WO2008023455A1

WO2008023455A1 - Tyrosinase inhibitor

Info

Publication number: WO2008023455A1
Application number: PCT/JP2007/000776
Authority: WO
Inventors: Masanori Sugiyama
Original assignee: Hiroshima University
Priority date: 2006-07-19
Filing date: 2007-07-19
Publication date: 2008-02-28
Also published as: JP5055540B2; JP2008024618A

Abstract

Disclosed is a tyrosinase inhibitor which has excellent tyrosinase-inhibiting activity, is safe and inexpensive, and is useful as a pharmaceutical, a cosmetic, a food or a food additive. The tyrosinase inhibitor comprises a triacyl glycerol represented by the general formula (1) as an active ingredient. (1) wherein R1, R2 and R3 independently represent a linear fatty acid residue having 10 to 18 carbon atoms.

Description

Specification

Tyrosinase inhibitor

Technical field

[0001] The present invention relates to a tyrosinase inhibitor useful as a pharmaceutical, food, or the like.

Background art

[0002] Melanin is a pigment related to the color of various parts of mammals, and one of the roles of melanin is to protect skin and tissues from ultraviolet rays. However, excessive melanin production in the skin is caused by over-colored spots, buckwheat, and spots. Such over-coloring depends on the activity of an enzyme that produces melanin, and tyrosinase is known as an enzyme that produces such melanin.

[0003] Tyrosinase (EC 1.1 4 4.1 8.1) requires copper ions to develop its activity, and uses tyrosine as a starting material for the hydroxylation of monophenol, two reactions related to melanin production. (Monoxygenase activity), catalyzes the reaction of o_diphenol to o_quinone (oxidase activity). Since o_quinone produced in this way has high reactivity, it is immediately polymerized into melanin. In addition, o_quinone reacts with amino acids and proteins to increase coloration. Therefore, tyrosinase inhibitors are thought to be effective as a material that suppresses melanin production and produces a whitening effect.

[0004] — On the other hand, tyrosinase is also known as an oxidant for polyphenols, and it is not only used for the production of melanin in mammals, but also for coloring in foods such as fruits, vegetables, mushrooms, shrimp, and salmon (brown and black ) Is also related. Coloring foods with tyrosinase reduces nutrition and commercial value, so preventing such coloring is a major challenge in the food industry.

[0005] From the above viewpoints, substances that inhibit the catalytic function of the enzyme tyrosinase that causes overcoloring are considered to be useful in the fields of cosmetics, pharmaceuticals, and food industry. Arbutin, kojic acid It has been reported that a wide variety of substances such as ellagic acid can be used as melanin production inhibitors (Patent Document 1, Non-Patent Document 1) or tyrosinase inhibitors (Patent Document 2_5).

However, the clinical effects of tyrosinase inhibitors and melanin production inhibitors disclosed so far are not clear, and some substances such as kojic acid are suspected to be carcinogenic. Development is desired

[0006] On the other hand, it has been empirically known that the hand of Mr. Tsuji who prepares sake is white and beautiful in spite of the cold and harsh working environment, and there are applications for cosmetics and bathing agents using sake lees or their extracts. There are few scientific arguments regarding the whitening effect (Patent Documents 6 and 7).

In addition, it has been reported that esters of unsaturated fatty acids and mono- or dihydric alcohols have tyrosinase inhibitory activity (Patent Document 8), and esters of fatty acids and trihydric alcohols are incorporated into whitening cosmetics. (Patent Documents 9, 10).

[0007] However, it has never been reported that triacylglycerol has a tyrosinase inhibitory action or a whitening effect.

Non-Patent Document 1: Maeda, K: FRAGRANG J0URNAK, September 1997 issue: 10-18

Patent Document 1: Japanese Patent Laid-Open No. 2 0 4 _5 9 4 9 6

Patent Document 2: Japanese Patent Laid-Open No. 2 0 0 1 _ 2 4 0 5 5 6

Patent Document 3: Japanese Patent Laid-Open No. 2 0 0 6 _ 6 9 9 5 4

Patent Document 4: Japanese Patent Application Laid-Open No. 5- 1 9 4 2 5 8

Patent Document 5: Japanese Patent Laid-Open No. 10-2 6 5 3 6 6

Patent Document 6: Japanese Patent Laid-Open No. 2 0 0 4 _ 3 4 6 0 4 5

Patent Document 7: Japanese Patent Laid-Open No. 11-1 9 6 8 4 9

Patent Document 8: Japanese Unexamined Patent Publication No. 6 3-2 8 4 1 0 9

Patent Document 9: Japanese Patent Laid-Open No. 2 0 0 4 _ 5 1 6 10

Patent Document 10: Japanese Patent Laid-Open No. 2 0 0 3 _ 2 6 1 4 3 1 Disclosure of the invention

Problems to be solved by the invention

[0008] The present invention relates to providing a tyrosinase inhibitor that has excellent tyrosinase inhibitory activity, is safer and cheaper, and is useful as a pharmaceutical, cosmetic, food, or food additive.

