JP2015020989A - Melanin formation inhibitor, and whitening agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a melanin formation inhibitor and a whitening agent, having an excellent whitening effect compared with an existing whitening agent, and also having an excellent safety and temporal stability.SOLUTION: The invention is "a melanin formation inhibitor and a whitening agent each containing 4-((E)-3-hydroxy-1-propenyl)phenylacetate as an active ingredient, and a method for preventing melanin formation by applying the inhibitor to the skin and a whitening method by applying the agent to the skin."

Description

  The present invention relates to a melanin production inhibitor, a whitening agent, a melanin production inhibition method, and a whitening method.

  Skin pigmentation such as stains, buckwheat, sunburn, and melasma is the result of an imbalance of melanin production and excretion, resulting in excessive accumulation of melanin in epidermal cells.

  Skin pigmentation is caused by the proliferation and activation of pigment cells (melanocytes) present in epidermal cells, which significantly increases the production of melanin pigments in melanocytes, producing more melanin than necessary, and the generated melanin pigment is adjacent to cells. It is caused by diffusing.

  The causes of proliferation and activation of melanocytes are various, such as inflammation, hormonal balance, genetic factors, etc., but are promoted by the influence of ultraviolet rays. In addition, melanocytes increase production of melanin by giving drugs such as theophylline as if exposed to ultraviolet rays.

  Therefore, as a method for preventing or improving skin spots, buckwheat and blackening, it is possible to suppress or inhibit tyrosinase activity, which is the rate-limiting enzyme for melanin production, inhibit the melanin production process, and turn over cells. It can be promoted to promote melanin excretion, and the produced melanin can be reduced and lightened, and various whitening components expected to have these actions have been proposed.

  For example, arbutin (tyrosinase activity inhibition), hydroquinone (tyrosinase activity inhibition, melanin bleaching), kojic acid (tyrosinase activity inhibition), glutathione (tyrosinase activity inhibition), ascorbic acid derivative (tyrosinase activity inhibition, melanin reduction), etc. It has been used as an ingredient in whitening cosmetic ingredients. However, none of them has a satisfactory whitening effect.

  Further, for example, arbutin is known to inhibit tyrosinase activity, but is a glycoside, and there is a problem that the glycoside is generally chemically unstable.

  Moreover, since hydroquinone is a carcinogenic substance, its use as a cosmetic is limited.

  Furthermore, ascorbic acid has a problem that the raw material itself is unstable, and the activity of the active ingredient decreases with time.

  As described above, all of the whitening agents known so far have some dissatisfaction.

JP 2011-173803 A

  An object of the present invention is to provide a melanin production inhibitor, a whitening agent, a melanin production inhibition method, and a whitening method, which have an excellent whitening effect as compared with existing whitening agents and are excellent in safety and stability over time. There is.

The following formula [A]

(In the formula, R ¹ , R ² , R ⁴ and R ⁵ represent hydrogen, a hydroxyl group, an alkyl group, or —O—C (═O) R ^6. R ⁶ represents an alkyl group. R ³ represents A collagen production promoter containing a compound represented by hydrogen, an alkyl group or —C (═O) R ⁷ , where R ⁷ represents an alkyl group) as an active ingredient is known (Patent Document 1).

As a result of intensive studies to solve the above-mentioned problems related to conventional melanin production inhibitors and whitening agents, the present inventors have found that among the compounds represented by the above formula [A], the following formula [1]

(In the formula, Ac represents an acetyl group.)
4-((E) -3-Hydroxy-1-propenyl) phenyl acetate (hereinafter abbreviated as “HPA”) is superior to conventional whitening agents as well as promoting collagen production. It has been found that it has an inhibitory effect on cellular melanin production. Further, since HPA has extremely low cytotoxicity at an effective concentration and high safety, it has been found that HPA is also useful as a whitening agent and a skin external preparation, and the present invention has been completed.

That is, the present invention has the following configuration.
(1) A melanin production inhibitor containing HPA as an active ingredient.
(2) A whitening agent containing HPA as an active ingredient.
(3) A method for inhibiting melanin production, which comprises applying HPA to the skin.
(4) A whitening method characterized by applying HPA to the skin.

  According to the present invention, it is possible to provide a melanin production inhibitor, a whitening agent, a melanin production inhibition method, and a whitening method that are excellent in a high melanin production inhibitory effect, safety to the living body, and stability over time.

