Proteasome activator containing podophyllotoxin

Abstract

A compound that is contained in common juniper extract and that promotes proteasome activation was discovered. The present invention provides: a cosmetic product, a food product, a medicinal product, or the like for creating, by using said compound, exterior beauty by activating proteasomes in cells including human skin cells; and / or a material to be blended in a cosmetic product, a food product, a medicinal product, or the like. This proteasome activator contains, as an active ingredient, at least one compound represented by formula (I) or formula (II) (in the formulae, R1 and R2 each independently represent a hydrogen atom, a hydroxy group, a methoxy group, an acetoxy group, or a glucosyl group, and each R3 independently represents a hydrogen atom, a hydroxy group, or a methoxy group), or a modification product thereof.

Description

Proteasome activators containing podophyllotoxins

[0001] The present invention relates to a proteasome activator containing a podophyllotoxin, and the like.

[0002] Proteasomes are complex proteases expressed intracellularly and selectively degrade proteins that have completed their role, proteins that have become dysfunctional, and exogenous proteins. It is known that proteasome activity weakens with age. Therefore, in aged cells, the degradation of proteins that have completed their role is delayed, and proteins oxidized by UV, stress, active enzymes, free radicals, physical stimuli, etc., structurally denatured proteins (e.g., carbonylated proteins), and aggregated proteins (e.g., lipofuscin) accumulate. The accumulation of carbonylated proteins and lipofuscin is known to impair skin clarity and cause pigmentation, which leads to dull skin (Patent Document 1).

[0003] Based on the above findings, it is expected that activating intracellular proteasomes and actively breaking down proteins that have completed their role within the cell, dysfunctional proteins, and foreign proteins will prevent the aging of the cellular tissues of individual organisms, and ultimately prevent the aging of the individual organism itself.

[0004] However, proteasome-activating compounds have not yet been thoroughly studied compared to proteasome-inhibiting compounds. Only a limited number of reports have been published on proteasome-activating compounds, such as 1'-acetoxychavicol acetate (Non-Patent Document 1) found in ginger (Alpinia galanga). Furthermore, these plant-derived natural products present challenges in their use. 1'-acetoxychavicol acetate is known to lack stability in aqueous solution (Patent Document 2).

[0005] Recently, a new plant extract, extracted from the Cupressaceae plant Juniperus communis L. (e.g., fruit), has been reported to activate the proteasome (Patent Document 3). However, the active ingredients contained in the Juniperus communis extract that activate the proteasome have not yet been identified. Cupressaceae plants contain a variety of compounds, including lignans, sesquiterpenes, diterpenes, flavonoids, and polyphenols. In particular, lignans with cytostatic activity have long been studied as anticancer agents. For example, a derivative of the lignan podophyllotoxin is used as the pharmaceutical drug etoposide (non-patent document 2). However, it was not known that podophyllotoxin or its analogs and derivatives activate the proteasome.

[0006] Planta Med “1'-Acetoxychavicol acetate increases Proteasome activity by activating cAMP-PKA signaling” (2018) 84(3):153-159.Molecules “Junipers of various origins as potential sources of the anticancer drug precursor podophyllotoxin” (2021) 26(17):5179.Heliyon “Insights about stabilization of sulforaphane through microencapsulation” (2019) 5(11):e02951.Chem Pharm Bull “The biological actions of podophyllotoxin (anthricin).Physiological activities and conformational analysis of deoxypodophyllotoxin” (1985) 33(2):704-709.Proc Natl Acad Sci USA “Mechanistic analysis of carbon-carbon bond formation by deoxypodophyllotoxin synthase” (2022) 119(1):e2113770119.

[0007] JP 2001-131044 A JP 2022-090958 A JP 2018-203725 A

[0008] The problem that the present invention aims to solve is to find a compound contained in Juniperus communis extract that activates proteasomes, and to provide cosmetics, foods, pharmaceuticals, etc., and / or materials to be incorporated into these, that can improve skin elasticity, dullness, and transparency by using this compound to activate intracellular proteasomes.

[0009] The present invention was made to solve the above-mentioned problems. The present inventors discovered that a specific compound fractionated and purified from a Juniperus communis extract using column chromatography or the like activates proteasomes, and thus the present invention was created based on this discovery. Specifically, the present invention includes the following embodiments: (1) A compound represented by the following formula (I): Or the following formula (II): (In the formula, R １ and R ２ are each independently a hydrogen atom, a hydroxy group, a methoxy group, an acetoxy group, or a glucosyl group, and R ３ (2) A proteasome activator comprising, as an active ingredient, one or more compounds represented by formula (I) or (II) or modified forms thereof, wherein each of the groups independently represents a hydrogen atom, a hydroxy group, or a methoxy group. Or the following formula (IV): (In the formula, R １ and R ２ is as described in (1). (3) A topical skin preparation for proteasome activation, containing the proteasome activator according to (1) or (2). (4) The topical skin preparation according to (3), containing at least one active ingredient at a concentration of 1 nM or more.

[0010] The proteasome activator of the present invention activates cellular proteasomes and decomposes dysfunctional proteins such as oxidized proteins, structurally denatured proteins, and aggregated proteins within the cells, and can provide cosmetics, foods, pharmaceuticals, etc., and / or materials to be incorporated into these, for improving skin elasticity, dullness, and transparency.

