JP2011184354A - Bleaching agent inhibiting melanin uptake into keratinocyte - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an inexpensive and safe serine protease inhibitor having a high effectiveness and consisting of a natural plant extract, and a cosmetic composition for bleaching and a medicinal composition for bleaching, having an effect of inhibiting melanin uptake into keratinocytes by the serine protease inhibitory effect. <P>SOLUTION: The serine protease inhibitor is provided by including an extract of Balanophora fungosa as an active ingredient, and the cosmetic composition for bleaching and medicinal composition for bleaching having the effect of inhibiting the melanin uptake into the keratinocytes by the serine protease inhibitory effect are also provided. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a serine protease inhibitor containing aspergillus plant extract as an active ingredient, and a whitening makeup having the effect of suppressing the uptake of melanin into keratinocytes by the serine protease inhibitory action, containing the inhibitor as an active ingredient The present invention relates to a coating composition and a whitening pharmaceutical composition.

  Many of the whitening cosmetics on the market have a function of inhibiting the activity of tyrosinase (or polyphenol oxidase) that works to produce melanin in melanocytes (also called epidermal melanocytes). As substances having tyrosinase inhibitory activity, kojic acid, arbutin, and L-ascorbic acid are well known. As substances having tyrosinase inhibitory activity, ferulic acid, caffeic acid, o-coumaric acid, p-coumaric acid, cinnamic acid and derivatives thereof have been reported for a long time (Non-patent Documents 1, 2, and 3). ). In addition, mushroom tyrosinase inhibitory activity of N, N'-diferloyl putrescine isolated from corn bran, melanin inhibitory activity in B16 melanoma cells (Non-patent Document 4), N- isolated from safflower seeds Mushroom tyrosinase inhibitory activity of feruloyl serotonin and N- (p-coumaroyl) serotonin and melanin production inhibitory activity in B16 melanoma cells have been reported (Non-patent Document 5). There is a published patent publication (Patent Document 1) of a coffee fruit cosmetic composition and method, and the preparation is a compound selected from the group consisting of coffee acid, coffee polyphenol, essential monosaccharide, coffee mucus polysaccharide, and trigonelline UV protection effect, skin whitening effect is mentioned. In connection with the present patent application, mushroom tyrosinase inhibitory action of plants from Okinawa Prefecture, such as leaves of Ryukyutsuritorimochi and leaves of moxena, has been reported (Patent Document 8, Non-Patent Document 6).

  As described above, various compounds and plant extracts have been studied as melanin production inhibitors. However, as described above, tyrosinase inhibition and melanin production inhibition in B16 cells have been the main researches so far. On the other hand, melanin production in the skin is first triggered by in vivo substances such as cell growth factors and hormones (for example, bFGF, endothelin-1, α-melanocyte stimulating hormone) that stimulate melanocytes. The search and chemical synthesis of substances that inhibit the action of these substances or the production of such cell growth factors and hormones are also widely performed.

  Further, inhibition of melanin production by the mechanism as described above is not always sufficient, and various whitening agents that suppress the movement of melanin to the epidermis have been studied. Melanin is synthesized and stored in vesicles called melanosomes that exist in melanocytes. The melanosomes that store the melanin pigment move in the cell and are taken up by the keratinocytes of the epidermis through melanocyte dendrites (dendrites). At this time, the protein Protease-Activated Receptor 2 (PAR-2) on the keratinocyte membrane surface is cleaved and activated by serine proteases such as trypsin and tryptase to activate phagocytosis (Non-patent Document 7). 8, Patent Document 2). Accordingly, various dendrite elongation inhibitors have been studied, and centaureidin and the like contained in yarrow have been reported (Non-patent Document 9).

  In addition, suppression of melanosome phagocytosis, compounds that act on the PAR-2 pathway (serine protease inhibition, PAR-2 antagonists), plant extracts (tougan, prune, amadokoroko, etc.), food-derived substances (casein hydrolysates), etc. Have been studied in various ways (Patent Documents 2 to 7).

