Agent containing araliadiol

Abstract

The purpose of the present invention is to discover a substance having an elastin production promotion effect and the like and to provide an agent and the like for use in elastin production promotion and the like.　The agent is used in fibroblast growth, MMP-1 production suppression, SPARC production, type IV collagen production, type I collagen production promotion, and / or elastin production promotion, the agent containing araliadiol and the like.

Description

Agents containing alariadiol

[0001] The present invention relates, for example, to an agent used to promote elastin production, a skin whitening agent, etc., for use in humans, etc.

[0002] As the frontline of the body directly affected by the external environment, the skin plays a crucial role in maintaining the internal environment of the organism. Therefore, while skin function never completely ceases, it gradually declines with age, UV exposure, and skin exposure to chemicals, leading to the manifestation of aging signs such as wrinkles, age spots, dullness, and sagging. For example, the presence or absence of skin firmness is considered one of the indicators for evaluating health and the degree of aging. Skin firmness can sometimes be divided into two types: firmness originating from the stratum corneum / epidermis and firmness originating from the dermis. Dermal firmness, in particular, refers to a state where the skin has elasticity that pushes back when pressed with a finger, and quickly returns to its original state when the finger is released; physically, this is also called viscoelasticity. While the detailed mechanisms causing a decrease in skin viscoelasticity are not always clear, one possible factor is, for example, thinning of the skin (Patent Document 1).

[0003] Elastin is a fiber that gives elasticity to skin tissue. Elastin production decreases with age, and its degradation and denaturation are accelerated by ultraviolet light. When normal elastin decreases, skin elasticity decreases, which can cause wrinkles and sagging. Therefore, it is thought that if elastin production can be promoted, wrinkles and sagging will become less likely to occur, and skin aging symptoms such as loss of firmness and decreased elasticity can be prevented and / or improved (Patent Document 1).

[0004] Furthermore, due to air pollution and ozone layer depletion, the amount of ultraviolet radiation reaching the epidermis is increasing year by year, and consequently, skin problems such as sunspots, freckles, and darkening caused by ultraviolet radiation are becoming more pronounced. Under these circumstances, various whitening agents are being developed to achieve fair and beautiful skin. Known whitening agents include ascorbic acid, ascorbic acid phosphate esters and ascorbic acid glucosides, ascorbic acid derivatives, hydroquinone glycosides and other hydroquinone derivatives, and tranexamic acid and tranexamic acid esters (Patent Document 2).

[0005] Japanese Patent Publication No. 7144878, Japanese Unexamined Patent Publication No. 2023-056457

[0006] The problem that this invention aims to solve is to find substances derived from natural sources (natural products) that have elastin production promoting effects and / or skin whitening effects, and to provide agents for use in promoting elastin production, etc.

[0007] In order to solve the above problems, the present inventors conducted diligent research and found that the above problems can be solved by using alariadiol or a predetermined combination of alariadiol. That is, the present invention includes the following embodiments.

[0008] [1] An agent containing alariadiol for use in promoting fibroblast proliferation, inhibiting MMP-1 production, producing SPARC, producing type IV collagen, promoting type I collagen production and / or promoting elastin production. [2] An agent containing gotu kola extract for use in promoting fibroblast proliferation, inhibiting MMP-1 production, producing SPARC, producing type IV collagen, promoting type I collagen production and / or promoting elastin production. [3] The agent according to [1] or [2], further containing a proteoglycan. [4] The agent according to [3], wherein the proteoglycan is a proteoglycan derived from salmon nasal cartilage. [5] The composition according to [4], wherein the molecular weight of the peak top of the proteoglycan is 300,000 to 800,000. [6] A method for producing gotu kola extract (alariadiol), comprising the steps of: extracting the gotu kola plant body with a mixed solvent of an organic solvent and water; passing the extract obtained in the extraction step through a column packed with a hydrophobic synthetic adsorbent to adsorb alariadiol onto the hydrophobic synthetic adsorbent; and eluting the components adsorbed onto the hydrophobic synthetic adsorbent with a mixed solvent of an organic solvent and water to obtain an eluted fraction containing alariadiol. [7] A method for producing a powder formulation containing alariadiol, comprising the steps of: mixing alariadiol, dextrin and cyclodextrin to obtain a mixture; homogenizing the mixture to prepare an emulsion; and drying the emulsion. [8] The method for producing a powder formulation according to [7], wherein the cyclodextrin contains γ-cyclodextrin. [9] A whitening agent containing alariadiol.

[10] A whitening agent containing gotu kola extract.

[11] The whitening agent according to [9] or

[10] , further comprising ceramide.

[12] The whitening agent according to

[11] , wherein the content ratio of alariadiol to ceramide is 0.01 to 0.1, preferably 0.02 to 0.08, and more preferably 0.04 to 0.07, with ceramide being 1.

[0009] According to the present invention, it is possible to provide agents for use in promoting elastin production, etc.

[0010] Figure 1 is a flow chart showing the manufacturing process of alariadiol in one embodiment. Figure 2 is a flow chart showing the manufacturing process of a powder formulation containing alariadiol in one embodiment.

[0011] Embodiments for carrying out the present invention will be described below. Unless otherwise specified, each disclosure can be referenced by the descriptions of other disclosures.

[0012] <Definition> (Derived from) In the specification of this application, the phrase "derived from" is used with the intention of encompassing the following (1) to (3): (1) being purified, (2) being isolated, and / or (3) being modified [this includes depolymerization and polymerization (polymerization)] or altered.

[0013] (Alariadiol) The formulation of this embodiment is the following formula (I): The formulation contains alariadiol (CAS No. 1354638-93-9), represented by formula (I), as an active ingredient. The compound represented by formula (I) above contains chiral carbon atoms at positions 3 and 8, and the double bond between carbon atoms at positions 9 and 10 has a cis (Z) conformation. Therefore, the formulation of this embodiment may contain isomers of alariadiol as an active ingredient. The isomers of alariadiol may include, for example, optical isomers with respect to these positions, racemates, and / or geometric isomers. Naturally occurring alariadiol has also been isolated from ethanol extracts of Aralia cordata leaves and has been reported to have inhibitory activity on the proliferation of human breast cancer cells (Cheng W-L et al. "Planta Medica" (2011); 77:164-168). Alariadiol can be produced from medicinal plants such as Gotu Kola and Aralia cordata by organic solvent extraction and purification by column chromatography.

[0014] Furthermore, this active ingredient may also be in the form of a salt or derivative. Examples of salts where the hydroxyl group of the compound represented by formula (I) has a suitable acidity include metal salts such as aluminum, alkali metal salts such as lithium, sodium, or potassium, alkaline earth metal salts such as calcium or magnesium, and ammonium or substituted ammonium salts, such as salts with lower alkylamines like triethylamine, hydroxyalkylamines such as 2-hydroxyethylamine, bis-(2-hydroxyethyl)-amine, or tri-(2-hydroxyethyl)-amine, or cycloalkylamines such as bicyclohexylamine, or salts with pyridine-type bases such as procaine, dibenzylpiperidine, N-benzyl-β-phenethylamine, dehydroabiethylamine, N,N'-bisdehydroabiethylamine, glucamine, N-methylglucamine, or pyridine, colidine, quinine, or quinoline. In addition, the hydroxyl group of the compound represented by formula (I) may be acylated, alkylated, phosphorylated, or borated derivatives. Accordingly, in this specification, the term "alariadiol" includes the compound represented by formula (I) above, its isomers or derivatives, or salts thereof.

[0015] The content of alariadiol in the formulation of this embodiment is not particularly limited, but it is sufficient to include an effective amount of alariadiol to exert a desired effect (e.g., elastin production effect). For example, when the entire formulation is considered to be 100% by mass: - The lower limit of the alariadiol content is preferably 1 ppm (0.0001% by mass) or more, more preferably 10 ppm (0.001% by mass) or more, and even more preferably 100 ppm (0.01% by mass) or more. - The upper limit of the alariadiol content is preferably 50% by mass or less, more preferably 40% by mass, and even more preferably 30% by mass or less. In the above examples of alariadiol content, the upper and lower limits can be arbitrarily combined. As a specific example, when the entire formulation is considered to be 100% by mass, the alariadiol content is preferably 0.0001 to 50% by mass, more preferably 0.001 to 40% by mass, and even more preferably 0.01 to 30% by mass.

