Caspase-14 activator and Anti-oxidizing agent

Abstract

To develop a novel plant extract having an excellent skin-conditioning effect.SOLUTION: Provided are a caspase-14 activator or an anti-oxidizing agent containing a Japanese apricot extract, and an external preparation for skin containing the caspase-14 activator or the anti-oxidizing agent.EFFECT OF THE INVENTION: According to the present invention, skin is protected from dryness, ultraviolet light and active oxygen which are harmful to skin by increasing the enzymatic activity of caspase-14 and deleting active oxygen, and a skin-conditioning effect that the external preparation for skin should include can be realized.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a caspase-14 activator and an antioxidant characterized by containing an extract of Prunus salicina Lindl. × Prunus mume Siebold & Zucc. [Background technology]

[0002] One of the functions that topical skin preparations should have is a skin conditioning effect, that is, the effect of regulating the skin condition by maintaining moisture and improving the skin texture, etc. In realizing such a function, it is considered important to protect the skin from harmful factors such as dryness, ultraviolet rays, and active oxygen.

[0003] Activation of caspase-14 is one of the ways to protect the skin from dryness and UV rays. Caspase-14 is a type of aspartic acid-specific cysteine ​​protease that uses profilaggrin as a substrate and contributes to the production of filaggrin, a profilaggrin degradation product that plays an important role in maintaining the skin's physical and chemical barrier function, and natural moisturizing factor (NMF), which is deeply involved in skin moisturization (Non-Patent Document 1).

[0004] Several methods for promoting the expression of the caspase-14 gene have been proposed. Examples include a method using an extract derived from witch hazel as an active ingredient (Patent Document 1), and a method using one or more active ingredients selected from yeast extract, fennel extract, wild thyme extract, wild strawberry extract, herb extract, Phellodendron bark extract, Coptis japonica extract, horsetail extract, and Chinese holly extract (Patent Document 2). Meanwhile, caspase-14 is an enzyme that exerts its function by being synthesized as a protein without protease activity called procaspase-14 and then converted to the active form, caspase-14 (Non-Patent Document 1). From the perspective of protecting skin from dryness and ultraviolet rays, its activity is more important than the level of gene expression. However, few methods are known for enhancing the enzymatic activity of caspase-14.

[0005] Reactive oxygen species generated in the environment and within the body can cause damage to molecules, tissues, and organs in the body. Reactive oxygen species include superoxide, hydroxyl radical, hydrogen peroxide, and singlet oxygen, with hydroxyl radicals in particular being known to be extremely reactive. One way to protect the skin from such reactive oxygen species is to use antioxidants, which have the ability to eliminate the generated reactive oxygen.

[0006] For example, chemically synthesized substances such as BHT (dibutylhydroxytoluene) and TBHQ (t-butylhydroquinone) are known to have high antioxidant properties (Non-Patent Document 2). However, several concerns have been raised about these compounds, including possible mutagenicity and suspected carcinogenicity. As a result, there are restrictions on their incorporation, and some people are concerned about applying products containing these compounds to the human body, making them difficult to use in general applications.

[0007] Therefore, safe and highly effective antioxidants have been sought, and plant extracts that can be used as antioxidants have been discovered. For example, an antioxidant containing an extract of a plant of the genus Glaux in the family Primulaceae as an active ingredient (Patent Document 3) and an antioxidant characterized by containing an extract of the mast tree (Polyalthia longifolia) of the genus Polyalthia in the family Annonaceae (Patent Document 4) have been disclosed. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Denecker, G., Ovaere, P., Vandenabeele, P., & Declercq, W. (2008). Caspase-14 reveals its secrets. The Journal of cell biology, 180(3), 451-458. [Non-patent document 2] Kajimoto, G., Takashima, R., & Murakami, C. (1998). Effect of antioxidants on rancidity of oils in the presence of acids and bases. Journal of Japan Oil Chemists' Society, 47(6), 591-597. [Patent documents]

