Use of indole-3-propionic acid and indole-3-acetic acid

Abstract

The present invention provides an agent which comprises IPA or IAA and which provides a novel physiological effect. An agent according to the present disclosure comprises a compound represented by general formula (1), a salt of the compound, and / or a conjugate of the compound. The agent can be used for anti-aging, hair growth, whitening, anti-obesity, corneal protection, brain function improvement, liver function improvement, blood flow improvement, gout improvement, as a cosmetic, as food / beverage, etc. [In general formula (1), n is 1 or 2.]

Description

Use of indole-3-propionic acid and indole-3-acetic acid

[0001] This disclosure relates to the use of indole-3-propionic acid and indole-3-acetic acid.

[0002] Indole-3-propionic acid (IPA) and indole-3-acetic acid (IAA) are known as tryptophan metabolites of intestinal bacteria, including lactic acid bacteria. In recent years, IPA has been shown to possess various physiological activities. For example, diverse effects of IPA, such as suppression of liver fibrosis, sepsis, obesity, diabetes-related kidney disease, hypertension, and promotion of intestinal barrier function, have been reported in Non-Patent Documents 1 to 6. On the other hand, while IAA is widely known as a plant hormone, there are few reports on its physiological activity in animals. For example, Non-Patent Document 7 reports that no anti-inflammatory effect on atopic dermatitis was observed.

[0003] Sehgal et al., Nutrients, 13, 3509, 2021Huang et al., Frontiers in Cellular and Infection Microbiology, 12:1015386, 2022Wang et al., Clinical and Translational Medicine, 14:e70053, 2024Geddo et al., International Journal of Molecular Sciences, 25, 3389, 2024Zeng et al., Journal of Advanced Research, 2024, DOI:10.1016 / j.jare.2024.08.018Chen et al., Molecular Medicine Reports, 30:189, 2024Yu et al., The Journal of Allergy and Clinical Immunology, 143, 6, 2109-2119, 2019

[0004] The full extent of the biological activity of IPA and IAA has not yet been grasped, and some aspects were previously unknown.

[0005] One aspect of this disclosure aims to provide an agent, etc., containing IPA or IAA, salts of these compounds, or conjugates of these compounds, that produces a novel physiological effect.

[0006] This disclosure includes the following aspects:

[0007] <1> An anti-aging agent comprising at least one of a compound represented by the following general formula (1), a salt of said compound, and a conjugate of said compound. [In general formula (1), n ​​is either 1 or 2.]

[0008] <2> A hair growth agent comprising at least one of the compound represented by the general formula (1), a salt of the compound, and a conjugate of the compound.

[0009] <3> A skin whitening agent comprising at least one of the compound represented by the general formula (1), a salt of the compound, and a conjugate of the compound.

[0010] <4> An anti-obesity agent comprising at least one of the compound represented by the general formula (1), a salt of the compound, and a conjugate of the compound.

[0011] <5> A corneal protective agent comprising at least one of the compound represented by the general formula (1), a salt of the compound, and a conjugate of the compound.

[0012] <6> A brain function improving agent comprising at least one of the compound represented by the general formula (1), a salt of the compound, and a conjugate of the compound.

[0013] <7> A liver function improving agent comprising at least one of the compound represented by the general formula (1), a salt of the compound, and a conjugate of the compound.

[0014] <8> A blood flow improving agent comprising at least one of the compound represented by the general formula (1), a salt of the compound, and a conjugate of the compound.

[0015] <9> A gout-improving agent comprising at least one of the compound represented by the general formula (1), a salt of the compound, and a conjugate of the compound.

[0016] <10> A cosmetic containing at least one selected from the group consisting of the anti-aging agent described in <1>, the hair growth agent described in <2>, the skin whitening agent described in <3>, the anti-obesity agent described in <4>, the corneal protective agent described in <5>, the brain function improving agent described in <6>, the liver function improving agent described in <7>, the blood flow improving agent described in <8>, and the gout improving agent described in <9>.

[0017] <11> A food or beverage containing at least one selected from the group consisting of the anti-aging agent described in <1>, the hair growth agent described in <2>, the skin whitening agent described in <3>, the anti-obesity agent described in <4>, the corneal protective agent described in <5>, the brain function improving agent described in <6>, the liver function improving agent described in <7>, the blood flow improving agent described in <8>, and the gout improving agent described in <9>.

[0018] According to one aspect of this disclosure, it is possible to provide an agent, etc., that contains IPA or IAA, salts of these compounds, or conjugates of these compounds, which provides a novel physiological effect.

[0019] The following describes in detail some examples of embodiments of this disclosure, but this disclosure is not limited to the embodiments described below, and various modifications may be made within the scope of the claims. Embodiments that combine the technical means described in different embodiments are also included in the technical scope of this disclosure.

[0020] Unless otherwise specified in this specification, "A to B" representing a numerical range means "greater than or equal to A and less than or equal to B". In this specification, indole-3-propionic acid is abbreviated as "IPA", and indole-3-acetic acid is abbreviated as "IAA".

[0021] [1. Agents of the Disclosure] The first aspect of the Disclosure is an anti-aging agent comprising at least one of the following general formula (1), a salt of the compound, and a conjugate of the compound. The second aspect of the Disclosure is a hair growth agent comprising at least one of the following general formula (1), a salt of the compound, and a conjugate of the compound. The third aspect of the Disclosure is a skin whitening agent comprising at least one of the following general formula (1), a salt of the compound, and a conjugate of the compound. The fourth aspect of the Disclosure is an anti-obesity agent comprising at least one of the following general formula (1), a salt of the compound, and a conjugate of the compound. The fifth aspect of the Disclosure is a corneal protective agent comprising at least one of the following general formula (1), a salt of the compound, and a conjugate of the compound. The sixth aspect of the Disclosure is a brain function improving agent comprising at least one of the following general formula (1), a salt of the compound, and a conjugate of the compound. A seventh aspect of this disclosure is a liver function improving agent comprising at least one of a compound represented by the following general formula (1), a salt of the compound, and a conjugate of the compound. A eighth aspect of this disclosure is a blood flow improving agent comprising at least one of a compound represented by the following general formula (1), a salt of the compound, and a conjugate of the compound. A ninth aspect of this disclosure is a gout improving agent comprising at least one of a compound represented by the following general formula (1), a salt of the compound, and a conjugate of the compound.

[0022] [In general formula (1), n ​​is either 1 or 2.]

[0023] These agents relating to one aspect of this disclosure may hereinafter be referred to as "the Agent." Furthermore, the compound represented by general formula (1) may hereinafter be referred to as "the Compound."

[0024] [1-1. The Compound] The compound is either IPA or IAA. The agent may contain either IPA or IAA, or both.

[0025] IPA is a compound represented by the following structural formula (2). The compound represented by structural formula (2) is a compound in which n is 2 in general formula (1).

[0026] Furthermore, IAA is a compound represented by the following structural formula (3). The compound represented by structural formula (3) is a compound wherein n is 1 in general formula (1).

[0027] Salts of the present compound refer to pharmaceutically acceptable salts of the present compound. Pharmaceutically acceptable salt means a salt that is medically free from excessive toxicity, irritation, allergic reaction and the like, and is suitable for use in contact with tissues of humans or other mammals.

[0028] The pharmaceutically acceptable salts of the present compound are well known in the art, and any thereof can be used. Examples of the pharmaceutically acceptable salts of the present compound include inorganic acid salts such as hydrochloride, hydrobromide, nitrate, sulfate, phosphate and the like; organic acid salts such as acetate, oxalate, maleate, fumarate, citrate, benzoate, methanesulfonate and the like; addition salts of lysine, glycine, phenylalanine, aspartic acid, glutamic acid and the like. The present agent may contain only one type of salt of the present compound, or may contain two or more types thereof.

[0029] Conjugates of the present compound include, for example, compounds obtained by adding a methyl group, an N-acetyl group or a water-soluble molecule to the present compound. In the case of IPA, examples include methylated IPA, N-acetylated IPA, or compounds obtained by adding a water-soluble molecule to IPA. Examples of water-soluble molecules capable of forming a conjugate include sulfuric acid, glucuronic acid, glutathione and amino acids. In one embodiment, the water-soluble molecule is sulfuric acid and / or glucuronic acid. In the conjugate, the total number of methyl groups, acetyl groups and / or water-soluble molecules that bind to one molecule of the present compound is not particularly limited. In one embodiment, the number of methyl groups, acetyl groups or water-soluble molecules that bind to one molecule of the present compound is 1. In the conjugate, the position at which the methyl group and / or water-soluble molecule binds to the present compound is not particularly limited.

[0030] When the conjugate of this compound is methylated IPA, for example, it may be a form in which the carboxyl group of IPA is substituted with a methyl ester. Also, when the conjugate of this compound is N-acetylated IPA, for example, it may be a form in which an acetyl group is added to the nitrogen atom of the indole ring of IPA. These may also apply to IAA. This agent may contain only one type of conjugate of this compound, or it may contain two or more types.

