JP4975069B2 - Tyrosinase inhibitor - Google Patents

Description

  The present invention relates to a tyrosinase inhibitor and a whitening agent useful as an active ingredient of a whitening external preparation. Furthermore, it is related with the whitening external preparation which uses the said tyrosinase inhibitor or whitening agent as an active ingredient, especially whitening cosmetics.

  Skin pigmentation after sunburn, buckwheat or sunburn is a result of markedly increased melanin production due to activation of pigment cells (melanocytes) present in the skin, and is one of the skin problems in middle-aged and elderly people It has become.

  In general, melanin is converted from tyrosine to dopa and from dopa to dopaquinone by the action of tyrosinase, an enzyme biosynthesized in pigment cells, and then formed through intermediates such as 5,6-dihydroxyindophenol. It is said that. Therefore, in order to prevent and treat skin darkness (skin pigmentation), it is effective to inhibit the melanin production process.

  Based on such an idea, various whitening components have been conventionally proposed. For example, as compounds that inhibit tyrosinase activity and suppress melanin production, sulfur compounds such as colloidal sulfuric acid and glutathione, kojic acid, arbutin, hydroquinone, and the like are known. It is also known that various plant extracts and plant components have a tyrosinase inhibitory action (for example, Patent Documents 1 and 2).

  Patent Document 3 describes a cosmetic containing, as an active ingredient, a fine powder of a fruit of the Gnetsum family Gnetsum (Merinjo), which describes that it has a whitening effect. Non-Patent Documents 1 and 2 describe that resveratrol contained in the gnetsum fruit has tyrosinase inhibitory activity and suppresses melanin production based on it.

  On the other hand, although resveratrol dimers such as gnetin C, genomonoside A, genomonoside C, and genomonoside D have been reported to have antibacterial activity and antioxidant activity (Patent Document 4), no whitening effect is known. .

JP 2002-201122 JP 2008-214272 A JP2008-100963 WO2006 / 030771

Takako Yokozawa et al., “Piceatannol Inhibits Melanogenesis by Its Antioxidative Actions,” Biol. Pharm. Bull. 30 (11) 2007-2011, 2007 Rishard A. Newton et al., “Post-Transcriptional Regulation of Melanin Biosynthetic Enzymes by cAMP and Resveratrol in Human Melanocytes,” Journal of Investigative Dermatology, Volume 127 (9) 2216-27, 2007

  The object of the present invention is to provide these new uses based on the newly discovered physiological activity of gnetin C. Specifically, it aims at providing the use as a tyrosinase inhibitor and the use as a whitening agent about gnetin C or its salt.

  Under the above-mentioned problems, the present inventors conducted extensive studies and found that Gnetin C has a significantly higher tyrosinase inhibitory activity than kojic acid and resveratrol, which have been conventionally known to inhibit tyrosinase. The present inventors have found that there is an action of suppressing the production of melanin which causes stains and freckles, that is, there is a whitening action, and it was confirmed that the gnetin C can be used alone as an active ingredient of a whitening agent. The present invention has been completed based on such findings and has the following configuration.

(1) A tyrosinase inhibitor containing gnetin C or a salt thereof isolated or purified as an active ingredient.
(2) A whitening agent containing, as an active ingredient, isolated or purified gnetin C or a salt thereof.
(3) A whitening external preparation comprising the tyrosinase inhibitor described in (1) or the whitening agent described in (2).

  Melanin pigments that cause skin coloring are produced by pigment cells (melanocytes) in the skin tissue. Pigmentation such as skin spots and buckwheat is caused by abnormal deposition of melanin as a result of activation of pigment cells due to stimulation by ultraviolet exposure from sunlight, hormonal abnormalities, or genetic factors. It is thought to do. Normally, melanin is metabolized by metabolism (turnover) of skin tissue, but a decrease in metabolism of skin tissue with aging also causes pigmentation.