Means for solving the problem

[0009] As a result of studies to solve the above problems, the present inventors have found that specific triacylglycerol has an excellent tyrosinase inhibitory activity.

[0010] That is, the present invention relates to the following inventions.

1) The following general formula (1):

[001 1] [Chemical 1]

H ₂ C-0-R

HC-0-R ² (1)

H ₂ C-0-R ³

[In the formula, R ¹ , R ² and R ³ each independently represent a linear fatty acid residue having 10 to 18 carbon atoms. ]

An oral inhibitor of triglyceride represented by the formula:

2) A whitening agent containing triacylglycerol represented by the general formula (1) as an active ingredient.

3) A food coloring preventing agent comprising triacylglycerol represented by the general formula (1) as an active ingredient.

4) The following general formula (1) for producing a tyrosinase inhibitor or whitening agent

[0013] [Chemical 2]

H ₂ C-0-R

HC-0-R ²

H ₂ C— 0— R ³ [Wherein, R ¹ , R ² and R ³ each independently represent a linear fatty acid residue having 10 to 18 carbon atoms. ]

Use of triacylglycerol represented by

The invention's effect

[0015] According to the tyrosinase inhibitor of the present invention, it is possible to suppress excessive production of melanin in the skin, and to prevent or ameliorate pigmentation, spots, and buckwheat. In addition, by applying the tyrosinase inhibitor of the present invention to foods such as fruits, vegetables, mushrooms, shrimp and persimmons, the coloring can be prevented.

Brief Description of Drawings

[0016] [Fig. 1] A graph showing the inhibitory effect of mushroom tyrosinase on the monooxygenase activity.

FIG. 2 is a graph showing the inhibitory effect of Streptomyces tyrosinase on the monooxygenase activity.

[Fig. 3] Graph showing the mode of inhibition of trilinolein on monooxygenase activity.

[Fig. 4] Graph showing the mode of inhibition of trilinolein on monooxygenase activity.

[Fig. 5] Graph showing the mode of inhibition of trilinolein on monooxygenase activity.

[Fig. 6] A graph showing the mode of inhibition of trilinolein on monooxygenase activity.

[FIG. 7] A graph showing the inhibitory effect of mushroom chisinase on oxidase activity.

FIG. 8 is a graph showing the inhibitory effect of Streptomyces tyrosinase on the monooxygenase activity.

FIG. 9 is a graph showing the mode of inhibition of trilinolein on oxidase activity.

FIG. 10 is a graph showing the mode of inhibition of trilinolein on oxidase activity. [Fig. 11] Graph showing the mode of inhibition of trilinolein on oxidase activity.

FIG. 12 is a graph showing the mode of inhibition of trilinolein on oxidase activity.

FIG. 13 is a photograph showing the inhibitory effect of trilinolein on recombinant tyrosinase (p ET-me I 2). Agar plates (a), (b), and (c) contain trilinolein 0 I, 15 I, and 30 I, respectively. Add 50 I distilled water to the left filter paper of the agar medium, and add 50 I 0.125 mM / ml I PTG aqueous solution to the right side.

FIG. 14 is a photograph showing the inhibitory effect of trilinolein on recombinant tyrosinase (p ET-me I 3). Agar plates (a), (b), and (c) contain trilinolein 0 I, 15 I, and 30 I, respectively. Add 50 I distilled water to the left filter paper of the agar medium, and add 50 I 0.125 mM / ml I PTG aqueous solution to the right side.

FIG. 15 is a graph showing the formation of crisp with Cu ² +.

FIG. 16 is a graph showing the formation of a chelate with Cu ^{2 +} .

[Fig. 17] Graph showing free radical scavenger activity by triolein.

FIG. 18 is a graph showing free radical-one scavenger activity by trilinolein.

