JP2007223919A - Skin cosmetic - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an anti-oxidizing agent which contains a substance having one or more actions selected from the group consisting of an SOD-like action, a peroxide-eliminating action, and a radical-eliminating action as an active ingredient, to provide an anti-inflammatory agent containing a substance having a hyaluronidase-inhibiting action and/or a hexosaminidase separation-inhibiting action as an active ingredient, to provide a bleaching agent or tyrosinase inhibitor containing a substance having a tyrosinase-inhibiting action as an active ingredient, and to provide an antiaging agent containing a substance having a fibroblast growth-stimulating action and/or an epidermal keratinocyte growth-stimulating action as an active ingredient. <P>SOLUTION: The anti-oxidizing agent, the anti-inflammatory agent, the bleaching agent, the tyrosinase inhibitor, the antiaging agent or the skin cosmetic is characterized by containing piceatannol 4'-O-β-D-glucopyranoside as an active ingredient. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to an antioxidant, an anti-inflammatory agent, a whitening agent, a tyrosinase inhibitor, an anti-aging agent and a skin cosmetic.

The active oxygen is generated by an energy metabolism process in a living cell, and includes superoxide (that is, superoxide anion generated by one-electron reduction of oxygen molecule: .O ₂ ⁻ ), hydrogen peroxide (H ₂ O ₂ ), hydroxy Radicals (.OH) and singlet oxygen. In the living body, radicals generated first based on oxygen are superoxide, and other radicals such as hydroxy radicals are generated via superoxide. Superoxide is converted into hydrogen peroxide by the action of superoxide dismutase (hereinafter referred to as “SOD”) produced in cells.

  The amount of SOD decreases with aging, and the intracellular concentration of superoxide increases as the amount of SOD decreases. In addition, the activity of catalase or the like, which is a detoxifying enzyme for active oxygen, also decreases, and superoxide causes damage to the living body. For example, tissue disorders such as rheumatoid arthritis and Behcet's disease, myocardial infarction, stroke, cataract, diabetes, arteriosclerosis, stiff shoulders, coldness, skin blemishes and wrinkles are brought about. As an SOD-like agent that prevents or treats such a disorder, a plant extract belonging to the genus Muranoceae (see Patent Document 1) is known.

  There are various causes and onset mechanisms of inflammatory diseases such as contact dermatitis (rash), psoriasis, pemphigus vulgaris, and various other skin diseases associated with rough skin, including hyaluronidase, histamine, and hexosa. The thing by minidase etc. is known.

  Hyaluronate that maintains affinity for living tissue is decomposed by ultraviolet rays, enzymes, and the like in a water-containing system, and the water retention effect decreases with a decrease in molecular weight. Hyaluronic acid is present in intercellular tissues and is also involved in vascular permeability. Furthermore, hyaluronidase that is present and activated in mast cells is said to be involved in degranulation from mast cells. Therefore, by inhibiting the activity of hyaluronidase, which is a hydrolase of hyaluronic acid, it can stabilize hyaluronic acid and prevent the release of various chemical mediators from mast cells. It is considered to be effective in preventing, treating or improving various inflammatory diseases such as dermatitis (rash), psoriasis, pemphigus vulgaris, and various other skin diseases associated with rough skin.

  Based on such an idea, as a substance having a hyaluronidase inhibitory action, for example, an extract of a plant belonging to the genus Osbecchia (see Patent Document 2), a Fuji tea extract (see Patent Document 3), rosemary, thyme, and Melissa Extracts (see Patent Document 4) and the like are known.

  Histamine is present in the mast cell, and the substance released by the degranulation reaction acts as a flammable substance. Since histamine in the cells is released and hexosaminidase is released at the same time, histamine release inhibitory action can be evaluated using hexosaminidase release as an index. Therefore, by inhibiting the release of hexosaminidase, it is also possible to inhibit the release of histamine at the same time, thereby causing various skin diseases such as contact dermatitis (rash), psoriasis, pemphigus vulgaris, and other rough skin. It is considered effective for prevention, treatment or improvement of inflammatory diseases.

  Based on such an idea, for example, red snow tea extract (see Patent Document 5) is known as one having an inhibitory action on hexosaminidase release.

  Melanin also plays a role in protecting the body from ultraviolet rays in the skin, but overproduction and uneven accumulation cause skin darkening and spots. In general, melanin is converted from tyrosine to dopa and from dopa to dopaquinone by the action of the enzyme tyrosinase biosynthesized in pigment cells, and then formed through intermediates such as 5,6-dihydroxyindophenol. ing. Therefore, in order to prevent, treat or improve skin darkness (skin pigmentation), spots, buckwheat, etc., it is conceivable to inhibit the activity of tyrosinase involved in the production of melanin.

  Conventionally, for the prevention, treatment or improvement of skin pigmentation, stains, buckwheat, etc., a treatment using a whitening agent containing a chemically synthesized product such as hydroquinone as an active ingredient has been performed. However, chemically synthesized products such as hydroquinone may cause side effects such as skin irritation and allergies. Therefore, it is desired to develop a whitening agent containing a highly safe natural raw material as an active ingredient. Examples of those having a tyrosinase inhibitory action include wisteria tea extract (see Patent Document 6), Yanagita extract (patent Reference 7) is known.

  The epidermis and dermis of the skin are composed of epidermal cells, fibroblasts, and an extracellular matrix such as collagen that is outside these cells and supports the skin structure. In young skin, the proliferation of fibroblasts is active, and moisture is retained in the skin by the interaction of skin tissues such as fibroblasts and collagen, and the flexibility and elasticity of the skin are secured. It is maintained in a fresh state with its tension and gloss.

