JPH0912471A - Skin preparation for external use - Google Patents

Abstract

PURPOSE: To obtain a skin preparation for external use, capable of normalizing and more activating a redox controlling mechanism in dermal tissues and having various physiological activities. CONSTITUTION: This skin preparation for external use contains a thioredoxin derived from a bacterium, a plant or an animal and a thioredoxin reductase. Furthermore, a reduced form nicotinamide adenine dinucleotide phosphate(NADPH) may be blended therein. The skin preparation for external use is capable of protecting the skin from an oxidation stress produced by ultraviolet rays, etc., activating dermal cells, manifesting excellent antioxidant and improving actions, inhibiting the biosynthesis of tyrosinases and thereby exhibiting excellent beautifying and whitening actions.

Description

Detailed Description of the Invention

[0001]

INDUSTRIAL APPLICABILITY The present invention protects the skin from oxidative stress caused by ultraviolet rays and the like, further promotes the proliferation of fibroblasts in the skin to prevent skin aging, and is excellent in the tyrosinase production inhibitory action. Can have a whitening effect,
The present invention relates to a skin external preparation having extremely high physiological activity. More specifically, the present invention relates to a skin external preparation containing a complex of thioredoxin and thioredoxin reductase, or containing the complex and reduced nicotinamide adenine dinucleotide (NADPH).

[0002]

2. Description of the Related Art Reactive oxygen species generated by ultraviolet rays, radiation, heavy metals, and the like cause various reactions in the living body of animals, and it is known that skin tissues are deeply involved in damage and aging of these. ing. In response to such oxidative stress, redox control is performed in vivo by various mechanisms. Attempts have also been made to apply such substances relating to redox control to external preparations for the skin, and the formulation of glutathione ester (Japanese Patent Publication No. 48-15).
05), application of thioredoxin to oxidative damage catalyzed by metal (Japanese Patent Laid-Open Nos. 62-246520 and 63-23).
0637) is known.

However, the above-mentioned conventional techniques mainly utilize the reducing action of glutathione and thioredoxin, and are not from the viewpoint of normalizing the redox control mechanism of the living body. .

[0004]

DISCLOSURE OF THE INVENTION In the present invention, by normalizing and more activating the redox control mechanism possessed by the living body in the skin tissue, the resistance to oxidative stress is improved, and various kinds of oxidative stress caused by oxidative stress are improved. The aim was to obtain a completely new type of external preparation for skin from the viewpoint of preventing injury reaction.

[0005]

In the present invention, we pay attention especially to a redox control system centered on thioredoxin, and by adding a complex of thioredoxin and its reductase thioredoxin reductase to a skin external preparation, In addition to improving resistance to oxidative stress, preventing skin damage caused by reactive oxygen species, and improving effect,
It was found that excellent physiological activity was obtained, and the present invention was completed. Further, reduced nicotinamide adenine dinucleotide (NADP) which is a hydrogen donor is further added.
It was found that the above physiological activity was further enhanced by adding H).

Thioredoxin has a molecular weight of 10,000-
An electron transfer protein with 13,000 thiol (SH) groups, which acts as a direct electron donor when ribonucleotide reductase reduces ribonucleotide diphosphate to deoxyribonucleotide diphosphate. In vivo, it forms a redox control system together with thioredoxin reductase. In the present invention, bacteria,
Plant and animal derived thioredoxin, thioredoxin reductase and NADPH can be used. Although thioredoxin and thioredoxin reductase may be purified, fractionated components having these activities prepared from bacteria or plants can be used as they are.

An example of the method for preparing the thioredoxin and thioredoxin reductase-containing fractions is shown below. First, 3 g of acetone powder obtained from guinea pig liver was extracted in 50 mM potassium phosphate buffer (pH 7.2) at 4 ° C. for 3 hours. The extract was treated with 0.5N hydrochloric acid to adjust the pH, and then heat treated (65 ° C), and then Red Sepharose.
Thioredoxin and thioredoxin reductase were separated on a CL-6B column. The thioredoxin reductase fraction was 0 on the DEAE-Sephacel column.
Further purification was performed by elution with potassium chloride having a concentration gradient of ˜0.3 M and then 0.3 to 1.0 M. On the other hand, the thioredoxin fraction was also eluted with a DEAE-Sephacel column using 0 to 0.3 M potassium chloride to obtain a fraction having insulin-reducing activity. As a result, thioredoxin fraction and thioredoxin reductase fraction as shown in Table 1 were obtained. Here, the thioredoxin reductase activity was expressed as 1 unit when 1 nmol of NADPH was oxidized in 1 minute.