[0011] Figure 1 shows the proteasome activation effect of juniper fruit extract (JBE) on HEK293T cells (1A), adult-derived human skin keratinocytes (1B), and neonatal-derived human skin keratinocytes (1C). The relative proteasome activity of each cell was determined based on the vehicle-only well (100). (1A) At a final JBE concentration of 1%, the proteasome activity was 245, 254, 152, and 129 after 2, 4, 8, and 24 hours, respectively. (1B) At a final JBE concentration of 0.5%, the proteasome activity was 143, 143, 109, and 104 after 2, 4, 8, and 24 hours, respectively. (1B) At a final JBE concentration of 1%, the proteasome activity was 178, 179, 153, and 120 after 2, 4, 8, and 24 hours, respectively. At a final JBE concentration of 0.5%, the lipofuscin accumulation was 147, 136, 122, and 104 after 2, 4, 8, and 24 hours, respectively. (1C) At a final JBE concentration of 1%, the lipofuscin accumulation was 168, 168, 126, and 103 after 2, 4, 8, and 24 hours, respectively. At a final JBE concentration of 0.5%, the lipofuscin accumulation was 139, 153, 123, and 101 after 2, 4, 8, and 24 hours, respectively. Figure 2 shows the results of evaluating the inhibitory effect of juniper fruit extract (JBE) on intracellular lipofuscin accumulation by Masson-Fontana staining using an inverted microscope (2A) and measuring the stained area (2B). In Figure 2B, the stained area of ​​the control cells (Control) was set at 100, and the relative value of the stained area of ​​the cells (JBE) cultured in the presence of juniper fruit extract was 37. Figure 3A shows the scheme for fractionation and purification of effective fractions from Juniperus communis fruit extract (hot water extract). Figure 3B shows the scheme for isolation and purification of Compounds 1 and 2 from Juniperus communis fruit extract (70% ethanol extract). Figure 4A shows the proteasome activation activity of Fractions 1 to 7 fractionated from Juniperus communis fruit extract (hot water extract). The relative proteasome activity of each fraction was determined as follows: Fraction 1 -1, Fraction 2 -4, Fraction 3 10, Fraction 4 1, Fraction 5 2, Fraction 6 -2, and Fraction 7 116, with the solvent-only well set as 0 and the JBE well set as 100. Figure 4B shows the proteasome activation activity of Fractions 7-1 to 7-5 fractionated from Fraction 7.The relative proteasome activity of each fraction was 93 for Fr. 7, 39 for Fr. 7-1, 51 for Fr. 7-2, 57 for Fr. 7-3, 91 for Fr. 7-4, and 60 for Fr. 7-5, with the well containing only solvent as 0 and the JBE as 100. Figure 4C shows the proteasome activation activity of Fr. 7-4-1 to 7-4-3 fractionated from Fr. 7-4. The relative proteasome activity of each fraction was 0 for the well containing only solvent, 0.3 for Fr. 7-4-2, and 102 for Fr. 7-4-3. Figure 4D shows the proteasome activation activity of H2-1-2 fractionated from Juniperus communis fruit extract (70% ethanol extract). The relative value of the proteasome activity of H2-1-2 was 127, with the proteasome activity of JBE set to 100. Figure 4E shows the proteasome activation activity of each compound isolated from Juniperus communis fruit extract (70% ethanol extract). The relative value of the proteasome activity of Compound 1 was 92, and the relative value of the proteasome activity of Compound 2 was 11, with the proteasome activity of JBE set to 100. Figure 5 shows the results of evaluating the proteasome activation activity of 4-deoxypodophyllotoxin (DPT), a compound 1 identified from peak A fractionated from juniper fruit extract (70% ethanol extract), podophyllotoxin (PT), a DPT analog, yatein, a compound 2 identified from peak B fractionated from juniper fruit extract (JBE), and β-peltatin, a DPT analog (5A), and the results of quantifying the concentrations of these compounds in juniper fruit extract (JBE) (5B). In Figure 5A, the relative values ​​of proteasome activity are as follows, with the activity of the control set at 100: DPT: 100nM was 383, 50nM was 454, 25nM was 361, 12.5nM was 224, 6nM was 164, 3nM was 131, 1.5nM was 119, and 0.75nM was 103. PT: 100nM was 403, 50nM was 439, 25nM was 249, 12.5nM was 173, 6nM was 124, 3nM was 118, 1.5nM was 115, and 0.75nM was 109.Yatein: 100 nM was 132, 50 nM was 133, 25 nM was 136, 12.5 nM was 133, 6 nM was 132, 3 nM was 129, 1.5 nM was 114, and 0.75 nM was 107. β-Pertatin: 100 nM was 406, 50 nM was 425, 25 nM was 250, 12.5 nM was 154, 6 nM was 120, 3 nM was 113, 1.5 nM was 114, and 0.75 nM was 104. Figure 6 shows the structural formulas of podophyllotoxin analogs such as DPT, PT, yatein, and β-peltatin. FIG. 7 shows the results of HPLC quantification of DPT and yatein contained in Juniperus communis fruit extract (JBE) after storage at 4° C., 25° C., and 40° C. under UV light for one month.

[0012] Next, various embodiments of the present invention will be described with reference to the drawings. Note that the various embodiments described below do not limit the scope of the invention as claimed, and not all of the elements and combinations thereof described in the various embodiments are necessarily essential to the solution of the present invention.

[0013] (Definition) As used herein, "proteasome" refers to a substance expressed in mammalian cells. Proteasomes are expressed throughout the body. That is, the proteasome activation effect of the compound confirmed in cultured human cells can be extrapolated to human tissues, including skin. Therefore, the compound is believed to improve skin elasticity, dullness, and transparency by activating proteasomes in skin cells, mainly in localized areas of human skin (such as the face, bust, lips, under-eye bags, back of the face and hands, etc.).

[0014] The "proteasome activator" in this embodiment (hereinafter sometimes simply referred to as "the agent of this embodiment") refers to a composition containing an active ingredient used in these applications, and is a material that can be added to cosmetics and pharmaceuticals. This agent may be in not only a liquid form but also a solid form, and specific applications include not only topical skin preparations but also oral compositions (which can be prepared as either solids or liquids, for example).

[0015] (Active ingredient) The proteasome activator of this embodiment is a compound represented by the following formula (I):

[0016] Or the following formula (II): (In the formula, R １ and R ２ are each independently a hydrogen atom, a hydroxy group, a methoxy group, an acetoxy group, or a glucosyl group, and R ３ are each independently a hydrogen atom, a hydroxy group, or a methoxy group. In the above formula (I) or (II), the wavy line represents R ２ indicates that the carbon atom bonded to the hydrogen atom or the aryl group may be in either R or S configuration. Therefore, the compounds shown in the above formula (I) or formula (II) include any enantiomers, diastereomers, racemates or any mixtures thereof.

[0017] The proteasome activator of this embodiment is further represented by the following formula (III), and for example, R １ is a hydrogen atom, R ２ Podophyllotoxin, where R is a hydroxy group １ is a hydrogen atom, R ２ is a hydrogen atom, and R １ is a hydroxy group, R ２ It contains β-peltatin, in which β is a hydrogen atom, as an active ingredient.

[0018]

[0019] The proteasome activator of this embodiment is further represented by the following formula (IV), and for example, R １ is a hydrogen atom, R ２ It contains yatein, which is a hydrogen atom, as an active ingredient.

[0020] In the above formula (IV), the wavy line represents R ２ This indicates that the carbon atom bonded to may be in either the R or S configuration. Therefore, the compound represented by the above formula (IV) includes any enantiomer, racemate or any mixture thereof.

[0021] These compounds may be extracted directly from Juniperus communis extracts, or may be synthesized by chemically modifying a parent compound extracted from a medicinal plant of the Cupressaceae family, such as Juniperus communis, for example, by glycosidation of the hydroxyl group at the 4-position of podophyllotoxin.