However, the growth of fibroblasts slows down due to the influence of certain external factors such as ultraviolet rays, drastic drying of air, excessive skin washing, etc., and aging progresses, and the moisture retention function and elasticity of the skin decrease. To do. And the skin loses its tension and gloss, and exhibits aging symptoms such as roughness and wrinkles. Therefore, it is considered that aging of the skin can be prevented or improved by promoting the proliferation of fibroblasts. Based on such an idea, as a substance having a fibroblast proliferation promoting action, for example, Kusunohagashi extract (see Patent Document 8) is known.
JP 2002-363087 A Japanese Patent Laid-Open No. 2003-55242 JP 2003-12532 A JP-A-8-333267 JP 2003-212789 A JP 2002-370962 A JP 2004-83488 A JP 2003-146837 A

  The present invention firstly finds a substance having one or more kinds of actions selected from the group consisting of SOD-like action, hydrogen peroxide scavenging action and radical scavenging action, and provides an antioxidant containing the substance as an active ingredient. The purpose is to provide.

  A second object of the present invention is to find a substance having a hyaluronidase inhibitory action and / or a hexosaminidase release inhibitory action, and to provide an anti-inflammatory agent containing the substance as an active ingredient.

  A third object of the present invention is to find a substance having a tyrosinase inhibitory action and to provide a whitening agent or a tyrosinase inhibitor containing the substance as an active ingredient.

  A fourth object of the present invention is to find a substance having a fibroblast proliferation promoting action and / or an epidermal keratinocyte proliferation promoting action, and to provide an anti-aging agent containing the substance as an active ingredient.

  Fifthly, the present invention provides SOD-like action, hydrogen peroxide scavenging action, radical scavenging action, hyaluronidase inhibiting action, hexosaminidase release inhibiting action, tyrosinase inhibiting action, fibroblast proliferation promoting action and epidermal keratinocyte proliferation promoting An object of the present invention is to find one having two or more kinds of actions selected from the group consisting of actions, and to provide a skin cosmetic containing the same.

  In order to solve the above problems, the antioxidant, anti-inflammatory agent, whitening agent, tyrosinase inhibitor and anti-aging agent of the present invention are piceatannol 4′-O-β-D-glucopyranoside (piceatannol 4′-O—). β-D-glucopyranoside) is contained as an active ingredient, and the skin cosmetic of the present invention is piceatannol 4′-O-β-D-glucopyranoside (piceatannol 4′-O-β-D). -glucopyranoside).

  In the antioxidant of the present invention, the compound preferably has one or more actions selected from the group consisting of SOD-like action, hydrogen peroxide scavenging action, and radical scavenging action. In the agent, the compound preferably has a hyaluronidase inhibitory action and / or a hexosaminidase release inhibitory action. In the anti-aging agent of the present invention, the compound has a fibroblast proliferation promoting action and / or epidermis. It preferably has a keratinocyte proliferation promoting action.

  According to the present invention, an antioxidant, an anti-inflammatory agent, a whitening agent, and tyrosinase containing piceatannol 4'-O-β-D-glucopyranoside as an active ingredient Inhibitors, anti-aging agents or skin cosmetics can be provided.

The present invention will be described below.
[Antioxidants, anti-inflammatory agents, whitening agents, tyrosinase inhibitors, anti-aging agents]
Antioxidant, anti-inflammatory agent, whitening agent, tyrosinase inhibitor or anti-aging agent of the present invention is piceatannol 4′-O-β-D-glucopyranoside (piceatannol 4′-O-β-D-glucopyranoside). Contains as an active ingredient.

  Piceatannol 4'-O-β-D-glucopyranoside (piceatannol 4'-O-β-D-glucopyranoside) is a kind of flavonoid derivative having a chemical structure represented by the following formula.

  Piceatannol 4′-O-β-D-glucopyranoside can be produced by isolation and purification from a plant extract containing piceatannol 4′-O-β-D-glucopyranoside. Can also be manufactured. When producing by synthesis, it can be synthesized by a known method.

  Plant extracts containing piceatannol 4'-O-β-D-glucopyranoside can be obtained by methods commonly used for plant extraction. Examples of plants containing piceatannol 4'-O-β-D-glucopyranoside include tenninka (scientific name: Rhodomyrtus tomentosa (Ait.) Hassk.).

  Tenninka (Rhodomyrtus tomentosa (Ait.) Hassk.) Is an evergreen shrub belonging to the family Myrtaceae distributed in the tropical and subtropical regions of Southeast Asia. In Japan, it grows naturally in Okinawa and other areas. It can be easily obtained. In addition to being eaten raw, tenninka fruit is also used as a raw material for juice and jam. In China, it is called Momokin daughter, and the fruit is privately used as anemia and hemostatic agent for pregnant women, and the leaves and roots are privately used for the treatment of headache and abdominal pain.

  Examples of the constituent parts of the tenninka that can be used as the raw material for extraction include above-ground parts such as leaves, branches, barks, trunks, stems, fruits, and flower parts, roots, or mixtures of these parts. Is preferably the above-ground part, root part or fruit part, or a mixture of these parts.

  The plant extract containing piceatannol 4′-O-β-D-glucopyranoside can be obtained by drying the raw material for extraction and then pulverizing it as it is or using a crusher and subjecting it to extraction with an extraction solvent. . Drying may be performed in the sun or using a commonly used dryer. Moreover, after performing pretreatment, such as degreasing, with a nonpolar solvent such as hexane, it may be used as an extraction material. By performing pretreatment such as degreasing, extraction treatment with a polar solvent of a plant can be performed efficiently.

  As the extraction solvent, it is preferable to use a polar solvent, and examples thereof include water and hydrophilic organic solvents. These may be used alone or in combination of two or more at room temperature or a temperature below the boiling point of the solvent. Is preferred.

  Examples of water that can be used as the extraction solvent include pure water, tap water, well water, mineral spring water, mineral water, hot spring water, spring water, fresh water, and the like, and those subjected to various treatments. Examples of the treatment applied to water include purification, heating, sterilization, filtration, ion exchange, osmotic pressure adjustment, buffering, and the like. Therefore, the water that can be used as the extraction solvent in the present invention includes purified water, hot water, ion-exchanged water, physiological saline, phosphate buffer, phosphate buffered saline, and the like.

