JP6767776B2 - Tyrosinase activity inhibitor - Google Patents

Description

The present invention relates to a tyrosinase activity inhibitor containing violacein, which is a natural blue pigment, as an active ingredient.

Natural pigments are often used to color products that come into direct contact with the human body, such as cosmetics. This is because it is relatively safe against synthetic pigments that may be harmful to the human body and the environment. Some of these natural dyes can be expected to have pharmacological actions as well as actions as dyes.

For example, it has recently been found that violacein, a natural blue pigment derived from bacteria, has antibacterial and anticancer effects. Patent Document 1 describes the marine bacterium Pseudoalteromonas sp. A protein kinase inhibitor containing violacein purified from a strain as an active ingredient is disclosed. Protein kinases are involved in many functions of the living body, and are expected to prevent or treat diseases by inhibiting the activity of protein kinases. Therefore, it has been clarified that the above-mentioned violacein inhibits the activity of protein kinase.

JP-A-2010-126469

There are still many unclear points about the pharmacological action of violasane. Therefore, it is an object of the present invention to clarify useful pharmacological effects in applying violasane to cosmetics and the like, and to provide cosmetics containing violasane that exerts such pharmacological effects.

The following inventions and the like are provided as means for solving the above problems. That is, a tyrosinase activity inhibitor characterized by containing violacein as an active ingredient is provided. Further, the present invention provides cosmetics, pharmaceuticals, and quasi-drugs characterized by containing the tyrosinase activity inhibitor.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a tyrosinase activity inhibitor capable of inhibiting the activity of tyrosinase, which is an enzyme that contributes to a melanin production reaction, and cosmetics, pharmaceuticals, and quasi-drugs containing such tyrosinase activity inhibitor.

The figure which shows the melanin production reaction with the catalytic action of tyrosinase Diagram showing the chemical structure of violasane A flow chart showing an example of the violasane production method of the present embodiment. Formulation example when preparing a cream containing violasane Formulation example when preparing an ointment containing violasane Absorbance measurement method when a sample is added to confirm the tyrosinase activity inhibitory effect of violacein Absorbance measurement method when no sample is added to confirm the tyrosinase activity inhibitory effect of violacein Results of absorbance measurement to confirm the tyrosinase activity inhibitory effect of violacein

Hereinafter, embodiments of the present invention will be described. The present invention should not be limited to these embodiments, and may be carried out in various embodiments without departing from the gist thereof.
<Embodiment>
<Overview>

Skin pigmentation such as stains and freckles is caused by the deposition of melanin produced in the epidermis on the epidermis and dermis. It is known that melanin is produced by reacting tyrosine, which is an amino acid contained in melanocytes, with the action of tyrosinase, which is an oxidase. That is, when the skin receives a stimulus such as exposure to ultraviolet rays, melanocyte-stimulating hormone (MSH), which is one of the signaling substances, is secreted from the pituitary gland and acts on the melanocytes of the epidermis. This activates tyrosinase.

FIG. 1 is a diagram showing a melanin production reaction together with a catalytic action of tyrosinase. First, tyrosine, which is a substrate, is oxidized by the monophenolase action of tyrosinase to become dopa (DOPA: Dihydroxyphenylalanine). Subsequently, dopa is oxidized by the action of diphenolase to become dopaquinone. Dopaquinone is further oxidized to dopachrome, which goes through a further reaction pathway to melanin.

Thus, in the melanin production reaction, the catalytic action of tyrosinase, which is an oxidase, plays an important role. In view of such circumstances, in the present embodiment, it is shown that violacein has an action of inhibiting the activity of tyrosinase, and a tyrosinase activity inhibitor containing violacein as an active ingredient is shown.
<Composition>

The present embodiment is a tyrosinase activity inhibitor characterized by containing violacein as an active ingredient, and is a cosmetic, pharmaceutical, or quasi-drug containing such a tyrosinase activity inhibitor. By inhibiting the activity of tyrosinase, it is possible to suppress the production of melanin and alleviate pigmentation such as stains and freckles.

"Violacein" is a bluish-purple pigment produced by violacein-producing bacteria. Examples of the violacein-producing bacterium include "Chromobacterium violaceum", "Chromobacterium violaceum", "Psedimentamentomas luteoviolacea", and "Janthinobacterium liverium". FIG. 2 is a diagram showing the chemical structure of violasane.

As a method for obtaining violacein, cultivate violacein-producing bacteria in a liquid medium containing peptone or the like, separate the cultured bacteria and violacein with a centrifuge or the like, and after separation, remove the supernatant and precipitate. There is a method of adding ethanol to the culture medium for extraction.