[0022] When the active ingredient of this embodiment is extracted from Juniperus communis, "Juniperus communis" refers to a plant of the Cupressaceae family and the Juniperus genus (scientific name: Juniperus communis). When producing Juniperus communis extract, for example, roots, rhizomes, leaves, stems, flowers, fruits, pericarp, seeds, the whole plant, or a mixture thereof can be used as the raw material. Juniperus communis extract is produced, for example, by extracting raw or dried raw materials with a solvent, either as is or after crushing. For example, 100 to 200 g of Juniperus communis fruit is immersed in 1 kg of a 10 to 100% 1,3-butylene glycol solution, and extracted for 5 to 10 days in an environment of about 10°C to about 30°C. Alternatively, 100 to 200 g of juniper fruit may be immersed in 1 kg of 10 to 100% ethanol solution and extracted for 5 to 10 days at a temperature of about 10°C to about 30°C. Alternatively, 100 to 200 g of juniper fruit may be immersed in 1 kg of warm water at a temperature of about 30°C to about 80°C and extracted for 5 to 24 hours. The solution obtained through this immersion is filtered using a predetermined filter material (such as a Glass Fiber Filter (Gf-75 manufactured by ADVANTEC) and Mixed Cellulose Ester (A045A047A manufactured by ADVANTEC)). The filtered solution is allowed to stand for 5 to 10 days at a temperature of about 0°C to about 10°C. The solution is again filtered using a predetermined filter material, and the resulting solution is used as a juniper extract.

[0023] (Isolation and Purification Procedure for Active Ingredients) The active ingredient of this embodiment can be isolated from the Juniperus communis extract by a method such as liquid-liquid extraction with a solvent, fractionation using chromatography in various separation modes (ion exchange, hydrophilic adsorption, hydrophobic adsorption, size exclusion, ligand exchange, affinity, etc.), molecular weight fractionation filtration using filter paper, membrane filters, ultrafiltration membranes, etc., pressurization or decompression, heating or cooling, drying, pH adjustment, deodorization, decolorization, long-term static storage, etc., and these methods can be arbitrarily selected and combined for processing. In one embodiment, it is preferable to combine column chromatography and preparative HPLC, etc., as used in the examples described below. The structure of the purified compound can be determined by NMR or mass spectrometry.

[0024] (Proteasome activator) The content ratio of each active ingredient in the agent of this embodiment is not particularly limited. For example, the agent of this embodiment may contain only one of podophyllotoxin, 4-deoxypodophyllotoxin, β-peltatin, and yatein, or may contain these in any ratio. The agent of this embodiment may also contain any compound in addition to these active ingredients. For example, Cupressaceae plants, including Juniperus communis, contain many types of lignans and their analogs (Non-Patent Document 3).

[0025] (Modified Form) The compounds represented by the above formulas (I) to (II) may be modified. In this specification, a modified form means a compound in which a part of the compound has undergone some kind of chemical modification or the like to the extent that the compound does not lose its proteasome activation ability. Examples of chemical modifications or the like include methylation, methoxylation, esterification, etherification, PEG modification, biotin modification, peptide modification, and sugar modification. The sugar modification is not limited to glucosylation, and one or more sugars (e.g., N-acetylgalactosamine, galactose, mannose, GlcNAc, glucose, fucose, or xylose) may be attached to any hydroxy group. The site that has undergone chemical modification or the like is represented by R １ ~R ３ The substituents are not limited to the above.

[0026] The modified compounds may be naturally occurring or non-naturally occurring. If they are naturally occurring, they may be directly extracted from medicinal plants or the like. If they are not naturally occurring, they may be synthesized by chemical modification using a compound extracted from a medicinal plant or the like as a parent compound, or they may be synthesized from a commercially available compound using organic synthesis techniques known in the art.

[0027] (Content of Active Ingredients Contained in Topical Skin Preparation) One application of the agent of this embodiment is as a topical skin preparation. The content of each active ingredient in the topical skin preparation is not particularly limited, as long as it is an amount that can promote proteasome activity in skin cells, taking into account the dosage form and administration method, etc. For example, the content of each active ingredient is preferably 0.1 nM (approximately 1 μg / L when used as an aqueous solution) or more, more preferably 0.2 nM or more, and even more preferably 0.4 nM or more, relative to the total weight of the topical skin preparation. Furthermore, in consideration of the cytotoxicity of podophyllotoxins, the content is preferably 10 mM or less, more preferably 1 mM or less, and even more preferably 0.1 mM or less. The content range of each active ingredient is preferably 0.1 nM or more to 10 mM or less, more preferably 0.2 nM or more to 1 mM or less, and particularly preferably 0.4 nM or more to 0.1 mM or less.

[0028] (Form of topical skin preparation) The topical skin preparation according to the present invention may be in an appropriate form for use, such as ampoules, capsules, powders, granules, liquids, gels, foams, emulsions, sheets, mists, sprays, etc., and may include 1) pharmaceuticals, 2) quasi-drugs, and 3) topical or whole-body topical skin preparations (for example, skin lotions, emulsions, creams, ointments, lotions, oils, packs, and other basic cosmetics, solid soaps, liquid soaps, hand washes, and other face washes and skin cleansers, massage preparations, cleansing preparations, hair removal preparations, depilatories, shaving preparations, aftershave lotions, preshave lotions, shaving creams, foundations, lipsticks, blushers, eye shadows, and eyeliners). , make-up cosmetics such as mascara, perfumes, nail tonics, nail enamel tonics, nail enamel removers, poultices, plasters, tapes, sheets, patches, aerosols, etc.); 4) medicinal and / or cosmetic preparations to be applied to the scalp and hair (for example, shampoos, rinses, hair treatments, pre-hair treatments, permanent solutions, hair dyes, hair styling products, hair tonics, hair growth and care products, poultices, plasters, tapes, sheets, aerosols, etc.); 5) bath additives to be added to bathwater; 6) other products such as underarm odor prevention agents, deodorants, antiperspirants, sanitary products, sanitary cotton, wet tissues, etc.

[0029] (Constituents of topical skin preparations) Furthermore, such preparations can be prepared by arbitrarily selecting and combining the components and additives exemplified below, as needed, within the range that does not impair the effects of the present invention. The amount of these to be added to the formulation is not particularly limited, but it is generally considered preferable to use about 0.0001 to 50%.

[0030] (1) Various oils and fats: avocado oil, almond oil, fennel oil, perilla oil, olive oil, orange oil, orange roughage oil, sesame oil, cacao butter, chamomile oil, carrot oil, cucumber oil, beef tallow fatty acids, kukui nut oil, safflower oil, shea butter, liquid shea butter, soybean oil, camellia oil, corn oil, rapeseed oil, persic oil, castor oil, cottonseed oil, peanut oil, turtle oil, mink oil, egg yolk oil, palm oil, palm kernel oil, Japan wax oil, coconut oil, beef tallow, lard, squalene, squalane, pristane, and hydrogenated products of these oils and fats (hardened oils, etc.).

