1230-1239 Zuk P.M. A. , Et al. , Tissue Engineering, 2001, 7, 211-228.

Among organs in which stem cells are present, skin is an organ that is routinely exposed to stimuli such as ultraviolet rays, and is significantly damaged.

Stem cells in the skin are considered to be present in the epidermis and dermis (see Non-patent Documents 6 and 7), and they exhibit pluripotency and can be differentiated into skin cells ( Non-patent document 8).
Bickenbach Jr. , Et al. , J. et al. Invest. Dermatol. , 1987, 88, 42-46. Chen FG. , Et al. , J. et al. Cell Sci. , 2007, 120, 2875-2883. Fuchs E.M. , Et al. Dev. Cell, 2001, Jul. 1 (1), 13-25, Review.

However, at present, the usefulness of stem cells and localization to the skin has been shown, but examination of materials that can restore skin damage by proliferating stem cells and inducing differentiation into epidermal and dermal cells. Has not been made.

Further, for the cherry cherry seeds, purple wheat seeds and hantaikai seeds used in the present invention, external skin preparations used alone are known (see Patent Documents 1 to 3), but the effect can be obtained by using them in combination. The enhancements are not being considered.
JP 11-312010 A JP2008-143827 JP-A-2005-194239

In view of such circumstances, the present invention provides a material having an effect of proliferating stem cells in the skin and differentiating into cells of the epidermis and dermis, containing them, improving rough skin and wrinkles, and comprehensively taking damage to the skin An object of the present invention is to provide an external preparation for skin that can be improved in an improved manner.

As a result of intensive studies aimed at solving the above problems, the present inventors have found that the excellent proliferation promoting effect of stem cells existing in the skin of the extract of the cherry tree seed belonging to the Rosaceae genus Rosa, The effect of promoting the induction of differentiation of stem cells present in the skin into excellent fibroblasts and the effect of promoting the induction of differentiation of stem cells present in the skin of keratinocytes into keratinocytes, Furthermore, the effect of improving rough skin and wrinkles was confirmed in the external preparation for skin containing them, and the present invention was completed.

That is, the present invention is as follows.
(1) An external preparation for promoting proliferation of stem cells present in the skin, comprising an extract of cherry cherry seeds.
(2) An external preparation for promoting differentiation induction of stem cells present in the skin into fibroblasts, comprising an extract of purple wheat seeds.
(3) An external preparation for promoting differentiation induction of stem cells existing in the skin into keratinocytes, characterized by containing an extract of hantaikai seeds.
(4) A skin external preparation characterized by containing these.

Hereinafter, the present invention will be described in more detail.

The cherry tree cherry used in the present invention is also called sugar beet, and its scientific name is Prunus avium L. It is. The main varieties include edible varieties such as American Cherry, Nishiki Sato, and Napoleon. Purple barley refers to barley with purple straw, berries, stems or leaves, and the scientific name is Hordeum vulgare L. It is. For example, barley varieties include OUC321, CI158, CI244 and the like. Hanta Taikai is a deciduous tree belonging to the family Aogiri, and its scientific name is Sterculia scaphigera WALL.

In preparing the extract from the plant, it is possible to use the plant body containing the seed as it is, and a portion selected from a flower, a leaf, a stem, a root, a bark or a fruit may be mixed and used. More preferably, only seeds are used. Moreover, a plant body may be used for extraction as it is, and you may extract after processing, such as drying, grinding | pulverization, and shredding.

Examples of the solvent to be extracted include water, lower alcohols (methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, etc.), liquid polyhydric alcohols (1,3-butylene glycol, propylene glycol). , Glycerin, etc.), ketones (acetone, methyl ethyl ketone, etc.), acetonitrile, esters (ethyl acetate, butyl acetate, etc.), hydrocarbons (hexane, heptane, liquid paraffin, etc.), ethers (ethyl ether, tetrahydrofuran, propyl ether) Etc.). Preferably, water, lower alcohol and liquid polyhydric alcohol are preferable, and water is particularly preferable. These solvents may be used alone or in combination of two or more. The extraction method is not particularly limited, but extraction by heating is preferable. Furthermore, the pH may be adjusted by adding an acid or alkali to the extraction solvent.

Each seed extract may be used as it is, or may be used after treatment such as concentration, dilution, filtration, decolorization or deodorization with activated carbon or the like, if necessary. In particular, the extracted solution is preferably subjected to treatment such as concentration to dryness, spray drying, freeze drying, etc., and used as a dried product.