BEST MODE FOR CARRYING OUT THE INVENTION

—In general formula (1), R ¹ , ² and ³ each represent a linear fatty acid residue having 10 to 18 carbon atoms, but the fatty acid side chain may be either saturated or unsaturated. Examples of fatty acid residues include force puric acid (decanoic acid), lauric acid (dodecanoic acid), tridecanoic acid, myristic acid (tetradecanoic acid), pentadecanoic acid, palmitic acid (hexadecanoic acid), heptadecanoic acid, stearic acid Residues such as (octadecanoic acid) and the like can be mentioned, and among these, the following residues are preferred: forcepuric acid, lauric acid, myristic acid, palmitic acid, stearic acid. [0018] The unsaturated fatty acid residue is preferably a monovalent to trivalent unsaturated fatty acid residue, such as decenoic acid, undecylenic acid, dodecenoic acid, tridecenoic acid, myristoleic acid (tetradecenoic acid). , Pentadecenoic acid, palmitoleic acid (hexadecenoic acid), heptadecenoic acid, oleic acid (octadedecenoic acid), hexadecadienoic acid, hexadedetrienoic acid, hexanodecatetraenoic acid, heptadecadienoic acid, linoleic acid (octadedecadiene) Residues such as acid (n _ 6)), bis-linolenic acid (octadecatrienoic acid (n-3)), γ-linolenic acid (octadecatotrienoic acid (η _ 6)), octadecatetraenoic acid, etc. Of these, the residues of palmitoleic acid, oleic acid, linoleic acid, bis-linolenic acid, and γ-linolenic acid are preferred, Rain residues, linoleic acid residues is particularly favorable preferable.

[0019] Among these, the one of R ^1, R ² and R ³ is a unsaturated fatty acid residue is preferably, R ^1, fatty acid residue of all unsaturated R ² and R ³ Is more preferred.

[0020] Preferable examples of the triacylglycose mouthpiece of the present invention include, for example, trilaurin, trimyristin, tri / remitin, tristearin, triolein, trilinolein, caproyl, myristol, palmito, and so on. Glyce Mouth, Caproyl-Dipalmi Toilel-Glycerol, Caproyl-Li Norreole, Myristol, Glycerol, Power Profile, Linoleo-Leur, Palmol, Toil, Glycerol, Laurel One linoleo oil, one pallet, one glycerol, a force profile, one glycerin, one linoleo one, one myristol, one palmi, one glycerol, one lauroyl, one gilol Glycerol, linoleoyl and dipal mint, glycerol and linole Emile Iris Eleol Leol Leol 1 Glycerol, Giole Leol Le Palmy Toy Leol Glycerol, Reno Leol Leol Leol Leil Le Palm Leol Leol Leol Leol Leol Leol Leol Leol Leol Glycerol, linoleoyl alcohol, glycerol, glycerol, dioleoyl, linoleoyl Examples include serol, linoleol, linoleo, luster alloy, glycerin and the like.

[0021] Such triacylglycerol can be synthesized by a condensation reaction of glycerol and a fatty acid according to a known method (5th edition, Experimental Science Course 16 Synthesis of Organic Compounds IV, edited by the Japan Society of Science). In addition, transesterify and refine oils such as corn oil, soybean oil, rapeseed oil, sunflower oil, safflower oil, olive oil, cocoa butter, palm oil, rice bran oil, cottonseed oil, fish oil, and tallow according to conventional methods. Can be obtained. Furthermore, as described in the Reference Example, the triacylglycose mouth of the present invention can also be obtained by extraction from sake lees with an organic solvent. For example, a hexane extract fraction of sake lees is used as the triacylglycerol of the present invention. You can also Commercial products can also be used.

[0022] The triacylglycerol of the present invention has a tyrosinase inhibitory activity, as shown in the examples described later, and therefore, fish and shellfish produced by oxidation of polyphenol by chininase overproduction of melanin in the skin. Blackening of fresh foods such as fruits and vegetables ■ It is thought that browning can be suppressed. Therefore, the triacylglycerol of the present invention can be used as a tyrosinase inhibitor, a whitening agent, or a food coloring inhibitor. The tyrosinase inhibitor and whitening agent can be used as pharmaceuticals, quasi-drugs, cosmetics, foods, etc. for preventing or improving the occurrence of browning of the skin and the occurrence of spots / sobacas. In addition, the food coloring inhibitor can be used as a food additive.

[0023] The dosage form when the tyrosinase inhibitor or whitening agent of the present invention is used as a pharmaceutical is, for example, oral administration by tablet, capsule, granule, powder, liquid, etc. or parenteral by injection, external preparation, etc. Any of administration may be sufficient. In order to prepare the pharmaceutical preparation, the triacylglycerol of the present invention may be used alone or in combination of two or more, and if necessary, other pharmaceutically acceptable excipients, binders, extenders, disintegrants, Surfactants, lubricants, dispersants, buffers, preservatives, flavoring agents, fragrances, film-forming agents, carriers, diluents, and the like can be used in appropriate combinations. The content of the triacylglycerol of the present invention in the preparation is from 0.001 to 1% by mass, in particular It is preferable to contain 0.005 to 0.02 mass%.