  HPA used in the present invention is a known compound and can be produced based on a known method. Alternatively, it can be produced by dissolving 1'-acetoxychabicol acetate or its analog in an aqueous solvent and heat-treating it according to the method described in Patent Document 1.

  For example, using 4-iodophenol as a raw material and acetylating a hydroxyl group, an allyl alcohol moiety is introduced by an organometallic reaction to obtain the desired compound.

  The HPA according to the present invention has an excellent melanin production inhibitory action as shown in Example 1 described later, and is useful as a melanin production inhibitor. Moreover, since it is excellent in safety, it is possible to provide a whitening agent and a whitening method for the purpose of whitening the skin using HPA.

  Below, the embodiment of the melanin production inhibitor using the HPA which concerns on this invention, and a whitening agent is demonstrated.

1. Melanin production inhibitor The melanin production inhibitor of the present invention contains HPA as an active ingredient.

  The blending ratio of HPA in the melanin production inhibitor of the present invention varies depending on the application site, age of application object, form of the melanin production inhibitor, etc., but is 0.1 to 1000 μM, preferably 0.3 to 600 μM with respect to the entire composition. .

  In addition, the melanin production inhibitor of the present invention may contain a pharmaceutically acceptable carrier, a known additive, or other pharmaceutically active ingredients as long as the effects of the present invention are achieved. it can.

  The melanin production inhibitor of the present invention can be made into an appropriate form according to a conventional method using a pharmaceutically acceptable carrier such as dextrin and cyclodextrin and other optional auxiliaries as desired. For example, it can be in the form of a dry powder, granule, tablet gel, cream, ointment, suspension, solution or the like.

  As the auxiliary agent at this time, for example, an excipient, a binder, a disintegrant, a lubricant, a stabilizer, a flavoring / flavoring agent and the like can be used.

  The melanin production inhibitor of the present invention can be contained, for example, in an external preparation for skin and used as an ointment, an external liquid, a patch, etc., for prevention of skin blackening and the like.

  Furthermore, since the melanin production inhibitor of this invention has the outstanding melanin production inhibitory effect, it can be used also as a reagent for the research relevant to the production | generation mechanism of melanin.

2. Whitening Agent The whitening agent of the present invention contains HPA as an active ingredient.

  The blending ratio of HPA in the whitening agent of the present invention varies depending on the type of compound, application site, age and characteristics of the application target, form of the cosmetic composition, etc., but is preferably 0.1 to 1000 μM with respect to the entire composition, preferably Is 0.3 to 600 μM. Alternatively, the content can be 0.000002 to 0.02 mass%, preferably 0.000006 to 0.006 mass%, based on the total amount of the composition.

  The whitening agent of the present invention may be composed of HPA itself, or other pharmacologically or hygienically acceptable carriers and additives as long as HPA is an active ingredient and does not impair the effects of the present invention. These components may further be included.

  The type and blending amount of such a carrier or additive can be appropriately set as long as the effects of the present invention are not impaired.

  For example, in addition to HPA, when other components having other whitening effect are blended with the whitening agent of the present invention, a more excellent whitening effect can be obtained, which is preferable. Examples of other ingredients having such a whitening effect include hydroquinone, arbutin, ellagic acid, vitamin C or a derivative thereof (for example, ascorbyl glucoside, sodium ascorbate phosphate, disodium ascorbate sulfate, ascorbic acid Phosphoric acid magnesium, ascorbic acid isopalmitic acid ester, ascorbic acid ethyl ester, etc.), biphenyl derivatives (e.g., dehydrodicreole, 2,2′-dihydroxy-5,5′-dipropylbiphenyl, etc.), 4- ( 4 monohydroxyphenyl) -2-butanol or derivatives thereof, placenta extract, fire spine extract, raspberry ketone glucoside, apricot extract, wheat germ extract, zinc-containing yeast extract, gentian extract, anise extract, oil-soluble licorice extract, saicin extract , Flaxseed extract, Dutch mustard extract, geoscorea composite overnight extract, elderberry extract, rock cabbage extract, chamomile extract, adenosine-5'-1-phosphate or its salt, linoleic acid derivative, vitamin B or its derivative, tranexamic acid, tranexam And acid salts and tranexamic acid derivatives.

