JP2013075850A - Composition - Google Patents

Abstract

An object of the present invention is to provide a whitening composition containing a compound that exhibits a whitening effect as an active ingredient in a water-soluble state and exhibiting high storage stability and high penetrability.
A whitening composition comprising protein particles and pterostilbene as active ingredients.
[Selection figure] None

Description

  The present invention relates to a composition containing pterostilbene and protein particles that are extremely useful for cosmetics and functional foods, and specifically to a whitening composition.

  Active oxygen generated by exposure to ultraviolet rays and various factors released from skin cells under the influence of the active oxygen enhance tyrosinase activity in melanocytes. Melanin involved in skin tone is produced by oxidation of tyrosine by tyrosinase in melanocytes. It is believed that when tyrosinase is activated by ultraviolet rays, melanin is excessively produced and delivered to epidermal cells, and the skin tone changes and blackens. Therefore, in order to obtain a whitening effect, it is required to inhibit tyrosinase activity and suppress melanin production.

  On the other hand, singlet oxygen is known as a kind of active oxygen generated by exposure to ultraviolet rays. Singlet oxygen is said to cause adverse effects such as tissue damage by reacting with biomolecules, and as a result, promotes skin pigmentation.

  Therefore, a search for a substance that removes singlet oxygen generated in a living body has been performed. Carotenoid compounds are useful as substances that remove singlet oxygen. However, singlet oxygen that cannot be completely removed is converted into free radicals in the living body, tyrosinase is activated, and pigmentation is greatly affected. Polyphenol compounds are said to be useful for removing these free radicals.

Hydroxystilbene, which is a polyphenol compound, has a tyrosinase inhibitory activity and is known to have a whitening effect. For example, Patent Document 1 describes a whitening cosmetic characterized by blending hydroxystilbene. Patent Document 2 describes a complex containing resveratrol (also referred to as 5-parahydroxystyrylresorcinol or 3,4 ', 5-stilbenetriol) and casein. It is described as being used for food or feed supplements. Furthermore, Patent Document 3 describes a matrix metalloprotease-2 (MMP-2) inhibitor containing resveratrol as an active ingredient. Patent Document 3 describes the use of resveratrol encapsulated in protein particles such as casein and gelatin as an MMP-2 inhibitor.
However, resveratrol itself has problems such as poor storage stability and coloring due to decomposition, low water solubility, and low permeability.

On the other hand, pterostilbene [4-[(E) -2- (3,5-dimethoxyphenyl) vinyl] phenol, 4-[(E) -3,5-dimethoxystyryl] phenol, (E) -3,5- Dimethoxy-4′-hydroxystilbene, or (E) -3 ′, 5′-dimethoxystilbene-4-ol, is also known to have antioxidant activity as resveratrol. As a product using pterostilbene, PteroWhite ^™ 90% (Sabinsa) is known.
However, pterostilbene alone is difficult to dissolve in an aqueous medium and difficult to formulate.

Japanese Patent No. 2502318 Special table 2010-500303 gazette JP 2011-46660 A

  This invention made it the subject which should be solved to provide the composition for whitening which contains the compound which exhibits the whitening effect as an active ingredient in the state water-solubilized, and shows high preservability and high permeability.

  As a result of diligent research to solve the above-mentioned problems, the present inventors can make pterostilbene water-soluble by mixing pterostilbene and protein particles, and simultaneously contain the obtained pterostilbene and protein particles. The solution was found to exhibit high storage stability and the present invention was completed.

That is, according to the present invention, a composition containing protein particles and pterostilbene is provided.
Preferably, the average particle size of the protein particles is 10 to 1000 nm.
Preferably, the composition of the present invention contains 0.1 to 100% by mass of pterostilbene based on the mass of the protein.
Preferably, the protein is at least one selected from the group consisting of collagen, gelatin, acid-treated gelatin, albumin, ovalbumin, casein, transferrin, globulin, fibroin, fibrin, laminin, fibronectin, or vitronectin.
Furthermore, according to the present invention, a melanin production inhibitor containing pterostilbene as an active ingredient is provided.
Furthermore, the composition of the present invention can be applied to an external preparation for skin.
Furthermore, the composition of the present invention can be applied to functional foods.

Further according to the present invention, there is provided a method of promoting skin whitening comprising administering to a subject a composition comprising protein particles and pterostilbene.
Furthermore, according to this invention, the method of suppressing the production of melanin including administering to a subject the composition containing a protein particle and pterostilbene is provided.
Further according to the present invention there is provided the use of protein particles and pterostilbene for the manufacture of a composition.
Furthermore, according to this invention, use of a protein particle and pterostilbene for manufacture of a melanin production inhibitor is provided.

