Classifications

• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
  • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
  • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
  • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
  • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
  • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
  • A61K31/365—Lactones
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
  • A61K36/13—Coniferophyta (gymnosperms)
  • A61K36/14—Cupressaceae (Cypress family), e.g. juniper or cypress
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
  • A61K8/49—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
  • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
  • A61K8/9755—Gymnosperms [Coniferophyta]
  • A61K8/9761—Cupressaceae [Cypress family], e.g. juniper or cypress
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
  • A61Q19/00—Preparations for care of the skin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
  • A61Q19/00—Preparations for care of the skin
  • A61Q19/08—Anti-ageing preparations

Definitions

• the present invention relates to a proteasome activator containing podophyllotoxins, etc.
• Proteasomes are complex proteases expressed within cells, and selectively degrade proteins that have completed their role within the cell, proteins that have become dysfunctional, and foreign proteins. Proteasome activity is known to weaken with age. For this reason, in aged cells, the decomposition of proteins that have completed their role is delayed, and proteins that have been 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 the transparency of the skin and cause pigmentation that leads to dull skin (Patent Document 1).
• structurally denatured proteins e.g., carbonylated proteins
• aggregated proteins e.g., lipofuscin
• Non-Patent Document 1 1'-acetoxychavicol acetate from ginger (Alpinia galanga)
• Non-Patent Document 2 1'-acetoxychavicol acetate is known to lack stability in aqueous solutions.
• Non-Patent Document 2 a derivative of the lignan podophyllotoxin is used as the drug etoposide (anticancer drug) (Non-Patent Document 2).
• etoposide anticancer drug
• 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).I.Physiological activities and conformational analysis of deoxypodophyllotoxin” (1985) 33(2):704-709. Proc Natl Acad Sci U S A “Mechanistic analysis of carbon-carbon bond formation by deoxypodophyllotoxin synthase” (2022) 119(1):e2113770119.
• 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, medicines, etc., and/or materials to be incorporated into these, that can improve skin elasticity, dullness, and clarity by using this compound to activate intracellular proteasomes.
• the present invention was made to solve the above-mentioned problems.
• the present invention was created based on the discovery that a specific compound fractionated and purified from a Juniperus communis extract using column chromatography or the like activates the proteasome. That is, the present invention includes the following embodiments.
• a proteasome activator comprising one or more compounds represented by the following formula (I) or modified forms thereof as an active ingredient.
• the proteasome activator of the present invention activates cellular proteasomes and degrades dysfunctional proteins such as oxidized proteins, structurally altered proteins, and aggregated proteins within cells, and can provide cosmetics, foods, pharmaceuticals, etc., and/or materials to be incorporated therein, for improving skin elasticity, dullness, and clarity.
• FIG. 1 shows the proteasome activation effect of Juniperus communis fruit extract (JBE) on HEK293T cells (1A), human skin keratinocytes derived from adults (1B), and human skin keratinocytes derived from newborns (1C).
• the relative proteasome activity of each cell was 245, 254, 152, and 129 after 2, 4, 8, and 24 hours, respectively, when the final concentration of JBE was 1%, with the value of the well to which only the solvent was added being taken as 100 (1A).
• the final concentration of JBE was 0.5%, the values were 143, 143, 109, and 104 after 2, 4, 8, and 24 hours, respectively.
• FIG. 2 shows the results of evaluating the inhibitory effect of Juniperus communis fruit extract (JBE) on intracellular lipofuscin accumulation by inverted microscopic observation after Masson-Fontana staining (2A) and measurement of the stained area (2B).
• FIG. 2B the stained area of the cells cultured in the presence of Juniperus communis fruit extract (JBE) was 37 relative to the stained area of the control cells (Control) which was set at 100.
• FIG. 3A shows a scheme for fractionating and purifying an effective fraction from Juniperus communis fruit extract (hot water extract).
• FIG. 3B shows a scheme for isolating and purifying compound 1 and compound 2 from Juniperus communis fruit extract (70% ethanol extract).
• FIG. 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 calculated based on the following: Fr. 1 -1, Fr. 2 -4, Fr. 3 10, Fr. 4 1, Fr. 5 2, Fr. 6 -2, and Fr. 7 116, with the well to which only the solvent was added being taken as 0 and the well to which JBE was taken as 100.
