Anti-aging agents for the skin

Abstract

To provide a skin anti-aging agent having anti-aging action.SOLUTION: A skin anti-aging agent contains an elastase activity inhibitor. The elastase activity inhibitor is a substance taken from Plukenetia volubilis nuts. A preferred method for taking the Plukenetia volubilis nuts is, for example, supercritical CO2 extraction. The elastase activity inhibitor can be at least one selected from the group consisting of α-tocopherol, γ-tocopherol, and δ-tocopherol.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a skin anti-aging agent, and particularly to a skin anti-aging agent having an elastase activity inhibitory effect.

Background Art

[0002] The skin consists of two layers, the epidermal layer and the dermal layer, and the underlying dermal layer retains moisture and supports the functions of the skin. This dermal layer is composed of intercellular matrices such as collagen and elastin and dermal fibroblasts. Collagen is known as a high-molecular-weight protein that functions as the main component of extracellular matrix in various tissues. Fibrous type I collagen maintains the firmness and elasticity of the skin, while non-fibrous type IV collagen and type VII collagen play roles in the formation of the basement membrane and the adhesion between the basement membrane and the dermis, respectively. Elastin, also called elastic fiber, is a flexible and stretchable fibrous protein. In the dermal layer, it connects collagen fiber bundles to each other and provides elasticity to the skin. It is known that elastin breaks down under the influence of aging and ultraviolet radiation, decreases with age, and causes wrinkle formation. This is caused by the activation of elastase along with aging and ultraviolet exposure, and inhibition of elastase activity can be expected to achieve skin anti-aging effects.

[0003] The present inventors have found that sacha inchi nut oil collected by various methods has an elastase activity inhibitory effect, and have completed the present invention.

Prior Art Literature

Patent Literature

[0004]

Patent Document 1

Summary of the Invention

Problem to be Solved by the Invention

[0005] This invention has been made in view of the above-mentioned problems, and its purpose is to provide a skin anti-aging agent having an elastase activity inhibitory function. [Means for solving the problem]

[0006] The anti-aging agent for skin of the present invention contains an elastase activity inhibitor, wherein the elastase activity inhibitor is an elastase activity inhibitor extracted from sacha inchi nuts, and / or at least one selected from the group consisting of α-tocopherol, γ-tocopherol, and δ-tocopherol.

[0007] In the present invention, it is preferable that the elastase activity inhibitor extracted from the sacha inchi nut is extracted by either a cold-pressing method or a supercritical fluid extraction method.

[0008] Furthermore, in the present invention, the elastase activity inhibitor is preferably supercritical CO2 extracted sacha inchi nut oil.

[0009] Here, it is preferable that the added concentration of the supercritical CO2-extracted sacha inchi nut oil is 0.1 mg / mL or higher.

[0010] In the present invention, the elastase activity inhibitor is cold-pressed sacha inchi nut oil, and it is preferable that the added concentration of the cold-pressed sacha inchi nut oil is 0.25 mg / mL or more and less than 1 mg / mL.

[0011] In the present invention, the elastase activity inhibitor is at least one selected from the group consisting of α-tocopherol, γ-tocopherol, and δ-tocopherol, and it is preferable that the content of the elastase activity inhibitor is 50 μg / mL or more.

[0012] The skin product of the present invention is characterized by containing any of the above-mentioned anti-aging agents for the skin. [Effects of the Invention]

[0013] According to the present invention, it is possible to provide a skin anti-aging agent that can promote the elastase activity inhibitory effect. Furthermore, according to the present invention, it is also possible to provide a skin product having an elastase activity inhibitory effect. [Brief explanation of the drawing]