[0031] Both IPA and IAA are known compounds. Therefore, a person skilled in the art can easily produce, extract, or purchase at least one of the compounds, salts of the compounds, or conjugates of the compounds to produce this product.

[0032] The lower limit of the total amount of the compound, salts of the compound, and conjugates of the compound contained in this product may be 0.0001% by weight or more, 0.0005% by weight or more, 0.001% by weight or more, 0.005% by weight or more, 0.01% by weight or more, 0.05% by weight or more, 0.1% by weight or more, 0.5% by weight or more, 1% by weight or more, 5% by weight or more, 10% by weight or more, 20% by weight or more, 30% by weight or more, 40% by weight or more, 50% by weight or more, 60% by weight or more, 70% by weight or more, 80% by weight or more, or 90% by weight or more, based on the total weight of the product as 100% by weight. The upper limit of the total amount of the compound, salts of the compound, and conjugates of the compound contained in this product may be 0.0001% by weight or less, 0.0005% by weight or less, 0.001% by weight or less, 0.005% by weight or less, 0.01% by weight or less, 0.05% by weight or less, 0.1% by weight or less, 0.5% by weight or less, 1% by weight or less, 5% by weight or less, 10% by weight or less, 20% by weight or less, 30% by weight or less, 40% by weight or less, 50% by weight or less, 60% by weight or less, 70% by weight or less, 80% by weight or less, 90% by weight or less, or 100% by weight or less, with the total weight of the product being 100% by weight. In one aspect of this disclosure, the product consists of at least one of the compound, salts of the compound, and conjugates of the compound.

[0033] This product can be manufactured according to conventional methods. Methods for incorporating at least one of the compound, a salt of the compound, or a conjugate of the compound into this product are well known to those skilled in the art.

[0034] [1-2. Other Components] This product may contain other components besides the compound, salts of the compound, and conjugates of the compound. These other components may consist of only one type or two or more types. The amount of other components in this product is not particularly limited.

[0035] Other ingredients include astringents, excipients, binders, disintegrants, lubricants, stabilizers, flavoring agents, deodorizing agents, moisture-proofing agents, preservatives, disinfectants, antibacterial agents, fortifiers, thickeners, emulsifiers, antioxidants, sweeteners, acidulants, seasonings, colorants, fragrances, humectants, oily components, fats and oils, waxes, hydrocarbons, fatty acids, UV absorbers, surfactants, cell activators, alcohols, esters, powder components, colorants, aqueous components, water, and skin nutrients. This product may contain only one of the above-mentioned other ingredients, or it may contain a combination of two or more substances.

[0036] [1-3. Effects of the Agent] This compound, its salts, and its conjugates have anti-aging, hair growth, and skin whitening effects.

[0037] Specifically, the inventors have novelly discovered that the compound has hyaluronic acid production promoting activity, epidermal keratinocyte proliferation promoting activity, elastase activity inhibitory activity, transglutaminase-1 (TGM1) mRNA expression promoting activity, serine palmitoyltransferase (SPT) mRNA expression promoting activity, and filaggrin (FLG) mRNA expression promoting activity. These activities are conventionally known to be effective in exerting anti-aging effects. The anti-aging effect of an anti-aging agent according to one aspect of this disclosure is exerted based on at least one of the hyaluronic acid production promoting activity, epidermal keratinocyte proliferation promoting activity, elastase activity inhibitory activity, transglutaminase-1 (TGM1) mRNA expression promoting activity, serine palmitoyltransferase (SPT) mRNA expression promoting activity, and filaggrin (FLG) mRNA expression promoting activity possessed by the compound, its salts, and conjugates.

[0038] Furthermore, the present inventors have novelly discovered that this compound has a testosterone 5-reductase activity inhibitory effect. This effect is conventionally known to be effective in exerting hair growth effects. The hair growth effect of a hair growth agent according to one aspect of this disclosure is exerted based on the testosterone 5-reductase activity inhibitory effect of this compound, its salt, and its conjugate.

[0039] Furthermore, the present inventors have novelly discovered that this compound has melanin production inhibitory activity and tyrosinase activity inhibitory activity. These activities are conventionally known to be effective in exerting a skin whitening effect. The skin whitening effect of a skin whitening agent according to one aspect of this disclosure is exerted based on at least one of the melanin production inhibitory activity and tyrosinase activity inhibitory activity possessed by this compound, a salt of this compound, and a conjugate of this compound.

[0040] Furthermore, the inventors have novelly discovered that this compound has a fat accumulation inhibitory effect and a UCP-1 mRNA expression promoting effect. These effects are conventionally known to be effective in exerting anti-obesity effects. The anti-obesity effect of an anti-obesity agent according to one aspect of this disclosure is exerted based on at least one of the fat accumulation inhibitory effect and the UCP-1 mRNA expression promoting effect of this compound, its salt, and its conjugate.

[0041] Furthermore, the inventors have novelly discovered that this compound has a corneal epithelial cell proliferation-promoting effect and a glutathione production-promoting effect in corneal epithelial cells. These effects are conventionally known to be effective in exerting corneal protective effects. The corneal protective effect of a corneal protective agent according to one aspect of this disclosure is exerted based on at least one of the corneal epithelial cell proliferation-promoting effect and the glutathione production-promoting effect in corneal epithelial cells possessed by this compound, a salt of this compound, and a conjugate of this compound.

[0042] Furthermore, the inventors have novelly discovered that this compound has an astrocyte proliferation-promoting effect and a microglia nitric oxide (NO) production-inhibiting effect. These effects are conventionally known to be effective in improving brain function. The brain function-improving effect of a brain function-improving agent according to one aspect of this disclosure is exerted based on at least one of the astrocyte proliferation-promoting effect and the microglia NO production-inhibiting effect of this compound, its salt, and its conjugate.

[0043] Furthermore, the inventors have novelly discovered that this compound has the effect of promoting ATP production in hepatocytes and promoting glutathione production in hepatocytes. These effects are conventionally known to be effective in improving liver function. The liver function improving effect of a liver function improving agent according to one aspect of this disclosure is exerted based on at least one of the effects of promoting ATP production in hepatocytes and promoting glutathione production in hepatocytes possessed by this compound, a salt of this compound, and a conjugate of this compound.

[0044] Furthermore, the inventors have novelly discovered that this compound has a blood flow promoting effect through the activation of endothelial cell-derived nitric oxide synthase (eNOS). This effect is conventionally known to be effective in exerting a blood flow improving effect. The blood flow improving effect of a blood flow improving agent according to one aspect of this disclosure is exerted based on the blood flow promoting effects of this compound, its salt, and its conjugate.

[0045] Furthermore, the inventors have novelly discovered that this compound has xanthine oxidase activity inhibitory activity. This activity is conventionally known to be effective in exerting gout-improving effects. The gout-improving effect of a gout-improving agent according to one aspect of this disclosure is exerted based on the xanthine oxidase activity inhibitory activity of this compound, its salt, and its conjugate.

[0046] Thus, this compound, its salts, and its conjugates can be suitably used as active ingredients in at least one of the following: anti-aging agents, hair growth agents, skin whitening agents, anti-obesity agents, corneal protective agents, brain function improving agents, liver function improving agents, blood flow improving agents, and gout improving agents.

[0047] Furthermore, this compound, its salts, and its conjugates can be suitably used as active ingredients in at least one agent selected from the group consisting of hyaluronic acid production promoters, epidermal keratinocyte proliferation promoters, elastase activity inhibitors, TGM1 mRNA expression promoters, SPT mRNA expression promoters, FLG mRNA expression promoters, testosterone 5-reductase activity inhibitors, melanin production inhibitors, tyrosinase activity inhibitors, fat accumulation inhibitors, UCP-1 mRNA expression promoters, corneal epithelial cell proliferation promoters, glutathione production promoters for corneal epithelial cells or hepatocytes, astrocyte proliferation promoters, microglia NO production inhibitors, hepatocyte ATP production promoters, blood flow promoters, and xanthine oxidase activity inhibitors.

[0048] In one embodiment of this disclosure, the subject to which the drug is administered is a mammal. In one embodiment of this disclosure, the subject is a human or a non-human mammal. Examples of non-human mammals include mice, rats, hamsters, dogs, cats, cattle, pigs, and monkeys.

[0049] Examples of administration methods for this drug include oral administration, parenteral administration, and topical administration.