  Substances that have a whitening effect have been studied on the basis of various principles, but those based on tyrosinase inhibition are the mainstream, and the search for whitening agents that suppress melanin migration and phagocytosis is still sufficiently conducted. There is always a need for a whitening agent that is derived from natural products and has high strength and safety.

Special Table 2007-532539 Special table 2001-502360 gazette JP 2006-124355 A JP 2006-273808 A JP 2006-273809 A JP 2006-347926 JP 2008-143796 JP 2009-227612 A

Journal of Agricultural and Food Chemistry, 1993, 41, pp. 526-531 Journal of Agricultural and Food Chemistry, 1996, 44, pp. 1668-1675 Journal of Agricultural and Food Chemistry, 2002, 50, pp. 1400-1403 Journal of Agricultural and Food Chemistry, 2007, 55, pp. 3920-3925 Biological & Pharmaceutical Bulletin, 2004, 27, pp. 1976-1978 Okinawa Industrial Technology Center Research Report, 2008, No. 10, pp. 61-63 Experimental Cell Research, 2000, 254, pp.25-32 Journal of Cell Science, 2000, 113, pp.3093-3101 Biochimica et Biophysica Acta, 2006, 1760, pp.487-494 Abstract of the 1999 annual meeting of the society of investigative dermatology., J. Invest. Dermatol., 1999, 112, p. 629

  An object of the present invention is to provide an inexpensive, safe and highly effective serine protease inhibitor comprising a natural plant extract, and a whitening cosmetic having an effect of suppressing melanin incorporation into keratinocytes by a serine protease inhibitory action It is to provide a composition and a whitening pharmaceutical composition.

  The present inventors use various subtropical plant extracts as materials, inhibit serine proteases, suppress melanin uptake into keratinocytes in experiments using a three-dimensional model of human skin, and exhibit whitening effects based on these effects. As a result of diligent search for the extract, particularly subtropical plants, it was found that the extract of the plant family plant has the action and completed the present invention. Heretofore, the serine protease inhibitory action and the suppressive action of melanin uptake into keratinocytes have not been known at all.

The present invention is as follows.
(1) An inhibitor of serine protease, which contains an extract of the plant family plant as an active ingredient.
(2) The inhibitor according to (1), wherein the plant is a Ryukyu-Turimochi.
(3) The inhibitor of (1) or (2), wherein the serine protease is trypsin and / or tryptase.
(4) A whitening cosmetic composition containing the inhibitor according to any one of (1) to (3) as an active ingredient.
(5) A whitening pharmaceutical composition comprising the inhibitor according to any one of (1) to (3) as an active ingredient.
(6) The whitening cosmetic composition of (4) or the whitening pharmaceutical composition of (5), which has an effect of suppressing melanin incorporation into keratinocytes by inhibiting serine protease.

  According to the present invention, an inexpensive, safe and highly effective serine protease inhibitor comprising a natural plant extract, and a whitening cosmetic composition having an effect of suppressing melanin incorporation into keratinocytes by serine protease inhibitory action And a whitening pharmaceutical composition.

FIG. 1 is a diagram in which inhibition of melanin production in a three-dimensional human skin model of Ryukyutsuritomochi extract was confirmed. The relative value of the amount of melanin was shown beside the skin model cup. FIG. 2 is a diagram showing that the extract of Ryukyutsuritorimochi suppresses the uptake of melanin into keratinocytes in a three-dimensional human skin model, and as a result, it is confirmed that melanin exists only in melanocytes.

  Hereinafter, although the said invention is demonstrated in detail, the range of this invention is not restrained by these description and can implement suitably in the range which does not impair the meaning of this invention except the following illustration.

  The present invention relates to a serine protease inhibitor containing a plant extract as an active ingredient, and a whitening cosmetic composition and a whitening pharmaceutical composition having an effect of suppressing melanin incorporation into keratinocytes due to serine protease inhibitory action About.