[0014] [Figure 1] This graph shows the elastase activity inhibitory effect (percentage of enzyme activity (%)) when sacha inchi nut oil extracted using different methods is added. [Figure 2] This graph shows the effect of different concentrations of supercritical CO2 extracted sacha inchi nut oil on inhibiting elastase activity (percentage of enzyme activity (%)). [Figure 3] This graph shows the effect of different concentrations of cold-pressed sacha inchi nut oil on inhibiting elastase activity (percentage of enzyme activity). [Figure 4] This graph shows the elastase activity inhibitory effect (percentage of enzyme activity) when α-tocopherol is added to the experimental system at different concentrations. [Figure 5] This graph shows the elastase activity inhibitory effect (percentage of enzyme activity (%)) when γ-tocopherol is added to the experimental system at different concentrations. [Figure 6] This graph shows the elastase activity inhibitory effect (percentage of enzyme activity (%)) when δ-tocopherol is added to the experimental system at different concentrations. [Figure 7] This graph shows the elastase activity inhibitory effect (percentage of enzyme activity (%)) when α-tocopherol, γ-tocopherol, and δ-tocopherol are added to the experimental system at the same concentration. [Modes for carrying out the invention]

[0015] The anti-aging agent for skin of the present invention contains an elastase activity inhibitor collected from sacha inchi nuts. The present inventors have found that sacha inchi nut oils collected by various methods contain an elastase activity inhibitor, and thus completed the present invention. For example, a composition containing sacha inchi nut oil collected by a supercritical extraction method, a cold pressing method or the like can be used as an anti-aging agent for skin.

[0016] The cold pressing method, hexane extraction method, ethanol extraction method and supercritical extraction method are described below. The cold pressing method is a method of squeezing out oil by applying pressure at a relatively low temperature without applying heat to the raw material. For example, pressure can be slowly applied to the raw material over time, with measures taken to suppress the generation of frictional heat, and oil can be collected while being controlled so that the temperature does not exceed 60°C at maximum. The cold pressing method is the most common method for collecting edible oil. Sacha inchi nut oil obtained by the cold pressing method (cold-pressed sacha inchi nut oil) is oil squeezed from sacha inchi seeds by applying pressure without applying heat.

[0017] The hexane extraction method is a method in which a raw material is immersed in n-hexane to elute the oil in the raw material, after which only highly volatile n-hexane is evaporated. Generally, this method has higher oil collection efficiency than the cold pressing method, but it is also considered concerning that solvent may remain. Sacha inchi nut oil obtained by n-hexane extraction (n-hexane-extracted sacha inchi nut oil) can be obtained, for example, by: stirring sacha inchi nut powder in n-hexane, filtering the mixture, passing the filtrate through a syringe filter, then allowing it to stand in an incubator, further evaporating n-hexane using a rotary evaporator, and recovering the remaining liquid.

[0018] The ethanol extraction method is a method of extraction using ethanol in place of n-hexane. While this is not a common method for collecting oil, it can collect oil based on the same principle as the hexane extraction method. Sacha inchi nut oil obtained by the ethanol extraction method (ethanol-extracted Sacha inchi nut oil) is obtained, for example, by stirring Sacha inchi nut powder in ethanol, filtering the mixture, passing it through a syringe filter, then evaporating ethanol using a rotary evaporator, centrifuging the liquid residue to separate components, and collecting the supernatant.

[0019] The supercritical CO₂ extraction method is an extraction method that uses supercritical CO₂ as a solvent. Said supercritical CO₂ is carbon dioxide brought to a supercritical state by applying temperature and pressure, and has attracted attention in recent years as a new solvent for extracting oil. Sacha inchi nut oil collected by the supercritical CO₂ extraction method (supercritical CO₂-extracted Sacha inchi nut oil) can be obtained, for example, by filling powdered Sacha inchi nuts into a supercritical extraction system to obtain supercritical CO₂-extracted Sacha inchi nut oil. The particle diameter of the Sacha inchi nut powder is preferably 10 to 10000 µm, more preferably 50 to 5000 µm. Powdered Sacha inchi nuts can be obtained, for example, by processing commercially available Sacha inchi nuts into a powder using a grinder or the like.

[0020] When obtaining an extract (Sacha inchi nut oil) using a supercritical CO₂ extraction system, it is necessary to set the temperature, pressure, and CO₂ flow rate to predetermined conditions. The temperature is in the range of 25°C to 150°C, preferably in the range of 32°C to 120°C, more preferably in the range of 40°C to 100°C. The pressure is in the range of 5 MPa to 40 MPa, preferably in the range of 7.5 MPa to 40 MPa, more preferably in the range of 15 MPa to 25 MPa. Furthermore, the CO₂ flow rate is preferably in the range of 1 mL / min to 40 mL / min. In the present invention, the supercritical CO₂ extract also includes subcritical CO₂ extract, as long as the effects of the present invention can be exerted.

