Anti-malassezia agent

Abstract

To provide an anti-Malassezia agent, as well as to provide a method for producing an active ingredient for the anti-Malassezia agent.SOLUTION: The present invention provides an anti-Malassezia agent containing any one of compounds (I), (II), and (III) respectively represented by the following formulas (I), (II), and (III), or a pharmaceutically acceptable salt thereof, or henna extract, and also provides a method for producing compound (I).SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an anti-Malassezia agent. [Background technology]

[0002] Among the normal fungi present on the scalp, Malassezia is known to be one of the fungi involved in dandruff and seborrheic dermatitis. Malassezia breaks down sebum and produces fatty acids. It has been reported that excessive growth of Malassezia can cause dandruff, folliculitis, dermatitis, and other conditions. It is said that the balance of scalp fungi, including Malassezia, is important, but the details have not been fully elucidated. For example, antifungal agents are widely used to improve dandruff, but there are many cases where antifungal agents do not completely cure the condition. Furthermore, recent reports have also shown that Malassezia can affect atopic dermatitis.

[0003] Henna (Lawsonia inermis) (also known as henna, tumbleweed, or finger flower) is a woody plant belonging to the genus Lawsonia in the family Lythraceae. Lawsone (2-hydroxy-1,4-naphthoquinone), a characteristic component of this plant, has been reported to have various pharmacological activities, including antioxidant and antibacterial effects (Patent Document 1).

[0004] Patent Document 2 describes that a compound of the following formula was isolated from an extract of henna flowers, and that the compound exhibits a neuron-like cell differentiation promoting effect and is useful for the prevention or treatment of neurological diseases.

[0005] [ka] [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Japanese Patent Application Publication No. 2024-7027 [Patent Document 2] Patent No. 5728105 Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide an anti-Malassezia agent and a method for producing an active ingredient of an anti-Malassezia agent. [Means for solving the problem]

[0008] In order to solve the above problems, the present invention includes the following inventions. [1] An anti-Malassezia agent comprising a compound (I) represented by the following formula (I) or a pharmaceutically acceptable salt thereof: [ka] [2] An anti-Malassezia agent comprising a compound (II) represented by the following formula (II) or a pharmaceutically acceptable salt thereof: [ka] [3] An anti-Malassezia agent comprising a compound (III) represented by the following formula (III) or a pharmaceutically acceptable salt thereof: [ka] [4] Anti-Malassezia fungus agent containing henna extract. [5] The anti-Malassezia fungus agent according to [4], wherein the henna extract contains at least one of compounds (I), (II) and (III) represented by the following formulas (I), (II) and (III), respectively, or a pharmaceutically acceptable salt thereof. [ka] [6] The anti-Malassezia fungus agent according to [4], wherein the henna extract is henna leaf extract. [7] A method for producing compound (I) represented by the following formula (I) or a pharmaceutically acceptable salt thereof, [ka] extracting henna leaves and obtaining the compound from the resulting extract. Manufacturing method. [Effects of the Invention]

[0009] The present invention provides an anti-Malassezia agent. Such an anti-Malassezia agent is useful in pharmaceutical compositions and cosmetics. The present invention also provides a novel method for producing compound (I), which can be used as an active ingredient of an anti-Malassezia agent. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 shows the steps for the preparation of henna leaf extract and the isolation of compounds 1-3. [Figure 2] 1 is a graph showing the results of an anti-Malassezia test of henna leaf extract (Malassezia survival rate). [Figure 3] 1 is a graph showing the results of an anti-Malassezia test of Compound 1 (survival rate of Malassezia). [Figure 4] 1 is a graph showing the results of an anti-Malassezia test of Compound 2 (survival rate of Malassezia). [Figure 5] 1 is a graph showing the results of an anti-Malassezia test of Compound 3 (survival rate of Malassezia). DETAILED DESCRIPTION OF THE INVENTION

[0011] [Anti-Malassezia agents] The present invention provides an anti-Malassezia agent. Malassezia is a yeast-like fungus that normally resides on the skin surface of mammals, including humans. Malassezia takes on both yeast and filamentous forms during its life cycle, but these are used interchangeably in the present invention. Malassezia requires lipids for growth. Examples of Malassezia include Malassezia globosa, Malassezia furfur, Malassezia restricta, and Malassezia obtusa. As used herein, the term "anti-Malassezia" refers to the ability to inhibit the proliferation of Malassezia, particularly in an environment favored by Malassezia (e.g., an environment in which lipids such as sebum are present). The anti-Malassezia effect can be confirmed by examining the growth of Malassezia in a medium containing a lipid component. The anti-Malassezia effect can be confirmed, for example, by the test method described in Example 2 below.

