Skin cell lysosome activator

Abstract

To discover a new material that can activate lysosome in skin cells to efficiently remove skin-accumulated melanin and provide compositions such as whitening cosmetics and pharmaceuticals.SOLUTION: The present invention provides an agent for activating lysosome in skin cells, a lysosomal enzyme gene expression accelerator, or a melanin degradation accelerator, each containing extract from Camellia japonica as an active ingredient.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a lysosome activator in skin cells, a lysosomal enzyme gene expression promoter, a melanin degradation promoter, and a whitening composition containing these agents.

Background Art

[0002] Generally, skin pigmentation such as freckles, age spots, and sunburn is considered to be caused by abnormal hormones or ultraviolet stimulation, which causes melanin-producing cells (melanocytes) present in the epidermal tissue of the skin to overproduce melanin pigment, which then deposits in the skin. As methods for preventing such pigmentation, methods for suppressing excessive production of melanin and methods for promoting epidermal turnover to promote excretion of melanin are known. For example, as prior arts for suppressing excessive production of melanin, arbutin, hydroquinone, kojic acid, tranexamic acid, and their derivatives are known. However, these whitening active ingredients have problems such as poor stability in the formulation system, resulting in decomposition and coloring, and generation of abnormal odors. Further, even if there is a whitening effect, inflammation and redness may occur in people with weak skin, and vitiligo due to cytotoxicity to melanocytes has been reported as a more serious side effect. Also, depending on their mechanism of action, people with certain diseases such as those at risk of thrombosis may need to be cautious when using them, and currently, long-term use and the amount of use are restricted from the perspective of safety.

[0003] In addition, in the skin clinical findings of the freckle area, melanin has also been detected in the dermis. In the dermis, since there is no rapid turnover like the epidermis, it is considered that the melanin that has fallen into the dermis exists in the dermis for a long time. Therefore, to eliminate freckles, not only the conventional approach to the epidermis but also an approach to the dermis is necessary.

[0004] Histological examination of the dermis in the area of ​​blemishes reveals a breakdown of the dermal matrix, an increase in blood vessels and inflammatory cells in the surrounding area. Furthermore, cells that have lost their migratory properties and are thought to be macrophages containing a large amount of melanin are also observed. This suggests that melanin deposited in the dermis is phagocytosed by macrophages and remains in place, making the blemishes difficult to remove (Non-Patent Literature 1). The main methods for removing melanin in the dermis involve cosmetic surgery techniques such as lasers and liquid nitrogen. These methods are painful and costly, placing a significant psychological and financial burden on the patient. Moreover, even if blemishes can be temporarily removed with lasers, recurrence is not uncommon. On the other hand, recent research has confirmed that fibroblasts in the dermis, like macrophages, possess the function of phagocytosing melanosomes containing melanin granules (Non-Patent Literature 2).

[0005] On the other hand, lysosomes are one of the intracellular organelles, and within them are various hydrolytic enzymes (lysosomal enzymes) that function under acidic pH conditions, hydrolyzing biomolecules taken in from inside and outside the cell by autophagy and endocytosis. Therefore, for example, it is thought that activating the hydrolytic enzymes contained in intracellular lysosomes present in the dermis can promote the breakdown of melanin accumulated in the dermis. To date, it has been reported that extracts from plants belonging to the genus Coix in the grass family and the genus Betula in the betulaceae family have the effect of promoting lysosome activity in epidermal cells and fibroblasts, and improving the ability to produce hyaluronic acid (Patent Document 1). [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Japanese Patent Publication No. 2017-206468 [Non-patent literature]

[0007] [Non-Patent Document 1] Fumio Kusuda, Toshiaki Kubo, Yukio Sudo, Tatsuo Kawabuchi, Atsushi Orikasa, Yoshisada Nakamura: Development of the functional cosmetic "ASTALIFT Whitening Essence", Fujifilm Research Report, (55), 33-37, 2010. [Non-Patent Document 2] Hideya Ando, ​​Sayumi Inoue, Kanoko Otsubo, Erina Ono, Takeshi Norimatsu, Masamitsu Ichihashi: "A comparative study on the melanosome phagocytic activity of keratinocytes and fibroblasts," Aesthetic Dermatology, 22(3):284-287, 2012. [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] In view of the circumstances described above, the present invention aims to discover a new, highly safe material that can efficiently remove melanin accumulated in the skin by activating lysosomes in skin cells, and to provide compositions such as whitening cosmetics and pharmaceuticals. [Means for solving the problem]

[0009] As a result of diligent research to solve the above problems, the inventors of the present invention have discovered that Camellia japonica, a highly safe natural plant material, can promote the expression of hydrolytic enzyme genes contained in lysosomes within skin cells, thereby activating lysosomes within skin cells and promoting melanin degradation, thus completing the present invention.