[0031] (2) Waxes: beeswax, carnauba wax, spermaceti, lanolin, liquid lanolin, reduced lanolin, hard lanolin, candelilla wax, montan wax, shellac wax, rice wax, etc.

[0032] (3) Mineral oils: liquid paraffin, petrolatum, paraffin, ozokeride, ceresin, microcrystalline wax, etc.

[0033] (4) Fatty Acids Natural fatty acids such as lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, oleic acid, linoleic acid, linolenic acid, docosahexaenoic acid, eicosapentaenoic acid, 12-hydroxystearic acid, undecylenic acid, tall oil, and lanolin fatty acids; and synthetic fatty acids such as isononanoic acid, caproic acid, 2-ethylbutanoic acid, isopentanoic acid, 2-methylpentanoic acid, 2-ethylhexanoic acid, and isopentanoic acid.

[0034] (5) Alcohols Natural alcohols such as ethanol, isopropanol, lauryl alcohol, cetanol, stearyl alcohol, oleyl alcohol, lanolin alcohol, cholesterol, phytosterol, and phenoxyethanol; and synthetic alcohols such as 2-hexyldecanol, isostearyl alcohol, and 2-octyldodecanol.

[0035] (6) Polyhydric Alcohols Ethylene oxide, ethylene glycol, diethylene glycol, triethylene glycol, ethylene glycol monoethyl ether, ethylene glycol monobutyl ether, diethylene glycol monomethyl ether, diethylene glycol monoethyl ether, polyethylene glycol, propylene oxide, propylene glycol, polypropylene glycol, 1,3-butylene glycol, pentyl glycol, glycerin, pentaerythritol, threitol, arabitol, xylitol, ribitol, galactitol, sorbitol, mannitol, lactitol, maltitol, and the like.

[0036] (7) Esters Isopropyl myristate, isopropyl palmitate, butyl stearate, hexyl laurate, myristyl myristate, oleyl oleate, decyl oleate, octyldodecyl myristate, hexyldecyl dimethyloctanoate, cetyl lactate, myristyl lactate, diethyl phthalate, dibutyl phthalate, lanolin acetate, ethylene glycol monostearate, propylene glycol monostearate, propylene glycol dioleate, and the like.

[0037] (8) Metallic soaps: aluminum stearate, magnesium stearate, zinc stearate, calcium stearate, zinc palmitate, magnesium myristate, zinc laurate, zinc undecylenate, etc.

[0038] (9) Gums, sugars or water-soluble polymer compounds Gum arabic, gum benzoin, gum dammar, guaiac butter, Irish moss, gum karaya, gum tragacanth, carob gum, quince seed, agar, casein, lactose, fructose, sucrose or its esters, trehalose or its derivatives, dextrin, gelatin, pectin, starch, carrageenan, carboxymethyl chitin or chitosan, hydroxyalkyl (C2-C4) chitin or chitosan to which alkylene (C2-C4) oxide such as ethylene oxide is added, low molecular weight chitin or chitosan, chitosan salt, sulfated chitin or chitosan, phosphorylated chitin or chitosan, alginic acid or its salt, hear Examples of suitable cellulose acetate copolymers include hydroxypropyl cellulose, ...

[0039] (10) Surfactants Anionic surfactants (alkyl carboxylates, alkyl sulfonates, alkyl sulfate ester salts, alkyl phosphate ester salts), cationic surfactants (alkylamine salts, alkyl quaternary ammonium salts), amphoteric surfactants: carboxylic acid type amphoteric surfactants (amino type, betaine type), sulfate ester type amphoteric surfactants, sulfonic acid type amphoteric surfactants, phosphate ester type amphoteric surfactants, nonionic surfactants (ether type nonionic surfactants, ether ester type nonionic surfactants, ester type nonionic surfactants, block polymer type nonionic surfactants, nitrogen-containing type nonionic surfactants), other surfactants (natural surfactants, derivatives of protein hydrolysates, polymer surfactants, surfactants containing titanium or silicon, fluorocarbon surfactants), etc.

[0040] (11) Various Vitamins Vitamin A group: retinol, retinal (vitamin A1), dehydroretinal (vitamin A2), carotene, lycopene (provitamin A), vitamin B group: thiamine hydrochloride, thiamine sulfate (vitamin B1), riboflavin (vitamin B2), pyridoxine (vitamin B6), cyanocobalamin (vitamin B12), folic acids, nicotinic acids, pantothenic acids, biotins, choline, inositols, vitamin C group: vitamin C acid or its derivatives, vitamin D group: ergocalciferol Examples of suitable vitamin supplements include hydroxybenzoates, hydroxybenzoates, hydroxybenzoates, hydroxybenzoates (hydroxybenzoates), ...

[0041] (12) Various Amino Acids: valine, leucine, isoleucine, threonine, methionine, phenylalanine, tryptophan, lysine, glycine, alanine, asparagine, glutamine, serine, cysteine, cystine, tyrosine, proline, hydroxyproline, aspartic acid, glutamic acid, hydroxylysine, arginine, ornithine, histidine, and the like, as well as their sulfates, phosphates, nitrates, citrates, and amino acid derivatives such as pyrrolidone carboxylic acid.

[0042] (13) Various additives derived from plant or animal raw materials These may be arbitrarily selected from various materials and provided after undergoing conventional processing (e.g., pulverization, milling, washing, hydrolysis, fermentation, refining, pressing, extraction, fractionation, filtration, drying, powdering, granulation, dissolution, sterilization, pH adjustment, deodorization, bleaching, etc.) according to the type and form of the product to which they are to be added.

[0043] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to these examples. In the following examples, the unit % used to indicate the amount of each component added means % by mass unless otherwise specified.

[0044] (Preparation of Juniper Fruit Extract) Juniper fruits (also known as juniper berries) were immersed in hot water at 80° C. for 5 hours. The solution obtained through this immersion (hot water extract) was filtered using predetermined filter materials (glass filter paper, Gf-75, manufactured by ADVANTEC, and cellulose mixed ester membrane filter, A045A047A, manufactured by ADVANTEC), and the resulting solution was adjusted to an evaporation residue of 0.5% (w / w) and a 1,3-butylene glycol concentration of 50% (w / w), and used as juniper fruit (juniper berry extract) extract (JBE).

[0045] (Proteasome Activation Effect of Juniperus communis Fruit Extract) Juniperus communis fruit extract was added to a human cell line, and after culturing for a certain period of time, the cells were lysed and the activity of the proteasome expressed in the cells was evaluated. Proteasome activity was measured using Cell-Based Proteasome-Glo™, which quantifies the activity based on luminescence intensity. ＴＭAssays kit (Promega G8660 (for measuring chymotrypsin-like activity)).

