JP2012067014A - Topical skin - Google Patents

Abstract

[PROBLEMS] To provide a skin external preparation, a whitening agent, an antioxidant, a matrix metalloproteinase (MMP) production inhibitor, and a testosterone-5α-reductase inhibitor characterized by containing polyumioside.
[Structure] The polyumioside of the present invention has an excellent antioxidant action, an MMP production inhibitory action, a melanin production inhibitory action and a testosterone-5α-reductase inhibitory action, and is also excellent in stability. In addition, the external preparation for skin containing polyumoside exhibited safe and excellent anti-aging, whitening and anti-acne effects.
[Selection diagram] None

Description

The present invention relates to a novel external preparation for skin having excellent anti-aging, whitening and anti-acne effects.

In recent years, the presence of free radicals and active oxygen has attracted attention as one of the causes of skin aging such as reduction of wrinkles and firmness of the skin. Free radicals and active oxygen decompose, crosslink, and oxidize living tissues such as collagen to damage the skin and promote aging of the skin.

In addition, the involvement of matrix metalloproteinases (MMPs; Matrix Metalloproteinases) has been pointed out as a factor inducing changes associated with skin aging. Among these MMPs, matrix metalloprotease-1 (MMP-1) is known as an enzyme that decomposes collagen, which is a main component of the dermal extracellular matrix of skin, but its expression is greatly increased by irradiation with ultraviolet rays. However, it is considered to be one of the causes of the decrease/denaturation of collagen, which is a major factor such as the formation of wrinkles on the skin and the decrease in elasticity. As an extract having such an MMP-1 activity inhibitory action, for example, an extract of bark of a pine genus Pinus bilobus is known (see Patent Document 1).

JP, 2003-277223, A

Further, MMP-2, which is an enzyme belonging to the gelatinase group, is known as an enzyme that decomposes type IV collagen and laminin-5, which are the main components of the basement membrane, but their expression and activity are greatly increased by irradiation with ultraviolet light. However, it has been clarified that ultraviolet rays cause a decrease in basement membrane components and a structural change in the basement membrane, which is a major factor in the formation of wrinkles and sagging in the skin (see Non-Patent Document 1).

Gary J. Fisher et al. , Nature, 1996, Vol. 379, No. 25, p. 335

Skin pigmentation, which is generally seen in spots, freckles, and sunburn, is that melanin pigment-producing cells present in the skin excessively produce melanin pigment due to hormonal abnormality or ultraviolet ray stimulation, which is deposited in the skin. Is believed to be the cause. As one of methods for preventing such pigmentation, a method for suppressing excessive production of melanin is known. Conventionally, treatment for pigmentation has been performed by externally applying hydroquinone, ascorbic acid (vitamin C), or the like.

In addition, it is considered that dihydrotestosterone generated from testosterone by 5α-reductase is involved in the generation and exacerbation of acne. If a drug having an inhibitory action on 5α-reductase can suppress or inhibit the production of dihydrotestosterone, it may cause acne. It is possible to suppress the occurrence and exacerbation of. Further, dihydrotestosterone is known to be involved in male pattern baldness (see Non-Patent Document 2).
J. Steroid Biochemistry, 11, 609 (1979)

However, polyumoside used in the present invention has not been investigated as a plant-derived natural raw material having these MMP production inhibitory effect, melanin production inhibitory effect and 5α-reductase inhibitory effect.

SOD used for the purpose of preventing skin aging or antioxidant is unstable and difficult to formulate, and it cannot be said that vitamin E is sufficiently effective. Further, since BHT and the like which are synthetic compounds have a problem in safety and the compounding amount is limited, a natural raw material which is stable and has few side effects is desired, not a chemically synthesized product. Similarly, it is preferable to have a safe and stable MMP production inhibitory action and a testosterone-5α-reductase inhibitory action for preventing aging and preventing and treating acne. Further, since ascorbic acid used as a whitening agent has drawbacks such that it is easily decomposed with time, a skin-derived external preparation derived from a natural product which is similarly highly stable and excellent in effect is desired.

From the above, there is a demand for a skin external preparation which is safe and excellent in stability, and has excellent anti-aging, whitening and anti-acne effects.

The present inventors have screened many compounds for various evaluation systems related to beauty as described above. In the process, polyumoside, which is a new ingredient in the cosmetics field, is effective for any action, is safe, and has a very stable property with little occurrence of precipitation, precipitation, discoloration and odor. We have found that it can be used as a functional beauty material. Under these circumstances, as a result of intensive investigations by the present inventors, polyumoside has an excellent active oxygen scavenging action, an MMP production inhibitory action, a melanin production inhibitory action and a testosterone-5α-reductase inhibitory action, and is also excellent in stability. I found that. Furthermore, they have found that an external preparation for skin containing polyumoside is safe and stable, and has excellent anti-aging, whitening and anti-acne effects, and completed the present invention.