  Examples of hydrophilic organic solvents that can be used as extraction solvents include lower aliphatic alcohols having 1 to 5 carbon atoms such as methanol, ethanol, propyl alcohol, and isopropyl alcohol; lower aliphatic ketones such as acetone and methyl ethyl ketone; 1,3-butylene. Examples thereof include polyhydric alcohols having 2 to 5 carbon atoms such as glycol, propylene glycol and glycerin.

  When using the liquid mixture of 2 or more types of polar solvents as an extraction solvent, the mixing ratio can be adjusted suitably. For example, when using a liquid mixture of water and a lower aliphatic alcohol, it is preferable to mix 1 to 90 parts by weight of a lower aliphatic alcohol with respect to 10 parts by weight of water. When using a mixed solution, it is preferable to mix 1 to 40 parts by mass of a lower aliphatic ketone with 10 parts by mass of water, and when using a mixed solution of water and a polyhydric alcohol, water 10 It is preferable to mix 10 to 90 parts by mass of polyhydric alcohol with respect to parts by mass.

  The extraction treatment is not particularly limited as long as the soluble component contained in the extraction raw material can be eluted in the extraction solvent, and can be performed according to a conventional method. For example, the extraction raw material is immersed in an extraction solvent 5 to 15 times (mass ratio) of the extraction raw material, the soluble components are extracted at room temperature or under reflux, and then filtered to remove the extraction residue. A liquid can be obtained. When the solvent is distilled off from the obtained extract, a paste-like concentrate is obtained, and when this concentrate is further dried, a dried product is obtained.

  The method for isolating and purifying piceatannol 4′-O-β-D-glucopyranoside from the extract obtained as described above, the concentrate of the extract or the dried product of the extract is particularly limited. It can be carried out by conventional methods. For example, a plant extract is subjected to column chromatography using a porous material such as silica gel or alumina, a porous resin such as styrene-divinylbenzene copolymer or polymethacrylate, and the order of water, alcohol and acetone. And can be obtained as a fraction eluted with alcohol. The alcohol used as the eluent in the column chromatography is not particularly limited, and examples thereof include lower aliphatic alcohols having 1 to 5 carbon atoms such as methanol, ethanol, propyl alcohol, isopropyl alcohol, and their aqueous solutions. Can be mentioned. Furthermore, any organic compound purification means such as reverse phase silica gel chromatography using ODS, recrystallization, liquid-liquid countercurrent extraction, column chromatography using ion exchange resin, etc., for the fraction obtained by column chromatography You may refine | purify using.

  The piceatannol 4′-O-β-D-glucopyranoside obtained as described above has SOD-like action, hydrogen peroxide scavenging action, radical scavenging action, hyaluronidase inhibiting action, hexosaminidase release inhibiting action, tyrosinase inhibition. Since it has an action, a fibroblast proliferation promoting action or an epidermal keratinocyte proliferation promoting action, it is effective to use antioxidants, anti-inflammatory agents, whitening agents, tyrosinase inhibitors, and anti-aging agents. Can be used as an ingredient. The plant extract obtained by the extraction treatment contains piceatannol 4′-O-β-D-glucopyranoside, and is used as it is as an antioxidant, anti-inflammatory agent, whitening agent, tyrosinase inhibitor or anti-aging agent. Although it can be used as an active ingredient, it is preferable to use a product obtained by purifying and increasing the purity of piceatannol 4′-O-β-D-glucopyranoside. Antioxidant, anti-inflammatory agent, whitening agent, tyrosinase inhibitor or more excellent in use effect by using as an active ingredient an increased purity of piceatannol 4′-O-β-D-glucopyranoside An anti-aging agent can be obtained. A plant extract containing piceatannol 4′-O-β-D-glucopyranoside includes an extract obtained by using a plant containing piceatannol 4′-O-β-D-glucopyranoside as an extraction raw material, the extraction A liquid dilute solution or a concentrated solution, a dry product obtained by drying the extract, or a crude product or a purified product thereof are included.

  Here, the antioxidant action of piceatannol 4′-O-β-D-glucopyranoside is, for example, one or more selected from the group consisting of SOD-like action, hydrogen peroxide scavenging action, and radical scavenging action. Based on the action of. However, the antioxidant action which piceatannol 4'-O-beta-D-glucopyranoside has is not limited to the antioxidant action exhibited based on the above action. Note that piceatannol 4′-O-β-D-glucopyranoside has an SOD-like action, a hydrogen peroxide scavenging action, and a radical scavenging action. It can be used as an active ingredient of a scavenger or radical scavenger.

  Moreover, the anti-inflammatory action which piceatannol 4'-O-beta-D-glucopyranoside has is exhibited based on, for example, a hyaluronidase inhibitory action and / or a hexosaminidase release inhibitory action. However, the anti-inflammatory action which piceatannol 4'-O-β-D-glucopyranoside has is not limited to the anti-inflammatory action exhibited based on the above-mentioned action. In addition, since piceatannol 4'-O- (beta) -D-glucopyranoside has a hyaluronidase inhibitory action and a hexosaminidase release inhibitory action, it uses these actions and a hyaluronidase inhibitor or a hexosaminidase release inhibitory effect. It can be used as an active ingredient of an agent.

  The whitening action of piceatannol 4'-O-β-D-glucopyranoside is exhibited based on, for example, a tyrosinase inhibitory action. However, the whitening action of piceatannol 4'-O-β-D-glucopyranoside is not limited to the whitening action exhibited on the basis of the above action.

