JP5184813B2 - Collagen production promotion and degradation inhibitor - Google Patents

Description

  The present invention relates to a collagen production promotion and degradation inhibitor.

It is known that the amount of collagen synthesis in dermal fibroblasts decreases with age, and the amount of synthesis of collagen metalloproteinase (MMP), which is a collagenolytic enzyme, increases. It is thought to cause a decrease in collagen (Non-Patent Documents 1 and 2). A decrease in collagen, which is a major component of the dermal extracellular matrix, is thought to lead to wrinkles and a decrease in skin elasticity. Conventionally, as a means of suppressing such skin symptoms, it has been the mainstream to apply a cosmetic compounded with collagen, but since it temporarily supplements the stratum corneum surface with moisture, it has a sufficient effect. Did not have. Accordingly, a highly safe active ingredient that improves the decrease in collagen production and the increase in degradation is desired.
Varani J, J Invest Dermatol, 114: 480-486, 2000. Chung J H, J Invest Dermatol, 117: 1218-1224, 2001.

Forkhead Box class O (FOXO) is one of a subfamily of transcription factors having a characteristic DNA-binding domain consisting of about 100 amino acids called forkhead, and more than 100 types of organisms ranging from yeast to humans. A gene family has been identified. There are four types of mammals: FOXO1, FOXO3, FOXO4, and FOXO6. Forkhead transcription factor is known to play an important role in sugar / lipid metabolism, cell cycle control, apoptosis, life span / aging, etc. (Non-Patent Documents 3 and 4), but collagen in dermal fibroblasts. There are no reports on the effects on the production and decomposition of sucrose.
Kaestner K H, Genes Dev, 14: 142-146, 2000. Acculi D, Cell, 117: 421-426, 2004.

  An object of the present invention is to provide a collagen production promoting and degrading inhibitor exhibiting an excellent improvement effect on collagen production reduction and degradation enhancement in dermal fibroblasts and collagen reduction caused thereby.

  As a result of intensive studies to solve such problems, the present inventors have promoted the production of collagen in dermal fibroblasts and suppressed the degradation of FOXO protein and / or FOXO protein production promoter. As a result, the present inventors have found an action that improves the decrease in collagen production and the increase in degradation due to aging, and prevents the decrease in collagen, thereby completing the present invention.

  Examples of the FOXO protein used in the present invention include FOXO1, FOXO3, FOXO4 and FOXO6, preferably FOXO1 and FOXO3. Since these proteins are present in cells of many tissues such as skeletal muscle, liver, adipose tissue, and skin, FOXO protein can be extracted from these cells. Commercial products are also available.

  The FOXO protein production promoter used in the present invention is not particularly limited as long as it increases the expression level of FOXO protein in cells.

  The beauty of the moon is a genus of cactiaceae peafowl cactus (Epiphyllum oxypetalum Haw.) And its related species, and it is possible to use whole plants and dried flowers of the cultivars.

  An extraction part is not specifically limited, All parts can be used, Preferably a flower is good. The extraction method is not particularly limited, and for example, it may be a heat extraction or a room temperature extraction.

  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). , Glycerol, etc.), ketones (acetone, methyl ethyl ketone, etc.), acetonitrile, esters (ethyl acetate, butyl acetate, etc.), hydrocarbons (hexane, heptane, etc.), ethers (ethyl ether, tetrahydrofuran, propyl ether, etc.), etc. Is mentioned. A polar solvent such as water or a lower alcohol is preferred. These solvents may be used alone or in combination of two or more.

  The above extract may be used as it is, or may be used after treatment such as concentration, dilution, filtration, decolorization with activated carbon, deodorization, ethanol precipitation or the like, if necessary. Further, the extracted solution may be subjected to a treatment such as concentration to dryness, spray drying, freeze drying, etc., and used as a dried product.

  For the collagen production promotion and degradation inhibitor of the present invention, the above FOXO protein and / or FOXO protein production promoter may be used as it is, and it is a component used for ordinary external preparations as long as the effect is not impaired. Certain fats and oils, waxes, hydrocarbons, fatty acids, alcohols, esters, surfactants, metal soaps, pH adjusters, preservatives, fragrances, moisturizers, powders, UV absorbers, thickeners, Components such as a dye, an antioxidant, a whitening agent, and a chelating agent can also be blended.

  The collagen production promotion and degradation inhibitor used in the present invention can be used in any of cosmetics, quasi-drugs, and pharmaceuticals. Examples of the dosage form include lotions, creams, emulsions, gels, and aerosols. , Ointments, poultices, pastes, plasters, essences, packs, cleaning agents, bath preparations, foundations, powders, lipsticks and the like.