[0012] The anti-Malassezia agent of the present invention contains the compound or henna extract described below. In this specification, the compound or henna extract described below is also collectively referred to as the "active ingredient of the anti-Malassezia agent."

[0013] The active ingredient of the anti-Malassezia agent of the present invention can be isolated and purified by preparing an extract from henna, or by further processing using a combination of solvent-based liquid extraction from the henna extract, fractionation using chromatography using various separation modes (e.g., ion exchange, hydrophilic adsorption, hydrophobic adsorption, size exclusion, ligand exchange, affinity), molecular weight fractionation filtration using filter paper, membrane filters, ultrafiltration membranes, etc., pressurization or decompression, heating or cooling, drying, pH adjustment, deodorization, bleaching, and prolonged static storage. For example, the active ingredient compound can be isolated by combining column chromatography and preparative HPLC as described in Example 1 below. The structure of the isolated compound can be determined by NMR or mass spectrometry, if necessary. The active ingredient compound can be used as an isolated compound, or it may be one contained in the henna extract.

[0014] The active ingredient of the anti-Malassezia agent of the present invention will be explained below.

[0015] 1.Compound In one aspect, the active ingredient of the anti-Malassezia agent of the present invention is a compound as described below: The structural formula of the compound of the active ingredient of the present invention is shown below.

[0016] [ka]

[0017] [ka]

[0018] [ka]

[0019] The above formulas (I), (II), and (III) are the structural formulas of inermioside A (Compound 1), apigenin 7-O-α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranoside (Compound 2), and luteolin 4'-O-β-D-glucopyranoside (Compound 3), respectively. The compounds represented by these formulas are referred to as Compounds (I), (II), and (III), respectively, and are also collectively referred to as the "compounds of the present invention."

[0020] The compounds of the present invention include stereoisomers, optical isomers and mixtures of these isomers, solvates, crystalline polymorphs, isotope-labeled compounds, and the like.

[0021] Some compounds of the present invention contain chiral carbon atoms. In the case of such compounds, stereoisomers of the compounds of the present invention exist. The present invention extends to all optical isomers, such as enantiomers, diastereomers, and mixtures thereof, including, for example, racemates, of the compounds of formulas (I), (II), and (III) (compounds 1, 2, and 3). The various stereoisomeric forms may be separated or resolved one from the other by conventional methods, or any isomer may be obtained by conventional stereoselective or asymmetric synthetic methods.

[0022] Some of the compounds of the present invention may exist in different tautomeric forms, and the present invention is understood to encompass all such tautomers.

[0023] The compounds of the invention may be prepared in crystalline or amorphous form, and, if crystalline, may be hydrated or solvated. This invention includes within its scope stoichiometric hydrates or solvates as well as compounds containing variable amounts of water and / or solvent.

[0024] The compounds of the present invention may take on crystalline polymorphic forms, which are also within the scope of the present invention.

[0025] The present invention also includes isotopically labeled compounds that are identical to the compounds described herein except that one or more atoms have been replaced by an atom having an atomic mass or mass number different from that normally found in nature. Isotopes that may be incorporated into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, iodine, or chlorine, for example: 3 H, 11 C. 14 C. 18 F, 123 I and 125 I. Compounds of the present invention that contain these isotopes and / or other isotopes of other atoms are within the scope of the present invention.

[0026] The compounds of the present invention also include ester and ether derivatives. It is understood by those skilled in the art that if a glycoside exhibits biological activity, an ester or ether derivative of the glycoside will have a similar effect. For example, when administered to the body or its surface, the ester or ether derivative is hydrolyzed by enzymes, acids, or the like under physiological conditions to be converted into the compounds of the respective formulae (compounds 1, 2, and 3), thereby exhibiting the desired activity. Examples of esters include C1-C6 alkyl esters having an alkyl group, such as methyl ester and ethyl ester. Examples of ethers include C1-C6 alkyl ethers having a C1-C6 alkyl group, such as methyl ether and ethyl ether.