  Furthermore, the anti-aging effect of piceatannol 4'-O-β-D-glucopyranoside is exhibited based on, for example, a fibroblast proliferation promoting action and / or an epidermal keratinocyte proliferation promoting action. However, the anti-aging action of piceatannol 4'-O-β-D-glucopyranoside is not limited to the anti-aging action exhibited based on the above action. Note that piceatannol 4′-O-β-D-glucopyranoside has a fibroblast proliferation promoting action and an epidermal keratinocyte proliferation promoting action. Therefore, using these actions, a fibroblast proliferation promoting agent or It can be used as an active ingredient of an epidermal keratinocyte proliferation promoter.

  The antioxidant, anti-inflammatory agent, whitening agent, tyrosinase inhibitor or anti-aging agent of the present invention may be composed solely of piceatannol 4′-O-β-D-glucopyranoside, or piceatannol. 4'-O-β-D-glucopyranoside may be formulated.

  Piceatannol 4′-O-β-D-glucopyranoside is prepared in the form of powder, granules, tablets, using a pharmaceutically acceptable carrier such as dextrin, cyclodextrin and any other auxiliary agent according to a conventional method. It can be formulated into any dosage form such as liquid. In this case, as an auxiliary agent, for example, an excipient, a binder, a disintegrant, a lubricant, a stabilizer, a flavoring / flavoring agent, and the like can be used. Piceatannol 4′-O-β-D-glucopyranoside can be used by blending it with other compositions (for example, skin cosmetics described later), and also as an ointment, a liquid for external use, a patch, etc. Can be used.

  In addition, the antioxidant, anti-inflammatory agent, whitening agent, tyrosinase inhibitor or anti-aging agent of the present invention may have an antioxidant action, SOD-like action, hydrogen peroxide scavenging action, radical scavenging action, anti-inflammation as necessary. Contains other natural extracts that have an action, hyaluronidase inhibitory action, hexosaminidase release inhibitory action, whitening action, tyrosinase inhibitory action, anti-aging action, fibroblast proliferation promoting action or epidermal keratinocyte proliferation promoting action It can be used as an active ingredient.

  The antioxidant of the present invention is one or more actions selected from the group consisting of SOD-like action, hydrogen peroxide scavenging action and radical scavenging action of piceatannol 4′-O-β-D-glucopyranoside. Prevent, treat, or improve various disorders involving active oxygen such as rheumatoid arthritis and Behcet's disease, myocardial infarction, stroke, cataract, diabetes, arteriosclerosis, stiff shoulders, coldness, skin blemishes and wrinkles Can do. However, the antioxidant of the present invention is significant in that it exhibits one or more actions selected from the group consisting of SOD-like action, hydrogen peroxide scavenging action and radical scavenging action in addition to these uses. It can be used for all applications.

  The anti-inflammatory agent of the present invention has contact dermatitis (rash), psoriasis, vulgaris through the hyaluronidase inhibitory action and / or hexosaminidase release inhibitory action of piceatannol 4′-O-β-D-glucopyranoside. Various inflammatory diseases such as pemphigus can be prevented, treated or ameliorated. However, the anti-inflammatory agent of the present invention can be used for all purposes other than these uses, which are meaningful for exerting a hyaluronidase inhibitory action and / or a hexosaminidase release inhibitory action.

  The whitening agent of the present invention can prevent, treat or ameliorate skin pigmentation, blemishes, freckles, etc. through the tyrosinase inhibitory action of piceatannol 4'-O-β-D-glucopyranoside. However, the whitening agent of the present invention can be used for all purposes other than these uses, which are meaningful for exerting a tyrosinase inhibitory action.

  The tyrosinase inhibitor of the present invention can prevent, treat or ameliorate skin pigmentation, blemishes, buckwheat and the like through the tyrosinase inhibitory action of piceatannol 4'-O-β-D-glucopyranoside. However, the tyrosinase inhibitor of the present invention can be used for all purposes that are meaningful for exerting a tyrosinase inhibitory action in addition to these uses.

  The anti-aging agent of the present invention is a skin wrinkle formation, a decrease in elasticity, etc. through the fibroblast proliferation promoting action and / or epidermal keratinocyte proliferation promoting action of piceatannol 4′-O-β-D-glucopyranoside. Can prevent, treat or ameliorate the aging phenomenon. However, the anti-aging agent of the present invention can be used for all purposes that are meaningful for exerting a fibroblast proliferation promoting action and / or an epidermal keratinocyte proliferation promoting action in addition to these uses.

[Skin cosmetic]
Piceatannol 4′-O-β-D-glucopyranoside is SOD-like action, hydrogen peroxide scavenging action, radical scavenging action, hyaluronidase inhibiting action, hexosaminidase release inhibiting action, tyrosinase inhibiting action, fibroblast proliferation promotion It has an action or an epidermal keratinocyte proliferation promoting action, and is excellent in usability and safety when applied to the skin, and is therefore suitable for blending into skin cosmetics. In this case, piceatannol 4′-O-β-D-glucopyranoside may be added to the skin cosmetic, or an antioxidant prepared from piceatannol 4′-O-β-D-glucopyranoside. An anti-inflammatory agent, a whitening agent, a tyrosinase inhibitor or an anti-aging agent may be added. By blending piceatannol 4′-O-β-D-glucopyranoside, the antioxidant, the anti-inflammatory agent, the whitening agent, the tyrosinase inhibitor or the anti-aging agent into the skin cosmetic, Can impart SOD-like action, hydrogen peroxide scavenging action, radical scavenging action, hyaluronidase inhibiting action, hexosaminidase release inhibiting action, tyrosinase inhibiting action, fibroblast proliferation promoting action or epidermal keratinocyte proliferation promoting action it can.

  The kind of skin cosmetics into which piceatannol 4′-O-β-D-glucopyranoside can be blended is not particularly limited. For example, ointment, cream, emulsion, lotion, pack, jelly, foundation, lip balm Lipsticks, bath additives and the like.