  In the composition of the present invention, pterostilbene can be water-solubilized by mixing pterostilbene and protein particles. The composition of the present invention exhibits high stability with respect to heat and pH without causing coloring or the like even when stored in an aqueous medium for a long period of time. In addition, the composition of the present invention exhibits high permeability and can exhibit an excellent whitening effect.

It is a figure which shows the result of having evaluated the permeability | transmittance about the resveratrol inclusion casein aqueous solution and the pterostilbene inclusion casein aqueous solution. It is a figure which shows the result of the coloring test about the resveratrol inclusion casein aqueous solution and the pterostilbene inclusion casein aqueous solution. It is a figure which shows the result of having investigated the temporal stability of the resveratrol inclusion casein aqueous solution. It is a figure which shows the result of having investigated the temporal stability of the pterostilbene inclusion casein aqueous solution. It is a figure which shows the result of having investigated the density | concentration dependence about the B16 melanin inhibitory effect of pterostilbene (PTER), arbutin (Arb), or resveratrol (Res). It is a figure which shows the result of having investigated IC50 value (mM) about the B16 melanin inhibitory effect of pterostilbene (PTER), arbutin (Arb), or resveratrol (Res).

Hereinafter, embodiments of the present invention will be described more specifically.
The composition of the present invention contains protein particles and pterostilbene.
The composition of the present invention can be used as a melanin production inhibitor.
The composition of the present invention contains pterostilbene as an active ingredient.

  Pterostilbene can be obtained commercially from Sabinsa and can be obtained in crude form from Wako Pure Chemical Industries, Ltd., Tokyo Kasei Kogyo Co., Ltd. or Sigma-Aldrich.

The composition of the present invention preferably contains 0.01 to 50% by mass of protein particles, more preferably 0.1 to 10% by mass of protein particles.
The content of pterostilbene is preferably 0.1 to 100% by mass, more preferably 0.1 to 50% by mass, based on the mass of the protein.

  In the present invention, pterostilbene may be added during the formation of protein particles, or may be added after the production of protein particles.

  The average particle size of the protein particles used in the present invention is usually 1-1000 nm, preferably 10-1000 nm, more preferably 10-500 nm, still more preferably 10-250 nm, and still more preferably. It is 10-100 nm, Most preferably, it is 20-40 nm.

The average particle size of the protein particles used in the present invention can be measured with a commercially available particle size distribution meter or the like.
Particle size distribution measurement methods include optical microscopy, confocal laser microscopy, electron microscopy, atomic force microscopy, static light scattering, laser diffraction, dynamic light scattering, centrifugal sedimentation, and electrical pulse measurement. Methods, chromatographic methods, ultrasonic attenuation methods, and the like are known, and devices corresponding to the respective principles are commercially available.

  In the particle size measurement of the protein particles of the present invention, it is preferable to apply the dynamic light scattering method from the particle size range and ease of measurement. As a commercially available measuring device using the dynamic light scattering method, Nanotrac UPA (Nikkiso Co., Ltd.), dynamic light scattering type particle size distribution measuring device LB-550 (Horiba, Ltd.), concentrated particle size Analyzer FPAR-1000 (Otsuka Electronics Co., Ltd.) etc. are mentioned.

The type of protein used in the present invention is not particularly limited, but a protein having a lysine residue and a glutamine residue is preferable, and a protein having a molecular weight of 10,000 to 1,000,000 is preferably used.
Specific examples are listed as proteins, but the present invention is not limited to these compounds. At least one selected from the group consisting of collagen, gelatin, acid-treated gelatin, albumin, ovalbumin, casein, transferrin, globulin, fibroin, fibrin, laminin, fibronectin, or vitronectin can be used.
The origin of the protein is not particularly limited, and any of cows, pigs, fish, plants and genetically modified organisms can be used.

As the genetically modified gelatin, for example, those described in EP1014176A2 and US Pat. No. 6,992,172 can be used, but are not limited thereto.
Preferred proteins are casein, acid-treated gelatin, collagen, or albumin, and most preferred is casein or acid-treated gelatin.
When casein is used in the present invention, the origin of casein is not particularly limited, and may be derived from milk or bean, α-casein, β-casein, γ-casein, κ-casein and theirs. Mixtures can be used. Casein can be used alone or in combination of two or more.

  The proteins used in the present invention may be used alone or in combination of two or more.