  As shown in the experimental examples to be described later, gnetin C exhibits superior inhibitory activity against tyrosinase, an enzyme involved in melanin synthesis, compared to kojic acid and resveratrol, which are known to inhibit tyrosinase, and produces melanin. Since it has an action to suppress odor, it has a whitening effect (improves pigmentation and dullness). For this reason, gnetin C is useful as an active ingredient of a tyrosinase inhibitor, a melanin production inhibitor, or a whitening agent, and is also useful as an active ingredient of a skin external preparation (whitening external preparation) having a whitening effect.

  The topical whitening agent containing the tyrosinase inhibitor or whitening agent of the present invention has an excellent whitening action, and has a skin-beautifying effect such as pigmentation after sunburn, lightening of spots, freckles, erythema, etc., and faintness. In addition, since healthy skin can be maintained, it is particularly useful as a whitening cosmetic, a beautiful skin cosmetic, and a skin care cosmetic.

In Experimental example 1, the result of having measured tyrosinase inhibitory activity (%) about each test sample (Gnetin C, trans-resveratrol, kojic acid) is shown. In Experimental example 2, the result of having measured the amount of melanin production in a cell about the case where each test sample (Gnetin C (15 micromol), kojic acid (1000 micromol)) is used is shown. (A) shows a photographic image of melanoma cells accumulated at the bottom of the tube. (B) shows the result of comparing the amount of melanin produced in the cells for each test sample (Gnetin C, Kojic acid).

(I) Tyrosinase inhibitor As described above, blemishes and buckwheat are generated when tyrosine is oxidized by tyrosinase, an enzyme in pigment cells, and a large amount of melanin is produced in the skin and abnormally deposited. That is, tyrosinase is an important enzyme for melanin synthesis. By inhibiting its activity, synthesis of melanin can be prevented, and as a result, a whitening effect can be obtained.

  The tyrosinase inhibitor in the present invention has an action of inhibiting the activity of such tyrosinase. As a result, it inhibits melanin synthesis in melanocytes and exerts a whitening effect.

  Such a tyrosinase inhibitor of the present invention is characterized by containing gnetin C as an active ingredient.

  Here, gnetin C is a dimer of resveratrol and is a kind of polyphenol represented by the following formula.

  Gnetin C can be used regardless of whether it is a home-made product or a commercial product. Here, the method for self-preparing gnetin C is not particularly limited, and is a method of extracting and isolating from a plant containing gnetin C, a method of producing and isolating it by microorganisms (for example, Adil E Bala et al., “Antifungal activity of resveratrol oligomers from Cyphostemma crotalarioides ", Pesticide Science, Vol. 55, Issue 2, Pages 206-208, etc.) and chemical synthesis methods.

 The plant containing gnetin C is not particularly limited, but preferably includes plants belonging to the family Gnetum (Ibrahim Iliya et al., “Stilbenoids from the stem of Gnetum latifolium (Gnetaceae)”, Phytochemistry, 2002 Dec; 61 (8): 959-61; Ibrahim Iliya et al., "Dimeric Stilbenes from Stem Lianas of Gnetum Africanum", VOL.57; NO.6; PAGE.1057-1062 (2002), http://sciencelinks.jp /j-east/article/200213/000020021302A0470840.php).

  Specifically, Gnetum latifolium, Gnetum Africanum, and Gnetum gnemon (Melinjo) belonging to the family Gnetum can be exemplified. Melinjo is preferred. The plant part to be used is not limited to parts such as fruits (or seeds), flowers and leaves as long as it contains a large amount of gnetin C, but is preferably fruit (or seeds), more preferably real endosperm. .

  Here, when extracting gnetin C from a plant, the method is not particularly limited, and examples thereof include a method of extracting using a polar solvent and a method of extracting using a supercritical extraction method using carbon dioxide or the like. When extracting with a polar solvent, examples of the extraction solvent to be used include water, lower alcohols (methanol, ethanol, n-propanol, isopropanol, etc.), ketones (acetone, methyl ethyl ketone, methyl butyl ketone, etc.), esters (acetic acid, etc.). Methyl, ethyl acetate, etc.), glycols (ethylene glycol, propylene glycol), glycerin, tetrahydrofuran, acetonitrile, acetic acid, propionic acid, acetamide, dimethylformamide, and dimethyl sulfoxide.

