JPH06199898A - New peptide and its use - Google Patents

Abstract

PURPOSE:To obtain a new peptide useful as medicine, cosmetic, food for beauty culture, agent for preventing browning of crustaceans, freshness preservative, etc., showing specific analyzed values of amino acids, having a specific amino acid sequence at the N end, exhibiting excellent inhibitory effect on tyrosinase activity. CONSTITUTION:A chrysalis of housefly is homogenized with 0.1 M acetic acid buffer solution (pH4.0), the filtrate is heated, centrifuged, the supernatant liquid is mixed with ammonium sulfate to give 80% saturated concentration, the precipitate is dialyzed against water, the dialyzed inner solution is purified by get filtration and HPLC to give a new peptide having analyzed values of amino acids (%) of 4.2 % Asp, 3.1 % Ser, 1.2% His, 4.1% Thr, 3.7% Pro, 8.8% Vat, 16.1% Cys, 7.3% Leu, 7.6% Lys, 4.5% Gluing, 19.4% Gly, 3.8% Arg, 6.5% Ala, 3.4% Tyr, 0% Met, 3.4% Ile and 1.4% Phe, containing four amino acids of formula I or formula II from N end, having 3,350+ or -900 molecular weight.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel bioactive peptide, and more particularly to a novel peptide having an inhibitory effect on tyrosinase activity.

[0002]

2. Description of the Related Art Tyrosinase is an enzyme that catalyzes the formation of melanin pigment and is widely present in the natural world. Those having an action of suppressing the tyrosinase activity are known to have various industrial uses. Has been.

For example, as disclosed in Japanese Patent Publication No. 61-144747, tyrosinase activity inhibitors are
From the viewpoint of beauty, it is used as a whitening agent for the prevention and treatment of melanin production. Further, regarding fruits, seeds and grains of plants, for example, JP-A-62-19 is used.
As disclosed in Japanese Patent No. 8372, browning occurs due to the production of melanin by tyrosinase, and a browning inhibitor is used for this.

In shellfish such as shrimp and crab, for example, as disclosed in JP-A-1-257427, it is used as a freshness-retaining agent for suppressing the activity of tyrosinase and maintaining the freshness. There is. Further, in pests, as disclosed in, for example, JP-A-1-132502, it is used as a pest control agent by suppressing melanin production.

However, the tyrosinase activity inhibitors used for these applications are not sufficiently satisfactory in terms of their tyrosinase activity inhibitory effect, and those having higher effects have been desired.

[0006]

From this point of view,
As a result of research on activity inhibitors of tyrosinase existing in animals, especially insects, we obtained the finding that a specific peptide has a very high tyrosinase activity inhibitory activity,
The present invention has been completed.

[0007]

The present invention provides a novel peptide having a very high tyrosinase activity inhibitory effect, and further provides a useful use thereof. That is, according to the present invention, the following amino acid analysis values are shown, and 4 amino acids from the N-terminus are H-
Disclosed is a peptide having a molecular weight of 3,350 ± 900 which is Glu-Ile- or H-Glu-Leu-, or a salt thereof, and a tyrosinase activity inhibitor characterized by using them as an active ingredient. Asp or Asn 4.2 ± 2% Glu or Gln 4.5 ± 2% Ser 3.1 ± 2% Gly 19.4 ± 2% His 1.2 ± 2% Arg 3.8 ± 2% Thr 4.1 ± 2% Ala 6.5 ± 2% Pro 3.7 ± 2% Tyr 3.4 ± 2% Val 8.8 ± 2% Met 0 Cys 16.1 ± 2% Ile 3.4 ± 2% Leu 7. 3 ± 2% Phe 1.4 ± 2% Lys 7.6 ± 2%

When amino acids, peptides and the like are represented by symbols in the present invention, symbols defined by IUPAC and IUP or ordinary symbols used in the field of peptide chemistry are used. Examples of symbols are as follows. Ala ... Alanine Arg ... Arginine Asn ... Asparagine Asp ... Aspartic acid Cys ... Cysteine Gln ... Glutamine Glu ... Glutamic acid Gly ... Glycine His ... Histidine Ile ... Isoleucine Leu ... Leucine Lys ... Lysine Met ... Methionine Phe ... Fernylalanine Pro ... Proline Ser ... Serine Thr ... Threonine Tyr ... Tyrosine Val ... Valine

Further, the peptide of the present invention may be a peptide in which at least one amino acid is deleted from the peptide as long as the effect of inhibiting tyrosinase activity is not lost.