[0010] In other words, the present invention encompasses the following inventions. (1) A lysosome activator for skin cells containing camellia extract as an active ingredient. (2) A lysosomal enzyme gene expression promoter in skin cells, containing camellia extract as an active ingredient. (3) A melanin decomposition promoter in skin cells, containing camellia extract as an active ingredient. (4) The agent according to any one of (1) to (3), wherein the skin cells are cells present in the dermis. (5) The agent according to any one of (1) to (4), wherein the variety of the camellia is Camellia japonica var decumbens. (6) A whitening composition comprising any of the agents described in (1) to (5). (7) The whitening composition according to (6), wherein the composition is a cosmetic, quasi-drug, pharmaceutical, or food or beverage. [Effects of the Invention]

[0011] The present invention provides a melanin decomposition accelerator that can efficiently remove melanin accumulated in the skin by activating lysosomes in skin cells. Therefore, the present invention is effective, for example, in improving or preventing age spots caused by melanin accumulated in the dermis. [Modes for carrying out the invention]

[0012] The present invention provides a lysosome activator for skin cells, a lysosomal enzyme gene expression promoter, and The melanin decomposition accelerator (hereinafter referred to as "the agent of the present invention") contains camellia extract as an active ingredient.

[0013] The camellia extract used in this invention refers to the extract of Camellia japonica, which belongs to the genus Camellia in the family Theaceae. The camellia varieties used in this invention include Camellia japonica var japonica, which includes wild species such as Camellia japonica and Camellia japonica; Camellia japonica var decumbens, which includes species adapted to heavy snowfall areas such as Camellia japonica var. 1999, Camellia japonica var. 1999, and Camellia japonica var. 1999; and Camellia japonica var. macrocarpa, which is adapted to southern regions and includes intermediate types between the two (such as Camellia japonica var. 1999), Camellia japonica var. 1999, Camellia japonica var. 1999, and Camellia japonica var. 1999. However, Camellia japonica var. decumbens is preferred. Furthermore, hybrids of the above varieties, as well as horticultural varieties resulting from crosses between the above varieties and closely related species, are also included. For example, some horticultural varieties of Camellia japonica var. decumbens include Otome Tsubaki, Agano Sato, Asazakura, Ariso, Ichiraku, Iwai no Sakazuki, Katsurahime, Kita no Yo, Gochi no Musume, Shima Chidori, Shima no Nishiki, Jakko, Yukihozan, Tashiro, Toyo no Hikari, Nishiki Kirin, Nihongami, Okinoka, Botan Yuki, Matsunami, Yukigeshiki, Yuki Komachi, Yukigoromo, Yoshun, Ogura no Sato, Shiratama, Tachiyama, Togashi Shiro, Hatsushigure, Hoju, Honpoji, Momosuzume, Yukiakari, Yukitoro, Kaga no Tsuru, and Yachiyo.

[0014] In the present invention, camellia extract refers to an extract of the entire plant, or an extract of a part of the plant such as leaves, stems, flowers, buds, fruits, seeds, bark, roots, or a mixture thereof, but seed and leaf extracts are preferred. Furthermore, these plant parts may be used as they are for extraction, or they may be subjected to processing such as drying, grinding, or finely chopping.

[0015] The extraction method of camellia extract is not particularly limited. For example, continuous extraction, immersion extraction, etc. can be mentioned. Also, a heating extraction method may be used, or a normal temperature or cold temperature extraction method may be used. Examples of the solvent used for extraction include water, lower alcohols (such as methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, etc.), liquid polyhydric alcohols (such as 1,3-butylene glycol, propylene glycol, glycerin, etc.), ketones (such as acetone, methyl ethyl ketone, etc.), acetonitrile, esters (such as ethyl acetate, butyl acetate, etc.), hydrocarbons (such as hexane, heptane, liquid paraffin, etc.), and ethers (such as ethyl ether, tetrahydrofuran, propyl ether, etc.). Among these solvents, water, lower alcohols, and liquid polyhydric alcohols are preferred, and water and ethanol are more preferred. These solvents can be used alone or in combination of two or more. For example, an aqueous ethanol solution of 30-70 v / v% can be used. Also, an acid or alkali can be added to the above extraction solvent to use a solvent with adjusted pH.

