JP2009280550A - Tyrosinase activity inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a tyrosinase activity inhibitor which uses a natural substance having high safety as a raw material, excels in tyrosinase activity inhibition, and can be widely used in the fields of foods and cosmetics. <P>SOLUTION: The tyrosinase activity inhibitor having excellent tyrosinase activity inhibition includes an olive leaf extract and enzyme-treated isoquercitrin or enzyme-treated rutin and is obtained from a natural substance having high safety, and can be widely used in the fields of foods and cosmetics. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

The present invention relates to a tyrosinase activity inhibitor. More specifically, the present invention relates to a tyrosinase activity inhibitor characterized by being able to significantly suppress the activity of tyrosinase that causes melanin production.

Melanin is a pigment that plays a role in protecting skin and tissues from ultraviolet rays. On the other hand, excessive melanin causes spots and buckwheat in the skin, and causes browning in foods such as mushrooms, crustaceans and fruits.

In general, melanin is biosynthesized using tyrosine, which is an amino acid, as a starting material. Tyrosinase catalyzes these reactions, although it is a substance produced from tyrosine via some intermediates such as dopa and dopaquinone. Therefore, the production of melanin can be suppressed by inhibiting the activity of tyrosinase.

Conventionally, ascorbic acid, glutathione, and the like are used in the cosmetics and food fields as representative substances that inhibit tyrosinase activity. However, these substances have problems such as being easily oxidized and having a characteristic odor.

Moreover, a tyrosinase activity inhibitor (for example, refer to Patent Document 1) characterized by containing a flavonoid-aldehyde polycondensate as an active ingredient, and a tyrosinase inhibitor (for example, patent) containing a rosehip extract as an active ingredient Obtained from a novel tyrosinase inhibitor (see, for example, Patent Document 3), an aerial extract of azalea, and a licorice root extract. Containing a tyrosinase activity inhibitor in a mixture of a licorice flavonoid, an extract of the root of Carrot family carrot, a leaf extract of Lamiaceae marjoram, and an extract of the flower part of the honeysuckle genocide as an active ingredient Tyrosinase activity inhibitor (see, for example, Patent Document 4), 4-position substituted resorcinol A tyrosinase activity inhibitor containing at least one specific flavonoid having a skeleton as an active ingredient (see, for example, Patent Document 5), a flavonoid having hydroxyl groups at the 2′-position and the 4′-position (however, A flavonoid such as a tyrosinase activity inhibitor (for example, see Patent Document 6) containing as an active ingredient a hydroxyl group at the 2-position and 4-position, or at least one of the 2'-position and the 4'-position) is used. Tyrosinase activity inhibitors have also been proposed.

However, a highly safe natural product that does not adversely affect the quality of the object to be added in terms of taste and odor and can be widely used in foods and cosmetics is strongly demanded.

JP2004-244335 JP2007-261987 JP2003-238425A JP 2004-083476 A JP-A-11-255637 JP-A-10-101543

This invention is made | formed in view of this present condition, and makes it a subject to solve the said various problems in the past and to achieve the following objectives. That is, an object of the present invention is to provide a tyrosinase activity inhibitor that is excellent in tyrosinase activity inhibitory activity and can be widely used in the field of foods and cosmetics from highly safe natural products.

As a result of intensive studies to solve the above-mentioned problems, the present inventor has shown that the combined use of olive leaf extract and enzyme-treated isoquercitrin or enzyme-treated rutin has an excellent tyrosinase activity inhibitory action. The headline, the present invention has been reached.

That is, the present invention is as follows.
Item 1. A tyrosinase activity inhibitor comprising an olive leaf extract and enzyme-treated isoquercitrin or enzyme-treated rutin.
Item 2. Item 2. The tyrosinase activity inhibitor according to Item 1, wherein the compounding ratio of the olive leaf extract and the enzyme-treated isoquercitrin is 40:60 to 95: 5 (solid conversion) (mass ratio).
Item 3. Item 2. The tyrosinase activity inhibitor according to Item 1, wherein the blending ratio of the olive leaf extract and the enzyme-treated rutin is 36:64 to 87:13 (solid conversion) (mass ratio).
Item 4. Item 2. The tyrosinase activity inhibitor according to Item 1, wherein the olive leaf extract is an extract extracted with an extraction solvent containing a hydrophilic solvent.
Item 5. A method for inhibiting tyrosinase activity, comprising an olive leaf extract and enzyme-treated isoquercitrin or enzyme-treated rutin.