The component content when each seed extract is used in the form of a solution is not particularly limited, but is preferably 0.00001 to 10% by weight as a dry product, and at a concentration of 0.0001 to 1% by weight. Most preferably it is used. If it is less than 0.00001% by weight, the effects of the present invention may not be sufficiently exerted.

Moreover, the skin external preparation of this invention can mix | blend suitably the component used for a normal pharmaceutical, a quasi-drug, cosmetics, etc. as needed within the range which does not impair an effect. For example, fats and oils, waxes, hydrocarbons, fatty acids, alcohols, esters, surfactants, pH adjusters, preservatives, moisturizers, powders, UV absorbers, thickeners, antioxidants, Components such as a whitening agent and a chelating agent can be blended.

The use of the external preparation for skin of the present invention is not particularly limited, but it is used for ointments, emulsions, gels, lotions, creams, poultices, aerosols, pastes, plasters, cleaning agents, etc. Can do. Moreover, the skin external preparation of this invention contains a pharmaceutical, a quasi-drug, and cosmetics.

In order to describe the present invention in detail, examples will be given, but the present invention is not limited to these examples.

Below, the manufacture example of the solvent extract using a cherry tree (Napoleon) seed, a purple wheat seed, and a Hantai Kai seed is shown.

Production Example 1 Hot water extract of Japanese cherry (Napoleon) seeds 500 mL of purified water was added to 25 g of pulverized Japanese cherry seeds, extracted at 95-100 ° C. for 2 hours, filtered, and the filtrate was concentrated. Lyophilized to obtain 2.0 g of a hot water extract of cherry cherry seeds.

Production Example 2 50% ethanol extract of Japanese cherry (Napoleon) seeds 500 mL of 50 (v / v)% ethanol was added to 25 g of pulverized Japanese cherry seeds, extracted at room temperature for 5 days, filtered, and the filtrate. Was concentrated to dryness to obtain 1.0 g of a 50% ethanol extract of Japanese cherry seeds.

Production Example 3 Ethanol extract of Japanese cherry (Napoleon) seeds 1 L of ethanol was added to 100 g of pulverized Japanese cherry seeds, extracted at room temperature for 7 days, filtered, and the filtrate was concentrated to dryness. 3.3 g of ethanol extract of cherry seeds was obtained.

Manufacture example 4 Hot water extract of purple wheat seeds 500 mL of purified water was added to 50 g of pulverized purple wheat seeds, extracted at 95-100 ° C. for 1 hour, filtered, and the filtrate was concentrated to dryness. 9.2 g of hot water extract of seeds was obtained.

Production Example 5 50% Ethanol Extract of Purple Wheat Seed 500 g of 50 (v / v)% ethanol was added to 50 g of pulverized purple wheat seed, extracted at room temperature for 5 days, filtered, and the filtrate was concentrated to dryness. Thus, 3.8 g of a 50% ethanol extract of purple wheat seeds was obtained.

Production Example 6 Purple Wheat Seed Ethanol Extract 500 g of ethanol was added to 50 g of pulverized purple barley seed, extracted at room temperature for 5 days, filtered, and the filtrate was concentrated to dryness to obtain an ethanol extract of purple wheat seed. 1.2g was obtained.

Production Example 7 Hot water extract of hantaikai seeds 800 ml of purified water was added to 20 g of dried hantaikai seeds, extracted at 95-100 ° C. for 2 hours, filtered, and the filtrate was concentrated and freeze-dried. 3.4 g of water extract was obtained.

Production Example 8 50% ethanol extract of hantaikai seeds 400 ml of 50 (v / v)% ethanol was added to 20 g of dried kaitai seeds, extracted at room temperature for 7 days, filtered, and the filtrate was concentrated to dryness. 3.7 g of a 50% ethanol extract of hantaikai was obtained.

Production Example 9 Hantaikai Seed Ethanol Extract 1 l of ethanol was added to 100 g of dried huntaikai seeds, extracted at room temperature for 7 days, filtered, the filtrate was concentrated to dryness, and 6.7 g of Hantaikai ethanol extract was obtained. Obtained.

Next, although the formulation example of the external preparation for skin using the seed extract used for this invention is given, this invention is not limited to this.