  These extraction solvents can be used alone or in a combination of two or more. Preferably, water, lower alcohol (preferably ethanol) or a mixture thereof (hydrous alcohol, preferably hydrous ethanol). In addition, when using water-containing alcohol (preferably water-containing ethanol) as an extraction solvent, although it does not restrict | limit, as ethanol concentration, 20-90 volume%, Especially 40-80 volume% can be mentioned suitably.

  Extraction is carried out by adding a part or all of the above plant to the solvent, followed by soaking or soaking for 30 minutes to 3 days at a temperature where the solvent boils, usually at a temperature of 100 ° C or lower, and then insoluble. This can be done easily by removing the object. The solvent extract thus obtained is further subjected to the isolation and purification treatment of gnetin C. Such isolation and purification treatment can be performed by combining one or more conventional methods. Examples of such treatment include a precipitation method, a liquid-liquid counter-current distribution method, an adsorption treatment with activated carbon, a resin, and the like, a purification treatment with column chromatography, ion exchange chromatography, and the like. In addition, isolation and purification of gnetin C can be performed using the following behavior as an index in addition to the molecular weight (454) and maximum absorption wavelength (320 nm) of gnetin C.

<Thin layer chromatography (TLC)>
The Rf value when analyzed under the following conditions is 0.61.

Silica gel 60F _254, developing solvent: chloroform containing 20 volume% methanol, detection wavelength: 254 nm.

<High performance liquid chromatography (HPLC)>
The retention time when analyzed under the following conditions is 33.5 minutes.
Column: Tosoh TSKgel ODS-100V, 5μm 4.6 × 150 mm,
Moving bed: A liquid: 1 volume% acetic acid containing water, B liquid: 1 volume% acetic acid containing methanol,
Gradient condition: 0 minutes → 10 minutes: A: B = 65: 35 (v / v) → 63: 37 (v / v), 10 minutes → 20 minutes: A: B = 63: 37 (v / v) → 56: 44 (v / v), 20 minutes → 40 minutes: A: B = 48: 52 (v / v),
Detection wavelength: 320 nm,
Flow rate: 0.8 ml / min.

  Note that purification does not need to be performed up to a purity of 100%. Gnetin C used in the present invention may have a purity of usually 50% or higher, preferably 80% or higher, more preferably 90% or higher.

  Moreover, when producing | generating gnetin C using microorganisms, also when acquiring by chemical synthesis, it is preferable to perform a refinement | purification process similarly to the above.

  Gnetin C may be used in a free state or in a salt state. Here, the salt is a salt formed by gnetin C and may be any salt that is acceptable as an external medicine. For example, salts with inorganic bases such as sodium, potassium, magnesium, calcium and aluminum; salts with organic bases such as methylamine, ethylamine and ethanolamine; salts and ammonium salts with basic amino acids such as lysine, ornithine and arginine Is mentioned. The salt may be an acid addition salt. Specific examples of the salt include hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, phosphoric acid and other mineral acids; formic acid, acetic acid, Organic acids such as propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, methanesulfonic acid, ethanesulfonic acid; and acidic amino acids such as aspartic acid, glutamic acid Examples include acid addition salts. Gnetin C also includes hydrates, pharmaceutically acceptable solvates and crystal polymorphs.

  The tyrosinase inhibitor of the present invention may consist only of the above isolated or purified gnetin C or a salt thereof (gnetin C or a salt thereof 100%), but the tyrosinase inhibitory action of gnetin C is not hindered. As long as other components (attachment components) are known, compounds known as tyrosinase inhibitors (for example, kojic acid, arbutin, hydroquinone), plant extracts, and the like may be blended. Further, it may contain carriers such as fats and oils, hydrocarbons, fatty acids, water, alcohols or esters, and additives such as antioxidants and bactericidal / antibacterial agents. The proportion of gnetin C in the tyrosinase inhibitor in the case of containing such other components may be a concentration of at least 3 μM where gnetin C exhibits tyrosinase inhibitory activity (see Experimental Example 1). 98% by weight can be exemplified.

  The form of the tyrosinase inhibitor of the present invention is not particularly limited. For example, the tyrosinase inhibitor may have a solid preparation form such as a powder form, a granule form, a tablet form, a capsule form, or a liquid form, a suspension form, You may have liquid formulation forms, such as emulsion.