ADVANTAGE OF THE INVENTION According to this invention, the tyrosinase activity inhibitor which can be widely used in the field | area of the foodstuffs and cosmetics excellent in the tyrosinase activity inhibitory action from a highly safe natural product can be provided.

1) Tyrosinase activity inhibitor The present invention is an tyrosinase activity inhibitor comprising an olive leaf extract and enzyme-treated isoquercitrin or enzyme-treated rutin.

The olive leaf extract used in the present invention is obtained by extracting from the leaves of Olea europaea L .. The olive leaf can be a plant itself, a dried product thereof, or a commercially available dry powder.

Although the extraction method is not particularly limited, for example, olive leaf can be extracted by being immersed or heated under reflux with an extraction solvent.

As said extraction solvent, if it is a solvent normally used, it can be used arbitrarily. For example, water, alcohols (for example, ethanol, methanol, propylene glycol, 1,3-butylene glycol, glycerin, etc.), hydrous alcohols (that is, a mixture of water and alcohols), other organic solvents (for example, chloroform) , Dichloroethane, carbon tetrachloride, acetone, ethyl acetate, hexane, etc.), supercritical carbon dioxide, etc. can be used singly or in combination, but at least a hydrophilic solvent can be used for food versatility. Suitable for obtaining excellent olive leaf extract. Examples of the hydrophilic solvent include water, ethanol, methanol, propanol, isopropanol, acetone, butanol, acetonitrile, ethyl acetate, and tetrahydrofuran.

In view of the excellent inhibitory activity when combined with enzyme-treated isoquercitrin or enzyme-treated rutin and the ability to efficiently extract a water-soluble tyrosinase inhibitor, it is preferably water or hydrous ethanol, more preferably 20 to 60 mass. It is preferable to use a% ethanol aqueous solution.

The olive leaf extract of the present invention includes an extract obtained by using olive leaves as an extraction raw material, a diluted or concentrated solution of the extract, and a dried product obtained by drying the extract. Alternatively, any of these crudely purified products or purified products is included. The dried extract can also be used by re-dissolving in a suitable solvent. When forming into powder or granules, dextrin or the like may be used as an excipient.

The enzyme-treated isoquercitrin used in the present invention is obtained by allowing glycosyltransferase to act on isoquercitrin in the presence of a sugar donor. Refers to a mixture with α-glycosyl isoquercitrin that has been converted to glycated.

（式中、Glcはグルコース残基を、ｎは０または１以上の整数を示す）

(In the formula, Glc represents a glucose residue, and n represents 0 or an integer of 1 or more)

Specifically, in the above formula, the enzyme-treated isoquercitrin includes isoquercitrin having an α-1,4-bonded glucose residue number (n) of 0 and an α-1,4-bonded glucose residue number (n ) Is 1 or more, usually 1 to 15, preferably 1 to 10 α-glycosylisoquercitrin.

The enzyme-treated isoquercitrin used in the present invention may be a mixture of various enzyme-treated isoquercitrins having different glucose group bond numbers (n), but the glucose group bond number (n) is single. It may be a kind of enzyme-treated isoquercitrin.

Such enzyme-treated isoquercitrin can be prepared by treating isoquercitrin with glucose transferase. Although not limited, enzyme-treated isoquercitrin is usually produced by glycosylation using a glucose residue transfer enzyme such as glucosidase or transglucosidase by transferring equimolar amounts of glucose residues to isoquercitrin. Can do.

The number of glucose groups bonded to the glucose residue of isoquercitrin (the number of n in the above formula (1)) is not particularly limited, but is usually 1 to 15, preferably 1 to 10 as described above. It can be arbitrarily adjusted to be within the range. Examples of such an adjustment method include a method of treating various amylases (α-amylase, β-amylase, glucoamylase, α-glucosidase, maltase, etc.) alone or in combination after producing enzyme-treated isoquercitrin. Can do. By carrying out like this, the number of glucose sugar chains in the enzyme-treated isoquercitrin molecule obtained by the above-mentioned method can be decreased to obtain enzyme-treated isoquercitrin having an arbitrary glucose sugar chain length.