[0046] HEK293T cells (manufactured by TAKARA), a cell line derived from human embryonic kidney cells commonly used in cell tests, were cultured at 1 x 10 cells per 96-well white plate using DMEM medium containing 10% FBS. ４ (cells) / well, and 5% CO ２ The plates were cultured in an incubator at 37°C for 16 hours. Juniperus communis fruit extract was added to the wells to a final concentration of 1% or 0.5%, and the plates were incubated under 5% CO ２ The cells were cultured in an incubator at 37°C for 2, 4, 8, and 24 hours. The medium in the wells was removed, and the wells were washed once with PBS, and then 50 μL of PBS was added to each well. ＴＭ Proteasome activity was assessed using the Juniperus communis Assays kit according to the manufacturer's recommended method. As a control for this experiment, wells were set up to contain 50% 1,3-butylene glycol, a solvent not containing Juniperus communis fruit extract, at a final concentration of 1% or 0.5%, and proteasome activity was measured. The proteasome activity in the wells containing the solvent alone was set at 100, and the percentage of proteasome activity (relative %) in the wells containing the Juniperus communis fruit extract was calculated.

[0047] The results are shown in Figure 1A (n = 4, ±SEM, ***p<0.001, **p<0.01, Dunnett's test vs. control). Juniperus communis fruit extract at 1% concentration activated proteasomes by 245%, 254%, 152%, and 129% after 2, 4, 8, and 24 hours, respectively (Figure 1A). Juniperus communis fruit extract at 0.5% concentration activated proteasomes by 143%, 143%, 109%, and 104% after 2, 4, 8, and 24 hours, respectively (Figure 1A). These results demonstrate that juniperus communis fruit extract activates the proteasome in HEK293T cells.

[0048] Human skin keratinocytes (manufactured by TOYOBO) derived from adults or newborns were cultured in a 96-well white plate at 1 x 10４ (cells) / well, and 5% CO ２ The plates were cultured in an incubator at 37°C for 16 hours. Juniperus communis fruit extract was added to the wells to a final concentration of 1% or 0.5%, and the plates were incubated under 5% CO ２ The cells were cultured in an incubator at 37°C for 2, 4, 8, and 24 hours. The medium in the wells was removed, and the wells were washed once with PBS, and then 50 μL of PBS was added to each well. ＴＭ Proteasome activity was assessed using the Juniperus communis Assays kit according to the manufacturer's recommended method. As a control for this experiment, wells were set up to contain 50% 1,3-butylene glycol, a solvent not containing Juniperus communis fruit extract, at a final concentration of 1% or 0.5%, and proteasome activity was measured. The proteasome activity in the wells containing the solvent alone was set at 100, and the percentage of proteasome activity (relative %) in the wells containing the Juniperus communis fruit extract was calculated.

[0049] The results are shown in Figures 1B and 1C (n = 4, ±SEM, ***p<0.001, **p<0.01, Dunnett's test vs. control). In adult human skin keratinocytes, a 1% concentration of Juniperus communis fruit extract resulted in 178%, 179%, 153%, and 120% proteasome activation after 2, 4, 8, and 24 hours, respectively (Figure 1B). A 0.5% concentration of Juniperus communis fruit extract resulted in 147%, 136%, 122%, and 104% proteasome activation after 2, 4, 8, and 24 hours, respectively (Figure 1B). In neonatal human skin keratinocytes, the addition of 1% Juniperus communis fruit extract resulted in 168%, 168%, 126%, and 103% proteasome activation after 2, 4, 8, and 24 hours, respectively (Fig. 1C). The addition of 0.5% Juniperus communis fruit extract resulted in 139%, 153%, 123%, and 101% proteasome activation after 2, 4, 8, and 24 hours, respectively (Fig. 1C). These results demonstrate that Juniperus communis fruit extract activates the proteasome in both adult and neonatal human skin keratinocytes.

[0050] (Inhibitory effect of Juniperus communis fruit extract on lipofuscin accumulation) It is known that oxidized proteins and structurally denatured proteins aggregate within cells as they age, resulting in the accumulation of oxidized aggregates called lipofuscin. We evaluated whether lipofuscin accumulation is inhibited as a result of proteasome activation using the Masson-Fontana staining method.

[0051] Human skin keratinocytes (manufactured by TOYOBO) derived from newborns were cultured in a medium recommended by the manufacturer, to which Juniperus communis fruit extract was added at a final concentration of 0.5%, under 5% CO ２ The cells were cultured in an incubator at 37°C for 14 days (excluding the day of subculture). The medium was changed twice daily and subcultured when the cells reached confluence. For subculture, the cells were plated in the manufacturer's recommended medium and cultured overnight to allow for cell adhesion. The medium was then changed to one containing Juniperus communis fruit extract. For comparison, cells were cultured in the same manner using medium supplemented with 50% 1,3-butylene glycol to a final concentration of 0.5%. The cells were plated on glass chamber slides (Matsunami Glass SCS-NO2) and, after adhesion, fixed in 10% neutral buffered formalin (Wako Pure Chemical Industries, Ltd., 062-01661). Ammoniacal silver used for Masson-Fontana staining was prepared by adding 28% aqueous ammonia to 10 mL of 10% silver nitrate, dropping 100 mL of purified water to form a thin, turbid solution, and allowing the solution to stand for 24 hours at room temperature in the dark. The supernatant was then collected. After the fixation, the cells were washed with purified water, and then ammoniacal silver solution was added and the cells were left to stand at room temperature in the dark for 24 hours. After washing the cells with purified water, 5% sodium thiosulfate was added and the cells were decolorized at room temperature for 2 minutes. After washing the cells with purified water, the cells were mounted using a mounting medium. The stained lipofuscin was detected using an inverted microscope (Olympus, main body: BX50, camera: DP70).

[0052] Masson-Fontana staining of the control cells (Control) revealed numerous dark brown dots due to the accumulation of lipofuscin (Figure 2A). In contrast, the number of dark brown dots was significantly lower in the cells cultured in the presence of juniper fruit extract (JBE) (Figure 2A). The stained area of ​​the control cells (Control) was set to 100, and the stained area of ​​the cells cultured in the presence of juniper fruit extract (JBE) was quantified (n = 20, ± SEM, ***p < 0.001, Student's t-test). The result was a significantly lower value of 37 (Figure 2B). These results demonstrate that the addition of juniper fruit extract inhibits intracellular lipofuscin accumulation.

[0053] (Fractionation of Juniperus communis fruit extract) Juniperus communis extract was fractionated according to the fractionation scheme shown in Figure 3A. When evaluating the proteasome activity of each fraction, it was prepared so that it contained an area comparable to that of the corresponding peak in the Juniperus communis fruit extract (JBE) (before fractionation).