Formulation Example 1 Ointment Formulation Formulation 1. Of cherry seeds
50% ethanol extract (Production Example 2) 0.5 part 50% ethanol extract of purple wheat seeds (Production Example 5) 0.5
3. 50% ethanol extract of hantaikai seeds (Production Example 8) 0.5
4). Polyoxyethylene cetyl ether (30E.O.) 2.0
5). Glycerol monostearate 10.0
6). Liquid paraffin 5.0
7. Cetanol 6.0
8). Methyl paraoxybenzoate 0.1
9. Propylene glycol 10.0
10. [Manufacturing method] Components 4-7 are heated and dissolved and mixed with purified water to 100, and kept at 70 ° C to obtain an oil phase. Ingredients 1 to 3 and 8 to 10 are dissolved by heating and mixed, and kept at 75 ° C. to obtain an aqueous phase. The aqueous phase is added to the oil phase to emulsify, and the mixture is cooled to 30 ° C. with stirring to obtain a product.

Comparative Examples 1-4 Conventional Ointment In Formulation Example 1, Comparative Example 1 containing only 50% ethanol extract of Japanese cherry seeds Comparative Example Comparative Example containing only 50% ethanol extract of purple wheat seeds 2. A sample containing 50% ethanol extract of hantaikai seeds was used as Comparative Example 3, and a sample obtained by removing all of them was used as Comparative Example 4.

Formulation Example 2 Emulsion formulation Hot water extract of Japanese cherry seed (Production Example 1) 0.001 part Hot water extract of purple wheat seed (Production Example 4) 0.001
3. Hot water extract of hantaikai seeds (Production Example 7) 0.001
4). Squalane 5.0
5). Olive oil 5.0
6). Jojoba oil 5.0
7. Cetanol 1.5
8). Glycerol monostearate 2.0
9. Polyoxyethylene cetyl ether (20E.O.) 3.0
10. Polyoxyethylene sorbitan monooleate (20E.O.) 2.0
11. Fragrance 0.1
12 Propylene glycol 1.0
13. Glycerin 2.0
14 Methyl paraoxybenzoate 0.2
15. Make the total volume 100 with purified water
[Manufacturing method] Components 4 to 10 are heated and dissolved, mixed, and kept at 70 ° C to obtain an oil phase. Ingredients 1 to 3 and 12 to 15 are dissolved by heating and mixed, and kept at 75 ° C. to obtain an aqueous phase. The aqueous phase is added to the oil phase to emulsify, and the mixture is cooled while stirring. The component 11 is added at 45 ° C., and further cooled to 30 ° C. to obtain a product.

Formulation Example 3 Gel formulation Formulation amount 1. 1.0 parts of ethanol extract of cherry tree seeds (Production Example 3) Ethanol extract of purple wheat seeds (Production Example 6) 1.0
3. Ethanol extract of hantaikai seed extract (Production Example 9) 1.0
4). Ethanol 5.0
5). Methyl paraoxybenzoate 0.1
6). Polyoxyethylene hydrogenated castor oil (60 EO) 0.1
7. Perfume
8.1,3-Butylene glycol 5.0
9. Glycerin 5.0
10. Xanthan gum 0.1
11. Carboxyvinyl polymer 0.2
12 Potassium hydroxide 0.2
13. [Production method] Components 4 to 7 and components 1 to 3 and 8 to 13 are uniformly dissolved in purified water to a total amount of 100, and both are mixed to obtain a product.

Formulation Example 4 Lotion preparation formulation Hot water extract of cherry tree seeds (Production Example 1) 0.1 part Hot water extract of purple wheat seeds (Production Example 4) 0.1
3. Hot water extract of hantaikai seed (Production Example 7) 0.1
4.1,3-Butylene glycol 8.0
5). Glycerin 2.0
6). Xanthan gum 0.02
7. Citric acid 0.01
8). Sodium citrate 0.1
9. Ethanol 5.0
10. Methyl paraoxybenzoate 0.1
11. Polyoxyethylene hydrogenated castor oil (40E.O.) 0.1
12 Perfume
13. [Manufacturing method] Components 1 to 8 and 13 and components 9 to 12 are uniformly dissolved in purified water, and the two are mixed and filtered to obtain a product.

Formulation Example 5 Cream formulation Formulation amount 1. Of cherry seeds
50% ethanol extract (Production Example 2) 0.05 part 50% ethanol extract of purple wheat seeds (Production Example 5) 0.05
3. 50% ethanol extract of hantaikai seeds (Production Example 8) 0.05
4). Squalane 5.5
5). Olive oil 3.0
6). Stearic acid 2.0
7. Beeswax 2.0
8). Octyldodecyl myristate 3.5
9. Polyoxyethylene cetyl ether (20E.O.) 3.0
10. Behenyl alcohol 1.5
11. Glycerol monostearate2.5
12 Fragrance 0.1
13. Methyl paraoxybenzoate 0.2
14 Ethyl paraoxybenzoate 0.05
15.1,3-Butylene glycol 8.5
16. [Manufacturing method] Components 4 to 11 are heated and dissolved and mixed with purified water to a total amount of 100, and kept at 70 ° C to obtain an oil phase. Ingredients 1 to 3 and 13 to 16 are dissolved by heating and mixed, and kept at 75 ° C. to obtain an aqueous phase. The aqueous phase is added to the oil phase to emulsify, and the mixture is cooled while stirring. The component 12 is added at 45 ° C., and further cooled to 30 ° C. to obtain a product.

