KR20100135703A - Wrinkle inhibiting composition and external skin composition - Google Patents

Abstract

The present invention provides a novel composition using fullerenes having a high anti-wrinkle ability. The anti-wrinkle composition of the present invention is characterized by containing a useful phase fullerene in which fullerenes are dissolved in squalane. The composition protects two chain breaks of DNA by lipid peroxide in human skin keratinocytes.

Description

Wrinkle inhibiting composition and skin external composition {ANTI-WRINKLE COMPOSITION AND EXTERNAL SKIN COMPOSITION}

The present invention relates to a composition for inhibiting wrinkles and a composition for external skin.

The external skin composition includes various formulations such as creams, emulsions, lotions, and oils, but UV-absorbers and UV-scattering agents are blended in any of the formulations for the purpose of UV protection.

On the other hand, in recent years, the development of cosmetics using nanotechnology is prevalent, but among these, cosmetics containing fullerenes such as C ₆₀ and C ₇₀ are known to exhibit various microscopic effects. For example, Patent Document 1 describes that a cosmetic product obtained by dissolving fullerene in oil has an ultraviolet absorbing effect. Patent Document 2 also discloses that the dispersion of fullerenes as ultrafine particles of carbon-burned residues in squalane shows a health promoting effect.

Moreover, the technique which solubilizes fullerene which is sparingly soluble in water is also developed (refer patent document 3), and the application to external skin compositions, such as cosmetics, is also utilized.

In addition, fullerenes are attracting attention as having an ability of scavenging active oxygen, and safety in external application can also be expected, and a fullerene-containing skin external composition used for the prevention of free radical disease has been proposed (patent document). 4).

Japanese Patent Laid-Open No. 09-278625 Japanese Patent Laid-Open No. 2001-316251 Japanese Patent Publication No. 2005-060380 Japanese Patent Publication No. 2006-160664

As mentioned above, fullerenes are expected to be applied to various external skin compositions such as cosmetics, but most of the functions and actions are expected to be unknown, and development for application of fullerenes is still in development stage. good.

Against this background, the present inventors have made detailed studies on the effect on skin cells in application to the skin. One of the problems which faced this examination was "the suppression of wrinkles".

This is also because the development of a technique for more effectively exerting the effect of "suppression of wrinkles" has been expected in the past.

This invention is made | formed in view of the above circumstances, and makes it a subject to provide the new composition using the fullerene which has a high wrinkle suppression ability.

The present invention is guided on the basis of completely new knowledge obtained from the results of detailed verification by the inventors on the basis of the formation of "wrinkles" in the human skin reconstituted tissue and the search for the means for "wrinkle suppression" based on this. It is completed.

In other words, lipid peroxide 2,4-nonadienal, which is the inducing factor of wrinkle formation, is an intermediate of peroxidation of fatty acids (mainly linoleic acid, arachidonic acid, etc.) at the 1,2-position of glycero lipid of UV-irradiated skin cells. It is relatively stable and stays in the skin, which causes wrinkle formation. In fact, subcutaneous treatment with 2,4-nonadienanal causes three types of wrinkle-related symptoms:

(1) The formation of uneven relief occurs on the skin surface. (Change from Fig. 1 (a) to Fig. 1 (b) showing the replica analysis using the silicone rubber replica method)

(2) Many keratin exfoliation occurs on the skin surface. (Change from Fig. 2 (a) to Fig. 2 (b) showing a scanning electron micrograph)

(3) Multi-layer separation and vertical atrophy of the dermis occurs on the cross section of the skin (change from Fig. 3 (a) to Fig. 3 (b) showing electron micrograph using DMSO freezing cutting method)

Incidentally, it was surprisingly found that all of the three types (1), (2) and (3) of wrinkle-related symptoms were protected by the application of fullerene on the skin surface in the form of oil-soluble fullerene dissolved in squalane. On the other hand, it was also confirmed that squalane alone is invalid.

In addition, the mechanism of the anti-wrinkle effect of fullerene in utility was also confirmed by defending the cleavage of DNA strands accompanied with cell membrane injury by lipid peroxide nonadienal in human skin keratinocytes.