[0016] There is no particular limitation on the amount of solvent used. For example, it may be 10 times or more, preferably 20 times or more, based on the above camellia (dry weight). However, for the convenience of operations in cases of concentration or isolation after extraction, it is preferably 100 times or less. Also, the extraction temperature and time depend on the type of solvent used. For example, it can be exemplified as 10-100 °C, preferably 30-90 °C, and 30 minutes to 24 hours, preferably 1-10 hours.

[0017] The extract may be used as the extracted solution as it is. However, if necessary, within the range not affecting its effect, treatments such as concentration (concentration by organic solvent, vacuum concentration, membrane concentration, etc.), dilution, filtration, decolorization with activated carbon, etc., deodorization, ethanol precipitation, etc. can be performed and then used. Furthermore, the extracted solution can be subjected to treatments such as concentration to dryness, spray drying, freeze drying, etc., and used as a dried product.

[0018] In the present invention, skin cells include epidermal keratinocytes (keratinocytes), fibroblasts, pigment cells (melanocytes), adipocytes, mast cells, plasma cells, Langerhans cells, α dendritic cells, Merkel cells, vascular and lymphatic endothelial cells, etc. Cells present in the dermis, specifically, dermal fibroblasts and macrophages (histiocytes) are preferred. Further, skin cells include not only differentiated cells but also their progenitor cells and stem cells. These skin cells may be of human origin or of other animals, such as rats, mice, rabbits, etc.

[0019] In the present invention, the "lysosomal enzyme gene" is not particularly limited as long as it is a hydrolase gene localized in the intracellular lysosomes of eukaryotes. Examples include the cathepsin D (CTSD) gene, which is an aspartic protease, the glucocerebrosidase (GBA) gene, neuraminidase 1 (NEU1), and the like.

[0020] The above-mentioned camellia extract has the effect of promoting lysosome activity by promoting the expression of lysosomal enzyme genes, and can efficiently decompose melanin in skin cells, particularly melanin granules that have dripped from the epidermis into the dermis, by lysosomes. Therefore, the camellia extract can be used as an active ingredient in lysosome activators, lysosomal enzyme gene expression promoters, and melanin decomposition promoters within skin cells. Thus, the agent of the present invention is effective, for example, in the treatment, improvement, and prevention of skin pigmentation caused by the accumulation of melanin in the dermis. Here, "pigmentation" refers to a condition in which the entire skin becomes dark, or a part of it becomes a darker brownish color than the surrounding skin, due to excessive melanin production caused by aging, ultraviolet rays, friction, hormonal imbalances, stress, lifestyle habits (sleep and smoking habits), etc., which increases the melanin content in the skin, and the excess melanin remains without being excreted. Skin diseases or conditions that exhibit pigmentation include, for example, senile lentigines (age spots), post-inflammatory hyperpigmentation, melasma, freckles, lentigines, seborrheic keratosis, nevus cells (nevus, pigmented nevi), acquired dermal melanocytosis (late-onset nevus of Ota), flat nevi, Riehl's melanosis, frictional melanosis, hereditary contralateral pigmentary disorders, Addison's disease, actinic lentigines, and fixed erythema pigmentosum.

[0021] The agent of the present invention can be used as is, but it can also be mixed with appropriate additives within a range that does not impair the effects of the present invention and incorporated into a whitening composition. Examples of compositional forms include cosmetics, pharmaceuticals, quasi-drugs, and food and beverages. When the agent of the present invention is used, for example, to remove accumulated melanin and for the purpose of whitening, it can be in the form of a topical skin composition such as a cosmetic. Furthermore, when the agent of the present invention is used for the purpose of treating, improving, and preventing pigmentation (spots), it is preferable to use it in the form of a pharmaceutical.