  There are no particular limitations on the plant family used in the present invention, and there is no particular limitation. Examples include Balanophora nipponica Makino), Balanophora tobiracola Makino, Balanophora yakushimensis Hatsu. Et Masam., And Balanophora yuwanensis Agusawa et Sakuta.

  Ryukyu Tsuchimochi is a parasitic plant belonging to the genus Tuchitorimochi, a hermaphroditic strain, about 10 cm in height, widely distributed in the main island of Okinawa and south of the Okinawa Islands. The plant belonging to the same genus, Balanophora japonica Mak., Is a folk remedy that is used for antipyretic and detoxification by taking it.

  There are no particular restrictions on the varieties and production areas of the plant family used in the present invention, but it is desirable to use those cultivated from the standpoint of nature conservation.

  Any of the root, stem, and leaf extracts may be included in the plant extract used in the present invention, but the higher the extract ratio, the better. The extract may be in any form such as liquid, powder, paste, solid or semi-solid, but is preferably powder. A powdery striatum plant extract can be obtained by the following method. That is, after the whole plant of roots, or roots, stems, and leaves are shredded or crushed finely with a mixer, etc., extracted by adding an extraction solvent and irradiated with ultrasonic waves for about 30 minutes, centrifuged, Filtration is performed and the supernatant or filtrate is recovered as an extract. Or an extract can also be obtained by performing solvent extraction using a high-speed solvent extraction apparatus. By drying the extract under reduced pressure, a plant extract can be obtained as a powder. As the extraction solvent, water or any organic solvent containing water can be used. As the organic solvent, for example, one or more of ethanol, methyl alcohol, ethyl alcohol, ethyl acetate, 1,3-butanediol and the like can be used. The organic solvent is used at a concentration of 30 to 70%, preferably 40 to 60%, more preferably 50%.

  As described in detail in the examples below, the plant extract of the genus Trichymeaceae has a serine protease inhibitory action and can be used as a serine protease inhibitor. In particular, the plant extract of Trichophyceae has trypsin and tryptase inhibitory activity as described in detail in the following examples, and can be used as a trypsin inhibitor and / or a tryptase inhibitor.

  Further, the plant extract of the genus Trichomyceae can suppress the uptake of melanin into keratinocytes when applied to the skin due to its serine protease inhibitory action. That is, the plant plant extract can be used as an inhibitor of melanin uptake into keratinocytes.

  In addition, the plant extract of the genus Trichymeaceae has a clear whitening effect due to its inhibitory action on the uptake of melanin into keratinocytes. “Whitening effect” means prevention or improvement of spots, freckles, dullness, etc. caused by melanin deposition in the skin.

  Therefore, the plant extract can be used as an active ingredient of a cosmetic composition or a pharmaceutical composition for improving, preventing or treating a condition, symptom or disease of a subject caused by melanin uptake and accumulation in keratinocytes. . Such conditions, symptoms or diseases include, but are not limited to, blackening of the skin, spots, freckles and the like.

  The extract of the plant family can be used as an active ingredient of a cosmetic composition or a pharmaceutical composition having a whitening effect in the following form.

  The plant plant extract can be used as an active ingredient of a whitening cosmetic composition having an action of suppressing melanin uptake into keratinocytes. As a cosmetic composition for whitening, the plant extract itself may be applied directly, or in the form of a solution, a solubilized form, an emulsified form, a powder form, a paste form, a mousse form, a gel form, As a lotion, emulsion, cream, pack, ointment, etc., it can be applied to the skin, for example, in the form of a skin external preparation.

  In addition, the whitening cosmetic composition is a component that is usually used in preparations of cosmetics, quasi-drugs, external medicines, etc., as long as the effects of the plant extract are not impaired as necessary. That is, purified water, alcohols, water-soluble polymers, oil agents, surfactants, gelling agents, humectants, vitamins, antibacterial agents, fragrances, salts, pH adjusters and the like can be added. The cosmetic composition for whitening containing the extract of the plantaceae plant extract may be one in which a label indicating that the whitening action of the skin is exhibited is attached to the package.