The enzyme-treated isoquercitrin thus obtained is mainly composed of α-glycosylisoquercitrin in which an equimolar amount or more of glucose is bound to the glucose residue of isoquercitrin (quercetin 3-0-monoglucoside). It is easy to dissolve in water.

The enzyme-treated rutin used in the present invention is obtained by acting a glycosyltransferase on rutin in the presence of a sugar donor, and is expressed by the following formula 2 and is α-glucosylated to various degrees with rutin. A mixture with glycosyl rutin.

（式中、Glcはグルコース残基を、Rhaはラムノース残基を、ｎは０または１以上の整数を示す）

(In the formula, Glc represents a glucose residue, Rha represents a rhamnose residue, and n represents 0 or an integer of 1 or more)

Specifically, in the above formula, the enzyme-treated rutin has an α-1,4-bonded glucose residue number (n) of 0 rutin and an α-1,4-bonded glucose residue number (n) of 1 or more. Usually, it is a mixture with 1-15, preferably 1-10 alpha-glycosyl rutin.

The enzyme-treated rutin used in the present invention may be a mixture of various enzyme-treated rutins having different glucose group bond numbers (n), but is a kind of enzyme having a single glucose group bond number (n). It may be treated rutin.

Such enzyme-treated rutin can be prepared by treating rutin with glucose transferase. Although not limited, usually, enzyme-treated rutin can be produced by using a glucose residue transferase such as glucosidase or transglucosidase to transfer a glucose residue to rutin in an equimolar amount or more for glycosylation.

The number of glucose groups bonded to the glucose residue of rutin (the number of n in the above formula (1)) is not particularly limited, but is usually in the range of 1 to 15, preferably 1 to 10 as described above. It can be arbitrarily adjusted to be. Examples of the adjustment method include a method of treating various amylases (α-amylase, β-amylase, glucoamylase, α-glucosidase, maltase, etc.) alone or in combination after producing the enzyme-treated rutin. . By doing so, the number of glucose sugar chains in the enzyme-treated rutin molecule obtained by the above-described method can be reduced to obtain an enzyme-treated rutin having an arbitrary glucose sugar chain length.

The enzyme-treated rutin thus obtained is mainly composed of α-glycosyl rutin in which equimolar amounts of glucose are further bound to the glucose residue of rutin and is readily water-soluble.

The tyrosinase activity inhibitor of the present invention is characterized by containing an extract of olive leaf and enzyme-treated isoquercitrin or enzyme-treated rutin, and the ratio of the olive leaf extract and enzyme-treated isoquercitrin is: It is 40: 60-95: 5 (solid conversion), Preferably it is 70: 30-86: 14 (solid conversion). The ratio of the olive leaf extract to the enzyme-treated rutin is 36:64 to 87:13 (solid conversion), preferably 57:43 to 76:24 (solid conversion).

The amount of the tyrosinase activity inhibitor of the present invention is not particularly limited. For example, when used for food, it is 0.0027 to 2600 ppm (solid conversion), preferably 13 to 1360 ppm, more preferably 27 to 270 ppm. . When the amount of the tyrosinase activity inhibitor of the present invention is less than 0.0027 ppm, sufficient tyrosinase inhibitory activity cannot be obtained, and when it exceeds 2600 ppm, problems such as coloring may occur, which is not preferable.

The tyrosinase activity inhibitor of the present invention includes, for example, saccharide sweeteners (such as sugars, fructose, glucose, tagatose, arabinose and other monosaccharides, lactose, oligosaccharides, maltose, trehalose, etc.) unless the purpose of the present invention is inhibited. Sugars, syrup, honey, dextrin, sugar alcohol, etc.), high-intensity sweeteners (sucralose, acesulfame K, stevia, etc.), polysaccharides such as starch, fats and oils, dairy products, stabilizers, emulsifiers, fragrances, pigments, acidity Ingredients, flavor ingredients (eggs, coffee, tea, cocoa, fruit pulp, yogurt, liquor, etc.), proteins, amino acids, dietary fiber, vitamins, minerals, glucosamine, yeast, eggshell membranes, lycopene, astaxanthin, other carotenoids, silk , Chondroitin, ceramide, placenta extract, shark fin extract, deep sea bream extract, squalene, γ- Minobutyric acid, raw chestnut skin extract, chestnut leaf extract, chestnut potato extract, chestnut pulp extract, chestnut bark extract, cabbage ferment extract, rose petal extract, grape leaf extract, grape seed extract , Apple polyphenol, chamomile extract, lychee seed extract, gotsukola extract, moon peach leaf extract, lotus germ extract, star fruit leaf extract, mulberry leaf extract, guava tea extract, catechin, raspberry ketone, low molecular weight alginic acid, ginkgo biloba extract Pine bark extract, chitosan, hyaluronic acid, methylsulfonylmethane, kojic acid, ellagic acid, glutathione, arbutin, lucinol, magnolignan, magnesium L-ascorbate phosphate, and the like can be used.

