JPH10287543A - Anti-pigmentation agent, and skin cosmetic and preparation for external use for skin containing the same agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an anti-pigmentation agent excellent in stability, safety, melanogenesis-suppressive action, etc., by including an extract from Hydrangea macrophylla Seringe as an active ingredient. SOLUTION: This anti-pigmentation agent is obtained by including an extract from Hydrangea macrophylla Seringe belonging to the genus Saxifraga and the extraction procedure is as follows: (i) rubbing and drying the leaves of the above plant, (ii) chopping the dried leaves, (iii) immersing the chopped leaves in a hydrophilic organic solvent, such as ethanol, and as necessary, heating for several hours to several days; (iV) filtering or centrifuging to separate a solid fraction, and (v) removing the extraction solvent from the resulting extract liquid. The extract has tyrosinase inhibitory action, dendrite formation suppressive action and melanogenesis-suppressive action, and can be used as an anti- pigmentation agent as it is. However, to use it as a cosmetic or preparation for external use for skin, each having skin-whitening action, it is preferably added at 0.0001-50 wt.% in terms of the extract from hydrangea macrophylla Seringe to the above cosmetic or preparation for external use for skin.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an anti-pigmentation agent which inhibits tyrosinase in human epidermis, suppresses dendritic formation of melanocytes and melanin production, and thereby prevents darkening of the skin. The present invention relates to a skin cosmetic and a skin external preparation contained therein.

[0002]

2. Description of the Related Art The occurrence of spots, freckles and pigmentation of skin after sunburn increases with aging and is difficult to disappear, so that it is a major problem for middle-aged and elderly people.
Although the detailed mechanism of the onset of these pigmentation disorders has not been elucidated, melanocytes are activated due to stimulation of ultraviolet rays from sunlight, abnormal hormone secretion, and genetic predisposition, resulting in excessive synthesis of melanin pigment. It is thought that it is caused by being done. Melanin is synthesized by the action of tyrosinase from the amino acid tyrosine in melanocytes existing in the basal layer of the epidermis. The synthesized melanin pigment is passed to the adjacent epidermal cells via the dendrites of the melanocytes, and it is thought that the skin darkens with the keratinization of the epidermal cells.

[0003] Conventionally, attempts have been made to use a substance that inhibits the activity of tyrosinase to suppress the production of melanin from tyrosine in melanocytes to prevent darkening of the skin.
As a result, the effectiveness of vitamin C, glutathione, arbutin, kojic acid, hydroquinone, and extracts from natural products such as various plant extracts and placenta extracts has been confirmed. Arbutin is also known as a substance that suppresses dendrite formation.

[0004] However, these substances have insufficient melanin formation inhibitory action and dendrite formation inhibitory action, and have some problems in safety and stability. It was not always satisfactory.

[0005]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide a pigmentation inhibitor which is excellent in all of stability, safety and action of inhibiting melanin production, and which is superior to conventional ones. .

[0006]

Means for Solving the Problems The present inventor has conducted a search mainly for natural products for substances having a superior melanin production inhibitory action. It has been found that a component having a tyrosinase inhibitory action, a dendritic formation inhibitory action and a melanin formation inhibitory action is contained therein. By applying this product to the skin, it was found that the production of melanin was suppressed, pigmentation could be prevented, and that the product was highly safe, and the present invention was completed.

That is, the present invention provides an anti-pigmentation agent characterized by containing an amateur extract as an active ingredient, and a skin cosmetic and a skin external preparation containing the same.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION An amateur extract, which is an active ingredient of the anti-pigmentation agent of the present invention, is obtained by extracting a crude drug herb with water, a hydrophilic organic solvent or a mixture thereof.

[0009] The herbal medicine armature is Saxifragaceae (Saxifrag
aceae) (Hydrangea macrophylla Seringe)
It is a dried product obtained by drying the leaves, which is already used as a sweetening and flavoring agent, and as an oral freshener. However, it is not known that the extract of the crude drug contains a substance having a tyrosinase inhibitory action, a dendritic formation inhibitory action and a melanin formation inhibitory action, and the present inventors have found for the first time. It is.

[0010] In the case of producing an amateur extract, a crude herb used as a raw material of a herbal medicine can be used as it is, which is generally used for herbal prescriptions. Among the solvents used for the extraction, preferred specific examples of the hydrophilic organic solvent include lower aliphatic alcohols such as methanol and ethanol, polyhydric alcohols such as 1,3-butylene glycol and glycerin, and acetone. Can be In the extraction, water and a hydrophilic organic solvent may be used alone, or may be used as a mixture.
The most preferred solvent used for the extraction is ethanol.