  In the present invention, the protein can be crosslinked during and / or after the formation of protein particles. An enzyme can be used for the above-described crosslinking treatment. The enzyme is not particularly limited as long as it has a known protein cross-linking effect, and transglutaminase is preferable among them.

  Transglutaminase may be derived from a mammal or a microorganism, and a genetic recombinant can be used. Specifically, Ajinomoto Co., Ltd. Activa series, mammal-derived transglutaminase released as a reagent, for example, Oriental Yeast Industry Co., Ltd., Upstate USA Inc. And guinea pig liver-derived transglutaminase, goat-derived transglutaminase, rabbit-derived transglutaminase, human-derived recombinant transglutaminase, etc. manufactured by Biodesign International.

  The amount of the enzyme used for the crosslinking treatment in the present invention can be appropriately set according to the type of protein, but is typically about 0.1 to 100% by mass with respect to the mass of the protein. 1-50 mass% can be preferably added.

  The time for the cross-linking reaction by the enzyme can be appropriately set according to the type of protein and the size of the protein particle, but it may be normally 1 to 72 hours, preferably 2 to 24 hours. That's fine.

  The temperature of the cross-linking reaction by the enzyme can be appropriately set according to the type of protein and the protein particle size, but it is typically 0 to 80 ° C., preferably 25 to 60 ° C. Good.

  The enzyme used for this invention can be used individually or in combination of 2 or more types.

  The protein particles used in the present invention are disclosed in JP-A-6-79168 or C.I. Although it can be produced according to the method described in Coester, Journal Microcapsulation, 2000, Vol. 17, p. 187-193, it is preferable to use an enzyme instead of glutaraldehyde as a crosslinking method.

Moreover, in this invention, it is preferable to perform an enzyme crosslinking process in an organic solvent. The organic solvent used here is preferably a water-soluble organic solvent such as ethanol, isopropanol, butylene glycol, or glycerol, which is added to a general skin external preparation.
Furthermore, in the present invention, it is preferable to disperse the organic solvent after the crosslinking treatment and disperse in water. Water may be added before the organic solvent is distilled off, or water may be added after the distillation.

  One or more components selected from lipids (phospholipids, etc.), anionic polysaccharides, cationic polysaccharides, anionic proteins, cationic proteins, or cyclodextrins can also be added to the composition of the present invention. The amount of lipid (such as phospholipid), anionic polysaccharide, cationic polysaccharide, anionic protein, cationic protein, and cyclodextrin is not particularly limited, but is generally 0.1 to 100 mass relative to the mass of the protein. % Can be added. In the composition of the present invention, the sustained release rate can be adjusted by changing the ratio of the above components to the protein.

  Specific examples are listed as phospholipids that can be used in the present invention, but the present invention is not limited to these compounds. Examples include phosphatidylcholine (lecithin), phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, diphosphatidylglycerol, sphingomyelin and the like.

  The anionic polysaccharide that can be used in the present invention is a polysaccharide having an acidic polar group such as a carboxyl group, a sulfate group, or a phosphate group. Specific examples are listed below, but the present invention is not limited to these compounds. Examples thereof include chondroitin sulfate, dextran sulfate, carboxymethyl cellulose, carboxymethyl dextran, alginic acid, pectin, carrageenan, fucoidan, agaropectin, porphyran, caraya gum, gellan gum, xanthan gum, and hyaluronic acid.

  The cationic polysaccharide that can be used in the present invention is a polysaccharide having a basic polar group such as an amino group. Specific examples are listed below, but the present invention is not limited to these compounds. Examples thereof include glucosamine such as chitin and chitosan and galactosamine as a constituent monosaccharide.

  Anionic proteins that can be used in the present invention are proteins and lipoproteins whose isoelectric point is more basic than physiological pH. Specific examples are listed, but the present invention is not limited to these compounds. Examples include polyglutamic acid, polyaspartic acid, lysozyme, cytochrome C, ribonuclease, trypsinogen, chymotrypsinogen, α-chymotrypsin and the like.

  The cationic proteins used in the present invention are proteins and lipoproteins whose isoelectric point is on the acidic side of physiological pH. Specific examples are listed, but the present invention is not limited to these compounds. Examples include polylysine, polyarginine, histone, protamine, and ovalbumin.

  Casein is preferably used as the protein particle of the present invention, and a form in which pterostilbene is encapsulated in the casein particle is preferable. Casein particles encapsulating pterostilbene are highly absorbable into the living body.