[0009] [Patent Document 1] Patent Publication No. 2018-203725 [Patent Document 2] Patent Publication No. 2022-144014 [Patent Document 3] Patent Publication No. 2011-168559 [Patent Document 4] Patent Publication No. 2011-037764 Summary of the Invention [Problem to be solved by the invention]

[0010] Although many plant extracts have become known that can contribute to protecting the skin from harmful dryness, ultraviolet rays, and active oxygen, the development of new plant extracts with superior effects that can fully fulfill the desired functions while also meeting the ever-diversifying needs of users remains a constant challenge. The object of the present invention is to invent a caspase-14 activator that enhances the enzymatic activity of caspase-14, and an antioxidant that has the effect of scavenging active oxygen, in accordance with the circumstances described in the [Background Art]. [Means for solving the problem]

[0011] The present inventors have investigated various plant extracts and found that an extract of Asiatic rosa var. asiaticus has excellent caspase-14 activation and antioxidant effects, leading to the completion of the present invention.

[0012] That is, the present invention relates to a caspase-14 activator and antioxidant characterized by containing an extract of Asiatic rosary, and also to an external skin preparation characterized by containing the caspase-14 activator and antioxidant. [Effects of the Invention]

[0013] According to the present invention, by increasing the enzymatic activity of caspase-14 and eliminating active oxygen, it is possible to protect the skin from dryness, ultraviolet rays, and active oxygen, which are harmful to the skin, and to achieve the skin-conditioning effect that a topical skin preparation should have. [Brief explanation of the drawings]

[0014] [Figure 1] Caspase-14 activation effect of the extract of Akane asiaticus [Figure 2] Antioxidant effect of Akane asiaticus extract [Figure 3] The moisturizing effect of madder plant extract on skin DETAILED DESCRIPTION OF THE INVENTION

[0015] The Tsuyuakane of the present invention is an interspecific hybrid between Japanese plum and Japanese apricot, "Tsuyuakane" (Prunus salicina Lindl. × Prunus mume Siebold & Zucc.), which was registered as a variety in 2009.

[0016] As long as the extract of the present invention contains the fruit of the Japanese laurel, it is acceptable for it to contain some parts other than the fruit (e.g., peel, seeds, trunk, branches, leaves) due to industrial production and operational reasons.

[0017] The solvent used to extract the rosa asiatica extract of the present invention is not particularly limited, but may be any suitable solvent, for example, extracted at low to elevated temperatures. Examples of suitable extraction solvents include water; lower monohydric alcohols such as methyl alcohol and ethyl alcohol; liquid polyhydric alcohols such as glycerin, propylene glycol, and 1,3-butylene glycol; ketones such as acetone and methyl ethyl ketone; alkyl esters such as ethyl acetate; hydrocarbons such as benzene and hexane; ethers such as diethyl ether; and halogenated alkanes such as dichloromethane and chloroform. Mixtures of one or more of water, ethyl alcohol, and 1,3-butylene glycol are particularly preferred, with water or a 50% by volume ethyl alcohol-water mixture being even more preferred.

[0018] The method for preparing the extract of the present invention is not particularly limited. For example, when using pure fruit of the Japanese laurel family that has not undergone significant changes in water content since harvest, for example, fruit stored at room temperature or frozen, the extract can be prepared by extracting at 0 to 100°C, preferably 20 to 40°C, for 1 hour to 7 days, more preferably 1 to 24 hours, using a solvent in an amount of 0.1 to 1000 times, more preferably 1 to 100 times, the mass of the fruit. When using dried fruit, the extract can be prepared by extracting at 0 to 100°C, preferably 20 to 40°C, for 1 hour to 7 days, more preferably 1 to 24 hours. Alternatively, the extract can be heated at 40 to 80°C for 1 to 6 hours. Furthermore, the above-mentioned solvent can be added to the juice obtained from the Japanese laurel family fruit, and then, if necessary, separation or other procedures can be performed to obtain the desired fraction as the Japanese laurel family extract.

[0019] The extract of Japanese laurel obtained under the above conditions may be used as the extracted solution, but if necessary, it can also be further processed, such as by solvent exchange or filtration, or concentrated or powdered and used as appropriate.