[Table 1]

Various physiological activities were observed in the above thioredoxin and thioredoxin reductase. These are shown below. As the thioredoxin reductase fraction, the (B) fraction in Table 1 was used.

(1) Protective effect against cell damage due to UV irradiation Mouse keratinocytes were modified by Kitano et al. MCDB1
After culturing in 53 medium at 37 ° C for 24 hours, washing twice with phosphate buffered saline, replacing with Hank's buffer, and adding 100 µl each of thioredoxin and thioredoxin reductase fractions to 100 µg / ml. And FL-2
Irradiation with 300 mJ / cm ² of medium wavelength ultraviolet (UVB) was carried out using the 0S · E lamp as a light source. After irradiation, the keratinocytes were incubated in MCDB153 medium at 37 ° C. for 24 hours, and the cell viability was determined by the neutral red method. The results in FIG. 1 show that the survival rate of the control culture system (1) not irradiated with UVB was 100%, and thioredoxin and thioredoxin reductase were not added to UV.
The results are shown in comparison with the system (2) irradiated with B and the system (3) irradiated with UVB by adding only 100 μg / ml NADPH.

From FIG. 1, in the system (4) to which the thioredoxin fraction and the thioredoxin reductase fraction were added, compared to the control culture system (2) in which they were not added, UV
The cell viability after B irradiation was increased to about 130%. In addition, in the system (3) in which only NADPH was added, no significant increase in cell viability was observed.

(2) Fibroblast proliferation promoting effect Human fibroblasts added with 10% by weight of fetal bovine serum D
Cultivated in MEM medium at 37 ℃ for 24 hours, 100μg / ml
Thioredoxin fraction and thioredoxin reductase fraction of 100 μg / ml NADPH for each 100 μ
1 and the cells were further cultured at 37 ° C. for 24 hours, and then the number of cells was controlled by adding no thioredoxin, etc. (1), and NAD.
FIG. 2 shows a comparison with the system (3) in which only PH was added.

From FIG. 2, it was confirmed that the system (4) containing the thioredoxin fraction and the thioredoxin reductase fraction had a cell growth promoting effect of 112% as compared with the control (1). Almost 12 in the system (5) to which NADPH is added.
0% cell growth promotion was seen. On the other hand, in the system (3) in which only NADPH was added, no significant cell growth was observed.

(3) Tyrosinase biosynthesis inhibitory effect: 100 μg / ml thioredoxin fraction and thioredoxin reductase fraction or further 100 μg / ml NADP
H is a suspension of mouse B16 melanoma cells (cell number 5
20,000) and cultured in DMEM medium containing 10% by weight of fetal bovine serum for 24 hours, and then the tyrosinase activity in the cells was measured by the following method. That is, 2 ml of 1/15 M phosphate buffer (pH 6.8) was mixed with 0.5 ml of 1.0 wt% dopa aqueous solution and 0.5 ml of culture cell solution, incubated at 37 ° C for 1 hour, and then at 405 nm. Absorbance (As) was measured. As a control, purified water was added and the cells were similarly cultured, and the cultured cell solution and the dopa aqueous solution were incubated to obtain the absorbance (Ab).
Was measured, and the tyrosinase biosynthesis inhibition rate was calculated by the formula (1). The tyrosinase biosynthesis inhibition rate was similarly measured for the positive control system (6) in which sodium lactate was added to a final concentration of 0.3 mM and the system (7) in which the thioredoxin fraction was added alone. did. The results are shown in Fig. 3.