The polyumoside used in the present invention is represented by the following general formula.
(Chemical formula 1) General formula (1)

The polyumosides used in the present invention can be extracted, isolated and purified from, for example, sardine (Erinus alpinus).

The sardine is a perennial that grows in rocky areas and grasslands of the Alpine belt of the Pyrenees and the European Alps, and is widely sold for gardening.

The extraction site is not particularly limited, and all sites can be used. The extraction method is not particularly limited, and may be, for example, heat extraction or room temperature extraction.

Examples of the solvent to be extracted include water, ethanol, methanol, and other water-soluble solvents, alone or as a mixture.

The polyumoside used in the present invention can be purified from the above extract by using column chromatography such as Sephadex LH-20, Diaion HP-20, ODS gel and the like, preparative high performance liquid chromatography and the like. The polyumoside used in the present invention does not necessarily have to be a purified product, and may be a crude purified product, or may be in a state containing a solvent.

In the external preparation for skin of the present invention, polyumoside may be used as it is, and oils and fats, waxes, hydrocarbons, fatty acids, alcohols which are components used in the external preparation, as long as the effect of polyumoside is not impaired. Components such as compounds, esters, surfactants, metal soaps, pH adjusters, preservatives, fragrances, humectants, powders, UV absorbers, thickeners, pigments, antioxidants, whitening agents, chelating agents, etc. It can be blended.

The external preparation for skin of the present invention can be used in any of cosmetics, quasi-drugs, and pharmaceuticals, and its dosage form includes, for example, lotion, cream, emulsion, gel, aerosol, essence, Examples thereof include those applied to the skin such as packs, detergents, bath agents, foundations, dusting powders, lipsticks, ointments and poultices.

The amount of polyumoside used in the present invention is 0.0001% by weight or more, preferably 0.001 to 10% by weight in terms of solid matter, based on the total amount of the skin external preparation of the present invention. If it is less than 0.0001% by weight, it is difficult to expect a sufficient effect. If the amount is more than 10% by weight, it is uneconomical to enhance the effect. The addition method may be added in advance or may be added during the production, and may be appropriately selected in consideration of workability.

In order to explain the present invention in detail, production examples, formulation examples and experimental examples of the extract used in the present invention will be given as examples, but the present invention is not limited thereto. The parts of the compounding amounts shown in the examples are parts by weight, and% is% by weight.

Production Example 1 4 L of methanol was added to 500 g of Porium oside sardine, and extraction was performed at room temperature for 1 week. After filtration, 4 L of methanol was added to the residue and the same extraction was performed. The obtained extracts were combined and concentrated to dryness to obtain 90.4 g of a methanol extract. The water-soluble fraction of this extract was subjected to Sephadex LH-20 column chromatography (column: 2×50 cm, eluent: 0-30% methanol aqueous solution). Fractions positive in color development with 4-aminoantipyrine solution (4-AA) were collected, and after methanol was distilled off, MCI gel CHP20P column chromatography (column: 1×30 cm, eluent: 0-50% aqueous methanol solution). I went to Then, 4-AA-positive fractions were collected, and methanol was distilled off, followed by freeze-drying to obtain 1.5 g of polyumoside.

Prescription example 1 lotion prescription compounding quantity (part)
1. Polium oside (Production Example 1) 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 (40EO) 0.1
10. Perfume proper amount 11. [Production method] Components 1 to 6 and 11 and components 7 to 10 each having a total amount of 100 with purified water are uniformly dissolved, and both are mixed and filtered to obtain a product.

Comparative Example 1 Conventional lotion The same lotion as in Formulation Example 1 except that polyumoside was replaced with purified water was used as a conventional lotion.

Prescription example 2 Cream prescription Compound amount (part)
1. Porium oside (Production Example 1) 0.05
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 (20EO) 3.0
8. Behenyl alcohol 1.5
9. Glycerin monostearate 2.5
10. Perfume 0.1
11. Methyl paraoxybenzoate 0.2
12. Ethyl paraoxybenzoate 0.05
13.1,3-Butylene glycol 8.5
14. [Manufacturing method] The total amount is 100 with purified water. Components 2 to 9 are dissolved by heating and mixed, and the mixture is kept at 70°C to form an oil phase. Ingredients 1 and 11 to 14 are heated to melt and mixed, and the mixture is kept at 75°C to form an aqueous phase. The aqueous phase is added to the oil phase to emulsify, the mixture is cooled with stirring, component 10 is added at 45°C, and further cooled to 30°C to obtain a product.