[0022] When the agent of the present invention is incorporated into cosmetics or quasi-drugs, the dosage form may be any of the following: aqueous solution, solubilized, emulsified, powder, powder dispersion, oil-liquid, gel, ointment, aerosol, water-oil two-layer system, or water-oil-powder three-layer system. Furthermore, the cosmetics or quasi-drugs may be manufactured by selecting and appropriately incorporating various components, additives, bases, etc., commonly used in topical skin compositions, along with the above-mentioned camellia extract, according to the methods known in the art. The form may be any of the following: liquid, emulsion, cream, gel, paste, spray, etc. The ingredients include, for example, oils and fats (olive oil, coconut oil, evening primrose oil, jojoba oil, castor oil, hydrogenated castor oil, etc.), waxes (lanolin, beeswax, carnauba wax, etc.), hydrocarbons (liquid paraffin, squalene, squalane, petrolatum, etc.), fatty acids (lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, etc.), higher alcohols (myristyl alcohol, cetanol, cetostearyl alcohol, stearyl alcohol, behenyl alcohol, etc.), and esters (isopropyl myristate, i) Examples of ingredients include sopropyl, cetyl octanoate, glyceryl trioctanoate, octyldodecyl myristate, octyl stearate, stearyl stearate, etc.), organic acids (citric acid, lactic acid, α-hydroxyacetic acid, pyrrolidone carboxylic acid, etc.), sugars (maltitol, sorbitol, xylobiose, N-acetyl-D-glucosamine, etc.), proteins and protein hydrolysates, amino acids and their salts, vitamins, plant and animal extracts, various surfactants, humectants, UV absorbers, antioxidants, stabilizers, preservatives, disinfectants, fragrances, etc.

[0023] Examples of cosmetics and quasi-drugs include lotions, emulsions, gels, serums, general creams, sunscreens, packs, masks, facial cleansers, cosmetic soaps, foundations, face powders, and body lotions.

[0024] When the above-mentioned camellia extract is incorporated into a pharmaceutical product, it can be mixed with pharmacologically and pharmaceutically acceptable additives and formulated into various formulations suitable for application to the affected area. Pharmacologically and pharmaceutically acceptable additives may include, depending on the dosage form and application, appropriately selected formulation bases or carriers, excipients, diluents, binders, lubricants, coatings, disintegrants or disintegration aids, stabilizers, preservatives, antiseptics, bulking agents, dispersants, wetting agents, buffers, solubilizers or solubilizers, isotonic agents, pH adjusters, propellants, colorants, sweeteners, flavoring agents, fragrances, etc., which can then be prepared into various formulations that can be administered orally or parenterally systemically or topically by various known methods. When providing the pharmaceutical product of the present invention in any of the above forms, it can be manufactured by methods commonly used by those skilled in the art, such as those indicated in the individual articles of the General Provisions for Formulations of the Japanese Pharmacopoeia [2].

[0025] Oral formulations may use, but are not limited to, excipients such as starch, glucose, sucrose, fructose, lactose, sorbitol, mannitol, crystalline cellulose, magnesium carbonate, magnesium oxide, calcium phosphate, or dextrin; disintegrants or disintegration aids such as carboxymethylcellulose, carboxymethylcellulose calcium, or hydroxypropylcellulose; binders such as hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, gum arabic, or gelatin; lubricants such as magnesium stearate, calcium stearate, or talc; coating agents such as hydroxypropylmethylcellulose, sucrose, polyethylene glycol, or titanium dioxide; and bases such as petrolatum, liquid paraffin, polyethylene glycol, gelatin, kaolin, glycerin, purified water, or hard fat.

[0026] Parenteral formulations may use, but are not limited to, solvents such as distilled water, physiological saline, ethanol, glycerin, propylene glycol, macrogol, alum solution, and vegetable oil; isotonic agents such as glucose, sodium chloride, and D-mannitol; and pH adjusters such as inorganic acids, organic acids, inorganic bases, or organic bases.

[0027] The form of the pharmaceutical product of the present invention is not particularly limited, but examples include oral preparations such as tablets, sugar-coated tablets, capsules, lozenges, granules, powders, liquids, pills, emulsions, syrups, suspensions, and elixirs; parenteral preparations such as injections (e.g., subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections), drip infusions, suppositories, transdermal preparations, transmucosal preparations, and patches. It may also be provided as a dried product that is redissolved before use, and in the case of injection preparations, it is provided in the form of unit dose ampoules or multi-dose containers.