The extraction using the above-mentioned solvent can be performed, for example, as follows. That is, the crude drug is first cut into small pieces, immersed in an extraction solvent, and heated for several hours to several days, if necessary. Thereafter, the solid is separated by filtration or centrifugation, and the extraction solvent is distilled off from the obtained extract. The obtained extract can be used as it is in the pigmentation inhibitor of the present invention, but if necessary, it is subjected to a purification treatment for decolorization, deodorization, etc. within a range that does not significantly impair its action. May be used.

[0012] The armature extract thus obtained can be used as it is as a pigmentation inhibitor,
Further, the melanin production inhibitor can be prepared as a powder dried by means such as freeze-drying or diluted with a suitable carrier. Examples of the carrier that can be used for dilution include alcohol, vegetable oil, petrolatum, and the like, and an emulsifier and the like may be further added thereto.

In the case of producing a skin cosmetic or a skin external preparation having a whitening effect by adding the pigmentation inhibitor of the present invention,
An appropriate amount of the pigmentation inhibitor of the present invention may be added at any stage of the production of cosmetics and external preparations by a conventional method. The amount of the pigmentation inhibitor added varies depending on the strength of the amacha extract used and the type of cosmetics or external preparation, but in many cases the amacha extract is about 0.0001 to 50% by weight, preferably 0% by weight. Good results can be obtained by adding about 0.001 to 10% by weight.

Examples of the skin cosmetic of the present invention include creams, emulsions, lotions, various other oily cosmetics, powder cosmetics, packs, bath preparations and the like. Examples of skin external preparations include ointments, plasters, liquids (alcoholic agents, tinctures, lotions, etc.), compresses (poultices, pastes), liniments, creams, emulsions and the like. .

When the skin cosmetic of the present invention is produced, in addition to the above-mentioned pigmentation inhibitor, nonionic surfactants, anionic surfactants, amphoteric surfactants and the like which are usually used in the production of cosmetics. Emulsifiers; vegetable oils, animal oils, higher fatty acids,
Oily substances such as higher alcohols, synthetic ester oils, waxes, silicone oils; water, fragrances, preservatives, pigments, skin nutrients, skin activators, moisturizers, UV inhibitors, pH regulators, etc. , Can be used as is,
There is no need to change the basic cosmetic formula.

[0016]

The pigmentation inhibitor of the present invention inhibits the production of melanin by the action of a substance having an inhibitory action on tyrosinase, an inhibitory action on dendritic formation and an inhibitory action on melanin production, which is present in an extract of crude herbs, In addition, it inhibits the migration of generated melanin to epidermal cells, thereby preventing pigment deposition comprehensively.

[0017]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited by these Examples.

EXAMPLE 1 100 g of shredded crude drug armature and 100 g of ethanol
0 ml was added, extraction was performed by immersion in a dark place at room temperature for 1 week, and thereafter, solid matter was removed, and the mixture was heated under reduced pressure to distill off ethanol. As a result, 9.1 g of an extract was obtained.

Example 2 Test for the inhibitory effect of tyrosinase activity:
The inhibitory effect of tyrosinase activity, the inhibitory effect of dendritic formation and the inhibitory effect of melanin production on the control kojic acid, (Sigma Chemical Co.) and arbutin (Wako Pure Chemical Industries) were measured by the following test methods.

(Test Method) B16 melanoma cultured cells (B16-F1 cells, purchased from Dainippon Pharmaceutical Co., Ltd.) derived from mice were placed in a culture dish (diameter, 150 mm) at 6 ×
10 ⁵ cells were dispensed, and Dulbecco's modified ME containing 10% fetal calf serum (Boehringer Mannheim), penicillin (100 U / ml), streptomycin (100 μg / ml) and amphotericin B (0.25 μg / ml)
The cells were cultured in a CO ₂ incubator (95% air, 5% CO ₂ ) at 37 ° C. in M medium (all manufactured by Sigma Chemical Co.). After 24 hours of the culture, the medium was replaced with a fresh medium containing 0.5 mM theophylline (manufactured by Sigma Chemical Co.), and the cells were further cultured for 3 days. Trypsin treatment, PBS
After washing with 0.5 ml of the collected cells (2 × 10 ⁷ )
Was suspended in 0.1 M phosphate buffer (pH 6.8), centrifuged (12,000 g, 10 minutes, 4 ° C.) of the cell homogenate obtained by freeze-thawing and sonication. The supernatant was used as a tyrosinase crude enzyme solution. .