Casein particles encapsulating pterostilbene can be produced by using a known method described in, for example, JP-A-2008-179551.
Specifically, there are a method in which casein is mixed in a basic aqueous medium and injected into an acidic aqueous medium, and a method in which casein is mixed in a basic aqueous medium and the pH is lowered while stirring.

As an example of the present invention, casein particles produced by the following steps (a) to (c) can be used.
(A) mixing casein into a basic aqueous medium having a pH of 8 or more and less than 11;
(B) adding pterostilbene to the solution obtained in step (a); and (c) injecting the solution obtained in step (b) into an acidic aqueous medium having a pH of 3.5 to 7.5;

As another example of the present invention, casein particles produced by the following steps (a) to (c) can be used.
(A) mixing casein into a basic aqueous medium having a pH of 8 or more and less than 11;
(B) a step of adding pterostilbene to the solution obtained in step (a); and (c) a step of lowering the pH of the solution obtained in step (b) to a pH separated from the isoelectric point by pH 1 or more;

  In the above, pterostilbene may be dispersed in a system not containing water, or can be dispersed by adding an aqueous medium after the dispersion. That is, in the above, step (b) may be performed before step (a).

  In the present invention, casein particles having a desired size can be produced. Further, the active ingredient can be included in the casein particles by utilizing the interaction between the hydrophobic active ingredient and the casein hydrophobic portion. Furthermore, it was found that these particles exist stably in an aqueous solution.

  As a method of mixing casein into a basic aqueous medium liquid and injecting it into an acidic aqueous medium, it is convenient and preferable to use a syringe, but any method that satisfies the injection speed, solubility, temperature, and stirring state can be used. There is no particular limitation. In general, the injection rate can be 1 mL / min to 100 mL / min.

Although the temperature of a basic aqueous medium can be set suitably, it can be 0-80 degreeC normally, Preferably it can be 25-70 degreeC. Although the temperature of an acidic aqueous medium can be set suitably, it can be normally 0-80 degreeC, Preferably it can be 25-60 degreeC.
The stirring speed can be appropriately set, but can be set to 100 to 3000 rpm, preferably 200 to 2000 rpm as a standard.

As a method of lowering the pH while mixing casein with a basic aqueous medium and stirring, it is simple and preferable to add an acid, but there is no particular limitation as long as the method satisfies the solubility, temperature, and stirring state. .
Although the temperature of a basic aqueous medium can be set suitably, it can be 0-80 degreeC normally, Preferably it can be 25-70 degreeC. The stirring speed can be set as appropriate, but it can be normally set to 100 to 3000 rpm, preferably 200 to 2000 rpm.

  Examples of the aqueous medium used for producing the casein particles include an aqueous solution of an organic acid or an organic base, an aqueous solution of an inorganic acid or an inorganic base, or a buffer solution. Specifically, citric acid, ascorbic acid, gluconic acid, carboxylic acid, tartaric acid, succinic acid, acetic acid, phthalic acid, trifluoroacetic acid, morpholinoethanesulfonic acid, 2- [4- (2-hydroxyethyl) -1- Piperazinyl] organic acids such as ethanesulfonic acid; organic bases such as tris (hydroxymethyl), aminomethane, ammonia; inorganic acids such as hydrochloric acid, perchloric acid, carbonic acid; sodium phosphate, potassium phosphate, calcium hydroxide, Although the aqueous solution using inorganic bases, such as sodium hydroxide, potassium hydroxide, and magnesium hydroxide, is mentioned, It is not limited to these.

The concentration of the aqueous medium described above is preferably about 10 mM to about 1M. More preferably, it is about 20 mM to about 200 mM.
The pH of the basic aqueous medium is preferably 8 or more, and preferably 8-12. More preferably, the pH is 9-11. If the pH is too high, there is concern about hydrolysis and danger in handling, so the above range is preferable.

In the present invention, the temperature at which casein is mixed with a basic aqueous medium having a pH of 8 or higher is preferably from 0 to 80 ° C, more preferably from 10 to 60 ° C. More preferably, it is 20-40 degreeC.
The pH of the acidic aqueous medium used in the present invention is preferably pH 3.5 to 7.5, more preferably pH 5 to 6, from the viewpoint of preventing rapid pH change.

  The skin external preparation of the present invention can further contain a carotenoid compound. It is expected that the carotenoid compound and pterostilbene are used in combination and supplemented to enhance the antioxidant network in the skin and promote the effects of the present invention.