[0010]

[Detailed Description of the Invention]

[Peptide Production Method] The peptide of the present invention can be produced by using a conventionally known peptide preparation method. As the preparation method, for example, methods such as an extraction method from a living body, a synthesis method, a gene recombination method and the like are known. In particular, a method utilizing an extraction method from a living body is preferable, and in that case, the above peptides can be produced from insects, especially flies, by combining various extraction and purification methods.

[0011]

[Use of Peptide] The tyrosinase activity inhibitory effect of the peptide thus obtained was investigated, and it was found that it has a significantly higher tyrosinase activity inhibitory effect than kojic acid. Goods,
It can be used in various applications such as cosmetics, foods, pest control agents, etc., which require a tyrosinase activity inhibitory effect.

As a drug, for example, a tyrosinase activity-suppressing effect is also exerted on a therapeutic agent for pigmentation that develops in the dermis portion of the skin such as spots, senile lentigines, endogenous melasma, and nevus. Further, it is useful as a quasi-drug or a cosmetic, for example, as a whitening agent.

[0013] As foods, for example, there are various uses for which tyrosinase activity suppressing action is required, such as an agent for preventing browning of crustaceans and fish and a freshness-retaining agent for fresh foods and vegetables.

The pest control agents include various pests, for example, hemiptera pests such as black leafhopper, lepidopteran pests such as Hansomoyoto and diamondback moth, as well as coleoptera pests, diptera pesticides, cockroach pests and arachnid pests. The use as a pesticide is mentioned.

The preferred use of the peptide of the present invention will be specifically described. The pharmaceutical composition containing the peptide of the present invention as an active ingredient, the quasi-drug and cosmetic dosage forms include various externally applicable forms such as creams, ointments, emulsions, injections, liniments, poultices, plasters,
Eye drops, paste, lotion, emulsion, essence,
In addition to known forms such as packs, gels, lipsticks, foundations, hair shampoos, hair rinses and hair tonics, various solid or liquid forms that can be orally administered, for example, capsules, tablets, granules, powders, syrups, It goes without saying that any liquid or solid raw material which can be freely formulated into a known form such as a drink and which is acceptable for skin and hair application or oral administration can be widely used.

As the active ingredient, in addition to the peptide of the present invention, known active ingredients such as ascorbic acid, hydroquinone, liquiritin and their derivatives, which are used as skin-whitening agents, kojic acid and kojic acid derivatives, arbutin, peripheral blood vessels. Cephalanthin, Vitamin E, Vitamin E nicotinate, nicotinic acid, nicotinic acid amide, benzyl nicotinate, ginger tincture, capsicum tincture, used as a dilator, camphor, menthol, used as a cooling agent, antibacterial agent Hinokitiol, benzalkonium chloride, undecylenic acid, anti-inflammatory lysozyme chloride, glycyrrhizin, allantoin, licorice extract, senburi extract, garlic extract, ginseng extract, o Gon'ekisu, rosemary extract,
One kind or two kinds or more selected from aloe extract, placenta extract, liver extract and the like can be appropriately selected and used freely.

At this time, various known additives such as antiseptics, fragrances, stabilizers, colorants, ultraviolet absorbers, antioxidants, humectants and thickeners can be added, if necessary.

<Uses as Pharmaceuticals, Quasi-drugs and Cosmetics> The amount of the peptide as the active ingredient of the present invention to be incorporated into pharmaceuticals, quasi-drugs and cosmetics is appropriate depending on the application site, degree of symptoms, dosage form and the like. Can be changed, but usually 0.
0001 to 10.00% by weight, preferably 0.1.
About 01 to 5.00% by weight is added to the formulation.

The peptide to be blended may be a purified one, but the extract or reaction solution can be used as it is.