Comparative Example 2 Conventional cream In Formulation example 2, the conventional cream was prepared by replacing the polyumoside with purified water.

Prescription example 3 Emulsion Prescription compounding amount (part)
1. Porium oside (Production Example 1) 0.05
2. Squalane 5.0
3. Olive oil 5.0
4. Jojoba oil 5.0
5. Cetanol 1.5
6. Glycerin monostearate 2.0
7. Polyoxyethylene cetyl ether (20EO) 3.0
8. Polyoxyethylene sorbitan monooleate (20EO) 2.0
9. Perfume 0.1
10. Propylene glycol 1.0
11. Glycerin 2.0
12. Methyl paraoxybenzoate 0.2
13. [Manufacturing method] The total amount is 100 with purified water. Components 2 to 8 are dissolved by heating and mixed, and maintained at 70°C to form an oil phase. Ingredients 1 and 10 to 13 are dissolved by heating and mixed, and the mixture is kept at 75°C to form an aqueous phase. The aqueous phase is added to the oil phase to emulsify, the mixture is cooled with stirring, component 9 is added at 45°C, and the mixture is further cooled to 30°C to obtain a product.

Prescription example 4 Gel formulation Prescription amount (part)
1. Polium oside (Production Example 1) 0.1
2. Ethanol 5.0
3. Methyl paraoxybenzoate 0.1
4. Polyoxyethylene hydrogenated castor oil (60EO) 0.1
5. Fragrance suitable amount 6.1,3-butylene glycol 5.0
7. Glycerin 5.0
8. Xanthan gum 0.1
9. Carboxy vinyl polymer 0.2
10. Potassium hydroxide 0.2
11. [Production method] Components 2 to 5 and components 1 and 6 to 11 each having a total amount of 100 with purified water are uniformly dissolved, and both are mixed to obtain a product.

Prescription example 5 Pack prescription Compound amount (part)
1. Polium oside (Production Example 1) 0.1
2. Polyvinyl alcohol 12.0
3. Ethanol 5.0
4.1,3-butylene glycol 8.0
5. Methyl paraoxybenzoate 0.2
6. Polyoxyethylene hydrogenated castor oil (20EO) 0.5
7. Citric acid 0.1
8. Sodium citrate 0.3
9. Fragrance suitable amount 10. [Manufacturing method] The total amount is 100 with purified water Components 1 to 10 are uniformly dissolved to obtain a product.

Prescription example 6 Foundation Prescription compounding quantity (part)
1. Polium oside (Production Example 1) 0.1
2. Stearic acid 2.4
3. Polyoxyethylene sorbitan monostearate (20EO) 1.0
4. Polyoxyethylene cetyl ether (20EO) 2.0
5. Cetanol 1.0
6. Liquid lanolin 2.0
7. Liquid paraffin 3.0
8. Isopropyl myristate 6.5
9. Carboxymethyl cellulose sodium 0.1
10. Bentonite 0.5
11. Propylene glycol 4.0
12. Triethanolamine 1.1
13. Methyl paraoxybenzoate 0.2
14. Titanium dioxide 8.0
15. Talc 4.0
16. Bengal 1.0
17. Yellow iron oxide 2.0
18. Perfume proper amount 19. [Manufacturing method] The total amount is 100 in purified water. Components 2 to 8 are dissolved by heating and kept at 80°C to form an oil phase. Ingredient 19 is swelled well with ingredient 9, and subsequently ingredients 1 and 10 to 13 are added and mixed uniformly. Components 14 to 17 crushed and mixed with a crusher are added to this, and the mixture is stirred with a homomixer and kept at 75°C to form an aqueous phase. The oil phase is added to this aqueous phase while stirring, and the mixture is cooled, component 18 is added at 45° C., and the mixture is cooled to 30° C. while stirring to obtain a product.

Prescription example 7 Bath agent Prescription compounding amount (part)
1. Polium oside (Production Example 1) 0.1
2. Sodium bicarbonate 50.0
3. Yellow No. 202 (1) Appropriate amount 4. Perfume proper amount 5. [Manufacturing method] The total amount is 100 with sodium sulfate. Components 1 to 5 are uniformly mixed to obtain a product.

