Carbonylation Inhibitors

Abstract

To provide an agent for inhibiting carbonylation useful for improving wrinkle or sagging, and suppressing skin yellowing.SOLUTION: There are provided cosmetic, a medicine, a quasi drug, and a food product containing an extract from cherry, comfrey, and Calendula officinalis Linne which prevents / improves skin drabness due to carbonylation, and is aimed to improve transparency of skin. The extract from cherry, comfrey, and Calendula officinalis Linne has excellent carbonylation inhibitory action, prevents carbonylation of protein or adduct formation of acrolein and protein, which shows effects of lightening skin drabness and improving transparency of skin.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a protein carbonylation inhibitor characterized by containing extracts of cherry blossom, comfrey and calendula officinalis. [Background technology]

[0002] The skin, located at the outermost layer of our body, is susceptible to oxidative stress caused by ultraviolet rays, air pollutants, and other factors. Living organisms naturally have mechanisms to eliminate reactive oxygen species generated by oxidative stress. However, when the ability to eliminate reactive oxygen species declines due to aging or other factors, changes such as oxidation, carbonylation, and glycation occur in skin proteins.

[0003] In particular, carbonylation is an irreversible oxidative modification that occurs non-enzymatically and non-specifically due to reactive oxygen species. Lipid peroxides are generated by oxidation of lipids by reactive oxygen species, and in recent years, it has been discovered that aldehydes, such as acrolein, which are decomposition products of lipid peroxides, can be added to proteins to carbonylate them (Non-Patent Document 1).

[0004] Acrolein is an environmental pollutant that is produced during lipid peroxidation (Non-Patent Document 2), but is also produced in vivo from polyamines (Non-Patent Document 3), and is also produced by burning plastics, cigarette smoke, exhaust fumes, and heating oils and fats. Thus, acrolein is produced from sources other than reactive oxygen species, and is a highly reactive substance involved in denaturation such as protein carbonylation and cytotoxicity. In recent years, it has been discovered that plant extracts such as rosehip extracts more directly supplement acrolein rather than reactive oxygen species, thereby preventing and improving skin aging (Patent Document 1).

[0005] Carbonylation occurs non-enzymatically and non-specifically, and therefore causes various diseases in all tissues. For example, it is known that the accumulation of abnormal proteins, such as carbonylated proteins, in the body with aging is involved in many diseases, such as Alzheimer's disease, Parkinson's disease, Lewy body disease, triplet repeat disease, amyotrophic lateral sclerosis, cataracts, arteriosclerosis, and diabetic nephropathy (Patent Document 2).

[0006] Carbonylated proteins cause dullness, wrinkles, sagging, and other skin problems. Recent reports have shown that carbonylated proteins in sun-exposed keratin in the epidermis reduce optical transparency (Non-Patent Document 4), and that carbonylated proteins in the dermis are involved in the yellowing of replicatively aged cultured normal human fibroblasts (Non-Patent Document 5). As these findings suggest, epidermal protein carbonylation is involved in the reduction of skin transparency, while dermal protein carbonylation is involved in skin yellowing. Therefore, preventing or reducing epidermal protein carbonylation is believed to be useful for improving dullness and enhancing skin clarity. Furthermore, preventing or reducing dermal protein carbonylation is believed to be useful for improving wrinkles and sagging and suppressing skin yellowing. Against this background, there is a need for the development of a formulation that inhibits protein carbonylation.

[0007] To date, carbonylation inhibitors known include a carbonylation inhibitor (Patent Document 3), an inhibitor of the carbonylation of epidermal proteins, and cosmetics and topical skin preparations containing the carbonylation inhibitor (Patent Document 4), and an anti-aging agent (Patent Document 5). However, safer and more effective formulations are desired.

[0008] Cherry blossoms are primarily deciduous trees of the genus Cerasus or the genus Prunus (subgenus Cerasus) of the subfamily Amygdaloideae of the family Rosaceae, and are widely appreciated for their beautiful spring flowers. Research on cherry blossoms has progressed in recent years, and topical skin preparations and bath preparations (Patent Document 6), firming and resilience-imparting compositions (Patent Document 7), and the like have been disclosed. However, nothing has been known about the carbonylation inhibitory effect of cherry blossoms.

[0009] Comfrey is a perennial plant of the genus Symphytum in the family Boraginaceae, native to Europe, and is distributed from Europe to Western Siberia and Central Asia. In recent years, research on comfrey has progressed, and hair dye compositions (Patent Document 8), whitening cosmetics (Patent Document 9), and the like have been disclosed. However, nothing has been known so far about the carbonylation inhibitory effect of comfrey.