• Figure 4B shows the proteasome activation activity of Fr. 7-1 to 7-5 fractionated from Fr. 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.
• 4C shows the proteasome activation activity of Fr. 7-4-1 to 7-4-3 fractionated from Fr. 7-4.
• the relative value of the proteasome activity of each fraction was ⁇ 15 for Fr. 7-4-1, 0.3 for Fr. 7-4-2, and 102 for Fr. 7-4-3, with the well to which only the solvent was added being set as 0 and that of Fr. 7 being set as 100.
• 4D shows the proteasome activation effect 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, taking the proteasome activity of JBE as 100.
• 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 being taken as 100.
• 5A shows the results of evaluating the proteasome activation activity of 4-deoxypodophyllotoxin (DPT), which is compound 1 identified from peak A fractionated from Juniperus communis fruit extract (70% ethanol extract), podophyllotoxin (PT), which is an analogue of DPT, yatein, which is compound 2 identified from peak B fractionated from Juniperus communis fruit extract (JBE), and ⁇ -peltatin, which is an analogue of DPT (5A), and the results of quantifying the concentrations of these compounds in Juniperus communis fruit extract (JBE) (5B).
• DPT 4-deoxypodophyllotoxin
• the relative values of proteasome activity are as follows, with the activity of the control taken as 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, 0.75nM was 103.
• Yatein 100nM was 132, 50nM was 133, 25nM was 136, 12.5nM was 133, 6nM was 132, 3nM was 129, 1.5nM was 114, 0.75nM was 107.
• ⁇ -peltatin 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.
• FIG. 6 shows the structural formulas of podophyllotoxin analogues such as DPT, PT, yatein, and ⁇ -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.
• proteasome refers to a substance expressed in cells derived from mammals. Proteasomes are expressed throughout the body. That is, the proteasome activation effect of the compound confirmed in cultured cells derived from humans can be extrapolated to human tissues, including skin. Therefore, the compound is considered to improve the elasticity, dullness, and transparency of skin by activating proteasomes in skin cells of localized skin (face, bust, lips, tear bags, back of face and hands, etc.) of humans, etc.
• the "proteasome activator” in this embodiment refers to a composition containing an active ingredient used for these applications, and is a material that can be added to cosmetics and pharmaceuticals.
• This agent can be in not only liquid form but also solid form, and specific applications include not only topical skin preparations but also oral compositions (which can be prepared in either solid or liquid form, for example).
• the proteasome activator of the present embodiment is represented by the following formula (I):
• R 1 and R 2 each independently represent a hydrogen atom, a hydroxyl group, a methoxy group, an acetoxy group, or a glucosyl group; R3 is independently a hydrogen atom, a hydroxyl group, or a methoxy group;
• the wavy line indicates that the carbon atom bonded to R 2 , a hydrogen atom, or an aryl group may be in either the R or S configuration.
• the compound shown in the above formula (I) or formula (II) includes any enantiomers, diastereomers, racemates, or any mixtures thereof.
• the proteasome activator of the present embodiment is further represented by the following formula (III), and contains, for example, podophyllotoxin in which R 1 is a hydrogen atom and R 2 is a hydroxy group, 4-deoxypodophyllotoxin in which R 1 is a hydrogen atom and R 2 is a hydrogen atom, and ⁇ -peltatin in which R 1 is a hydroxy group and R 2 is a hydrogen atom as an active ingredient.
• the proteasome activator of the present embodiment further contains yatein as an active ingredient, which is represented by the following formula (IV) in which, for example, R 1 is a hydrogen atom and R 2 is a hydrogen atom.
• the wavy line indicates that the carbon atom bonded to R2 may be in either the R or S configuration.
• the compound shown in the above formula (IV) includes any enantiomer, racemate, or any mixture thereof.
• These compounds can be extracted directly from, for example, Juniperus communis extracts, or can be synthesized by chemical modification of parent compounds extracted from medicinal plants of the Cupressaceae family, such as Juniperus communis.
• the hydroxyl group at the 4-position of podophyllotoxin can be converted to a glucosidyl group.
• Juniperus communis is a plant of the Cupressaceae family and Juniperus genus (Juniperus communis (scientific name: Juniperus communis)).
• Juniperus communis extract for example, roots, rhizomes, leaves, stems, flowers, fruits, pericarp, seeds, whole plants, or mixtures thereof can be used as materials.