  The content of the plant extract (dry weight) in the whitening cosmetic composition (total weight) is preferably 0.05% by weight to 20% by weight, and more preferably 0.1% by weight to 0.4% by weight.

  In the case of using a plant extract, as the active ingredient of a whitening cosmetic composition, the dosage of the extract is not particularly limited. For example, when the extract is applied directly to the skin, the extract is used. Of 0.1 to 1% by weight of a cosmetic composition may be administered by applying 1 to 4 times per day.

  The plant extract can also be used as an active ingredient of a whitening pharmaceutical composition having an action of suppressing melanin uptake into keratinocytes.

  As the whitening pharmaceutical composition, the plant extract itself may be applied directly, or may be formulated in combination with a known pharmacologically acceptable carrier. Examples of such carriers include various types commonly used for ordinary drugs, such as excipients, binders, disintegrants, lubricants, diluents, solubilizers, suspending agents, isotonic agents, pH. Examples include regulators, buffers, stabilizers, colorants, flavoring agents, and flavoring agents.

  The dosage form of the whitening pharmaceutical composition is not particularly limited and is appropriately selected as necessary. Generally, transdermal absorbents, transmucosal absorbents, ointments, patches, suppositories, inhalants, injections, It is used as parenterals such as drops, or as oral preparations such as tablets, capsules, granules, fine granules, powders, solutions, syrups, suspensions, emulsions and elixirs. The transdermal absorption agent is, for example, components used in preparations such as external medicines, that is, purified water, alcohols, water-soluble polymers, oils, surfactants, gelling agents, moisturizers, vitamins, antibacterial agents, A fragrance | flavor, salts, a pH adjuster, etc. can be added. Oral preparations are produced according to a conventional method using excipients such as starch, lactose, sucrose, mannitol, carboxymethylcellulose, corn starch, and inorganic salts.

  The content of the plant extract (dry weight) in the whitening pharmaceutical composition varies depending on various conditions such as the type, size and range of the application target of the composition, the administration route, or the dosage form. The pharmaceutical composition (total weight) contains 0.05 wt% to 40 wt%. The dose of the plant extract (dry weight) is not particularly limited. For example, the degree of skin blackening, spots, freckles, etc., the patient's age, sex, weight, degree of symptoms, or administration It can be determined appropriately according to the method and the like. For example, in the case of percutaneous absorption administration, it is preferably applied 1 to 4 times per day.

  In the whitening cosmetic composition or whitening pharmaceutical composition of the present invention, the amount of melanin produced in melanocytes is 10 as compared with the case where the whitening cosmetic composition or whitening pharmaceutical composition was not applied. %, 20%, 30%, 40%, 45%, 50% or more.

  Further, the whitening cosmetic composition or whitening pharmaceutical composition of the present invention has a melanin incorporation into keratinocytes in comparison with the case where the whitening cosmetic composition or whitening pharmaceutical composition is not applied. 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more can be suppressed.

  Furthermore, the extract of plants is a topical agent for the improvement of pruritus, wrinkles, sagging, rough skin due to the inhibitory action of tryptase, systemic anaphylaxis disease, aspirin-sensitive asthma, asthma, interstitial lung disease, interstitial bladder Inflammation, irritable bowel syndrome, allergic disease, atopic disease, skin blistering, hypersensitivity, pain, pruritus, gingivitis, edema, psoriasis, pulmonary fibrosis, rheumatoid arthritis, arthritis, periodontal disease, blood External preparations, medications or foods and drinks for the treatment or prevention of diseases selected from the group consisting of coagulation disorders, renal interstitial fibrosis, increased vascular permeability or pulmonary edema as a side effect of X-ray contrast media, and hay fever It can also be used as an active ingredient of a composition. It is known that tryptase inhibitors are useful for the treatment or prevention of the above diseases (Japanese Patent Laid-Open No. 2007-186457). The external preparation, medication or food / beverage composition is usually used for the above-mentioned whitening in combination with ingredients used in preparations such as cosmetics, quasi-drugs, external medicines, or pharmacologically acceptable carriers. It can be prepared in the same manner as a cosmetic composition or a whitening pharmaceutical composition.