  In addition, the whitening agent includes components that are usually blended in pharmaceuticals, quasi drugs, skin cosmetics, and the like as necessary, such as humectants, ultraviolet absorbers, ultraviolet scattering agents, antioxidants, anti-inflammatory agents, etc. May be included.

  As the humectant, 1,3-butylene glycol, glycerin, hyaluronic acid, squalane, cholesterol, 1,2-propanediol, polyglutamic acid, serine, glycine, threonine, alanine, collagen, hydrolyzed collagen, hydronectin, fibronectin, Keratin, elastin, royal jelly, chondroitin sulfate, heparin, glycerophospholipid, glyceroglycolipid, sphingophospholipid, sphingoglycolipid, linoleic acid or esters thereof, eicosapentaenoic acid or esters thereof, pectin, bifidobacteria fermentation product, lactic acid fermentation , Yeast extract, litchi mycelium culture or extract thereof, wheat germ oil, avocado oil, rice germ oil, jojoba oil, soybean phospholipid, γ-oryzanol, belode oyster extract, yokuinin extract, geowex , To total extract, seaweed extract, Aloe arborescens extract, burdock root extract, Ganoderma wax extract, arnica extract, wheat bran polyhydric alcohol, sugar alcohol, and the like.

  Examples of ultraviolet absorbers include p-aminobenzoic acid derivatives, salicylic acid derivatives, anthranilic acid derivatives, coumarin derivatives, amino acid compounds, benzotriazole derivatives, tetrazole derivatives, imidazoline derivatives, pyrimidine derivatives, dioxane derivatives, camphor derivatives, furan derivatives, Examples include pyrone derivatives, nucleic acid derivatives, allantoin derivatives, nicotinic acid derivatives, vitamin B6 derivatives, oxybenzone, benzophenone, guaiazulene, shikonin, baicalin, baicalein, and berberine.

  Examples of the ultraviolet scattering agent include Cosmeserve WP-40TK (Dainippon Kasei Co., Ltd. trade name), fine particle titanium oxide, fine particle zinc oxide and the like.

  Antioxidants include dibutylhydroxytoluene, ascorbic acid and its salts, stearic acid ester, tocopherol and its ester derivatives, nordihydrogua cetelenic acid, butylhydroxytoluene (BHT), butylhydroxyanisole (BHA), parahydroxyanisole Propyl gallate, sesamol, sesamorin, gossypol and the like.

  Examples of the anti-inflammatory agent include melanin metabolism improving agents such as niacinamide, lactic acid bacteria fermentation liquid, and α-hydroxy liquid, diphenhydramine hydrochloride, glycyrrhizic acids, and derivatives thereof.

  In addition, emulsifiers such as DN Basic CP (trade name of Dainippon Kasei Co., Ltd.), glyceryl stearate, sorbitan stearate, glyceryl (behenic acid / eicosandioic acid), dilauryl glutamic acid (phytosteryl / octyldodecyl), cetanol, Emulsification stabilizers such as phytosteryl hydroxystearate, behenyl alcohol, glycol monostearate, isostearyl alcohol, solubilizers such as polyoxyethylene hydrogenated castor oil, cetyl palmitate, neopentyl glycol dicaprate, cholesteryl hydroxystearate, xanthan gum, Carboxyvinyl polymer, quince seed extract dispersion, PEG-9 polydimethylsiloxyethyl dimethicone, microcrystalline wax, disteardimonium hectorite, etc. Thickeners, antibacterial and antifungal agents, parabens, methyl parabens, phenoxyethanol, triethylhexanoin paraoxybenzoic acid propyl, paraoxybenzoic acid methyl, propylparaben and other preservatives, isostearic acid polyglyceryl-2 dispersing agents, dimethicone, etc. Water repellent and glossing agents, lubricants such as phenyl trimethicone, lubricants such as methyl methacrylate crosspolymer, softeners such as cyclopentasiloxane, pH adjusters, anti-discoloring agents, surfactants, alcohols, water-soluble A polymer oil component, powder component, colorant (pigment, pigment, etc.), cleaning agent, drug, fragrance, resin, various skin nutrients, and the like are appropriately blended as necessary within the range not impairing the object of the present invention. be able to.

  The whitening agent of the present invention can be prepared by formulating a whitening agent containing HPA or HPA and an additive into a target form (dosage form) together with an appropriate medium and carrier according to a conventional method.

  The form of the whitening agent of the present invention is not particularly limited as long as HPA can be stably blended, and can be set according to the dosage form, form, use, etc. of the applied product.