[0050] [2. Composition] The agent may also be in the form of a composition. In other words, one aspect of the present disclosure is an anti-aging composition containing the above-mentioned anti-aging agent. One aspect of the present disclosure is a hair growth composition containing the above-mentioned hair growth agent. One aspect of the present disclosure is a skin whitening composition containing the above-mentioned skin whitening agent. One aspect of the present disclosure is an anti-obesity composition containing the above-mentioned anti-obesity agent. One aspect of the present disclosure is a corneal protective composition containing the above-mentioned corneal protective agent. One aspect of the present disclosure is a brain function improving composition containing the above-mentioned brain function improving agent. One aspect of the present disclosure is a liver function improving composition containing the above-mentioned liver function improving agent. One aspect of the present disclosure is a blood flow improving composition containing the above-mentioned blood flow improving agent. One aspect of the present disclosure is a gout improving composition containing the above-mentioned gout improving agent. These compositions relating to one aspect of the present disclosure may hereinafter be referred to as "the composition".

[0051] [2-1. Forms of Composition] As an example, this drug is distributed to manufacturers as a material that provides a specific physiological function (such as an active pharmaceutical ingredient or functional material), and this composition is distributed to consumers as a specific product containing this drug (such as a pharmaceutical product, quasi-drug, food or beverage, or cosmetic). However, the above is merely an example, and in reality, this drug and this composition may be manufactured and distributed in a wide variety of forms.

[0052] The lower limit of the total amount of the agent contained in this composition may be 0.0001% by weight or more, 0.0005% by weight or more, 0.001% by weight or more, 0.005% by weight or more, 0.01% by weight or more, 0.05% by weight or more, 0.1% by weight or more, 0.5% by weight or more, 1% by weight or more, 5% by weight or more, 10% by weight or more, 20% by weight or more, 30% by weight or more, 40% by weight or more, 50% by weight or more, 60% by weight or more, 70% by weight or more, 80% by weight or more, or 90% by weight or more, with the total weight of this composition being 100% by weight. The upper limit of the total amount of the agent contained in this composition may be 0.0001% by weight or less, 0.0005% by weight or less, 0.001% by weight or less, 0.005% by weight or less, 0.01% by weight or less, 0.05% by weight or less, 0.1% by weight or less, 0.5% by weight or less, 1% by weight or less, 5% by weight or less, 10% by weight or less, 20% by weight or less, 30% by weight or less, 40% by weight or less, 50% by weight or less, 60% by weight or less, 70% by weight or less, 80% by weight or less, 90% by weight or less, or 100% by weight or less, with the total weight of this composition being 100% by weight. In one aspect of this disclosure, this composition consists solely of the agent.

[0053] This composition may contain other components in addition to the present agent. Examples of other components are those exemplified in Section [1-2]. The content of other components in this composition is not particularly limited. In other words, the present agent may be in the form of a composition containing such other components.

[0054] Examples of administration routes for this composition include oral administration, parenteral administration, and topical administration. Below, specific forms of this composition are illustrated according to these administration routes. Note that these specific forms may be classified as pharmaceuticals, quasi-drugs, food and beverages, cosmetics, or general merchandise, etc., according to laws, administrative regulations, or customary practices.

[0055] Examples of this composition for topical application include ointments, lotions, emulsions, creams, serums, gels, beauty oils, masks, jellies, lip balms, lipsticks, foundations, bath additives, soaps, body washes, astringents, hair tonics, hair lotions, hair creams, hair liquids, pomades, shampoos, rinses, conditioners, eye drops, and eye washes.

[0056] Examples of parenterally administered compositions include injections, intravenous infusions, and suppositories.

[0057] Examples of this composition for oral administration include: beverages (tea beverages, soft drinks, carbonated drinks, nutritional drinks, fruit drinks, lactic acid drinks, alcoholic beverages, coffee beverages, coffee-flavored soft drinks, etc.); concentrated stocks and powders for preparing these beverages; frozen desserts (ice cream, ice sherbet, and shaved ice, etc.); noodles (soba, udon, vermicelli, gyoza wrappers, shumai wrappers, Chinese noodles, and instant noodles, etc.); confectionery (candy, gum, chocolate, tablets, snacks, biscuits, jelly, jam, cream, baked goods, and bread, etc.); marine products (crab, salmon, clams, tuna, sardines, shrimp, bonito, mackerel, whale, oyster, saury, squid, ark clam, scallop, abalone, sea urchin, salmon roe, and sea snail, etc.); and processed marine and livestock products (fish cake, ham, and sausage, etc.). Examples of quasi-drugs include: dairy products (processed milk and fermented milk, etc.); oils and fats and processed oils and fats (salad oil, tempura oil, margarine, mayonnaise, shortening, whipped cream and dressings, etc.); seasonings (sauces and dips, etc.); retort pouch foods (curry, stew, oyakodon, porridge, rice gruel, chukadon, katsudon, tempuradon, unadon, hayashi rice, oden, mapo tofu, gyudon, meat sauce, egg soup, omurice, gyoza, shumai, hamburgers and meatballs, etc.); prepared foods (salads and pickles, etc.); health, beauty and nutritional supplements in various forms; pharmaceuticals and quasi-drugs (tablets, powders, capsules, granules, extracts, syrups, drinks, lozenges and mouthwash, etc.); oral fresheners for use in the mouth (mouth fresheners and bad breath preventatives, etc.); and toothpaste.

[0058] This composition may also be used as a research reagent. For example, the anti-aging composition according to one aspect of this disclosure may be used to study the function of genes related to aging symptoms such as skin, or to study the mechanism by which aging symptoms are improved.

[0059] These compositions can be manufactured according to conventional methods. Methods for incorporating the agent into these compositions are well known to those skilled in the art.

[0060] [2-2. Function of the Composition] This composition contains the agent comprising at least one of the compound, a salt of the compound, and a conjugate of the compound. As a result, the anti-aging composition according to one aspect of this disclosure exerts an anti-aging effect on the administered subject. The hair growth composition according to one aspect of this disclosure exerts a hair growth effect on the administered subject. The skin whitening composition according to one aspect of this disclosure exerts a skin whitening effect on the administered subject. The anti-obesity composition according to one aspect of this disclosure exerts an anti-obesity effect on the administered subject. The corneal protective composition according to one aspect of this disclosure exerts a corneal protective effect on the administered subject. The brain function improving composition according to one aspect of this disclosure exerts a brain function improving effect on the administered subject. The liver function improving composition according to one aspect of this disclosure exerts a liver function improving effect on the administered subject. The blood flow improving composition according to one aspect of this disclosure exerts a blood flow improving effect on the administered subject. The gout improving composition according to one aspect of this disclosure exerts a gout improving effect on the administered subject. These effects and the subjects are as described in Section [1-3].

[0061] Furthermore, one aspect of this disclosure also includes compositions for promoting hyaluronic acid production, epidermal keratinocyte proliferation, elastase activity inhibition, TGM1 mRNA expression promotion, SPT mRNA expression promotion, FLG mRNA expression promotion, testosterone 5-reductase activity inhibition, melanin production suppression, tyrosinase activity inhibition, fat accumulation suppression, UCP-1 mRNA expression promotion, corneal epithelial cell proliferation promotion, glutathione production promotion by corneal epithelial cells or hepatocytes, astrocyte proliferation promotion, microglia NO production suppression, hepatocyte ATP production promotion, blood flow promotion, and xanthine oxidase activity inhibition.

[0062] [3. Cosmetics] [3-1. Form of Cosmetics] A cosmetic according to one aspect of the present disclosure contains at least one selected from the group consisting of anti-aging agents, hair growth agents, skin whitening agents, anti-obesity agents, corneal protective agents, brain function improving agents, liver function improving agents, blood flow improving agents, and gout improving agents according to one aspect of the present disclosure, and may further contain other ingredients as needed. The cosmetic may hereinafter be referred to as "this cosmetic."

[0063] The lower limit of the amount of this product contained in this cosmetic product may be 0.0001% by weight or more, 0.0005% by weight or more, 0.001% by weight or more, 0.005% by weight or more, 0.01% by weight or more, 0.05% by weight or more, 0.1% by weight or more, 0.5% by weight or more, 1% by weight or more, 5% by weight or more, 10% by weight or more, 20% by weight or more, 30% by weight or more, 40% by weight or more, 50% by weight or more, 60% by weight or more, 70% by weight or more, 80% by weight or more, or 90% by weight or more, based on the total weight of this cosmetic product as 100% by weight. The upper limit of the amount of this product contained in this cosmetic product may be 0.0001% by weight or less, 0.0005% by weight or less, 0.001% by weight or less, 0.005% by weight or less, 0.01% by weight or less, 0.05% by weight or less, 0.1% by weight or less, 0.5% by weight or less, 1% by weight or less, 5% by weight or less, 10% by weight or less, 20% by weight or less, 30% by weight or less, 40% by weight or less, 50% by weight or less, 60% by weight or less, 70% by weight or less, 80% by weight or less, 90% by weight or less, or 100% by weight or less, with the total weight of this cosmetic product being 100% by weight.

[0064] In this cosmetic composition, a higher content of this agent is preferable. Furthermore, this cosmetic composition may be at least one of the following: anti-aging agents, hair growth agents, skin whitening agents, anti-obesity agents, corneal protective agents, brain function improving agents, liver function improving agents, blood flow improving agents, and gout improving agents.