When using the tyrosinase inhibitor or whitening agent of the present invention as a pharmaceutical product

The daily dose per adult is preferably, for example, 1 mg to 10 g, particularly 5 to 20 O mg, as triacylglycerol.

[0024] In addition, when the tyrosinase inhibitor or whitening agent of the present invention is used as a quasi-drug or a cosmetic, various dosage forms such as lotion, milky lotion, cream, and pack can be used. Such a preparation is prepared by mixing the triacylglycerol of the present invention singly or in combination of two or more, and appropriately blended in quasi-drugs, cosmetics and detergents, oily ingredients, humectants, powders, pigments, It can be prepared by appropriately combining emulsifiers, solubilizers, detergents, ultraviolet absorbers, thickeners, medicinal ingredients, fragrances, resins, plant extracts, alcohols, antioxidants, and the like. Examples of medicinal ingredients include other whitening ingredients such as kojic acid, arbutin, and bracenta extract.

The content of the triacylglycerol of the present invention in the quasi-drug and cosmetic is preferably 0.001 to 1 mass ⁰ / o, particularly 0.005 to 0.0. It is preferable to set it as 2 mass%.

[0025] When the tyrosinase inhibitor or whitening agent of the present invention is used as a food, in addition to various foods such as pans, cakes, rice cakes, confectionery, jelly, frozen foods, dairy products, beverages, tablets And supplement preparations in the form of capsules or liquids. Preparations of various forms of food include preparation of triacylglycerol of the present invention alone or in combination of two or more, and other food ingredients, solvents, softeners, oils, emulsifiers, preservatives, fragrances, stabilizers, coloring agents. UV absorbers, antioxidants, humectants, thickeners, etc. can be used in appropriate combinations.

[0026] When the tyrosinase inhibitor or food coloring inhibitor of the present invention is used as a food additive, it can be in the same form as the above oral preparation. That is, if necessary, other additives are mixed or dissolved, and processed into a powder, granule, pellet, tablet, or various liquid forms. The food additive of the present invention is added to fruits, vegetables, seafood and processed foods such as pickles, etc. Can be used to suppress color.

[0027] Since many of the triacylglycerols according to the present invention are insoluble in water, the above-mentioned preparations can be made water-soluble by using an emulsifier generally used in the field of food and cosmetics. Sex can be imparted. Examples of such emulsifiers include glycerin fatty acid esters such as acetic acid monoglyceride, lactic acid monoglyceride, citrate monoglyceride, dicetyltartaric acid monoglyceride, succinic acid monoglyceride, polyglycerin fatty acid ester, and polyglycerin condensed linosyl acid ester. Saponins obtained by extraction from mosquito bark, quince bark, soybean seeds, chia seeds, etc., higher fatty acids such as sucrose and stearic acid, palmitic acid, oleic acid or lower fatty acids such as acetic acid and isobutyric acid And phospholipids such as plant lecithin obtained from rape and soybean seeds or animal lecithin extracted from egg yolk. Among them, it is preferable to use a biosurfactant produced by microorganisms, particularly yeast.

[0028] In addition, alkylbenzene sulfonates, alkyl sulfates, alkyl ether sulfates, monoalkyl phosphates, alpha olefin sulfonates, alkane sulfonates, nonionic surfactants that are anionic surfactants Surfactant sorbitan fatty acid ester, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene fatty acid ester, fatty acid alkanolamide, polyoxyethylene alkyl ether, n_octyl-S-D-glucoside, polyoxyethylene alkylphenol Nitrile, zwitterionic surfactant alkyl alkylamino fatty acid salt, alkyl betaine, alkyl amine oxide, cationic surfactant alkyl trimethylammonium salt, dialkyldimethyl Nmoniumu salts, alkyl dimethyl benzyl ammonium Niu unsalted, well as N- methyl-bis-hydroxy E chill § Min fatty E ester hydrochloride may be used.

Example

[0029] (1) Reagents, etc. Freeze-dried sake lees were obtained from China Brewing Co., Ltd. Mushroom tyrosina

—Ze, triolein, ((DP PH (2,2-di pheny I -1 -pi cry I hydrazy I))) was purchased from Sigma (USA) and used without purification. 3, 4-dihy droxy-L-phenylalanine) and trilinolein were obtained from Nacalai Tesque Co., Ltd. (Japan), L-tyrosine-kojic acid (5-hydroxy-2- (hydroxymethy I) -4H-pyron- 4-one) And all other reagents and solvents were purchased from Wako Pure Chemical Industries, Ltd. (Japan).