<Use as whitening food> The form of the whitening food containing the peptide of the present invention as an active ingredient is, for example, jelly, drink, jam, biscuits, candy, chocolate, or any of various publicly known processed foods. The following forms can be used.

<Use as an anti-browning agent and a freshness-retaining agent> When the peptide is used as a browning-preventing agent and a freshness-retaining agent, the peptide should be used in a known manner for vegetables, marine products, feeds and the like. You can

In this case, other known tyrosinase activity inhibitors that can be used as foods as required, such as ascorbic acid and kojic acid, as well as preservatives, perfumes, stabilizers, colorants, emulsifiers, and antioxidants. , Various seasoning agents, thickeners, and various other known additives such as alcohol, protamine, red No. 2, yellow No. 4, BHA, carrageenan, natural flavor, β
Carotene monoglyceride, sodium glutamate, sorbitol, stevia, fructose, citric acid etc. can be added.

The amount of the peptide as the active ingredient of the present invention to be added may be appropriately changed depending on the form of the food or the like to be added, but it is usually about 0.0001 to 10.00% by weight, preferably 0.01 to 5%. It is advisable to add about 0.000% by weight.

The peptide to be blended may be a purified one, but the extract or reaction solution can be used as it is.

<Use as a Pest Control Agent> As the form of the pest control agent containing the peptide of the present invention as an active ingredient, various known forms such as paste, solution and powder can be used.

In that case, various known additives such as other known pest control agents, preservatives, fragrances, stabilizers, colorants, emulsifiers, antioxidants, extenders, thickeners, etc., may be added, if necessary. ,
Kojic acid, diflubenzolone, sodium dodecylbenzene sulfonate, dimethylformamide, methanol, ethanol, isopropyl alcohol, polyoxyethylene nonylphenyl ether, kaolin, silicic acid and the like can also be added.

The amount of the peptide which is the active ingredient of the present invention may be appropriately changed depending on the form of the pest control agent and the like, but it is usually about 0.0001 to 10.00% by weight, preferably 0.01 to 5.00. It is advisable to mix the pesticide with about% by weight.

The peptide to be blended may be a purified one, but the extract or reaction solution can be used as it is.

Next, examples of the method for producing the peptide which is the active ingredient of the present invention, pharmaceuticals, quasi drugs, cosmetics, foods and other examples and test examples of their effects will be shown, but these do not limit the present invention in any way. Not something to do.

<Production Example of Peptide> As an example of production of the novel peptide of the present invention, an extraction method from housefly will be shown. This peptide was prepared by preparing a crude extract from housefly, fractionation using SP-Toyopearl 550C column, Sephadex G-25.
Purification was carried out in four steps: fractionation using a column and fractionation using a YMC C8 HPLC column.

Elution chromatograms in the fractionation of the peptide using various columns are shown in the figures. At this time, the present tyrosinase activity-inhibiting fraction is shown by a dotted line, and the protein mass fraction is shown by a diagram in practice.

The degree of inhibition of the tyrosinase activity inhibition fraction was measured by the following method. That is, using tyrosinase prepared from flies as the enzyme solution, the final concentration was 2 mM.
DOPA and each fraction are added so that the total volume becomes 3 ml.
Add 50 mM phosphate buffer and incubate at 25 ° C,
The degree of tyrosinase activity inhibitory effect was determined by following the absorbance at 470 nm. The protein mass of each fraction is Lowr
y et al. [Journal of the Biological
Chemistry, 193, 265-275, 1951].

(1) Preparation of crude extract from housefly 6 L of 0.1 M acetate buffer (pH 4.0) in 1.5 Kg of housefly pupae
Was added and homogenized at 4 ° C. The homogenate solution was filtered with gauze, the filtrate was heated at 70 ° C. for 30 minutes, and a clear supernatant was obtained by centrifugation. Solid ammonium sulfate was added to the supernatant so that the concentration became 60%, and the mixture was centrifuged. Further, solid ammonium sulfate was added to the supernatant so that the concentration became 80%, and the mixture was centrifuged. The precipitate was dialyzed against water. The dialyzed solution was centrifuged to obtain a clear supernatant as a crude extract.