[0020] When the caspase-14 activator or antioxidant of the present invention is incorporated into a topical skin preparation, the amount of the Asiatic iris extract to be incorporated is not particularly limited as long as an effective amount is present, but a range of 0.0001 to 100% by mass, particularly 0.001 to 10% by mass, calculated as the evaporated dry matter in the composition of the caspase-14 activator or antioxidant, is preferred, and a range of 0.000001 to 100% by mass, particularly 0.001 to 1.0% by mass, is preferred, calculated as the evaporated dry matter in the composition of the topical skin preparation.

[0021] The caspase-14 activator, antioxidant, or topical skin preparation of the present invention may contain ingredients other than the extract of Citrus oryzae, as long as the effects of the present invention are not impaired, such as oils and fats, waxes, hydrocarbon oils, ester oils, higher alcohols, silicone oils, UV absorbers, UV scattering agents, moisturizers, surfactants, water-soluble polymers, thickeners, powders, various active ingredients, skin protectants, anti-aging agents, plant extracts, preservatives, anti-inflammatory agents, pH adjusters, sequestering agents, and antioxidants.

[0022] The dosage form of the caspase-14 activator, antioxidant, or topical skin preparation of the present invention is not particularly limited as long as it is physiologically acceptable, and may be any of an ointment, cream, emulsion, lotion, powder, essence, pack, gel, hair tonic, aerosol, etc. Furthermore, the caspase-14 activator and antioxidant of the present invention can be used in foods and beverages in the form of capsules, powder, granules, spheres, tablets, solids, liquids, gels, etc. [Example]

[0023] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. Unless otherwise specified, the blending amounts are expressed in mass %.

[0024] <Preparation of practical extract of Japanese laurel (Sample No. 1)> Purified water was added to roughly chopped daisy fruit in an amount five times its mass, and the mixture was extracted for 6 hours at 60°C. The solids were then removed by filtration, and a portion of the filtrate was taken to measure the loss on drying. Water was then added to the filtrate so that the dry matter was 1%, to obtain the experimental daisy extract (sample number 1).

[0025] <Preparation of comparative plum extract (sample number 2)> Dried and chopped Japanese plum fruit was mixed with purified water in an amount 40 times its mass and extracted for 6 hours at 60°C. The solids were then removed by filtration, and a portion of the filtrate was taken and measured for loss on drying. Water was then added to the filtrate so that the dry matter was 1%, to obtain a comparative plum extract (sample number 2).

[0026] <Preparation of comparative plum extract (sample number 3)> Dried and chopped plums were mixed with purified water in an amount 40 times their mass, and extracted for 6 hours at 60° C. The solids were then removed by filtration, and a portion of the filtrate was taken and measured for loss on drying. Water was then added to the filtrate so that the dry matter content was 1%, to obtain a comparative plum extract (sample number 3).

[0027] <Preparation of comparative plum extract (sample number 4)> After freezing, chopped plum fruit was added with purified water in an amount 40 times its mass, and extracted at 60°C for 6 hours. The solids were then removed by filtration, and the solvent was removed from the filtrate using an evaporator, followed by freeze-drying. A 50% by volume mixture of ethyl alcohol and purified water was added to the fruit so that the freeze-dried product was 1%, and the mixture was dissolved and filtered to obtain a comparative plum extract (sample number 4).

[0028] <Preparation of practical extract of Japanese laurel (Sample No. 5)> Purified water was added to crushed and damaged fruits of the Japanese laurel tree in an amount 40 times its mass, and the fruit was extracted at 40°C for 6 hours. The solids were then removed by filtration, and the solvent was removed from the filtrate using an evaporator, followed by freeze-drying. A 50% by volume mixture of ethyl alcohol and purified water was added to the fruit so that the freeze-dried product was 1%, and the fruit was dissolved and filtered to obtain the Japanese laurel tree extract for use (sample number 5).