[0027] As used herein, "alkyl group" may have branched, cyclic, and / or unsaturated bonds. The C1-C6 in the C1-C6 alkyl group is defined to refer to a straight-chain or branched group having 1, 2, 3, 4, 5, or 6 carbons. Thus, C1-C6 alkyl groups specifically include, but are not limited to, methyl, ethyl, ethenyl, Z-ethylene, n-propyl, isopropyl, cyclopropyl, propenyl, n-butyl, isobutyl, tert-butyl, 1-butenyl, pentyl, hexyl, and cyclohexyl groups.

[0028] Esters and ethers can be produced using commercially available reagents or known methods. For example, ester derivatives can be obtained by the Fischer ester synthesis reaction using sulfuric acid as a catalyst. Specifically, ester derivatives can be obtained by dehydration condensation of the hydroxyl group in formulas (I), (II), and (III) with an oxoacid such as carboxylic acid, sulfuric acid, phosphoric acid, or nitric acid. Alternatively, esters can be obtained by the Schotten-Baumann reaction using an acid anhydride such as acetic anhydride or an acid halide. Ether derivatives can be obtained by dehydration condensation of alcohols with the hydroxyl group in formulas (I), (II), and (III). Ethers can also be obtained by reacting sodium alkoxides of the hydroxyl group in formulas (I), (II), and (III) with alkyl halides. Alternatively, ethers can be obtained by alkylating metal alcoholates of the hydroxyl group in formulas (I), (II), and (III) with sulfate esters, alkylating alcohols with alkyl halides in the presence of a tertiary amine, or by the Williamson ether synthesis method.

[0029] When the compounds of the present invention have a basic group, they can form acid addition salts. Examples of such salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid; and acid addition salts formed with organic acids such as succinic acid, maleic acid, formic acid, acetic acid, trifluoroacetic acid, propionic acid, fumaric acid, citric acid, tartaric acid, benzoic acid, p-toluenesulfonic acid, methanesulfonic acid, and naphthalenesulfonic acid. Furthermore, when the compounds herein contain an acidic functional group, such as a carboxyl group, the counterion may be selected not only from an organic base but also from inorganic salts selected from sodium, potassium, lithium, calcium, magnesium, and the like. These salts should be pharmaceutically acceptable. The compounds of the present invention may also include those that form acid addition salts with one or more equivalents of the above acids. The present invention encompasses all possible stoichiometric and non-stoichiometric forms.

[0030] Pharmaceutically acceptable salts of the compound of the present invention can also be used as an active ingredient of the anti-Malassezia fungus agent. The pharmaceutically acceptable salts are not particularly limited as long as they maintain the efficacy of the active ingredient and do not have any adverse effects on the human body. Examples of the pharmaceutically acceptable salts include acetic acid, propionic acid, butyric acid, formic acid, trifluoroacetic acid, maleic acid, tartaric acid, citric acid, stearic acid, succinic acid, ethylsuccinic acid, malonic acid, lactobionic acid, gluconic acid, glucoheptonic acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, paratoluenesulfonic acid (tosylic acid), Examples of the salt include salts with acids such as lauryl sulfate, malic acid, aspartic acid, glutamic acid, adipic acid, cysteine, N-acetylcysteine, hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, hydroiodic acid, nicotinic acid, oxalic acid, picric acid, thiocyanic acid, undecanoic acid, acrylic acid polymers, and carboxyvinyl polymers; salts with inorganic bases such as lithium salts, sodium salts, potassium salts, and calcium salts; salts with organic amines such as morpholine and piperidine; and salts with amino acids.

[0031] 2. Henna extract In another aspect, the active ingredient of the anti-Malassezia agent of the present invention is henna extract. Henna (Lawsonia inermis) (also known as henna, jasmine, or jasmine flower) is a woody plant belonging to the genus Lawsonia in the family Lythraceae. Henna grows on dry, well-drained hills. Known sources of henna include Egypt, India, North Africa, and Iran, but any suitable growing environment is acceptable. Henna is an evergreen shrub or small tree about 3 to 6 meters tall, producing small, white or pink flowers with four petals, approximately 7 mm in diameter, and elliptical to lanceolate leaves, approximately 2 to 4 cm long and 1 to 2 cm wide. Henna extract can be prepared as described below.