[0016] (Gotu Kola Extract) Gotu Kola (Centella asiatica; also known as Centella asiatica) is a herb native to India, Indonesia, China, and Southeast Asia, and is a plant used in Ayurveda, the traditional Indian system of medicine. In this embodiment, the Gotu Kola used may be the flower, inflorescence, fruit, stem, leaf, petiole, branch, branch leaves, rhizome, root, seed, or whole plant, but other species of the same genus may also be used. For extraction, the plant can be extracted with water or an organic solvent (such as an ethanol solution), and a mixed solvent of organic solvent and water is preferred. Lower alcohols such as methanol and ethanol, acetonitrile, and acetone can be used as organic solvents, but ethanol and methanol are preferred for safety reasons. The mixing ratio of alcohol to water is preferably alcohol:water = 10:90 to 60:40. If the alcohol concentration is too high, it will be difficult to retain it on the carrier in the next step, and if it is less than 10%, the active ingredient will not dissolve sufficiently. Preferably, a 50% aqueous ethanol solution is used as the extraction solvent. In one embodiment, it is preferable to use an ethanol extract from the whole plant of Gotu Kola.

[0017] One of the active ingredients in gotu kola extract is given by the following formula (I): The compound represented by formula (I) is alariadiol (CAS No. 1354638-93-9). The compound represented by formula (I) contains chiral carbon atoms at positions 3 and 8, and the double bond between carbon atoms at positions 9 and 10 has a cis (Z) conformation. Therefore, the formulation of this embodiment may contain isomers of alariadiol as the active ingredient. The isomers of alariadiol may include, for example, optical isomers with respect to these positions, racemates, and / or geometric isomers. Furthermore, alariadiol may also be in the form of the salts described above, and examples of salts when the hydroxyl group of the compound represented by formula (I) has a suitable acidity include metal salts such as aluminum, alkali metal salts such as lithium, sodium, or potassium, alkaline earth metal salts such as calcium or magnesium, and ammonium or substituted ammonium salts.

[0018] Other active ingredients in gotu kola extract include, for example, the triterpenoids listed below, and preferably, triterpenic acids such as madecassic acid and asiatic acid, and their glycosides, can be used as analytes.

[0019]

[0020] Other active ingredients contained in gotu kola extract include, for example, flavonoids such as quercetin and kaempferol, as listed below, which can be used as analytes.

[0021] A method for analyzing the components of gotu kola extract is described, for example, in Japanese Patent Publication No. 2024-35131.

[0022] (Method for producing gotu kola extract and alariadiol) The present invention provides a method for producing gotu kola extract and alariadiol. Figure 1 is a flow chart showing the process for producing alariadiol. This method includes an extraction step (S01) in which the gotu kola plant body is extracted with a mixed solvent of an organic solvent and / or water; a column adsorption step (S02) in which the obtained extract is passed through a column packed with a hydrophobic synthetic adsorbent to adsorb alariadiol thereon; a column elution step (S03) in which the components adsorbed on the hydrophobic synthetic adsorbent are eluted with a mixed solvent of an organic solvent and water to obtain an eluted fraction containing alariadiol; and a concentration step (S04) in which the obtained extract is concentrated.

[0023] The Gotu Kola plant material used in the extraction process (S01) can be extracted from the flowers, inflorescences, fruits, stems, leaves, petioles, branches, branch leaves, rhizomes, roots, seeds, or the whole plant, but other species of the same genus can also be used. The extraction method can be water or an organic solvent (such as an ethanol solution) or a mixture thereof. For example, a 30-90% aqueous ethanol solution, preferably a 50% aqueous ethanol solution, is used as the extraction solvent. In this embodiment, it is preferable to use an ethanol extract using the whole Gotu Kola plant.

[0024] The adsorbent used in the column adsorption step (S02) is not particularly limited as long as it is a hydrophobic synthetic adsorbent capable of adsorbing highly hydrophobic alariadiol, but it is preferable to use an aromatic synthetic adsorbent. A "hydrophobic synthetic adsorbent" is a hydrophobic adsorbent composed of a crosslinked polymer having a porous structure, and has the property of adsorbing various organic substances in solution through physical interaction between the pores and the substance to be adsorbed. The type of hydrophobic synthetic adsorbent is not particularly limited as long as it can achieve the objective of the present invention, but it is preferable to use an aromatic synthetic adsorbent because it improves the efficiency of impurity removal. An "aromatic synthetic adsorbent" is a synthetic adsorbent in which the crosslinked polymer constituting the adsorbent has an aromatic ring group such as a benzene ring. An example of an aromatic synthetic adsorbent is a porous body made of styrene-divinylbenzene copolymer. Examples of aromatic synthetic adsorbents include Diaion HP20, Diaion HP21, Sepabeads SP825, Sepabeads SP850, Sepabeads SP700, Sepabeads SP270 (all product names of Mitsubishi Chemical Corporation) and Amberlite XAD4, Amberlite XAD16HP, Amberlite XAD1180, Amberlite XAD2000 (all product names of Rohm & Haas).

[0025] (Method for producing a powder formulation containing alariadiol) When using alariadiol, for example, it is used as a formulation containing alariadiol (such as a powder formulation containing an excipient such as dextrin). Figure 2 is a flow chart showing the process for producing a powder formulation containing alariadiol. This method includes an excipient addition step (S11) in which dextrin and an excipient (dextrin, cyclodextrin, etc.) are added to an alariadiol solution to obtain a mixture; a homogenization step (S12) in which the mixture obtained in S11 is homogenized to prepare an emulsion; a drying step (S13) in which the emulsion obtained in S12 is dried; and a recovery step (S14) in which the powder formulation after drying is recovered.

[0026] The excipient addition step (S11) can be achieved by mixing the oil phase, in which alariadiol has been added and dissolved, with the aqueous phase in which the excipient has been dissolved.

[0027] In the homogenization step (S12), the above-mentioned mixed solution is homogenized to prepare an emulsion. The emulsification method is not particularly limited, and various emulsifiers conventionally used in food and beverages, etc., for example, the above-mentioned polysaccharides, surfactants, etc. are added, and an emulsion excellent in stability can be obtained by performing an emulsification treatment using a homomixer, a colloid mill, a rotary disk type homogenizer, a high-pressure homogenizer, etc.

[0028] The emulsion thus obtained is then subjected to drying in the drying step (S13). As the drying method applicable to this production method, usually, any method that can be used for this application may be used, and examples include spray drying, freeze drying, vacuum drying, shelf drying, belt drying, drum drying, etc. Among these, from the viewpoint of handling of the powder, spray drying and freeze drying are preferable.

[0029] (Proteoglycan) "Proteoglycan" means a molecule (glycoprotein) in which a protein (core protein) and a glycosaminoglycan (GAG, also referred to as "polysaccharide" or "sugar chain") are covalently bonded. The proteoglycan exists, for example, as an extracellular matrix such as skin, organs, and cartilage. The glycosaminoglycan is usually known as a sugar chain having a long-chain structure without a branched structure. Examples of the proteoglycan include aggrecan, versican, decorin, testican, brevican, biglycan, serglycin, syndecan, perlecan, dystroglycan, agrin, clustrin, glypican, lumican, keratocan, and neurocan. The proteoglycan can be classified into chondroitin sulfate proteoglycan, dermatan sulfate proteoglycan, heparan sulfate proteoglycan, or keratan sulfate proteoglycan, for example, depending on the type of GAG bound to the protein.

[0030] Examples of the aforementioned GAGs include chondroitin, chondroitin sulfate, dermatan sulfate (chondroitin sulfate B), heparan sulfate, heparin, and keratan sulfate. Examples of the aforementioned chondroitin include O-type sugar chains whose main disaccharide structure is a disaccharide structure of glucuronic acid and acetylgalactosamine, and iO-type sugar chains whose main structure is a disaccharide structure of iduronic acid and acetylgalactosamine (hereinafter also referred to as "chondroitin sulfate O" and "chondroitin sulfate iO," respectively). The aforementioned chondroitin sulfate has a structure in which a sulfate group is added to a sugar chain in which the disaccharides of glucuronic acid and acetylgalactosamine are repeated. For example, the aforementioned chondroitin sulfate is gluc Examples include chondroitin sulfate A (Type A), which has a disaccharide structure mainly consisting of iduronic acid and acetylgalactosamine 4-sulfate; chondroitin sulfate iA (Type iA), which has a disaccharide structure mainly consisting of iduronic acid and acetylgalactosamine 4-sulfate; chondroitin sulfate C (Type C), which has a disaccharide structure mainly consisting of glucuronic acid and acetylgalactosamine 6-sulfate; and chondroitin sulfate iC (Type iC), which has a disaccharide structure mainly consisting of iduronic acid and acetylgalactosamine 6-sulfate.