(Equation 1)

As is clear from FIG. 3, in the system (4) to which both the thioredoxin fraction and the thioredoxin reductase fraction were added, the amount of tyrosinase in the cells was reduced to about 60% of that of the control (1). In addition, N
Similar inhibition of tyrosinase biosynthesis was observed in the system (5) containing ADPH. On the other hand, in the system (7) in which the thioredoxin fraction was added alone, no significant decrease in the amount of tyrosinase biosynthesis was observed as compared with the control (1).

In the present invention, thioredoxin and thioredoxin reductase are added to the external preparation base as an aqueous solution or in the state of the extract fractions of plants and animals containing them to prepare a skin external preparation. The external preparation for skin may be in the form of lotion, gel, emulsion, ointment or the like. In addition, softening lotion, astringent lotion, lotion such as cleaning lotion, emollient cream, moisturizing cream, massage cream, cleansing cream, cream such as makeup cream, emollient emulsion, moisturizing emulsion, nourishing emulsion, It can also be provided as cosmetics in various formulation forms such as milky lotions such as cleansing emulsions, jelly-like packs, peel-off packs, packs such as powder packs, and face wash. The amount of the thioredoxin and thioredoxin reductase contained in an aqueous solution containing 0.5 μg / ml of each is preferably about 0.01 to 10.0% by weight of the total amount of the external preparation for skin. The amount of NADPH that can be mixed with thioredoxin and thioredoxin reductase is also preferably about 0.01 to 10.0% by weight.

[0016]

In the external preparation for skin according to the present invention, thioredoxin and thioredoxin reductase have cytoprotective action, fibroblast proliferation promoting action and tyrosinase biosynthesis inhibiting action, thereby preventing and improving skin inflammation and aging symptoms due to ultraviolet rays, The skin-whitening effects such as accelerating wound healing of the skin, elimination of wrinkles and sagging of the skin, restoration of skin elasticity, and activation of skin cells, improvement of spots and freckles, prevention of sunburn, and improvement are observed.

[0017]

EXAMPLES Further, the features of the present invention will be described in detail with reference to examples.

Example 1 Skin Cream (1) Stearic Acid 1.00 (wt%) (2) Stearyl Alcohol 4.00 (3) Glyceryl Monostearate 3.00 (4) Hydrogenated Castor Oil 7.00 (5) Liquid paraffin 14.00 (6) Safflower oil 2.00 (7) Sorbitan sesquioleate 1.00 (8) Sodium hydroxide 0.05 (9) Carboxyvinyl polymer 0.10 (10) Thioredoxin Thioredoxin reductase 0.10 aqueous solution (0.5 mg / ml each) (11) Methyl paraoxybenzoate 0.10 (12) Perfume 0.20 (13) Purified water 67.45 Production method: oils of (1) to (7) The phases are melted by heating to 75 ° C. on the other hand,
Add (8) and (11) to a part of (13) and heat to dissolve at 75 ℃.
After the above oil phase was added to this and emulsified, (9) which was dissolved in the rest of (13) and made thickened was added, and then cooled, 40
Add (10) and (12) at ℃ and cool to room temperature.

[Example 2] Liquid external preparation for skin (1) Glycerin 5.0 (% by weight) (2) Propylene glycol 4.0 (3) Ethanol 10.0 (4) Thioredoxin / thioredoxin reductase 0.2 aqueous solution ( 0.5 mg / ml each) (5) Methyl paraoxybenzoate 0.1 (6) Purified water 80.7 Manufacturing method: Dissolve (5) in (3) and add to (6), and add (1), (2 ) And (4) are added in order and mixed and homogenized.

[Example 3] Liquid external preparation for skin (1) Glycerin 5.0 (% by weight) (2) Ethanol 10.0 (3) Thioredoxin / thioredoxin reductase 0.2 aqueous solution (each 0.5 mg / ml) ( 4) NADPH 0.2 (5) Methyl paraoxybenzoate 0.1 (6) Purified water 84.5 Production method: (5) is dissolved in (2) and added to (6), and (1), (3) , (4) are added sequentially and mixed and homogenized.