[0032] In one embodiment, the henna extract as an active ingredient of the anti-Malassezia agent of the present invention is a henna extract containing the compound of the present invention or a pharmaceutically acceptable salt thereof. That is, the henna extract as an active ingredient contains at least one of compounds (I), (II), and (III) represented by the following formulas (I), (II), and (III), respectively, or a pharmaceutically acceptable salt thereof. Details of the compound are as described above.

[0033] [ka]

[0034] The compounds of formulas (I), (II), and (III), respectively, inermioside A (Compound 1), apigenin 7-O-α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranoside (Compound 2), and luteolin 4'-O-β-D-glucopyranoside (Compound 3), have been confirmed to be present in henna leaf extract by chromatographic fractionation of the extract, as described below. Compound (I) has also been found in an extract of henna flowers (Patent Document 2).

[0035] Henna extract can be obtained by a process of extracting a henna plant or a part thereof. For example, henna extract can be obtained by drying a henna plant or a part thereof and extracting the dried matter with a solvent. There are no particular restrictions on the origin or growth level of the henna plant used for extraction. The part used for henna extraction can be the leaves or flowers of henna, or both, with the leaves being preferred. In one embodiment, the henna extract is a henna leaf extract. There are no particular restrictions on the drying method, and for example, freeze drying, spray drying, etc. can be used. There are no particular restrictions on the extraction solvent, and for example, alcohols such as methanol and ethanol can be used. Examples of suitable solvents include butylene glycol (BG), propylene glycol (PG), and 1,3-propanediol, which are commonly used in the production of alcohol and cosmetics, but methanol is preferred. The extraction temperature is not particularly limited as long as it is a temperature commonly used in henna extraction. For example, hot extraction (approximately 80°C) is used. The extraction time is not limited as long as the active ingredients can be extracted, and can be determined according to the extraction temperature. If necessary, procedures such as crushing, filtration, re-extraction, and solvent distillation may be performed. The henna extract may be concentrated by freeze-drying, spray-drying, or other methods. The henna extract may be in either liquid or powder form.

[0036] [Method for producing compound (I)] The present invention also provides a method for producing Compound (I) or a pharmaceutically acceptable salt thereof. The production method of the present invention includes the steps of extracting henna leaves and obtaining the compound from the resulting extract. Compound (I) was originally discovered in henna flowers (Patent Document 2), but the present inventors have discovered that it can also be produced from henna leaves. Because small buds of about 1 mm can be used as the henna flowers used for extraction, and because the proportion of leaves in the henna plant is significantly greater than that of flowers, the production method of the present invention allows for the mass production of Compound (I), which was newly discovered in henna flowers, compared to production from flowers. The production method of the present invention is advantageous for inexpensive and large-scale production of the compound, making it economically valuable.

[0037] The method for extracting henna leaves is as described above in the section "2. Henna Extract." Compound (I) can be obtained from this extract. To obtain Compound (I) from the extract, the compound of formula (I) can be isolated from the henna leaf extract, or, if desired, subjected to an etherification reaction or an esterification reaction without isolation. Isolation of the compound can be achieved by any combination of treatments, such as liquid-liquid extraction with a solvent, fractionation using chromatography using various separation modes (e.g., ion exchange, hydrophilic adsorption, hydrophobic adsorption, size exclusion, ligand exchange, affinity), molecular weight fractionation filtration using filter paper, membrane filter, or ultrafiltration membrane, pressurization or decompression, heating or cooling, drying, pH adjustment, deodorization, bleaching, and prolonged static storage. For example, the method described in Example 1 below can be used. The etherification reaction and esterification reaction are as described above in the section "1. Compound."