  Although the compounding quantity of FOXO protein used for this invention is not specifically limited, The range of 0.00001 to 10 weight% is preferable, More preferably, it is 0.0001 to 1 weight%. The blending amount of the FOXO protein production promoter used in the present invention is not particularly limited, but it is preferably in the range of 0.00001 to 10% by weight, more preferably 0.0001 to 5% by weight as a dry product. In addition, the addition method may be added in advance or during the production, and may be appropriately selected in consideration of workability.

  INDUSTRIAL APPLICABILITY The present invention prevents the decrease in collagen generation and the increase in MMP generation, thereby suppressing the decrease in collagen and preventing and improving the formation of wrinkles and elasticity due to aging. Production of FOXO protein and / or FOXO protein A collagen production promotion and degradation inhibitor characterized by containing a promoter, a skin firmness, elasticity improving agent, and a skin external preparation characterized by containing the collagen production promotion and degradation inhibitor.

  Next, in order to describe the present invention in detail, examples of obtaining the protein used in the present invention, examples of producing the extract, formulation examples and experimental examples are given as examples, but the present invention is not limited thereto. The compounding amount shown in the examples indicates wt%.

Obtaining Example 1 FOXO protein FOXO1 and FOXO3 proteins were obtained commercially from Abnova Corporation (Taiwan).

Production Example 1 Tsukishita Beauty Extract 1
3 L of purified water was added to 100 g of a dried product of the beauty of the moon, and extracted at 95-100 ° C. for 2 hours. After filtration, the filtrate was concentrated under reduced pressure and further freeze-dried to obtain 48 g of a moon moon beauty extract.

Production Example 2 Tsukishita Beauty Extract 2
1 L of a 30% aqueous ethanol solution was added to 100 g of a dry product of the above-ground whole grass of Tsukishita Bijin, and extracted at room temperature for 1 week. After filtration, the filtrate was concentrated under reduced pressure, and further freeze-dried to obtain 9 g of a beautiful moon extract.

  Next, the prescription of the Example which concerns on this invention is shown.

Formulation Example 1 Cream 1
Formulation amount (% by weight)
1. FOXO1 protein 0.1
2. Squalane 5.5
3. Olive oil 3.0
4). Stearic acid 2.0
5. Beeswax 2.0
6). Octyldodecyl myristate 3.5
7). Polyoxyethylene cetyl ether (20E.O.) 3.0
8). Behenyl alcohol 1.5
9. Glycerol monostearate2.5
10. Fragrance 0.1
11.1,3-butylene glycol 8.5
12 Ethyl paraoxybenzoate 0.05
13. Methyl paraoxybenzoate 0.2
14 Purified water 68.05
[Production Method] Components 2 to 9 are heated and mixed, and kept at 70 ° C. to obtain an oil phase. Ingredients 11-14 are dissolved by heating and mixed, and kept at 75 ° C. to form an aqueous phase. The aqueous phase is added to the oil phase to emulsify, and the mixture is cooled while stirring. Then, component 10 is added at 45 ° C., and further cooled to 30 ° C., and component 1 is added to obtain a product.

Formulation Example 2 Cream 2
In Formulation Example 1, cream 2 was prepared by replacing FOXO1 protein with FOXO3 protein.

Formulation Example 3 Cream 3
In Formulation Example 1, Cream 3 was prepared by replacing FOXO1 protein with Tsukishita Beauty Extract 1 (Production Example 1).

Comparative Example 1 Conventional Cream In Formulation Example 1, FOXO1 protein was replaced with purified water to obtain a conventional cream.

Formulation Example 4 Lotion Formulation Amount (wt%)
1. FOXO1 protein 0.1
2. 1,3-butylene glycol 8.0
3. Glycerin 2.0
4). Xanthan gum 0.02
5. Citric acid 0.01
6). Sodium citrate 0.1
7). Ethanol 5.0
8). Methyl paraoxybenzoate 0.1
9. Polyoxyethylene hydrogenated castor oil (40E.O.) 0.1
10. Fragrance 0.1
11. Purified water 84.47
[Production method] Components 1 to 6 and 11 and components 7 to 10 are uniformly dissolved, and both are mixed and filtered to obtain a product.

Formulation Example 5 Emulsion Formulation Amount (wt%)
1. FOXO3 protein 0.05
2. Squalane 5.0
3. Olive oil 5.0
4). Jojoba oil 5.0
5. Cetanol 1.5
6). Glycerol monostearate 2.0
7). Polyoxyethylene cetyl ether (20E.O.) 3.0
8). Polyoxyethylene sorbitan monooleate 2.0
9. Fragrance 0.1
10. Propylene glycol 1.0
11. Glycerin 2.0
12 Methyl paraoxybenzoate 0.2
13. Purified water 73.15
[Manufacturing method] Components 2 to 8 are dissolved by heating and mixed, and kept at 70 ° C to obtain an oil phase. Ingredients 10 to 13 are dissolved by heating and mixed, and kept at 75 ° C. to form an aqueous phase. The aqueous phase is added to the oil phase to emulsify, and the mixture is cooled while stirring. Then, component 9 is added at 45 ° C., and further cooled to 30 ° C., and component 1 is added to obtain a product.