(2) Fractionation using SP-Toyopearl 550C column This crude extract was added to a 440 ml column of SP-Toyopearl 550C equilibrated with 50 mM phosphate buffer, and the sodium chloride concentration was changed from 0M to 0.6M. The tyrosinase activity inhibitory fraction was eluted with a gradient of. The elution chromatogram is shown in FIG. The fraction inhibiting tyrosinase activity was eluted at a sodium chloride concentration of around 0.5M.

(3) Fractionation using Sephadex G-25 column The fraction inhibiting tyrosinase activity was lyophilized and dissolved in water. The solution was added to a Sephadex G-25 column (diameter 2.8 cm x length 110 cm) equilibrated with 50 mM phosphate buffer, and the tyrosinase activity inhibitory fraction was eluted. The elution chromatogram is shown in FIG. The tyrosinase activity inhibitory fraction was detected in the eluate from 250 ml to 400 ml. The fractions were collected, dialyzed, lyophilized, and the dried product was dissolved in water.

(4) Fractionation using YMC C8 HPLC column The fractions were separated by YMC C8 HPLC column (diameter 4.6 cm x length 250 m).
Fractionation was performed using m). That is, fractionation was performed with a gradient of 12% to 24% acetonitrile containing 0.1% trifluoroacetic acid at a flow rate of 1 ml / min. The elution chromatogram is shown in FIG. The retention time of the tyrosinase activity inhibition fraction was about 47 minutes.

<Analysis Result> The analysis result of the peptide of the present invention thus obtained is shown below. (1) Amino acid analysis The amino acid analysis of the peptide of the present invention was performed by the following method. The purified peptide was dissolved in 6N hydrochloric acid containing 0.05% 2-mercaptoethanol, decompressed, and sealed in a glass tube. Place the glass tube at 110 ° C for 24 hours.
The peptide was hydrolyzed by heating for 48 hours and 72 hours. By using the Wako Pack WS-PTC column (manufactured by Wako Pure Chemical Industries, Ltd.) by the PTC-labeling method,
The amino acid analysis value of the peptide of the present invention was determined. The cysteine content was determined from cysteic acid produced by oxidation with performic acid.

The amino acid analysis values are as follows. Asp or Asn 4.2 ± 2% Glu or Gln 4.5 ± 2% Ser 3.1 ± 2% Gly 19.4 ± 2% His 1.2 ± 2% Arg 3.8 ± 2% Thr 4.1 ± 2% Ala 6.5 ± 2% Pro 3.7 ± 2% Tyr 3.4 ± 2% Val 8.8 ± 2% Met 0 Cys 16.1 ± 2% Ile 3.4 ± 2% Leu 7. 3 ± 2% Phe 1.4 ± 2% Lys 7.6 ± 2%

(2) Measurement of N-terminal amino acid sequence The amino acid sequence of the peptide of the present invention was determined by the double coupling method using 4-N, N-dimethylaminoazobenzene-4'isothiocyanate and phenylthiocyanate. The amino acid sequence of the peptide of the present invention is
H-Gl analyzed as 4 amino acids from N-terminus
It was u-Ile- or H-Glu-Leu-.

(3) Measurement of molecular weight The molecular weight of the peptide of the present invention was determined by the Laemmli method (Nature, 227) using a separation gel of 12.5% acrylamide.
Vol. 680-685, 1970) and performed by SDS polyacrylamide gel electrophoresis. The molecular weight of this peptide was 3,550 ± 900.

(4) Stability The tyrosinase activity residual ratio of the peptide of the present invention under various conditions was 60% under the condition of 80 ° C. for 1 hour.

(5) pH stability The tyrosinase activity inhibitory effect of the peptide of the present invention is
It was stable at 4 to 10.