[0038] [Application] Malassezia is a yeast-like fungus that normally resides on the skin surface of mammals, including humans, and typically exists as a parasitic fungus in the normal skin flora. Malassezia is also involved in diseases such as Malassezia folliculitis, Malassezia intertrigo, seborrheic dermatitis, dandruff, and tinea versicolor. Malassezia is also said to be an aggravating factor for atopic dermatitis. Furthermore, excessive fatty acids produced by the overgrowth of Malassezia on the scalp raise concerns about their effects on the scalp, potentially leading to scalp troubles. Due to its anti-Malassezia effect, the anti-Malassezia agent of the present invention is useful for preventing or treating diseases associated with Malassezia, or for preventing or suppressing adverse effects that may result from the overgrowth of Malassezia.

[0039] The anti-Malassezia agent of the present invention can be applied orally or parenterally and can be used in products or compositions in which an anti-Malassezia effect is desired. Such products or compositions include pharmaceuticals (pharmaceutical compositions), quasi-drugs, cosmetics, functional foods, etc. Such products or compositions are within the scope of the present invention.

[0040] The product or composition according to the present invention can be formulated by a conventional method, but is not limited to this.

[0041] Dosage forms include, for example, dermatological preparations and oral preparations. Dermatological preparations include, for example, liniments, patches, and sprays. Liniments include, for example, ointments, creams, liquids, gels, lotions, and patches. Patches include, for example, poultices, plasters, tapes, and patches. Sprays include, for example, aerosols. Oral preparations include, for example, tablets, powders, fine granules, granules, coated tablets, capsules, syrups, and lozenges. For formulation, commonly used excipients, binders, lubricants, colorants, flavorings, and, if necessary, stabilizers, emulsifiers, absorption enhancers, surfactants, pH adjusters, preservatives, antioxidants, and the like can be used. The formulations can be prepared by conventional methods by blending ingredients commonly used as raw materials for pharmaceuticals, quasi-drugs, cosmetics, functional foods, and the like.

[0042] The anti-Malassezia agent of the present invention can be used as cosmetics or quasi-drugs for application to the skin, and examples thereof include, but are not limited to, hair cosmetics, shampoos, rinses, conditioners, hair growth agents, scalp care products, skin care products, lotions, cosmetic oils, emulsions, beauty serums, creams, foundations, packs, cleansers, facial washes, all-in-one gels, body lotions, etc. The anti-Malassezia agent of the present invention may be in the form of a food or drink for oral administration, and may be used as, for example, a functional food (health food) such as a food for specified health uses, a food with functional claims, or a food with nutrient function claims, or may be incorporated into ordinary food or drink.

[0043] These components include, for example, animal and vegetable oils such as soybean oil, beef tallow, and synthetic glycerides; hydrocarbons such as liquid paraffin, squalane, and solid paraffin; ester oils such as octyldodecyl myristate and isopropyl myristate; higher alcohols such as cetostearyl alcohol and behenyl alcohol; silicone resins; for example, silicone oils; surfactants such as polyoxyethylene fatty acid esters, sorbitan fatty acid esters, glycerin fatty acid esters, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene hydrogenated castor oil, and polyoxyethylene polyoxypropylene block copolymers; water-soluble polymers such as hydroxyethyl cellulose, polyacrylic acid, carboxyvinyl polymers, polyethylene glycol, polyvinylpyrrolidone, and methylcellulose; lower alcohols such as ethanol and isopropanol; polyhydric alcohols such as glycerin, propylene glycol, dipropylene glycol, and sorbitol; sugars such as glucose and sucrose; inorganic powders such as silicic anhydride, aluminum magnesium silicate, and aluminum silicate; and purified water. Examples of excipients include water, purified water, alcohol, glycerin, lactose, corn starch, sucrose, glucose, mannitol, sorbitol, crystalline cellulose, and silicon dioxide. Examples of binders include polyvinyl alcohol, polyvinyl ether, methylcellulose, ethylcellulose, gum arabic, tragacanth, gelatin, shellac, hydroxypropylmethylcellulose, hydroxypropylcellulose, polyvinylpyrrolidone, polypropylene glycol-polyoxyethylene block polymer, and meglumine. Examples of disintegrants include starch, agar, gelatin powder, crystalline cellulose, calcium carbonate, sodium bicarbonate, calcium citrate, dextrin, pectin, and calcium carboxymethylcellulose. Examples of lubricants include magnesium stearate, talc, polyethylene glycol, silica, and hydrogenated vegetable oil. Colorants that are permitted for addition to pharmaceuticals or cosmetics are used. Examples of flavoring agents that can be used include cocoa powder, peppermint, aromatic powder, peppermint oil, borneol, and cinnamon powder.