Formulation Example 6 Ointment Formulation Amount (% by weight)
1. Moonlight Beauty Extract 1 (Production Example 1) 1.0
2. Polyoxyethylene cetyl ether (30E.O.) 2.0
3. Glycerol monostearate 10.0
4). Liquid paraffin 5.0
5. Cetanol 6.0
6). Methyl paraoxybenzoate 0.1
7). Propylene glycol 10.0
8). Purified water 65.9
[Manufacturing method] Components 2 to 5 are dissolved by heating and mixed, and kept at 70 ° C to obtain an oil phase. Ingredients 1 and 6 to 8 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.

Formulation Example 7 Foundation Formulation Amount (% by weight)
1. Moonlight Beauty Extract 2 (Production Example 2) 1.0
2. Stearic acid 2.4
3. Polyoxyethylene sorbitan monostearate 1.0
(20 EO)
4). Polyoxyethylene cetyl ether (20E.O.) 2.0
5. Cetanol 1.0
6). Liquid lanolin 2.0
7). Liquid paraffin 3.0
8). Isopropyl myristate 6.5
9. Butyl paraoxybenzoate 0.1
10. Sodium carboxymethylcellulose 0.1
11. Bentonite 0.5
12 Propylene glycol 4.0
13. Triethanolamine 1.1
14 Methyl paraoxybenzoate 0.2
15. Titanium dioxide 8.0
16. Talc 4.0
17. Bengala 1.0
18. Yellow iron oxide 2.0
19. Fragrance 0.1
20. Purified water 60.0
[Manufacturing method] Components 2 to 9 are heated and dissolved and kept at 80 ° C to obtain an oil phase. Swell component 10 well with component 20, then add components 1 and 11-14 and mix uniformly. To this, components 15 to 18 pulverized and mixed with a pulverizer are added, and the mixture is stirred with a homomixer and kept at 75 ° C. to obtain an aqueous phase. The oily phase is added to the aqueous phase with stirring, cooled, and component 19 is added at 45 ° C, and cooled to 30 ° C with stirring to give a product.

Formulation Example 8 Bath preparation formulation Formulation amount (% by weight)
1. Moonlight Beauty Extract 2 (Production Example 2) 5.0
2. Sodium bicarbonate 50.0
3. Yellow No. 202 (1) 0.05
4). Fragrance 0.25
5. Anhydrous sodium sulfate 44.7
[Production method] Components 1 to 5 are uniformly mixed to obtain a product.

  Next, experimental examples will be given to explain the effects of the present invention in detail.

Experimental Example 1 Improvement test of FOXO1 and FOXO3 production reduction by aging The improvement effect on the production reduction of FOXO1 and FOXO3 by aging was evaluated using the mRNA expression level as an index.

Method for measuring FOXO1 and FOXO3 mRNA expression levels Normal human dermal fibroblasts were repeatedly subcultured 10 times in a DMEM medium containing 10% FCS at 37 ° C. and 5% CO ₂ to induce cell senescence. After becoming confluent, the cells were further cultured for 24 hours in a DMEM medium supplemented with a sample having a concentration of 1 μg / ml, and then total RNA was extracted. ISOGEN (Nippon Gene) was used for extraction of total RNA. Based on the total RNA extracted from fibroblasts, FOXO1 and FOXO3 mRNA expression levels were measured by real-time RT-PCR. TaKaRa SYBR ExScript RT-PCR Kit was used for the real-time RT-PCR method, and 5 'TCATGGATGAGACATCATTGGATT3' (SEQ ID NO: 1) and 5 'CCAGCTATGTGTCGGTTGTCTTGA3 (SEQ ID NO: 1) were used as primers for FOXO1. As primers for FOXO3, 5′TGGATGCTGATGGGTTGGA3 ′ (SEQ ID NO: 3) and 5′CCCCACGTTCAAACCAACA3 ′ (SEQ ID NO: 4) were used. GAPDH was used as an internal standard. Other operations were performed in accordance with a prescribed method, and the FOXO1 and FOXO3 mRNA expression levels were determined as a ratio to the internal standard GAPDH mRNA expression level. In senescent cells, FOXO1 and FOXO3 mRNA expression levels were decreased, and the rate of improvement in FOXO1 and FOXO3 production reduction was calculated from the value and the value of FOXO1 and FOXO3 mRNA expression levels when the sample was added.

  The test results are shown in Table 1. As a result, the extract of Tsukishita Bijin was found to have an excellent effect of reducing the production of FOXO1 and FOXO3 by aging, that is, the effect of promoting the production of FOXO protein in aged cells.