Next, the effect of the tyrosinase activity inhibition test will be shown. <Test example> Using tyrosinase prepared from flies as an enzyme solution, add DOPA at a final concentration of 1.25 mM to 5.0 mM and the peptide of 1.5 nM to 6.0 nM to a total volume of 50 ml of a 50 mM phosphate buffer solution. Was added and incubated at 25 ° C., and the absorbance of 470 nm of the produced dopachrome was traced. Using the initial production rate of dopachrome, Lineweaver
-Burk plot and Dixon plot were performed to calculate the type of tyrosinase activity inhibition and the inhibition constant. The results are shown in Table 1. As is clear from this result, the peptide of the present invention was found to have a very high tyrosinase activity inhibitory effect.

Next, more specific examples of useful uses of the present invention will be shown with formulation examples. In the prescription example, "appropriate amount" is 10 in total.
It means a blending amount of 0% by weight. <Formulation Example 1> Cream (% by weight) A Monostearic acid 2.0 Polyethylene glycol (40.EO) Self-emulsifying glyceryl monostearate 5.0 Stearic acid 5.0 Behenyl alcohol 1.0 Liquid paraffin 10.0 Trioctanoic acid Glyceryl 10.0 B Glycerin 5.0 Ethylparaben 0.1 Peptide of the present invention 1.0 Purified water Appropriate amount A component belonging to A is dissolved by heating. Separately, the components belonging to B are melted by heating. B was added to A, and the mixture was stirred, emulsified, and cooled to produce a cream.

<Formulation Example 2> Emulsion (wt%) Peptide of the present invention 0.5 A Polyoxyethylene sorbitan monostearate (20.EO) 1.0 Polyoxyethylene sorbit monostearate (60.EO) 0. 5 Lipophilic glyceryl monostearate 1.0 Stearic acid 0.5 Behenyl alcohol 0.5 Avocado oil 4.0 Glyceryl trioctanoate 4.0 B 1,3-Butylene glycol 5.0 Methylparaben 0.2 Purified water The ingredients to which it belongs are heated and dissolved. Separately, the components belonging to B are melted by heating. B was added to A, a peptide as an active ingredient was added, and the mixture was stirred, emulsified, and cooled to prepare an emulsion.

<Formulation Example 3> Lotion (% by weight) Peptide of the present invention 0.5 Allantoin 0.1 Polyoxyethylene hydrogenated castor oil (60.EO) 8.0 Ethanol 15.0 Ethylparaben 0.1 Citric acid 0.1 Sodium citrate 0.3 1,3-Butylene glycol 4.0 Disodium edetate 0.01 Purified water Appropriate amount The above components were mixed, uniformly stirred and dissolved to produce a lotion.

<Formulation Example 4> Cream pack (% by weight) Peptide of the present invention 1.0 A Bagum 5.0 Squalene 2.0 Propylene glycol 5.0 Vitamin B ₁₂ 0.05 Purified water Amount B Zinc oxide 10.0 C Ethanol 5.0 Mix components belonging to A, swell by stirring, and add B little by little. C was gradually added to this, and the peptide of the present invention was further added and kneaded until a paste was formed to prepare a cream pack.

<Formulation Example 5> Essence (wt%) Peptide of the present invention 0.2 1% carboxyvinyl polymer solution 10.0 Glycerin 20.0 Hyaluronic acid 0.5 Ethanol 1.0 Purified water Appropriate amount of each of the above components Essence was manufactured by mixing, stirring and dissolving uniformly.

<Formulation Example 6> Hydrophilic ointment (% by weight) Peptide of the present invention 0.1 Ascorbic acid 0.5 A Polyoxyethylene cetyl ether 2.0 Glycerin monostearate 10.0 Liquid paraffin 10.0 Vaseline 4 0.0 Cetanol 5.0 B Propylene glycol 10.0 Methylparaben 0.1 Purified water Appropriate amount The components belonging to A are dissolved by heating. Separately, the components belonging to B are melted by heating. B was added to A, the peptide of the present invention as an active ingredient was added, and the mixture was stirred, emulsified, and cooled to produce a hydrophilic ointment.

Formulation Example 7 Aerosol (wt%) Peptide of the present invention 2.0 Benzyl nicotinate 0.01 Vitamin E acetate 0.05 Cetanol 1.2 Propylene glycol 4.0 Ethanol 8.0 Purified water proper amount Above The ingredients are uniformly mixed and dissolved, placed in an aerosol container, and 1 per 100% by weight of the mixture is prepared by a conventional method.
0.0% by weight of Freon 123 / 141b (57:43)
Was filled in a container to produce an aerosol.