[0054] Ethanol was added to a hot water extract of Juniperus communis fruit to prepare a 10% ethanol solution, which was then crudely fractionated on a column packed with Diaion HP-20 (Mitsubishi Chemical Corporation). The resulting fractions were: a flow-through fraction (Fr. 1), a 10% ethanol-eluted fraction (Fr. 2), a 20% ethanol-eluted fraction (Fr. 3), a 30% ethanol-eluted fraction (Fr. 4), a 40% ethanol-eluted fraction (Fr. 5), a 50% ethanol-eluted fraction (Fr. 6), and a 100% ethanol-eluted fraction (Fr. 7). These fractions were evaluated for proteasome activation (2 hours after sample addition), and activity was confirmed in Fraction 7 (Figure 4A). Subsequently, the fractions were separated using preparative HPLC (condition 1), yielding five fractions, Fractions 7-1 to 7-5. These fractions were evaluated for proteasome activation (2 hours after sample addition), and activity was confirmed in Fraction 7-4 (Figure 4B). Therefore, Fr. 7-4 was further fractionated using HPLC under the same conditions to obtain three fractions, Fr. 7-4-1 to 7-4-3. The proteasome activation activity of these fractions was evaluated (2 hours after sample addition), and Fr. 7-4-3 exhibited activity nearly equivalent to that of the Juniperus communis fruit extract (Figure 4C). These results demonstrated that the active component is present in the less polar fraction.

[0055] To obtain a large amount of the active ingredient, a 70% ethanol extract of Juniperus communis fruit was prepared using 70% ethanol. The 70% ethanol extract of Juniperus communis fruit was fractionated using the fractionation scheme shown in Figure 3B. Water was added to make a 50% ethanol solution, which was then crudely fractionated using a column packed with Diaion HP-20 (Mitsubishi Chemical Corporation), and the 90% ethanol eluted fraction was collected. This recovered solution was concentrated once, adjusted to a 30% ethanol solution, and then loaded onto a column packed with Diaion HP-20. The column was washed with 50% ethanol and 60% ethanol to elute the unwanted fraction, and the fraction eluted with 80% ethanol was collected. This eluted fraction was concentrated using an evaporator and extracted with hexane (3 x 700 mL) to obtain the hexane fraction. The hexane fraction (6.9 g) was concentrated using an evaporator, dissolved in 60% aqueous ethanol (200 g), and then applied to a column (70 mmφ x 350 mmL) packed with Diaion HP-20. The liquid eluted with ethanol was analyzed by HPLC, yielding two fractions (H-1 and H-2). H-2 (4.8 g) was concentrated using an evaporator, dissolved in 50% aqueous ethanol (200 g), and applied to ODS gel chromatography (Seppak® Vac 35 cc (10 g) C18 Cartridges) and eluted with a 1:1 volumetric ratio ethanol:water mixture. The eluate was analyzed by HPLC (condition 1), yielding five fractions (HFr. 2-1 to 2-5). After concentrating HFr.2-1 using an evaporator, it was dissolved in a minimum amount of 30% ethanol in water and subjected to ODS gel chromatography (Seppak® Vac 35 cc (10 g) C18 Cartridges) and eluted with a mixed solvent of ethanol:water (volume ratio: 3:7). The eluate was analyzed by HPLC (condition 1), and five fractions (H-2-1-1 to 2-1-5) were obtained. Of these, H2-1-2 exhibited proteasome activity (2 hours after sample addition) almost equivalent to that of Juniperus communis fruit extract (JBE) (Figure 4D). Finally, H-2-1-2 (23.4 mg) was subjected to preparative HPLC (conditions 2 and 3).Compound 1 (Compound 1, 3.5 mg, purity 98.39%, retention time 18.9 min) was obtained by preparative HPLC (condition 2), and compound 2 (Compound 2, 1.7 mg, purity 94.50%, retention time 19.7 min) was obtained by preparative HPLC (condition 3). Compound 1 exhibited proteasome activity (2 hours after sample addition) almost equivalent to that of the Juniperus communis fruit extract (Figure 4E). Compound 2 also exhibited weak proteasome activity (2 hours after sample addition) (Figure 4E). From these results, we concluded that Compounds 1 and 2 are the main compounds contributing to the proteasome activation effect of Juniperus communis fruit extract.

[0056] (Structural analysis of isolated compounds) Compound 1 (3.5 mg, purity 98.39%) was dissolved in deuterated chloroform and subjected to FT-NMR analysis. It was also dissolved in methanol and subjected to LC-MS / MS analysis. Compound 2 (1.7 mg, purity 94.50%) was dissolved in deuterated chloroform and subjected to FT-NMR analysis. It was also dissolved in methanol and subjected to LC-MS / MS analysis.

[0057] The purity of the target compound in the sample used in the above structural analysis was confirmed by analysis using high performance liquid chromatography (HPLC) (condition 1).

[0058] <Preparative High Performance Liquid Chromatography (Preparative HPLC)> Condition 1 Apparatus: SHIMADZU LC-20ADxr Column: Mightysil RP-18GP 250 x 4.6 mm (particle size 5 μm) Flow rate: 1.0 mL / min Detector wavelength: 210 nm Gradient program: CH ３ CN:H ２ O = (10:90) → (100:0) / 30 min <Preparative High Performance Liquid Chromatography (Preparative HPLC)> Condition 2 Apparatus: 1260 Infinity III (Agilen Technologies) Column: Develosil ODS (20 mmφ×250 mmL) Flow rate: 10.0 mL / min Detector wavelength: 210 nm Gradient program: MeOH:H ２O = 57:43 (40 min) Condition 3 Apparatus: SHIMADZU LC-40Dxr Column: Mightysil RP-18GP 250 x 4.6 mm (particle size 5 μm) Flow rate: 1.0 mL / min Detector wavelength: 210 nm Gradient program: MeOH:H ２ O = 57:43 (20 min) <High performance liquid chromatography (Analytical HPLC)>

[0059] <Transformation nuclear magnetic resonance apparatus (FT-NMR)> Equipment: JEOL EC 500MHz (JEOL, TOKYO, JAPAN) Solvent: Chloroform-d (CAS865-49-6)

[0060] <Ultrafast triple quadrupole LC-MS / MS (LC-MS / MS)> Apparatus: SHIMADZU LCMS-8050 Ion mode: positive ion mode Mass range: 100 ＋ ~1000 ＋

[0061] The active compounds were measured as above. １３ C-NMR spectrum, １ The structure estimated from the H-NMR spectrum and LC-MS spectrum is shown below.