Then, this invention provides the composition characterized by the following in order to solve the subject as mentioned above.

 The 1st: The composition for wrinkle suppression containing the oil-soluble fullerene which melt | dissolved fullerene in squalane.

Second 2: The first anti-wrinkle composition, characterized in that it contains at least one selected from C ₆₀ , C ₇₀ and salts or derivatives thereof as fullerenes.

Third 3: DNA double strand / one chain cleavage and / or by oxidative stress comprising lipid peroxide and / or free radicals in human skin keratinocytes and / or human skin fibroblasts and / or skin tissues comprising them The anti-wrinkle composition according to claim 1 or 2, which protects against DNA injury including DNA base damage.

Fourth: The composition for external application of skin, comprising the composition of any one of the first to the third.

(Effects of the Invention)

According to the present invention, it is possible to effectively suppress "wrinkle", which has been difficult until now, by the useful fullerenes (which may be referred to as "lipofullerenes") dissolved in squalanes. As a result, new horizons of microscopic effects due to fullerenes as a composition for external application of skin, which have not been previously expected at all, have been explored.

BRIEF DESCRIPTION OF THE DRAWINGS It is a figure which shows the replica image of the 3D skin tissue model surface in Example 1, and the analysis result by the NIH-image.
2 is a scanning electron micrograph of the surface shape of a three-dimensional skin tissue model in Example 1. FIG.
3 is a scanning electron micrograph of a cross-sectional structure of a three-dimensional skin tissue model in Example 1. FIG.
It is a figure which showed the inhibitory effect of the cell death of human skin keratinocytes in Example 2. FIG.
5 is a scanning electron micrograph of the surface shape of a human skin normal tissue model in Example 4. FIG.
6 is a scanning electron micrograph of the cross-sectional structure of the human skin normal tissue model in Example 4. FIG.
7 is a fluorescence micrograph of a cross section of a human skin normal tissue model segment in Example 5. FIG.
It is a graph which shows the evaluation result of the wrinkle area ratio change amount in the randomized double-blind matched pair comparison test of Example 6. FIG.
9 is a scanning electron micrograph of the surface shape of a human skin normal tissue model in Example 7. FIG.
It is a figure explaining the NIH-image analysis method of the surface SEM image of the human skin normal tissue model in Example 7. FIG.
It is a figure which shows the analysis result by NIH-image of the surface SEM image of the human skin normal tissue model in Example 7. FIG.
12 is a scanning electron micrograph of a cross-sectional structure of a human skin normal tissue model in Example 7. FIG.
It is a figure which shows the analysis result by NIH-image of the cross-sectional SEM image of the human skin normal tissue model in Example 7. FIG.
14 is a replica image of a human skin normal tissue model in Example 7. FIG.
It is a figure which shows the analysis result by NIH-image of the replica image of the human skin normal tissue model in Example 7. FIG.

Hereinafter, the present invention will be described in detail.

The useful phase fullerene (abbreviation: lipo fullerene) used in this invention is a thing which fullerene dissolved in squalane. As fullerenes, C ₃₂ , C ₄₄ , C ₅₀ , C ₆₀ , C ₅₈ , C ₆₀ , C ₇₀ , C ₇₆ , C ₇₈ , C ₈₂ , C ₈₄ , C ₉₀ , C ₉₆ and salts or derivatives thereof may be used. Among them, C ₆₀ , C ₇₀ and salts or derivatives thereof are preferably used. Moreover, you may use the mixture of 2 or more types in these fullerenes.

As for squalane, any one or a mixture of squalane and squalene may be used. It may be of vegetable origin, or of animal or fish.

In order to dissolve fullerenes in squalane, what is necessary is just below the solubility amount of fullerenes, and the amount can be determined according to the application form of the composition of this invention. At the time of melt | dissolution, you may stir and may heat. As a composition, it can be set as an external composition for skin.