(2) Preparation of tyrosinase derived from actinomycetes (S. castaneoslobisporus)

The actinomycete (S. castaneoslobisporus) tyrosinase was expressed in large amounts using Idaos containing pAKM1 (Appl. Microbiol. Biotechnol. 45, 80-85, 1996). TSB medium (10% sucrose, 30 g / L tryptone sorb broth) Cu S04 was added to 40 OmL to a final concentration of 8 mM, and tiostrepton was added to a final concentration of 45 mg / L. In addition, a spring was placed in front of the medium autoclave to prevent actinomycetes from aggregating. 400 L of S. I ividan S spore suspension was added and cultured at 28 ° C. for 46 hours. All subsequent purification steps were performed at 4 ° C. After incubation, the cells were removed by centrifugation at 27, 000 Xg, 20 minutes, 4 ° C, and the supernatant was equilibrated with 1 OmM phosphate buffer (pH 6.8) DEAE cellulose Melanin was removed by passing through a column (Whatman, wet volume 40 OmL). Na CI was added to the eluted solution to a final concentration of 4M and sodium phosphate to a final concentration of 1 OmM, and pH was adjusted to 6.8. When this sample was passed through a Fernsefalose column (Amersham, wet volume 5 OmL) equilibrated with 4M NaCI, 1 OmM phosphate buffer (pH 6.8), 1 OmM phosphate buffer was immediately obtained. The solution (pH 6.8) was used for dissolution. The eluate was concentrated using MACROS EP 10 K (PA LL), and the resulting concentrated solution was subjected to melanin removal operation twice using an appropriate amount of DEAE cellulose. The eluate was filtered through an acrodisc syringe filter (0.45; um HT Tuffry Membrane, PALL) and then flattened with 4MNa CI, 1 OmM phosphate buffer (pH 6.8). Simpsonated Husier Cephaloska Lamb (Amersham, wet volume 1 OmL). The column was washed with a 10 bed amount of 4MNaCl, 10 mM phosphate buffer (pH 6.8). Tyrosinase was eluted by a gradient from 4 to OMN a CI. The purified tyrosinase was confirmed using SDS-PAGE and concentrated with MA CRO SEP 10 K (PA LL). Tyrosinase was stored at 4 ° C.

[0031] Reference Example 1 Extraction, purification and identification of tyrosinase inhibitory active substance

The strongest tyrosinase inhibition was confirmed from hexane extract of freeze-dried sake lees. Thus, freeze-dried sake lees (700 g) were extracted at room temperature for 3 hours and 7 times with 3 L of hexane. The extract was concentrated at 40 ° O under reduced pressure to obtain 22.64 g.

[0032] The obtained extract was applied to a small amount of silica gel column chromatography and eluted with hexane: ethyl acetate changed to 1: 0, 32: 1, 1 6: 1, 8: 1, 0: 1, Five fractions were obtained. Among these, the fraction with the strongest inhibitory activity: hexane: ethyl acetate = 32: 1 was placed in a silica gel column chromatography (diameter 5 cm x 20 cm) and hexane: ethyl acetate 8: 1. And purified into four fractions. Among the four fractions, the second fraction was placed in a small amount of silica gel column chromatography, washed with hexane until no spots were confirmed, and then purified with hexane: CHC I ₃ = 9: 1 To obtain 1.81 7 g of inhibitor (R _f = 0.4, CHC I ₃ ).

The material was found to be a mixture of triacyl glycerol from ¹ HNMR, ¹³ CNMR, GCMS and IR.

[0033] IR (N EAT): 301 1, 2955, 2855, 1 747, 1 464, 1 21 5, 1 1 63, 1 099, 760 cm- ¹

[0034] Table 1 shows the ¹ H-NMR spectrum of triacylglycerol obtained from freeze-dried sake lees, and Table 2 shows the ¹³ C-NMR spectrum. The mass spectrum was measured in both EI mode and CI mode. GCMS revealed that this mixture contained several types of triacylglycerol (Table 3). The ingredients of the mixture are shown in Table 4. [0035] [Table 1]

NMR spectra of triacylglycerol mixtures

[0037]