  Examples thereof include liquid forms such as lotions, emulsion forms such as gels, emulsions and creams, sheet forms, stick forms, ointments, and powder forms using suitable excipients. The emulsified product may be any of water-in-oil type, oil-in-water type, and multi-emulsion.

  Further, using the whitening agent of the present invention, various cosmetics and cosmetics such as lotion, lotion, essence (whitening serum), tonic, milky lotion, cream, ointment, pack, lipstick, foundation, bathing agent, hair conditioner, etc. Can also be produced according to conventional methods.

  The application part of the whitening agent of the present invention is not particularly limited as long as the effect of the present invention is exhibited, and can be used for various parts such as for face, body, and hair. For example, by applying the whitening agent of the present invention to the skin, a whitening action is exhibited, and skin pigmentation and accompanying stains, freckles, etc. can be prevented or improved.

  Examples of the method for applying the whitening agent to the skin include application to the skin, application to the skin, and other methods similar to these. For example, it is used by methods such as liquids, ointments, emulsions, packs, haps, sprays, soaps, creams, rinses, and baths.

  The above-described whitening agent additives and dosage forms can also be applied to the melanin production inhibitor of the present invention.

  In addition, although the melanin production inhibitor and the whitening agent of the present invention are suitably applied to humans, they can also be applied to animals other than humans as long as their effects are exhibited.

  Hereinafter, the present invention will be specifically described with reference to production examples and examples, but the present invention is not limited thereto.

Synthesis example 1
4-Iodophenol 75 g (341 mmol) and triethylamine 68.99 g (682 mmol) were dissolved in 375 mL of toluene, and 32.11 g (409 mmol) of acetyl chloride was added dropwise under ice cooling. After 1 hour, the completion of the reaction was confirmed by TLC, 150 mL of ion exchange water was added, and the mixture was stirred for 10 minutes and then separated. Washing and separation were carried out in the order of 3.5% hydrochloric acid 150 mL, ion-exchanged water 150 mL × 2, 5% NaHCO ₃ aqueous solution 150 mL, and ion-exchanged water 150 mL × 2, and the organic layer was dried over magnesium sulfate and concentrated. The residue was purified by silica gel column chromatography (eluent: toluene / ethyl acetate = 20/1) to obtain 78.93 g of acetyl compound (yield 88.4%).

¹ H-NMR (CDCl ₃ ) δ: 7.68ppm (2h, dt, J = 2.9 and 9.0Hz), 6.86 ppm (2h, dt, J = 2.9 and 9.0Hz), 2.29ppm (3h, s)

The obtained acetyl compound 80 g (305 mmol), palladium acetate 3.43 g (15.3 mmol), silver acetate 50.96 g (305 mmol), triphenylphosphine 16.02 g (61.1 mmol), allyl alcohol 53.19 g (91.6 mmol) were suspended in 640 mL of DMF. It became cloudy and stirred at 70 ° C. for 7 hours under a nitrogen atmosphere. To the reaction solution was added 100 mL of toluene, filtered through Celite, and concentrated under reduced pressure. To the residue are added 300 mL of ethyl acetate and 100 mL of 5% hydrochloric acid, and the mixture is separated, then washed and separated in the order of ion-exchanged water 100 mL × 2, 5% NaHCO ₃ aqueous solution 150 mL, 5% brine 150 mL × 2, and the organic layer is sulfuric acid. After drying with magnesium, it was concentrated. The residue was purified by silica gel column chromatography (elution solvent: ethyl acetate / hexane = 1/5) to obtain 14 g of crude crystals. The crude crystals were dissolved in 28 mL of ethyl acetate, crystallized with 140 mL of hexane, stirred for 1 hour, filtered and dried, and 11.8 g of the desired 4-((E) -3-hydroxy-1-propenyl) phenyl acetate ( Yield 20.1%).

  Melting point: 102.6 ° C.

¹ H-NMR (CDCl ₃ ) δ: 7.38 ppm (2h, d, J = 8.6Hz), 7.04 ppm (2h, d, J = 8.6Hz), 6.60ppm (1h, d, J = 16Hz), 6.31 ppm (1h, dt, J = 5.6 and 16Hz), 4.32 ppm (2h, d, J = 5.6 Hz), 2.29 ppm (3h, s)

Example 1. Examination of melanin production inhibitory action in mouse melanoma cells (1) Examination of melanin production inhibitory action
Matsuda H. et al., Melanogenesis inhibitors from the rhizomes of Alpinia officinarum in B16 melanoma cells, Bioorg. Med. Chem., 17 , pp. According to the method described in 6048-6053 (2009), the inhibitory action of HPA on the melanogenesis of mouse melanoma cells was examined by the following method.