  When piceatannol 4′-O-β-D-glucopyranoside is blended into the skin cosmetic, the blending amount can be adjusted as appropriate according to the type of skin cosmetic, but the preferred blending ratio is It is about 0.0001 to 10% by mass in terms of a typical extract, and a particularly suitable blending ratio is about 0.001 to 1% by mass in terms of a standard extract.

  The skin cosmetics of the present invention include an SOD-like action, hydrogen peroxide scavenging action, radical scavenging action, hyaluronidase inhibiting action, hexosaminidase release inhibiting action, tyrosinase, which piceatannol 4′-O-β-D-glucopyranoside has Main agents, auxiliaries or other components used in the production of normal skin cosmetics, such as astringents, bactericides and antibacterial agents, as long as they do not interfere with the inhibitory action, fibroblast proliferation promoting action or epidermal keratinocyte proliferation promoting action , Whitening agent, UV absorber, moisturizer, cell activator, anti-inflammatory / anti-allergic agent, antioxidant / reactive oxygen scavenger, fats and oils, waxes, hydrocarbons, fatty acids, alcohols, esters, surface activity An agent, a fragrance | flavor, etc. can be used together. By using in this way, it becomes a more general product, and a synergistic action with the above-mentioned components used in combination may bring about an excellent effect that is usually expected.

  Note that the antioxidant, anti-inflammatory agent, whitening agent, tyrosinase inhibitor, anti-aging agent or skin cosmetic of the present invention is suitably applied to humans, and has the respective effects. As long as it can be applied to animals other than humans.

  Hereinafter, although a manufacture example, a test example, and a compounding example are shown and this invention is demonstrated concretely, this invention is not restrict | limited to each following example at all.

[Production Example 1] Production of piceatannol 4′-O-β-D-glucopyranoside 1000 mL of 80% by weight ethanol (mass ratio of water to ethanol = 1: 4) was added to 100 g of dried fruit of the ground portion of tenninka. In addition, the mixture was heated and extracted at 80 ° C. for 2 hours with a reflux extractor and filtered while hot. The obtained extract was concentrated under reduced pressure and dried to obtain 6.6 g of Tenninka fruit part extract.

The obtained Tenninka fruit part extract was attached to a porous adsorption resin (product name: Diaion HP-20, manufactured by Mitsubishi Chemical Corporation) and eluted in the order of 3000 mL of water, 1000 mL of methanol, and 1000 mL of acetone. The obtained methanol elution part (solid content: 2.6 g) was fractionated using a silica gel column (manufactured by Fuji Silysia Ltd., mobile phase; chloroform: methanol: water = 10: 5: 1 (mass ratio)), Fraction 1 (solid content: 360 mg), fraction 2 (solid content: 390 mg), fraction 3 (solid content: 110 mg), fraction 4 (solid content: 320 mg), and fraction 5 were obtained. The obtained fraction 4 was applied to an ODS column (mobile phase: 30% by mass methanol) and further applied to a recycle HPLC (product name: LC-908 recycle preparative HPLC, manufactured by Nihon Analytical Industrial Co., Ltd., mobile phase: methanol). And purified by separation to obtain 42 mg of purified product (Sample 1). The results of analyzing this purified product by ¹ H-NMR and ¹³ C-NMR are shown below.

< ¹ H-NMR chemical shift δ (assigned hydrogen)>
7.03 (1H, d, J = 8.5Hz, 5'-H), 6.92 (1H, d, J = 1.5Hz, 2'-H), 6.81 (1H, d, J = 16.4Hz, β-H), 6.80 (1H, dd-like, 6'-H), 6.72 (2H, d, J = 2.2Hz, 2,6-H), 6.08 (1H, br s, 4-H), 4.68 (1H, d, (J = 7.3Hz, 1``-H)

< ¹³ C-NMR chemical shift δ (assigned carbon)>
159.6 (3,5-C), 149.2 (3'-C), 146.7 (4'-C), 140.8 (1-C), 134.5 (1'-C), 129.0 (β-C), 128.6 (α -C), 119.1 (6'-C), 118.5 (5'-C), 114.8 (2'-C), 105.9 (2,6-C), 104.1 (1 ''-C), 103.3 (4- C), 78.2 (3``-C), 77.5 (5 ''-C), 74.8 (2 ''-C), 71.2 (4 ''-C), 62.4 (6 ''-C)

  From the above results, it was confirmed that the obtained purified product was piceatannol 4'-O-β-D-glucopyranoside.

[Test Example 1] SOD-like action test For piceatannol 4'-O-β-D-glucopyranoside obtained in Production Example 1, SOD was performed as follows. The effect was tested.

  In a test tube, 0.05 mL / L sodium bicarbonate buffer (pH 10.2) 2.4 mL, 3 mmol / L xanthine 0.1 mL, 3 mmol / L EDTA 0.1 mL, 50 μg / mL bovine serum albumin 0.1 mL And 0.1 mL of 0.75 mmol / L nitroblue tetrazolium were added, 0.1 mL of the sample solution was added thereto, and the mixture was allowed to stand at 25 ° C. for 10 minutes.

  Thereafter, 0.1 mL of a xanthine oxidase solution was added and stirred rapidly, and reacted at 25 ° C. for 20 minutes. Then, 0.1 mL of 6 mmol / L copper chloride was added to stop the reaction, and the absorbance at a wavelength of 560 nm was measured. A blank test was conducted in the same manner. From the obtained results, the superoxide elimination rate (%) was calculated based on the following formula.

Superoxide erasure rate (%) = {1- (St-Sb) / (Ct-Cb)} × 100
In the formula, St represents “absorbance of the sample solution at a wavelength of 560 nm”, Sb represents “absorbance of the sample solution at a wavelength of 560 nm”, Ct represents “absorbance of the control solution at a wavelength of 560 nm”, and Cb represents “control”. The absorbance of the solution blank at a wavelength of 560 nm is expressed.