FIG. 3 is a flow chart showing an example of a method for producing violasane. First, in the "pre-culture step" (S0301), the violacein-producing bacteria are pre-cultured. Since violasane is often not produced when the isolated bacteria are suddenly inoculated into a large-volume medium, the first step is to increase the number of bacteria by putting them in a small amount of medium, and then perform this as a pre-stage of the main culture. In the preculture, for example, ordinary bouillon: 1.8 g, agar: 1.5 g, pure water: 1000 ml is used as a medium, and Chromobacterium violaceum VP-2 strain, which is a violacein-producing bacterium, is inoculated for one loopful and the culture temperature is adjusted. Incubate at 25 ° C. for about 2 days.

Next, in the "main culture step" (S0302), the pre-cultured violacein-producing bacteria are main-cultured. For example, _{polypeptone: 2.0g, K 2 HPO 4:} 0.15g, MgSO 4 · 7H 2 O: 0.15g, glycerine 1.25 g, pure water: King B medium (pH 7.2 ± plus 100 ml 0. 2) is inoculated with pre-cultured violacein-producing bacteria, the culture temperature is set to 25 ° C., and the cells are shake-cultured for about 4 days.

Next, in the "extraction step" (S0303), after the main culture, the cells and pigments were precipitated by centrifugation, the supernatant was removed, ethanol was added to the precipitate, and violacein was extracted. Centrifugation was performed at RCF: 8000 × g, temperature: 4 ° C., time: 5 minutes.

Next, in the "filtration step" (S0304), the violacein extract obtained in the extraction step is filtered. Then, in the "drying step" (S0305), ethanol, which is the solvent of the filtered filtrate, is dried by a rotary evaporator.

Next, in the "reprecipitation step" (S0306), the dried-solidified filtrate is recrystallized. Then, in the "freeze-drying step" (S0307), pure water is added to the reprecipitate obtained in the reprecipitation step and centrifuged, and then the precipitate from which the supernatant has been removed is freeze-dried. At this time, it is preferable to set the temperature of the pure water to be added to about 80 ° C. and add it in three portions. When violasane was collected at the stage after the completion of the previous recrystallization step, the collected amount was only about 30 mg per 100 ml of the medium, but by going through this freeze-drying step, the collected amount became about 80 mg per 100 ml of the medium. Improved to. Deoxyviolacein can also be produced in the above-mentioned production process of violacein, but even if it is contained together and is used as a tyrosinase activity inhibitor, it does not interfere with the tyrosinase activity inhibitory action.

In the tyrosinase activity inhibitor of the present embodiment, in addition to using violacein alone, components usually used or permitted to be used according to a desired purpose such as cosmetics, pharmaceuticals, quasi-drugs, etc. It can be appropriately blended.

Cosmetics containing a tyrosinase activity inhibitor containing violacein as an active ingredient include, for example, skin care cosmetics such as beauty essences, creams, lotions, washing pigments, milky lotions, lotions, ointments, cleansing and sunscreens, and makeup cosmetics. It can be a foundation or eye shadow.

For example, in the case of a beauty liquid, in addition to violacein, water, rice bran oil, pentylene glycol, glycerin, squalane, cetyl palmitate, dimer dilinoleic acid, etc. are the main components, and sodium hyaluronate and hydrogenated rapeseed oil. Additives include alcohol, carbomer, xanthan gum, potassium hydroxide, dimethicone, polysorbate-60, glyceryl stearate, hydrogenated castor oil, phenoxyethanol, urea, arginine, albutin, and linoleic acid. Then, each component is divided into a water-soluble raw material and an oil-soluble raw material and dissolved, and then they are heated to be mixed and emulsified. Additives such as extracts are added while cooling this, and when the temperature becomes lower, highly volatile substances such as essential oils and fragrances are added. After that, a predetermined safety inspection (bacteria, pH, temperature stability, viscosity, etc.) can be performed, and the product can be provided as a product by filling it in a bottle or the like.

Further, in addition to the tyrosinase activity inhibitor of the present embodiment, a component having an action of inhibiting the activity of tyrosinase and a component of suppressing the production of melanin may be further blended to make cosmetics, pharmaceuticals, and quasi-drugs. Examples of the former component include arbutin, hydroquinone, ellagic acid, kojic acid, licorice extract, vitamin C derivative, rhododenol, 4MSK, and resveratrol. The latter component includes tranexamic acid, chamomile ET, rucinol, linoleic acid, nicotinamide, magnolignan and the like.