In each well of a 96-well plate, 25 μl of distilled water, 25 μl of crude tyrosinase enzyme, 25 μl of ethanol, and samples prepared at graded concentrations were taken and mixed. 37.5 μl of 0.4 M-HEPES buffer (pH 6.8) previously incubated at 37 ° C., 37.5 μl of 10 mM L-dopa solution
Was added to each well, and after stirring, the mixture was incubated at 37 ° C. for 15 minutes. Before and after the reaction, the absorbance at 475 nm was measured using a microplate reader (manufactured by Bio-tek),
The tyrosinase inhibition rate was calculated from the following equation (1). The inhibitory effect on tyrosinase activity was expressed by this value by further calculating the 50% inhibitory concentration (IC ₅₀ ) from the calculated inhibition rate.
Table 1 shows the results.

Calculation formula: A: Absorbance before incubation with sample added B: Absorbance after incubation with sample added C: Absorbance before incubation with no sample added D: After incubation without sample added Absorbance of

[0023]

[Table 1] ---------------------------------------------- ----- Sample Inhibitory effect of tyrosinase activity (IC ₅₀ , μg / ml) ------------------------------ --------------------- amateur extract 186 kojic acid 36 arbutin 1246 ------------------ ---------------------------------

Example 3 Dendritic formation inhibitory effect test: 1 × 10 ⁵ mouse-derived B16 melanoma cultured cells were dispensed into each well of a 6-well culture plate, and 10% fetal bovine serum, penicillin ( 100 U / ml), streptomycin (100 μg
/ Ml) and amphotericin B (0.25 μg / m
In a Dulbecco's modified MEM medium containing 1), the cells were cultured in a CO ₂ incubator (95% air, 5% CO ₂ ) at 37 ° C. After 5 hours of culture, the medium was replaced with a fresh medium without fetal calf serum, and various concentrations of samples (ethanol solution) and a control ethanol (final concentration of 1%) were added. After further culturing for 19 hours, photographs of the cells were taken with a phase-contrast microscope (manufactured by Olympus), and the number of dendritic cells (cells having dendrites longer than the cell diameter or shorter diameter) was counted. The number of projection-bearing cells / the total number of cells was determined. After photographing, the number of cells collected by trypsin treatment was measured with a Coulter counter (manufactured by Coulter Corporation) to confirm that there was no cytotoxicity. The results are shown as numbers with the measured value of the control taken as 100. Table 2 shows the results.

[0025]

[Table 2] ---------------------------------------------- ----------------- Sample (concentration, μg / ml) Number of dendritic cells / total number of cells -------------- ------------------------------------------------- versus Illuminated 0.0 100 ± 4 armature extract 3.3 82 ± 13 6.6 72 ± 13 Kojic acid 14.2 84 ± 5 28.4 73 ± 6 Arbutin 27.2 89 ± 4 54.4 86 ± 1- -------------------------------------------------- ------------ Numerical values are mean ± standard deviation (N = 3)

Example 4 Melanin production inhibitory effect test: 1 × 10 ⁵ B16 melanoma cultured cells derived from a mouse were dispensed into each well of a 6-well culture plate, and 10% fetal calf serum and penicillin (100 U / ml), streptomycin (100 μg
/ Ml) and amphotericin B (0.25 μg / m
In a Dulbecco's modified MEM medium containing 1), the cells were cultured in a CO ₂ incubator (95% air, 5% CO ₂ ) at 37 ° C. After 24 hours of culture, the medium was replaced with fresh medium containing 0.5 mM theophylline, and various concentrations of samples (ethanol solution) and control ethanol (final concentration 1%) were added. After further culturing for 3 days, the total melanin content and cell protein content were measured by the following methods.

(Measurement method of melanin and protein)
After removing the medium from the wells and performing trypsin treatment, washing with PBS, and washing with ethanol, the air-dried cells were dissolved in 1M-NaOH by heating at 80 ° C. for 30 minutes. The amount of intracellular melanin was determined from the absorbance at 475 nm to 660 nm of the solution and a synthetic melanin (Sigma Chemical Co.) solution dissolved in the same manner. In addition, the amount of protein in the cells was determined from the amount of protein in the solution measured using a BCA protein assay kit (Pierce).
Further, the medium removed from the wells was centrifuged (700
g for 10 minutes at 4 ° C.).
0.36 M-HEPES buffer solution containing ethanol (pH 6.
After adding 8) and adjusting the pH, the amount of extracellular melanin was determined from the absorbance at 475 nm to 660 nm of this solution and the synthetic melanin solution of which the pH was similarly adjusted. The sum of the amount of intracellular melanin and the amount of extracellular melanin was defined as the total amount of melanin. The results are shown as numbers with the measured value of the control taken as 100. Table 3 shows the results.