[0010] Calendula officinalis is a plant of the genus Calendula in the subfamily Asteroideae of the family Asteraceae, native to Europe, and is cultivated throughout Japan. In recent years, research on calendula officinalis has progressed, and prostaglandin E2 production inhibitors (Patent Document 10), antioxidants, anti-glycation agents, hyaluronidase inhibitors, and cosmetics (Patent Document 11) have been disclosed. However, nothing has been known about the carbonylation inhibitory effect of calendula officinalis. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] Patent Publication No. 2006-160630 [Patent Document 2] Patent Publication No. 2011-148715 [Patent Document 3] Patent Publication No. 2012-246226 [Patent Document 4] Patent Publication No. 2021-17407 [Patent Document 5] Patent Publication No. 2018-150262 [Patent Document 6] JP 8-245409 [Patent Document 7] Patent Publication No. 2013-180969 [Patent Document 8] JP 8-165227 [Patent Document 9] Patent Publication No. 2002-179547 [Patent Document 10] Patent Publication No. 2020-164481 [Patent Document 11] Patent Publication No. 2020-70258 [Non-patent literature]

[0012] [Non-Patent Document 1] Ichiro Iwai et al., Journal of Cosmetic Technology, 42(1), 16-21(2008) [Non-patent document 2] Koji Uchida, Journal of the Japan Oil Chemists Society, 47(11), 29-37(1998) [Non-patent document 3] Houen G et al,Acta Chem.Scand.,48(1),52-60(1994) [Non-patent document 4] Iwai I et al,Int.J.Cosmet.Sci.,30(1),41-46(2008) [Non-patent document 5] Kotone Eguchi et al., Journal of the Japanese Society of Cosmetic Chemistry, 44(2), 92-98(2020) Summary of the Invention [Problem to be solved by the invention]

[0013] The problem to be solved by the present invention is to find a plant-derived component that inhibits carbonylation, and to provide an excellent carbonylation inhibitor and acrolein-protein adduct formation inhibitor that uses this as an active ingredient and has a clear site of action. [Means for solving the problem]

[0014] As a result of intensive research aimed at solving the above problems, the present inventors have found that extracts of cherry blossom, comfrey, and calendula officinalis have excellent inhibitory effects on carbonylation and on the formation of adducts of acrolein and proteins. Furthermore, the present inventors have found that a composition containing extracts of cherry blossom, comfrey, and calendula officinalis has an excellent effect of improving skin transparency, leading to the completion of the present invention.

[0015] That is, the present invention relates to a carbonylation inhibitor, an inhibitor of the formation of adducts of acrolein and proteins, and an agent for improving skin clarity, which are characterized by containing extracts of cherry blossom, comfrey, and calendula officinalis.

[0016] The present invention includes the following inventions. (1) A protein carbonylation inhibitor characterized by containing an extract of one or more plants selected from cherry blossom, comfrey, and calendula officinalis. (2) An inhibitor of the formation of adducts between acrolein and proteins, characterized by containing an extract of one or more plants selected from cherry blossom, comfrey, and calendula officinalis. (3) The inhibitor according to claim 1 or 2, wherein the protein is an epidermal protein. (4) The inhibitor according to claim 1 or 2, wherein the protein is a keratin protein. (5) The inhibitor according to claim 1 or 2, wherein the protein is a protein in keratinocytes. (6) A skin clarity improver characterized by containing an extract of one or more selected from cherry blossom, comfrey, and calendula officinalis. [Effects of the Invention]

[0017] The cherry blossom, comfrey, and calendula extracts of the present invention were found to have excellent carbonylation inhibitory effects.Furthermore, carbonylation inhibitors, acrolein-protein adduct formation inhibitors, and skin clarity enhancers containing these extracts were found to be safe and to have excellent effects in preventing and improving skin dullness by inhibiting carbonylation. [Brief explanation of the drawings]

[0018] [Figure 1] 1 shows micrographs of stained carbonylated proteins in Experimental Example 1, in which human keratin collected with tape strips was carbonylated with acrolein and simultaneously extracts of cherry blossom, comfrey, and calendula were added.

[0019] [Figure 2] This is a micrograph of the appearance of three-dimensional human cultured epidermis in Experimental Example 2, when carbonylation was induced with acrolein and cherry blossom extract was added at the same time.