Experimental Example 2 Collagen production decrease and degradation enhancement improvement test with aging Collagen (I), MMP-1 and MMP-2 mRNA have an improvement effect on collagen production decrease and degradation enhancement due to decrease in FOXO1 and FOXO3 production due to aging The expression level was evaluated as an index.

Method for measuring expression levels of collagen (I), MMP-1 and MMP-2 mRNA Normal human skin fibroblasts are repeatedly subcultured 10 times in a DMEM medium containing 10% FCS at 37 ° C. and 5% CO _2. And induced cellular senescence. After becoming confluent, the cells were further cultured for 24 hours in a DMEM medium to which a sample was added, and then total RNA was extracted. ISOGEN (Nippon Gene) was used for extraction of total RNA. Based on total RNA extracted from fibroblasts, the expression levels of collagen (I), MMP-1 and MMP-2 mRNA were measured by real-time RT-PCR. TaKaRa SYBR ExScript RT-PCR Kit was used for the real-time RT-PCR method, and 5′GCTACCCACTACTGCTCTTCATG3 ′ (SEQ ID NO: 5) and 5′TTCTTTGCAGTGGGTAGGGTGATTGTC3 ′ were used as primers for collagen (I). As primers for MMP-1, 5′GGGAGATCATCGGGGACAACTC3 ′ (SEQ ID NO: 7) and 5′TGAGCATCCCCCTCCAATACC3 ′ (SEQ ID NO: 8) were used. As a primer for MMP-2, 5′CCGTCGCCCATCATCAA3 ′ (SEQ ID NO: 9) and 5′CTTCTGCCATCTTCTTTAGTGGTCCTT3 ′ (SEQ ID NO: 10) were used. GAPDH was used as an internal standard. Other operations were performed according to a predetermined method, and the mRNA expression levels of collagen (I), MMP-1 and MMP-2 were determined as a ratio to the GAPDH mRNA expression level as an internal standard. In senescent cells, a decrease in the expression level of collagen (I) mRNA and an increase in the expression level of MMP-1 and MMP-2 mRNA were observed, and the values and collagen (I), MMP-1 and MMP when the sample was added. -2 Collagen production reduction improvement rate and MMP production enhancement improvement rate were calculated from the value of mRNA expression level.

  The test results are shown in Tables 2 and 3. As a result, the FOXO1 and FOXO3 proteins and the extract of Tsukishita Bijin were found to have an excellent action for improving collagen degradation and degradation due to aging. That is, the FOXO1 and FOXO3 proteins and the extract of Tsukishita Bijin were observed to promote collagen production and suppress degradation in aged cells.

Experimental Example 3 Use Test For 2 months for 30 women (40 to 50 years old) suffering from wrinkles and reduced skin elasticity using the creams of Formulation Examples 1 to 3 and the conventional cream of Comparative Example 1 A use test was conducted. Before and after use, the elasticity of the skin was measured using a cute meter (Courage + Khazaka). The value after use when the value of elasticity before use was taken as 100% was determined to evaluate the effect of improving elasticity. In addition, after the use, a questionnaire survey was conducted on the effects of improving skin firmness and elasticity. The evaluation criteria of the questionnaire were evaluated as “excellent” for valid, “good” for slightly effective, “good” for slightly effective, and “impossible” for invalid.

  The results of the elasticity improvement effect are shown in Table 4. The cream formulated with the FOXO1 protein of Formulation Example 1, the cream formulated with the FOXO3 protein of Formulation Example 2, and the cream containing the extract of the beauty of the moon of Formulation Example 3 showed excellent skin elasticity improving effects.

  The results of the questionnaire survey are shown in Table 5. The creams of Formulation Examples 1, 2 and 3 showed excellent skin firmness and elasticity improvement effect. During the test period, there was no skin problem and there was no problem with safety.

  When the usage tests of the lotions, emulsions, ointments, foundations, and bath preparations of Formulation Examples 4 to 8 were conducted, all showed safe and excellent skin firmness and elasticity improvement effects.

  As an application example of the present invention, it can be used for any of cosmetics, quasi drugs, and pharmaceuticals. Examples of the dosage form include lotions, creams, emulsions, gels, aerosols, ointments, poultices, pastes, plasters, essences, packs, detergents, bath preparations, foundations, powders, lipsticks, etc. By promoting the production of collagen and suppressing the degradation, it is possible to prevent the decrease of collagen due to aging, and to prevent and improve the formation of wrinkles and the decrease in elasticity seen in aging skin.

Claims (3)

A collagen production promotion and degradation inhibitor comprising forkhead box class O 1 and / or fork headbox class O3. A skin elasticity and elasticity improving agent comprising the collagen production promoting and degrading inhibitor according to claim 1. An external preparation for skin comprising the collagen production promoting and degrading inhibitor according to claim 1.