[Example 4] Lotion (1) 1,3-butylene glycol 3.0 (% by weight) (2) sorbitol 2.0 (3) ethanol 2.0 (4) sodium pyrrolidonecarboxylate 3.0 (5) Thioredoxin-containing fraction of placenta extract 1.0 (6) Thioredoxin reductase-containing fraction of placenta extract 1.0 (7) Carboxyvinyl polymer 1.0% by weight aqueous solution 2.0 (8) Paraoxybenzoic acid Methyl 0.1 (9) Perfume 0.2 (10) Purified water 85.7 Manufacturing method: (8) is dissolved in (3) and added to (10), (1), (2), (4)
~ (7) and (9) are added in order to mix and homogenize.

Example 5 O / W Emulsion for Skin (1) White Vaseline 25.0 (wt%) (2) Stearyl Alcohol 25.0 (3) Sodium Lauryl Sulfate 1.0 (4) Thioredoxin / Thioredoxin Reductase 0.5 aqueous solution (0.5 mg / ml each) (5) NADPH 0.5 (6) Methyl paraoxybenzoate 0.1 (7) Purified water 47.9 (1)-(3) oil phase components Mix and dissolve to homogeneity, 75 ℃
Heat to On the other hand, the aqueous phase components (6) and (7) are mixed, dissolved and heated to 75 ° C. Then, the oil phase component is added to the aqueous phase component to emulsify, and after cooling, (4) and (5) are added and mixed at 40 ° C., and further cooled.

Example 6 Emulsion (1) Squalane 5.0 (wt%) (2) White petrolatum 2.0 (3) Beeswax 0.5 (4) Sorbitan sesquioleate 0.8 (5) Polyoxy Ethylene (20E.O) sorbitan oleate 1.2 (6) Propylene glycol 5.0 (7) Ethanol 5.0 (8) Thioredoxin-containing fraction of placenta extract 1.5 (9) Thioredoxin reductase of placenta extract Fractions contained 1.5 (10) NADPH 0.1 (11) 1.0 wt% aqueous solution of carboxyvinyl polymer 20.0 (12) Methyl paraoxybenzoate 0.1 (13) Potassium hydroxide 0.1 (14) Fragrance 0.2 (15) Purified water 57.0 Production method: Mix and dissolve the oil phase components (1) to (5) to homogenize,
Heat to 75 ° C. On the other hand, the water phase components (6), (12) and (15) are mixed and dissolved and heated to 75 ° C., the oil phase component is added thereto to pre-emulsify, and (11) is added thereto. After that, homogenize with a homomixer. After cooling, (13) is added to adjust the pH, and then (7) to (10) and (14) are added and mixed at 40 ° C. and further cooled.

In the above examples, the effect of preventing skin aging due to ultraviolet rays, the effect of wound healing and the effect of whitening were evaluated.

First, regarding the effect of preventing skin aging by ultraviolet rays, one group consisting of 20 male and female panelists aged 20 to 50 working outdoors was used, and each Example and Comparative Example was blinded to the face and hands. Wrinkles and changes in skin elasticity were evaluated by visual observation, photography, and measurement of skin elasticity. The period of use was 6 months from April to September, when the amount of UV irradiation was high, and it was used twice a day in the morning and in the evening. The evaluation is "decrease", "slightly decrease" for wrinkles,
There are 4 levels of "no change" and "increase". For skin elasticity, there are "rise", "slightly rise", "no change" and "decrease".
The number of panelists who obtained each evaluation was shown in Table 2. In each Example, Comparative Examples 1 to 6 were obtained by substituting purified water for the thioredoxin / thioredoxin reductase aqueous solution or the thioredoxin-containing fraction and the thioredoxin reductase-containing fraction.

[Table 2]

From Table 2, regarding the wrinkles, only one panel was evaluated in the group used in Example of the present invention as "no change" in the group used in Example 4, and in the group used in other Examples. In each case, a decreasing trend was seen. Particularly in the use groups of Example 2, Example 3 and Example 5 in which the amount of thioredoxin / thioredoxin reductase aqueous solution added was large,
The wrinkle reducing effect was remarkable. In addition, in the use groups of Examples 4 and 6 which are cosmetics containing the thioredoxin fraction and the thioredoxin reductase fraction, 3
There was a clear reduction in wrinkles in the 5% and 40% panelists.