[0020] [Figure 3] 1 is a micrograph of stained carbonylated proteins in human keratin collected with a tape strip in Experimental Example 3. DETAILED DESCRIPTION OF THE INVENTION

[0021] In the present invention, protein carbonylation refers to a reaction in which carbonyl compounds such as aldehydes generated by lipid peroxidation or the like nonenzymatically and nonspecifically modify proteins. When lipids are exposed to reactive oxygen, lipid peroxides are formed, which are further decomposed to produce highly reactive aldehydes such as acrolein, 4-hydroxy-2-nonenal, and malondialdehyde. These intermediates are highly reactive and modify proteins.

[0022] Acrolein in the present invention is a type of aldehyde that is produced from lipid peroxidation products generated in the body by ultraviolet rays or oxidative stress, is produced from polyamines in the body, and is also contained in cigarette smoke, environmental pollutants, etc.

[0023] In the present invention, the formation of an adduct between acrolein and a protein is caused by acrolein binding to a protein, resulting in protein denaturation and cytotoxicity.

[0024] The skin clarity enhancer of the present invention is an agent that improves skin clarity from a reduced (poor) state to a better state (normal state or a state better than normal state), or from a normal state to a better state. The protein of the stratum corneum, the outermost layer of skin, is mainly composed of keratin, and when keratin is carbonylated, the moisture content of the stratum corneum surface decreases, and the light reflected from the skin surface decreases, causing a loss of skin clarity. Skin clarity can be achieved by increasing the moisture content of the stratum corneum surface and increasing the light reflected from the skin surface.

[0025] The cherry trees used in the present invention belong to the genus Cerasus or the genus Prunus subgenus Cerasus in the family Rosaceae, subfamily Amygdaloideae, and include, for example, Somei-Yoshino and Oshima-zakura. The parts of the cherry tree used in the present invention may be extracted from parts of the plant, such as flowers, fruits, seeds, leaves, stems, and roots, or from the whole plant, preferably leaves. The extraction method is not particularly limited, and may involve, for example, heat extraction or room temperature extraction. For extraction, the plant may be used as is, or may be subjected to processing such as drying, crushing, or shredding.

[0026] The comfrey used in the present invention is a member of the Boraginaceae family, genus Symphytum, and its scientific name is Symphytum officinale L. It is native to Europe and grows wild in Siberia. The Japanese name "comfrey" comes from the fact that the shape of the leaves creeping along the stems at their base resembles a fish fin. The extract may be obtained from any part of the plant, such as flowers, fruits, seeds, leaves, stems, or roots, or from the whole plant, with leaves being preferred. The extraction method is not particularly limited, and may be, for example, heated or room temperature. For extraction, the plant may be used as is, or may be subjected to processing such as drying, crushing, or shredding.

[0027] The calendula used in the present invention is a member of the genus Calendula in the subfamily Asteroideae of the family Asteraceae, native to the Mediterranean coast. It produces orange or yellow flowers with a diameter of approximately 10 cm, and the flowers vary in appearance, from single to double, with some even having a black spot in the center. The extract may be obtained from any part of the plant, such as the flower, fruit, seed, leaf, stem, or root, or from the whole plant, with the flower being preferred. The extraction method is not particularly limited, and may involve, for example, heat extraction or room temperature extraction. For extraction, the plant may be used as is, or may be processed by drying, crushing, shredding, or other processes.

[0028] The extraction method is not particularly limited, but can be carried out using water, hot water, or a mixed solvent of water and an organic solvent, by stirring or column extraction. Examples of extraction solvents include water, lower alcohols (methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, etc.), liquid polyhydric alcohols (1,3-butylene glycol, propylene glycol, glycerin, etc.), ketones (acetone, methyl ethyl ketone, etc.), acetonitrile, esters (ethyl acetate, butyl acetate, etc.), hydrocarbons (hexane, heptane, liquid paraffin, etc.), and ethers (ethyl ether, tetrahydrofuran, propyl ether, etc.). Polar solvents such as water, lower alcohols, and liquid polyhydric alcohols are preferred, with water, ethanol, 1,3-butylene glycol, and propylene glycol being particularly preferred. These solvents may be used alone or in combination. Particularly preferred extraction solvents include water or a water-ethanol mixed polar solvent. The amount of solvent used is not particularly limited, and may be, for example, 10 times or more, preferably 20 times or more, the dry weight of cherry leaves, comfrey leaves, or calendula flowers, but is preferably 100 times or less for ease of concentration or isolation after extraction. The extraction temperature and time can be appropriately selected depending on the type of solvent used, the pressure during extraction, etc.