[0021] Sacha inchi nut oil, extracted by methods such as cold pressing and supercritical fluid extraction, is an elastase inhibitor that inhibits elastase activity and is used as an ingredient in anti-aging cosmetics. In particular, sacha inchi nut oil extracted by supercritical CO2 extraction has the advantage of being highly effective in inhibiting elastase activity. For example, supercritical CO2 extracted sacha inchi nut oil can efficiently inhibit elastase activity, and therefore, when used alone or in combination with other active ingredients, it can be a promising ingredient in anti-aging cosmetics.

[0022] The anti-aging agent for skin containing the elastase activity inhibitor of the present invention can achieve its effects by direct application to the skin, for example, by incorporating the anti-aging agent into a skin product, anti-aging of the skin can be achieved.

[0023] In this invention, "skin products" refers to products (cosmetics) applied to the skin of the human body, and includes not only cosmetics such as basic cosmetics, makeup cosmetics, facial soaps, body soaps, and body lotions, but also medicated cosmetics classified as quasi-drugs. [Examples]

[0024] The present invention will be specifically described below with reference to examples, but these examples do not limit the present invention in any way, and various applications are possible without departing from the technical scope of the present invention.

[0025] (Sample preparation) (1) Cold-pressed sacha inchi nut oil Sacha Inchi nuts (from Thailand) were commercially obtained from Imagawa Confectionery Co., Ltd. These sacha inchi nuts were pressed using a cold-pressing machine, and the extracted crude oil was centrifuged at 4000 rpm for 10 minutes. The supernatant was passed through a 1.0 μm syringe filter, and then through a 0.22 μm syringe filter again to obtain the liquid which was used as cold-pressed sacha inchi nut oil.

[0026] (2) Supercritical CO2 extracted sacha inchi nut oil Sacha Inchi nuts (from Thailand) were commercially obtained from Imagawa Confectionery Co., Ltd. These sacha inchi nuts were ground in a grinder, and 3.0 g of this sacha inchi nut powder was loaded into a supercritical extraction system manufactured by JASCO Corporation. The system was treated with supercritical CO2 as the extraction solvent under the conditions of 40°C, 20 MPa pressure, and a CO2 flow rate of 3.0 mL / min. The resulting liquid was used as supercritical CO2 extracted sacha inchi nut oil.

[0027] (3) Ethanol-extracted sacha inchi nut oil Sacha Inchi nuts (from Thailand) were commercially obtained from Imagawa Confectionery Co., Ltd. These sacha inchi nuts were ground in a grinder, and 30.0 g of the sacha inchi nut powder was placed in a container. 90 mL of ethanol (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added, and the mixture was stirred at room temperature for three nights. After stirring, the supernatant was filtered through filter paper, and the liquid was further passed through a syringe filter with a pore size of 0.22 μm before the ethanol was evaporated using a rotary evaporator. Of the liquid and solid residues, the liquid was centrifuged at 4000 rpm for 10 minutes, and the supernatant was recovered as ethanol-extracted sacha inchi nut oil.

[0028] (4) Hexane-extracted sacha inchi nut oil Sacha Inchi nuts (from Thailand) were commercially obtained from Imagawa Confectionery Co., Ltd. These sacha inchi nuts were ground in a grinder, and 30.0 g of the powder was placed in a container. 90 mL of n-hexane (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added, and the mixture was stirred at room temperature for two nights. After stirring, the supernatant was filtered through filter paper, and the liquid was further passed through a syringe filter with a pore size of 0.22 μm. The mixture was then left to stand overnight in a 40°C incubator, and subsequently, the n-hexane was evaporated using a rotary evaporator. The liquid remaining after the n-hexane evaporation was recovered as hexane-extracted sacha inchi nut oil.