B16 melanoma 4A5 (RCB0557) derived from mouse melanoma, which was distributed from the Cell Bank, RIKEN BioResource Center, was obtained from DMEM (4500 mg / mL) L glucose) medium (Sigma-Aldrich) was cultured (5% CO ₂ , 37 ° C.).

Next, the cultured cells were seeded in a DMEM medium having the same composition as described above on a 24-well multiplate at 2.0 × 10 ⁴ cells / 400 μL / well, cultured for 24 hours, the medium was removed, and Synthesis Example 1 A medium supplemented with HPA synthesized in step 1 and theophylline (Sigma-Aldrich, final concentration 1 mM) was added to the cells. In addition, HPA was added to the culture medium so that the final concentration of the solution dissolved in DMSO to 0.1% would be the concentration described in Table 1 below.

  After culturing for 72 hours, trypsinization was performed, and the cells were detached from the multiplate and collected. The collected cells were washed twice with PBS, 1 mol / L NaOH aq. Was added at 120 μL / well, and treated at 80 ° C. for 30 minutes to lyse the cells. The obtained cell lysate was fractionated (100 μL / well) into a 96-well microplate, and the absorbance of the produced melanin was measured with a microplate reader (SH-1000, manufactured by Corona Electric Co., Ltd.) (measurement wavelength) : 405nm).

  Based on the measured values obtained, the inhibitory effect of HPA on the melanin production of B16 melanoma 4A5 was determined by the following formula.

Inhibition (%) = [(AB) / A] / (C / 100) x 100
[A: Absorbance when HPA is not added (control), B: Absorbance when HPA is added, C: Cell viability (%)]

  In addition, arbutin (manufactured by Sigma-Aldrich) was used instead of HPA as a comparison object, and the absorbance of melanin produced by the cells was measured by the same method to determine the inhibitory effect of arbutin on melanin production of B16 melanoma 4A5. . Arbutin was added to the medium so that the final concentration was as shown in Table 2 below.

(2) Cytotoxicity test (cell viability)
The cultured mouse melanoma-derived B16 melanoma 4A5 (RCB0557) was seeded in a 96-well microplate using the DMEM medium having the above composition (5.0 × 10 ³ cells / 0.1 mL / well) and further cultured for 24 hours. Thereafter, HPA synthesized in Synthesis Example 1 and theophylline (final concentration 1 mM) were added to the medium, followed by culturing for 70 hours, followed by reaction for 2 hours using Cell Counting Kit-8 (Dojin). In addition, HPA was added to the medium so that the final concentration of the solution dissolved in DMSO to 0.1% was as shown in Table 1 below.

  Cytotoxicity was determined by the following formula.

Cell viability (%) = [B / A] × 100
[A: Number of cells with no HPA added (control), B: Number of cells with HPA added]

  Moreover, the cytotoxicity test was done by the same method, using arbutin instead of HPA as a comparison object. Arbutin was added to the medium so that the final concentration was as shown in Table 2 below.

(3) Results Table 1 also shows the examination results (Inhibition (%)) of the melanin production inhibitory action of HPA and the cytotoxicity test results (cell viability, Cell viability (%)).

  The examination result (Inhibition (%)) of the melanin production inhibitory effect of arbutin and the cytotoxicity test result (Cell viability (%)) are shown together in Table 2.

As is apparent from Table 1, 1.0 μM HPA significantly inhibited the melanin production of B16 melanoma 4A5 induced by theophylline stimulation. Further, the IC ₅₀ value of the melanin production inhibitory action of HPA was 0.3 μM (Table 1). On the other hand, as is apparent from Table 2, the IC ₅₀ value of the melanin production inhibitory action of arbutin conventionally used as a whitening agent was 174 μM.

  From the above, it can be seen that HPA has a remarkable inhibitory action on melanin production, which is much higher than arbutin.