Regarding skin elasticity, no panelists whose skin elasticity was not improved were not recognized in the group used in the examples. In particular, in the group used in Example 2, 85%, in the group used in Example 3, 95%, and in all the panelists in the group used in Example 5, a clear increase in skin elasticity was observed.

On the other hand, in the group used in Comparative Example, no panelists were observed in which wrinkles and skin elasticity were observed to improve, and wrinkles increased, skin elasticity decreased, skin aging symptoms were promoted, and skin symptoms deteriorated. The panelists were also allowed.

Next, the wound healing effect and the anti-inflammatory effect of each of the examples of the present invention and the above-mentioned Comparative Examples 1 to 6 were evaluated. Evaluation was carried out by using 5 mice each artificially inducing a wound or inflammation, applying 0.5 g of each sample of the Example and Comparative Example to the wound site or the inflamed site twice a day for 7 days, and then for 7 days. The condition of the wound or the site of inflammation was observed in the eye. Wound healing was evaluated in three stages of "complete healing", "almost healing" and "incomplete healing", and anti-inflammatory action was evaluated in three stages of "effective", "slightly effective" and "ineffective". Here, when it progressed to about 70 to 80% of the wound healing process, it was evaluated as "almost healed", and when improvement of inflammation was recognized by about 70 to 80%, it was evaluated as "somewhat effective". The results are shown in Table 3 in terms of the number of mice obtained in each evaluation.

[Table 3]

In Table 3, in the application group of the present invention, no mouse was found to have incomplete wound healing. The number of mice in which no effective anti-inflammatory effect was observed was also 0. Particularly in the Example 5 coating group,
Complete wound healing was observed in 4 out of 5, and effective anti-inflammatory effect was observed in all mice. Also in the application groups of Example 4 and Example 6, which are cosmetics in which the thioredoxin fraction and the thioredoxin reductase fraction of the bovine placenta extract were blended as they were, the tendency to heal wounds was observed in all the mice, and in improving inflammation. Was also quite effective.

On the other hand, in the group to which the comparative example was applied, none of the mice showed complete cure, and the anti-inflammatory effect was evaluated to be almost ineffective.

Subsequently, the skin whitening effect in humans was evaluated by a use test. Use test is for stains, freckles,
20 female panelists in their thirties to fifties in which pigmentation such as sunburn is remarkably observed are set as one group, and each sample of Examples and Comparative Examples is blinded in the morning and evening for each group.
Once, it was used by continuously using it for 3 months at the site where pigmentation was observed. The whitening effect was evaluated by observing the appearance of the pigmentation site after the use test. In each of Examples 1 to 3 and 5, 1.0 mg of 0.5 mg / ml thioredoxin / thioredoxin reductase aqueous solution was added.
/ Ml of the thioredoxin aqueous solution was used as Comparative Examples 7 to 9 and Comparative Example 11, respectively, and in Example 4 and Example 6, the thioredoxin fraction was mixed in a double amount without the thioredoxin reductase fraction. Those were designated as Comparative Example 10 and Comparative Example 12. Table 4 shows the results.

[Table 4]

As is clear from Table 4, in the group used in Examples of the present invention, no change was observed in only one case in the group used in Example 4, and in other cases, improvement in pigmentation symptoms was observed. Particularly in the groups used in Examples 2, 3 and 5, the disappearance of pigmentation was observed in 75% to 90% of the panelists. Complete disappearance of pigmentation was also observed in 30% and 35% of the panelists even in the use groups of Examples 4 and 6 which were cosmetics containing a small amount of thioredoxin and thioredoxin reductase.

On the other hand, although a slight improvement in pigmentation is recognized in the group used in Comparative Examples, the degree of improvement is lower than that in the group used in Examples, and the panelists who do not show any change in all groups used in Comparative Examples. In particular, in the group using Comparative Example 10 and the group using Comparative Example 12, no improvement in pigmentation symptoms was observed in more than half of the panelists.