The above-mentioned erasure rate was measured by gradually reducing the sample concentration, and the sample concentration IC ₅₀ (ppm; μg / mL) at which the superoxide erasure rate was 50% was determined by interpolation. As a result of the above test, the IC ₅₀ of piceatannol 4′-O-β-D-glucopyranoside was 81.7 μg / mL. From this, it was confirmed that piceatannol 4′-O-β-D-glucopyranoside has an excellent SOD-like action.

[Test Example 2] Hydrogen peroxide scavenging action test For piceatannol 4′-O-β-D-glucopyranoside obtained in Production Example 1, the following was performed. The hydrogen peroxide scavenging action was tested.

  After adding 10 μL of the sample solution to 10 μL of the 1.5 mmol / L hydrogen peroxide solution and reacting at 37 ° C. for 20 minutes, the coloring solution (containing 100 μmol / L DA-64, 0.5% Triton X-100) 2.98 mL of 100 mols / mL peroxidase was added to 100 mL of 0.1 mol / L PIPES buffer (pH 7.0) and reacted at 37 ° C. for 5 minutes. After completion of the reaction, absorbance at a wavelength of 727 nm was measured. A blank test was conducted in the same manner. From the obtained results, the hydrogen peroxide elimination rate (%) was calculated based on the following formula.

Hydrogen peroxide elimination rate (%) = {1− (St−Sb) / (Ct−Cb)} × 100
In the formula, St represents “absorbance of the sample solution at a wavelength of 727 nm”, Sb represents “absorbance of the sample solution blank at a wavelength of 727 nm”, Ct represents “absorbance of the control solution at a wavelength of 727 nm”, and Cb represents “control”. The absorbance of the solution blank at a wavelength of 727 nm is expressed.

The above-mentioned erasure rate was measured by gradually decreasing the sample concentration, and the sample concentration IC ₅₀ (ppm; μg / mL) at which the erasure rate of hydrogen peroxide was 50% was determined by interpolation. As a result of the above test, the IC ₅₀ of piceatannol 4′-O-β-D-glucopyranoside was 63.9 μg / mL. From this, it was confirmed that piceatannol 4′-O-β-D-glucopyranoside has an excellent hydrogen peroxide scavenging action.

[Test Example 3] Radical scavenging action test With respect to piceatannol 4'-O-β-D-glucopyranoside obtained in Production Example 1, radicals were treated as follows. The erasing action was tested.

  3 mL of the sample solution was added to 3 mL of 150 μmol / L DPPH (diphenyl-p-picrylhydrazyl) ethanol solution, sealed, shaken and allowed to stand for 30 minutes. After standing, the absorbance at a wavelength of 520 nm was measured. In addition, a blank test was performed and corrected in the same manner. From the obtained results, radical scavenging rate (%) relative to DPPH was calculated based on the following formula.

DPPH erasure rate (%) = {C− (St−Sb)} / C × 100
In the formula, St represents “absorbance of the sample solution at a wavelength of 520 nm”, Sb represents “absorbance of the sample solution blank at a wavelength of 520 nm”, and C represents “absorbance of the control solution at a wavelength of 520 nm”.

The above-mentioned erasure rate was measured by decreasing the sample concentration stepwise, and the sample concentration IC ₅₀ (ppm; μg / mL) at which the radical erasure rate was 50% was determined by interpolation. As a result of the above test, the IC ₅₀ of piceatannol 4′-O-β-D-glucopyranoside was 159.7 μg / mL. From this, it was confirmed that piceatannol 4′-O-β-D-glucopyranoside has an excellent radical scavenging action.

[Test Example 4] Hyaluronidase inhibitory action test For piceatannol 4'-O-β-D-glucopyranoside obtained in Production Example 1, hyaluronidase was treated as follows. Inhibitory effects were tested.

  0.2 mL of sample solution (sample concentration: 400 μg / mL) in which sample 1 was dissolved in 0.1 mol / L acetate buffer (pH 3.5) and hyaluronidase solution (Type IV-S (from bovine testis), SIGMA) And 400 unit / mL) was mixed with 0.1 mL and reacted at 37 ° C. for 20 minutes. Further, 0.2 mL of 2.5 mmol / L calcium chloride was added as an activator and reacted at 37 ° C. for 20 minutes to activate the enzyme. Thereafter, 0.5 mL of 0.4 mg / mL potassium hyaluronate solution (from rooster comb) was added and reacted at 37 ° C. for 40 minutes, and 0.2 mL of 0.4 mol / L sodium hydroxide solution was added and cooled with ice. The reaction was stopped.

  0.2 mL of boric acid solution was added to each reaction solution, heated in a boiling water bath for 3 minutes, and then ice-cooled for 10 minutes. Next, 6 mL of p-DABA reagent (10 g of p-dimethylaminobenzaldehyde dissolved in 12.5 mL of 10N hydrochloric acid and 87.5 mL of acetic acid and diluted 10-fold with acetic acid) was added, and the mixture was added at 37 ° C. for 20 minutes. Reacted. Thereafter, the absorbance at a wavelength of 585 nm was measured. A blank test was performed and corrected in the same manner. From the obtained results, the hyaluronidase inhibition rate (%) was calculated based on the following formula.

Hyaluronidase inhibition rate (%) = {1− (St−Sb) / (Ct−Cb)} × 100
In the formula, St represents “absorbance of the sample solution at a wavelength of 585 nm”, Sb represents “absorbance of the sample solution at a wavelength of 585 nm”, Ct represents “absorbance of the control solution at a wavelength of 585 nm”, and Cb represents “control”. The absorbance of the solution blank at a wavelength of 585 nm is expressed.

  As a result of the above test, the hyaluronidase inhibition rate of piceatannol 4′-O-β-D-glucopyranoside was 34.2%. This confirmed that piceatannol 4'-O-β-D-glucopyranoside has an excellent hyaluronidase inhibitory action.

