JPH10265403A - Tyrosinase activity inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject inhibitor useful in the fields of medicines, cosmetics, foods, etc. by including sericin. SOLUTION: This tyrosinase activity inhibitor contains sericin (preferably natural sericin, which is obtained e.g. by partially hydrolyzing cocoon or silk with an acid, an alkali or an enzyme, extracting the hydrolysate, adjusting the pH of the obtained aqueous solution to 3-5 with an organic acid or an inorganic acid, adding an organic coagulant or an inorganic coagulant to deposit the sericin, filtering out the sericin and subsequently drying the sericin. The inhibitor has high safety for human bodies and high heat stability, and can advantageously be produced. The inhibitor is added to cosmetics, medicines, etc., in an amount of about 0.1-50 wt.%, preferably 0.5-5 wt.%, or to foods in an amount of 0.1-100 wt.%, preferably 0.5-50 wt.%. The inhibitor is administered as a medicine at a daily dose of e.g. about 10-100 g.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel tyrosinase activity inhibitor which can be used in a wide range of fields such as pharmaceuticals, quasi-drugs, cosmetics, and foods.

[0002]

2. Description of the Related Art Tyrosinase has an enzyme number of 1.14.1.
It is a kind of enzyme having monophenol oxidase activity represented by 8.1. Enzymes of this type include melanocytes, houseflies, mushrooms, potatoes, apples,
It is widely distributed in animals, plants, and microorganisms such as Neurospora. In addition, this type of enzyme has been highly purified from various materials in recent years. For example, mushroom fruit bodies have been purified by methods such as acetone treatment, ammonium sulfate fractionation, ion exchange, and gel filtration. The molecular weight is 110,000 for mushroom enzyme and 14.4 for higher plant enzyme.
In the case of Neurospora enzyme, it differs considerably depending on the origin, which is 33,000. These are all copper enzymes but have different copper contents. The substrate is monophenol, o-
Enzymes specific for diphenols, especially animals, show high activity on tyrosine, DOPA, and are deeply involved in the first stage of melanin biosynthesis. This is represented by the following reaction equation. L-tyrosine + dihydroxy-L-phenylalanine (DOPA) + O ₂ → DOPA + DOPA-quinone +
H ₂ O

[0003] In humans, melanin is a pigment that is distributed in hair, skin, and the like, and determines the color thereof.
The formation of melanin in the skin is also one of human defense functions. Melanin has a function of absorbing and scattering excess light, and increases the amount of light inside the skin, thereby preventing the adverse effects of ultraviolet rays such as sunburn. In animals, melanin is biosynthesized as follows. First, when exposed to ultraviolet light, tyrosinase in melanosomes in melanocytes is activated, and from tyrosine as shown in the above-mentioned reaction formula,
DOPA and also DOPA-quinone are biosynthesized. D
After OPA-quinone, it undergoes polycondensation by non-enzymatic oxidation, decarboxylation, and coupling reaction to form melanin. When released from UV radiation, melanin falls off the stratum corneum due to skin metabolism and returns to skin color. On the other hand, when there is a stimulus such as exposure to a large amount of ultraviolet rays, the melanin-producing function is locally maintained, and the skin in that portion remains black, causing pigmentation and spots.

On the other hand, in the field of food, for example, when a captured crustacean is killed, the crustacean of the crustacean changes color within a short time. In addition, when peeling or cutting fruits and vegetables such as apples, yams and lettuce, the insides of the fruits and vegetables change color in a short time. This phenomenon is caused by activation of tyrosinase present in crustaceans or fruits and vegetables such as apples, yams and lettuce, resulting in the formation of melanin as a reaction product. Pigmentation of the skin due to these melanin formation,
BACKGROUND ART Tyrosinase activity inhibitors for inhibiting melanin biosynthesis have been used to prevent spots, promote whitening effects, or prevent discoloration of foods. For example, synthetic tyrosinase activity inhibitors include sulfites, and natural tyrosinase activity inhibitors include koji acid, placenta extract, vitamin C, cysteine, and extracts having a tyrosinase activity inhibiting function extracted from plants and the like. Had been.