[0031] The proteoglycan derived from salmon nasal cartilage is a proteoglycan obtained by extraction from salmon nasal cartilage. Here, salmon is, for example, a fish belonging to the genus Oncorhynchus, but preferably salmon with the scientific name "Oncorhynchus keta" is selected from the viewpoint of efficiently regulating the immune response. The proteoglycan contained in the composition of the present invention is prepared, for example, by the method described in the publication (Japanese Patent Publication No. 6317053). Furthermore, the amount of proteoglycan contained in the composition of the present invention is preferably such that it is 5 mg to 50 mg per human daily intake, for example from the viewpoint of effectively exhibiting the desired effect. The molecular weight of the peak top of the proteoglycan is preferably 300,000 to 800,000.

[0032] The proteoglycans used in this invention, such as PG derived from salmon nasal cartilage, can be produced by methods described in, for example, Japanese Patent Publications (Japanese Patent No. 6875701, Japanese Patent No. 6317053, and Japanese Patent No. 7295572).

[0033] Commercially available proteoglycans include, for example, proteoglycans derived from salmon nasal cartilage, such as proteoglycans derived from salmon nasal cartilage (manufactured by Fujifilm Wako Pure Chemical Corporation (product codes: 162-22131, 168-22133)), Proteoglycan IPC (registered trademark) (manufactured by Ichimaru Pharcos Co., Ltd.), and Proteoglycan F (registered trademark) (manufactured by Ichimaru Pharcos Co., Ltd.).

[0034] (Promotion of elastin production) By increasing the amount of elastin in the living body (promotion of elastin production), the strength and elasticity of the dermis, ligaments, tendons, blood vessel walls, bones, cartilage, etc. can be enhanced. Agents for elastin production are used for the prevention and / or improvement of various disorders associated with elastin reduction (for example, for the treatment or prevention of vascular diseases such as arteriosclerosis caused by the lack of normal elastin), and for the prevention and / or improvement of cosmetic problems (for example, for the prevention and / or improvement of skin wrinkles or sagging caused by elastin reduction due to aging, ultraviolet rays, reactive oxygen species, stress, etc., or for the prevention and / or improvement of decreased skin elasticity or firmness).

[0035] (Extracellular matrix) The extracellular matrix (ECM: Extracellular Matrix) is a complex of sugars and proteins that underlies tissues and exists in the intercellular spaces. Structures formed by ECM include, for example, basement membranes, glial scars, and perineuronal nets.

[0036] (Collagen) Collagen is a major fibrous component that supports the structure of living tissues such as skin, bones, and tendons, and is an essential protein that exists abundantly in the animal body. It is known that types I to XIII of collagen exist. In the said collagen, one molecule forms a triple helix structure consisting of three polypeptide chains, and each polypeptide chain is called an α-chain. One molecule of the said collagen may be composed of one type of α-chain or may be composed of a plurality of types of α-chains encoded by different collagen genes. The type I collagen mainly exists in the skin, bones, and tendons.

[0037] (Hyaluronic Acid) Hyaluronic acid is a polysaccharide with a structure in which disaccharide units of glucuronic acid and N-acetylglucosamine are linked. Furthermore, there are no particular limitations on the salt of hyaluronic acid; any salt that is acceptable in food or pharmaceutical terms is acceptable, such as sodium salt, potassium salt, calcium salt, zinc salt, magnesium salt, and ammonium salt. The molecular weight of hyaluronic acid is not particularly limited.

[0038] (Promotion of Hyaluronic Acid Production) In this invention, "promotion of hyaluronic acid production" is not limited to the mechanism as long as it primarily promotes the synthesis of hyaluronic acid in keratinocytes, etc., of the epidermis (especially the stratum corneum). Hyaluronic acid production promotion is preferably achieved by promoting the synthesis of hyaluronic acid, for example, by promoting the expression of genes encoding HAS2 or HAS3, which are mammalian hyaluronic acid synthases. In this invention, the hyaluronic acid production promoter can be used not only as a hyaluronic acid production promoter, but also, for example, as an expression promoter of hyaluronic acid synthases (e.g., a gene expression promoter for hyaluronic acid), and in particular, specifically as an expression promoter of the HAS2 gene or the HAS3 gene. The hyaluronic acid production promoter of this invention can prevent various disorders, diseases, and functional declines associated with a decrease in hyaluronic acid in the body, especially in the skin. For example, it can promote skin hydration, improve skin firmness and elasticity, and provide moisture, thereby preventing or improving skin problems such as rough skin, wrinkles, and dryness, as well as preventing or improving joint disorders and joint damage.

[0039] (Improvement of skin elasticity) "Improvement of skin elasticity" includes not only "suppressing the decrease in skin elasticity (preventing the decrease in skin elasticity, etc.)", but also, for example, "improvement of the symptoms of decreased skin elasticity after it has occurred (increased skin thickness, etc.) by administering a specified composition (oral administration, etc.)".

[0040] (Fibroblasts) Fibroblasts are mesoderm-derived cells scattered throughout the connective tissue of the body. They are also called fibroblasts. In the skin, they are located in the dermis and perform metabolism, including the synthesis and breakdown of extracellular matrix, including collagen and elastin fibers, in the dermis or basement membrane. In addition, fibroblasts construct and organize the extracellular matrix they produce. When tissue is damaged, fibroblasts migrate and proliferate from the surrounding tissue and synthesize collagen, thus promoting healing. After attaching a collected skin tissue sample to a petri dish with the epidermal side up and the dermal side down, and continuing to culture it with culture medium, fibroblasts migrate from the tissue sample toward the surface of the petri dish and proliferate. Cells that have proliferated or passaged in this way can be used for culture experiments in the laboratory. Experiments using cultured cells have revealed that the number of cell divisions of fibroblasts is finite, that this number of divisions is negatively correlated with the age of the cell donor, and that it is negatively correlated with the lifespan of the animal species from which the fibroblasts originated. Cells with a high passage number (in vitro aged cells) obtained by repeatedly dividing and proliferating fibroblasts collected from young individuals in the laboratory are thought to have undergone more cellular senescence compared to cells before division. This is because cells with a high passage number show similar changes in morphology and cellular activity levels to cells collected from older individuals (in vivo aged cells). It has been shown that with age, collagen fibers in the dermis become thinner and the amount of collagen decreases, and in vitro aged fibroblasts show a decrease in collagen synthesis ability and an increase in collagenase mRNA levels. Human fibroblast proliferation refers to the proliferation of human fibroblasts.

[0041] (Inhibition of MMP-1 production) MMP (matrix metalloproteinase) is an enzyme that specifically degrades extracellular matrix components. It has a zinc ion (Zn) at its active site. ２＋ ) possesses calcium ions (Ca ２＋) is necessary. Fibroblasts, keratinocytes (epidermal keratinocytes), etc. produce this enzyme, and its mRNA expression is enhanced by UV exposure and inflammation. Mainly, MMP-1 acts as a degrading enzyme for type I collagen, MMP-2 and MMP-9 as a degrading enzyme for type IV collagen and gelatin in the basement membrane, and MMP-3 as a degrading enzyme for proteoglycans, etc. Inhibition of MMP-1 production means suppressing the production of MMP-1.

[0042] (SPARC Production) SPARC (Secreted Protein Acid and Rich in Cysteine) is a 43 kDa matrix cellular protein that has various functions in cell adhesion, differentiation, angiogenesis, and cell-cell matrix interactions. In the skin, it has been reported that the diameter of collagen fiber bundles is reduced and mechanical strength is decreased in SPARC-deficient mice. Furthermore, in vitro studies have reported that it directly binds to collagen and regulates collagen fiber formation (Japanese Patent Publication No. 2014-118392). SPARC production is the production of SPARC.