  Since the tyrosinase inhibitor of the present invention has an action of suppressing melanin production by inhibiting tyrosinase in pigment cells (see Experimental Example 2), whitening (including prevention and reduction of spots and dullness) is effective. It can be suitably used as an active ingredient in skin external preparations (whitening external preparations), particularly whitening cosmetics.

(2) Whitening agent The whitening agent of the present invention is characterized by containing gnetin C, which is effective as a tyrosinase inhibitor, as an active ingredient.

  Gnetin C may be used in a free state or in a salt state. Here, as described above, the salt may be a salt that is formed by gnetin C and that is acceptable as a pharmaceutical product. Gnetin C also includes hydrates, pharmaceutically acceptable solvates and crystal polymorphs.

  The whitening agent of the present invention may comprise only the isolated or purified gnetin C or a salt thereof (gnetin C or a salt thereof 100%), but the tyrosinase inhibitory action of gnetin C or melanin production thereby As long as the whitening action based on the inhibitory action is not hindered, as other ingredients (attachment ingredients), compounds known as whitening agents (for example, ascorbic acid or derivatives thereof, sulfur, placenta extract, ellagic acid or derivatives thereof, koji Acid or its derivatives, Glucosamine or its derivatives, Azelain or its derivatives, Arbutin or its derivatives, Hydroxycinnamic acid or its derivatives, Glutathione, etc., Various plant extracts (Arnica extract, Ogon extract, Ogon extract, Senkyu extract, Sahakuhi Extract, Psycho extract, Bow Ueki , Cinnamon humus extract, Candida mycelium culture or extract thereof, Gymnema extract, Linden extract, Peach leaf extract, Ages extract, Kujin extract, Chiyu extract, Toki extract, Yakuinin extract, Oyster leaf extract, Daio extract, Buttonpi extract, Hamamelis extract , Marronnier extract, hypericum extract, licorice extract) and beekeeping products (e.g., processed products such as royal jelly, propolis, pollen, or chinoko (e.g., extract)). Further, it may contain carriers such as fats and oils, hydrocarbons, fatty acids, water, alcohols or esters, and additives such as antioxidants and bactericidal / antibacterial agents. When such other components are included, the proportion of gnethine C in the whitening agent may be at least 3 μM in concentration of gunetin C exhibiting a whitening action based on tyrosinase inhibitory activity (see Experimental Example 1). For example, 1 to 98% by weight can be exemplified.

  The form of the whitening agent of the present invention is not particularly limited. For example, the whitening agent may have a solid preparation form such as powder, granule, tablet, capsule or the like, and may be liquid, suspension, emulsion. You may have liquid formulation forms, such as a shape.

  Since the whitening agent of the present invention has an action of suppressing the production of melanin based on the tyrosinase inhibitory activity of gnetin C, the skin external preparation (including whitening prevention and reduction) is effective. Agent), particularly as an active ingredient of whitening cosmetics.

(3) Whitening external preparation In the present invention, the whitening external preparation means a skin external preparation used for the purpose of whitening (including prevention and reduction of spots and dullness). The external preparation for skin includes external medicines and cosmetics. Whitening cosmetics are preferred.

  The whitening external preparation of the present invention is characterized by containing the above-described tyrosinase inhibitor or whitening agent containing gnetin C as a whitening component as an active ingredient.

  Such a whitening external preparation can be prepared by blending the aforementioned tyrosinase inhibitor or whitening agent in the base of the external preparation and formulating it into an arbitrary dosage form according to a conventional method. Here, the dosage forms of the whitening external preparation are aqueous solution system, solubilization system, emulsification system, powder system, oil liquid system, gel system, ointment system, aerosol system, water-oil two-layer system, and water-oil-powder 3 A wide range of dosage forms such as layer systems can be adopted. Examples of the form include lotion, milky lotion, cream, ointment, gel, foam, essence, cosmetic liquid, pack, mask and the like.