FIG. 4 shows one of specific compounding examples when preparing a cream as a cosmetic product. As shown in the figure, violacein having a tyrosinase activity inhibitory action, resveratrol having an antioxidant action and a tyrosinase activity inhibitory action, monostearic acid glyceride and monolauric acid polyoxyethylene sorbitan which are emulsifying components, and cetanol which is a stabilizing component. And liquid paraffin, jojoba oil as an emollient component, propylene glycol as a moisturizing component, and a fragrance and water. Since violasane has an antiseptic effect, it may not be necessary to add a separate preservative, and it can also function as a blue pigment.

Further, FIG. 5 shows a formulation example for adjusting an ointment as an external medicine. As shown, violasane, glycyrrhetinic acid as a rough skin improving ingredient, sulfur as a stratum corneum softening ingredient, beeswax and liquid paraffin as stabilizing ingredients, lanolin polysorbate 80 as an emulsifying ingredient, olive oil as an emollient ingredient, and , Preservatives and water. Since violasane has an antiseptic effect, it may not be necessary to add a separate preservative, and it can also function as a blue pigment.

The drug or quasi-drug containing the tyrosinase activity inhibitor of the present embodiment is preferably an external drug because it acts on the reaction of producing melanin generated in the skin, but it prevents the drug from being taken internally. It's not a thing. When used as an internal medicine, violasane is powdered or granulated and filled into capsules by a conventional method, or excipients, binders, disintegrants, etc. are added and used as tablets using a tableting machine or the like. Can be manufactured. It may also be used as a supplement or health food. In that case, it may be provided in the form of capsules or tablets like an internal medicine, or may be provided in the form of being added to various foods such as beverages, seasonings and confectionery. May be good.
<Test tyrosinase activity inhibitory effect>

A test was conducted to confirm the tyrosinase activity inhibitory action of violacein. Violasane solutions having different concentrations were added to a solution containing mushroom tyrosinase (mushroom-derived tyrosinase) using L-tyrosine as a substrate, and the absorbance of the solution after the reaction at 484 nm was measured. 484 nm is the absorption wavelength of melanin. In addition, as a comparison target, a sample in which β-arbutin having a known tyrosinase activity inhibitory action was added in place of violacein was also prepared. Β-Arbutin is a plant-derived phenolic glycoside and is used in cosmetics and the like.

FIG. 6 is a diagram showing a test method in which the above-mentioned absorbance measurement was performed. As shown in the figure, 1.5 ml (2.5 mM) of L-tyrosine, 1 / 15M phosphate buffer, and 0.05 ml of mushroom tyrosinase are mixed with violacein solutions (A.1 μg / ml, B.10 μg) having different concentrations. /Ml, C.20 μg / ml, D.50 μg / ml), or β-arbutin solution having a different concentration like violacein was added, and a 5% DMSO (dimethyl sulfoxide) solution was used as a reaction solvent, and they were used. Each sample was kept warm at 37 ° C., and the absorbance (484 nm) was measured for 25 minutes with a microplate reader. In addition, the same absorbance measurement was performed on the sample to which neither violacein nor β-arbutin was added as a control, and the sample to which only DMSO was added.

FIG. 7 is a graph showing the results of the above test. As shown in the figure, the violacein solution had a lower absorbance than the sample to which only the control and DMSO were added, and the ability to inhibit tyrosinase activity could be confirmed. In addition, the violacein solution showed excellent ability to inhibit tyrosinase activity against β-arbutin at the same concentration at any concentration.

FIG. 8 is a graph showing the tyrosinase activity inhibition rate of the violacein solution (50 μg / ml) and the β-arbutin solution (50 μg / ml) calculated with the tyrosinase activity inhibition rate of the DMSO 5% solution as 0%. The violacein solution was 58.5%, which was an excellent result, compared with 29.6% of the β-arbutin solution.

Based on the above test results, obtained an IC ₅₀ value for the tyrosinase activity of Viola Thane and β- arbutin (50% inhibitory concentration). _{The IC 50} values of Viola Sein is 37.5 / ml, _{an IC 50} value of β- arbutin was 68.8μg / ml. From this result, it was found that violacein has a tyrosinase activity inhibitory ability 1.8 times that of β-arbutin.
<Effect>

According to this embodiment, it is possible to provide a tyrosinase activity inhibitor containing violacein as an active ingredient. In addition, it is possible to provide cosmetics, pharmaceuticals, and quasi-drugs characterized by containing such a tyrosinase activity inhibitor.

Claims (4)

A tyrosinase activity inhibitor characterized by containing violacein as an active ingredient. A cosmetic product for inhibiting tyrosinase activity, which comprises the tyrosinase activity inhibitor according to claim 1. Tyrosinase activity inhibition pharmaceuticals, characterized in that blended with tyrosinase activity inhibitor according to claim 1. A quasi-drug for inhibiting tyrosinase activity, which comprises the tyrosinase activity inhibitor according to claim 1.