The tyrosinase activity inhibitor of the present invention may be any of powder, granule, and liquid, and these preparations can be produced according to conventional methods.

In addition, the tyrosinase activity inhibitor of the present invention is a composition containing, as other components, auxiliary agents such as antioxidants and chelating agents, and diluents, carriers or other additives as long as the effect is not inhibited. There may be.

For example, when used in foods, antioxidants can be widely exemplified as those used as food additives without any particular limitation. For example, ascorbic acids such as L-ascorbic acid and sodium L-ascorbate Ascorbic acid esters such as L-ascorbic acid stearate and L-ascorbic acid palmitate; erythorbic acids such as erythorbic acid and sodium erythorbate; sodium sulfite, sodium hyposulfite, sodium pyrosulfite and potassium pyrosulfite Sulphites and the like; Tocopherols such as α-tocopherol and mixed tocopherol; Dibutylhydroxytoluene (BHT) and butylhydroxyanisole (BHA); Ethylenediaminetetraacetate calcium disodium and ethyl Ethylenediaminetetraacetic acid such as diaminetetraacetic acid disodium; gallic acid such as gallic acid and propyl gallate; aoi flower extract, Aspergillus terreus extract, licorice oily extract, edible canna extract, glove extract, essential oil-removed fennel Extract, horseradish extract, sage extract, seri extract, tea extract, tempeh extract, dokudami extract, fresh coffee bean extract, sunflower seed extract, pimenta extract, grape seed extract, blueberry leaf Extract, Propolis extract, Hego Ginkgo biloba extract, Pepper extract, Spinach extract, Bayberry extract, Eucalyptus leaf extract, Gentian root extract, Rutin extract (Red beans whole plant, Enju, Buckwheat whole plant extract ), Rosemary extract and other plant extracts; quercetin, rutin enzyme degradation product ( Isoquercitrin), enzyme-decomposed apple extract, sesame oil extract, rapeseed oil extract, rice bran oil extract, rice bran enzyme digest, and the like.

The tyrosinase inhibitor thus obtained can be applied not only to various foods and drinks but also to orally ingested products such as pharmaceuticals and general industrial products such as cosmetics and hair washing products.

In addition, what is necessary is just to select suitably the addition time of the said tyrosinase inhibitor to the food / beverage products of this invention in the step of a manufacturing process according to the characteristic and objective of each food / beverage product.

2) Method for Inhibiting Tyrosinase Activity The present invention is a method for inhibiting tyrosinase activity characterized by using an olive leaf extract and enzyme-treated isoquercitrin or enzyme-treated rutin, and the blending ratio is as described above. . For example, in the case of foods such as fruits, the amount of olive leaf extract used may be 0.0019 to 1560 ppm (solid conversion), preferably 9 to 950 ppm, and more preferably 19 to 190 ppm. If it is less than 0.0019 ppm, even if it is used together with enzyme-treated isoquercitrin or enzyme-treated rutin, the inhibitory effect on tyrosinase activity is insufficient, and if it exceeds 1560 ppm, coloring problems may occur, which may be undesirable.

For example, in the case of foods such as fruits, the enzyme-treated isoquercitrin may be used in an amount of 0.0008 to 1040 ppm (solid conversion), preferably 4 to 410 ppm, and more preferably 8 to 81 ppm. If it is less than 0.0008 ppm, the inhibitory effect on tyrosinase activity is insufficient even when used with olive leaf extract, and if it exceeds 1040 ppm, coloring problems may occur, which may be undesirable.