[0029] <Preparation of practical extract of Japanese laurel (Sample No. 6)> After freezing, the fruits were chopped into approximately 1 cm cubes, and a 50% by volume ethyl alcohol-purified water mixture (40 times the mass of the fruit) was added to the fruit, followed by extraction at 40°C for 3 hours. The solids were then removed by filtration, and the solvent was removed from the filtrate using an evaporator. A portion of the filtrate was then freeze-dried to determine the percentage of the evaporated dry matter in the filtrate. Based on the results, a 50% aqueous solution of ethyl alcohol was added so that the evaporated dry matter was 1%, and the mixture was dissolved and filtered to obtain the experimental extract of the fruit (sample number 6).

[0030] <Preparation of comparative plum extract (sample no. 7)> Dried and chopped plums were mixed with a 50% by volume ethyl alcohol-purified water mixture in an amount 40 times the mass of the fruit, and extracted for 6 hours at 60°C. The solids were then removed by filtration, and a portion of the filtrate was taken to measure the loss on drying. Water was then added to the mixture so that the dry matter content was 1%, to obtain a comparative plum extract (sample number 7).

[0031] <Preparation of comparative plum extract (sample number 8)> A 50% by volume mixture of ethyl alcohol and purified water was added to chopped plum fruit after freezing, and the fruit was extracted for 6 hours at 60°C. The solids were then removed by filtration, and the solvent was removed from the filtrate using an evaporator, followed by freeze-drying. A 50% by volume mixture of ethyl alcohol and purified water was added to the fruit so that the freeze-dried product was 1%, and the fruit was dissolved and filtered to obtain a comparative plum extract (sample number 8).

[0032] <Caspase-14 activation effect> The synthetic substrate acetyl-L-tryptophyl-L-glutamyl-L-histidyl-L-aspartic acid α-(4-methylcoumaryl-7-amide) emits fluorescence when decomposed by the enzymatic activity of caspase-14. The caspase-14 activation effect of each extract was measured as follows. (1) A tape measuring 1.8 cm in width and 5 cm in adhesive area was prepared, and the stratum corneum was collected by tape stripping. (2) 0.1 mol / L tris(hydroxymethyl)aminomethane, 0.14 mol / L sodium chloride, and 0.1 wt% polyoxyethylene sorbitan monosodium laurate were dissolved in distilled water, and the pH was adjusted to 8.0 with 1N hydrochloric acid to prepare a solution for extracting caspase-14. (3) The tape with the stratum corneum attached from (1) was cut into small pieces and immersed in 1 mL of caspase-14 extraction solution, then ultrasonicated on ice and centrifuged to collect the supernatant as a caspase-14 solution. (4) 50 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid, 60 mM sodium chloride, 5 mM 2,2',2'',2'''-(ethane-1,2-diyldinitrilo)tetraacetic acid, 2 mM dithiothreitol, and 1.5 M sodium citrate were dissolved in distilled water to prepare a measurement solution. (5) 1 mM acetyl-L-tryptophyl-L-glutamyl-L-histidyl-L-aspartic acid α-(4-methylcoumaryl-7-amide) was dissolved in DMSO (dimethyl sulfoxide) to prepare a substrate solution. (6) 130 μL of test solution, 30 μL of caspase-14 solution, 20 μL of substrate solution, and 20 μL of sample or distilled water were mixed in a 96-well microplate and left to stand at 37°C for approximately 30 hours to form a test group. To subtract the effect of the sample or distilled water on the measurement, a control test group was also prepared in which only 130 μL of test solution, 20 μL of substrate solution, and 20 μL of sample or distilled water were mixed. It was previously confirmed that replacing the distilled water added in place of the sample in this test system with a 50% ethyl alcohol-purified water mixture by volume did not affect the interpretation of the results.