In addition, as a base for external preparations, any substance may be used as long as it does not interfere with the melanin production inhibitory activity based on the tyrosinase inhibitory activity of gnetin C. Can do. For example, as a base for external preparations, the following oils and fats, hydrocarbons, fatty acids, water, alcohols or esters can be exemplified:
Fats and oils: soybean oil, linseed oil, tung oil, sesame oil, nutka oil, cottonseed oil, rapeseed oil, safflower oil, corn oil, olive oil, coconut oil, almond oil, castor oil, peanut oil, cacao oil, coconut oil, palm oil, palm Nuclear oil, beef tallow, mink oil, egg yolk oil, jojoba oil, evening primrose oil, horse oil.

  Waxes: carnauba wax, candelilla wax, beeswax, salasy beeswax, whale wax, serax, lanolins.

  Hydrocarbons: liquid paraffin, petrolatum, microcrystalline wax, ceresin, squalane, polyethylene powder.

  Fatty acids: stearic acid, linoleic acid, lauric acid, myristic acid, palmitic acid, hebenic acid, lanolinic acid, oleic acid, undecylenic acid, isostearic acid.

  Alcohols: lauryl alcohol, cetyl alcohol, stearyl alcohol, lanolin alcohol, hydrogenated lanolin alcohol, oleyl alcohol, hexadecyl alcohol, 2-octyldodecanol, glycerin, sorbitol, propylene glycol, 1,3-butylene glycol, ethylene glycol and Its polymer, glucose, sucrose, cholesterol, phytosterol, cetostearyl alcohol.

  Esters: Decyl oleate, butyl stearate, myristyl myristate, hexyl laurate, isopropyl palmitate, isopropyl myristate, octyldodecyl myristate, hexyldecyl dimethyloctanoate, propylene glycol dioleate, diethyl phthalate, monostearic acid Propylene glycol, ethylene glycol monostearate, glyceryl monostearate, glyceryl trimyristate, lanolin acetate, cetyl lactate.

  Further, the whitening external preparation of the present invention includes components usually used in cosmetics and external preparations, such as moisturizers, astringents, cell activators, antioxidant / active oxygen scavengers, ultraviolet absorbers, ultraviolet cut agents, and interfaces. An activator, a thickener, a powder component, a coloring material, a bactericidal / antibacterial agent, an anti-inflammatory / antiallergic agent, various skin nutrients, a fragrance and the like can be appropriately blended as necessary.

  The ratio of the tyrosinase inhibitor or the whitening agent contained in the whitening external preparation of the present invention is not particularly limited as long as the whitening external preparation exhibits a whitening effect, but in terms of the amount of Gunnetin C, C is preferably blended at a ratio of 3 μM or more. As shown in Experimental Example 1 (FIG. 1), gnetin C has tyrosinase inhibitory activity at 3 μM or more, and tyrosinase inhibitory activity increases as the concentration increases, and becomes almost plateau at 10 μM or more. As can be seen from this, the upper limit of the amount of gnetin C is not particularly limited from the viewpoint of exerting a whitening effect. From practicality, a concentration of 0.003 mM to 66 mM, preferably 0.1 mM to 40 mM, more preferably 0.2 mM to 20 mM, particularly preferably 1 mM to 10 mM (about 0.05 to 0.5% by weight) can be mentioned.

  Further, as shown in Experimental Example 1 (FIG. 1), gnetin C has a conventionally known tyrosinase inhibitory action at a relatively low concentration, for example, 3 to 60 μM, preferably 7.5 to 30 μM, more preferably 15 to 30 μM. And high tyrosinase inhibitory activity. Thus, the tyrosinase inhibitor of the present invention is characterized by having a high tyrosinase inhibitory activity even when it contains gnetin C in the low concentration range.

  The form of the whitening external preparation is not particularly limited. For example, when the whitening external preparation is a basic cosmetic, examples of the form include skin lotion, milky lotion, cream, gel, essence, cosmetic liquid, pack, and mask. Other forms include face wash, massage agent, cleansing agent, after shave lotion, pre-shave lotion, shaving cream, body soap, soap and the like.

  EXAMPLES Hereinafter, although an experiment example and an Example demonstrate this invention in detail, this invention is not limited at all by these Examples.