For example, in the case of foods such as fruits, the amount of the enzyme-treated rutin is 0.0015 to 1944 ppm (solid conversion), preferably 8 to 766 ppm, and more preferably 15 to 151 ppm. If it is less than 0.0015 ppm, the inhibitory effect on tyrosinase activity is insufficient even when used together with olive leaf extract, and if it exceeds 1944 ppm, coloring problems may occur, which may be undesirable.

The tyrosinase inhibitor of the present invention is highly safe and can be blended in cosmetics, foods and the like, and can also be used for food processing for the purpose of preventing discoloration and the like. For example, confectionery (chewing gum, candy, tablets, chocolate, jelly, etc.), frozen confectionery, starch-based foods such as noodles, powdered foods, beverages (soup, coffee, teas, juice, cocoa, alcoholic beverages) , Jelly drinks, etc.), bakery foods (bread, biscuits, etc.), fat and oil foods (margarine, shortening, fat spread, etc.), high salt content foods (miso, soy sauce, pickles, boiled, salted, ham, etc.), etc. It can also be used in foods such as milk, cream, lotion, packs, cleaning products, makeup cosmetics, dispersions, ointments, liquids, aerosols, patches, poultices, liniments, external medicines, etc. .

Furthermore, when the tyrosinase activity inhibitor of the present invention is used as a food browning inhibitor, the target food includes fruits such as apples and bananas, mushrooms such as mushrooms and shiitake, crustaceans such as crabs and shrimps, potato Root vegetables such as burdock, leaf vegetables such as cabbage and lettuce, and cut vegetables containing these. As its use method, the tyrosinase activity inhibitor of the present invention is diluted with an appropriate solvent, and this solution can be sprayed or immersed in an object to prevent browning of the food surface.

EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated, this invention is not limited to these Examples. Unless otherwise specified, “part” means “part by mass” and “%” means “% by mass”.

(Preparation Example 1: Olive leaf hot water extract)
Extract 1 was obtained by adding 10 g of olive leaf powder to 90 g of ion-exchanged water, heating and stirring at 80 ° C. for 3 hours, and filtering. The solid content of the obtained extract 1 was 3.1%.

(Preparation Example 2: Olive leaf 50% aqueous ethanol extract)
The extract obtained by adding 10 g of olive leaf powder to 90 g of 50% aqueous ethanol, heating and stirring at 60 ° C. for 3 hours, and filtering was designated as extract 2. The resulting extract 2 had a solid content of 3.3%.

(Preparation Example 3: Olive leaf 99% ethanol extract)
Extract 3 was obtained by adding 10 g of olive leaf powder to 90 g of 99% ethanol, heating and stirring at 60 ° C. for 3 hours, and filtration. In addition, the solid content of the obtained extract 3 was 2.5%.

(Preparation Example 4: Enzyme-treated isoquercitrin aqueous solution)
Enzyme-treated isoquercitrin was prepared by the method of patent application by the present applicant (see the preparation example of Japanese Patent No. 3510717).

(1) Preparation of isoquercitrin After 6 kg of Enju bud, which is a leguminous plant, was immersed in 60 kg of hot water (95 ° C. or higher) for 2 hours, the filtrate obtained by filtration was obtained as a “first extract”. On the other hand, the filtered residue was further immersed in hot water and extracted to obtain a “second extract”. These first and second extracts were combined, cooled to 30 ° C. or lower and the precipitated components were filtered off, and the precipitate was washed with water, recrystallized and dried to obtain 547 g of rutin having a purity of 95% or more. .

Rutin (500 g) was dispersed in 100 L of water, and 50 g of naringinase (Amano Enzyme Co., Ltd., trade name: naringinase “Amano”) was added thereto. The pH of this system was 6. This was kept at 50 ° C. for 5 hours and then concentrated to 50 L. Upon cooling, isoquercitrin precipitated. The precipitate was collected by filtration and dried to obtain 320 g of isoquercitrin.

(2) Preparation of enzyme-treated isoquercitrin 100 L of water was added to 320 g of isoquercitrin obtained above, 800 g of dextrin was added and homogenized, and CGTase (Amano Enzyme Co., Ltd., trade name Contizyme) 200 ml And maintained at a temperature of 55 ° C. and a pH of 7.0 for 24 hours. This solution is added to an adsorption resin column (Diaion HP-20 manufactured by Mitsubishi Chemical Corporation), and then a 50% aqueous ethanol solution is applied to the column. The obtained eluate is concentrated under reduced pressure, freeze-dried, 395 g of enzyme-treated isoquercitrin (rutin equivalent content: 80.2%), which is a mixture of ercitrin and α-glycosylisoquercitrin, was obtained. Using the enzyme-treated isoquercitrin thus obtained, a 20 mg / mL solution was prepared with ion-exchanged water.