[0033] <Calculation of caspase-14 activation effect> (1) The fluorescence intensity f at 465 nm was measured with an excitation wavelength of 360 nm. (2) The fluorescence intensity of the test area was measured as f s , the fluorescence intensity of the target test area is f c The caspase-14 activation effect of the sample compared to distilled water was calculated using the following formula:

[0034]

number

[0035] As shown in Figure 1, the experimental Japanese laurel extract (sample number 1) showed higher caspase-14 activity than distilled water, and its activation effect was confirmed to be higher than that of the comparative plum extract (sample number 2), comparative plum extract (sample number 3), and comparative plum extract (sample number 4). As mentioned in

[0004] , little is known about methods for increasing the enzymatic activity of caspase-14, so it is not possible to discuss the strength of the caspase-14 activating effect of Tsuyuakane extract by comparing it with a positive control. However, according to a study that measured the ratio of activated caspase-14 concentration to total caspase-14 concentration in the stratum corneum of patients with atopic dermatitis who exhibit skin conditions such as dryness and poor barrier function, and in non-patients (Yamamoto, M., Kamata, Y., Iida, T., Fukushima, H., Nomura, J., Saito, M., ... & Hibino, T. (2011). Quantification of activated and total caspase-14 with newly developed ELISA systems in normal and atopic skin. Journal of dermatological science, 61(2), 110-117.), the ratio in non-patients was about 3 to 5 times that of patients, and the effect of the practical extract of Japanese laurel (Sample No. 1) in Figure 1, which was about 5.5 times that of distilled water, is comparable to the difference between patients and non-patients. In other words, the caspase-14 activation effect of the extract of Japanese laurel is at a strong level that can be effective in improving skin conditions such as dryness and poor barrier function.

[0036] <Antioxidant effect test> To evaluate the antioxidant effect of the Japanese laurel extract, the highly reactive radical DPPH (2,2-diphenyl-1-picrylhydrazyl) radical changes color from purple to yellow when reduced by reaction with antioxidants that have radical scavenging ability. The radical scavenging ability was measured as follows. (1) MES (2-morpholinoethanesulfonic acid) was dissolved in purified water to prepare a 400 mM MES solution. (2) DPPH was dissolved in 100 mL of ethyl alcohol to prepare a 0.4 mM DPPH solution. (3) DPPH:MES:purified water were mixed in a ratio of 4:1:3 to prepare a DPPH test solution. (4) 10 μL of the sample or positive control ascorbic acid solution prepared by the above method and 190 μL of the DPPH test solution were added to a 96-well microplate, and the plate was shaken for 20 minutes using a plate mixer. (5) The absorbance at 540 nm, A540, was measured using a microplate reader.

[0037] <Calculation of antioxidant effect> (1) Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) was dissolved in DMSO to prepare a 10 mM Trolox solution, which was then diluted with a 50% (v / v) ethyl alcohol-purified water mixture to prepare a dilution series. (2) The absorbance A540 of the serially diluted Trolox solution (1) that had been reacted with the DPPH test solution in the same manner as the sample and ascorbic acid solution in the above <Antioxidant Effect Test> was measured, and a regression equation was obtained that clearly expressed the relationship between A540 and the Trolox concentration in the Trolox solution. (3) The absorbance A540 of the sample and the ascorbic acid solution was substituted into the regression equation obtained in (2) to calculate the antioxidant effect in terms of Trolox equivalent (unit: mM).

[0038] As shown in Figure 2, both the practical Tsuyuakane extract (sample number 5) and the practical Tsuyuakane extract (sample number 6) exhibited clear antioxidant effects, and these effects were superior to those of the comparative plum extract (sample number 3), comparative plum extract (sample number 7), comparative plum extract (sample number 4), and comparative plum extract (sample number 8).

[0039] <Skin conditioning effect test> In order to evaluate the function of the extract of A. asiaticus as a topical skin agent, a lotion containing the extract of A. asiaticus was prepared and applied for 5 days, and the changes in skin moisture were measured.

[0040] <Preparation of test lotion> The following formulation was prepared in a conventional manner. The contents are in mass %.

[0041] [Table 1]

[0042] <Use and measurement of test lotion> Three 2 x 3 cm test areas were set up on the inner forearms of both sides, for a total of six areas. After washing the test areas with facial cleanser, the test areas were left for 15 minutes before measuring the stratum corneum moisture content W0. Then, for five days, an appropriate amount of the test lotion was applied to two areas on each test area, morning and evening. After washing the test areas again with facial cleanser, the test areas were left for 15 minutes before measuring the stratum corneum moisture content W1.