  EXAMPLES Hereinafter, the present invention will be specifically described by way of examples, but these do not limit the scope of the present invention.

(Example 1) Method for producing extract of Ryukyutsurimochi After cutting the whole plant of Ryukyutsurimochi to an appropriate size, each is dried at 60 ° C for 12 hours, and this is centrifuged (MRK-Retsch, ZM- 100). The powder that passed through the 0.5 mm screen was collected, and this powder was subjected to solvent extraction using a high-speed solvent extraction device (ASE-200, manufactured by Nippon Dionex). That is, a dry weight of 2.0 g of powder was loaded into an extraction cell together with 8.0 g of diatomaceous earth, 50% ethanol, extraction temperature of 80 ° C., extraction time of 10 minutes, pressure of 1500 psi, and the number of extractions twice, for a total of 50 mL. An extract was obtained. The extract was dried under reduced pressure using a centrifugal evaporator to obtain 1.3 g of extract.

(Example 2) Serine protease (trypsin) inhibitory activity of Ryukyutsuritomochi extract Using porcine pancreatic trypsin (purchased from Wako Pure Chemical Industries) as a serine protease, and BAPA (benzoyl-arginine-p-nitroanilide) as a synthetic substrate The experiment was conducted using a 96-well microplate. Place 10 μl of the test sample solution (dissolved in 0.25 to 2.0 mg / ml of the extract of Ryukyu Torimochi obtained in Example 1 or distilled water as a control) into each well of the microplate. 50 μl of 30 μg / ml porcine pancreatic trypsin (dissolved in 50 mM Tris-HCl buffer (pH 8.0) containing 10 mM CaCl ₂ ) was added and shaken at 25 ° C. for 5 minutes. Next, 100 μl of 0.75 mM BAPA (dissolved in 50 mM Tris-HCl buffer (pH 8.0) containing 10 mM CaCl ₂ , 1.0% (v / v) DMSO) was added and shaken at 25 ° C. for 20 minutes. After stopping the reaction by adding 50 μl of 45% acetic acid, the absorbance at 405 nm of paranitroaniline produced by the decomposition of BAPA was measured with a microplate reader. In this experiment performed at n = 4, the concentration of the test sample that inhibits the activity of trypsin by 50% was defined as the IC ₅₀ value. Ryukyutsuritomochi extract inhibited the activity of trypsin, and the IC ₅₀ value was 75 μg / ml.