  Carotenoid compounds are naturally occurring yellow to red terpenoid pigments that can be found in plants, algae, and bacteria. Examples of the carotenoid compounds include hydrocarbons (carotenes) and oxidized alcohol derivatives thereof (xanthophylls). As these, actinioelisrol, astaxanthin, bixin, canthaxanthin, capsanthin, capsorbin, β-8′-apo-carotenal (apocarotenal), β-12′-apo′-carotenal, α-carotene, β-carotene, “ Carotenes "(mixtures of [alpha]-and [beta] -carotenes), [gamma] -carotene, [beta] -cryptoxanthin, lutein, lycopene, biolerithrin, zeaxanthin, and esters of those containing hydroxyl or carboxyl. Many carotenoid compounds occur naturally in the form of cis and trans isomers, but the composites are often racemic mixtures. Carotenes are generally extracted from plant materials.

  In the present invention, the carotenoid compound preferably contains at least one of astaxanthin and lycopene, and more preferably contains both astaxanthin and lycopene. As content, it is preferable to contain 0.1-100 mass% carotenoid type compound with respect to the mass of protein. When both astaxanthin and lycopene are included, it is preferable that the ratio contains 0.1 to 1000 parts by mass of lycopene with respect to 100 parts by mass of astaxanthin. By containing astaxanthin and lycopene in specific amounts, decomposition of astaxanthin and lycopene and precipitation in the system of lycopene crystals are effectively suppressed. As a result, the composition of the present invention has excellent storage stability.

Lycopene (sometimes referred to as “lycopene”) is a carotenoid of the chemical formula C ₄₀ H ₅₆ (molecular weight 536.87) and is absorbed by 474 nm (acetone) belonging to one of the carotenoids of carotenoids. It is a red pigment showing maximum.
In lycopene, there are also cis- and trans- isomers of a conjugated double bond at the center of the molecule, and examples include all-trans-, 9-cis and 13-cis isomers. In the present invention, Any of these may be used.

Lycopene may be contained in the composition of the present invention as a lycopene-containing oil or lycopene-containing paste separated and extracted from a natural product containing it.
Lycopene is naturally contained in tomatoes, strawberries, watermelons, and pink grapefruits, and the above-mentioned lycopene-containing oil may be separated and extracted from these natural products. There are four known product types: oil type, emulsion type, paste type, and powder type.
In addition, the lycopene used in the present invention may be the above-described extract, a product obtained by appropriately purifying the extract as necessary, or a synthetic product.

A particularly preferred form of lycopene in the present invention is a fat-soluble extract extracted from tomato pulp. The fat-soluble extract extracted from the tomato pulp is particularly preferable from the viewpoints of stability, quality, and productivity in the composition.
Here, the fat-soluble extract extracted from tomato pulp is an extract extracted from a pulp-like solid obtained by distance-separating a pulverized product obtained by pulverizing tomato using an oily solvent Means a thing.
As the lycopene which is a fat-soluble extract, a tomato extract widely marketed as lycopene-containing oil or paste can be used, for example, Lyc-O-Mato 15% sold by Sunbright Co., Ltd. Examples include Lyc-O-Mato 6% and lycopene 18 sold by Kyowa Hakko Kogyo Co., Ltd.

The composition of the present invention can be applied to an external preparation for skin.
The composition of the present invention can promote skin whitening. Since the composition of the present invention is excellent in safety and permeability when applied to the skin, it is suitable for use as a skin external preparation.
Moreover, the composition of this invention can be used as a melanin production inhibitor. For example, the composition of the present invention can be used by blending with a skin cosmetic. Only the composition of this invention may be mix | blended with skin cosmetics, and you may mix | blend with another active ingredient in combination.

  The external preparation for skin of the present invention may further contain an additive. Although it does not specifically limit as an additive, an antioxidant component, an active oxygen remover, an anti-inflammatory agent, an anti-aging agent, a collagen synthesis promoter, an anti-wrinkle agent, a vitamin agent, a mineral, an amino acid, an antibacterial agent, an ultraviolet ray Use one or more selected from absorbents, moisturizers, softeners, transdermal absorption enhancers, soothing agents, preservatives, antioxidants, pigments, thickeners, fragrances, or pH adjusters can do.

  Although the dosage form of the external preparation for skin of the present invention is not particularly limited, for example, liquid, poultice, coating agent, gel, ointment, cream, aerosol, lotion, essence, powder, foam, lotion, Examples include body soaps, soaps, and cosmetics. When used in a dosage form such as an ointment or cream, a suspension or stabilizer may be added as necessary.

The composition of the present invention can be applied to functional foods.
The composition of the present invention can promote skin whitening.