[0043] <Calculation of changes in skin moisture> The change in skin moisture was calculated using the following formula:

[0044]

number

[0045] As shown in Figure 3, both the test lotions containing asiatic rosa extract, Formula No. 1 and Formula No. 2, showed a greater increase in skin moisture than Formula No. 3, which did not contain asiatic rosa extract. Furthermore, in the test areas where either Formula No. 1 or Formula No. 2 was applied, an improvement in the moisturized feel was observed, as well as an improvement in the feel of smooth skin texture. Therefore, it can be said that topical skin preparations containing asiatic rosa extract have excellent skin conditioning effects.

[0046] Examples of formulations for the external skin preparation according to the present invention are shown below. The contents are in mass %. The preparation method is a conventional method. In all formulation examples, an improvement in the moisturized feel was observed, as well as an improvement in the feel of smooth skin texture.

[0047] <Formulation example 1: Lotion> Contents 1,3-butylene glycol 8.00 Glycerin 5.00 Polyoxyethylene sorbitan monolaurate (20E.O.) 0.15 Ethyl alcohol 5.00 Red laurel extract (sample no. 1) 2.00 Preservatives (appropriate amount) Fragrance (appropriate amount) Purified water remainder Total 100

[0048] <Formulation Example 2 Emulsion> Content Red laurel extract (sample number 5) 5.00 Squalane 8.00 Vaseline 2.00 Beeswax 0.50 Sorbitan sesquioleate 0.80 Polyoxyethylene oleyl ether (20E.0.) 1.20 Carboxyvinyl polymer 0.20 Propylene glycol 0.50 Potassium hydroxide 0.10 Ethyl alcohol 7.00 Preservatives and antioxidants (appropriate amount) Fragrance (appropriate amount) Purified water remainder Total 100

[0049] <Formulation Example 3 Powder> Content Liquid paraffin 2.00 Vaseline 1.00 Diisostearyl malate 1.00 Polyoxybutylene Polyoxyethylene Polyoxypropylene Glyceryl Ether (3B.O.) (8E.O.) (5P.O.) 0.20 Lauryl PEG-9 Polydimethylsiloxyethyl Dimethicone 0.50 PEG-9 Polydimethylsiloxyethyl Dimethicone 0.20 Decamethylcyclopentasiloxane 4.00 Octyl methoxycinnamate 2.60 Diethylaminohydroxybenzoylhexyl benzoate 1.00 Sericite 20.0 Zinc Laurate 2.00 Boron nitride 8.00 Lyophilized extract of Akane asiaticus (sample no. 5) 0.01 Preservatives (appropriate amount) Talc Remainder Total 100

[0050] <Formulation Example 4: Tablets> Content Lyophilized extract of Akane asiaticus (sample no. 6) 0.03 Xylitol 25.0 Sugar Esters 3.00 Polydextrose 7.00 Fragrance (appropriate amount) Palatinose Remainder Total 100

[0051] <Formulation Example 5 Candy> Content Red laurel extract (sample no. 6) 10.0 Maltitol 50.0 Starch syrup 30.0 Citric acid 1.0 Strawberry 1 / 5 concentrated juice 1.0 Dye appropriate amount Fragrance (appropriate amount) Purified water remainder Total 100 [Industrial Applicability]

[0052] As described above, Tsuyuakane extract has caspase-14 activation and antioxidant effects, protecting the skin from harmful dryness, ultraviolet rays, and active oxygen, and when incorporated into topical skin preparations, it achieves the skin-conditioning effects that topical skin preparations should have.

Claims

1. A caspase-14 activator characterized by containing an extract of Akamagasaki acutifolia.

2. An antioxidant characterized by containing an extract of Japanese laurel.

3. A topical skin preparation comprising the caspase-14 activator or antioxidant according to claim 1 or 2.

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