[0029] (5) Various tocopherols α-tocopherol was commercially obtained from Fujifilm Wako Pure Chemical Industries, Ltd., while γ-tocopherol and δ-tocopherol were commercially obtained from Sigma-Aldrich, respectively.

[0030] Test Example 1 (Measurement of elastase activity inhibitory effect) Each of the samples prepared in the "Sample Preparation" procedure described above—supercritical CO2 extracted sacha inchi nut oil, cold-pressed sacha inchi nut oil, hexane-extracted sacha inchi nut oil, and ethanol-extracted sacha inchi nut oil—was dissolved in dimethyl sulfoxide (DMSO) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) and added to 0.2 M Tris-HCl (pH 8.0) to achieve the desired concentration (the amount of DMSO added to 0.2 M Tris-HCl (pH 8.0) was kept constant regardless of the concentration of the sacha inchi nut oil). This was mixed in a 2:1:1 ratio with 1 mM N-succinyl-Ala-Ala-Ala-p-nitroanilide (Sigma-Aldrich) and 0.75 U / mL elastase derived from pig pancreas (Fujifilm Wako Pure Chemical Industries, Ltd.) in the wells of a 96-well colorimetric plate. Specifically, sacha inchi nut oil:N-succinyl-Ala-Ala-Ala-p-nitroanilide:elastase derived from pig pancreas at a predetermined concentration was mixed in a 2:1:1 ratio, and after reacting at 37°C, the absorbance at 405 nm of the test solution (each sample addition group) was measured using a microplate reader (CHIROMATE MODEL 4300, Awareness Technology Inc., FL). In addition, the absorbance of a control solution (control group) was measured in the same manner, prepared by mixing 0.2M Tris-HCl (pH 8.0) containing only DMSO, N-succinyl-Ala-Ala-Ala-p-nitroanilide, and elastase derived from pig pancreas in a ratio of 3:1:1, and reacting it at 37°C. However, both the control group and each sample-added group consisted of 5 wells per group. Furthermore, absorbance was measured for the blank (BLK) solution from which only the enzyme was removed, as well as from the test solutions to which the above-mentioned supercritical CO2 extracted sacha inchi nut oil, cold-pressed sacha inchi nut oil, hexane-extracted sacha inchi nut oil, and ethanol-extracted sacha inchi nut oil were added, and the elastase activity inhibitory effect was calculated using the following formula to determine the percentage of enzyme activity.

[0031] Enzyme activity percentage (%) = {(Absorbance of each test solution - Absorbance of BLK in each test solution) / (Absorbance of control - Absorbance of control BLK)} × 100

[0032] (Statistical processing) First, the Hartley test was used to confirm the equal variance of the values ​​in each group. Next, a one-way analysis of variance was performed, and for groups where a potential difference was indicated, the Tukey method or Dunnett method was used to further confirm whether there was a significant difference between the groups. In addition, the same statistical processing was performed for all subsequent test examples in the examples provided.

[0033] Figure 1 shows the percentage of enzyme activity (%) as an elastase activity inhibitory effect when supercritical CO2 extracted sacha inchi nut oil, cold-pressed sacha inchi nut oil, hexane-extracted sacha inchi nut oil, and ethanol-extracted sacha inchi nut oil are each added at a concentration of 0.5 mg / mL using the method described above. Figure 1 shows that the group of sacha inchi nut oil extracted using supercritical CO2 showed a significant and remarkable effect compared to the control group, and significantly reduced elastase activity compared to the group of cold-pressed sacha inchi nut oil, indicating superior elastase inhibitory effects. The group of cold-pressed sacha inchi nut oil significantly reduced elastase activity compared to the control group, but the effect was smaller, indicating that its elastase inhibitory effect was inferior to that of supercritical CO2 extracted sacha inchi nut oil. On the other hand, hexane-extracted sacha inchi nut oil and ethanol-extracted sacha inchi nut oil were both almost equivalent to the control, and no significant difference was observed. Furthermore, significant differences were observed between the supercritical CO2 extracted sacha inchi nut oil group and the cold-pressed sacha inchi nut oil group compared to the hexane-extracted sacha inchi nut oil group and the ethanol-extracted sacha inchi nut oil group.