3) GCMS data of triacylglycerol contained in sake lees

[Table 4] Triacylglycerol in sake lees

No Carbon number: Unsaturated number

1 10: 0 14: 0 16: 0 523 [M-COOC ₉ H ₁₉ ] ⁺ , 467 [M-COOC ₁₃ H ₂₇ ] ⁺ , 439 [M COOC ₁₅ H ₃₁ ] ⁺

2 10: 0 16: 0 16: 0 551 [M-COOC ₉ H _l9 ] ⁺ , 467 [M-COOC ₁₅ H ₃₁ ] ⁺

3 10: 0 14: 0 〖8: 2 547 [M-COOC ₉ H ₁₉ ] ⁺ , 491 [M-COOC ₁₃ H ₂₇ ] 439 [M-CO ₁₇ H ₃₁ ] ⁺

4 10: 0 16: 0 18: 2 575 [M-C00C ₉ H, ₉ ] ⁺ , 491 [M-C00C, ₅ H ₃₁ ] ⁺ , 467 [M-C00C ₁₇ H ₃₁ ] ⁺

5 12: 0 16: 0 〖8: 2 575 [M-COOC „H ₂₃ ] +, 519 M- COOC ₁₅ H ₃₁ ] +, 495 [M-COOC ₁₇ H ₃₁ ] +

6 10: 0 18: 2 18: 2 599 [M-C00C ₉ H ₁₉ ] ⁺ , 491 [M-C00C ₁₇ H ₃₁ ] ⁺

7 14: 0 16: 0 18: 2 575 [M-C00C ₁₃ H ₂₇ ] ⁺ , 547 [M-C00C ₁₆ H ₃₁ ] ⁺ , 523 [M-C00C _l7 H ₃₁ ] ⁺

8 12: 0 18: 2 18 : 2 599 [M-C00C "H 23] +, 519 [M- C00C 17 H 3l] +

9 16: 0 16: 0 18 : 2 575 [M-COOC 15 H 31] +, 551 [M_COOC 17 H 3l] +

10 14: 0 18: 1 18: 2 601 [M-COOC ₁₃ H ₂₇ ] ⁺ , 547 M- C00C ₁₇ H ₃₃ ] ⁺ , 549 [M-COOC ₁₇ H ₃₁ ] ^T

11 16: 0 18: 1 18: 1 603 [M-COOC ₁₅ H ₃₁ ] ⁺ , 577 [M-C00C ₁₇ H ₃₃ ] ⁺

12 16: 0 18: 1 18: 2 601 [M-COOC ₁₅ H ₃₁ ] ⁺ , 575 [M- C00C ₁₇ H ₃₃ ] ⁺ , 577 [M-COOC ₁₇ H ₃₁ ] ^T

13 16: 0 18: 2 18: 2 599 [M-COOC ₁₅ H ₃₁ ] ⁺ , 575 [M-C00C ₁₇ H ₃₁ ] ⁺

14 16: 1 18: 1 18: 3 599 [M-COOC ₁₅ H ₂₉ ] ⁺ , 571 [M- C00C ₁₇ H ₃₃ ] +, 575 [M- C00C _l7 H ₂₉ ] —

15 18: 1 18: 1 18: 2 601 [M-C00C ₁₇ H ₃₃ ] ⁺ , 603 [M- C00C ₁₇ H ₃₁ ] +

16 18: 0 18: 2 18: 3 597 [M-COOC ₁₇ H ₃₅ ] ⁺ , 601 [M- C00C ₁₇ H ₃₁ ] ⁺ , 603 [M-C00C _l7 ¾ ₉ ] ÷

17 18: 2 18: 2 18: 2 599 [M- C00C ₁₇ H ₃₁ ] + Example 1 Inhibitory activity against monooxygenase

96-well plate with 100 I of 1 mM L tyrosine for monooxygenase activity as a substrate for measurement of inhibitory activity, and 100 I of 10 mM L-dope solution for oxidase activity described below 74 1 of 0.1 M phosphate buffer (pH 6.8) was added. This mixed solution was incubated at 25 ° C. for 5 minutes, and then DMSO containing 6 UI sample and DMSO not containing 6 UI sample were mixed with the cultured mixture. Thereafter, 20 UI of 200 units / m I tyrosinase aqueous solution was added, and a linear increase in absorbance was measured at 475 nm for 5 minutes. The inhibitory activity was expressed as IC ₅₀ which is the concentration of the inhibitor when inhibiting tyrosinase activity by 50%. Control experiments were evaluated with a linear increase in DMSO without sample. Kojic acid, well known as a tyrosinase inhibitor, was used as a positive control. The inhibition mode was determined by adding 20 I of 400 unit / mI tyrosinase aqueous solution to various concentrations of L-tyrosine and L-doper solution, and using the Lineweaver Bark plot and Dixon plot.