In this case, the composition for external application on the skin does not impair its effect, and various components commonly used in cosmetics, quasi-drugs, medicines, and the like, such as water, oils, hydrocarbons, higher fatty acids, higher alcohols, silicones, and anions Surfactants, cationic surfactants, amphoteric surfactants, nonionic surfactants, preservatives, sugars, metal ion sequestrants, polymers such as water-soluble polymers, thickeners, powder components, ultraviolet absorbers, ultraviolet shielding agents, humectants, fragrances, pH adjusters and the like. It can mix. In addition, other active ingredients such as vitamins, skin activators, blood circulation promoters, active oxygen scavengers, anti-inflammatory agents, whitening agents, fungicides, and the like can be blended.

Specific examples of fats and oils include camellia oil, evening primrose oil, macadamia nut oil, olive oil, rapeseed oil, corn oil, sesame oil, jojoba oil, germ oil, wheat germ oil, and liquid fats such as glycerin trioctanoate, cocoa fat, palm oil and hardened palm oil. Solid oils such as palm oil, palm kernel oil, wax, wax kernel oil, hardened oil, hardened castor oil, beeswax, candelia beeswax, cotton beeswax, nukarou, lanolin, lanolin acetate, liquid lanolin, beeswax beeswax do.

Specific examples of the hydrocarbons include liquid paraffin, squalene, squalane, microcrystalline wax, and the like.

Specific examples of higher fatty acids include lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and the like.

Specific examples of the higher alcohols include straight chain alcohols such as lauryl alcohol, stearyl alcohol, cetyl alcohol, cetostearyl alcohol, monostearyl glycerin ether, lanolin alcohol, cholesterol, phytosterol, and branched chain alcohols such as octyldodecanol. do.

As a specific example of silicone, the dimethyl polysiloxane of linear polysiloxane, methylphenyl polysiloxane, the decamethyl cyclopentasiloxane of cyclic polysiloxane, etc. are mentioned.

Specific examples of the anionic surfactant include fatty acid salts such as sodium laurate, higher alkyl sulfate ester salts such as sodium lauryl sulfate, alkyl ether sulfate ester salts such as POE lauryl sulfate triethanolamine, N-acyl sarcosine acid, sulfosuccinate salt, N-acylamino acid salt etc. are mentioned.

Specific examples of the cationic surfactant include alkyltrimethylammonium salts such as stearyltrimethylammonium chloride, benzalkonium chloride, benzetonium chloride and the like.

Specific examples of the amphoteric surfactant include betaine-based surfactants such as alkyl betaine and amide betaine.

Specific examples of the nonionic surfactant include sorbitan fatty acid esters such as sorbitan monooleate, cured castor oil derivatives, and the like.

Specific examples of the preservative include methyl paraben, ethyl paraben and the like.

Specific examples of the metal ion blocking agent include edetate salts such as ethylenediamine tetraacetic acid disodium acetate, edetic acid, and edetic acid sodium salt.

Specific examples of the polymer include gum arabic, tragacanth, galactan, guar, carrageenan, pectin, agar, queens seed, dextran, pullulan, carboxymethyl starch, collagen, casein, gelatin, methylcellulose, methylhydroxypropyl And vinyl polymers such as cellulose, hydroxyethyl cellulose, sodium carboxymethyl cellulose (CMC), sodium alginate, and carboxyvinyl polymer.

Specific examples of the thickener include carrageenan, tragacanth rubber, queen's seed, casein, dextrin, gelatin, CMC, hydroxyethyl cellulose, hydroxypropyl cellulose, carboxyvinyl polymer, guar rubber, xanthan rubber, bentonite, and the like.

Specific examples of the powder component include talc, kaolin, mica, silica, zeolite, polyethylene powder, polystyrene powder, cellulose powder, inorganic white pigment, inorganic red pigment, titanium oxide coated mica, titanium oxide coated talc, and colored titanium oxide coated mica Pearl pigments, such as these, and organic pigments, such as red 201 and red 202, etc. are mentioned.

Specific examples of the ultraviolet absorber include paraaminobenzoic acid, phenyl salicylate, paramethoxycinnamic acid isopropyl, paramethoxycinnamic acid octyl, 2,4-dihydroxybenzophenone and the like.