[0028] When the pharmaceutical product of the present invention is used to treat, improve, or prevent pigmentation (spots), a suitable form is a topical preparation, such as an ointment, cream, gel, liquid, patch, foam, spray, or aerosol. An ointment is a homogeneous, semi-solid topical preparation and includes oily ointments, emulsion ointments, and water-soluble ointments. A gel is a topical preparation in which a water-insoluble component containing a water-holding compound is suspended in an aqueous solution. A liquid preparation is a liquid topical preparation and includes lotions, suspensions, emulsions, liniments, and the like.

[0029] The amount of the cosmetic, quasi-drug, or pharmaceutical product used or administered according to the present invention can be appropriately determined according to its type and form, the age, sex, weight, and severity of symptoms of the person using or administering it. For example, when administered orally to an adult, the amount of camellia extract is 0.1 to 1000 mg / day, preferably 1 to 500 mg / day, more preferably 5 to 300 mg / day, administered once to several times a day. In some cases, an amount less than the above dosage range may be sufficient, and in other cases, it may be necessary to administer an amount exceeding the range.

[0030] When the camellia extract is incorporated into the above-mentioned cosmetics, quasi-drugs, and pharmaceuticals, the amount is not particularly limited, but it is preferably 0.001 to 30% by weight (w / w) of the camellia extract as dry solids relative to the total weight of the formulation (composition), and more preferably 0.01 to 10% by weight (w / w). Below 0.001% by weight (w / w), the effect is low, and above 30% by weight (w / w), a significant increase in effect is unlikely to be observed. Furthermore, regarding the method of adding the active ingredient in formulation, it may be added in advance or added during manufacturing, and the appropriate method should be chosen considering workability.

[0031] Furthermore, the above-mentioned camellia extract can also be incorporated into food and beverages. In this invention, "food and beverages" refers to general food and beverages, as well as foods other than pharmaceuticals that can be consumed for the purpose of maintaining or promoting health, such as health foods, functional foods, health functional foods, or foods for special dietary uses. Health foods include foods provided under names such as nutritional supplements, health supplements, and supplements. Health functional foods are defined by the Food Sanitation Act or the Food Promotion Act and include Foods for Specified Health Uses and Foods with Nutrient Function Claims, which can display specific health effects, the functions of nutritional components, or the reduction of disease risk. The form of food and beverages may be any form suitable for consumption, such as solid, liquid, granular, granular, powder, capsule, cream, or paste.

[0032] Examples of food and beverage products include, but are not limited to, bread, noodles, confectionery, dairy products, processed seafood and livestock products, oils and fats and processed oils and fats products, seasonings, various beverages (soft drinks, carbonated drinks, beauty drinks, nutritional drinks, fruit drinks, dairy drinks, etc.), and concentrated liquids and powders for adjusting such beverages.

[0033] The food and beverages of the present invention may contain additives that are commonly used depending on their type. Any additive that is acceptable from a food hygiene perspective can be used, but examples include sweeteners such as glucose, sucrose, fructose, isomerized liquid sugar, aspartame, and stevia; acidulants such as citric acid, malic acid, and tartaric acid; excipients such as dextrin and starch; binders, diluents, flavorings, colorings, buffers, thickeners, gelling agents, stabilizers, preservatives, emulsifiers, dispersants, suspending agents, and antiseptics.

[0034] The amount of the camellia extract used in the food and beverage of the present invention should be sufficient to exert its lysosome-activating effect and melanin-degrading-promoting effect within skin cells. However, it should be set appropriately considering the general intake amount of the food and beverage, the form of the food and beverage, its efficacy and effects, taste, palatability, and cost. [Examples]

[0035] The present invention will be described in more detail below with reference to examples. However, the present invention is not limited to these examples.

[0036] [Example 1] (Manufacturing Example 1) Preparation of hot water extract of Camellia japonica seeds 30 g of dried oil residue from Camellia japonica var. decumbens seeds was mixed with 300 mL of purified water, extracted at 95-100°C for 2 hours, filtered, concentrated the filtrate, and freeze-dried to obtain 3.2 g of hot water extract of Camellia japonica seeds.