[0044] For example, in the case of dermatological preparations, the manufacturing method is not particularly limited and they can be manufactured by conventional methods. Oral preparations, for example, are prepared by adding the active ingredient compound or its derivatives or hydrates thereof and excipients, and further, if necessary, binders, disintegrants, lubricants, colorants, flavorings, etc., and then, by conventional methods, are made into, for example, powders, fine granules, granules, tablets, coated tablets, capsules, etc. In the case of tablets and granules, they can of course be coated with sugar or other coatings as needed. In the case of syrups and injectable preparations, for example, pH adjusters, solubilizers, isotonicity agents, etc., and, if necessary, solubilizers, stabilizers, etc., and are formulated by conventional methods. The base raw materials used can be various raw materials commonly used in pharmaceuticals, quasi-drugs, cosmetics, functional foods, etc., such as animal and vegetable oils, mineral oils, ester oils, waxes, higher alcohols, fatty acids, silicone oils, surfactants, phospholipids, alcohols, polyhydric alcohols, water-soluble polymers, clay minerals, purified water, etc., and may contain pH adjusters, antioxidants, chelating agents, antiseptics and fungicides, colorants, fragrances, etc. Furthermore, ingredients such as blood flow promoters, bactericides, anti-inflammatory agents, cell activators, vitamins, amino acids, moisturizers, keratolytic agents, etc., may also be added as needed.

[0045] The amount of the active ingredient of the anti-Malassezia agent of the present invention contained in the product or composition of the present invention is not particularly limited, and the amount used can be adjusted appropriately depending on the purpose. For example, the amount used is a dose sufficient to exert an anti-Malassezia effect. The content of the active ingredient of the anti-Malassezia agent of the present invention in the pharmaceutical composition formulation varies depending on the form of the formulation, etc., but in the case of the compound or a pharmaceutically acceptable salt thereof, it can be, for example, 0.001% by mass or more, 0.005% by mass or more, or 0.01% by mass or more, or, for example, 30% by mass or less, 20% by mass or less, or 10% by mass or less, based on the total amount of the formulation.

[0046] The dosage of the pharmaceutical composition of the present invention varies depending on, for example, the severity of symptoms, age, sex, body weight, dosage form, type of salt, and specific type of disease, but typically, for adults, the active ingredient of the anti-Malassezia fungus agent of the present invention is administered per day in an amount of approximately 30 μg to 10 g, preferably 100 μg to 5 g, and more preferably 100 μg to 100 mg, as a compound or a pharmaceutically acceptable salt thereof, either once or in divided doses.

[0047] The amount of the active ingredient of the anti-Malassezia agent of the present invention contained in the quasi-drug, cosmetic or functional food of the present invention and the number of times of use are similar to those of the pharmaceutical composition, but are adjusted appropriately according to the purpose.

[0048] The anti-Malassezia agent of the present invention may be used in combination with one or more compounds known to be useful for treating or preventing skin diseases associated with Malassezia or their symptoms.

[0049] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. [Example]

[0050] The present invention will be described in more detail below with reference to specific examples, but the present invention is not limited thereto. Unless otherwise specified, the compounds and reagents used in the examples of the present invention are readily available commercially and can be used, or can be synthesized by methods known in the art.

[0051] Example 1: Preparation of henna leaf extract and isolation of compounds A henna leaf extract was prepared and each compound was isolated from the henna leaf extract according to the procedure shown in Figure 1. The details are described below.

[0052] Dried leaves (10.0 kg) of henna (Lawsonia inermis) grown in Kyoto were extracted with methanol (MeOH) at 80°C. The extract was filtered, and MeOH was added to the residue. The same extraction procedure was repeated three times. The MeOH extracts were combined and the solvent was distilled off under reduced pressure to obtain a MeOH extract (3522.7 g, yield 35.2%). (This extract was used as "henna leaf extract" in Example 2 below.)