• Juniperus communis extract is produced, for example, by extracting raw or dried materials with a solvent, either as is or after crushing.
• 100 to 200 g of Juniperus communis fruit is immersed in 1 kg of 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.
• 100 to 200 g of Juniperus communis fruits are immersed in 1 kg of 10 to 100% ethanol solution and extracted for 5 to 10 days in an environment of about 10°C to about 30°C.
• 100 to 200 g of Juniperus communis fruits may be immersed in 1 kg of warm water at about 30°C to about 80°C and extracted for 5 to 24 hours.
• the solution obtained through this immersion is filtered using a specified filter material (such as a Glass Fiber Filter (Gf-75 manufactured by ADVANTEC) and Mixed Cellulose ester (A045A047A manufactured by ADVANTEC)).
• a specified 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 left to stand in an environment of about 0°C to about 10°C for 5 to 10 days. It is again filtered using a specified filter material, and the resulting solution is used as the Juniperus communis extract.
• the active ingredient of this embodiment can be extracted from Juniperus communis extract by liquid separation 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 filter, ultrafiltration membrane, etc., pressurization or decompression, heating or cooling, drying, pH adjustment, deodorization, decolorization, long-term static storage, etc., and these can be arbitrarily selected and combined for processing. As one embodiment, it is preferable to combine column chromatography and preparative HPLC, etc., used in the examples described below.
• the structure of the purified compound can be determined by NMR or mass spectrometry.
• the content ratio of each active ingredient in the agent of this embodiment is not particularly limited.
• the agent of this embodiment may contain only one of podophyllotoxin, 4-deoxypodophyllotoxin, ⁇ -peltatin, and yatein, or may contain them in any ratio.
• the agent of this embodiment may contain any compound other than these active ingredients.
• Cupressaceae plants, including Juniperus communis contain many types of lignans and their analogues (Non-Patent Document 3).
• modified form The compounds represented by the above formulas (I) to (II) may be modified.
• a modified form means that a part of the compound has been subjected to some kind of chemical modification or the like to the extent that the proteasome activation ability of the compound is not lost.
• the chemical modification 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 may be one in which one or more sugars (e.g., N-acetylgalactosamine, galactose, mannose, GlcNAc, glucose, fucose, or xylose) are attached to any hydroxy group.
• sugars e.g., N-acetylgalactosamine, galactose, mannose, GlcNAc, glucose, fucose, or xylose
• the site that has been subjected to the chemical modification or the like is not limited to the substituents of R 1 to R 3 .
• the modified compounds may be naturally occurring or may not be naturally occurring. If they are naturally occurring, they may be directly extracted from medicinal plants, etc. If they are not naturally occurring, they may be synthesized by chemical modification using a parent compound extracted from a medicinal plant, etc., or may be synthesized from a commercially available compound using organic synthesis techniques known in the art.
• composition of active ingredients in topical skin preparations One of the uses of the agent of this embodiment is a skin external preparation.
• the content of each active ingredient in the skin external preparation is not particularly limited, as long as it is an amount that can obtain the promotion effect of proteasome activity in skin cells, taking into consideration its administration form and administration method.
• the content of each active ingredient is preferably 0.1 nM (about 1 ⁇ g/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, based on the total weight of the skin external preparation.
• 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.
• topical skin preparations are in suitable forms for use, such as ampoules, capsules, powders, granules, liquids, gels, foams, emulsions, sheets, mist, sprays, etc., and include 1) medicines, 2) quasi-drugs, and 3) topical or whole-body topical skin preparations (for example, basic cosmetics such as skin lotions, milky lotions, creams, ointments, lotions, oils, and packs, face washes and skin cleansers such as solid soaps, liquid soaps, and hand washes, massage preparations, cleansing preparations, hair removal preparations, depilatories, shaving treatment preparations, aftershave lotions, preshave lotions, shaving creams, foundations, lipsticks, blushers, eyeshadows, and eyeliners).
• basic cosmetics such as skin lotions, milky lotions, creams, ointments, lotions, oils, and packs
• face washes and skin cleansers such as solid soaps, liquid soaps, and hand was
• make-up cosmetics such as mascara, perfumes, nail beautifying agents, nail beautifying enamel, nail beautifying enamel remover, poultices, plasters, tapes, sheets, patches, aerosols, etc.