[0028]

[Table 3] ---------------------------------------------- ---------------------- Sample (concentration, μg / ml) Total melanin (%) Cellular protein (%) ------ -------------------------------------------------- ------------ Control 0.0 100 ± 2 100 ± 11 Armature extract 3.3 84 ± 8 106 ± 5 6.6 75 ± 298 98 ± 12 16.5 56 ± 7 89 ± 16 Kojic acid 14.2 90 ± 4 108 ± 4 28.4 83 ± 7 108 ± 67 11.1 30 ± 13 107 ± 8 Arbutin 13.6 81 ± 7 103 ± 8 27.2 74 ± 4 106 ± 9 54.4 72 ± 11 100 ± 7 -------------------------------------- ------------------------------ Numerical values are mean ± standard deviation (N = 4 to 6)

As described above, the effect of inhibiting tyrosinase activity was observed in the amacha extract. However, the effect was about 5-fold lower than the control kojic acid, but about 7-fold higher than arbutin (Example 2). On the other hand, the inhibitory effect of amacha extract on melanin production and dendritic formation related to extracellular secretion of melanin measured using cultured B16 mouse melanoma cells were at least 4 times higher than those of the control kojic acid and arbutin. (Examples 3 and 4).

As is evident from the results, the amacha extract is slightly inferior to kojic acid in inhibiting tyrosinase activity, but has an inhibitory effect on melanin production and an inhibitory effect on dendritic formation involved in extracellular secretion of melanin. Is superior to kojic acid and arbutin, and in the overall pigmentation prevention effect, amacha extract is arbutin,
Better than Kojic acid.

Example 5 A lotion having the following composition was produced by a conventional method. Amacha extract 0.5% by weight Ethanol 10.0% by weight 1,3-butylene glycol 2.0% by weight Polyoxyethylene hydrogenated castor oil (50EO) 0.05% by weight Methyl paraoxybenzoate 0.1% by weight % Perfume 0.1% by weight Refined water balance

The obtained lotion had an excellent whitening effect and a refreshing feeling. The storage stability was also good.

Example 6 An emulsion having the following composition was produced by a conventional method. Amacha extract 1.0% by weight Stearic acid 2.0% by weight Liquid paraffin 6.0% by weight Squalane 2.0% by weight Sorbitan monostearate 1.5% by weight Polyoxyethylene sorbitan monostearate 2.0% by weight ( 20EO) Methyl p-hydroxybenzoate 0.05% by weight 1,3-butylene glycol 0.1% by weight Fragrance 0.15% by weight Refined water balance

The obtained emulsion had excellent whitening effect and had a moist feeling. Also, it had excellent storage stability.

Example 7 A cream having the following composition was produced by a conventional method. Amacha extract 1.0% by weight Liquid paraffin 23.0% by weight Vaseline 7.0% by weight Behenyl alcohol 1.0% by weight Stearic acid 2.0% by weight Beeswax 2.0% by weight Sorbitan monostearate 1.5% by weight Poly Oxyethylene sorbitan monostearate 2.5% by weight (20EO) Butyl parahydroxybenzoate 0.05% by weight 1,3-butylene glycol 3.0% by weight Methyl paraoxybenzoate 0.1% by weight Flavoring agent 0.1 15% by weight Refined water balance

The obtained cream had an excellent whitening effect and a good feeling upon use. Also, the storage stability was excellent.

Example 8 An ointment having the following composition was produced by a conventional method. Amacha extract 5.0% by weight Japanese Pharmacopoeia hydrophilic ointment 95.0% by weight

This ointment is applied to the spots twice a day (2 m
g / cm ² ), an excellent whitening effect was observed, and the storage stability was also good.

[0039]

The pigmentation inhibitor of the present invention comprising an amateur extract as an active ingredient has an excellent whitening effect based on an inhibitory effect on tyrosinase and an inhibitory effect on dendrite formation. Further, as is apparent from the fact that amateurs have been drinking for a long time, the pigmentation preventing agent of the present invention is excellent in safety, and also excellent in use feeling and stability. Therefore, this pigmentation inhibitor can be advantageously used as an additive or compounding ingredient for skin cosmetics or external preparations for the purpose of whitening effect. that's all

 ──────────────────────────────────────────────────の Continuing on the front page (72) Inventor Makoto Owaki Yakult Honsha, 1-1-1 Higashishimbashi, Minato-ku, Tokyo

Claims (5)

[Claims] 1. An anti-pigmentation agent comprising an amateur extract as an active ingredient. 2. The pigmentation inhibitor according to claim 1, which is caused by an inhibitory action on tyrosinase, an inhibitory action on dendrite formation or an inhibitory action on melanin production. 3. A skin cosmetic comprising the pigmentation inhibitor according to claim 1 or 2. 4. The skin cosmetic according to claim 3, which is a whitening cosmetic. 5. An external preparation for skin, comprising the pigmentation inhibitor according to claim 1 or 2.