[0037] (Manufacturing Example 2) Preparation of hot water extract of Camellia japonica leaves 30 g of dried leaves of Camellia japonica var. decumbens were added to 300 mL of purified water, extracted at 95-100°C for 2 hours, filtered, the filtrate was concentrated, and freeze-dried to obtain 4.4 g of hot water extract of Camellia japonica leaves.

[0038] (Manufacturing Example 3) Preparation of a 50% ethanol extract of Camellia japonica seeds 20 g of dried oil residue from Camellia japonica var. decumbens seeds was mixed with 100 mL of 50% (w / w) ethanol, extracted at room temperature for 3 days, filtered, concentrated the filtrate, and freeze-dried to obtain 2.6 g of a 50% ethanol extract of Camellia japonica seeds.

[0039] (Manufacturing Example 4) Preparation of a 50% ethanol extract of Camellia japonica leaves 20 g of dried leaves of Camellia japonica var. decumbens were added to 100 mL of 50% (w / w) ethanol, extracted at room temperature for 3 days, filtered, concentrated the filtrate, and freeze-dried to obtain 2.4 g of a 50% ethanol extract of Camellia japonica leaves.

[0040] (Manufacturing Example 5) Preparation of ethanol extract of Camellia japonica seeds 20 g of dried oil residue from Camellia japonica var. decumbens seeds was mixed with 100 mL of ethanol, extracted at room temperature for 3 days, filtered, concentrated the filtrate, and freeze-dried to obtain 1.4 g of ethanol extract from Camellia japonica seeds.

[0041] (Manufacturing Example 6) Preparation of ethanol extract of Camellia japonica leaves 20 g of dried leaves of Camellia japonica var. decumbens were added to 100 mL of ethanol, extracted at room temperature for 3 days, filtered, concentrated the filtrate, and freeze-dried to obtain 1.0 g of ethanol extract of Camellia japonica leaves.

[0042] [Example 2] (Experimental Example 1) Intracellular lysosome activation effect of Camellia japonica extract (1) Lysosomal activation effect within dermal stem cells Commercially available human dermal fibroblasts (Toyobo Co., Ltd.) were used as dermal-derived cells, and dermal stem cells were isolated using NGFR (nerve growth factor receptor: Genbank number: Nucleotide NM_002507.3; Protein NP_002498.1) as an indicator, in accordance with the method described in Japanese Patent Publication No. 2017-093383. Dermal stem cells maintained in 15% FBS-containing αMEM medium (Thermo Inc.) were divided into 2 × 10⁶ cells. 4 Seeds were seeded in 24-well plates (Falcon) to form individual plants. After 24 hours of incubation, the test samples (extracts of Camellia japonica prepared in Example 1, from Production Examples 1-6) were added to a final concentration of 100 μg / mL, and the plants were incubated for another 24 hours.

[0043] After culturing, the cells were harvested, and the lysosomal activation effect within the cells was evaluated using the expression levels of the lysosomal enzyme genes CTSD and GBA as indicators.

[0044] Gene expression analysis was performed as follows: After washing the collected cells twice with PBS(-), RNA was extracted from the cells using RNAIso+ (Takara Bio Inc.). The extracted RNA was reverse transcribed into cDNA using the High Capacity RNA to cDNA Kit (Thermo Inc.), and then PCR was performed using the following primer set with SYBR Select Master Mix (Thermo Inc.) (initial denaturation at 95°C for 2 minutes, followed by 95°C for 15 seconds and 60°C for 30 seconds, 40 cycles) to confirm the gene expression levels of CTSD and GBA. Other procedures were performed according to the prescribed method.

[0045] CTSD Primer Set: 5'-CCGAGGTGCTCAAGAACTACA-3' (Sequence ID 1) 5'-CGTCCCGATGCCAATCT-3' (SEQ ID NO: 2) GBA Primer Set: 5'-GGTCCTACCCTCGCCAACA-3' (Sequence ID 3) 5'-AAGCGTTGGTCATCCAGCAT-3' (Sequence ID 4) Primer set for GAPDH (internal standard): 5'-TGCACCACCAACTGCTTAGC-3' (Sequence ID 5) 5'-TCTTCTGGGTGGCAGTGATG-3'(Sequence No. 6)

[0046] The expression of CTSD and GBA genes was evaluated by calculating the relative expression levels of CTSD and GBA mRNA in cells without the test sample as a ratio to the expression level of GAPDH mRNA, which is an internal standard. The relative expression levels of CTSD and GBA genes (CTSD gene expression level / GAPDH gene expression level, GBA gene expression level / GAPDH gene expression level) were set to 100%. In contrast, the relative expression levels of CTSD and GBA genes in cells cultured with the test sample were calculated and evaluated.