• medicinal and/or cosmetic preparations applied to the scalp and hair e.g. shampoos, rinses, hair treatments, pre-hair treatments, permanent solutions, hair dyes, hair styling products, hair tonics, hair growth and tonics, poultices, plasters, tapes, sheets, aerosols, etc.
• bath agents used by being added to the bath 6) other products such as underarm odor prevention agents and deodorants, antiperspirants, sanitary products, sanitary cotton, wet tissues, etc.
• compositions of topical skin preparations can be produced by arbitrarily selecting and combining the components and additives exemplified below as necessary 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 specified, but it is generally considered preferable to add about 0.0001 to 50%.
• Waxes beeswax, carnauba wax, spermaceti, lanolin, liquid lanolin, reduced lanolin, hard lanolin, candelilla wax, montan wax, shellac wax, rice wax, etc.
• Mineral oils liquid paraffin, vaseline, paraffin, ozokerite, ceresin, microcrystalline wax, etc.
• 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 acid; and synthetic fatty acids such as isononanoic acid, caproic acid, 2-ethylbutanoic acid, isopentanoic acid, 2-methylpentanoic acid, 2-ethylhexanoic acid, and isopentanoic acid.
• 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.
• 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.
• 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 ester, trehalose or its derivatives, dextrin, gelatin, pectin, starch, carrageenan, carboxymethyl chitin or chitosan, hydroxyalkyl (C2-C4) chitin or chitosan to which an alkylene (C2-C4) oxide such as ethylene oxide has been added, low molecular weight chitin or chitosan, chitosan salt, sulfated chitin or chitosan, phosphorylated chitin or chitosan, alginic acid or its salt, hyaline
• suitable cellulose acetate hydrochloride include hydroxy
• Surfactants Anionic surfactants (alkyl carboxylates, alkyl sulfonates, alkyl sulfates, alkyl phosphates), cationic surfactants (alkylamine salts, alkyl quaternary ammonium salts), amphoteric surfactants: carboxylate type amphoteric surfactants (amino type, betaine type), sulfate type amphoteric surfactants, sulfonic acid type amphoteric surfactants, phosphate 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, polymeric surfactants, surfactants containing titanium/silicon, fluorocarbon surfactants), etc.
• 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 acid, nicotinic acid, pantothenic acid, biotin, choline, inositol, Vitamin C group: vitamin C acid or its derivatives, Vitamin D group: ergocalciferol (vitamin D2), cholecalciferol (vitamin D3), dihydrotachysterol, vitamin E group: vitamin E or a derivative thereof, ubiquinones, vitamin K group: phytonadione (vitamin K1), menaquinone (vitamin K2), menadione (vitamin K3), menad
• 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 amino acid derivatives thereof such as sulfates, phosphates, nitrates, citrates, or pyrrolidone carboxylic acid.
• Juniperus communis fruit extract (Preparation of Juniperus communis fruit extract) Juniperus communis fruits (also known as juniper berries) were immersed in hot water at 80° C. for 5 hours. The solution (hot water extract) obtained through this immersion was filtered using a predetermined filter material (glass filter paper, Gf-75, manufactured by ADVANTEC and cellulose mixed ester membrane filter, A045A047A, manufactured by ADVANTEC), and the evaporation residue was adjusted to 0.5% (w/w) and the 1,3-butylene glycol concentration was adjusted to 50% (w/w), and the solution was used as juniperus communis fruit (juniper berry extract) extract (JBE).
• a predetermined filter material glass filter paper, Gf-75, manufactured by ADVANTEC and cellulose mixed ester membrane filter, A045A047A, manufactured by ADVANTEC
• 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 proteasomes expressed in the cells was evaluated. Proteasome activity was measured using a Cell-Based Proteasome-Glo TM Assays kit (G8660 (for measuring chymotrypsin-like activity) manufactured by Promega), which quantifies the activity based on luminescence intensity.
• G8660 for measuring chymotrypsin-like activity
• HEK293T cells manufactured by TAKARA
• TAKARA TAKARA
• a cell line derived from human embryonic kidney cells commonly used in cell tests were seeded at 1x104 (cells)/well in a 96-well white plate using DMEM medium containing 10% FBS, and cultured for 16 hours at 37°C in a 5% CO2 incubator.
• Juniperus communis fruit extract was added to the wells to a final concentration of 1% or 0.5%, and cultured for 2, 4, 8, and 24 hours at 37°C in a 5% CO2 incubator.