(Preparation Example 5: Enzyme-treated rutin aqueous solution)
500 mL of water is added to 5 g of rutin obtained by the same method as in Preparation Example 4, 25 g of dextrin is added and homogenized, and 5 g of CGTase (Amano Enzyme Co., Ltd., trade name Contizyme) is added thereto at a temperature of 55 ° C. It was kept at pH 7.0 for 24 hours. This solution is added to an adsorption resin column (Diaion HP-20 manufactured by Mitsubishi Chemical Corporation), and then a 50% ethanol aqueous solution is applied to the column. The obtained eluate is concentrated under reduced pressure, and freeze-dried to obtain rutin. 9.6 g (rutin equivalent content: 42.9%) of enzyme-treated rutin, which is a mixture of and α-glycosyl rutin, was obtained. Using the enzyme-treated rutin thus obtained, a 20 mg / mL solution was prepared with ion-exchanged water.

Experimental Example A: Measurement of inhibition of tyrosinase activity obtained from melanin production (absorbance method)
By the following method, the inhibition rate of tyrosinase activity of the preparation of the olive leaf extract of the present invention and the enzyme-treated isoquercitrin (hereinafter referred to as “EMIQ”) alone or in combination was examined.

Extracts 1 to 3 and EMIQ aqueous solution or enzyme-treated rutin aqueous solution obtained in the above preparation examples were adjusted so as to have the formulations shown in Tables 1 and 2, and 3.0 mM L- 0.95 mL of DOPA (Wako Pure Chemical Industries, Ltd.) was added, and 0.1 M phosphate buffer (pH 6.8) was further added to make 1.95 mL, which was incubated at 30 ° C. for 10 minutes. Thereafter, 0.05 mL of 600 U / mL tyrosinase (SIGMA-ALDRICH) solution previously heated to 30 ° C. was added, incubated at 30 ° C. for 30 minutes, and absorbance was measured at a wavelength of 475 nm, which is an indicator of the amount of melanin. This value was _designated as S _Abs475 . The case where no sample was added was used as a control, and this absorbance was _defined as C _Abs475 . When L-DOPA was not added, the sample blank, the absorbance was SB _Abs475 , when the sample and L-DOPA were not added were the control blank, and this absorbance was CB _Abs475, and the tyrosinase activity inhibition rate was calculated. As a positive control, a 20 mg / mL aqueous solution of kojic acid (Kishida Chemical Co., Ltd.) was used.

Tyrosinase activity inhibition rate (%) = {1− (S _Abs475 −SB _Abs475 ) / (C _Abs475 −CB _Abs475 )} × 100

From the above, when olive leaf extract and EMIQ or enzyme-treated rutin are combined, tyrosinase activity inhibitory action is higher and synergistic activity than when each is used alone, and is conventionally known as a tyrosinase activity inhibitor. There was a higher tyrosinase activity inhibition effect than kojic acid.

In addition, the following experiment was conducted in order to confirm the optimum blending ratio of olive leaf extract and EMIQ or enzyme-treated rutin by the same method as described above.

From the above, in the mixing ratio of olive leaf extract and EMIQ, 40:60 to 95: 5 (solid conversion), and in the mixing ratio of olive leaf extract and enzyme-treated rutin, 36:64 to 87:13 (solid conversion) It was confirmed that the compound has excellent tyrosinase activity inhibition when formulated in the above.

Experimental Example B: Measurement of inhibition of tyrosinase activity determined from the remaining amount of substrate (L-DOPA) (HPLC method)
According to the following method, the tyrosinase activity inhibitory action of the combination of the olive leaf extract of the present invention and EMIQ was examined.

As a sample, a preparation in which the extract 1 obtained in Preparation Example 1 and the EMIQ aqueous solution obtained in Preparation Example 4 were mixed at a ratio of 7: 3 (in solid conversion) was used, and the concentration in the reaction system was 1.3, 0.00. Adjustments were made to be 67, 0.27, 0.027, and 0.0027 ppm.