[0047] (2) Lysosomal activation effect within macrophages Human monocytic leukemia cell line THP1 2 × 10 5 Seeds were seeded into 24-well plates (Falcon) to form macrophages, and cultured for 72 hours in RPMI medium (Thermo) containing 10% FBS with PMA added to a final concentration of 100 nM to induce differentiation into macrophages. Subsequently, the test samples (extracts of Camellia japonica prepared in Example 1, from Production Examples 1 to 6) were added to a final concentration of 100 μg / mL, and cultured for 24 hours. After the culture period, gene expression analysis of CTSD and GBA in macrophages was performed in the same manner as in (1).

[0048] (3) Activation effect of lysosomes in fibroblasts Using commercially available human dermal fibroblasts (manufactured by Toyobo Co., Ltd.), dermal stem cells were isolated using NGFR (nerve growth factor receptor: Genbank number: Nucleotide NM_002507.3; Protein NP_002498.1) as an indicator, in accordance with the method described in Japanese Patent Publication No. 2017-093383. At that time, 2 × 10⁶ dermal fibroblasts that were not isolated as dermal stem cells were collected.4 Seeds were seeded in 24-well plates (Falcon) using 10% FBS-containing DMEM medium (Nacalai). After 24 hours of incubation, the test samples (extracts of Camellia japonica prepared in Example 1, from Production Examples 1-6) were added to a final concentration of 100 μg / mL, and the cells were incubated for another 24 hours. After incubation, gene expression analysis of CTSD and GBA in fibroblasts was performed in the same manner as in (1).

[0049] The results of (1) to (3) above are shown in Table 1.

[0050] [Table 1]

[0051] As shown in Table 1, the expression level of lysosomal enzyme genes was increased in cells to which Camellia japonica extract (Production Examples 1-6) was added, indicating that Camellia japonica extract has an intracellular lysosome activating effect. In addition to the above-mentioned cells (dermal stem cells, macrophages, fibroblasts), the intracellular lysosome activating effect of Camellia japonica extract was similarly observed in keratinocytes and melanocytes, which are cells present in the skin.

[0052] (Experimental Example 2) Effect of Camellia japonica extract on promoting intracellular melanin degradation (1) Preparation of melanosomes Normal human epidermal melanocytes NHEM (manufactured by Kurabo Corporation) 1 × 10 6Cells were seeded in 100 mm petri dishes (TPP) to form individual cells and cultured for 72 hours in Medium 254 medium (Thermo) supplemented with HMGS (Thermo). Subsequently, they were cultured for 96 hours in 10% FBS-containing Medium 254 medium (Thermo) supplemented with 10 nM ET-1 (Sigma) and 500 μM dbcAMP (Santa Crutz). After the culture period, the cells were detached using a cell scraper and collected along with the culture supernatant, and centrifuged at 25°C and 2000 g for 10 minutes. The supernatant was collected and centrifuged at 25°C and 15000 g for 5 minutes. The supernatant was filtered through a 5 μm filter (Membrane Solutions), centrifuged at 25°C and 15000 g for 5 minutes, and the resulting pellet was suspended in PBS and used as a melanosome suspension.

[0053] (2) Effect of promoting melanin decomposition in dermal stem cells Commercially available human dermal fibroblasts (Toyobo Co., Ltd.) were used as dermal-derived cells, and dermal stem cells were isolated using NGFR (nerve growth factor receptor: Genbank number: Nucleotide NM_002507.3; Protein NP_002498.1) as an indicator, in accordance with the method described in Japanese Patent Publication No. 2017-093383. Dermal stem cells maintained in 15% FBS-containing αMEM medium (Thermo Inc.) were divided into 1 × 10⁶ cells. 5 The cells were seeded in a 6-well plate (Falcon) to a concentration of 100 μg / mL. After 72 hours of incubation, the melanosome suspension obtained in (1) above and the test sample (extract of Camellia japonica prepared in Example 1) were added to achieve a final concentration of 100 μg / mL, and the cells were incubated for 48 hours. After incubation, the cells were detached using 1 M NaOH and heated at 60°C for 30 minutes, and the wavelength of 450 nm was detected using a microplate reader (Molecular Devices). The amount of melanin was calculated from a melanin calibration curve created using melanin pigment (Sigma-Ace), and the amount of melanin without the test sample was used as the control, with the relative amount of melanin being analyzed with the control set to 100 (%).