(Example 3) Serine protease (tryptase) inhibitory activity of Ryukyutsuritorimochi extract Experiment using human lung tryptase (purchased from Sigma) as serine protease and BAPA (benzoyl-arginine-p-nitroanilide) as synthetic substrate Went. Place 10 μl of the test sample solution (Ryukyu-tori-mochi extract obtained in Example 1 in distilled water at a concentration of 0.25 to 1.0 mg / ml or distilled water as a control) in a 500 μl microtest tube. 50 μl of 60 μg protein / ml human lung tryptase (purchased from Sigma, dissolved in 40 mM Tris-HCl buffer (pH 8.1) containing 0.15 M NaCl, 10 mM CaCl ₂ , 50 μg / ml heparin) Mix and incubate at 30 ° C. for 5 minutes. Next, add 100 μl of 0.75 mM BAPA (0.15 M NaCl, 10 mM CaCl ₂ , 50 μg / ml heparin, dissolved in 40 mM Tris-HCl buffer (pH 8.1) containing 1.0% (v / v) DMSO). Incubated at 30 ° C for 60 minutes. The reaction was stopped by adding 50 μl of 45% acetic acid, and paranitroaniline produced by the decomposition of BAPA was quantified by high performance liquid chromatography (HPLC). HPLC uses a μBondasphere 5μC ₁₈ 300 mm column (3.9 × 150 mm; manufactured by Waters) as a column, and 0 to 63% (V / V) acetonitrile aqueous solution containing 0.1% trifluoroacetic acid as an eluent. This was carried out by detecting the ultraviolet absorption at 405 nm under the conditions of a linear concentration gradient (20 minutes) and a flow rate of 1 ml / min. In this experiment conducted with n = 2, the concentration of the test sample that inhibits the activity of tryptase by 50% was defined as the IC ₅₀ value. Ryukyutsuritomochi extract inhibited the activity of tryptase, and the IC ₅₀ value was 44 μg / ml. In the same test, 6 μg / ml leupeptin tested as a positive control substance inhibited the activity of tryptase by 96%.

(Example 4) 3D human skin three-dimensional model of Ryukyutsuritorimochi extract suppresses melanin transfer to the epidermis and inhibits melanin production Normal human skin three-dimensional model purchased from Kurabo Industries Co., Ltd. (MEL-300-B kit, Black donor) was used for the test. 0.9 ml of a long-term maintenance medium EPI-100LLMM (containing bFGF and α-melanocyte stimulating hormone, purchased from Kurabo Corporation) was added to each well of the 6-well plate. The skin model cup was transferred to the 6-well plate with tweezers and cultured at 37 ° C. for 1 hour in a humidified state in the presence of 5% CO _{2 in} a carbon dioxide incubator. Next, two stainless steel washers were placed in the center of each hole of another 6-well plate, and 5 ml of the long-term maintenance medium EPI-100LLMM was added thereto. On this washer, the skin model cup was transferred with tweezers, and the test sample solution (12.5% (v / v) 0.5 to 2 mg / ml Ryukyu-tori-mochi extract, 12.5% (v / v) Add 100 μl of 1 mg / ml arbutin dissolved in aqueous glycerol and 12.5% (v / v) aqueous glycerol as a control) in a humidified state in the presence of 5% CO ₂ in a carbon dioxide incubator at 37 ° C. For 10 days. During the culture period, the test sample solution and the medium were changed every two days. On day 10 of culture, the skin model cup was transferred to a 24-well plate with tweezers, each cup was washed three times with Dulbecco-PBS solution, and after removing the washing solution, a photograph of the cups arranged on the 24-well plate was taken. Micrographs were also taken. They are shown in FIGS.

  Next, melanin in the skin model cup is extracted as follows according to a known method (Abstract of the 1999 annual meeting of the society of investigative dermatology., J. Invest. Dermatol., 1999, 112, p. 629). Quantified. Transfer the skin model cup to a 24-well plate, add 0.45 ml of 10 mM Tris-HCl buffer (pH 6.8) containing 1% SDS and 0.05 mM EDTA to each skin model cup, and add 5 mg / ml. 20 μl of protease K was added, sealed and reacted at room temperature for 3 hours. The solution and cells in the cup were all collected in a 1.5 ml tube and allowed to react overnight at 45 ° C. Next, for the purpose of washing and removing the dye contained in a trace amount in the Ryukyutsurimochi extract, all 1.5 ml tubes including the control group were centrifuged at 15000 rpm for 15 minutes, and the supernatant was discarded. To this tube, 0.45 ml of 10 mM Tris-HCl buffer solution (pH 6.8) containing 0.05 mM EDTA was added, stirred, centrifuged again under the same conditions, and the supernatant was discarded. To this tube, add 0.45 ml of 10 mM Tris-HCl buffer (pH 6.8) containing 1% SDS and 0.05 mM EDTA, add 20 μl of 5 mg / ml protease K, and react at 45 ° C for 4 hours. It was. Next, 50 μl of 500 mM sodium carbonate was added, then 10 μl of 30% aqueous hydrogen peroxide was added, reacted at 80 ° C. for 30 minutes, and then cooled. Next, 100 μl of chloroform: methanol (2: 1) was added and centrifuged at 10,000 × g for 10 minutes. 100 μl of the supernatant was collected, transferred to a 96-well plate, and the absorbance at 405 nm was measured with a microplate reader. The amount of melanin is shown next to the skin model cup in FIG. 1 as a relative value (average value of n = 3 ± standard deviation) with the amount of melanin in the control group (12.5% (v / v) glycerol aqueous solution) as 100%. Moreover, when a significant difference was recognized between the test sample addition group and the control group, it was indicated as * p <0.05, ** p <0.01, *** p <0.001.