When HPA and arbutin were compared for cytotoxicity in the vicinity of the concentration showing the IC ₅₀ value of the melanin production inhibitory effect, as shown in Table 1, in the case of HPA, 0.33 showing the IC ₅₀ value of melanin production inhibition. Cell viability at μM was 111.6 ± 3.3%.

On the other hand, the concentration of arbutin showing an IC ₅₀ value for suppressing melanin production was 174 μM, but the cell viability at this concentration is of course the cell viability when the arbutin concentration is 100 μM, that is, 78.1 ± Lower than 1.9% (Table 2) (data not shown).

  From the above, it can be seen that HPA is excellent in safety against cytotoxicity at an effective concentration of melanin production inhibitory action.

Reference Example 1 Tyrosinase inhibitory activity test (1) Tyrosinase inhibitory activity test
L-DOPA [L-3,4-dihydroxyphenylalanine, 2.5 mM, 0.1 M phosphate buffer (pH 6.8)] Enzyme solution (Sigma-Aldrich: Mushroom derived, 80.5 units / mL) in a mixed solution of 70 μL and HPA 20 μL ) Was added and incubated at 25 ° C for 5 minutes. Absorbance at 405 nm was measured after the reaction. In addition, HPA was added to the culture medium so that the final concentration of the solution dissolved in DMSO to 0.1% was the concentration described in Table 3 below.

  Based on the obtained measured value, the tyrosinase activity inhibition rate was calculated | required by the following formula.

Inhibition (%) = {1- (B / A)} × 100
[A: Absorbance when HPA is not added (control), B: Absorbance when HPA is added]

  In addition, kojic acid (manufactured by Wako Pure Chemical Industries, Ltd.) was used as a comparison object instead of HPA, and the absorbance was measured by the same method to determine the tyrosinase activity inhibition rate.

(2) Results Table 3 shows the test results of the obtained tyrosinase inhibitory activity (tyrosinase activity inhibition rate, Inhibition (%)).

  As is apparent from Table 3, HPA showed almost no mushroom tyrosinase inhibitory activity. The tyrosinase inhibitory activity of kojic acid, which has been used as a whitening agent, was 22.3% ± 2.1% with 10 μM kojic acid.

Reference Example 2 Inhibition of nitric oxide production by lipopolysaccharide (LPS) stimulation in RAW264.7 cells (1) Confirmation of inhibition of nitric oxide production
In accordance with the method described in Hegazy ME.F. et al., Chem. Pharm. Bull., 60 , 363-370 (2012), RAW264.7 cells activated by LPS stimulation (ACTT No. The inhibitory action of HPA on nitric oxide (NO) production in TIB-71, a mouse macrophage-like cell line) was confirmed.

RAW264.7 cells were cultured in 10% fetal bovine serum (FBS), 100 units / mL penicillin, 100 μg / mL streptomycin-containing DMEM medium (4500 mg / mL glucose) at 37 ° C in 5% carbon dioxide gas phase . Next, the cultured RAW264.7 cells are detached from the culture vessel using a cell scraper, adjusted to 2.5 × 10 ⁵ cells / well using a DMEM medium having the same composition as described above, and 100 μL in a 96-well microplate. Each seed was seeded and pre-cultured (5% CO _2, 37 ° C.) for 6 hours. After washing RAW264.7 cells adhering to a 96-well microplate with PBS (−), 100 μL of medium containing HPA in DMEM medium having the same composition as above is used, so that the final concentration of HPA becomes the concentration shown in Table 4 below. After further incubation for 10 minutes, 100 μL of DMEM medium containing LPS ( Escherichia coli- derived lipopolysaccharide, Lipopolyisaccharide, Sigma) was added (LPS final concentration: 10 μg / mL).

After culturing the cells for 18 hours, NO ₂ ⁻ accumulated in the supernatant of the medium was quantified by the Griess method to obtain the amount of NO production. That is, the culture supernatant was taken into a tube, the same amount of Griess reagent (1% sulfanilamide / 0.1% N -1-naphthylethylenediamine dihydrochloride / 2.5% phosphoric acid) was added and mixed, and allowed to stand at room temperature for 10 minutes. Thereafter, the absorbance (measurement wavelength: 570 nm, reference wavelength: 655 nm) was measured with a microplate reader. NO ₂ ⁻ accumulated in the culture supernatant was quantified using NaNO ₂ of known concentration diluted in DMEM medium as a standard. Nitric oxide production inhibition rate (%) was determined by the following formula.