[0040] Fig. 1 shows the results of examining the inhibitory effect of mushroom tyrosinase on the monooxygenase activity using triolein, trilinolein and kojic acid as a positive control. Trilinolein showed a stronger inhibitory effect on mushroom tyrosinase than triolein. Trilinolein inhibits mushroom tyrosinase at 10, 50, 100, and 200 M, 8.3%, 32.9%, 42.8%, and 75.4%, respectively, and IC ₅₀ is 1 2 2 UM. Since the measurement solution was based on water, triolein was poorly soluble and did not reach a concentration at which IC ₅₀ could be measured. 100 and 200 M triolein inhibited monooxygenase activity by 1.4 <½ and 27.5%, respectively. Kojic acid had an IC ₅₀ of 8. OM.

Figure 2 shows the inhibitory effect of S. castaneoglobisDorus tyrosinase on monooxygenase activity. Despite the kojic acid IC ₅₀ of 2.7; UM, triolein and trilinolein showed only weak inhibition. [0041] The mode of inhibition of trilinolein on monooxygenase activity, which is the oxidation of L-tyrosine, was examined with various concentrations of L-tyrosine and trilinolein. Figures 3 and 4 show the mode of inhibition against pine schulm mutyrosinase, and Figures 5 and 6 show the mode of inhibition of tyrosinase from S. castaneoslobi §β ^ Οϋ. In Fig. 3 and Fig. 5, the horizontal axis 1 / [L-tyrosine] is the reciprocal of the concentration of the substrate L-tyrosine, and the vertical axis 1 / V is the reaction rate indicating the activity of tyrosinase. It is the reciprocal number. The 1 / V Rheinwiber-Bark plot for 1 / [L-tyrosine] showed that the V _{ma χ} of tyrosinase varied with the inhibitor concentration, but K _m did not. In other words, trilinolein was found to be a non-competitive inhibitor from the results of the lineweaver plot. In Fig. 4 and Fig. 6, the inhibition pattern was examined using Dixon plot. From 1 / V to [I] at various concentrations of substrate, it was confirmed that trilinolein acts as a non-competitive inhibitor in the monooxygenase activity of tyrosinase.

[0042] Example 2 Inhibitory activity against oxidase

Inhibition of the oxidase activity of pine mushroom synthase was evaluated using triolein, trilinolein and kojic acid as a positive control (Figure 7). Trilinolein inhibited pine mushroom synthase at 5, 10, 25, and 50 mg at 17.6%, 25.0%, 55.6%, and 70.8%, respectively. Triolein is difficult to dissolve in the measurement solution based on water, and its IC ₅₀ was predicted from three concentrations at which triolein can be completely dissolved. When 25 M and 50 M triolein were added to the measurement solution containing L-dopa, the inhibition rates were 19.4% and 26.4%, respectively. In addition, when 100 イン triolein was added, the inhibition rate increased linearly to 34.4%, so the IC ₅₀ is estimated to be 431; UM. The IC ₅₀ values for trilinolein and kojic acid were 22.1 and 14.1 M, respectively.

[0043] The inhibition rate of S. castaneoslobis Dorus for tyrosinase is shown in FIG. Trilinolein showed a higher inhibition rate than kojic acid, a positive control. 5 M trilinolein showed an inhibition rate of 85.5% Was inhibited by 2.2%, 33.8%, 45.8%, and 66.7% at 5 M, 10 Μ, 25 M, and 50 それぞれ, respectively. In other words, the IC ₅₀ of triolein, trilinolein, and kojic acid for the oxidase activity of S. castaneoglobisDorus is 30.0, 2.9, and 7.8 M, respectively. 2.5 times stronger inhibition than acid.

[0044] The mode of inhibition of trilinolein on oxidase activity, which is an oxidation of L_dopa by a mushroom cinnacinase and. Castaneog I ob i SDorus tyrosinase, was investigated. Fig. 9 and Fig. 10 are the Lineweaver _ _ Bark plot and Dickson plot, respectively, when using mushroom chiro synthase. The mode of inhibition of castaneog lob isDorus tyrosinase by trilinolein is shown in Figs. 11 and 12. While you decrease V _{ma x} did not change K _m by 1 Z [S] against concentration of trilinolein from line Ui one server eleven Bakupuro' Bok is 1 / V. From these results, it was revealed that trilinolein is non-competitive inhibitory not only in monooxygenase activity but also in oxidase activity from the lineweigh bark plot.