Specific examples of the ultraviolet shielding agent include titanium oxide, talc, carmine, betonite, kaolin, zinc oxide and the like.

Specific examples of the moisturizing agent are polyethylene glycol, propylene glycol, dipropylene glycol, 1,3-butylene glycol, 1,2-pentanediol, glycerin, diglycerin, polyglycerin, xylitol, maltitol, maltose, sorbitol, glucose, fructose And sodium chondroitin sulfate, sodium hyaluronate, sodium lactate, pyrrolidonecarboxylic acid, cyclodextrin and the like.

The composition of the present invention can be applied, for example, in the form of an aqueous solution, an emulsion, an emulsion, a suspension such as a suspension, a semisolid agent such as a gel or a cream, or a solid agent such as a solid. Conventionally, it is prepared in these forms by a well-known method, and can be various formulations, such as a lotion, an emulsion, a gel, a cream, an ointment, a warning, a pape, an aerosol, These are apply | coated to a body, a patch, spraying, etc. Applicable In particular, among these formulations, lotions, emulsions, creams, ointments, warnings, papes, aerosols and the like are suitable for external skin compositions.

The composition of the present invention is dispersed in an aqueous solvent such as a mixture of water and an aqueous solvent, for example, an alcohol and water, and thus develops more effectively as a practical technique for external preparations such as cosmetics.

Dispersion in an aqueous solvent is embodied, for example, as an O / W emulsion or the like, but for the production of this aqueous dispersion, treatment with ultrasonic waves, vortex mixers, homogenizers, or the like is more effective.

A nonionic surfactant is also effectively used for the dispersion of the fullerene fat solution in the aqueous solvent. For example, it is a nonionic surfactant mainly having a polyoxyethylene ether structure. It is preferable to make the usage-amount of addition of these nonionic surfactants into the range of 0.01-2 mass% of whole quantity.

As a homogenizer, a Potter type homogenizer is considered as a preferable thing. This is combined with a glass cylinder and a rotating rod made of a fluorine resin fitted into the tube lumen to atomize the particles by sliding contact between the rotation and the piston movement of both. Since air is hard to wind up, it is effective for atomization.

It is preferable to perform under disperse bubble conditions as a dispersion process by these means, More preferably, a water dispersion can be applied as an external preparation, and the following is considered about the form, for example.

1.Application amount

The concentration of fullerenes in the composition for external application of skin is, for example, 0.00001 to 30% by mass, and 5% by mass or less is preferable in consideration of the feeling of use. When applied to the skin, the amount of the external composition is, for example, 0.001-20 ml of liquid per 1 square meter of skin area, preferably 0.01-5.0 ml, and is preferably applied by external application, wet foam or spraying.

2. Application form

Examples of the form of the composition for external application of skin are not particularly limited, and may take the form of all external preparations such as water soluble agents, ointments, emulsions, creams, gels, packs, solvents, detergents, papes, dispersions, and the like. There is no restriction | limiting in particular also regarding a paste form, a mousse form, a gel form, a powder form, a solution type, a solubilization type, and an emulsification type. In particular, aqueous solutions, emulsions, ointments, gels, water-soluble agents, cosmetics, and packs are applied externally to promote the penetration of fullerenes into the skin by using a humidification introducer, vibration introducer, ion introducer, sound wave introducer, and electromagnetic wave introducer. It can make a greater effect.

In the case of a liquid formulation, all physically possible methods, such as spray, a patch, a compress, a dipping, and a mask, can be used.

When the composition of the present invention is used as a cosmetic, skin cosmetic such as lotion, milky lotion, cream, pack, makeup base lotion, makeup cream, milky lotion, creamy or ointment foundation, hand cream, leg cream, body lotion, etc. It can be made into a body cosmetic.

Example

Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited to these Examples at all. In addition, in the following,% display represents the mass% unless there is particular notice.

≪ Example 1 >

Using the three-dimensional skin tissue model (Test skin LSE-high, Toyobo Co., Ltd.) in accordance with the procedure of Table 1 below, a test sample was produced, and wrinkle suppression test by lipofullerene was performed.