[0054] (3) Effect of promoting melanin degradation within macrophages Human monocytic leukemia cell line THP1 5 × 10 5 Cells were seeded into 6-well plates (Falcon) to form macrophages, and cultured for 72 hours in RPMI medium containing 10% FBS (Thermo), to which PMA was added to a final concentration of 100 nM, to induce differentiation into macrophages. Subsequently, the melanosome suspension obtained in (1) above and the test sample (extract of Camellia japonica prepared in Example 1) were added to a final concentration of 100 μg / mL, and cultured for 48 hours. After the culture period, the cells were detached using 1 M NaOH and heated at 60°C for 30 minutes, and the wavelength of 450 nm was detected using a microplate reader (Molecular Devices). The amount of melanin was calculated from a melanin calibration curve created using melanin pigment (Sigma-Ace), and the amount of melanin without the addition of the test substance was used as a control, and the relative amount of melanin was analyzed with the control set to 100 (%).

[0055] (4) Effect of promoting melanin degradation in fibroblast cells Using commercially available human dermal fibroblasts (manufactured by Toyobo Co., Ltd.), dermal stem cells were isolated using NGFR (nerve growth factor receptor: Genbank number: Nucleotide NM_002507.3; Protein NP_002498.1) as an indicator, in accordance with the method described in Japanese Patent Publication No. 2017-093383. At that time, 1 × 10⁶ dermal fibroblasts that were not isolated as dermal stem cells were collected. 5Cells were seeded in 6-well plates (Falcon) using 10% FBS-containing DMEM medium (Nacalai). After 72 hours of incubation, the melanosome suspension obtained in (1) above and the test sample (extract of Camellia japonica prepared in Example 1) were added to achieve a final concentration of 100 μg / mL, and the cells were incubated for 48 hours. After incubation, the cells were detached using 1 M NaOH and heated at 60°C for 30 minutes, and the wavelength of 450 nm was detected using a microplate reader (Molecular Devices). The amount of melanin was calculated from a melanin calibration curve created using melanin pigment (Sigma-Ace), and the amount of melanin without the addition of the test substance was used as a control, with the relative amount of melanin being analyzed with the control set to 100 (%).

[0056] The results of (2) to (4) above are shown in Table 2.

[0057] [Table 2]

[0058] As shown in Table 2, the extract of Camellia japonica was found to have an effect of promoting melanin degradation within cells. In addition to the various cells mentioned above (dermal stem cells, macrophages, and fibroblasts), the effect of Camellia japonica extract on promoting melanin degradation within cells was similarly observed in keratinocytes and melanocytes, which are cells present in the skin. [Industrial applicability]

[0059] This invention can be used in the manufacturing fields of cosmetics, quasi-drugs, pharmaceuticals, and food and beverages for the purpose of whitening, treating, improving, or preventing hyperpigmentation (spots).

Claims

1. A lysosome activator for cells present in the dermis, containing the oil extraction residue or leaf extract of Camellia japonica var. decumbens as an active ingredient.

2. A lysosomal enzyme gene expression promoter in cells present in the dermis, containing the oil extraction residue or leaf extract of Camellia japonica var. decumbens as an active ingredient.

3. A melanin decomposition accelerator present in dermal cells, containing the oil extraction residue or leaf extract of Camellia japonica var. decumbens as an active ingredient.

4. A composition for treating, improving, and preventing skin pigmentation caused by the accumulation of melanin in the dermis, comprising the agent according to any one of claims 1 to 3.

5. The composition for treating, improving, and preventing skin pigmentation caused by the accumulation of melanin in the dermis, as described in claim 4, wherein the composition is a cosmetic, quasi-drug, pharmaceutical, or food or beverage.

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