[0029] The extract may be used as the extracted solution as is, or, if necessary, may be subjected to treatments such as concentration (vacuum concentration, membrane concentration, etc.), dilution, filtration, decolorization with activated carbon, deodorization, ethanol precipitation, etc., within the scope of the effects of the present invention. Furthermore, the extracted solution may be concentrated to dryness, spray-dried, freeze-dried, etc., and used as a dried product. The cherry blossom, comfrey, and calendula used in the present invention are naturally occurring plants, and the components extracted from cherry blossom, comfrey, and calendula are mixtures in which many compounds with diverse structures exist simultaneously. Therefore, it is difficult to clarify the structures or properties of all of the components contained therein, and it is preferable to treat them as extracts.

[0030] In the present invention, the extract may be used as is, or may contain ingredients such as oils and fats, waxes, hydrocarbons, fatty acids, alcohols, esters, surfactants, metal soaps, pH adjusters, preservatives, fragrances, moisturizers, powders, UV absorbers, thickeners, pigments, antioxidants, whitening agents, chelating agents, excipients, coating agents, sweeteners, and acidulants, which are used in cosmetics, quasi-drugs, pharmaceuticals, foods, etc., within a range that does not impair the effects of the extract.

[0031] The present invention can be used for any of cosmetics, quasi-drugs, pharmaceuticals, and foods, and examples of dosage forms thereof include lotions, creams, emulsions, gels, aerosols, essences, packs, cleansers, bath additives, foundations, dusting powders, lipsticks, ointments, poultices, tablets, capsules, chocolates, gums, candies, beverages, powders, granules, tablets, sugar-coated tablets, syrups, pills, suspensions, liquids, emulsions, suppositories, and injectable solutions.

[0032] For external use, the content of the extract used in the present invention is preferably 0.0001% by weight or more, more preferably 0.001 to 10% by weight, calculated as solid matter. Furthermore, 0.01 to 5% by weight is most preferable. If it is less than 0.0001% by weight, it is difficult to expect a sufficient effect. If it exceeds 10% by weight, it is difficult to see an enhancement of the effect, which is uneconomical.

[0033] For internal use, the dosage varies depending on age, body weight, symptoms, therapeutic effect, administration method, treatment time, etc. Generally, the daily dosage per adult is preferably 5 mg or more, more preferably 10 mg to 5 g, and most preferably 20 mg to 2 g.

[0034] Next, in order to explain the present invention in detail, production examples, formulation examples, and experimental examples of the extract used in the present invention are given as examples, but the present invention is not limited to these. In the production examples, % means % by weight, and in the formulation examples, parts of the content means parts by weight. [Example]

[0035] Cherry extracts were produced as follows: In Production Examples 1 to 4, cherry leaves were used as the extraction material.

[0036] (Production Example 1) Preparation of hot water extract of cherry blossoms 200 mL of water was added to 10 g of dried cherry blossoms, and the mixture was extracted for 2 hours at 95-100° C. The resulting extract was filtered, and the filtrate was concentrated and freeze-dried to obtain 1.5 g of hot water cherry blossom extract.

[0037] (Production Example 2) Preparation of 50% ethanol extract of cherry blossoms 10 g of dried cherry blossoms were soaked in 200 mL of 50% ethanol solution at room temperature for 7 days to perform extraction. The resulting extract was filtered and then concentrated to dryness using an evaporator to obtain 1.1 g of 50% ethanol extract of cherry blossoms.

[0038] (Production Example 3) Preparation of ethanol extract of cherry blossoms 10 g of dried cherry blossoms were soaked in 200 mL of ethanol at room temperature for 7 days for extraction. The resulting extract was filtered and then concentrated to dryness using an evaporator to obtain 0.6 g of cherry blossom ethanol extract.

[0039] (Production Example 4) Preparation of 1,3-butylene glycol extract of cherry blossoms 10 g of dried cherry blossoms were soaked in 200 mL of 1,3-butylene glycol at room temperature for 7 days to perform extraction. The resulting extract was filtered to obtain 201 g of 1,3-butylene glycol cherry blossom extract.

[0040] Comfrey extract was produced as follows: In Production Examples 5 to 8, comfrey leaves were used as the extraction material.