  A clear melanin production inhibitory effect was recognized in the group added with Ryukyutsuritorimochi extract and the group added with arbutin by comparing the photograph of the skin model cup in FIG. 1 and the extraction / quantification of melanin. In addition, as shown in the micrograph of FIG. 2, melanin diffuses around the melanocytes in the control group, and even in the arbutin-added group, the amount of melanin is small but diffused around. On the other hand, it was clarified that melanin was present only in melanocytes in the group added with Ryukyu-tori-mochi extract, and that Ryukyu-tuchi-mochi extract suppressed the movement of melanin to the epidermis.

Next, for the purpose of examining the cytotoxicity of the test sample solution, a test sample (4 mg / ml of Ryukyutsuri Trimochi dissolved in 12.5% (v / v) glycerol aqueous solution was separately prepared in the skin model cup by the same method as above. The extract, 1 mg / ml arbutin dissolved in 12.5% (v / v) glycerol aqueous solution, and 100 μl of 12.5% (v / v) glycerol aqueous solution as a control (both n = 3) were added and cultured for 10 days. Put 300 μl of MTT solution (MTT-100 kit, purchased from Kurabo Industries Co., Ltd.) into each hole of the 24-well plate, transfer the skin model cup washed 3 times with Dulbecco-PBS solution with tweezers, and in a carbon dioxide incubator It was allowed to stand at 37 ° C. for 3 hours in a humidified state in the presence of 5% CO ₂ . Next, the skin model cup was gently washed with a PBS solution and then transferred to another 24-well plate. 2 ml of MTT extract (MTT-100 kit, purchased from Kurabo Industries, Inc.) was placed in each hole, and extracted at room temperature for 2 hours while shaking. 200 μl of this extract was put in each hole of a 96-well plate, and the absorbance at 570 nm was measured with a microplate reader. The relative absorbance of the 4 mg / ml Ryukyu Trimochi extract added group with the absorbance at 570 nm of the control group as 100% is 107 ± 4%, and the relative absorbance of the 1 mg / ml arbutin added group is 98. It was ± 7%, and no cytotoxicity was observed in the Ryukyutsuritomochi extract even at a concentration higher than that in FIG.

  According to the present invention, it is possible to provide a serine protease inhibitor, a whitening cosmetic composition and a whitening pharmaceutical composition which are composed of natural plant extracts and are inexpensive, safe and highly effective.

Claims (6)

  An inhibitor of serine proteases, which contains an extract of the plant family plant as an active ingredient.   The inhibitor according to claim 1, wherein the plant is a Ryukyu moth.   The inhibitor according to claim 1 or 2, wherein the serine protease is trypsin and / or tryptase.   A whitening cosmetic composition comprising the inhibitor according to any one of claims 1 to 3 as an active ingredient.   The whitening pharmaceutical composition which contains the inhibitor of any one of Claims 1-3 as an active ingredient.   The whitening cosmetic composition according to claim 4 or the whitening pharmaceutical composition according to claim 5, which has an effect of inhibiting melanin incorporation into keratinocytes by inhibiting serine protease.

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