  As the form of the functional food of the present invention, not only general foods such as nutrition drinks, nourishment tonics, palatability drinks, frozen desserts, but also nutritional supplements in the form of tablets, granules, capsules, etc. are suitable. It can be mentioned, but is not limited to these.

  Examples of the method for administering the composition of the present invention include transdermal, transmucosal and oral administration.

The dosage of the whitening composition of the present invention can be appropriately set according to the weight of the user, the state of the disease, etc., but in general, the dosage of pterostilbene which is an active ingredient per one administration A dose of about 1 μg to 50 mg / cm ² can be administered, and preferably about 2.5 μg to 10 mg / cm ² can be administered.

  The following examples further illustrate the present invention, but the scope of the present invention is not limited to these examples.

Example 1 Preparation of Casein Particles Encapsulating Pterostilbene Pterostilbene was dissolved in 1,3-butylene glycol so as to be 5% (w / w) to obtain a pterostilbene solution.
Similarly, resveratrol was dissolved with 1,3-butylene glycol so as to be 5% (w / w) to obtain a resveratrol solution.
Casein was dispersed in the same amount of 1,3-butylene glycol, and 1% (w / w) of about 50 mM Na citrate adjusted to pH 11 was added to obtain a casein dispersion. The pH of this casein nanodispersion was 7.2.

  A 1/10 amount (mass ratio) of a pterostilbene solution or resveratrol solution was added to the casein dispersion obtained above, and the mixture was well stirred and dispersed with a magnetic stirrer. The obtained solution was a solution containing 0.9% by mass of casein and about 0.03% by mass of pterostilbene or resveratrol. Then, it was applied to a filter of about 0.22 μm to obtain a casein particle-containing dispersion enclosing more transparent and uniform pterostilbene or resveratrol. The appearance immediately after preparation of the obtained casein particle-containing dispersion was uniformly transparent, and the average particle size was 27.5 nm. The aqueous solution containing 0.03% by mass of resveratrol was already cloudy (absorbance <OD 700 nm> at 700 nm was 0.1 or more) immediately after preparation.

  The average particle size of the casein particles is a value measured using a concentrated particle size analyzer FPAR-1000 (Otsuka Electronics Co., Ltd.), and specifically, a value measured as follows is adopted. That is, the particle diameter is measured by using a quartz cell after diluting with pure water so that the concentration of casein contained in the sample is 0.1% by mass. The particle size was determined as the volume average diameter when 1.333 (pure water) was set as the refractive index of the dispersion medium and the viscosity of pure water was set as the viscosity of the dispersion medium. The casein particles encapsulating pterostilbene immediately after production could be stably water-solubilized.

Example 2: Inclusion rate test Resveratrol (Res) and pterostilbene (PTER) inclusion casein solution prepared in Example 1 was weighed in an amount of 1 g and ultracentrifuged (HITACHI 100,000 G, 60 minutes). Unbound content in the aqueous solution was quantified by quantifying the supernatant on which encapsulated casein was precipitated by HPLC. The ratio with the prescription application amount was calculated, and the leakage rate (%) is shown in Table 1. The binding rate was calculated as 100-leakage rate (%).
HPLC condition column: C18 5 μm 4.6 mm × 250 mm
Flow rate: 1 ml / min
Column temperature: 40 ° C
Detection wavelength: 310 nm
Developing solution: acetonitrile / water = 20/80 (volume ratio including 0.1 volume (volume)% acetic acid and 0.1 volume (volume)% triethanolamine)

Example 3: Penetration rate analysis (permeability evaluation)
The permeability was evaluated by a skin permeation test.
For the skin of the subject, hairless rats (8 weeks old) were purchased and used (Ishikawa Experimental Animal, Saitama). A 27 mm diameter biopsy punched out of the abdomen of a hairless rat in a circle was fixed to a Franz cell (Hanson Research). After refluxing with PBS for 1 hour, the receptor solution was replaced with 7 mL of PBS containing 30 v / v% ethanol. 1 mL of a preparation prepared by adding 0.1 ml of 10 times concentrated PBS (pH 7.2-7.4) to 0.9 mL of pterostilbene-encased casein particles and resveratrol-encapsulated casein particles prepared in Example 1 was used as a donor solution. Added. The preparation was prepared by adjusting the salt strength of donor and receptor fluids with PBS.

  While stirring the receptor liquid, it was kept at 32 ° C. using a water jacket, and the receptor liquid was collected over time. This receptor solution was analyzed by high performance liquid chromatography (HPLC) under the following conditions to quantify the permeated pterostilbene and resveratrol.