[0041] (Production Example 5) Preparation of hot water extract of comfrey 200 mL of water was added to 10 g of dried comfrey, and the mixture was extracted for 2 hours at 95-100° C. The resulting extract was filtered, and the filtrate was concentrated and freeze-dried to obtain 1.8 g of a hot water extract of comfrey.

[0042] (Production Example 6) Preparation of 50% ethanol extract of comfrey 10 g of dried comfrey was soaked in 200 mL of 50% ethanol solution at room temperature for 7 days to extract it. The resulting extract was filtered and then concentrated to dryness using an evaporator to obtain 1.3 g of 50% ethanol extract of comfrey.

[0043] (Production Example 7) Preparation of ethanol extract of comfrey 10 g of dried comfrey was soaked in 200 mL of ethanol at room temperature for 7 days to perform extraction. The resulting extract was filtered and then concentrated to dryness using an evaporator to obtain 0.5 g of ethanol extract of comfrey.

[0044] (Production Example 8) Preparation of 1,3-butylene glycol extract of comfrey 10 g of dried comfrey was soaked in 200 mL of 1,3-butylene glycol at room temperature for 7 days to extract it, and the resulting extract was filtered to obtain 181 g of 1,3-butylene glycol extract of comfrey.

[0045] Calendula officinalis extract was produced as follows: In Production Examples 9 to 12, Calendula officinalis flowers were used as the extraction material.

[0046] (Production Example 9) Preparation of hot water extract of Calendula officinalis 200 mL of water was added to 10 g of dried calendula officinalis, and extraction was carried out for 2 hours at 95-100° C. The obtained extract was filtered, and the filtrate was concentrated and freeze-dried to obtain 1.3 g of a hot water extract of calendula officinalis.

[0047] (Production Example 10) Preparation of 50% ethanol extract of Calendula officinalis 10 g of dried calendula officinalis was soaked in 200 mL of 50% ethanol solution at room temperature for 7 days to perform extraction. The resulting extract was filtered and then concentrated to dryness using an evaporator to obtain 1.2 g of a 50% ethanol extract of calendula officinalis.

[0048] (Production Example 11) Preparation of ethanol extract of Calendula officinalis 10 g of dried calendula officinalis was soaked in 200 mL of ethanol at room temperature for 7 days to perform extraction. The resulting extract was filtered and then concentrated to dryness using an evaporator to obtain 0.7 g of an ethanol extract of calendula officinalis.

[0049] (Production Example 12) Preparation of 1,3-butylene glycol extract of Calendula officinalis 10 g of dried calendula officinalis was soaked in 200 mL of 1,3-butylene glycol at room temperature for 7 days to perform extraction. The resulting extract was filtered to obtain 192 g of 1,3-butylene glycol extract of calendula officinalis. [Example]

[0050] (Formulation example 1) Lotion Prescription Content (parts) 1. Cherry blossom ethanol extract (Production Example 3) 2.0 2.1,3-Butylene Glycol 8.0 3. Glycerin 2.0 4. Xanthan gum 0.02 5. Citric acid 0.01 6. Sodium citrate 0.1 7. Ethanol 5.0 8. Methyl parahydroxybenzoate 0.1 9. Polyoxyethylene hydrogenated castor oil (40E.O.) 0.1 10.Fragrance (appropriate amount) 11. Add purified water to make the total volume 100 [Manufacturing method] Components 1 to 6 and 11 are dissolved uniformly, and components 7 to 10 are dissolved uniformly. The mixture is then mixed and filtered to obtain the product.

[0051] (Prescription Example 2) Cream Prescription Content (parts) 1. 50% ethanol extract of comfrey (Production Example 6) 1.0 2. Squalane 5.5 3. Olive Oil 3.0 4. Stearic Acid 2.0 5. Beeswax 2.0 6. Octyldodecyl myristate 3.5 7. Polyoxyethylene cetyl ether (20E.O.) 3.0 8. Behenyl alcohol 1.5 9. Glyceryl monostearate 2.5 10.Fragrance 0.1 11. Methyl parahydroxybenzoate 0.2 12.1,3-butylene glycol 8.5 13. Add purified water to make the total volume 100 [Manufacturing Method] Heat, dissolve, and mix ingredients 2-9, then maintain at 70°C to form the oil phase. Heat, dissolve, and mix ingredients 1 and 11-13, then maintain at 75°C to form the water phase. Add the water phase to the oil phase and emulsify, then cool with stirring. Add ingredient 10 at 45°C, then cool further to 30°C to form the final product.