[0065] [3-2. Other Ingredients] This cosmetic composition may further contain other ingredients as necessary, to the extent that they do not impair the purpose and effects of this disclosure. Examples of other ingredients are those exemplified in Section [1-2]. The amount of other ingredients in this cosmetic composition is not particularly limited.

[0066] There are no particular restrictions on the content of other ingredients in this cosmetic composition, as long as they do not impair the effects of this disclosure, and they can be appropriately selected depending on the purpose.

[0067] [3-3. Uses of Cosmetics] There are no particular restrictions on the uses of these cosmetics, and they can be appropriately selected from among general cosmetics. For example, carriers that are acceptable as cosmetics, as exemplified in Section [2-1] when administered externally, can be used.

[0068] This cosmetic composition may be prepared by incorporating this drug into any cosmetic composition in a manner that does not interfere with its activity. Alternatively, this cosmetic composition may be a cosmetic composition with this drug as its main ingredient. Furthermore, this cosmetic composition may be this drug itself.

[0069] The subjects for this cosmetic product are as described in Section [1-3].

[0070] Because this cosmetic product contains this agent, it is useful in that, when applied to the skin, it exhibits at least one of the following effects: excellent anti-aging, hair growth, skin whitening, and corneal protective effects. Furthermore, when the active ingredient of this agent is absorbed into the body transdermally, it may also exhibit at least one of the following effects: anti-obesity, brain function improvement, liver function improvement, blood flow improvement, and gout improvement. Furthermore, this cosmetic composition exerts at least one of the following effects selected from the group consisting of hyaluronic acid production promoting effect, epidermal keratinocyte proliferation promoting effect, elastase activity inhibitory effect, TGM1 mRNA expression promoting effect, SPT mRNA expression promoting effect, FLG mRNA expression promoting effect, testosterone 5-reductase activity inhibitory effect, melanin production suppression effect, tyrosinase activity inhibitory effect, fat accumulation suppression effect, UCP-1 mRNA expression promoting effect, corneal epithelial cell proliferation promoting effect, glutathione production promoting effect of corneal epithelial cells or hepatocytes, astrocyte proliferation promoting effect, microglia NO production suppression effect, hepatocyte ATP production promoting effect, blood flow promoting effect, and xanthine oxidase activity inhibitory effect.

[0071] [4. Food and Beverages] [4-1. Form of Food and Beverages] Food and beverages relating to one aspect of this disclosure contain at least one selected from the group consisting of anti-aging agents, hair growth agents, skin whitening agents, anti-obesity agents, corneal protective agents, brain function improving agents, liver function improving agents, blood flow improving agents, and gout improving agents relating to one aspect of this disclosure, and may further contain other ingredients as necessary. Such food and beverages may be referred to as "the Food and Beverages" below.

[0072] The lower limit of the amount of this drug contained in this food and beverage may be 0.0001% by weight or more, 0.0005% by weight or more, 0.001% by weight or more, 0.005% by weight or more, 0.01% by weight or more, 0.05% by weight or more, 0.1% by weight or more, 0.5% by weight or more, 1% by weight or more, 5% by weight or more, 10% by weight or more, 20% by weight or more, 30% by weight or more, 40% by weight or more, 50% by weight or more, 60% by weight or more, 70% by weight or more, 80% by weight or more, or 90% by weight or more, based on the total weight of this food and beverage as 100% by weight. The upper limit of the amount of this drug contained in this food and beverage may be 0.0001% by weight or less, 0.0005% by weight or less, 0.001% by weight or less, 0.005% by weight or less, 0.01% by weight or less, 0.05% by weight or less, 0.1% by weight or less, 0.5% by weight or less, 1% by weight or less, 5% by weight or less, 10% by weight or less, 20% by weight or less, 30% by weight or less, 40% by weight or less, 50% by weight or less, 60% by weight or less, 70% by weight or less, 80% by weight or less, 90% by weight or less, or 100% by weight or less, based on the total weight of this food and beverage as 100% by weight.

[0073] In this food and beverage, a higher content of this drug is preferable. Furthermore, this food and beverage may be at least one of the following selected from the group consisting of anti-aging agents, hair growth agents, skin whitening agents, anti-obesity agents, corneal protective agents, brain function improving agents, liver function improving agents, blood flow improving agents, and gout improving agents.

[0074] [4-2. Other Ingredients] This food and beverage may further contain other ingredients as necessary, to the extent that they do not impair the purpose and effects of this disclosure. Examples of other ingredients are those exemplified in Section [1-2]. The amount of other ingredients in this food and beverage is not particularly limited.

[0075] There are no particular restrictions on the content of other ingredients in this food and beverage, as long as they do not impair the effects of this disclosure, and they can be appropriately selected depending on the purpose.

[0076] [4-3. Uses of Food and Beverages] There are no particular restrictions on the uses of these food and beverages, and they can be appropriately selected from among general food and beverages. For example, the food and beverages exemplified in Section [2-1] as being administered orally can be used.

[0077] This food or beverage may be a mixture of this drug and other food or beverages in which this drug is incorporated without interfering with its activity. Alternatively, this food or beverage may be a food or beverage with this drug as its main ingredient. Furthermore, this food or beverage may be this drug itself.

[0078] The subjects of this food and beverage test are as described in Section [1-3].

[0079] Because this food and beverage contains this drug, it exerts at least one of the effects selected from the group consisting of anti-aging effects, hair growth effects, skin whitening effects, anti-obesity effects, corneal protective effects, brain function improvement effects, liver function improvement effects, blood flow improvement effects, and gout improvement effects. Furthermore, this food and beverage exerts at least one of the effects selected from the group consisting of hyaluronic acid production promoting effects, epidermal keratinocyte proliferation promoting effects, elastase activity inhibitory effects, TGM1 mRNA expression promoting effects, SPT mRNA expression promoting effects, FLG mRNA expression promoting effects, testosterone 5-reductase activity inhibitory effects, melanin production suppression effects, tyrosinase activity inhibitory effects, fat accumulation suppression effects, UCP-1 mRNA expression promoting effects, corneal epithelial cell proliferation promoting effects, glutathione production promoting effects of corneal epithelial cells or hepatocytes, astrocyte proliferation promoting effects, microglia NO production suppression effects, hepatocyte ATP production promoting effects, blood flow promoting effects, and xanthine oxidase activity inhibitory effects.

[0080] [5. Other Expressions] One aspect of the present disclosure may also be expressed as follows: That is, one aspect of the present disclosure is a method comprising administering an effective amount of at least one of the compound, a salt of the compound, and a conjugate of the compound to a subject, one or more methods selected from the group consisting of a method for inhibiting aging, a method for promoting hair growth, a method for skin whitening, a method for inhibiting obesity, a method for protecting the cornea, a method for improving brain function, a method for improving liver function, a method for improving blood flow, and a method for improving gout.

[0081] Furthermore, one aspect of this disclosure is the use of at least one of the Compound, a salt of the Compound, and a conjugate of the Compound for producing one or more agents selected from the group consisting of anti-aging agents, hair growth agents, skin whitening agents, anti-obesity agents, corneal protective agents, brain function improving agents, liver function improving agents, blood flow improving agents, and gout improving agents.

[0082] One embodiment of this disclosure is described below.

[0083] [1. Anti-aging effect] [1-1. Hyaluronic acid production promotion effect test (epidermal keratinocytes)] <Test method> Normal human neonatal epidermal keratinocytes (NHEK) were cultured in normal human epidermal keratinocyte proliferation medium (KGM) and then harvested by trypsin treatment. The harvested cells were 1 × 10⁻⁶ ５ After diluting with KGM to a concentration of cells / mL, 100 μL was seeded per well in a 96-well plate and cultured for 24 hours. After the culture period, 100 μL of the test sample dissolved in KGM was added to each well and cultured for 7 days. After culturing, the amount of hyaluronic acid in the culture medium of each well was measured using a sandwich method with hyaluronic acid-binding protein (HABP).

[0084] The method for calculating the hyaluronic acid production promotion rate is as follows:

[0085] • Hyaluronic acid production promotion rate (%) = A / B × 100 A: Absorbance at a wavelength of 405 nm when the test sample is added B: Absorbance at a wavelength of 405 nm when the test sample is not added

[0086] <Results>

[0087] IPA was found to have a strong effect in promoting hyaluronic acid production in epidermal keratinocytes.

[0088] [1-2. Hyaluronic Acid Production Promotion Test (Skin Fibroblasts)] <Test Method> Normal human fibroblasts (NB1RGB) were cultured in DMEM containing 10% FBS and then harvested by trypsin treatment. The harvested cells were 1.6 × 10⁶ ５ After diluting the above medium to a concentration of cells / mL, 100 μL was seeded per well into a 96-well microplate and incubated overnight. After incubation, 100 μL of the test sample dissolved in 0.25% FBS-containing DMEM was added to each well and incubated for 3 days. After incubation, the amount of hyaluronic acid in the medium of each well was measured using a sandwich method with hyaluronic acid-binding protein (HABP).