As a test sample, a three-dimensional skin tissue model sample of control (administered PBS (-) only), 500 μM of lipid peroxide 2,4-nonadienal (NDA), 500 μM of NDA and squalane, and 500 μM of NDA and lipofullerene was prepared. The evaluation of the image | photographing by the stereomicroscope of the replica using the silicone rubber duplication method, analysis of the wrinkle degree, etc. was performed. Lipofullerene was adjusted to dissolve fullerene (including C ₆₀ , C ₇₀ ) in vegetable squalane (Uniquema) to contain 500 ppm as C ₆₀ concentration.

The result is shown in FIG. In addition, in experimental verification, human skin tissue samples were incubated for 42 hours in a medium containing 500 µM of 2,4-nonadienal (NDA), and lipofullerene was administered to the medium 5 hours before NDA administration. In FIG. 1, (a) control (PBS (-)), (b) administration of NDA only (500 micrometers), and (c) show the comparison result when administering vegetable squalane instead of lipo fullerene. As shown in the figure, the lipofullerene administration sample suppressed wrinkle formation on the skin surface even when compared to the NDA administration sample and the control and squalane administration samples. That is, as shown in Fig. 1 (c), the surface irregularities were hardly improved in the case of squalane alone. As shown in Fig. 1 (d), the surface irregularities were almost improved in the case of lipofullerene administration.

In addition, the surface shape of the three-dimensional skin tissue model was observed with a scanning electron microscope (Fig. 2) for the same test sample as described above, and the cross-sectional structure of the three-dimensional skin tissue model was observed with an electron microscope using DMSO freezing cutting method. (FIG. 3). In FIGS. 2 and 3, (a) is control (PBS (-)), (b) is administered only NDA (500 μM), (c) is administered vegetable squalane instead of lipofullerene, (d) is lipofullerene The result at the time of administration is shown.

In the case of lipofullerene, as shown in FIG. 2 (d), a large number of keratin exfoliation occurring on the skin surface is controlled, and as shown in FIG. 3 (d), it is also possible to prevent multilayer separation and atrophy of the dermis in the skin cross section. Confirmed. As shown in Fig. 2 (c) and Fig. 3 (c), squalane alone had a weak effect.

<Example 2>

Fullerene dissolved in squalane, that is, lipofullerene, was used to confirm the effect of cell death (apoptosis) inhibition of human skin keratinocytes (HaCaT) by NDA by TUNEL staining.

Fig. 4 shows the result, in which the control, the administration of NDA (30 μM) only, and the administration of lipofullerene when the vegetable squalane was administered instead of the lipofullerene are shown in order from the left. In lipofullerene (4 ppm: 20 ppm as C ₆₀ ), it was confirmed that cleavage of the two DNA strands was remarkably suppressed. On the other hand, such a thing was almost invalid by squalane alone.

<Example 3>

The ratio of C ₆₀ fullerene was dissolved in C ₆₀ specimen of approximately 95% in vegetable squalane, followed by dispersion in (one of O to screen a reduced pressure in the aspirator reduces the bubble) Potter-type homo makes cold Four conditions by homogenizer O / W emulsion was formed.

Each of these emulsions was added to a human skin keratinocyte: HaCaT Cell culture system to evaluate cell viability. As a result, it was confirmed that the improvement was large.

<Example 4>

A 24 mm diameter human skin normal tissue model with the dermis, basement membrane, epidermis and stratum corneum was produced in the following procedure.

Culture of collagen gels using the collagen gel culture kit (Nitta Gelatin Inc.) was based on Nitta Gelatin Inc.'s protocol (see http://www.nitta-gelatin.co.jp/products/labo/column_1.html). I did it. To the pellets containing the centrifuged recovered fibroblasts OUMS-36 5 × 10 ⁵ / well, a cooled Type I collagen gel mixed solution was added and mixed to be uniform. The type I collagen mixed solution containing the fibroblast OUMS-36 was dispensed into a culture dish, and allowed to stand for 30 minutes at 37 ° C. in an incubator to gel.

Next, type I collagen gel was cultured in a liquid medium in a medium (DMEM / 10% FCS). After 5 days the gel contracted.