In the examples of the present invention, no panelists were found to have any skin irritation or sensitization during the use test.

[0036]

INDUSTRIAL APPLICABILITY As described in detail above, according to the present invention, it is possible to prevent skin aging and inflammation due to ultraviolet rays, and to have excellent wound healing action and anti-inflammatory action by virtue of good skin activation action. A skin external preparation having a whitening effect could be obtained.

[Brief description of the drawings]

FIG. 1 is a diagram showing a cell damage prevention action by ultraviolet rays.

FIG. 2 is a view showing a fibroblast proliferation promoting action.

FIG. 3 is a view showing an inhibitory effect on tyrosinase biosynthesis.

[Explanation of symbols]

1 Control 2 System containing only Hank's buffer 3 System containing only NADPH 4 System containing thioredoxin fraction and thioredoxin reductase fraction 5 System containing thioredoxin fraction, thioredoxin reductase fraction and NADPH 6 Lactate System containing sodium 7 System containing only thioredoxin fraction

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[Procedure amendment]

[Submission date] July 28, 1995

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0001

[Correction method] Change

[Correction contents]

[0001]

INDUSTRIAL APPLICABILITY The present invention protects the skin from oxidative stress caused by ultraviolet rays and the like, further promotes the proliferation of fibroblasts in the skin to prevent skin aging, and is excellent in the tyrosinase production inhibitory action. Can have a whitening effect,
The present invention relates to a skin external preparation having extremely high physiological activity. More specifically, it relates to a skin external preparation comprising a complex of thioredoxin and thioredoxin reductase, or a complex containing the complex and reduced nicotinamide adenine dinucleotide phosphate (NADPH).

[Procedure amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0005

[Correction method] Change

[Correction contents]

[0005]

In the present invention, we pay attention especially to a redox control system centered on thioredoxin, and by adding a complex of thioredoxin and its reductase thioredoxin reductase to a skin external preparation, In addition to improving resistance to oxidative stress, preventing skin damage caused by reactive oxygen species, and improving effect,
It was found that excellent physiological activity was obtained, and the present invention was completed. In addition, reduced nicotinamide adenine dinucleotide phosphate (N
It was found that the physiological activity was further enhanced by adding ADPH).

[Procedure 3]

[Document name to be amended] Statement

[Correction target item name] 0013

[Correction method] Change

[Correction contents]

(3) Tyrosinase biosynthesis inhibitory effect 100 μg / ml thioredoxin fraction and thioredoxin reductase fraction or further 100 μg / ml NADP
H is a suspension of mouse B16 melanoma cells (cell number 5
20,000) and cultured in DMEM medium containing 10% by weight of fetal bovine serum for 24 hours, and then the tyrosinase activity in the cells was measured by the following method. That is, 2 ml of 1/15 M phosphate buffer (pH 6.8) was mixed with 0.5 ml of 1.0 wt% dopa aqueous solution and 0.5 ml of culture cell solution, incubated at 37 ° C for 1 hour, and then at 405 nm. Absorbance (As) was measured. As a control, purified water was added and the cells were similarly cultured, and the cultured cell solution and the dopa aqueous solution were incubated to obtain the absorbance (Ab).
Was measured, and the tyrosinase biosynthesis inhibition rate was calculated by the formula (1). The tyrosinase biosynthesis inhibition rate was similarly measured for the positive control system (6) in which sodium lactate was added to a final concentration of 0.3 mM and the system (7) in which the thioredoxin fraction was added alone. did. The results are shown in Fig. 3.

(Equation 1)

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display area A61K 38/44 ADA A61K 37/50 ADA

Claims (4)

[Claims] 1. An external preparation for skin, comprising thioredoxin and thioredoxin reductase. 2. A skin external preparation containing thioredoxin and thioredoxin reductase, and reduced nicotinamide adenine dinucleotide phosphate (NADPH). 3. The external preparation for skin according to claim 1, wherein the external preparation for skin is a cosmetic. 4. The external preparation for skin according to claim 2, wherein the external preparation for skin is a cosmetic.