[0052] (Formulation Example 3) Emulsion Prescription Content (parts) 1. 50% ethanol extract of cherry blossoms (Production Example 2) 0.01 2. Squalane 5.0 3. Olive oil 5.0 4. Jojoba oil 5.0 5. Cetyl alcohol 1.5 6. Glyceryl Monostearate 2.0 7. Polyoxyethylene cetyl ether (20E.O.) 3.0 8. Polyoxyethylene sorbitan monooleate (20E.O.) 2.0 9.Fragrance 0.1 10. Propylene Glycol 1.0 11. Glycerin 2.0 12. Methyl parahydroxybenzoate 0.2 13. Add purified water to make the total volume 100 [Manufacturing Method] Heat, dissolve, and mix ingredients 1-8, then maintain at 70°C to form the oil phase. Heat, dissolve, and mix ingredients 10-13, then maintain at 75°C to form the water phase. Add the water phase to the oil phase and emulsify, then cool with stirring. Add ingredient 9 at 45°C, then cool further to 30°C to form the final product.

[0053] (Prescription Example 4) Pack Prescription Content (parts) 1. 1,3-butylene glycol extract of cherry blossoms (Production Example 4) 5.0 2. Polyvinyl alcohol 12.0 3. Ethanol 5.0 4.1,3-Butylene Glycol 8.0 5. Methyl parahydroxybenzoate 0.2 6. Polyoxyethylene hydrogenated castor oil (20E.O.) 0.5 7. Citric acid 0.1 8. Sodium citrate 0.3 9.Fragrance (appropriate amount) 10. Add purified water to make the total volume 100 [Manufacturing method] Dissolve ingredients 1 to 10 uniformly to produce the product.

[0054] (Comparative Formulation Example 1) Conventional Pack In Formulation Example 4, the 1,3-butylene glycol extract of cherry blossoms was replaced with purified water to create a conventional pack.

[0055] (Formulation Example 5) Gel Prescription Content (parts) 1. 1,3-butylene glycol extract of comfrey (Example 8) 1.0 2. Ethanol 5.0 3. Methyl parahydroxybenzoate 0.1 4. Polyoxyethylene hydrogenated castor oil (60E.O.) 0.1 5.Fragrance (appropriate amount) 6. 1,3-Butylene Glycol 5.0 7. Glycerin 5.0 8. Xanthan gum 0.1 9. Carboxyvinyl polymer 0.2 10. Potassium hydroxide 0.2 11. Add purified water to make the total volume 100 [Manufacturing method] Components 2 to 5, 1, and 6 to 11 are each dissolved uniformly, and then mixed to form the product.

[0056] (Formulation example 6) Foundation Prescription Content (parts) 1. 1,3-butylene glycol extract of calendula officinalis (Production Example 12) 1.0 2. Stearic acid 2.4 3. Polyoxyethylene sorbitan monostearate (20E.O.) 1.0 4. Polyoxyethylene cetyl ether (20E.O.) 2.0 5. Cetyl alcohol 1.0 6. Liquid Lanolin 2.0 7. Liquid Paraffin 3.0 8. Isopropyl myristate 6.5 9. Sodium carboxymethylcellulose 0.1 10. Bentonite 0.5 11. Propylene Glycol 4.0 12. Triethanolamine 1.1 13. Methyl parahydroxybenzoate 0.2 14. Titanium dioxide 8.0 15. Talc 4.0 16. Bengala 1.0 17. Yellow Iron Oxide 2.0 18.Fragrance (appropriate amount) 19. Add purified water to make the total volume 100 [Manufacturing Method] Heat and dissolve ingredients 2-8 and maintain at 80°C to form the oil phase. Ingredient 9 is thoroughly swelled in ingredient 19, followed by addition of ingredients 1 and 10-13 and homogenous mixing. To this, ingredients 14-17, which have been pulverized and mixed in a grinder, are added, and the mixture is stirred in a homomixer and maintained at 75°C to form the water phase. The water phase is added to this oil phase while stirring, and emulsified. After cooling, ingredient 18 is added at 45°C, and the mixture is cooled to 30°C while stirring to form the final product.

[0057] (Formulation Example 7) Bath additive Prescription Content (parts) 1. Comfrey ethanol extract (Production Example 7) 1.0 2. Sodium bicarbonate 50.0 3. Yellow No. 202 (1) appropriate amount 4.Fragrance (appropriate amount) 5. Add sodium sulfate to make the total volume 100 [Manufacturing method] Mix ingredients 1 to 5 uniformly to produce the product.