To each sample, 0.95 mL of 3.0 μM L-DOPA (Wako Pure Chemical Industries, Ltd.) that had been heated to 30 ° C. in advance was added, and 0.1 M phosphate buffer (pH 6.8) was further added. Add to 1.95 mL and incubate at 30 ° C. for 10 minutes. Thereafter, 0.05 mL of a 0.6 U / mL tyrosinase (SIGMA-ALDRICH) solution that had been heated to 30 ° C. was added and incubated at 30 ° C. for 30 minutes. The area value of L-DOPA was determined according to the following analysis conditions. And this was designated as S. The case where no sample was added was taken as a control, and the area value was taken as C. The sample and the case where tyrosinase was not added were defined as a control blank, and this area value was defined as CB.

Tyrosinase activity inhibition rate (%) = {1- (CB-S) / (CB-C)} × 100

<HPLC analysis conditions>
Column: Luna 5u C18 (2) (250 mm × 2.0 mm, 5 μm, Phenomenex), column temperature: 30 ° C., mobile phase: 0.1% phosphoric acid, flow rate: 0.2 mL / min, detector: UV detector , Wavelength 280 nm, injection volume: 50 μL, analysis time: 20 minutes

From the above, the addition of 0.0027 ppm (solid basis) or more of the combination of olive leaf extract and EMIQ had a tyrosinase activity inhibitory effect.

Example 1: Inhibition of browning of apple grated fruit juice An aqueous solution containing 0.5% of a preparation prepared by mixing the extract 1 obtained in Preparation Example 1 and the EMIQ aqueous solution obtained in Preparation Example 4 at 6: 4 (in solid conversion) ( The apple cut into about 1.5 cm square was immersed in solid conversion (130 ppm) for 2 minutes, wiped off with water, then grated and filtered, and the absorbance at 475 nm was measured. For comparison, it was also measured for apple juice soaked in water to which no preparation was added. The measurement was started 2 minutes after the start of grated, and the browning state of apple juice was confirmed by absorbance, and the change is shown in FIG.

From FIG. 1, it was confirmed that browning of apple juice could be suppressed by using a combination of olive leaf extract and enzyme-treated isoquercitrin. In addition, when an aqueous solution containing 10% of the preparation used in this experiment (solid conversion 2600 ppm) was used, it was desirable to use 10% or less (solid conversion 2600 ppm or less) because of the effects of coloring.

Example 2: Mushroom blackening suppression An aqueous solution containing 0.1% of a preparation prepared by mixing the extract 1 obtained in Preparation Example 1 and the EMIQ aqueous solution obtained in Preparation Example 4 at 6: 4 (solid conversion) (solid conversion) 26 ppm), 8 sliced mushrooms were immersed for 1 minute, packaged so as not to dry, and stored at 15 ° C. For comparison, mushrooms soaked in water to which no preparation was added were also stored at 15 ° C.

One hour later, when an appearance inspection was performed, all eight mushrooms treated with water to which the preparation was not added turned black, whereas those immersed in an aqueous solution to which the preparation was added turned black. I was not able to admit.

The tyrosinase inhibitor of the present invention is made from a highly safe natural product and does not adversely affect the taste and odor of the addition object in the standard usage, so it can be used in foods, seafood feeds, etc. It can be used in a wide range of fields such as discoloration prevention, quality-preserving agents and cosmetic compositions.

Graph showing the browning of apple juice in terms of absorbance

Claims (5)

A tyrosinase activity inhibitor comprising an olive leaf extract and enzyme-treated isoquercitrin or enzyme-treated rutin. The tyrosinase activity inhibitor according to claim 1, wherein the blending ratio of the olive leaf extract and the enzyme-treated isoquercitrin is 40:60 to 95: 5 (solid conversion) (mass ratio). The tyrosinase activity inhibitor according to claim 1, wherein the blending ratio of the olive leaf extract and the enzyme-treated rutin is 36:64 to 87:13 (solid conversion) (mass ratio). The tyrosinase activity inhibitor according to claim 1, wherein the olive leaf extract is an extract extracted with an extraction solvent containing a hydrophilic solvent. A method for inhibiting tyrosinase activity, comprising an olive leaf extract and enzyme-treated isoquercitrin or enzyme-treated rutin.

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