PBS solution as a negative control at the time of the test, and a solution in which pterostilbene and resveratrol were solubilized with PEG40 hydrogenated castor oil (HCO-40; Nikko Chemical) as a general solubilizer as a control (hereinafter referred to as HCO-40 solubilization) Used). The HCO-40 solubilizate was adjusted to contain about 0.03% by weight pterostilbene and resveratrol.
HPLC condition column: C18 5 μm 4.6 mm × 250 mm
Flow rate: 1 ml / min
Column temperature: 40 ° C
Detection wavelength: 310 nm
Developing solution: acetonitrile / water = 20/80 (volume ratio including 0.1 volume (volume)% acetic acid and 0.1 volume (volume)% triethanolamine)

  The skin permeability in each preparation was determined by the following skin permeation count (P value). The formula for calculating the P value is as follows. In the calculation formula, D = diffusion coefficient, K = distribution coefficient, C = drug concentration, L = film thickness, J = flux, and P = permeation coefficient. FIG. 1 shows the permeation results of pterostilbene and resveratrol in the receptor fluid over time.

  As shown in FIG. 1, the skin permeation rate of the aqueous skin external preparation using pterostilbene-encased casein significantly increased compared with the solubilized product of Comparative Example HCO-40. Moreover, it turns out that it has very high permeability compared with the resveratrol inclusion casein similarly included in casein.

Example 4: Coloring test (accelerated storage over time → coloring compared with absorbance)
The pH of resveratrol (RES) and pterostilbene (PTER) -containing casein prepared in Example 1 was adjusted to 6N hydrochloric acid and 8N sodium hydroxide at pH 6, pH 7 or pH 8, which are generally preferred as external preparations for skin. The color condition was observed at 50 ° C. for 3 months. The degree of coloring was measured over time by the change in absorbance at an absorption wavelength of 400 nm. The results are shown in FIG.

  Within the range of pH 6-8, which is preferred as an external product, the resveratrol-encased casein aqueous solution became more colored as the pH increased, and was inappropriate as a product. Table 2 shows values obtained by calculating the time required to reach OD400 nm = 0.1 or more, which can be seen with the naked eye, from an approximate curve. The results in Table 2 also indicate that pterostilbene encased casein is preferable as a skin external preparation.

Example 5: Stability test (HPLC)
The resveratrol-encased casein solution and pterostilbene-encased casein solution prepared in Example 1 were aged for 36 days in a thermostatic bath at each temperature. The decomposition at this time was quantified by high performance liquid chromatography (HPLC) analysis under the above conditions. The results are shown in FIGS.
As shown in FIG. 3 and FIG. 4, the pterostilbene-encased casein solution showed higher stability than the resveratrol-encased casein solution.

Example 6: Melanin production inhibition test B16 melanoma cells (mouse melanoma) 10% (v / v) FBS-containing MEM medium was seeded in a 12-well culture plate at 2.5 × 10 ⁵ cells / well. This was precultured for 24 hours by the method. After the preculture, the medium was changed to a test medium to which an evaluation sample (pterostilbene (PTER), arbutin (Arb), or resveratrol (Res)) was added, and the cells were cultured for 72 hours. As the test medium, a medium in which theophylline was added to 0.25 mmol / L and dimethyl sulfoxide to 0.5% (v / v) to the pre-culture medium was used. After completion of the culture, the cells were washed with PBS, 1 mol / L sodium hydroxide aqueous solution containing 10% (v / v) dimethyl sulfoxide was added, sonicated at 50 ° C. for 30 minutes, and then the wavelength of the lysate. Absorbance at 400 nm was measured and used as the amount of melanin in the well. At the same time, the number of viable cells was measured by MTT assay on another culture plate. In the MTT assay, after culturing in a separate plate under the same conditions, the cells were washed with PBS, 0.5 mg / mL MTT solution was added at 500 μL / well, and the mixture was incubated in an incubator for 2 hours. Thereafter, 1.5 mL of dimethyl sulfoxide was added, and the absorbance at a wavelength of 570 nm was measured and used as an index of the number of living cells. The cell viability (%) when the evaluation sample was added was determined with the number of living cells in the case where the evaluation sample was not added being 100.

Finally, the amount of melanin in the well was normalized by the cell viability (%) by the MTT assay to obtain the amount of melanin per unit cell.
The melanin production suppression curve when the evaluation sample was added was determined with the amount of melanin when the evaluation sample was not added being 100 (FIG. 5). FIG. 6 shows the IC50 value for inhibiting melanin production by 50%, obtained from this inhibition curve.