[0058] (Prescription Example 8) Ointment Prescription Content (parts) 1. Calendula officinalis ethanol extract (Production Example 11) 5.0 2. Polyoxyethylene cetyl ether (30E.O.) 2.0 3. Glyceryl monostearate 10.0 4. Liquid Paraffin 5.0 5. Cetyl alcohol 6.0 6. Methyl parahydroxybenzoate 0.1 7. Propylene Glycol 10.0 8. Add purified water to make the total volume 100 [Manufacturing Method] Heat, dissolve, and mix ingredients 2-5, then maintain at 70°C to form the oil phase. Heat, dissolve, and mix ingredients 1 and 6-8, then maintain at 75°C to form the water phase. Add the water phase to the oil phase and emulsify, then cool to 30°C while stirring to form the final product.

[0059] (Prescription Example 9) Powder Prescription Content (parts) 1. Hot water extract of cherry blossoms (Production Example 1) 0.5 2.Dry cornstarch 39.0 3. Microcrystalline cellulose 60.5 [Manufacturing method] Mix ingredients 1 to 3 to form a powder.

[0060] (Prescription Example 10) Tablets Prescription Content (parts) 1. Comfrey hot water extract (Production Example 5) 2.5 2.Dry cornstarch 25.0 3. Calcium carboxymethylcellulose 20.0 4. Microcrystalline cellulose 40.0 5. Polyvinylpyrrolidone 7.0 6. Talc 5.5 [Manufacturing Method] Components 1 to 4 are mixed, and then an aqueous solution of component 5 is added as a binder to form granules. Component 6 is added to the formed granules and compressed into tablets. Each tablet weighs 0.52 g.

[0061] (Prescription Example 11) Tablets Prescription Content (parts) 1. Hot water extract of calendula officinalis (Production Example 9) 1.0 2. Dry cornstarch 49.8 3. Erythritol 40.0 4. Citric Acid 5.0 5. Sucrose fatty acid ester 3.0 6.Fragrance 0.1 7. Purified water 1.1 [Manufacturing method] Mix ingredients 1 to 4 and 7 and form into granules. Add ingredients 5 and 6 to the formed granules and compress into tablets. Each tablet weighs 1.0 g.

[0062] (Formulation Example 12) Beverage Prescription Content (parts) 1. 50% ethanol extract of Calendula officinalis (Production Example 10) 0.025 2. Stevia 0.05 3. Malic acid 5.0 4.Fragrance 0.1 5.Purified water 94.825 [Manufacturing Method] Dissolve ingredients 2 and 3 in a small amount of water. Then add ingredients 1, 4, and 5 and mix.

[0063] Next, experimental examples will be given to explain the effects of the present invention in detail. [Example]

[0064] (Experimental Example 1) Effects of cherry, comfrey, and calendula extracts on the induction of carbonylated proteins by acrolein in human stratum corneum Keratin samples were collected from the inner right upper arm of three male subjects (3 individuals in their 20s and 30s) using 10 x 30 mm transparent double-sided tape (Nitto). Half of the tape (10 x 15 mm) was immersed for 3 hours in a 30 mM aqueous solution of acrolein (Tokyo Chemical Industry Co., Ltd.) containing cherry blossom extract (Production Example 4), comfrey extract (Production Example 8), and calendula extract (Production Example 12) at a final concentration of 1 mg / mL. After rinsing, the entire tape was reacted with 20 μM fluorescent hydrazide (fluorescein-5-thiosemicarbazide, Chemodex) in 100 mM MES-Na buffer (pH 5.5) at room temperature for 1 hour. After the reaction, the tape was thoroughly washed and the green fluorescence was observed under a fluorescence microscope. Photographs were taken of both the unimmersed area and the area immersed in acrolein (or acrolein + extract). The fluorescence intensity per area of ​​the keratin in the photographed images was determined by image analysis. The amount of carbonylated protein was calculated from the fluorescence intensity of the acrolein (or acrolein + extract) immersion site relative to the unimmersed site for each subject, and statistical analysis was performed.