Inhibition (%) = [(A−B) / (A−C)] × 100
AC: Nitrous acid concentration (μg / mL)
[A: Absorbance when LPS is added and HPA is not added, B: Absorbance when LPS is added and HPA is added, C: Absorbance when LPS is not added and HPA is not added]

  In addition, CAPE (caffeic acid phenethyl ester, manufactured by Wako Pure Chemical Industries, Ltd.) was used instead of HPA as a comparison target, and the inhibitory action of CAPE on nitric oxide production was determined by the same method.

(2) Results Table 4 shows the test results of the obtained nitric oxide production inhibitory action (inhibition rate of nitric oxide production, Inhibition (%)).

As is clear from Table 4, HPA had almost no inhibitory effect on nitric oxide production. On the other hand, the IC ₅₀ value for the inhibitory effect of CAPE on nitric oxide production was 3.1 μM (data not shown). That is, it was found that activation of macrophages, that is, bactericidal action of macrophages and the like were not inhibited by using nitric oxide production ability as an index.

Example 2
A lotion containing HPA synthesized in Synthesis Example 1 was prepared, and the stability and usability evaluation of the preparation over time were tested.
(1) Preparation of HPA-containing lotion To the container, component A described in the column of “Example 2” in Table 5 below was added and dissolved uniformly. In a separate container, component B was uniformly dissolved. Next, the A component was slowly added to the dissolved B component to solubilize it. Stir until the whole is uniform and fill the container.
The concentration of HPA in the prepared lotion is about 1 μM.

(2) Stability Evaluation of Formulation After the skin lotion prepared in (1) above was stored at 40 ° C. for 1 month, the state of the skin lotion was visually observed to confirm odor.

(3) Usability evaluation The female panelists (10 persons) used the lotion prepared in the above (1) in the usual usage, and for each item in Table 5 regarding usability, “5: Very good 4 : Good, 3: Normal, 2: Slightly bad, 1: Very bad ”. The average score of the obtained evaluation was used as an evaluation score, and a determination code was attached according to the following criteria.

A: Evaluation score 5 to 4.5
○: Evaluation point 4.5 to 3.5
(Triangle | delta): Evaluation point 3.5-2.5
×: Evaluation score 2.5 or less

(4) Results Table 5 shows the results obtained.

Examples 3-4
According to the description in the column of “Example 3” or “Example 4” in Table 5, a lotion containing HPA was prepared in the same manner as in Example 2.

  The concentrations of HPA in the lotions prepared in Examples 3 to 4 are about 300 μM (Example 3) and about 1000 M (Example 4), respectively.

  In addition, the stability evaluation and usability evaluation of the preparation were performed in the same manner as in Example 2.

  The obtained results are also shown in Table 5.

Comparative Examples 1-2
(1) Preparation of ascorbic acid-containing lotion, and stability and usability evaluation of the formulation According to the description in the column of “Comparative Example 1” or “Comparative Example 2” in Table 5, the same method as in Example 2 was used. A lotion containing ascorbic acid instead of HPA according to the present invention was prepared.

  In addition, the stability evaluation and usability evaluation of the preparation were performed in the same manner as in Example 2.

  The obtained results are also shown in Table 5.

(2) Results As is clear from the results of Examples 2 to 4 in Table 5, the skin lotion of the present invention containing HPA has no discoloration or off-flavor even when stored at 40 ° C. for 1 month, and the stability of the preparation. It was an excellent solubilizing lotion. Moreover, the lotion of the present invention prepared in Examples 2 to 4 had no irritation at the time of use, and was excellent in whitening feeling, skin gloss improvement effect, and fine wrinkle reduction effect.

  On the other hand, as is clear from the results of Comparative Examples 1 and 2 in Table 5, the skin lotion containing ascorbic acid also blended as a quasi-drug main ingredient is discolored and has a strange odor when stored at 40 ° C. for 1 month. It was confirmed that the stability of the preparation over time was poor. In order to maintain ascorbic acid activity, the pH of the system must be kept low. Therefore, the pH values of the lotions prepared in Comparative Example 1 and Comparative Example 2 were pH 3.41 and pH 2.95, respectively, which were suitable for exhibiting the activity of ascorbic acid. It is considered that sufficient pH could not be maintained, which affected the stability of the preparation. Moreover, since a use effect is not obtained unless it mix | blends much, on the contrary, the sticky feeling came out.