[0034] Test Example 2 In Test Example 1, the sample was supercritical CO2 extracted sacha inchi nut oil, and the addition concentrations were changed to 0.1 mg / mL, 0.25 mg / mL, 0.5 mg / mL, and 1.0 mg / mL, respectively. The procedure was the same as in Test Example 1, and the percentage of enzyme activity was determined as the elastase activity inhibitory effect. The results are shown in Figure 2.

[0035] Figure 2 shows that in all groups with supercritical CO2-extracted sacha inchi nut oil concentrations of 0.1 mg / mL, 0.25 mg / mL, 0.5 mg / mL, and 1.0 mg / mL, elastase activity was significantly reduced compared to the control group (indicated as 0 mg / mL). Furthermore, the elastase activity inhibitory effect of supercritical CO2-extracted sacha inchi nut oil was concentration-dependent up to 0.5 mg / mL, significantly inhibiting elastase activity compared to 0 mg / mL (inhibition rate at 0.5 mg / mL was 54.4%), but the inhibitory effect appeared to plateau beyond that point.

[0036] Test Example 3 In Test Example 2, the elastase activity inhibitory effect (percentage of enzyme activity) was determined in the same manner as in Test Example 2, except that the sample was changed from supercritical CO2 extracted sacha inchi nut oil to cold-pressed sacha inchi nut oil. The results are shown in Figure 3.

[0037] Figure 3 shows that cold-pressed sacha inchi nut oil significantly inhibited elastase activity in the 0.25 mg / mL group compared to the 0 mg / mL control group (inhibition rate: 19.5%), but the inhibitory effect appeared to plateau at concentrations above this level. In the 0.5 mg / mL group, the inhibition rate increased slightly (inhibition rate: 22.4%), but was almost the same as the 0.25 mg / mL group, while no significant difference was observed in the 1 mg / mL group.

[0038] Test Example 4 In Test Example 1, the elastase activity inhibitory effect (percentage of enzyme activity) was determined in the same manner as in Test Example 1, except that the sample was α-tocopherol and its addition concentration was changed to 50 μg / mL, 100 μg / mL, and 150 μg / mL. The results are shown in Figure 4.

[0039] Figure 4 shows that in all groups, with α-tocopherol concentrations of 50 μg / mL, 100 μg / mL, and 150 μg / mL, elastase activity was significantly reduced compared to the control group (indicated as 0). Furthermore, it was found that the elastase activity inhibitory effect of α-tocopherol is concentration-dependent.

[0040] Test Example 5 In Test Example 4, the elastase activity inhibitory effect (ratio of enzyme activity) was determined in the same manner as in Test Example 4, except that the sample was changed from α-tocopherol to γ-tocopherol. The results are shown in Figure 5.

[0041] Figure 5 shows that in all groups, with γ-tocopherol concentrations of 50 μg / mL, 100 μg / mL, and 150 μg / mL, elastase activity was significantly reduced compared to the control group (indicated as 0). Furthermore, it was found that the elastase activity inhibitory effect of γ-tocopherol is concentration-dependent.

[0042] Test Example 6 In Test Example 4, the elastase activity inhibitory effect (ratio of enzyme activity) was determined in the same manner as in Test Example 4, except that the sample was changed from α-tocopherol to δ-tocopherol. The results are shown in Figure 6.

[0043] Figure 6 shows that in all groups with δ-tocopherol concentrations of 50 μg / mL, 100 μg / mL, and 150 μg / mL, elastase activity was significantly reduced compared to the control group (indicated as 0). Furthermore, it was found that the elastase activity inhibitory effect of δ-tocopherol is concentration-dependent.

[0044] Test Example 7 In Test Example 2, the elastase activity inhibitory effect (ratio of enzyme activity) was determined in the same manner as in Test Example 2, except that the sample was changed from supercritical CO2 extracted sacha inchi nut oil to α-tocopherol, γ-tocopherol, and δ-tocopherol, respectively, and the concentration of each was changed to 150 μg / mL. The results are shown in Figure 7.