[0062] NMR data for Compound 1: 1 H NMR (CDCl3) δ 2.74 (br s, 2H), 2.77 (m, 1H), 3.08(m, 1H), 3.75 (s, 6H), 3.81 (s, 3H), 3.93 (m, 1H), 4.46 (m, 1H),4.61 (br s, 1H), 5.95 (2H, J =11), 6.35 (s, 2H), 6.52 (s, 1H), 6.67 (s, 1H); 1313C NMR (CDCl3) 32.7(CH), 33.1 (CH2), 43.7 (CH), 47.5 (CH), 56.2 (CH3), 60.7 (CH3),72.1 (CH2), 101.2 (OCH2O), 108.2 (CH), 108.5 (CH), 110.4 (CH), 128.3 (C), 130.6 (C), 136.3 (C), 136.9 (C), 146.7 (C), 147.0 (C), 152.5 (C), 175.0 (C); mass spectrum m / z (relative intensity) 399.15 (M+H); MS caled for C 22 H 23 O7 399.15 (M+H), found 399.1444.

[0063] NMR data of Compound 2: 1 1H NMR (CDCl3) δ 2.49 (m, 1H), 2.55 (dd, J =8.0, 12.0, 1H), 2.62 (dd, J =6.5, 13.5, 1H), 2.89(dd, J =7.0, 14.0, 1H), 2.93(dd, J =5.5, 14.0, 1H), 3.83 (s, 3H), 3.83 (s, 6H), 3.90 (dd, J =7.5, 9.0, 1H), 4.19 (dd, J =7.5, 9.0,1H), 5.94 (J =1.5, 1H), 5.95 (J =1.5, 1H), 6.36 (s, 2H), 6.46 (s, 2H), 6.70 (d, J =8.0, 1H); 13C NMR (CDCl3) 35.2(CH2), 38.4 (CH2), 41.0 (CH), 46.5 (CH), 56.1 (CH3), 60.9 (CH3),71.2 (CH2), 101.1 (OCH2O), 106.1 (CH), 108.3 (CH), 108.8 (CH), 121.6 (CH), 131.5 (C), 133.3 (C), 146.4 (C), 147.9 (C), 153.2 (C), 178.7 (C); mass spectrum m / z (relative intensity) 402.20 (M+H); MS caled for C 22 H 23 O7 401.16 (M+H), found 400.16.

[0064] (Evaluation of Proteasome Activation of Purified Fractions) Addition of the purified fractions to human cells was examined to determine whether intracellular proteasomes were activated. Each purified fraction used in this evaluation was prepared to contain an area comparable to that of the corresponding peak in Juniperus communis fruit extract (JBE). HEK293T cells (manufactured by TAKARA), a human embryonic kidney cell-derived cell line commonly used in cell testing, were used, and the assay was carried out according to the method described above. Juniperus communis fruit extract or purified fractions were added to wells to a final concentration of 1% or 0.5%. As a control for this experiment, wells were set up to contain 30% ethanol, the solvent for the purified fractions, to a final concentration of 1 or 0.5%, and proteasome activity (2 hours after sample addition) was measured. Proteasome activity in wells to which only the solvent was added was 0, and in wells to which the Juniperus communis fruit extract (JBE) or Fr. The proteasome activity of well 7 was set as 100, and the ratio (relative value) of the proteasome activity of the well to which the purified fraction was added was calculated.

[0065] The results are shown in Figure 4. First, purified fraction Fr. 7 exhibited proteasome activity equivalent to that of Juniperus communis fruit extract (n = 4, ± SEM, ***p < 0.001, Dunnett's test vs. control) (Figure 4A). When Fr. 7 was divided into five fractions, Fr. 7-1 to 7-5, Fr. 7-4 fraction exhibited high proteasome activity (n = 4, ± SEM, ***p < 0.001, Dunnett's test vs. control) (Figure 4B). When Fr. 7-4 fraction was further divided into three fractions, Fr. 7-4-1 to 7-4-3, Fr. Only fraction 7-4-3 exhibited proteasome activity equivalent to that of juniper fruit extract (n = 5, ± SEM, ***p < 0.001, Dunnett's test vs. control) (Figure 4C). Therefore, the peak contained in fraction 7-4-3 was prepared in large quantities from juniper fruit extract using 70% EtOH (Figure 3, Fraction 80% EtOH). The main peak was extracted with hexane and purified by gel chromatography and HPLC. As a result, fraction H2-1-2 exhibited proteasome activity equivalent to that of juniper extract (n = 8, ± SEM, ***p < 0.001, Dunnett's test vs. control) (Figure 4D). The fraction H2-1-2 was subjected to preparative HPLC, yielding two peaks, A and B. Peak A showed activity almost equivalent to that of the common juniper fruit extract, and proteasome activation was also observed in Peak B (n = 8, ±SEM, *p<0.05, ***p<0.001, Dunnett's test vs. control) (Figure 4E). From these results, we concluded that Peak A and Peak B are the main compounds contributing to the proteasome activation activity of the common juniper fruit extract. Structural analysis revealed that Compound 1 derived from Peak A is 4-deoxypodophyllotoxin (see Figure 6). Furthermore, structural analysis revealed that Compound 2 is yatein (see Figure 6).

[0066] (Evaluation of proteasome activation by compounds) Many analogs of 4-deoxypodophyllotoxin and yatein have been reported (Non-Patent Document 3) (Figure 6). Therefore, the proteasome activation activity (2 hours after sample addition) of 4-deoxypodophyllotoxin, yatein, and their analogs podophyllotoxin and β-peltatin was evaluated (Figure 5A).

[0067] 4-Deoxypodophyllotoxin (hereinafter referred to as DPT) (D249500, Toronto Research Chemicals), yatein (CAY 29766, Cayman), and their analogs podophyllotoxin (hereinafter referred to as PT) (161-20901, Fujifilm Wako Pure Chemical Industries), and β-peltatin (SC-478230, Santa Cruz Animal Health) were each dissolved in DMSO to give 10 mM solutions. A 2-fold dilution series was prepared using DMSO, and the solution was added to cells to a final DMSO concentration of 0.1%. HEK293T cells (TAKARA), a cell line derived from human embryonic kidney cells commonly used in cell tests, were used to evaluate proteasome activity according to the method described above. Compounds were added to the wells at final concentrations of 100 nM, 50 nM, 25 nM, 12.5 nM, 6 nM, 3 nM, 1.5 nM, and 0.75 nM. As a control for this experiment, wells were set up to receive compound-free DMSO at a final concentration of 0.1%, and proteasome activity was measured in the same manner. The proteasome activity in the wells to which only DMSO was added was set at 100, and the relative proteasome activity in the wells to which the compounds were added was calculated.