[0043] (Type IV Collagen Production) Type IV collagen is a major component of the compact layer of the basement membrane and, unlike fibrous collagens such as type I and type II, is a protein called basement membrane collagen. Type IV collagen production is the production of type IV collagen.

[0044] (Ceramide) Ceramide has a structure in which a fatty acid is bound to sphingosine, a long-chain base. Keratinocytes in the later stages of differentiation (granular layer) synthesize various ceramides, and the diversity of these molecules is due to the diversity of hydroxylation (non-hydroxy, 2-hydroxy, and terminal ω-hydroxy) of the fatty acid and sphingosine base that constitute ceramide. In addition to the diversity of hydroxylation, there is also diversity in the chain length of the fatty acid and sphingosine base. These ceramides are found in living organisms in addition to the skin, in the blood, brain, spinal cord, and nerve tissue, and in plants they are found in pineapple, peach, konjac, wheat, rice, soybean, millet, spinach, mushrooms, etc. (Japanese Patent No. 6959204). In the present invention, ceramide includes, for example, glucosylceramide (a type of sphingoglycolipid in which ceramide is bound to glucose).

[0045] Methods for extracting ceramides from plant materials include, for example, extracting them from plant materials such as seeds of pineapple, peach, konjac, yuzu, grapes, and cherries, wheat, rice, and soybeans using an alcohol-based solvent (e.g., ethanol) (Japanese Patent Publication No. 4108069), and extracting them from plant materials such as wheat, soybeans, corn, and rice bran after hydrolysis with an alkaline ethanol solvent using a mixed solvent of hexane, acetone, and water (e.g., Japanese Patent Publication No. 2002-030093, Japanese Patent Publication No. 2006-232699). Plant-derived ceramides have the advantage of being gentle on the body and less likely to cause irritation when incorporated into cosmetics and supplements because they are plant-derived. However, because they are plant-derived components, their structure is subtly different from ceramides found in human skin.

[0046] Methods for extracting glucosylceramide from plant materials include, for example, extraction by the method described in Japanese Patent Publication No. 2001-097983, or by using commercially available rice-derived glucosylceramide (for example, from Nagara Science Co., Ltd.).

[0047] (Rice and rice-derived extracts) Rice refers to cultivated varieties of the genus Oryza, specifically Oryza sativa (Oryza sativa LINNE), Oryza glaverrima, and their hybrids, among other annual plants of the genus Oryza. The rice used in this invention may be Japonica, Javanica, or Indica varieties, and may be non-glutinous rice, glutinous rice, sake rice, or feed rice. The rice variety used in this invention is not particularly limited and is not limited to any variety that is commonly cultivated as a crop. The rice used in this invention may also be rice bred using hybrid technology. Rice bred using hybrid technology is a first-generation hybrid obtained by crossing two different strains of rice varieties, and generally possesses hybrid vigor (generally resulting in increased yield potential, improved resistance to biological and abiotic stressors, etc.) which is superior to either of the parents. In order to produce ceramide (a ceramide-containing agent) according to the present invention, rice seeds can preferably be used, taking into consideration the ceramide content.

[0048] For obtaining rice-derived extracts, organic solvents such as water, alcohols (e.g., anhydrous ethanol, ethanol, propylene glycol, 1,3-butylene glycol, etc.), ketones such as acetone, and esters such as diethyl ether, dioxane, and ethyl acetate can be used individually or in any combination of two or more. The solvent extracts can also be used in their combined state. Ethanol is preferably used. The amount of solvent used is 1 to 100 parts by weight, preferably 10 to 100 parts by weight, per 1 part by weight of the plant material for extraction.

[0049] Alternatively, alkaline organic solvents can be used by adding potassium hydroxide, sodium hydroxide, etc., to these alcohol-based solvents to make them alkaline. The lower limit of the alkali concentration is preferably 0.01 mol / l, and more preferably 0.1 mol / l. The upper limit of the alkali concentration is preferably 1 mol / l, more preferably 0.5 mol / l, and even more preferably 0.4 mol / l.

[0050] There are no particular restrictions on the extraction method, but generally, any method within the boiling point range of the solvent at room temperature and atmospheric pressure is acceptable, and filtration is sufficient after extraction. Any commonly used extraction method may be arbitrarily selected and used.

[0051] The rice-derived extract used in the present invention can be prepared, for example, by the manufacturing method described in Production Example 1 below. The rice-derived extract described in the following examples is the rice-derived extract prepared by Production Example 1 below.

[0052] (Rice Extract Production Example 1) Method for Producing Rice-Derived Extract 881 g of rice Oryza sativa Linne (Gramineae) seeds were extracted by adding 0.2 N potassium hydroxide-ethanol. The extract was purified by silica gel column, neutralized, and desalted to obtain 20 g of concentrated rice extract. 800 g of excipient (dextrin) was added to 200 g of rice extract and dried to obtain 1000 g of a white to pale yellow powder.

[0053] (Recommended intake) The daily intake of the rice-derived extract used in the present invention (when taken as an oral composition) can be appropriately adjusted depending on the form of intake, purpose of use, age, body weight, etc. However, in order for the desired effect (skin whitening, etc.) to be achieved, the daily intake of the rice-derived extract per adult human is preferably 20 mg / day or more, more preferably 40 mg / day or more, and even more preferably 60 mg / day or more, when converted to dry matter. From the viewpoint of safety in humans, etc., when taking such intake, it is preferably 480 mg / day or less, more preferably 360 mg / day or less, and even more preferably 180 mg / day or less.

[0054] (Skin Whitening) Skin whitening refers to the prevention and / or improvement of pigmentation, more specifically, to the prevention and / or improvement of pigmentation symptoms caused by increased melanin production, excessive accumulation, and abnormal deposition, such as age spots, dullness, freckles, sunburn, and darkening due to skin inflammation or irritation, as well as pigmentation symptoms caused by diseases that result in pigmentation, such as skin melanosis caused by drugs such as steroids.

[0055] (Forms of use of the agent, e.g., oral compositions) Forms of use of the present invention (e.g., oral compositions containing the present invention) include, for example, food and beverages (including functional foods, foods for specified health uses, supplements, etc.), pharmaceuticals, etc.

[0056] For example, if the oral composition is a food or beverage, the form of the food or beverage may include various foods and beverages such as bread, cakes, noodles, confectionery, jellies, frozen foods, ice cream, dairy products, and beverages, as well as forms similar to those of the oral administration preparations described above (tablets, capsules, syrups, etc.). Foods in various forms can be prepared by using the active ingredient of the present invention alone, or by combining it with other food ingredients, solvents, softeners, oils, emulsifiers, preservatives, flavorings, stabilizers, colorants, antioxidants, humectants, thickeners, etc., as appropriate.

[0057] For example, if the oral composition is a pharmaceutical, it is generally easy to assemble a convenient daily dosing regimen that can be adjusted according to the degree of discomfort, but the form of the pharmaceutical may be, for example, a solid or a liquid. Examples of the solid form include powders, tablets, pills, capsules, cachets, lozenges, suppositories, and dispersible granules. For example, in the case of a powder, the carrier is generally a finely ground solid that is a mixture with the finely ground active ingredient. For example, in the case of a tablet, the active ingredient is generally mixed in an appropriate proportion with a carrier having the necessary binding ability and molded into the desired shape and size. Suitable carriers may, but are not limited to, magnesium carbonate, magnesium stearate, talc, sugars, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, low-melting-point waxes, cocoa butter, etc. The pharmaceutical product may also contain, as necessary, excipients, stabilizers, preservatives, binders, disintegrants, hydrocarbons, fatty acids, alcohols, esters, pH adjusters, antiseptics, etc., to the extent that it does not impair the desired effect.