[0045] Figures 10 and 12 analyzed the mode of inhibition from the Dickson plot for various concentrations of substrate. The 1 / V Dixon plot for [I] was confirmed to be non-competitive inhibition consistent with lineweaver for the oxidase activity of chinose synthase.

Example 3 Inhibitory Effect on Melanin Synthesis → Inhibitory Effect on Recombinant Tyrosinase FIGS. 13 and 14 show that p E T_m e I 2 or p E T_me I 3 is a plasmid that produces tyrosinase. E. coli BL 2 1 (DE 3)-p Lys S (Protein Expr. Pur if., 34, 202-207,2004) is cultured on agar medium containing trilinolein (inhibitor) Therefore, the inhibition is confirmed. Figures 13 and 14 (a), (b), and (c) contain trilinolein at 0 I, 15 I, and 30 μ \, respectively. Among the two filter papers on the agar medium, 50 I distilled water was added on the left side, and 50 I 0.125 mM / m IIP TG aqueous solution was added on the right side. The area around the filter paper with distilled water is black Although it is not connected, it is dark where I PTG aqueous solution is added. In other words, the reason for the blackening is due to the secreted tyrosinase induced by I PTG. Fig. 13 shows photographs of Enterobacteriaceae BL 21 (DE3) with p ET_me I 2 — p Lys S cultured for 0, 21, 24 hours, and Fig. 14 shows p E Tm e I E. coli BL 21 (DE 3) containing 3 is a photograph of p Lys S cultured for 0, 24, 28 hours. As can be seen in Fig. 13 and Fig. 14, it was confirmed that the blackening time was delayed depending on the concentration of trilinolein.

Example 4 Inhibitory activity mechanism

1) Clear formation

In order to evaluate whether the inhibition by triacylglycerol was due to chelate formation with Cu ²⁺ , a UV-Vis spectrum of 240-540 nm was observed. 1. 0.1 ml (0.005 mM) of the sample solution was added to a mixture of 8 ml 0.1 M phosphate buffer (pH 6.8) and 1.0 ml distilled water. Then, 0.1 m I tyrosinase aqueous solution (100 units) or Cu SO ₄ aqueous solution (125 M) was added and incubated at 25 ° C for 30 minutes, and the change in the spectrum was measured. The presence or absence of vasochromic shift due to the presence or absence of tyrosinase and Cu S0 ₄ was examined. As shown in Figs. This result suggests that no chelation was formed between tyrosinase and copper ions.

[0048] 2) Free radical-one scavenger activity

Antioxidant ability was investigated by investigating the effect of triacyl glyceride on radicals. To 0.1 ml of a triacylglyce mouth solution (DMSO), 0.1 ml of 0.1 mM DPPH ethanol solution was added. This mixture was mixed and allowed to stand in a dark room at room temperature for 30 minutes, and the decrease in absorbance at 517 nm was measured. Figures 17 and 18 show the change in absorption at 51 7 nm. Since there is almost no change from the blank, there is no antioxidant activity.

By the way, copper ions are essential for tyrosinase to show activity, As shown in Example (7), triacylglycerol did not form a chelate with copper ions and did not exhibit antioxidant activity. In other words, triacylglycerol appears to exhibit inhibitory activity by binding to the hydrophobic region of thiocinase and causing a structural change. Furthermore, the degree of unsaturation of such an acyl group is thought to affect the solubility and to increase the inhibitory activity.

Claims

Claim [1] The following general formula (1):

[Chemical 1]

H ₂ C— 0-R ¹

HC-0-R ² (1)

H ₂ C— 0— R ³

[Wherein R ¹ , R ² and R ³ each independently represent a linear fatty acid residue having 10 to 18 carbon atoms. ]

The tyrosinase inhibitor which uses the triacylglyce mouth represented by this as an active ingredient.

[2] The following general formula (1):

[Chemical 2]

H ₂ C-0-R

HC-0-R ² (!)

H ₂ C-0-R ³

The whitening agent which uses the triacylglycerol represented by this as an active ingredient.

[3] The following general formula (1):

[Chemical 3]

H ₂ C-0-R ¹

HC-0-R ² (1)

H ₂ C— 0— R ³

The food coloring prevention agent which uses the triacylglycerol represented by this as an active ingredient.

[4] The following general formula (1) for producing a tyrosinase inhibitor or whitening agent: [Chemical 4]

H ₂ C-0-R

HC-OR ² (i)

H ₂ C-0-R ³

[Wherein, R ¹ , R ² and R 3 each independently represent a linear fatty acid residue having 10 to 18 carbon atoms. ]

Use of triacylglycerol represented by