[0068] The results are shown in Figure 5A (n = 4, ±SEM, *p<0.05, ***p<0.001, Dunnett's test vs. control). DPT, PT, and β-peltatin all increased proteasome activity in a compound concentration-dependent manner, and at a concentration of 50 nM, they demonstrated a proteasome activation effect of 400% or more. The EC ５０ The value was 16 nM, and the EC ５０ The value was 27 nM, and the EC ５０The calculated value was 28 nM. On the other hand, the proteasome activation activity of yatein saturated at approximately 6 nM, approximately 130%, suggesting that it was a partial effect. The results of quantifying the contents of these compounds in juniper fruit extract (JBE) are shown in Figure 5B. It was found that JBE contained approximately 1000 nM of DPT and approximately 600 nM of yatein. Although the peaks for PT and β-peltatin could not be separated by HPLC, it was found that the two compounds were contained in total at approximately 100 nM. When juniper fruit extract (JBE) was added to cells at a final concentration of 1%, the final concentrations of each compound were approximately 10 nM for DPT, approximately 6 nM for yatein, and approximately 1 nM for PT and β-peltatin combined. The proteasome activation activity corresponding to these concentrations is indicated by a triangle in Figure 5A. The proteasome activity at the concentrations indicated by the triangles was 234% for DPT (12.5 nM), 115% for PT (1.5 nM), 132% for yatein (6 nM), and 114% for β-peltatin (1.5 nM) (Figure 5A). This result correlates well with the 245% increase in proteasome activity observed when Juniperus communis fruit extract (JBE) was added to cells at a final concentration of 1% (Figure 1A). This indicates that the compounds responsible for the proteasome activation of Juniperus communis fruit extract (JBE) are DPT, PT, yatein, and β-peltatin. This also suggests that other podophyllotoxins (Non-Patent Document 3) (Figure 6) may also have proteasome activation effects. Optical isomers of DPT have been reported (Non-Patent Documents 4 and 5), and it has been suggested that these may also have proteasome activating activity.

[0069] The stability of DPT and yatein in juniper fruit extract (JBE) was evaluated. After storing JBE at 4°C, 25°C, and 40°C under UV light for one month, the DPT and yatein contents were quantified by HPLC (Figure 7). The peak area and peak height of DPT in JBE stored at 4°C were 99.7% and 97.9%, respectively, while those stored at 25°C were 98.4% and 94.9%, respectively, and those stored under UV light were 95.4% and 89.2%, respectively. If the peak area and peak height of yatein contained in Juniperus communis fruit extract (JBE) stored at 4°C were taken as 100%, respectively, they were 99.0% and 93.0% after storage at 25°C, 104.7% and 86.8% after storage at 40°C, and 101.4% and 89.3% after storage under UV light. In other words, there was almost no decrease in the peaks during storage for one month, indicating that DPT and yatein exist stably in aqueous solution without structural changes or decomposition.

[0070] (Evaluation of proteasome activation by plant-derived extracts) Proteasome activation was evaluated by adding the following solutions. - Luna White B (Ichimaru Pharcos Co., Ltd.): A solution prepared by adding 1,3-butylene glycol solution to an extract obtained from the seeds of evening primrose (Oenothera biennis Linne(') (Onagraceae) and dissolving, storing in a cool place, adjusting, and filtering. The composition of this extract was 49.5% 1,3-butylene glycol solution, 49.5% water, and 1.0% evening primrose seed extract. The extract contained 0.3-0.7 w / v% polyphenols (as tannic acid equivalents). - Sakura Extract B (Ichimaru Pharcos Co., Ltd.): Sakura Extract B was prepared as follows. Leaves of Oshima cherry (Prunus lannesiana (Carr.) Wilson var. speciosa (Koidz.) Makino) or other species of the Prunus subgenus were taken and immersed in 30% ethanol solution and extracted at room temperature for one week. After filtration, the filtrate was collected and evaporated to dryness under reduced pressure. A 50% 1,3-butylene glycol solution was added to the dried product, dissolved, and filtered to produce an extract. The composition of this solution was 49.0% 1,3-butylene glycol solution, 49.0% water, and 2.0% cherry leaf extract.

[0071] Evening primrose extract and cherry leaf extract were each dissolved in DMSO to prepare 10 mM solutions. A 2-fold dilution series was prepared using DMSO and added to cells to a final DMSO concentration of 0.1%. Proteasome activity was assessed using HEK293T cells (manufactured by TAKARA), a human embryonic kidney cell-derived cell line commonly used in cell assays, according to the method described above. The extracts were added to wells to final concentrations of 1% and 0.5%. As a control for this experiment, wells were set up to receive a 50% 1,3-butylene glycol solution containing no extract at a final concentration of 1%, and proteasome activity was measured in the same manner. The proteasome activity in wells receiving only 50% 1,3-butylene glycol solution was set at 100, and the relative proteasome activity in wells receiving the compound was calculated.

[0072] The calculated results are shown in Tables 1 and 2 below (n=4, ±SEM, *p<0.05, ***p<0.001, Dunnett's test vs. control). It was confirmed that the addition of Luna White B and Sakura Extract B resulted in proteasome activation.

[0073]

[0074] This application claims priority based on Japanese Patent Application No. 2023-035167, filed March 8, 2023, and Japanese Patent Application No. 2023-151616, filed September 19, 2023, the disclosures of which are incorporated herein in their entireties.

[0075] The present invention, through activation of the proteasome, promotes the degradation of proteins that have completed their role within cells, primarily in humans and other organisms, or dysfunctional proteins (e.g., oxidized proteins, structurally denatured proteins, aggregated proteins, etc.), and exogenous proteins (e.g., proteins derived from pathogens such as viruses that have invaded cells), thereby preventing the aging of cellular tissues, including the skin, and maintaining the health of individual organisms.

Claims

1. The following formula (I): 【Chemistry 1】 Or the following formula (II): 【Chemistry 2】 (In the formula, R １ and R ２ each independently represents a hydrogen atom, a hydroxyl group, a methoxy group, an acetoxy group, or a glucosyl group, R ３ each independently represents a hydrogen atom, a hydroxyl group, or a methoxy group; 2. An agent for suppressing lipofuscin accumulation, comprising as an active ingredient one or more compounds represented by the following formula:

2. The compound has the following formula (III): 【Chemistry 3】 Or the following formula (IV): 【Chemistry 4】 (In the formula, R １ and R ２ The lipofuscin accumulation inhibitor according to claim 1, wherein the compound is selected from the group consisting of phenylalanine, ...

3. A skin external preparation for inhibiting lipofuscin accumulation, comprising the lipofuscin accumulation inhibitor according to claim 1 or 2.

4. The topical skin preparation according to claim 3 , wherein at least one of the active ingredients is contained at a concentration of 1 nM or more.

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