[0058] (Forms of use of the agent, e.g., topical skin preparations) The forms of use of the present invention (for example, topical skin preparations containing the present invention) include 1) pharmaceuticals, 2) quasi-drugs, and 3) topical or systemic skin preparations in forms suitable for use, such as ampoules, capsules, powders, granules, liquids, gels, bubbles, emulsions, sheets, mists, sprays, etc. (e.g., basic cosmetics such as lotions, emulsions, creams, ointments, oils, and packs; facial cleansers and skin cleansers such as solid soaps, liquid soaps, and hand washes; massage agents, cleansing agents, depilatory agents, hair removal agents, shaving agents, aftershave lotions, preshave lotions, shaving creams, foundations, lipsticks, blushes, and eyeshadows) (c) Makeup cosmetics such as eyeliner and mascara, perfumes, nail care products, nail enamel, nail enamel removers, poultices, plasters, tapes, sheets, adhesive patches, aerosols, etc.) (4) Medicinal and / or cosmetic preparations applied to the scalp and hair (for example, shampoos, conditioners, hair treatments, pre-hair treatments, permanent solutions, hair dyes, hair styling products, hair tonics, hair growth and nourishing products, poultices, plasters, tapes, sheets, aerosols, etc.) (5) Bath additives used by adding them to bathwater (6) Other products include deodorants and antiperspirants, antiperspirants, hygiene products, sanitary cotton, wet wipes, etc. Furthermore, such agents can be manufactured by arbitrarily selecting and using certain additives (various oils and fats, waxes, mineral oils, fatty acids, alcohols, polyhydric alcohols, esters, metal soaps, gums, sugars, water-soluble polymer compounds, surfactants, various vitamins, various amino acids, additives, etc.) as needed, within the limits that do not impair the effects of the present invention.

[0059] The present invention will now be described in more detail with reference to examples, but the present invention is not limited in any way to these examples. In the following examples, the unit % indicating the amount of each component added means mass %.

[0060] [Example 1] Preparation of Gotu Kola Extract 6 g of dried and pulverized Gotu Kola leaves, stems, or whole plant were each added to 75 mL of 30-90% ethanol, and extraction was carried out at room temperature for 7 days with occasional stirring. The resulting extract was filtered and used as a test sample. Alariadiol in these test samples was analyzed under the following conditions.

[0061] Column: Mightysil RP-18 GP 250-4.6 (5 μm) (manufactured by Kanto Chemical Co., Ltd.) Eluten: CH ３ CN: 0.1% phosphoric acid = 52:48; Flow rate: 1.0 mL / min; Analysis temperature: 40°C; Detection: UV 210 nm

[0062] The alariadiol content in gotu kola extract varied depending on the origin and part of the gotu kola plant, but the 50% ethanol extract showed the highest recovery rate. In addition, other components such as phenylpropanoids, flavonoids, and triterpenoids were also extracted simultaneously from the 50% ethanol extract of gotu kola. Under the above HPLC analysis conditions, it was found that alariadiol was eluted at a retention time of approximately 24 minutes. In the following experiment, samples of gotu kola extract purified and stored under various conditions were analyzed by HPLC under the same conditions as above, and the alariadiol concentration in the samples was quantified by comparing it with the peak area of ​​the standard.

[0063] [Example 2] Purification of Gotu Kola Extract (Preparation of Alariadiol Concentrate) 1500 g of 50% ethanol was added to 100 g of whole Gotu Kola plants, and the extract was extracted at room temperature for 7 days. The extract was then filtered to obtain the extract. This extract was placed on a column packed with approximately 500 mL of aromatic synthetic adsorbent (e.g., Diaion HP20), and other components were eluted with approximately 1500 mL of 50% ethanol. Alariadiol was then eluted with 55% ethanol. The fraction containing alariadiol was concentrated to obtain 100 mL of concentrate. This concentrate contained approximately 750 μg / mL of alariadiol. The ethanol and water in this concentrate were evaporated to dryness in a 50°C water bath, and the concentrate was dissolved again in ethanol. The alariadiol peak was measured by HPLC, and the concentration of alariadiol was found to be 296.2 μg / mL.

[0064] [Test Example 1] Measurement of Storage Stability of Gotu Kola Extract The Gotu Kola extract obtained in Example 1 was further concentrated and dried into a powder, which was then placed in a resealable bag, heat-sealed, and stored. Samples (the sealed powder) stored for one month and three months at -20°C and 40°C were dissolved in ethanol and analyzed by HPLC under the same conditions as in Example 1. The amount of alariadiol remaining in the samples one month and three months after the start of storage is shown in Table 1 below, with the amount of alariadiol in the sample immediately after the start of storage (day 0) set to 100. In Table 1, "-20°C" indicates the group in which the sample was stored at -20°C, and "40°C" indicates the group in which the sample was stored at 40°C.

[0065] As shown in Table 1, the amount of alariadiol in the sample after 3 months of storage at 40°C decreased to 79.3. It is thought that the decomposition of alariadiol is accelerated at higher storage temperatures.

[0066] [Test Example 2] Accelerated Degradation Test of Alariadiol Concentrate, Part 1 An accelerated degradation test was conducted at 40°C using the following compositions (Powder 1, Powder 2). The test results are shown in Table 2 below.

[0067] - Powder 1: Powder 1 obtained by mixing 0.1% by mass of the alaria diol concentrate obtained in Example 2 with 99.9% by mass of highly branched chain dextrin (product name: Cluster Dextrin, manufactured by Nippon Shokuhin Kako Co., Ltd.). - Powder 2: Powder 2 obtained by mixing 0.1% by mass of the alaria diol concentrate obtained in Example 2 with 99.9% by mass of starch hydrolysate (Pinex #100 (DE 3.8), manufactured by Matsutani Chemical Industry Co., Ltd.).

[0068] In Table 2, the amount of alariadiol in the sample immediately after the start of storage (day 0) was set to 100. As shown in Table 2 (values ​​of 17.2 and 19.3 after one month from the start of storage), simply adding common excipients using conventional technology, such as highly branched cyclic dextrin or starch hydrolysates, was not enough to solve the problem of improving the storage stability of alariadiol.

[0069] [Test Example 3] Accelerated Degradation Test of Alariadiol Purified Product, Part 2 The following Table 3 shows the results of an accelerated degradation test at 60°C using the following compositions (Composition 1, Composition 2, Composition 3). In Table 3, the amount of alariadiol in the sample immediately after the start of storage (day 0) was set to 100 (100%).

[0070] • Composition 1: A mixture of the alaria diol concentrate obtained in Example 2 (0.1% by mass) and Dexypearl SD-20 (99.9% by mass). This Dexypearl SD-20 (manufactured by Shiozuiko Sugar Refining Co., Ltd.) is a product containing cyclodextrin and dextrin. • Composition 2: A mixture of the alaria diol concentrate obtained in Example 2 (0.1% by mass) and cyclodextrin-β (99.9% by mass). The cyclodextrin-β is "product name Celldex B-100, manufactured by Nippon Shokuhin Kako Co., Ltd." • Composition 3: A mixture of the alaria diol concentrate obtained in Example 2 (0.1% by mass) and olive oil (99.9% by mass). The olive oil is olive oil manufactured by Kanto Chemical Co., Ltd.

[0071] This 60°C accelerated degradation test is based on the Arrhenius equation and assumes the following (Shigeyuki Sakagami, Akito Kawase: Storage Stability Testing of Pharmaceuticals, SCAS NEWS, 2000-1, pp. 7-11): • 14 days from the start of storage: In the 40°C accelerated degradation test, it is assumed to be 2 years from the start of storage.

[0072]

[0073] As shown in Table 3, when a mixture of cyclodextrin and dextrin was used as an excipient, the amount of alariadiol remaining was 90% or more in the group that had been stored for 14 days compared to the group that had been stored for 0 days (90% or more after 2 years at 40°C).

[0074] [Example 3] Preparation of Formulation 1 A powder formulation was prepared using the alariadiol concentrate obtained in Example 2 and various excipients. First, predetermined amounts of the aqueous components of polyglycerin fatty acid ester, sucrose fatty acid ester, cyclodextrin, gum arabic, and dextrin shown in Table 4 were mixed and dissolved in purified water heated to 85°C. Next, predetermined amounts of the oily components of alariadiol concentrate, rice oil, and tocopherol shown in Table 4 were mixed and dissolved by heating to 50°C. The aqueous component mixture and the oily component mixture prepared in this way were mixed and diluted with water to approximately 100 mL, and then homogenized using a precision emulsification disperser, Creamix (manufactured by M-Technique Co., Ltd.). The supernatant obtained by centrifugation at 13,000 rpm for 5 minutes was concentrated until water was removed, and then freeze-dried using a freeze-dryer (FDU-2100, Tokyo Rikakikai Co., Ltd.). The following day, the obtained freeze-dried product was pulverized with a lab mill and stored in a resealable bag under predetermined conditions.