Prescription example 8 Acne treatment ointment Prescription compounding amount (part)
1. Porium oside (Production Example 1) 0.05
2. Polyoxyethylene cetyl ether (30EO) 2.0
3. Glycerin monostearate 10.0
4. Liquid paraffin 5.0
5. Cetanol 6.0
6. Methyl paraoxybenzoate 0.1
7. Propylene glycol 10.0
8. [Production method] Components 2 to 5 having a total amount of 100 with purified water are dissolved by heating and mixed, and maintained at 70°C to form an oil phase. Ingredients 1 and 6 to 8 are dissolved by heating and mixed, and the mixture is kept at 75°C to form an aqueous phase. The water phase is added to the oil phase to emulsify, and the product is cooled to 30°C while stirring.

Comparative Example 3 Conventional Ointment In Formulation Example 8, the conventional ointment was prepared by replacing polyumoside with purified water.

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

Experimental Example 1 Free radical scavenging and removing action The free radical scavenging and removing action was evaluated. Ascorbic acid was used as a positive control. As a model of free radical, α,α-diphenyl-β-picrylhydrazyl (hereinafter referred to as DPPH), which is a stable free radical, is used, and the amount of the radical is decreased by reacting with the sample for a certain period of time. Was measured from the decrease in absorbance at a wavelength of 517 nm.

Method for measuring free radical scavenging and removing action To 2 mL of 0.1 M acetic acid buffer (pH 5.5) in which porium oside and ascorbic acid were added to a final concentration of 10 μM, 2 mL of anhydrous ethanol and 1 mL of 0.5 mM DPPH anhydrous ethanol solution were added. To obtain a reaction solution. In the case of an oil-soluble sample, the sample was added to 2 mL of absolute ethanol to prepare a reaction solution. Then, the mixture was reacted at 37°C for 30 minutes, and the absorbance (A) at a wavelength of 517 nm was measured using water as a control. Further, the absorbance (B) was measured using purified water instead of the sample as a blank. The free radical trapping/removing rate was calculated by the following formula.
Free radical trapping/removing rate (%)=(1−A/B)×100

The results of these tests are shown in Table 1. It was confirmed that the polyumosides of the present invention have a stable and excellent free radical trapping/scavenging action. In addition, ascorbic acid was inactivated by heat treatment at 100° C. for 1 hour, but the activity of polyumoside of the present invention was not changed.

Experimental Example 2 MMP production inhibitory action The MMP production inhibitory action was evaluated using the expression levels of MMP-1 and MMP-2 mRNA as an index.

Method for Measuring MMP-1 and MMP-2 mRNA Expression Level Normal human skin fibroblasts were cultured in DMEM medium containing 10% FCS at 37° C. under 5% CO ₂ conditions, and when a confluent state was reached, the sample (final After further culturing in a DMEM medium supplemented with a concentration of 10 μM) for 24 hours, total RNA was extracted. ISOGEN (Nippon Gene) was used for extraction of total RNA. Based on the total RNA extracted from the fibroblasts, the expression levels of MMP-1 and MMP-2 mRNA were measured by the real-time RT-PCR method. TaKaRa SYBR ExScript RT-PCR Kit was used for the real-time RT-PCR method, and 5′GGGAGATCATCGGGGACAACTC3′ (SEQ ID NO: 1) and 5′TGAGCATCCCCCTCCATACATC3′ (SEQ ID NO: 2) were used as primers for MMP-1. 5'CCGTCGCCCCATCATCAA3' (SEQ ID NO: 3) and 5'CTTCTGCATCTTCTTTTAGTGTGTCTC3' (SEQ ID NO: 4) were used as the primers for MMP-2. GAPDH was used as the internal standard, and 5'TGCACCACCAACTGCTTAGC3' (SEQ ID NO: 5) and 5'TCTTCTGGGTGGCAGTGATG3' (SEQ ID NO: 6) were used as the primers for GAPDH. Other operations were carried out according to a predetermined method, and the mRNA expression levels of MMP-1 and MMP-2 were determined as a ratio with respect to the expression level of GAPDH mRNA which is an internal standard.

The results of these tests are shown in Table 2. It was confirmed that the polyumioside of the present invention has an excellent MMP production inhibitory action.