[0089] The calculation method for the hyaluronic acid production promotion rate is as follows: • Hyaluronic acid production promotion rate (%) = A / B × 100 A: Amount of hyaluronic acid when the test sample is added B: Amount of hyaluronic acid when the test sample is not added

[0090] <Results>

[0091] IPA was found to promote hyaluronic acid production in skin fibroblasts.

[0092] [1-3. Test for Promoting Epidermal Keratinocyte Proliferation] <Test Method> Normal human neonatal epidermal keratinocytes (NHEK) were cultured in normal human epidermal keratinocyte proliferation medium (KGM), and then harvested by trypsin treatment. The harvested cells were 3.0 × 10⁻⁶ ４After diluting with KGM to a concentration of cells / mL, 100 μL was seeded per well into a collagen-coated 96-well plate and cultured overnight. After culturing, 100 μL of the test sample dissolved in KGM was added to each well and cultured for 3 days. The epidermal keratinocyte proliferation-promoting effect was measured using the MTT assay method. After culturing, the medium was removed, and 100 μL of MTT dissolved in PBS(-) at a final concentration of 0.4 mg / mL was added to each well. After culturing for 2 hours, the blue formazan produced in the cells was extracted with 100 μL of 2-propanol. After extraction, the absorbance at a wavelength of 570 nm was measured. Simultaneously, the absorbance at a wavelength of 650 nm was measured as turbidity, and the difference between the two was used as the amount of blue formazan produced.

[0093] The method for calculating the rate of promotion of epidermal keratinocyte proliferation is as follows: • Rate of promotion of epidermal keratinocyte proliferation (%) = A / B × 100 A: Amount of blue formazan produced in cells when the test sample is added B: Amount of blue formazan produced in cells without the test sample

[0094] <Results>

[0095] IPA was found to have an effect of promoting the proliferation of epidermal keratinocytes.

[0096] [1-4. Elastase Activity Inhibition Test] <Test Method> In a 96-well plate, 50 μL of the test sample prepared in 0.2 mol / L Tris-HCl buffer (pH 8.0) was mixed with 50 μL of 20 μg / mL elastase type III solution. Then, 100 μL of 0.4514 mg / mL N-SUCCINYL-ALA-ALA-ALA-p-NITROANILIDE prepared in the above buffer was added and the mixture was reacted at 25°C for 15 minutes. After the reaction was complete, the absorbance at a wavelength of 415 nm was measured. A blank test (without enzyme) was performed in the same manner and the measurement results were corrected.

[0097] The calculation method for the elastase activity inhibition rate is as follows: Elastase activity inhibition rate (%) = {1 - (C - D) / (A - B)} × 100 A: Absorbance at 415 nm with no test sample and with enzyme added B: Absorbance at 415 nm with no test sample and without enzyme added C: Absorbance at 415 nm with test sample and with enzyme added D: Absorbance at 415 nm with test sample and without enzyme added

[0098] <Results>

[0099] IPA was found to have an elastase activity inhibitory effect.

[0100] [1-5. TGM1 mRNA Expression Enhancement Test] <Test Method> Normal human neonatal epidermal keratinocytes (NHEKs) were cultured in normal human epidermal keratinocyte growth medium (KGM), and then harvested by trypsin treatment. The harvested cells were divided into 1.5 × 10⁻⁶ cells. ５ After diluting with KGM to a concentration of cells / mL, 2 mL was seeded per well in a 6-well plate and stored at 37°C in 5% CO2. ２ The cultures were incubated overnight under the following conditions. After incubation, the culture medium was replaced with KBM (KBM) without added growth factors. After 24 hours, the culture medium was discarded, and 2 mL of the test sample, dissolved to the required concentration in KBM, was added to each well. The cultures were incubated at 37°C and 5% CO2. ２ The cells were incubated under the following conditions for 24 hours. After incubation, the culture medium was discarded, and total RNA was extracted using ISOGEN II (NIPPON GENE). The amount of RNA was calculated from the absorbance at a wavelength of 260 nm, and total RNA was prepared to 200 ng / μL.

[0101] Using this total RNA as a template, the expression levels of TGM1 and GAPDH mRNA (the internal standard) were measured. Detection was performed using a real-time PCR apparatus Thermal Cycler Dice (Registered trademark of Japan) Real Time System III (TaKaRa) by two-step real-time RT-PCR reaction with PrimeScript (Registered trademark of Japan) RT Master Mix (Perfect Real Time) and TB Green (Registered trademark of Japan) Fast qPCR Mix (TaKaRa). The expression level of TGM1 was calculated after correction with the expression level of GAPDH mRNA.

[0102] The calculation method of TGM1 mRNA expression promotion rate is as follows. ・TGM1 mRNA expression promotion rate (%) = A / B×100 A: Corrected value when test sample is added B: Corrected value when no test sample is added

[0103] <Results>

[0104] IPA was found to have a TGM1 mRNA expression promoting effect.

[0105] [1-6. SPT mRNA expression promotion effect test] <Test method> Normal human neonatal epidermal keratinocytes (NHEK) were cultured using normal human epidermal keratinocyte growth medium (KGM), and then collected by trypsin treatment. The collected cells were 1.5×10 ５ After diluting with KGM to a concentration of cells / mL, 2 mL per well was seeded into a 6-well plate, 37°C, 5% CO ２ Culture was performed overnight under the above conditions. After culturing, the medium was replaced with 1.8 mL of growth factor-free medium (KBM). After 24 hours, 0.2 mL of the test sample dissolved to the required concentration in KBM was added to each well, 37°C, 5% CO ２ Culture was performed for 24 hours under the above conditions. After culturing, the culture medium was discarded, total RNA was extracted with ISOGEN II (NIPPON GENE), the RNA amount was calculated from the absorbance at a wavelength of 260 nm, and total RNA was prepared to a concentration of 200 ng / μL.

[0106] Using this total RNA as a template, the expression levels of SPT and the internal standard GAPDH mRNA were measured. Detection was performed using a two-step real-time RT-PCR reaction with PrimeScript RT Master Mix (Perfect Real Time) and TB Green Fast qPCR Mix (TaKaRa) on a Thermal Cycler Dice Real Time System III (TaKaRa) real-time PCR instrument. SPT expression levels were corrected for GAPDH mRNA expression levels and then calculated.

[0107] The calculation method for the SPT mRNA expression enhancement rate is as follows: • SPT mRNA expression enhancement rate (%) = A / B × 100 A: Corrected value when the test sample is added B: Corrected value when the test sample is not added

[0108] <Results>

[0109] IPA was found to promote SPT mRNA expression.

[0110] [1-7. FLG mRNA Expression Enhancement Test] <Test Method> Normal human neonatal epidermal keratinocytes (NHEKs) were cultured in normal human epidermal keratinocyte growth medium (KGM) and then harvested by trypsin treatment. The harvested cells were divided into 1.5 × 10⁻⁶ cells. ５ After diluting with KGM to a concentration of cells / mL, 2 mL was seeded per well in a 6-well plate and stored at 37°C in 5% CO2. ２ The cultures were incubated overnight under the following conditions. After incubation, the culture medium was replaced with 1.8 mL of KBM (KBM) without growth factors. After 24 hours, 0.2 mL of the test sample, dissolved to the required concentration in KBM, was added to each well, and the cultures were incubated at 37°C and 5% CO2. ２ The cells were incubated under the following conditions for 24 hours. After incubation, the culture medium was discarded, and total RNA was extracted using ISOGEN II (NIPPON GENE). The amount of RNA was calculated from the absorbance at a wavelength of 260 nm, and total RNA was prepared to 200 ng / μL.

[0111] Using this total RNA as a template, the expression levels of FLG and the internal standard GAPDH mRNA were measured. Detection was performed using a two-step real-time RT-PCR reaction with PrimeScript RT Master Mix (Perfect Real Time) and TB Green Fast qPCR Mix (TaKaRa) on a Thermal Cycler Dice Real Time System III (TaKaRa) real-time PCR instrument. FLG expression levels were calculated after correcting for GAPDH mRNA expression levels.

[0112] The calculation method for the FLG mRNA expression enhancement rate is as follows: • FLG mRNA expression enhancement rate (%) = A / B × 100 A: Corrected value when the test sample is added B: Corrected value when the test sample is not added

[0113] <Results>

[0114] IPA was found to promote FLG mRNA expression.

[0115] [1-8. Summary] As described above, this compound has been shown to have the following effects: hyaluronic acid production promoting effect, epidermal keratinocyte proliferation promoting effect, elastase activity inhibitory effect, TGM1 mRNA expression promoting effect, SPT mRNA expression promoting effect, and FLG mRNA expression promoting effect. Each of these effects has been reported as an anti-aging effect. In other words, it has been suggested that this drug and composition containing at least one of this compound, a salt of this compound, or a conjugate of this compound have an anti-aging effect based on these effects.