[0075]

[0076] [Example 4] Preparation of Formulation 2 Formulation 2 was prepared in the same manner as in Example 3, except that ghatti gum was used instead of gum arabic, and the composition shown in Table 5 below was prepared.

[0077]

[0078] [Example 5] Preparation of Formulation 3 Formulation 3 was prepared in the same manner as in Example 3, with the composition shown in Table 6 below.

[0079]

[0080] The product names and manufacturers of the various excipient components used in Examples 3 to 5 are shown in Table 7 below.

[0081]

[0082] [Test Example 4] The results of the accelerated degradation test conducted using formulations 1 to 3 manufactured in Examples 3 to 5 in the same manner as in Test Example 3 are shown in Table 8 below.

[0083] This 60°C accelerated degradation test is based on the Arrhenius equation and assumes the following (Shigeyuki Sakagami, Akito Kawase: Storage Stability Test of Pharmaceuticals, SCAS NEWS 2000-1 pp. 7-11): The start of storage was set to day 0 (indicated as day 0 in Table 8). ・7 days from the start of storage: In the 40°C accelerated degradation test, it is assumed that storage has been in storage for 1 year. Indicated as day 7 in Table 8. ・14 days from the start of storage: In the 40°C accelerated degradation test, it is assumed that storage has been in storage for 2 years. Indicated as day 14 in Table 8.

[0084] In Table 8, the amount of alariadiol in the sample immediately after the start of storage (day 0) was set to 100. In Table 8, the group using Formulation 1 as the sample was designated "Formulation 1," the group using Formulation 2 as the sample was designated "Formulation 2," and the group using Formulation 3 as the sample was designated "Formulation 3."

[0085]

[0086] As shown in Table 8, in all groups, more than 90% of the alariadiol remained on day 14 compared to day 0.

[0087] [Test Example 5] Elastin and Type I Collagen Gene Expression (Detection by qRT-PCR) The expression of the said genes was confirmed as follows.

[0088] (Materials used in the test) ・Normal human dermal fibroblasts: Neonatal dermal fibroblasts (KURABO, KF-4009) ・Culture medium: DMEM medium containing 5% FBS (low glucose, Fujifilm Wako Pure Chemical Industries, Ltd.) ・Test medium: DMEM medium containing 0.25% FBS (low glucose, Fujifilm Pure Chemical Industries, Ltd.) ・Formulation 3: Alariadiol-containing formulation. Composition shown in Table 6. ・Proteoglycan: Proteoglycan, derived from salmon nasal cartilage (Fujifilm Wako Pure Chemical Industries, Ltd., (product codes: 162-22131, 168-22133)) ・Primer information used to confirm gene expression described below is shown in Table 9.

[0089]

[0090] (Gene expression confirmation test) Normal human dermal fibroblasts 2 x 10 ５ cells / mL~5×10 ５seeded in a 75 cm flask so that the cell density was cells / mL. After seeding, the cells were cultured in a culture medium at 37 °C and 5% CO ２ until they reached 80% confluence. After the culture, the cells were harvested using TrypLE Select (manufactured by Gibco). The harvested cells were seeded in a 24-well cell culture plate so that the cell density was 2 × 10 ２ cells / well. After this seeding, the cells were cultured in a culture medium at 37 °C and 5% CO ４ until they reached 100% confluence. After the culture, the culture medium was replaced with a test medium, and the cells were further cultured at 37 °C and 5% CO ２ for 24 hours. ２ After the 24-hour culture, the medium was replaced with the following media for each group: - Control group: culture medium - Sample 1 group: culture medium containing proteoglycan at a final concentration of 100 μg / mL and formulation 3 at a final concentration of 8 μg / mL. - Sample 2 group: culture medium containing proteoglycan at a final concentration of 100 μg / mL and formulation 3 at a final concentration of 40 μg / mL.

[0091] After replacing the medium for each group, the cells were cultured at 37 °C and 5% CO

[0092] [[ID=十六]] ２ ​The cells were cultured for 24 hours under the following conditions. After the culture, the culture supernatant was removed, and the lysate of the cells after the 24-hour culture was collected according to the RNeasyMiniKit (QIAGEN) protocol, and mRNA was purified. The purified mRNA was subjected to a reverse transcription reaction according to the Prime Script® RT Master Mix (TaKaRaBio) protocol, and the reaction product was subjected to an RT-PCR reaction using TB Green Premix EX Taq II (TaKaRaBio). RT-PCR was performed using the LightCycle® 96 system (Roche), and the expression level of the target gene was quantified using the accompanying software. This measurement was performed on three samples each for each group (Sample 1 group and Sample 2 group). Table 10 below shows the relative values ​​when the average value of the sample 1 group is set to 1, using the average values ​​of three samples calculated for each group. As shown in Table 10, the addition of alariadiol and proteoglycan was confirmed to improve collagen and elastin expression.

[0093]

[0094] [Example 6] Preparation of Gotu Kola Extract 2 (Preparation of a solution containing 41.21 μg / mL of alariadiol) 10 g of whole Gotu Kola plant was mixed with 150 g of ethanol and extracted at room temperature for 7 days. The mixture was filtered to obtain 135 g of extract. 90 g of water was added to this extract to obtain 225 g of a 60% ethanol solution. This solution was placed on a column packed with approximately 100 g of aromatic synthetic adsorbent (e.g., Diaion HP20) and eluted with approximately 1000 g of 60% ethanol. The fraction containing alariadiol was concentrated to dryness, and the dry fraction was dissolved in 27 g of 50% 1,3-butylene glycol. The supernatant obtained by centrifugation was designated as Gotu Kola Extract 2. The alariadiol peak was measured in Gotu Kola Extract 2 by HPLC, and the concentration of alariadiol was found to be 41.21 μg / mL.

[0095] [Test Example 6]: In vitro test: Using normal human dermal fibroblasts, the following effects were confirmed by the addition of alariadiol, etc.: • Presence or absence of fibroblast proliferation • Presence or absence of MMP-1 production inhibition • Presence or absence of SPARC production effect • Presence or absence of type IV collagen production effect

[0096] (Preparation of samples used in the test) The following were prepared: - Horse serum: Horse Serum, New Zealand origin (Thermofisher Scientific) - DMEM medium (low glucose): Fujifilm Wako Pure Chemical Corporation - Normal human dermal fibroblasts: Neonatal-derived dermal fibroblasts (KURABO, KF-4009)

[0097] (Test method, human fibroblast proliferation effect) Using DMEM medium containing 20% ​​horse serum, normal human dermal fibroblasts were seeded into 96-well plates at a cell count of 8000 cells / well. After seeding, the cells were incubated at 37°C and 5% CO2. ２ The cells were cultured for 24 hours under these conditions. After the 24-hour culture, the culture medium was replaced with DMEM medium containing 2% horse serum. After the medium replacement, the cells were incubated at 37°C and 5% CO2. ２ The cultures were incubated for 24 hours under the following conditions. After incubation, the culture medium was replaced with a fresh DMEM medium containing 2% horse serum, and Gotu Kola extract 2 or Alariadiol was added to the replaced medium to the concentrations shown in Table 11. The "Control group" in Table 11 is the group to which neither Gotu Kola extract 2 nor Alariadiol was added. After the addition, the cultures were incubated at 37°C and 5% CO2. ２ The cells were cultured for 48 hours under the specified conditions. After 48 hours, the number of viable normal human dermal fibroblasts in the culture medium was measured using Cell Counting Kit-8 (manufactured by DOJINDO).

[0098] (Measurement Results, Human Fibroblast Proliferation Effect) The measurement results are shown in Table 11. The values ​​listed in Table 11 are relative values ​​with the measured value of the "Control group" set to 100. "*" indicates data in which a significant difference (p < 0.001) was observed compared to the control group (value set to 100) in the Dunnett test. The addition of alariadiol, etc., showed a tendency towards human fibroblast proliferation.