• the medium in the wells was removed, and the wells were washed once with PBS, and 50 ⁇ L of PBS was added per well.
• Proteasome activity was then evaluated using a Cell-Based Proteasome-Glo TM Assays kit according to the method recommended by the manufacturer.
• wells were set up to which 50% 1,3-butylene glycol, a solvent not containing juniper fruit extract, was added to a final concentration of 1% or 0.5%, and proteasome activity was measured.
• the proteasome activity in the wells to which only the solvent was added was set at 100, and the ratio (relative %) of proteasome activity in the wells to which the juniper fruit extract was added was calculated.
• Human skin keratinocytes (TOYOBO) derived from adults or newborns were seeded at 1 x 104 (cells)/well in a 96-well white plate using the medium recommended by the manufacturer, and cultured at 37°C in a 5% CO2 incubator for 16 hours. Juniperus communis fruit extract was added to the wells to a final concentration of 1% or 0.5%, and cultured at 37°C in a 5% CO2 incubator for 2, 4, 8, and 24 hours. The medium in the wells was removed, and the wells were washed once with PBS, and 50 ⁇ L of PBS was added per well.
• TOYOBO Human skin keratinocytes
• Proteasome activity was then evaluated using a Cell-Based Proteasome-Glo TM Assays kit according to the method recommended by the manufacturer.
• wells were set up to which 50% 1,3-butylene glycol, a solvent not containing juniper fruit extract, was added to a final concentration of 1% or 0.5%, and proteasome activity was measured.
• the proteasome activity in the wells to which only the solvent was added was set at 100, and the ratio (relative %) of proteasome activity in the wells to which the juniper fruit extract was added was calculated.
• Juniperus communis fruit extract activates the proteasome in human skin keratinocytes derived from newborns.
• the cells were plated on a glass chamber slide (Matsunami Glass SCS-NO2), and after adhesion, the cells were fixed with 10% neutral buffered formalin (Wako Pure Chemical Industries, Ltd. 062-01661).
• the ammoniacal silver used in Masson-Fontana staining was prepared by dropping 28% ammoniacal water into 10 mL of 10% silver nitrate, adding 100 mL of purified water when it became a thin turbid liquid, and leaving it at room temperature in the dark for 24 hours, and then preparing the supernatant.
• the cells after fixation were washed with purified water, and then ammoniacal silver solution was added, and the mixture was left at room temperature in the dark for 24 hours.
• the cells were washed with purified water, and then 5% sodium thiosulfate was added, and the mixture was decolorized at room temperature for 2 minutes.
• the cells were washed with purified water, and then mounted using a mounting medium.
• the stained lipofuscin was detected using an inverted microscope (Olympus, main body: BX50, camera: DP70).
• Juniperus communis extract was fractionated according to the fractionation scheme shown in Figure 3 A. When evaluating the proteasome activity of each fraction, it was prepared so as to contain an area equivalent to that of the corresponding peak in the Juniperus communis fruit extract (JBE) (before fractionation).
• Ethanol was added to the hot water extract of Juniperus communis fruits to obtain a 10% ethanol aqueous solution, which was then crudely fractionated using a column packed with Diaion HP-20 (Mitsubishi Chemical Corporation). The following fractions were obtained: a pass-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).
• a 70% ethanol extract of Juniperus communis fruit was prepared again 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 aqueous 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 liquid was concentrated once and adjusted to a 30% ethanol aqueous solution, and then applied to a column packed with Diaion HP-20. The column was washed with 50% ethanol and 60% ethanol to elute unnecessary fractions, 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% ethanol in water (200 g), and then applied to a column (70 mm diameter x 350 mmL) packed with Diaion HP-20.
• the liquid eluted with ethanol was analyzed by HPLC, and two fractions (H-1 and H-2) were obtained.
• the eluate was analyzed by HPLC (condition 1), and five fractions (HFr. 2-1 to 2-5) were obtained.
• 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 showed proteasome activity (2 hours after sample addition) almost equal to that of the extract of juniper fruit ( Figure 4E).
• Compound 2 also showed weak proteasome activity (2 hours after sample addition) ( Figure 4E). From these results, it was concluded that Compound 1 and Compound 2 are the main compounds that contribute to the proteasome activation effect of the extract of juniper fruit.