Experimental Example 3 Melanin production inhibition test using B16 mouse melanoma B16 mouse melanoma in logarithmic growth phase was seeded with 3×10 ⁴ cells in φ60 mm dish, and Eagles' MEM (10% containing a final concentration of 1 μM or 10 μM sample) A medium containing fetal bovine serum) was added, and the cells were cultured under the conditions of 37°C and 5% CO ₂ . After 5 days of culturing, the cells were detached from the dish, ultrasonically disrupted, added with 4N NaOH, treated at 60° C. for 2 hours, and analyzed with a spectrophotometer. D. 475 nm was measured. In addition, the cell lysate after ultrasonic treatment was quantified for protein by the Lowry method (J. Biol. Chem., 193, 265-275, 1951), and melanin production was suppressed by comparing the melanin amount per protein amount. It was used as an index of effect.

The results of these tests are shown in Table 3. It was confirmed that the polyumioside of the present invention has an excellent melanin production inhibitory action.

Experimental Example 4 5α-reductase activity inhibition test Measurement was carried out using rat liver microsome-derived 5α-reductase under the conditions in the following reaction system. After dissolving testosterone (final concentration 0.49 mM) in DMSO, NADPH (final concentration 0.97 mM) dissolved in 0.1 M Tris-hydrochloric acid buffer (pH 7.2) was added, and then the sample (final concentration 0.1 mM or 0.2 mM) and 5α-reductase were added in that order, and the mixture was reacted at 37° C. for 30 minutes. After the reaction, an equal amount of dichloromethane was added to stop the reaction, and then the dichloromethane layer was extracted. Then, the dichloromethane layer was distilled off under reduced pressure, silylation was performed using BSTFA, and then the reaction amount was measured by a gas chromatograph mass spectrometer by a conventional method. Regarding the inhibition rate, the one using purified water instead of the sample was used as a control, the reaction rate of the control was regarded as 100% (inhibition rate 0%), and the inhibition rate was calculated by calculating the reaction amount including the sample. The formula is as follows.

Inhibition rate (%)=(1-(b′/a′)/(b/a))×100
a: Peak area of testosterone (control)
b: Peak area of dihydrotestosterone (control)
a': peak area of testosterone (sample addition)
b': peak area of dihydrotestosterone (sample added)

The results are summarized in Table 4. The polyumioside of the present invention showed an excellent 5α-reductase activity inhibitory effect.

Experimental example 5 Usage test 1
Using the lotion of Prescription Example 1, the cream of Prescription Example 2, the conventional lotion of Comparative Example 1 and the conventional cream of Comparative Example 2 for 20 women (aged 22 to 51) for one month. The test was conducted. After use, the effect of improving skin wrinkles and tarmi was evaluated by questionnaire.

The results of these tests are shown in Table 5. As a result, the external preparation for skin containing the extract of the present invention showed an excellent effect of improving wrinkles and tarmi. During the test period, there was no skin trouble and there was no problem in safety. Also, there was no problem with the deterioration of the prescription ingredients.

Experimental example 6 Usage test 2
Using the lotion of Formulation Example 1, the cream of Formulation Example 2, the conventional lotion of Comparative Example 1 and the conventional cream of Comparative Example 2, targeting 20 women (22 to 51 years old) suffering from spots and freckles. A usage test for one month was conducted. After use, the effect of improving spots and freckles was evaluated by a questionnaire.

The results of these tests are shown in Table 6. As a result, the external preparation for skin containing the extract of the present invention showed an excellent effect of improving spots and freckles. During the test period, there was no skin trouble and there was no problem in safety. Also, there was no problem with the deterioration of the prescription ingredients.

Experimental example 3 Usage test 3
Using the ointment for treating acne of Formulation Example 8 and the conventional ointment of Comparative Example 3, a one-month use test was conducted on 15 men (19 to 29 years old) who suffer from acne. After use, the effect of improving (preventing and treating) acne was evaluated by a questionnaire.

The results of these tests are shown in Table 7. As a result, the external preparation for skin containing the extract of the present invention showed an excellent acne improving effect. During the test period, there was no skin trouble and there was no problem in safety. Also, there was no problem with the deterioration of the prescription ingredients.

From the above, polyumioside had an excellent antioxidant action, MMP production inhibitory action, melanin production inhibitory action and testosterone-5α-reductase inhibitory action, and was also excellent in stability. Furthermore, the external preparation for skin containing polyumoside showed safe and excellent anti-aging, whitening and anti-acne effects. Therefore, the external preparation for skin containing the polyumioside of the present invention is particularly effective as an external preparation for aging, whitening or anti-acne.

Claims (5)

An external preparation for skin characterized by containing porium oside. An antioxidant characterized by containing porium oside. A matrix metalloprotease production inhibitor characterized by containing porium oside. A whitening agent characterized by containing porium oside. A testosterone-5α-reductase inhibitor characterized by containing polioside.