[0099]

[0100] (Test method, MMP-1 production inhibitory effect) Normal human dermal fibroblasts were seeded in a 96-well plate at a cell count of 8000 cells / well using DMEM medium containing 20% ​​horse serum. After seeding, the plate was heated at 37°C and 5% CO2. ２ The cells were cultured for 24 hours under these conditions. After the 24-hour culture, the culture medium was replaced with DMEM medium containing 2% horse serum. After the medium replacement, the cells were incubated at 37°C and 5% CO2. ２ The cultures were incubated for 24 hours under the following conditions. After incubation, the culture medium was replaced with a fresh DMEM medium containing 2% horse serum, and Gotu Kola extract 2 or alariadiol was added to the replaced medium to the concentrations shown in Table 12. The "Control group" in Table 12 is the group to which neither Gotu Kola extract 2 nor alariadiol was added. After the addition, the cultures were incubated at 37°C and 5% CO2. ２ The cells were cultured for 48 hours under the specified conditions. After 48 hours, the concentration of MMP-1 in the culture medium was confirmed using the HUMAN MMP1 ELISA KIT (AB215083) (manufactured by ABCAM). The amount of MMP-1 produced was corrected using the number of viable cells obtained with CellCountingKit-8 (manufactured by DOJINDO).

[0101] (Measurement Results, MMP-1 Production Inhibitory Effect) The measurement results are shown in Table 12. The values ​​listed in Table 12 are relative values ​​with the measured value of the "Control group" set to 100. "**" indicates data in which a significant difference (p < 0.01) was observed compared to the control group (value set to 100) in the Dunnett test. The addition of alariadiol and other substances showed a tendency for human fibroblasts to proliferate.

[0102]

[0103] (Test method, SPARC production effect) Normal human dermal fibroblasts were seeded in a 24-well plate at a cell count of 40,000 cells / well using DMEM medium containing 20% ​​horse serum. After seeding, the cells were incubated at 37°C and 5% CO2. ２ The cells were cultured for 24 hours under these conditions. After the 24-hour culture, the culture medium was replaced with DMEM medium containing 2% horse serum. After the medium replacement, the cells were incubated at 37°C and 5% CO2. ２The cultures were incubated for 24 hours under the following conditions. After incubation, the culture medium was replaced with fresh DMEM medium containing 2% horse serum, and Gotu Kola extract 2 was added to the replaced medium to the concentrations shown in Table 13. The "Control group" in Table 13 is the group to which Gotu Kola extract 2 was not added. After the addition, the cultures were incubated at 37°C and 5% CO2. ２ The cells were cultured for 48 hours under the specified conditions. After 48 hours, the concentration of Secreted Protein Acid and Rich in Cysteine ​​(SPARC) was confirmed using the Human SPARC ELISA Kit (ab220654, Abcam). Furthermore, a cell suspension was prepared by homogenizing the cells with PassiveLysisBuffer (Promega) containing protease and phosphatase inhibitors after the culture. The amount of protein in the cell suspension was determined using the Bradford method, and the amount of SPARC produced was corrected.

[0104] (Measurement Results, SPARC Production Effect) The measurement results are shown in Table 13. The values ​​listed in Table 13 are relative values ​​with the measured values ​​of the "Control group" set to 100. "**" indicates data in which a significant difference (p < 0.01) was observed compared to the control group (value set to 100) in the Dunnett test. The addition of Gotu Kola Extract 2 tended to promote SPARC production.

[0105]

[0106] (Test method, type IV collagen production effect) Using DMEM medium containing 20% ​​horse serum, normal human dermal fibroblasts were seeded into 24-well plates at a cell count of 40,000 cells / well. After seeding, the plates were heated at 37°C and 5% CO2. ２ The cells were cultured for 24 hours under the following conditions. After the 24-hour culture, the culture medium was replaced with DMEM medium containing 2% horse serum. After the medium replacement, the cells were cultured at 37°C and 5% CO2. ２ The cultures were incubated for 24 hours under the following conditions. After incubation, the culture medium was replaced with fresh DMEM medium containing 2% horse serum, and Gotu Kola extract 2 was added to the replaced medium to the concentrations shown in Table 14. The "Control group" in Table 14 is the group to which Gotu Kola extract 2 was not added. After the addition, the cultures were incubated at 37°C and 5% CO2.２ Cells were cultured for 48 hours under the specified conditions. After 48 hours, the concentration of Type IV collagen in the culture medium was evaluated using Human Collagen Type IV (COL IV) ELISA Kit (CSB-E17116h, CUSABIO). Furthermore, a cell suspension was prepared by homogenizing the cultured cells with Passive Lysis Buffer (Promega) containing protease and phosphatase inhibitors. The amount of protein in the cell suspension was determined using the Bradford method, and the amount of Type IV collagen produced was corrected.

[0107] (Measurement Results, Type IV Collagen Production Effect) The measurement results are shown in Table 14. The values ​​listed in Table 14 are relative values ​​with the measured values ​​for the "Control group" set to 100. The addition of Gotu Kola Extract 2 tended to increase the production of type IV collagen.

[0108]

[0109] [Test Example 7: Confirmation of the components of the agent used in Test Example 8 (containing rice-derived extract)] The content of a predetermined glucosylceramide in the agent containing the rice-derived extract prepared by the above-described Rice Extract Production Example 1 was measured. The composition of the agent (powder) is 20% of the rice-derived extract and 80% of dextrin.

[0110] Approximately 50 mg of the agent was accurately weighed, dissolved in 3 mL of water, and then 17 mL of methanol was gradually added while stirring. Chloroform was added to this solution to make exactly 50 mL, and the solution was filtered through a membrane filter (pore size 0.45 μm) to obtain the filtrate, which was used as the test solution. Separately, 6 mg of rice-derived glucosylceramide (manufactured by Nagara Science Co., Ltd.) was accurately weighed, dissolved in a chloroform / methanol mixture (2:1) to make exactly 50 mL, and used as the standard solution. 3 μL of the test solution and 1, 2, 3, 4, and 5 μL of the standard solutions were measured, and liquid chromatography was performed under the following operating conditions. Next, the peak area of ​​glucosylceramide in each standard solution was measured, and a calibration curve was created. The concentration of glucosylceramide in the test solution was determined from the calibration curve, and the glucosylceramide content in the product was measured.

[0111] Measurements taken under the following conditions confirmed that the rice extract production example 1 contained 10.0% or more glucosylceramide.

[0112] Operating conditions: Detector: Light scattering detector Column packing material: 2.2 μm silica gel for liquid chromatography Column tube: Stainless steel tube with an inner diameter of 3.0 mm and a length of 75 mm Column temperature: Constant temperature around 35°C Mobile phase 0 min → 5 min Chloroform / 95 vol% methanol solution mixture (99:1) → (75:25) 5 min → 6.5 min Chloroform / 95 vol% methanol solution mixture (75:25) → (10:90) 6.5 min → 8 min Chloroform / 95 vol% methanol solution mixture (10:90) → (99:1) Flow rate: 0.8 mL / min

[0113] [Test Example 8] Melanin Production Inhibition Test A melanin production inhibition test was performed using B16 mouse melanoma cells (melanoma cells) as follows.

[0114] (Materials used in the test) ・B16 mouse melanoma cells (melanoma cells): B16 melanoma 4A5, RIKEN CELLBANK ・Pre-culture medium for the cells: MEM medium containing 5% fetal bovine serum ・MEM medium: E-MEM (051-07615), Fujifilm Wako Pure Chemical Corporation ・Fetal bovine serum: Thermofisher Scientific ・Main culture medium: MEM medium containing 0.036% deforin ・Theophylline (1,3-dimethylxanthine): Tokyo Chemical Industries Ltd., CAS RN: 58-55-9, Product code: T0179 ・Formulation 3: Contains alariadiol, composition shown in Table 6. ・Rice extract production example 1: Contains 10.0% or more glucosylceramide. DMSO: Dimethyl sulfoxide, Fujifilm Wako Pure Chemical Corporation.

[0115] (Melanin Production Inhibition Test) By adding the sample to designated cells (the melanoma cells mentioned above), it was confirmed whether there was a change in melanin production in the designated cells. B16 mouse melanoma cells were used as the cells to which the sample was added.

[0116] (1) Pre-culture step of melanoma cells The above-mentioned melanoma cells are cultured using the above-mentioned pre-culture medium (MEM medium containing 5% fetal bovine serum) and 5% CO2 ２ The cells were incubated at 37°C for 24 hours.

[0117] (2) Main culture step with added sample After pre-culture in (1), 8.0 × 10 ４ Melanoma cells were seeded at a cell-per-well rate into each well (wells containing the culture medium, 24-well plate). After seeding, 5% CO2 was added. ２ The cells were incubated at 37°C for 24 hours.