  The pterostilbene (PTER) of the present invention was concentration-dependent and showed a remarkable melanin production inhibitory effect. Moreover, it turned out that the effect is strong even if compared with arbutin (Arb) and resveratrol (Res) which are the existing whitening active ingredients.

Example 13: Whitening emulsion (component) (%)
Pterostilbene encased casein 1.3
Squalane 8.0
Jojoba oil 7.0
Glyceryl paraaminobenzoate 1.0
Ascorbyl magnesium phosphate 0.5
Cetyl alcohol 1.5
Glycerol monostearate 2.0
Polyoxyethylene cetyl ether 3.0
Polyoxyethylene soorbitan monooleate 2.0
1,3-butylene glycol 1.0
Glycerin 2.0
1,2-pentanediol 3.0
Collagen 1.0
Sodium citrate 1.0
Perfume Appropriate amount of purified water Remaining amount

Example 14: Whitening cream (ingredient) (%)
Pterostilbene encased casein 1.5
Cetostearyl alcohol 3.0
Glycerin fatty acid ester 2.0
Polyoxyethylene (20) sorbitan monooleate 1.0
Sorbitan monostearate 1.0
N-stearoyl-N-methyltaurine sodium 0.5
Vaseline 5.0
Dimethylpolysiloxane (100 mPa · s) 3.0
Glyceryl tri-2-ethylhexanoate 20.0
Astaxanthin 0.05
Ellagic acid 0.05
Lactic acid 1.0
Dipropylene glycol 10.0
Arbutin 3.0
Sodium citrate 0.5
Ascorbyl magnesium phosphate 0.1
Titanium oxide 0.1
Perfume proper amount edetate disodium 0.03
Ethyl paraoxybenzoate 0.05
Purified water remaining

Example 15: Jerry whitening serum (component) (%)
Butylene glycol 5.0
Pterostilbene encased casein 1.1
Glycerin 7.0
(PEG-240 / decyltetradeceth-20 / HDI) copolymer 1.0
Astaxanthin (Hematococcus alga extract) 0.05
Ceramide 1.0
Collagen 0.5
Water-soluble collagen 0.5
Ascorbyl palmitate trisodium 0.01
Damask rose flower oil PEG60 hydrogenated castor oil appropriate amount vitamin E 0.01
Tri (caprylic / capric) glyceryl 0.1
Na phosphate 0.8
Sodium hydroxide appropriate amount Phenoxyethanol 0.1
Water remaining

Example 16: Tablet (component) (%)
Lactose 48.0
Resulting cellulose 31.0
Starch degradation product 9.0
Sucrose fatty acid ester 5.0
Pterostilbene encased casein 6.0
Sodium citrate 1.0

Example 17: Soft capsule (component) (%)
Edible soybean oil 41.4
Glycerin fatty acid ester 12.0
Beeswax 5.0
Pterostilbene encased casein 1.3
Sodium citrate 0.3

Example 18: Soft Drink (Ingredient) (%)
Fructose dextrose liquid sugar 30.0
Orange juice 20.0
Pterostilbene casein 2.0
Sodium citrate 0.5
Emulsifier 0.5
Perfume Appropriate amount of purified water Remaining amount

Claims (13)

A composition comprising protein particles and pterostilbene. The composition according to claim 1, wherein the protein particles have an average particle size of 10 to 1000 nm. The composition of Claim 1 or 2 containing 0.1-100 mass% pterostilbene with respect to the mass of protein. The protein according to any one of claims 1 to 3, wherein the protein is at least one selected from the group consisting of collagen, gelatin, acid-treated gelatin, albumin, ovalbumin, casein, transferrin, globulin, fibroin, fibrin, laminin, fibronectin, or vitronectin. The composition according to item. The composition according to any one of claims 1 to 4, wherein the protein is casein. The skin external preparation according to any one of claims 1 to 5, comprising casein particles encapsulating pterostilbene. Furthermore, the composition of any one of Claims 1-5 containing a carotenoid type compound. The composition according to any one of claims 1 to 6, which is used as a whitening agent. The composition according to any one of claims 1 to 6, which is used as a melanin production inhibitor. A whitening agent containing protein particles and pterostilbene as active ingredients. A melanin production inhibitor containing protein particles and pterostilbene as active ingredients. The skin external preparation containing the composition of any one of Claims 1-9. The functional food containing the composition of any one of Claims 1-9.

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