[0116] [2. Hair growth effect] [2-1. Testosterone 5α-reductase activity inhibition test] <Test method> In a V-bottomed test tube with a lid, 20 μL of 4.2 mg / mL testosterone prepared with propylene glycol and 825 μL of 5 mmol / L Tris-HCl buffer (pH 7.13) containing 1 mg / mL NADPH were mixed. To this, 80 μL of the test sample prepared with ethanol and 75 μL of S-9 (rat liver homogenate, Oriental Yeast Co., Ltd.) were added and mixed again. After reacting at 37°C for 60 minutes, 1 mL of methylene chloride was added to stop the reaction. This was centrifuged (1600 × g, 10 min), and the methylene chloride layer was analyzed by gas chromatography under the following conditions.

[0117] To determine the conversion rate of testosterone to 3α-androstanediol and dihydrotestosterone, ethanol solutions of 3α-androstanediol, dihydrotestosterone, and testosterone standards were first analyzed by gas chromatography to determine the peak areas of these three compounds. The relative ratio of the peak areas of 3α-androstanediol, dihydrotestosterone, and testosterone after the reaction with enzyme solution S-9 to the peak areas of the standards was determined according to Equation 1 below. Subsequently, the conversion rate of the test sample was determined according to Equation 2 below. Based on this conversion rate, the testosterone 5α-reductase activity inhibition rate was determined according to Equation 3 below.

[0118] [Equation 1] Relative ratio = Peak area of ​​test sample / Peak area of ​​standard [Equation 2] Conversion rate (%) = (A + B) / (A + B + C) × 100 A: Relative ratio of 3α-androstanediol B: Relative ratio of dihydrotestosterone C: Relative ratio of testosterone [Equation 3] Testosterone 5α-reductase activity inhibition rate (%) = (1 - E / D) × 100 D: Conversion rate without test sample E: Conversion rate with test sample

[0119] (Gas chromatography conditions) Equipment used: Shimadzu GC-2014 Column: DB-1701 (φ0.53 mm × 30 m, film thickness: 1.0 μm) Column / injection temperature: 240°C / 300°C Detector: FID Carrier gas: Nitrogen gas

[0120] <Results>

[0121] IPA was found to have an inhibitory effect on testosterone 5α-reductase activity.

[0122] [2-2. Summary] As described above, this compound has been shown to have a testosterone 5α-reductase activity inhibitory effect. Testosterone 5α-reductase activity inhibitory effect has been reported to be effective in promoting hair growth. In other words, it is suggested that this drug and composition, which contain at least one of this compound, a salt of this compound, or a conjugate of this compound, have a hair growth effect based on the said testosterone 5α-reductase activity inhibitory effect.

[0123] [3. Whitening effect] [3-1. Test of melanin production inhibitory effect on melanoma cells] <Test method> B16 melanoma cells were cultured in DMEM containing 10% FBS and then harvested by trypsin treatment. The harvested cells were treated with 10% FBS and DMEM containing 1 mmol / L theophylline in a 2.4 × 10⁶ solution. ５ After diluting to a concentration of cells / mL, 300 μL was seeded per well in a 48-well plate and cultured for 6 hours. After the culture period, 300 μL of the test sample dissolved in 10% FBS and 1 mmol / L theophylline-containing DMEM was added to each well and cultured for 4 days. After the culture period, the culture medium was removed from each well, 200 μL of 2 mol / L NaOH solution was added, and the cells were destroyed using an ultrasonic disruptor. The absorbance at a wavelength of 475 nm was measured. From the measured absorbance values, the amount of melanin was calculated based on a calibration curve created using synthetic melanin.

[0124] Furthermore, to measure cell viability, cells were similarly cultured, washed with 400 μL of PBS(-), and 200 μL of neutral red dissolved in 10% FBS-containing DMEM at a final concentration of 0.05 mg / mL was added to each well. After 2.5 hours of incubation, the neutral red solution was discarded, and 200 μL of ethanol-acetic acid solution (ethanol:acetic acid:water = 50:1:49) was added to each well to extract the dye. After extraction, the absorbance at a wavelength of 540 nm was measured. As a blank test, cells cultured in 10% FBS and 1 mmol / L theophylline-containing DMEM alone were tested in the same manner.

[0125] The calculation methods for the melanin production inhibition rate and cell viability rate are as follows: • Melanin production inhibition rate (%) = {1 - (B / D) / (A / C)} × 100 • Cell viability rate (%) = (D / C) × 100 A: Amount of melanin without the test sample B: Amount of melanin with the test sample added C: Absorbance at 540 nm without the test sample added D: Absorbance at 540 nm with the test sample added

[0126] <Results>

[0127] IPA was found to have an inhibitory effect on melanin production.

[0128] [3-2. Tyrosinase Activity Inhibition Test] <Test Method> 0.2 mL of McIlvaine buffer (pH 6.8), 0.06 mL of 0.3 mg / mL tyrosine solution, and 0.18 mL of 25% DMSO solution of the test sample were added to a 48-well plate and allowed to stand at 37°C for 10 minutes. 0.02 mL of 800 units / mL tyrosinase solution was then added and the mixture was reacted at 37°C for 15 minutes. After the reaction was complete, the absorbance at a wavelength of 475 nm was measured. A blank test (without tyrosinase solution) was performed in the same manner and corrected.

[0129] The method for calculating the tyrosinase activity inhibition rate is as follows: • Tyrosinase activity inhibition rate (%) = {1 - (A - B) / (C - D)} × 100 A: Absorbance of the test sample solution at a wavelength of 475 nm B: Absorbance of the test sample solution (blank) at a wavelength of 475 nm C: Absorbance of the solution without the test sample at a wavelength of 475 nm D: Absorbance of the solution without the test sample (blank) at a wavelength of 475 nm

[0130] <Results>

[0131] IPA was found to have tyrosinase activity inhibitory effects.

[0132] [3-3. Summary] As described above, this compound has been shown to have melanin production inhibitory activity and tyrosinase activity inhibitory activity. These activities have been reported to be effective for skin whitening. In other words, it is suggested that this agent and composition, which contain at least one of this compound, a salt of this compound, or a conjugate of this compound, have a skin whitening effect based on these activities.

[0133] [4. Anti-obesity effect] [4-1. Fat accumulation inhibitory effect test using human visceral-derived adipocytes] <Test method> Human visceral-derived preadipocytes were placed in a 75 cm² area. ２ In a flask, using the PGM2 Bullet Kit growth medium, at 37°C and 5% CO2, ２ The cells were pre-cultured under the following conditions and collected by trypsin treatment. Using growth medium, 2.5 × 10⁶ cells were placed on a collagen-coated 96-well plate (IWAKI). ３ Seeds were sown in 200 μL / well increments at 37°C and 5% CO2. ２ The cells were cultured under the following conditions until confluence was reached. After culturing, the culture medium was discarded and replaced with differentiation induction medium at 37°C and 5% CO2. ２ The culture was carried out for 7 days. The culture supernatant was discarded, and 100 μL / well of the test sample, dissolved in the growth medium to the required concentration, was added, and the culture was continued for another 7 days.

[0134] After culturing was complete, the culture medium was discarded, and intracellular triglycerides (TG) were measured using AdipoRed Assay Reagent (LONZA, USA). Subsequently, proteins were extracted using M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific Inc.), and protein quantification was performed using the BCA Protein Assay Reagent Kit (Thermo Fisher Scientific Inc.).

[0135] The results for TG amount, protein amount, and TG amount per protein are shown as percentages relative to the values ​​for the sample without additive (= 0 μmol / L).

[0136] <Results>

[0137] IAA was found to have an inhibitory effect on fat accumulation.

[0138] [4-2. Test on the promotion of UCP-1 mRNA expression in human visceral adipocytes] <Test method> Human visceral adipocytes were divided into 75 cm². ２ In a flask, using the PGM2 Bullet Kit growth medium, at 37°C and 5% CO2, ２ The cells were pre-cultured under the following conditions and collected by trypsin treatment. Using growth medium, 7.5 × 10⁶ cells were placed on a collagen-coated 6-well plate (IWAKI). ４ Sow cells / well, at 37°C and 5% CO2. ２ The cells were cultured under the following conditions until confluence was reached. After culturing, the culture medium was discarded and replaced with differentiation induction medium at 37°C and 5% CO2. ２ The culture was carried out for 13 days. The culture supernatant was discarded, and 2 mL / well of the test sample, dissolved in growth medium to the required concentration, was added, and the culture was continued for another 24 hours.