[Test Example 5] Hexosaminidase release inhibitory action test About piceatannol 4'-O-β-D-glucopyranoside obtained in Production Example 1 (piceatannol 4'-O-β-D-glucopyranoside), Thus, the inhibitory effect on hexosaminidase release was tested.

Rat basophil leukemia cells (RBL-2H3) were cultured in S-MEM medium containing 15% FBS, and then cells were collected by trypsin treatment. The collected cells are diluted to a cell density of 4.0 × 10 ⁵ cells / mL, and after adding DNP-specific IgE so that the final concentration of DNP-specific IgE is 0.5 μg / mL, 96 100 μL per well was seeded on a well plate and cultured overnight. After completion of the culture, the medium was removed and washing was performed twice with 100 μL of Silaganian buffer.

Next, 30 μL of Silaganian buffer and 10 μL of a sample solution (sample final concentration: 400 μg / mL) dissolved in the same buffer were added, and the mixture was allowed to stand at 37 ° C. for 10 minutes. Thereafter, 10 μL of a 100 ng / mL DNP-BSA solution was added, and the mixture was allowed to stand at 37 ° C. for 15 minutes to release hexosaminidase. Thereafter, the release of hexosaminidase was stopped by allowing the 96-well plate to stand on ice. 10 μL of cell supernatant in each well and 10 μL of 1 mmol / L p-nitrophenyl-N-acetyl-α-D-glucosaminide (p-NAG) solution were added to a new 96-well plate and reacted at 37 ° C. for 1 hour. I let you. After completion of the reaction, 250 μL of 0.1 mol / L Na ₂ CO ₃ / NaHCO ₃ was added to each well, and the absorbance at a wavelength of 415 nm was measured. Similarly, the absorbance of a mixed solution of 10 μL of cell supernatant in a well to which 10 μL of Silaganian buffer was added without adding the sample and 250 μL of 0.1 mol / L Na ₂ CO ₃ / NaHCO ₃ was measured. Similarly, the sample was added and the absorbance was measured without adding p-NAG. From the obtained results, the hexosaminidase release inhibition rate (%) was calculated based on the following formula.

Inhibition rate of hexosaminidase release (%) = {1− (B−C) / A} × 100
In the formula, A indicates “absorbance at a wavelength of 415 nm when no sample is added”, B indicates “absorbance at a wavelength of 415 nm when a sample is added”, and C indicates “at a wavelength of 415 nm when a sample is added and p-NAG is not added”. Absorbance ".

  As a result of the above test, the inhibition rate of hexosaminidase release of piceatannol 4′-O-β-D-glucopyranoside was 17.8%. From this, it was confirmed that piceatannol 4′-O-β-D-glucopyranoside has an excellent hexosaminidase release inhibitory action.

[Test Example 6] Tyrosinase inhibitory action test The piceatannol 4'-O-β-D-glucopyranoside obtained in Production Example 1 was subjected to tyrosinase as follows. Inhibitory effects were tested.

  To a 48-well plate, 0.2 mL of Mcllvaine buffer (pH 6.8), 0.06 mL of a 0.3 mg / mL tyrosine solution and 0.18 mL of a 25% DMSO solution of a sample were added and allowed to stand at 37 ° C. for 10 minutes. . To this, 0.02 mL of 2500 unit / mL tyrosinase solution was added, followed by reaction at 37 ° C. for 15 minutes. After completion of the reaction, the absorbance at a wavelength of 475 nm was measured. A blank test was performed and corrected in the same manner. From the obtained results, the tyrosinase inhibition rate (%) was calculated based on the following formula.

Tyrosinase inhibition rate (%) = {1− (St−Sb) / (Ct−Cb)} × 100
In the formula, St represents “absorbance of the sample solution at a wavelength of 475 nm”, Sb represents “absorbance of the sample solution blank at a wavelength of 475 nm”, Ct represents “absorbance of the control solution at a wavelength of 475 nm”, and Cb represents “control”. The absorbance of the solution blank at a wavelength of 475 nm is expressed.

The inhibition rate was measured by gradually reducing the sample concentration, and the sample concentration IC ₅₀ (ppm; μg / mL) that inhibits the activity of tyrosinase by 50% was determined by interpolation. As a result of the above test, the IC ₅₀ of piceatannol 4′-O-β-D-glucopyranoside was 114.4 μg / mL. This confirmed that piceatannol 4′-O-β-D-glucopyranoside has an excellent tyrosinase inhibitory action.

[Test Example 7] Fibroblast proliferation promoting action test About piceatannol 4'-O-β-D-glucopyranoside obtained in Production Example 1 Thus, the fibroblast proliferation promoting effect was tested.

Human normal skin fibroblasts (NB1RGB) were cultured using α-MEM containing 10% FBS, and then cells were collected by trypsin treatment. The collected cells were diluted with α-MEM containing 5% FBS to a concentration of 7.0 × 10 ⁴ cells / mL, then seeded at 100 μL per well in a 96-well plate, and cultured overnight. After completion of the culture, 100 μL of a sample solution (sample concentration: 400 μg / mL) dissolved in α-MEM containing 5% FBS was added to each well and cultured for 3 days. Fibroblast proliferation was measured using the MTT assay. After completion of the culture, 100 μL of the medium was removed from each well, and 20 μL of MTT dissolved in PBS (−) at a final concentration of 5 mg / mL was added to each well. After culturing for 4.5 hours, 100 μL of a 0.01 mol / L hydrochloric acid solution in which 10% SDS was dissolved was added to each well and incubated overnight, and then the absorbance at a wavelength of 570 nm was measured. At the same time, 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. In addition, a blank test was performed and corrected in the same manner. From the obtained results, the fibroblast proliferation promotion rate (%) was calculated by the following formula.