[0005] However, in the case of the synthetic tyrosinase activity inhibitor, there is a problem regarding safety to the human body.
For example, there have been concerns that sulfites have an allergic effect on the human body and formaldehyde is generated during storage when used in crustaceans. Sodium sulfite, sodium bisulfite, etc., which are designated as food additives, also have use standards, and there are also restrictions on their remaining in foods. In the case of a natural tyrosinase activity inhibitor, for example, kojic acid is difficult to produce or purify, and is expensive. Specifically, a strain having a kojic acid-producing ability was cultured, and kojic acid was extracted and further crystallized from a fermentation solution containing kojic acid as a main component. On the other hand, it has been known that some bacterial strains having a kojic acid-producing ability produce aflatoxins having strong carcinogenicity, and there was a problem in safety. Production or purification of placenta extract extracted from placenta was also difficult and expensive, similar to kojic acid. In the case of vitamin C, the enzymatic oxidation reaction by tyrosinase (the above reaction formula) is suppressed by its own reducing power.
Therefore, over time, ascorbic acid changed to dehydroascorbic acid, and ascorbic acid decreased, so that the inhibitory activity was not sustained. In the case of cysteine, the inhibitory activity was not maintained because cysteine itself was oxidized similarly to vitamin C. In addition, cysteine has a disadvantage that it changes color to black when oxidized. The extract having a tyrosinase activity inhibitory function extracted from a plant or the like had a difficult chemical composition and low purity. In addition, there was a concern about quality variations among lots of the extract. In addition, most of the tyrosinase activity inhibitors conventionally used are fat-soluble substances, and water-soluble tyrosinase activity inhibitors are limited.

[0006]

SUMMARY OF THE INVENTION An object of the present invention is that it is derived from a natural product, has high safety to the human body, does not inactivate even when heated, has a long inhibitory activity, and is easy to produce or purify. To provide a novel tyrosinase activity inhibitor which can be produced at low cost and is single-purity and water-soluble.

[0007]

The present invention relates to a tyrosinase activity inhibitor containing sericin as an active ingredient. As sericin used in the present invention, usually, high-purity sericin (including a hydrolyzate) derived from a cocoon or raw silk is preferably used. Sericin as a non-hydrolysate according to the present invention can be obtained from a cocoon or raw silk by an extraction method generally performed. For example, the protein can be extracted in the state of a single protein having a high purity of 90% or more as follows.

[0008] That is, sericin contained in a cocoon or raw silk is extracted with water, and for example, the following (1), (2),
Alternatively, sericin as a non-hydrolysate is obtained by the method as in (3). In addition, the hydrolyzate of sericin according to the present invention can be obtained from a cocoon or raw silk by an extraction method generally performed. For example, it can be extracted in the state of a protein chamber (peptide) with a high purity of 90% or more as follows. That is, sericin contained in a cocoon or raw silk is partially hydrolyzed and extracted with an acid, an alkali, or an enzyme and then extracted, for example, by the following method (1), (2), or (3). To obtain a hydrolyzate of (1) After adjusting the pH of the aqueous sericin solution to 3 to 5 with an organic acid or an inorganic acid, an organic coagulant or an inorganic coagulant is added to precipitate sericin, which is filtered and dried to obtain solid sericin. (2) A sericin aqueous solution is mixed with a water-soluble solvent such as methanol, ethanol or dioxane to precipitate sericin, and then filtered and dried to obtain solid sericin. (3) After removing the substance that has passed through the dialysis membrane in the sericin aqueous solution, the substance that has not passed through the dialysis membrane is dried to obtain a sericin solid.

[0009] Sericin as a non-hydrolysate or hydrolyzate obtained as described above may be dissolved in a solid or solvent as it is, depending on the intended use as a tyrosinase activity inhibitor. An appropriate amount may be dissolved in water and used in an appropriate form such as an aqueous solution. Applications include cosmetics, food, food additives, topical drugs, quasi-drugs,
It can be used in medicines and the like in the same manner as conventional tyrosinase activity inhibitors. For example, the amount of sericin added to cosmetics, food additives, external medicines, quasi-drugs, etc. is usually 0.1 to 5
It is about 0% by weight, preferably about 0.5 to 5% by weight. Sericin is usually added in foods in an amount of about 0.1 to 100% by weight, preferably about 0.5 to 50% by weight. Sericin is non-toxic and excellent in water solubility, so that no particular problem occurs even if it is added or taken in large amounts. Dosage forms for cosmetics and external medicines include creams, emulsions, foundations, packs, lotions, gels, solutions, and sticks. Further, these may contain appropriate components such as oils, humectants, thickeners, preservatives, emulsifiers, pigments, pH adjusters, other medicinal components, ultraviolet absorbers, fragrances, and the like. It can also be administered orally as a pharmaceutical. The dose in this case is not particularly limited, either.
About 00 g / day is administered. Next, the present invention will be specifically described with reference to examples.