[0139] After culturing, the culture medium was discarded, and total RNA was extracted using the QuickGene-Mini480 nucleic acid isolation system (Kurabo). The amount of RNA was calculated from the absorbance at a wavelength of 260 nm, and total RNA was prepared to 80 ng / μL. Using this total RNA as a template, the expression levels of UCP-1 and the internal standard GAPDH mRNA were measured. Detection was performed using a two-step real-time RT-PCR reaction with a Thermal Cycler Dice Real Time System III (TaKaRa) using PrimeScript RT Master Mix (Perfect Real Time) and TB Green Premix Ex Taq II FAST qPCR (TaKaRa). The expression level of UCP-1 mRNA was corrected for the expression level of GAPDH mRNA.

[0140] The method for calculating the UCP-1 mRNA expression enhancement rate is as follows: • UCP-1 mRNA expression enhancement rate (%) = A / B × 100 A: Corrected value when the test sample is added B: Corrected value when the test sample is not added

[0141] <Results>

[0142] IPA and IAA were found to promote UCP-1 mRNA expression.

[0143] [4-3. Summary] As described above, this compound has been shown to have lipid accumulation inhibitory effects and UCP-1 mRNA expression promoting effects. These effects have been reported to be effective against obesity. In other words, it is suggested that this drug and composition, which contain at least one of this compound, a salt of this compound, or a conjugate of this compound, have anti-obesity effects based on these effects.

[0144] [5. Corneal protective effect] [5-1. Corneal epithelial cell proliferation promoting effect test] <Test method> Normal human corneal epithelial cells (HCECs) were cultured using proliferation medium (OcuLife Comp kit; Kurabo) and then harvested by trypsin treatment. The harvested cells were 15.0 × 10⁻⁶ ４After adjusting the concentration to cells / mL, 100 μL was seeded per well into a collagen-coated 96-well plate and cultured overnight. After culturing, 100 μL of the test sample lysed in growth medium was added to each well and cultured for 3 days.

[0145] The corneal epithelial cell proliferation-promoting effect was measured using the MTT assay method. Specifically, after the culture period, 100 μL of MTT dissolved in PBS(-) at a final concentration of 0.4 mg / mL was added to each well. After 2 hours of culture, the blue formazan produced in the cells was extracted with 100 μL of 2-propanol. After extraction, the absorbance at a wavelength of 570 nm was measured. Simultaneously, the absorbance at a wavelength of 650 nm was measured as turbidity, and the difference between the two was used to determine the amount of blue formazan produced.

[0146] The method for calculating the corneal epithelial cell proliferation promotion rate is as follows: • Corneal epithelial cell proliferation promotion rate (%) = A / B × 100 A: Amount of blue formazan produced in cells when the test sample is added B: Amount of blue formazan produced in cells without the test sample

[0147] <Results>

[0148] IPA and IAA were found to have a corneal epithelial cell proliferation-promoting effect.

[0149] [5-2. Glutathione Production Promotion Test Using Corneal Epithelial Cells] <Test Method> Normal human corneal epithelial cells (HCECs) were cultured in growth medium (OcuLife Comp kit; Kurabo) and then harvested by trypsin treatment. The harvested cells were divided into 5.0 × 10⁻⁶ cells. ４ After adjusting the concentration to cells / mL, 200 μL was seeded per well into a collagen-coated 48-well plate and cultured for 48 hours. After the culture period, 200 μL of the test sample dissolved in growth medium was added to each well and cultured for a further 24 hours.

[0150] After culturing was complete, the culture medium was removed from each well, washed with 300 μL of PBS(-), and the cells were lysed with 150 μL of M-PER (Thermo Fisher Scientific Inc.). Of this, 100 μL was used to quantify total glutathione. Specifically, 100 μL of cell extract dissolved in a 96-well plate, 50 μL of 0.1 mol / L phosphate buffer, 25 μL of 2 mmol / L NADPH, and 25 μL of glutathione reductase (final concentration 17.5 units / mL) were added and heated at 37°C for 10 minutes. Then, 25 μL of 10 mmol / L 5,5'-dithiobis(2-nitrobenzoic acid) was added, and the absorbance at a wavelength of 412 nm was measured up to 5 minutes later to determine ΔOD / min. The total glutathione concentration was calculated based on a calibration curve created using oxidized glutathione.

[0151] The obtained values ​​were corrected to the amount of glutathione per unit of total protein, and the glutathione production promotion rate was calculated according to the following formula: Glutathione production promotion rate (%) = A / B × 100 A: Amount of glutathione per unit of total protein in cells when the test sample was added B: Amount of glutathione per unit of total protein in cells when the test sample was not added

[0152] <Results>

[0153] IPA and IAA were found to promote glutathione production in corneal epithelial cells.

[0154] [5-3. Summary] As described above, this compound has been shown to have corneal epithelial cell proliferation promoting effects and glutathione production promoting effects of corneal epithelial cells. These effects have been reported to be effective in protecting the cornea. In other words, it is suggested that this drug and this composition, which contain at least one of this compound, a salt of this compound, or a conjugate of this compound, have corneal protective effects based on these effects.

[0155] [6. Brain function improvement effect] [6-1. Astrocyte culture cell proliferation promotion test] <Test method> Mouse-derived astrocyte culture cells (C8-S) were cultured in DMEM containing 10% FBS and then harvested by trypsin treatment. The harvested cells were 2.5 × 10⁶ ４ The sample was diluted with 10% FBS-containing DMEM to a concentration of cells / mL, then seeded at a rate of 100 μL per well in a 96-well plate and incubated for 6 hours. After incubation, 100 μL of the test sample dissolved in 10% FBS-containing DMEM was added to each well and incubated for 4 days.

[0156] The cell proliferation-promoting effect was measured using the MTT assay method. After the culture period, the culture medium was removed, and 100 μL of MTT dissolved in PBS(-) at a final concentration of 0.4 mg / mL was added to each well. After 2 hours of incubation, the blue formazan produced in the cells was extracted with 100 μL of 2-propanol. After extraction, the absorbance at a wavelength of 570 nm was measured. Simultaneously, the absorbance at a wavelength of 650 nm was measured as turbidity, and the difference between the two was used to determine the amount of blue formazan produced.

[0157] The method for calculating the astrocyte proliferation promotion rate is as follows: • Astrocyte proliferation promotion rate (%) = A / B × 100 A: Amount of blue formazan produced in cells when the test sample is added B: Amount of blue formazan produced in cells without the test sample

[0158] <Results>

[0159] IPA and IAA were found to have an astrocyte proliferation-promoting effect.

[0160] [6-2. Test of Nitric Oxide Production Inhibition in Microglia Culture Cells] <Test Method> Mouse-derived microglia culture cells (C8-B4) were cultured in DMEM containing 10% FBS, and then harvested by trypsin treatment. The harvested cells were 1.0 × 10⁶ ５The sample was diluted to cells / mL in 10% FBS-containing DMEM, then seeded at a rate of 100 μL per well in a 96-well plate and cultured until confluence. After culturing, the culture medium was removed, and 100 μL of the test sample dissolved in 10% FBS-containing DMEM containing 0.5% DMSO was added to each well. Subsequently, 100 μL of lipopolysaccharide (LPS, E. coli O111;B4, SIGMA) and 5 ng / mL interferon-gamma (IFN-γ from mouse, R&D systems), dissolved in 10% FBS-containing DMEM, were added, and the cultures were incubated for 24 hours.

[0161] NO production is due to nitrite ions (NO ２ － The amount was used as an indicator for measurement. After the culture was completed, the same amount of Gris reagent (1% sulfanilamide, 0.1% N-1-naphthyl ethylenediamine dihydrochloride in 5% phosphoric acid solution) was added to 100 μL of culture medium in each well and reacted at room temperature for 10 minutes. After the reaction, the absorbance at a wavelength of 540 nm was measured. NO ２ － A calibration curve was created using this as an indicator, and the amount of NO produced in the culture supernatant was determined.

[0162] The method for calculating the NO production inhibition rate is as follows: NO production inhibition rate (%) = {(B - A) / B} × 100 A: Amount of NO when the test sample is added B: Amount of NO when the test sample is not added

[0163] <Results>

[0164] IAA was found to have an inhibitory effect on NO production in microglia.

[0165] [6-3. Summary] As described above, this compound has been shown to have an astrocyte proliferation-promoting effect and an inhibitory effect on microglia NO production. These effects have been reported to be effective in improving brain function. In other words, it is suggested that this drug and composition, which contain at least one of this compound, a salt of this compound, or a conjugate of this compound, have brain function-improving effects based on these effects.

[0166] [7. Liver function improvement effect] [7-1. Glutathione production promotion effect test using normal human liver cells] <Test method> Normal human liver cells were cultured in DMEM containing 10% FBS and then harvested by trypsin treatment. The harvested cells were 1.0 × 10⁶ ５ The sample was diluted with 10% FBS-containing DMEM to a concentration of cells / mL, then seeded at 200 μL per well in a 48-well plate and incubated overnight. After incubation, 200 μL of the test sample dissolved in 1% FBS-containing DMEM was added to each well and incubated for a further 24 hours.