[0118] After this culture, the culture medium in each well was replaced with fresh culture medium. The following samples were added to the replaced medium (containing the cells): (Samples) - Control group: 0.075% DMSO solution at a final concentration. - Sample 1 (Comparative Example) group: Rice extract preparation example 1 at a final concentration of 10 ppm. - Sample 2 group: Formulation 3 as alariadiol-containing gotu kola extract at a final concentration of 200 ppm. - Sample 3 group: Rice extract preparation example 1 at a final concentration of 10 ppm and Formulation 3 at a final concentration of 200 ppm.

[0119] After the addition, 5% CO ２ The samples were incubated at 37°C for 72 hours (3 days). After this 72-hour incubation period, the following measurements were taken.

[0120] (Melanin content measurement) For this measurement, the cultured samples were harvested by trypsin treatment. The harvested cells were dissolved in a solution (1 mol / l NaOH, containing 10% DMSO). The absorbance of the dissolved solution (control group, and absorbance of samples 1 to 3) was measured at 420 nm. These measured values ​​were defined as the melanin content.

[0121] The measurement results are shown as "melanin production amount (average value of measurement results for n=4 in each group)" and "production rate (for the average value, the value for each group is a relative value compared to the control group, with the control group value set to 100)." • Control group: Melanin production amount 0.483, melanin production rate 100. • Sample 1 (Comparative Example) group: Melanin production amount 0.361, melanin production rate 74.75 (*). • Sample 2 group: Melanin production amount 0.306, melanin production rate 63.31 (*). • Sample 3 group: Melanin production amount 0.251, melanin production rate 51.84 (*). * indicates a significant difference (p < 0.01) when compared with the melanin production rate of the control group (value set to 100) in the Student t-test. In particular, suppression of melanin production was confirmed in the groups from Sample 2 to Sample 3.

[0122] [Test Example 9] Human Monitor Study A human monitor study was conducted with a healthy male in his 40s (N=1) as the subject. The test method and other details are described below.

[0123] (Preparation of Compositions) Compositions with the compositions listed in Table 15 (control and Examples 7-9) were prepared. The composition in Test Example 9 is an oral gummy candy composition. Specifically, this composition was prepared by the following steps. The compositions listed in Table 15 are the compositions at the time of preparation. The units % indicated for the content of the components of the composition mean mass %.

[0124] Gelatin was dissolved in water by heating. While heating this dissolved mixture to approximately 80°C, proteoglycan (if added) and sugar were added in that order to prepare a liquid gummy candy composition. The resulting gummy candy composition was filled into plastic molds. These molds were left to stand at 25°C for 30 minutes to 1 hour, and then cooled and solidified at 4°C. The solidified material was used as a sample for the monitoring test.

[0125] The terms listed in Table 15 have the following meanings: • Residual: The amount of water (purified water) included in the composition, added so that the total amount in the composition becomes 100%.

[0126] In the preparation of the composition, the following were used: • Proteoglycan: Proteoglycan F (prepared by extraction from salmon nasal cartilage with an aqueous acetic acid solution, Ichimaru Falcos) • Gotu Kola extract: Alariadiol-containing Gotu Kola extract as described in Formulation 3 above, with the composition shown in Table 6. • Ceramide: Rice extract production example 1 • Sugar: Kururumark granulated sugar, Wellneo sugar • Gelatin: Zerice (gelatin powder), Maruha Nichiro

[0127]

[0128] (Evaluation method for the compositions in Table 15) The following items were measured and sensory evaluated by a subject (N=1).

[0129] (Evaluation: Measurement method for mechanical properties) The mechanical properties were measured as follows, based on the description in the literature (Aichi Industrial Science and Technology General Center News, March 2025 issue, p. 5, Analysis of fracture strength of food using a rheometer). A sample with a height of 18 mm was fixed to the base surface of a rheometer (Sun RHEOMETER CR3000EX-S, manufactured by Sun Science Co., Ltd.), and a cylindrical plunger with a diameter of 20 mm was pressed against the sample from above in the vertical direction. A load of 200 N was applied at a predetermined speed (30 mm / min), and the elasticity (mm) and hardness (N) were measured using the amount of deformation and load until fracture.

[0130] (Method of sensory evaluation) The following evaluation was conducted: - Softness ○: Smooth overall, △: Some parts are not smooth, ×: Not smooth overall - Jiggle when eaten ○: Jiggle, △: Jiggle but with a firm core, ×: Not jiggle and has a firm core

[0131] (Evaluation results and mechanical properties of the compositions in Table 15) The results (elasticity (mm) and hardness (N)) are listed in Table 15.

[0132] (Results of sensory evaluation of the compositions in Table 15) The results (softness and bounciness) are shown in Table 15. Compared to the control, Examples 7 to 9 had the required softness and bounciness. It was also confirmed that the compositions of Examples 7 to 9 could be taken orally.

[0133] Although embodiments of the present invention (including examples) have been described above with reference to the drawings, the specific configuration of the present invention is not limited thereto, and any design changes, etc., that do not depart from the spirit of the present invention are still included.

[0134] This application claims priority based on Japanese Patent Application No. 2025-008558 filed on 21 January 2025, Japanese Patent Application No. 2025-010961 filed on 24 January 2025, and Japanese Patent Application No. 2025-099779 filed on 13 June 2025, and incorporates all of their disclosures herein.

[0135] <Notes> Some or all of the above embodiments and examples may be described as follows, but are not limited to the following. (Note 1) An agent containing alariadiol for use in promoting fibroblast proliferation, inhibiting MMP-1 production, producing SPARC, producing type IV collagen, promoting type I collagen production and / or promoting elastin production. (Note 2) An agent containing gotu kola extract for use in promoting fibroblast proliferation, inhibiting MMP-1 production, producing SPARC, producing type IV collagen, promoting type I collagen production and / or promoting elastin production. (Note 3) The agent according to Note 1 or Note 2, further containing a proteoglycan. (Note 4) The agent according to Note 3, wherein the proteoglycan is a proteoglycan derived from salmon nasal cartilage. (Note 5) The composition according to Note 4, wherein the molecular weight of the peak top of the proteoglycan is 300,000 to 800,000. (Note 6) A method for producing gotu kola extract (alariadiol), comprising the steps of: extracting the gotu kola plant body with a mixed solvent of an organic solvent and water; passing the extract obtained in the extraction step through a column packed with a hydrophobic synthetic adsorbent to adsorb alariadiol onto the hydrophobic synthetic adsorbent; and eluting the components adsorbed on the hydrophobic synthetic adsorbent with a mixed solvent of an organic solvent and water to obtain an eluted fraction containing alariadiol. (Note 7) A method for producing a powder formulation containing alariadiol, comprising the steps of: mixing alariadiol, dextrin and cyclodextrin to obtain a mixture; homogenizing the mixture to prepare an emulsion; and drying the emulsion. (Note 8) The method for producing a powder formulation according to [7], wherein the cyclodextrin contains γ-cyclodextrin. (Note 9) A whitening agent containing alariadiol. (Note 10) A whitening agent containing gotu kola extract. (Note 11) The whitening agent according to Note 9 or Note 10, further comprising ceramide. (Note 12) The whitening agent according to Note 11, wherein the content ratio of alariadiol to ceramide is 0.01 to 0.1, preferably 0.02 to 0.08, and more preferably 0.04 to 0.07, of which ceramide is 1.

[0136] The present invention may be used as a topical skin preparation (such as a cosmetic), a functional food (such as a supplement), and the like.

Claims

1. An agent containing alariadiol for use in promoting fibroblast proliferation, inhibiting MMP-1 production, producing SPARC, producing type IV collagen, promoting type I collagen production, and / or promoting elastin production.

2. An agent containing Gotu Kola extract for use in promoting fibroblast proliferation, inhibiting MMP-1 production, producing SPARC, producing type IV collagen, promoting type I collagen production, and / or promoting elastin production.

3. The agent according to claim 1 or 2, further comprising a proteoglycan.

4. The agent according to claim 3, wherein the proteoglycan is a proteoglycan derived from salmon nasal cartilage.

5. A skin whitening agent containing alariadiol.

6. A skin whitening agent containing gotu kola extract.

7. The whitening agent according to claim 5 or 6, further comprising ceramide.

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