[0053] 2525.5 g of the obtained MeOH extract was partitioned and extracted with ethyl acetate (EtOAc) and water (HO), and the obtained HO-soluble fraction was further partitioned and extracted with n-butanol (BuOH). The solvent of each soluble fraction was distilled off under reduced pressure to obtain an EtOAc-soluble fraction (1001.0 g, yield 14.0%), an n-BuOH-soluble fraction (683.3 g, yield 9.5%), and an HO-soluble fraction (841.2 g, yield 11.7%).

[0054] The resulting n-BuOH transition fraction (600.0 g) was fractionated by normal phase silica gel column chromatography [3.0 kg, CHCl3:MeOH:H2O = (30:3:1) → (20:3:1) → (10:3:1) → (7:3:1) → (6:4:1) → (5:5:1) → MeOH] to give Fr. B1 (5.3 g), Fr. B2 (22.2 g), Fr. B3 (50.3 g), Fr. B4 (256.9 g), Fr. B5 (182.6 g), and Fr. B6 (3. (Here, Fr. B1 means the first fraction obtained by fractionating the n-BuOH transition part, Fr. B2 means the second fraction, Fr. B3 means the third fraction, Fr. B4 means the fourth fraction, Fr. B5 means the fifth fraction, and Fr. B6 means the sixth fraction. Hereinafter, fractions derived from each fraction will be indicated with a hyphen. For example, Fr. B5-6-4 means the fourth fraction obtained by further fractionating the sixth fraction obtained by fractionating the fifth fraction of the n-BuOH transition part.)

[0055] Fractionation of Fr. B5 (182.6 g) by normal-phase silica gel column chromatography [1.5 kg, CHCl3:MeOH = (7:1) → (5:1) → (3:1) → (2:1) → (1:1) → MeOH] yielded Fractions B5-1 (1.8 g), B5-2 (1.6 g), B5-3 (6.5 g), B5-4 (5.9 g), B5-5 (17.9 g), B5-6 (36.2 g), B5-7 (27.5 g), B5-8 (34.3 g), B5-9 (16.5 g), and B5-10 (23.3 g).

[0056] Fractionation of Fr. B5-6 (36.2 g) by reversed-phase ODS column chromatography [400.0 g, MeOH:HO = (10:90) → (20:80) → (30:70) → (40:60) → (50:50) → (60:40) → (80:20) → MeOH] yielded Fr. B5-6-1 (2646.4 mg), Fr. 5-6-2 (14178.8 mg), Fr. 5-6-3 (2071.5 mg), Fr. B5-6-4 (5135.8 mg), and Fr. B 5-6-5(1126.6mg),Fr.B5-6-6(2503.7mg),Fr.B5-6-7(3700.5mg),Fr.B5-6-8(2089.0mg),Fr.B5-6-9(907.7mg),Fr.B5 -6-10 (336.8mg), Fr.B5-6-11 (359.1mg), Fr.B5-6-12 (545.1mg), Fr.B5-6-13 (237.6mg) and Fr.B5-6-14 (361.4mg) were obtained.

[0057] Fr. B5-6-4 (189.7 mg) was purified by HPLC [column: COSMOSIL 5C 18 The mixture was separated and purified using HCl-PAQ, eluent: CH3CN:H2O=(10:90), to isolate inermioside A (Compound 1, 18.9 mg, 0.00814%).

[0058] Fraction B5-6-8 (430.4 mg) was purified by HPLC [column: COSMOSIL 5C 18The resulting mixture was purified using HCl-PAQ (eluent: CHCN:HO (20:80)) to isolate apigenin 7-O-α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranoside (compound 2, 10.1 mg, 0.00078%) and luteolin 4'-O-β-D-glucopyranoside (compound 3, 14.9 mg, 0.00115%).

[0059] The structural formulas of the three isolated compounds are shown below.

[0060] [ka]

[0061] Example 2: Anti-Malassezia Effect of Henna Leaf Extract and Compounds 1 to 3 1. Materials and Methods (Malassezia culture) The Malassezia fungus used was a strain owned by the NPR Medical Resources Research Institute (derived from scalp dander from a human male in his 40s). Sequence analysis of the D1 / D2 region of 26S rDNA confirmed that the fungus was Malassezia globosa [601 / 601 (100%) match]. Malassezia fungus was cultured using Chromoagar Malassezia / Candida Raw Medium (Kanto Chemical Co., Ltd.).