[0167] After culturing was complete, the culture medium was removed from each well, washed with 400 μL of PBS(-), and the cells were lysed with 150 μL of M-PER (Thermo Fisher Scientific Inc.). Of this, 100 μL was used to quantify total glutathione. Specifically, 100 μL of cell extract dissolved in a 96-well plate, 50 μL of 0.1 mol / L phosphate buffer, 25 μL of 2 mmol / L NADPH, and 25 μL of glutathione reductase (final concentration 3.2 units / mL) were added and heated at 37°C for 10 minutes. Then, 25 μL of 10 mmol / L 5,5'-dithiobis(2-nitrobenzoic acid) was added, and the absorbance at a wavelength of 412 nm was measured up to 5 minutes later to determine ΔOD / min. The total glutathione concentration was calculated based on a calibration curve created using oxidized glutathione.

[0168] The obtained values ​​were corrected to the amount of glutathione per unit of total protein, and the glutathione production promotion rate was calculated according to the following formula: Glutathione production promotion rate (%) = A / B × 100 A: Amount of glutathione per unit of total protein in cells when the test sample was added B: Amount of glutathione per unit of total protein in cells when the test sample was not added

[0169] <Results>

[0170] IPA was found to promote glutathione production in hepatocytes.

[0171] [7-2. ATP Production Promotion Test Using Normal Human Hepatocytes] <Test Method> Normal human hepatocytes were cultured in DMEM containing 10% FBS and then harvested by trypsin treatment. The harvested cells were 2.0 × 10⁶ ５ After diluting with 10% FBS-containing DMEM to a concentration of cells / mL, 100 μL was seeded per well into a 96-well plate and incubated overnight.

[0172] After the culture period, the culture medium was removed, and 100 μL of the test sample dissolved in 10% FBS-containing DMEM was added to each well and cultured for 2 hours. The ATP production promoting effect was assessed by measuring the amount of ATP in the cells using the firefly luciferase luminescence method. Specifically, after the culture period, 100 μL of "ATP Measurement Reagent for Cells" (Toyo B-Net Co., Ltd.) was added to each well. After the reaction, the amount of chemiluminescence was measured.

[0173] The method for calculating the ATP production promotion rate is as follows: ATP production promotion rate (%) = A / B × 100 A: Chemiluminescence amount in cells to which the test sample was added B: Chemiluminescence amount in cells without the test sample added

[0174] <Results>

[0175] IPA and IAA were found to promote ATP production in hepatocytes.

[0176] [7-3. Summary] As described above, this compound has been shown to have glutathione production-promoting and ATP production-promoting effects in hepatocytes. These effects have been reported to be effective in improving liver function. In other words, it is suggested that this drug and this composition, which contain at least one of this compound, a salt of this compound, or a conjugate of this compound, have liver function-improving effects based on these effects.

[0177] [8. Blood Flow Improvement Effect] [8-1. Blood Flow Promotion Effect Test Using Activation of Endothelial Cell-Derived Nitric Oxide Synthase as an Indicator] <Test Method> Normal human umbilical vein endothelial cells (HUVECs) were cultured in vascular endothelial cell proliferation medium (EGM-2) containing 5% FBS, and then harvested by trypsin treatment. The harvested cells were divided into 1.5 × 10⁻⁶ cells. ５After diluting the above medium to a concentration of cells / mL, 200 μL was seeded per well into a collagen-coated 48-well microplate and incubated overnight. After incubation, the medium was replaced with 0.25% FBS-containing EGM-2 and incubated for 4 hours. Then, 200 μL of the test sample, prepared at twice the final concentration using 0.25% FBS-containing EGM-2, was added to each well and incubated at 37°C for 30 minutes.

[0178] After culturing, the amount of phosphorylated (activated) eNOS was evaluated using the RayBio Human Phospho-eNOS (Ser1177) ELISA Kit (RayBiotech). Similarly, the amount of phosphorylated eNOS was also evaluated for dimethyl sulfoxide, which was used to dissolve the test samples as a control.

[0179] The eNOS activation rate is calculated as follows: eNOS activation rate (%) = A / B × 100 A: Measured value of phosphorylated eNOS when the test sample is added B: Measured value of phosphorylated eNOS when the test sample is not added

[0180] <Results>

[0181] IPA and IAA were found to have eNOS activating effects.

[0182] [8-2. Summary] As described above, this compound has been shown to have a blood flow promoting effect via eNOS activation. This effect has been reported to be effective in improving blood flow. In other words, it is suggested that this drug and composition, which contain at least one of this compound, a salt of this compound, and a conjugate of this compound, have a blood flow improving effect based on the blood flow promoting effect via eNOS activation.

[0183] [9. Gout-improving effect] [9-1. Xanthine oxidase activity inhibitory effect test] <Test method> 75 μL of 0.1 mol / L phosphate buffer (pH 7.4), 30 μL of distilled water, 5 μL of methanol, and 15 μL of the test sample solution were added to a 96-well quartz microplate. 25 μL of xanthine oxidase solution (0.12 U / mL, enzyme) was added and the mixture was heated at 37°C for 15 minutes. After heating, 150 μL of 0.1 mol / L xanthine solution was added and quickly stirred, and the mixture was reacted at 37°C for 5 minutes. After the reaction, the absorbance at a wavelength of 295 nm was measured. A blank test (without enzyme) was performed in the same manner and corrected.

[0184] The method for calculating the xanthine oxidase activity inhibition rate is as follows: Xanthine oxidase activity inhibition rate (%) = {1 - (A - B) / (C - D)} × 100 A: Absorbance at 295 nm with test sample and enzyme added B: Absorbance at 295 nm with test sample and no enzyme added C: Absorbance at 295 nm without test sample and with enzyme added D: Absorbance at 295 nm without test sample and no enzyme added

[0185] <Results>

[0186] IPA and IAA were found to have xanthine oxidase activity inhibitory effects.

[0187] [9-2. Summary] As described above, this compound has been shown to have xanthine oxidase activity inhibitory activity. This activity has been reported to be effective in improving gout. In other words, it is suggested that this drug and this composition, which contain at least one of this compound, a salt of this compound, or a conjugate of this compound, have a gout-improving effect based on this xanthine oxidase activity inhibitory activity.

[0188] This disclosure can be used, for example, as an anti-aging agent, hair growth agent, skin whitening agent, anti-obesity agent, corneal protective agent, brain function improving agent, liver function improving agent, blood flow improving agent, and gout improving agent.

Claims

1. An anti-aging agent comprising at least one of a compound represented by the following general formula (1), a salt of said compound, and a conjugate of said compound. [In general formula (1), n ​​is either 1 or 2.] 2. A hair growth agent comprising at least one of the compound represented by the following general formula (1), a salt of said compound, and a conjugate of said compound. [In general formula (1), n ​​is either 1 or 2.] 3. A skin whitening agent comprising at least one of a compound represented by the following general formula (1), a salt of said compound, and a conjugate of said compound. [In general formula (1), n ​​is either 1 or 2.] 4. An anti-obesity agent comprising at least one of a compound represented by the following general formula (1), a salt of said compound, and a conjugate of said compound. [In general formula (1), n ​​is either 1 or 2.] 5. A corneal protective agent comprising at least any one of a compound represented by the following general formula (1), a salt of said compound and a conjugate of said compound. [In general formula (1), n is 1 or 2.] 6. A brain function improving agent comprising at least one of a compound represented by the following general formula (1), a salt of said compound, and a conjugate of said compound. [In general formula (1), n ​​is either 1 or 2.] 7. A liver function improving agent comprising at least one of a compound represented by the following general formula (1), a salt of said compound, and a conjugate of said compound. [In general formula (1), n ​​is either 1 or 2.] 8. A blood flow improving agent comprising at least one of a compound represented by the following general formula (1), a salt of said compound, and a conjugate of said compound. [In general formula (1), n ​​is either 1 or 2.] 9. A gout-improving agent comprising at least one of a compound represented by the following general formula (1), a salt of said compound, and a conjugate of said compound. [In general formula (1), n ​​is either 1 or 2.] 10. A cosmetic composition containing at least one selected from the group consisting of the anti-aging agent described in claim 1, the hair growth agent described in claim 2, the skin whitening agent described in claim 3, the anti-obesity agent described in claim 4, the corneal protective agent described in claim 5, the brain function improving agent described in claim 6, the liver function improving agent described in claim 7, the blood flow improving agent described in claim 8, and the gout improving agent described in claim 9.

11. A food or beverage containing at least one selected from the group consisting of the anti-aging agent described in claim 1, the hair growth agent described in claim 2, the skin whitening agent described in claim 3, the anti-obesity agent described in claim 4, the corneal protective agent described in claim 5, the brain function improving agent described in claim 6, the liver function improving agent described in claim 7, the blood flow improving agent described in claim 8, and the gout improving agent described in claim 9.

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