Preparation Example 1 Preparation of Gnetin C Gnetin C was isolated according to the following method.

  (i) According to the description in paragraph [0024] of JP-A-2009-013123, a crushed product of dried fruit of Merinjo is immersed in hydrous ethanol at room temperature, and the resulting extract is concentrated under reduced pressure to obtain a solid content. A meringo extract paste containing 63.2% by weight was obtained.

(ii) 2.5 g of the meringo extract paste obtained above was subjected to column chromatography (gel type: Cosmosil 75C ₁₈ -PREP (manufactured by Nacalai Tesque), column size: φ3 × 26.5 cm) and methanol as the eluent. Stepwise elution was sequentially performed using water-containing methanol having a content of 10%, 25%, 40%, 80%, and 100% by volume (each of the eluents 600 mL was poured and 100 mL was fractionated). When each component obtained was confirmed by HPLC under the following conditions, it was confirmed that Gnetin C was eluted at a retention time of 33.5 minutes (water-containing methanol elution fraction containing 1% by volume acetic acid and 80% by volume methanol).

<HPLC conditions>
Column: Tosoh TSKgel ODS-100V, 5μm 4.6 × 150 mm,
Moving bed: A liquid: 1 volume% acetic acid-containing water, B liquid: 1 volume% acetic acid-containing methanol,
Gradient condition: 0 minute → 10 minutes: A: B = 65: 35 (v / v) → 63: 37 (v / v), 10 minutes → 20 minutes: A: B = 63: 37 (v / v) → 56: 44 (v / v), 20 minutes → 40 minutes: A: B = 48: 52 (v / v)
Detection wavelength: 320 nm,
Flow rate: 0.8 ml / min.

   (iii) After concentrating the above fractions, the fractions were sequentially eluted by medium pressure column chromatography under the following conditions.

<Medium pressure column chromatography conditions>
Gel type: Silica gel, Daisogel IR-60-40 / 63-W,
Column size: φ2 × 7.48 cm,
Detection wavelength: 320 nm
Moving layer: A liquid: methanol, B liquid: chloroform,
Gradient condition: 0 minute → 2 minutes: A: B = 11: 89 (v / v), 2 minutes → 8 minutes: A: B = 11: 89 (v / v) → 18: 82 (v / v) 8 minutes → 12 minutes: A: B = 18: 82 (v / v), 12 minutes → 14 minutes: A: B = 21: 79 (v / v), 14 minutes → 20 minutes: A: B = 21 : 79 (v / v) → 29: 71 (v / v), 20 minutes → 24 minutes: A: B = 29: 71 (v / v), 24 minutes → 30 minutes: A: B = 29: 71 ( v / v) → 36:64 (v / v), 30 minutes → 34 minutes: 36:64 (v / v),
Flow rate: 60 ml / min.

  The eluate was fractionated 60 mL each, and the 29th to 30th fractions having an absorption peak at a detection wavelength of 320 nm were concentrated by an evaporator to obtain gnetin C (35.2 mg, purity 97%).

Experimental Example 1 Measurement of Tyrosinase Inhibitory Activity (1) Test Method The tyrosinase inhibitory activity of each test sample (Gnetin C, trans-resveratrol, kojic acid) was evaluated by measuring the dopa oxidation activity of tyrosinase.
Specifically, first, in each well of a 96-well plate, as a test sample, Gunnetin C (obtained from Hosoda SHC), trans-resveratrol (Sigma), kojic acid (Sigma), or DMSO (Sigma) as a control. 50 μL of mushroom-derived tyrosinase solution (prepared with 50 mM phosphate buffer (pH 6.8)) prepared at 40 U / ml, stirred for 1 minute, and then at 37 ° C. for 10 minutes Incubated. The reaction mixture (1.1 mM L-DOPA, 6.7 mM MBTH [3-methyl-2-benzothiazolinone hydrazone] (Sigma), 2.7% N, N-dimethylformamide (Sigma) / 17 mM phosphate buffer (pH 6.8)) was added at 130 μL and incubated at 37 ° C. for 10 minutes, and then the absorbance at 515 nm was measured and used as the measured value (test sample measured value, control measured value). At the same time, the case where 50 mM phosphate buffer (pH 6.8) was added instead of tyrosinase was measured, and this was used as the background value. The average value of 3 wells was taken as one data, and 3 independent tests were performed.