[0062] (Preparation of YM medium) 10 g of D-glucose, 5 g of peptone, 3 g of yeast extract, 3 g of malt extract, and 0.1 g of penicillin were dissolved in 1 L of distilled water, filtered, and used in the experiment.

[0063] (positive control drug) The positive control drugs used were streptomycin sulfate, amorolfine hydrochloride, and itraconazole. Streptomycin sulfate is known as an antibiotic, but it was previously reported to have bacteriostatic activity against yeast (fungi) [Nature, 209, 536 (1966)], so it was used as one of the positive controls.

[0064] (Anti-Malassezia test) Henna leaf extract, Compounds 1-3, and each positive control were used as test substances in anti-Malassezia tests. 0.5 mL of Malassezia fungal solution prepared in McFarland No. 3 was added to a tube. The prepared sample, oleic acid, and YM medium were mixed in the tube, and the Malassezia fungi were cultured at 37°C for 22 hours. The samples were then stained using a Microbial Viability Assay Kit-WST (Dojindo Laboratories, Inc.). After 2 hours of incubation at 37°C, the absorbance was measured and the survival rate of Malassezia fungi was calculated. Results were expressed as a percentage (%) of the negative control ("control": no test substance added), with a lower value indicating a stronger anti-Malassezia effect. The values ​​obtained in the experiment were expressed as mean ± standard error. The Dunnett method was used to test for significant differences between the mean values ​​of the test substance-treated and negative control groups. The significance level was set at 5% or 1%.

[0065] 2.Results The results of the anti-Malassezia test (Malassezia survival rate (%)) for henna leaf extract and compounds 1 to 3 are shown in Tables 1 to 4 below and Figures 2 to 5 (n=4. In the figures, "*" indicates a significant difference compared to the negative control, * is p<0.05, ** is p<0.01).

[0066] [Table 1]

[0067] [Table 2]

[0068] [Table 3]

[0069] [Table 4]

[0070] Both henna leaf extract and Compounds 1 to 3 inhibited the survival rate of Malassezia fungi as the added amount increased.

[0071] The results for the positive control drugs were as follows: At 10 μM, streptomycin sulfate showed a 105.9% survival rate for Malassezia, indicating a tendency toward proliferation, and a concentration of 137 μM or higher was required to reduce survival by 20%. Furthermore, the antifungal drug amorolfine hydrochloride required 282.5 μM to reduce Malassezia survival by 20% or more, and itraconazole required a concentration of around 10 μM to reduce Malassezia survival by around 20%.

[0072] The anti-Malassezia activity of compounds 1, 2 and 3 was comparable to or stronger than that of these positive control drugs.

[0073] The present invention is not limited to the above-described embodiments and examples, and various modifications are possible within the scope of the claims. The technical scope of the present invention also includes embodiments obtained by appropriately combining the technical means disclosed in different embodiments. Furthermore, all academic literature and patent documents described in this specification are incorporated herein by reference.

Claims

1. An anti-Malassezia agent comprising a compound (I) represented by the following formula (I) or a pharmaceutically acceptable salt thereof: 【Chemistry 1】

2. An anti-Malassezia agent comprising a compound (II) represented by the following formula (II) or a pharmaceutically acceptable salt thereof: 【Chemistry 2】

3. An anti-Malassezia agent comprising a compound (III) represented by the following formula (III) or a pharmaceutically acceptable salt thereof: 【Transformation 3】

4. Anti-Malassezia agent containing henna extract

5. The anti-Malassezia agent according to claim 4, wherein the henna extract contains at least one of compounds (I), (II) and (III) represented by the following formulas (I), (II) and (III), respectively, or a pharmaceutically acceptable salt thereof. 【Chemistry 4】

6. The anti-Malassezia agent according to claim 4, wherein the henna extract is a henna leaf extract.

7. A method for producing compound (I) represented by the following formula (I) or a pharmaceutically acceptable salt thereof, 【Transformation 5】 extracting henna leaves and obtaining the compound from the resulting extract. Manufacturing method.

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