[0010]

【Example】

Example 1 1 kg of silk fabric consisting of raw silk was treated in 50 l of water at 95 ° C. for 2 hours to extract sericin. The obtained extract was filtered through a filter having an average pore size of 0.2 μm to remove aggregates, and the filtrate was dialyzed using a dialysis membrane having a molecular weight cutoff of 3500 to remove permeated substances and to remove non-permeated substances. By separating the substance, a colorless and transparent aqueous sericin solution having a concentration of 0.2% was obtained. The aqueous solution was concentrated to a sericin concentration of about 2% using an evaporator, and then lyophilized to obtain 100 g of a sericin solid having a purity of 95% or more.
I got

Example 2 1 kg of silk fabric made of raw silk
With 0.2% aqueous sodium carbonate (pH 11 to 12) 50
The mixture was treated at 95 ° C. for 2 hours in 1 L to extract the sericin hydrolyzate. The obtained extract was filtered through a filter having an average pore size of 0.2 μm to remove aggregates, and the filtrate was dialyzed using a dialysis membrane having a molecular weight cutoff of 3500 to remove permeated substances and to remove non-permeated substances. By separating the substance, a concentration of 0.
A 2% clear and colorless sericin hydrolyzate extract was obtained. The extract was subjected to a sericin concentration of about 2% using an evaporator.
After concentration, freeze drying was performed to obtain 100 g of a sericin hydrolyzate solid having a purity of 90% or more.

Test Example 1 0.5 mg / ml (1/15 M phosphate buffer pH 6.8) in 1/15 M phosphate buffer (pH 6.8) containing sericin or sericin hydrolyzate.
8) Tyrosinase (mushroom-derived, Sigma) 0.1 ml and 1/15 M phosphate buffer (pH
6.8) 0.9 ml was added and incubated at 25 ° C. for 10 minutes. Then 0.3 mg / ml DOPA (1/1
After adding 1 ml of 5M phosphate buffer (pH 6.8) and incubating at 25 ° C. for 5 minutes, the absorbance (D1) at 475 nm was measured. Separately, the same operation was performed using the enzyme inactivated by heating (D2), and DOPA without sericin was added.
The amount of chromium (D3) was determined and the inhibition rate was calculated. Inhibition rate (%) = ((D3-D1) / (D3-D2)) ×
100 As shown in Table 1, it was confirmed that both sericin and sericin hydrolyzate had the ability to inhibit tyrosinase activity. Particularly at a final concentration of 0.5% or more, the ability to inhibit tyrosinase activity was definitely present.

[Test Example 2] Here, the final concentration of sericin and sericin hydrolyzate was set to 0.5%. For comparison, the same experiment as in Test Example 1 was performed using bovine serum albumin (final concentration: 0.5%). Was. Furthermore, the sericin and the sericin hydrolyzate solution dissolved in the phosphate buffer were boiled for 2 hours, and the ability to inhibit tyrosinase activity after heating was also examined. Comparison of the tyrosinase activity inhibition rate between sericin and sericin hydrolyzate and other general proteins (bovine serum albumin) showed that the ability of bovine serum albumin to inhibit tyrosinase activity was almost nonexistent. Superiority was confirmed. On the other hand, the ability of sericin and sericin hydrolyzate to inhibit tyrosinase activity was maintained almost unchanged by heating. Therefore, it was confirmed that the ability of sericin and sericin hydrolyzate to inhibit tyrosinase activity was considerably stable even with heat.

[0014]

Industrial Applicability The tyrosinase activity inhibitor of the present invention is water-soluble and can be used for products having a low fat content. In addition, the tyrosinase activity inhibitor of the present invention has properties as a protein or peptide derived from a natural product, has a good affinity for the skin, is easily hydrolyzed by a protease, has no accumulation property, and has high safety. Have
It can be used in a wide range of fields such as pharmaceuticals, quasi-drugs, cosmetics, and foods. In addition, the tyrosinase activity is not deactivated by heat treatment, and the tyrosinase activity is stable. Furthermore, the tyrosinase activity inhibitor of the present invention can be easily extracted from a solvent extract of cocoon or raw silk with high purity as a single protein or peptide, so that it can be obtained at low cost and the color of the aqueous solution is colorless and transparent. Therefore, there is a great advantage that no decoloring is required and no complicated processing steps are required.

[0015]

[Table 1]

[0016]

[Table 2]

────────────────────────────────────────────────── ─── Continued on the front page (51) Int.Cl. ⁶ Identification code FI A61K 7/00 A61K 7/00 J X 7/48 7/48 37/18 ADA

Claims (3)

[Claims] 1. A tyrosinase activity inhibitor comprising sericin as an active ingredient. 2. The tyrosinase activity inhibitor according to claim 1, wherein the sericin is natural sericin extracted from cocoons or raw silk. 3. The tyrosinase activity inhibitor according to claim 1, wherein the sericin is a hydrolyzate of natural sericin.