The tyrosinase inhibitory activity (%) was derived from the following formula, and the IC _{50 of} each test sample was determined from the obtained inhibitory activity (%).

(2) Test results Table 1 shows the tyrosinase inhibitory activity (%) of each test sample and the IC ₅₀ obtained therefrom.

  As can be seen from the above, it has been found that Gunnetin C has a significantly stronger tyrosinase inhibitory activity than resveratrol and kojic acid, which are conventionally known to have tyrosinase inhibitory activity. Further, as can be seen from FIG. 1, it was found that gnetin C exhibits a strong tyrosinase inhibitory activity particularly at a low concentration of at least about 3 μM.

Experimental Example 2 Inhibitory effect on cell melanin production (1) Test method
Mouse melanoma B16 cells (DS Pharma Biomedical) were seeded at a rate of 5 × 10 ⁴ cells / well on Eagle MEM medium (Nissui) in each well of a 96-well plate, at 37 ° C. and 5% CO ₂ . Incubated under conditions for 24 hours. Then, theophylline (Sigma) was added to a final concentration of 500 μM, and to this was added gnetin C (15 μM), kojic acid (1000 μM) as a test sample, and solvent (DMSO) as a control, at 37 ° C., 5% CO _2. For 3 days. After culture, the medium was removed, 500 μL of trypsin-EDTA solution (Gibco) was added to detach the cells, and the resulting cell suspension was collected in a tube.

When collecting the cells in a tube, a small amount of the cell suspension was taken and the number of cells was measured using a CellTiter-Glo ^™ kit (Promega). The tube from which the cell suspension was collected was centrifuged at 15,000 rpm for 10 minutes, and the cells were collected at the bottom of the tube. Then, the supernatant was removed and a photograph was taken (FIG. 2 (a)). After shooting, add 1N NaOH in an amount corresponding to the number of cells, incubate at 95 ° C for 60 minutes, extract melanin, measure the absorbance at 405 nm, and use it as the measurement value (test sample measurement value, control measurement value) . At the same time, the absorbance of 1N NaOH was measured and used as a background value (test sample BG value, control BG value).

  From the obtained measured value, the amount of melanin was calculated | required about each test sample and control using the following Formula. Three independent tests were performed, and the amount of melanin in the test sample was calculated with the amount of control melanin obtained in each test as 100.

(2) Test results Fig. 2 (a) shows a photographic image of cells accumulated at the bottom of the tube, and Fig. 2 (b) shows the results of contrasting melanin levels for each test sample (Gnetin C, Kojic acid) and control. .

  In Example 1, it was shown that gnetin C has tyrosinase inhibitory activity. From the results shown in FIG. 2, it is shown that gnetin C has an action of suppressing melanin production in pigment cells based on the tyrosinase inhibitory activity. Was confirmed. And as shown in Example 1, it was confirmed that the effect is significantly higher than that of kojic acid.

Formulation Example 1 : Whitening agent having a lotion form Using each component shown in Table 2 below, a whitening external preparation having a lotion form was prepared according to a conventional method. Here, 97% purity prepared in Preparation Example 1 is used as Gnetin C (the same applies to Formulation Examples 2 and 3).

Formulation Example 2 : Whitening agent having a cosmetic liquid form Using each of the components shown in Table 3 below, a whitening external preparation having a cosmetic liquid form was produced according to a conventional method.

Formulation example 3 : Whitening agent having emulsion form An emulsion (whitening preparation for external use) was produced according to a conventional method using each component (% by weight) shown in Table 4 below.

Claims (3)

  A tyrosinase inhibitor comprising gnetin C or a salt thereof isolated or purified as an active ingredient.   A whitening agent containing, as an active ingredient, isolated or purified gnetin C or a salt thereof.   A whitening external preparation containing the tyrosinase inhibitor according to claim 1 or the whitening agent according to claim 2 as an active ingredient.

Images