Subsequently, the gel for base film formation containing type IV collagen, laminin, and entaxin was layered on the type I collagen gel.

Subsequently, 5 × 10 ⁵ / well of human skin keratinocytes (HaCaT) are seeded on the basement membrane-forming gel, which is then cultured with medium (DMEM / Ham's F12 (1: 1) + 5% FBS + 15% Knock out serum replacement (SR). , Introgen Inc.)) was incubated for 1 day.

Subsequently, liquid culture was performed for 4 days by replacing with medium (DMEM / Ham's F12 (1: 1) + 1% FBS + 15% Knock out serum replacement).

Subsequently, a gel layer of human skin keratinocytes was layered on a medium (DMEM / Ham's F12 (1: 1) + 15% Knock out serum replacement), and the human skin keratinocytes were incubated for 14 days by air exposure. . This formed the epidermis and the stratum corneum on the base film.

The test sample was produced according to the procedure of following Table 2 using the obtained human skin normality roughness model, and the evaluation test of the NDA protective effect by lipo fullerene was done.

As a test sample, a human skin normal tissue model sample of control (administered PBS (-) only), NDA 450 μM administration, NDA 450 μM + squalane administration, NDA 450 μM + lipofullerene (2-500 ppm as C ₆₀ ) administration was prepared, and the surface shape thereof was prepared. It was observed with a scanning electron microscope (FIG. 5), and the cross-sectional structure was observed with an electron microscope using DMSO freezing cutting method (FIG. 6). As shown in FIG. 5, FIG. 6, in the NDA administration sample and the NDA + squalane administration sample, the structure of the skin was injured by NDA. On the other hand, administration of lipo fullerene suppressed the injury of the skin by NDA, and the protective effect increased depending on the fullerene content.

<Example 5>

Using the same sample as in Example 4, the NDA defense effect (cell nucleus retention effect) by lipofullerene was examined by fluorescence microscopy using Hoechst33342 nuclear staining. 150 μl / well of squalane or lipofullerene (2, 50, 200 ppm as C ₆₀ ) was administered to a human skin normal tissue model (Φ 24 mm) and incubated (5 hours, 37 ° C.). It was then exchanged with a medium containing 450 μM NDA / well and incubated (42 hours, 37 ° C., 5% CO ₂ ). After rinsing three times with 200 µl of PBS (-), Hoechst33342 nuclear staining was performed on the skin sections (5 mm thick), and observed under a fluorescence microscope.

Nuclear staining of the human skin normal tissue model was performed in the following procedure.

The fluorescence micrograph of the cross section of a human skin normal tissue model section (thickness 5 micrometers) is shown in FIG. In control, the nucleus was healthy. In NDA administered samples and NDA + squalane administered samples, DNA adiabatic, nuclear condensation and modification, which are one of the symptoms of apoptosis in epidermal cells, were caused by NDA. On the other hand, administration of lipofullerene prevented CDA-induced injury to the cell nucleus in a concentration-dependent manner of C ₆₀ .

<Example 6>

For lipofullerene blended cosmetics, a randomized double blind matched pair comparison test was performed. To 23 subjects, cosmetics (cream, left: test article, fullerene 1.8 µg / day, right: placebo, fullerene 0.0 µg / day) shown in Table 4 were applied. In group I (11), subjects were applied to the right eye and placebo was applied to the left eye. In group II (12 patients), placebo was applied to the right eye and the subject was left. Applied inside.

The subject's selection criterion was adult females having the same wrinkle grade as 2-3 (left and right same grade). The cosmetics were prepared twice a day in the morning and at night, after cleansing the skin with a designated skin care preparation, and then applied to the half of the designated side before makeup. The used cosmetics were taken every day (about 0.3g for half eye) according to the sample and placed in four places (forehead, cheeks, chin, and eyes) of the face and absorbed enough to half the face. Eyes gently absorbed and penetrated firmly. Use period was eight weeks.