[0045] Figure 7 shows that in all three groups—α-tocopherol, γ-tocopherol, and δ-tocopherol—elastase activity was significantly reduced compared to the control group (indicated as 0), indicating that the elastase activity inhibitory effects of these three types of tocopherols are equivalent.

[0046] The results above show that supercritical CO2-extracted sacha inchi nut oil exhibits excellent elastase activity inhibitory effects, and that cold-pressed sacha inchi nut oil also significantly reduces elastase activity, albeit weakly. Furthermore, ethanol-extracted and hexane-extracted sacha inchi nut oils did not show any elastase activity inhibitory effects. This suggests that the active ingredients in sacha inchi nut oil that contribute to elastase activity inhibitory effects vary depending on the extraction method. Although the specific components and their proportions are not clear, academic literature has reported that sacha inchi nut oil contains α-tocopherol, γ-tocopherol, and δ-tocopherol. When the elastase activity inhibitory effect of these three types of tocopherols was investigated, it was revealed for the first time in this application that they possess an elastase activity inhibitory effect. In other words, it is possible that these three types of tocopherols are the active ingredients of supercritical CO2 extracted sacha inchi nut oil and cold-pressed sacha inchi nut oil, and that the difference in their content may be reflected in the difference in the elastase activity inhibitory effect of supercritical CO2 extracted sacha inchi nut oil and cold-pressed sacha inchi nut oil. Furthermore, it is possible that the content of the above three types of tocopherols is the reason why hexane extracted sacha inchi nut oil and ethanol extracted sacha inchi nut oil do not show an elastase activity inhibitory effect. Therefore, based on the results of this application, we believe that the above three types of tocopherols may be usable as elastase activity inhibitors, either individually or in appropriate combinations.

[0047] Sacha inchi is a plant of the Euphorbiaceae family native to South America. Its seeds yield a high-quality oil and have been known as a healthy food. However, there have been no academic reports to date on the elastase activity inhibitory effect of sacha inchi nut oil. This application is the first to discover this effect and reveal a new function of sacha inchi nut oil. Furthermore, the use of supercritical fluid extraction as a method for extracting sacha inchi nut oil is also a first for this invention. Moreover, it is also a first for this application to discover that sacha inchi nut oil obtained by supercritical fluid extraction exhibits a particularly excellent elastase activity inhibitory effect. These effects can be expected even when sacha inchi nut oil is taken orally, but it is anticipated that applying it directly to the skin will produce faster results. As described above, this application discloses sacha inchi nut oil extracted by various methods. For example, it has been found that sacha inchi nut oil extracted using supercritical CO2 yields a particularly significant elastase activity inhibitory effect. It is presumed that the ratio of active ingredients in sacha inchi nut oil that yield the elastase activity inhibitory effect will vary depending on the extraction method, or that the presence or absence of active ingredients may change depending on the extraction method. Furthermore, this application suggests that α-tocopherol, γ-tocopherol, and δ-tocopherol may be active ingredients that yield the elastase activity inhibitory effect. It is also suggested that any of these three types of tocopherols may be used individually, in combination, or even in combination with supercritical CO2 extracted sacha inchi nut oil as elastase activity inhibitors. In other words, not only supercritical CO2 extracted sacha inchi nut oil and cold-pressed sacha inchi nut oil, but also α-tocopherol, γ-tocopherol, and δ-tocopherol have significant elastase activity inhibitory effects, and are expected to be utilized as ingredients in anti-aging cosmetics in the future.

Claims

1. An anti-aging agent for the skin, characterized in that it contains an elastase activity inhibitor extracted from sacha inchi nuts, wherein the elastase activity inhibitor is extracted by a supercritical CO2 extraction method.

2. The anti-aging agent for skin according to Claim 1, characterized in that the concentration of the substance collected by the supercritical CO2 extraction method is 0.1 mg / mL or more.

3. A skin product characterized by containing the anti-aging agent for skin described in any one of claims 1 or 2.

4. A method for producing an anti-aging agent for skin containing an elastase activity inhibitor, characterized in that the elastase activity inhibitor is obtained from sacha inchi nuts by a supercritical CO2 extraction method.

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