<Formulation Example 8> Pap agent (% by weight) Peptide of the present invention 10.0 A Polyacrylic acid 30.0 Sorbitan monooleate 1.0 Purified water 30.7 B Sodium polyacrylate 7.0 Aluminum chloride 0.3 Concentrated glycerin 20.0 Titanium oxide 1.0 A component belonging to A is dissolved by heating. Separately, the components belonging to B are melted by heating. B was added to A, the peptide of the present invention as an active ingredient was added, and the mixture was stirred, emulsified, and cooled to prepare a poultice.

<Formulation Example 9> Tablet confectionery (% by weight) Citric acid 1.0 Skim milk powder 15.0 Sucrose fatty acid ester 1.0 Flavor 0.8 Peptide of the present invention 0.1 Kojic acid 0.05 Granulated sugar 20 0.0 Lactose 61.65 The above raw materials were uniformly mixed, granulated and tableted to produce.

<Formulation Example 10> Candy (% by weight) A Powder Marvit 83.0 Citric acid 0.4 Water 14.8 B Flavor 1.0 Yellow No. 4 0.001 Ingredient belonging to peptide 0.05 A of the present invention Are mixed and heated in a vacuum pan under reduced pressure to condense until the water content becomes 2 to 5%. This is transferred to a cooling plate, then the components belonging to B are sequentially added and cooled to about 60 ° C. Was molded by a roller or a stamping machine.

<Formulation Example 11> Pest control agent (% by weight) Peptide of the present invention 0.5 Silica anhydride 5.0 Sodium dodecylbenzenesulfonate 2.0 Kaolin 73.0 The above components are mixed and finely pulverized with a mixer. Then, it was manufactured as a 20% wettable powder.

<Formulation Example 12> Pest control agent (wt%) Peptide of the present invention 1.0 Dimethylformamide 40.0 Methanol 20.0 Ethanol 10.0 Isopropyl alcohol 10.0 Polyoxyethylene nonylphenyl ether 10.0 Above The ingredients were uniformly mixed and dissolved to obtain an emulsion.

[0056]

INDUSTRIAL APPLICABILITY According to the present invention, a novel peptide having a high tyrosinase activity inhibitory effect is provided, and this peptide has a tyrosinase activity inhibitory effect on drugs, quasi drugs, cosmetics, foods and pest control agents. It can be effectively used for various required applications.

[Brief description of drawings]

FIG. 1 SP-Toyopearl 5 is a crude extract from the housefly.
It is a graph which shows the result of having fractionated using a 50C column.

FIG. 2: Sephadex G- was used as a crude extract from housefly.
It is a graph which shows the result of having fractionated using 25 columns.

FIG. 3: HP of YMC C8 with a crude extract from housefly
It is a graph which shows the result of having fractionated using an LC column.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical display location A61K 7/00 K 9164-4C 7/48 9051-4C 37/64 ADA 8314-4C C07K 3/20 8318-4H C12N 9/99

Claims (3)

[Claims] 1. The following amino acid analysis values are shown, and N
Molecular weight 3,350 ± 90 in which 4 amino acids from the end are H-Glu-Ile- or H-Glu-Leu-
0 peptides or salts thereof. Asp or Asn 4.2 ± 2% Glu or Gln 4.5 ± 2% Ser 3.1 ± 2% Gly 19.4 ± 2% His 1.2 ± 2% Arg 3.8 ± 2% Thr 4.1 ± 2% Ala 6.5 ± 2% Pro 3.7 ± 2% Tyr 3.4 ± 2% Val 8.8 ± 2% Met 0 Cys 16.1 ± 2% Ile 3.4 ± 2% Leu 7. 3 ± 2% Phe 1.4 ± 2% Lys 7.6 ± 2% 2. The peptide according to claim 1, which is deficient in one or more constituent amino acids. 3. A tyrosinase activity inhibitor comprising the peptide according to claim 1 or 2 or a salt thereof as an active ingredient.