• Each purified fraction used in this evaluation was prepared to contain an area equivalent to that of the peak in the Juniperus communis fruit extract (JBE).
• the cells were HEK293T cells (manufactured by TAKARA), a cell line derived from human embryonic kidney cells commonly used in cell tests, and the above-mentioned method was used.
• Juniperus communis fruit extract or purified fraction was added to the wells to a final concentration of 1% or 0.5%.
• DPT 4-deoxypodophyllotoxin
• yatein CAY 29766, Cayman
• PT analogues podophyllotoxin
• SC-478230 Santa Cruz Animal Health
• Proteasome activity was evaluated using HEK293T cells (TAKARA), a cell line derived from human embryonic kidney cells that is widely used in cell tests, 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 which DMSO containing no compound was added at a final concentration of 0.1%, and proteasome activity was measured in the same manner. The proteasome activity of the wells to which only DMSO was added was set as 100, and the ratio (relative value) of proteasome activity of the wells to which the compound was added was calculated.
• TAKARA HEK293T cells
• DPT, PT, and ⁇ -peltatin all increased proteasome activity depending on the compound's concentration, and showed a proteasome activation effect of 400% or more at a concentration of 50 nM.
• the EC 50 value of DPT was calculated to be 16 nM, the EC 50 value of PT to be 27 nM, and the EC 50 value of ⁇ -peltatin to be 28 nM.
• the proteasome activation effect of yatein was saturated at about 6 nM and was about 130%, suggesting that it was a partial effect.
• Juniperus communis fruit extract JBE
• JBE Juniperus communis fruit extract
• FIG. 5B The results of quantifying the contents of these compounds in Juniperus communis fruit extract (JBE) are shown in FIG. 5B. It was found that JBE contained about 1000 nM of DPT and about 600 nM of yatein. Although the peaks of PT and ⁇ -peltatin could not be separated by HPLC, it was found that the two compounds were contained in a total of about 100 nM. That is, when Juniperus communis fruit extract (JBE) was added to the cells at a final concentration of 1%, the final concentrations of each compound were about 10 nM for DPT, about 6 nM for yatein, and about 1 nM for PT and ⁇ -peltatin combined.
• proteasome activation effects corresponding to these concentrations are shown by ⁇ in Figure 5A.
• the proteasome activity at the concentrations shown by ⁇ was 234% for DPT (12.5 nM), 115% for PT (1.5 nM), 132% for yatein (6 nM), and 114% for ⁇ -peltatin (1.5 nM) ( Figure 5A).
• This result correlates well with the result that the proteasome activation effect was 245% when Juniperus communis fruit extract (JBE) was added to cells at a final concentration of 1% ( Figure 1A), and indicates that the compounds that contribute to the proteasome activation effect of Juniperus communis fruit extract (JBE) are DPT, PT, yatein, and ⁇ -peltatin.
• Non-Patent Document 3 Figure 6
• Optical isomers of DPT have been reported (Non-Patent Documents 4 and 5), and it is indicated that these may also have a proteasome activation effect.
• ⁇ Sakura Extract B (Ichimaru Pharcos Co., Ltd.): This Sakura Extract B was prepared as follows. Leaves of Oshimazakura (Prunus lannesiana (Carr.) Wilson var. speciosa (Koidz.) Makino) or other Prunus subgenus were taken and soaked in 30% ethanol solution and extracted at room temperature for one week. After filtration, the filtrate was taken and evaporated to dryness under reduced pressure. A 50% 1,3-butylene glycol solution was added to the dried product, dissolved, and filtered to prepare an extract. The composition of this liquid was 49.0% 1,3-butylene glycol solution, 49.0% water, and 2.0% cherry leaf extract.
• the present invention through activation of the proteasome, can promote the degradation of proteins that have completed their role within cells, mainly in humans, or proteins that have become dysfunctional (e.g., oxidized proteins, structurally denatured proteins, aggregated proteins, etc.) and foreign proteins (e.g., proteins derived from pathogens such as viruses that have invaded cells), thereby making it possible to prevent the aging of cellular tissues, including the skin, and maintain the health of individual organisms.
• proteins that have become dysfunctional e.g., oxidized proteins, structurally denatured proteins, aggregated proteins, etc.
• foreign proteins e.g., proteins derived from pathogens such as viruses that have invaded cells

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