Wrinkle area ratio was made into an evaluation item, and the evaluation method was based on the cosmetic functional evaluation method guideline (Japan Cosmetics Society 2006). The wrinkles before and after the use of the cosmetics were irradiated with parallel light in a constant direction (30 ° from the horizontal plane) in the replica of the eye area and the eye area under the image. Here, the length and depth of the shaded area were calculated. Similarly, the shadow area and its length were also calculated from the standard scale, a calibration curve was produced, and the numerical value of the wrinkled eye replica was corrected.

The evaluation results of the wrinkle area ratio and the wrinkle area percentage change amount are shown in Table 5 and FIG. 8.

The wrinkle area ratio decreased in the test subjects at 4 and 8 weeks as compared to before use. Placebo increased in both 4 and 8 weeks. Then, the t-test corresponding to the test article and the placebo in the wrinkle area ratio change amount was found to be significant (p = 0.021) at 8 weeks. In addition, no harmful event that is a safety problem was expressed.

In the above, it was confirmed that the lipofullerene blended cream has an anti-wrinkle action to reduce wrinkles.

<Example 7>

In the same manner as in Example 4, a human skin normal tissue model having the dermis, basement membrane, epidermis and stratum corneum was produced.

Using this human skin normal tissue model, a test sample was prepared according to the procedure shown in Table 6 below, and lipo fullerene and radical sponge [Radical Sponge (R), Vitamin C60 Bio Research Corporation, mixed fullerene (C ₆₀ , C ₇₀ ) were prepared. The inclusion of 10% polyvinylpyrrolidone and then dissolved in water and C ₆₀ 200 ppm or more] were compared against the anti-wrinkle effect.

Human skin normal tissue model with control (administered PBS (-) only), NDA 500 μM administration, NDA 500 μM + radical sponge (4 ppm as C ₆₀ , 30 ppm) administration, NDA 500 μM + lipofullerene (4 ppm as C ₆₀ , 30 ppm) The sample was prepared and the surface shape was observed with the scanning electron microscope (FIG. 9). Moreover, as shown in FIG. 10, the analysis by the NIH-image of the SEM image of the surface shape of a human skin normal tissue model was performed. Specifically, a line histogram is created by NIH-image 1.6.3 for three regions of the SEM image, a line is drawn in the middle of the unevenness, the upper and lower areas are summed, the average value is obtained, and the wrinkle degree of each treatment section (µm ² / Realm). The result is shown in FIG.

9-11, it was shown that the lipofullerene reduces the skin irritation by the irritating substance (NDA) rather than an equivalent amount of radical sponge.

In addition, the cross-sectional structure of the same human skin normal tissue model sample was observed by scanning electron microscopy using DMSO freezing cutting method (FIG. 12), and the NIH-image of the SEM image of the cross-sectional shape of the human skin normal tissue model was observed. Analysis was performed (FIG. 13).

It is shown from FIG. 12, FIG. 13 that the lipo fullerene reduces skin irritation by an irritating substance (NDA) rather than an equivalent amount of radical sponge.

Moreover, the imaging | photography by the stereomicroscope of the replica using the silicone rubber duplication method was performed about the same human skin normal tissue model sample (FIG. 14), and the NIH-image of the replica image was analyzed (FIG. 15).

From FIG. 14, FIG. 15, the lipo fullerene suppressed wrinkle formation of the skin surface rather than an equivalent amount of radical sponge.

As mentioned above, as a fullerene containing agent, it became clear that lipofullerene is a more promising anti-wrinkle agent than a radical sponge.

Claims (4)

Wrinkle suppression composition characterized by containing the oil-soluble fullerene which melt | dissolved fullerene in squalane. The method of claim 1,
Wrinkle suppression composition characterized in that it contains at least one selected from C ₆₀ , C ₇₀ and salts or derivatives thereof as the fullerenes. The method according to claim 1 or 2,
DNA double strand / one chain cleavage and / or DNA base damage by oxidative stress comprising lipid peroxide and / or free radicals in human skin keratinocytes and / or human skin fibroblasts and / or skin tissues comprising them Wrinkle suppressing composition, characterized in that to defend against DNA injury comprising a. It consists of the composition for wrinkle suppression in any one of Claims 1-3, The external composition for skin characterized by the above-mentioned.

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