Below, the experimental example and the result of the proliferation promotion effect of a stem cell using the extract of the cherry tree seeds of the manufacture examples 1-3 shown in Example 1 are shown.

Experimental Example 1 Evaluation of Stem Cell Proliferation Promoting Effect Using human stem cell culture solution (manufactured by TOYOBO), 1 × 10 ⁵ human somatic stem cells (DS Pharma Biomedical) were seeded in a 6 cm dish, and Extracts (Production Examples 1 to 3) were added to a final concentration of 0.001%, and the culture was continued for 3 days. Next, the cells were washed three times with PBS (−) and then collected with a rubber policeman, and the number of each cell was counted.
Calculation of increase / decrease (%) in the number of cells when adding extracts of cherry cherry seeds (Production Examples 1 to 3) when the total number of cells when no extract is added is a control and the control is 100 (%) Then, the cell proliferation promoting effect of stem cells was evaluated.
In this experimental example, a stem cell factor (SCF: stem cell growth factor) 10 ng / mL (manufactured by Pepro Tech), which is generally used as a proliferation promoting factor for stem cells, was set as a positive control and used as a comparison target. .

The test results are shown in Table 1. As a result, all the extracts of cherry cherry seeds (Production Examples 1 to 3) were found to have a remarkable effect of promoting stem cell proliferation. From the above, the extremely excellent stem cell growth promoting effect of the cherry tree seed extract was clarified, and the growth promoting effect of the cherry tree seed extract was confirmed. The proliferation effect of these cherry cherry seeds was significantly higher than that of SCF, which is currently generally used as a stem cell proliferation promoting factor.
In addition to the stem cells used in this experimental example, a similar test was performed on embryonic stem cells (ES cells), and a remarkable effect of promoting cell proliferation of stem cells was observed.

Below, the differentiation induction promotion effect to the fibroblast of the stem cell using the extract of the purple wheat seed of the manufacture examples 4-6 shown in Example 1 is evaluated, The stem cell to a fibroblast of this extract is evaluated. The experiment example which confirms the performance as a differentiation-induction promoter and the result are shown.

Experimental Example 2 Evaluation of the effect of promoting differentiation induction of stem cells into fibroblasts Seed 5x10 ³ human somatic stem cells (DS Pharma Biomedical) cultured in human stem cell culture medium (manufactured by TOYOBO) on a 96-well plate. Then, purple wheat seed extract (Production Examples 4 to 6) was added to a final concentration of 0.001%, and the cultivation was continued for 7 days. Seven days later, the differentiation induction efficiency from stem cells to fibroblasts was evaluated. As an index of the effect of inducing differentiation into fibroblasts, type 1 collagen gene expression (the higher the expression, the more differentiated into fibroblasts) was measured. Specifically, mRNA was extracted from the cells using TaqMan GENE Expression Cells-to-Ct ^™ kit (Applied Biosystems), and the gene expression of type 1 collagen was measured by real-time PCR.
The increase / decrease (%) in collagen expression when the extract of purple wheat seeds (Production Examples 4 to 6) was added when the expression of the collagen gene when no extract was added was taken as a control (100%) was calculated from the stem cells. The effect of promoting differentiation induction into fibroblasts was evaluated.
In this experimental example, a basic-fibroblast growth factor (bFGF: fibroblast growth factor) 10 ng / mL (manufactured by Pepro Tech), which is generally used as a factor for inducing differentiation of fibroblasts, is set as a positive control. And it was set as a comparison target.

The test results are shown in Table 2. As a result, all the extracts of purple wheat seeds (Production Examples 4 to 6) showed a remarkable effect of promoting differentiation induction into fibroblasts. From the above, the effect of promoting differentiation induction into fibroblasts, which was extremely excellent for the extract of purple wheat seeds, was clarified, and the effect of the extract of purple wheat seeds as a differentiation induction promoter was confirmed. The effect of promoting differentiation induction of these purple wheat seed extracts was significantly higher than that of bFGF, which is currently generally used as a fibroblast differentiation inducer.
In addition to the stem cells used in the present experimental example, embryonic stem cells (ES cells) and other undifferentiated cells (somatic stem cells) were tested, and similar effects were confirmed.