[0065] The amount of carbonylated protein (average value for three subjects) calculated from the fluorescence brightness of the acrolein (or acrolein + extract) immersion site relative to the unimmersed site is shown in Table 1, and representative stratum corneum images are shown in Figure 1. Acrolein increased the amount of carbonylated protein. Furthermore, adding cherry extract, comfrey extract, or calendula extract simultaneously with acrolein suppressed the increase in carbonylated protein amount due to acrolein. Similar effects were observed with cherry extract (Production Examples 1-3), comfrey extract (Production Examples 5-7), and calendula extract (Production Examples 9-11) obtained using other extraction methods.

[0066] [Table 1]

[0067] (Experimental Example 2) Effects of acrolein carbonylation and cherry blossom extract on the optical properties of three-dimensional human cultured epidermis Human-derived keratinocytes were seeded onto cell culture inserts and cultured in KG-2 medium (KURABO) for two days. Differentiation induction was performed using differentiation-inducing medium (CellTec) at the air-liquid interface to reconstruct epidermal tissue, which was then harvested on the seventh day after the start of air-liquid interface culture. The resulting three-dimensional human cultured epidermis was then immersed for four hours in a 30 mM aqueous solution of cherry blossom extract (Production Example 4) containing a final concentration of 1 mg / mL acrolein. After washing, the epidermis was placed on a cover glass and the optical properties of the three-dimensional human cultured epidermis were photographed.

[0068] The effects of acrolein and cherry blossom extract on epidermal carbonylation are shown in Figure 2. Carbonylation induced by acrolein caused the appearance of three-dimensional human cultured epidermis to become opaque and cloudy, but cherry blossom extract inhibited this change. From the above, it was revealed that cherry blossom extract has a high carbonylation inhibitory effect and prevents and improves the loss of skin transparency caused by carbonylation. Furthermore, similar effects were observed in cherry blossom extracts (Production Examples 1-3), comfrey extracts (Production Examples 5-7), and calendula extracts (Production Examples 9-11) obtained using other extraction methods.

[0069] (Experimental Example 3) Usage test A use test was conducted on 11 male subjects (aged 20-50) using the pack of Formulation Example 4 and the conventional pack of Comparative Formulation Example 1. For one month, the packs of Formulation Example 4 and Comparative Formulation Example 1 were applied to half of the face (e.g., Subject 1 used the pack of Formulation Example 4 on the left half of the face and the pack of Comparative Formulation Example 1 on the right half of the face; Subject 2 used the pack of Comparative Formulation Example 1 on the left half of the face and the pack of Formulation Example 4 on the right half of the face). After use, the degree of dullness of the cheeks was assessed by questionnaire. Carbonylated proteins were also examined before and after the use test by collecting keratinous tissue with tape strips. Specifically, keratinous tissue was collected from the subjects' cheeks using 10 x 15 mm transparent double-sided tape. The tissue was immersed in a 30 mM acrolein solution for 3 hours, washed, and then reacted with 20 μM fluorescent hydrazide in 100 mM MES-Na buffer (pH 5.5) at room temperature for 1 hour. After the reaction was completed, the plate was thoroughly washed and the green fluorescence was observed under a fluorescence microscope.

[0070] As a result of the usage test, more people responded that the degree of dullness in the cheeks was reduced when the half face used with the pack of Formulation Example 4 was used than when the half face used with the conventional pack of Comparative Formulation Example 1 was used. Also, as shown in Figure 3, the pack of Formulation Example 4 reduced carbonylated proteins in the cheeks. Furthermore, not a single person experienced any skin troubles during the test period, and there were no safety issues. There were also no problems with the deterioration of the prescription ingredients. [Industrial Applicability]

[0071] The carbonylation inhibitor, acrolein-protein adduct formation inhibitor, and skin clarity enhancer according to the present invention, which contain extracts of cherry blossom, comfrey, and calendula officinalis, exhibit excellent improving effects for the purposes of each agent. Therefore, it is possible to provide cosmetics, pharmaceuticals, quasi-drugs, and foods that aim to prevent and improve dull skin by inhibiting carbonylation.

Claims

1. A protein carbonylation inhibitor characterized by containing an extract of one or more species selected from the group consisting of cherry blossom, comfrey and calendula officinalis.

2. An inhibitor of the formation of adducts between acrolein and proteins, characterized by containing an extract of one or more species selected from the group consisting of cherry blossom, comfrey and calendula officinalis.

3. 3. The inhibitor according to claim 1, wherein the protein is an epidermal protein.

4. 3. The inhibitor according to claim 1, wherein the protein is a keratin protein.

5. The inhibitor according to claim 1 or 2, wherein the protein is a protein in keratinocytes.

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