Fibroblast proliferation promotion rate (%) = (St−Sb) / (Ct−Cb) × 100
In the formula, St represents “absorbance in cells to which a sample was added”, Sb represents “absorbance in a blank test to which a sample was added”, Ct represents “absorbance in cells to which no sample was added”, and Cb It represents “absorbance of blank test without adding sample”.

  As a result of the above test, the rate of promotion of fibroblast proliferation of piceatannol 4′-O-β-D-glucopyranoside was 115.2 ± 1.3%. It was. From this, it was confirmed that piceatannol 4′-O-β-D-glucopyranoside has an excellent fibroblast proliferation promoting action.

[Test Example 7] Epidermal keratinocyte proliferation-promoting action test About piceatannol 4'-O-β-D-glucopyranoside obtained in Production Example 1 (piceatannol 4'-O-β-D-glucopyranoside), Thus, the epidermal keratinocyte proliferation promoting action was tested.

Normal human neonatal capsular keratinocytes (NHEK) were cultured using normal human epidermal keratinocyte long-term culture growth medium (EpiLife-KG2), and then cells were collected by trypsin treatment. The collected cells were diluted with Epilife-KG2 to a concentration of 1.5 × 10 ⁴ cells / mL, then seeded at 100 μL per well in a collagen-coated 96-well plate, and cultured overnight. After the purification, 100 μL of a sample solution (sample concentration: 12.5 μg / mL) in which sample 1 was dissolved in EpiLife-KG2 was added to each well and cultured for 3 days.

  Epidermal keratinocyte proliferation was measured using the MTT assay. After completion of the culture, the medium was removed, and 100 μL of MTT dissolved in PBS (−) at a final concentration of 0.4 mg / mL was added. After culturing for 2 hours, 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. At the same time, 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. From the obtained results, the epidermal keratinocyte proliferation promotion rate (%) was calculated based on the following formula.

Epidermal keratinocyte proliferation promotion rate (%) = St / Ct × 100
In the formula, St represents “absorbance in cells to which a sample was added”, and Ct represents “absorbance in cells to which no sample was added”.

  As a result of the above test, the epidermal keratinocyte proliferation promotion rate of piceatannol 4′-O-β-D-glucopyranoside was 112.7 ± 3.8%. there were. From this, it was confirmed that piceatannol 4'-O-β-D-glucopyranoside has an excellent epidermal keratinocyte proliferation promoting action.

[Formulation Example 1]
An emulsion having the following composition was produced by a conventional method.
Piceatannol 4′-OD-glucopyranoside (Production Example 1) 0.01 g
Jojoba oil 4.0g
Placenta extract 0.1g
Olive oil 2.0g
Squalane 2.0g
Cetanol 2.0g
2.0g glyceryl monostearate
Polyoxyethylene cetyl ether (20E.O.) 2.5g
Oleic acid polyoxyethylene sorbitan (20E.O.) 2.0g
1,3-butylene glycol 3.0 g
Hinokitiol 0.15g
Fragrance 0.05g
Purified water remainder (total amount is 100 g)

[Formulation Example 2]
A cream having the following composition was produced by a conventional method.
Piceatannol 4′-OD-glucopyranoside (Production Example 1) 0.1 g
Liquid paraffin 5.0g
Salamichi Michirou 4.0g
Cetanol 3.0g
Squalane 10.0g
Lanolin 2.0g
Stearic acid 1.0g
Oleic acid polyoxyethylene sorbitan (20E.O.) 1.5g
3.0 g of glyceryl monostearate
1,3-butylene glycol 6.0 g
1.5 g of methyl paraoxybenzoate
Fragrance 0.1g
Purified water remainder (total amount is 100 g)

[Composition Example 3]
A lotion having the following composition was produced by a conventional method.
Piceatannol 4′-OD-glucopyranoside (Production Example 1) 0.1 g
Glycerin 3.0g
1,3-butylene glycol 3.0 g
Oleic acid polyoxyethylene sorbitan (20E.O.) 2.0g
Methyl paraoxybenzoate 0.15g
Citric acid 0.1g
Sodium citrate 0.1g
Oil soluble licorice extract 0.1g
Seaweed extract 0.1g
Kujin extract 0.1g
Xylobiose Mixture 0.05g
Fragrance 0.05g
Purified water remainder (total amount is 100 g)

The antioxidant of the present invention is used for the prevention, treatment or amelioration of various disorders involving active oxygen, and the anti-inflammatory agent of the present invention is used for the prevention, treatment or amelioration of various inflammatory diseases. The inhibitor can greatly contribute to the prevention, treatment or improvement of skin pigmentation, stains, etc., and the anti-aging agent of the present invention can greatly contribute to the prevention, treatment or improvement of skin aging.

Claims (9)

  An antioxidant comprising piceatannol 4'-O-β-D-glucopyranoside as an active ingredient.   2. The antioxidant according to claim 1, wherein the compound has one or more actions selected from the group consisting of an SOD-like action, a hydrogen peroxide scavenging action, and a radical scavenging action.   An anti-inflammatory agent characterized by containing piceatannol 4'-O-β-D-glucopyranoside as an active ingredient.   The anti-inflammatory agent according to claim 3, wherein the compound has a hyaluronidase activity inhibitory action and / or a hexosaminidase release inhibitory action.   A whitening agent comprising piceatannol 4'-O-β-D-glucopyranoside as an active ingredient.   A tyrosinase activity inhibitor characterized by containing piceatannol 4'-O-β-D-glucopyranoside as an active ingredient.   An anti-aging agent characterized by containing piceatannol 4'-O-β-D-glucopyranoside as an active ingredient.   The anti-aging agent according to claim 7, wherein the compound has a fibroblast proliferation promoting action and / or an epidermal keratinocyte proliferation promoting action. A skin cosmetic characterized by containing piceatannol 4′-O-β-D-glucopyranoside (piceatannol 4′-O-β-D-glucopyranoside).