Hereinafter, the effect of promoting the induction of differentiation of stem cells into keratinocytes using the seed extract of Hantaikai of Production Examples 7 to 9 shown in Example 1 is evaluated, and differentiation induction of the extract into keratinocytes is induced. The effect as an accelerator was confirmed.

Experimental Example 3 Evaluation of stem cell keratinocyte differentiation induction promoting effect MCDB153 culture solution (manufactured by Sigma), ITS-X supplement (diluted 100 times, manufactured by GIBCO), hydrocortisone (0.4 ng / mL, Sigma) 1 × 10 ⁶ stem cells isolated from mouse epidermal tissue using a medium prepared by adding penicillin (100 unit / mL, Sigma) and streptomycin (100 μg / mL, Sigma). After seeding, each extract (Production Examples 7 to 9) was added to a final concentration of 0.001%, and the culture was continued for 3 days. Next, the cells were washed three times with PBS (−), collected with a rubber policeman, counted with a hemocytometer, and then extracted with CelLytic (Sigma) to extract keratinocytes. The differentiation state was measured according to the report of Toyooka et al. (See Non-Patent Document 9).
That is, the expression level of involucrin protein showing the differentiation state into keratinocytes was quantitatively analyzed by Western blotting, and the differentiation induction promoting effect from stem cells to keratinocytes was evaluated using this as an index. In this case, the amount of involucrin protein expressed in cells cultured without adding the seed extract of Hantaikai was used as a control (100%), and the involucrin when each extract (Production Examples 7 to 9) was added and cultured. The expression level of the protein was calculated as a relative value (%), and compared with the control, the effect of promoting differentiation induction into keratinocytes (%) was evaluated. In this experimental example, Epidermal Growth Factor (EGF: keratinocyte growth factor, see Non-Patent Document 10) 10 ng / mL (manufactured by Pepro Tech), which is generally used as a differentiation inducing factor for keratinocytes, is used. It was set as a positive control and used for comparison.
Yayoi Toyooka, Extraordinary issue of Molecular Medicine, Regenerative Medicine, 2003, 106-115 Rheinwald. JD et al. , Nature, 1977, 265, 421-424.

These test results are shown in Table 3. As a result, the remarkable effect of promoting the differentiation of stem cells into keratinocytes was observed in all the extracts of Hankaikai seeds (Production Examples 7 to 9). From the above, the effect of promoting the induction of differentiation of hantaikai seeds into keratinocytes was clarified, and the effect of the extract of hantaikai seeds as a differentiation induction promoter was confirmed. The effect of promoting the induction of differentiation of these hunting kai seed extracts was significantly higher than that of EGF, which is generally used as a differentiation-inducing factor for keratinocytes.
In addition to the stem cells used in the present experimental example, embryonic stem cells (ES cells) and other undifferentiated cells (somatic stem cells) were tested, and similar effects were confirmed.

Experimental Example 4 Clinical Test A 25-month clinical test was conducted on 25 women (40 to 60 years old) with rough skin and wrinkles confirmed using the ointment of Formulation Example 1. Twenty-five people were randomly divided into five groups of five, and in each group, the ointment of Prescription Example 1 or Comparative Examples 1 to 4 was used. After use for 2 months, it was evaluated by visual observation, scored in 6 levels from 5 (effective) to 0 (no effect), and evaluated by comparing each group.

The results of the clinical trial are shown in Table 4. The ointment of Formulation Example 1 significantly improved the effect on rough skin and wrinkles as compared with the comparative example. During the test period, there was no skin problem and there was no problem with safety.

A similar clinical test was conducted on Formulation Examples 2 to 5, and all showed safe and excellent improvement effects on rough skin and wrinkles.

  The extract of cherry cherry seed of the present invention, the extract of purple wheat seed, and the extract of hantaikai seed are excellent in promoting the proliferation of stem cells present in the skin, the effect of promoting differentiation induction into fibroblasts, and the horn, respectively. The skin external preparation containing them has an effect of promoting differentiation induction into cells, and is excellent in the effect of improving rough skin and wrinkles, and can be used as pharmaceuticals, quasi drugs and cosmetics.

Claims (1)

An external preparation for skin, comprising an extract of cherry cherry seeds, an extract of purple wheat seeds and an extract of hantaikai seeds.