Example 5
An emulsion containing HPA synthesized in Synthesis Example 1 was prepared, and a test for evaluating the stability and usability of the preparation over time was performed.

(1) Preparation of HPA-containing emulsion A component shown in Table 6 below was added to a container, and the mixture was heated and stirred to dissolve uniformly. The B component was weighed into an emulsification pot in which the entire amount was charged, and heated and stirred to make it uniform. While stirring the B component, the oil phase (A component) obtained above was slowly added to carry out emulsification. The homomixer was operated to make the whole uniform, and then cooled under reduced pressure. When the bulk temperature reached 40 ° C., component C previously dissolved in another container was added. When the bulk temperature became 30 ° C. or lower, the cooling and stirring were stopped, the pressure was returned to normal pressure, and then the container was filled.

  The concentration of HPA in the prepared emulsion is about 260 μM.

(2) Evaluation of stability of formulation and usability evaluation In the same manner as in Example 2, the stability of the obtained emulsion formulation was evaluated. In addition, usability evaluation by female panelists (10 persons) was performed in the same manner as in Example 2.

(3) Results
The emulsion of the present invention containing HPA has a good formulation stability, and even when stored at 40 ° C. for 1 month, there is no separation or precipitation of the emulsion, and there is no discoloration or off-flavor. .

  Moreover, the emulsion of the present invention had no stickiness during use and no irritation during use. Furthermore, the skin after one month using the emulsion of the present invention had a slight whitening feeling, improved skin gloss, and an effect of reducing fine lines.

Example 6
A face cream containing HPA synthesized in Synthesis Example 1 was prepared, and the stability and usability evaluation of the formulation over time were tested.

(1) Preparation of HPA-containing face cream A component shown in Table 7 below was added to a container, and the mixture was heated and stirred to dissolve uniformly. The B component was weighed into an emulsification pot in which the entire amount was charged, and heated and stirred to make it uniform. While stirring the B component, the oil phase (A component) obtained above was slowly added to carry out emulsification. The homomixer was operated to make the whole uniform, and then cooled under reduced pressure. When the bulk temperature reached 40 ° C., component C previously dissolved in another container was added. When the bulk temperature fell below 30 ° C, cooling and stirring were stopped and the pressure was returned to normal pressure. The obtained face cream was filled in a container.

  The concentration of HPA in the prepared face cream is about 520 μM.

(2) Stability evaluation and usability evaluation of the preparation In the same manner as in Example 2, the stability of the obtained face cream preparation was evaluated. In addition, usability evaluation by female panelists (10 persons) was performed in the same manner as in Example 2.

(3) Results
The face cream of the present invention containing HPA has good stability of the formulation, and even when stored at 40 ° C. for 1 month, the emulsion does not separate or precipitate, and there is no discoloration or off-flavor, and it is in a stable formulation state. It was.

  Moreover, the face cream of the present invention had no stickiness during use and no irritation during use. Furthermore, the skin after one month using the face cream of the present invention had a slight whitening feeling, improved skin gloss, and an effect of reducing fine lines.

Example 7
An essence containing HPA synthesized in Synthesis Example 1 was prepared, and a test for evaluating the stability and usability of the preparation over time was performed.

(1) Preparation of HPA-containing essence A component shown in Table 8 below was added to a container and dissolved uniformly. The component B was weighed into a production kettle that was charged with the entire amount and dissolved uniformly. Next, the C component was added to the dissolved B component to make it uniform, and then the A component was added and stirred until it became uniform. Next, after adding D component and fully stirring it, it was filled with the container.

  The concentration of HPA in the prepared essence is about 520 μM.

(2) Evaluation of Stability and Usability of Formulation In the same manner as in Example 2, the stability of the obtained essence formulation was evaluated. In addition, usability evaluation by female panelists (10 persons) was performed in the same manner as in Example 2.

(3) Results
The essence of the present invention containing HPA also has good stability of the formulation, and there is no change in the viscosity of the gel even after storage at 40 ° C. for 1 month, no separation / precipitation, no discoloration, no strange odor, and stable It was in the formulation state.

  In addition, the essence of the present invention was not sticky during use and there was no irritation during use. Furthermore, the skin after one month using the essence of the present invention had a slight whitening, improved skin gloss, and an effect of reducing fine lines.

Example 8
A BB cream containing HPA synthesized in Synthesis Example 1 was prepared, and the stability and usability evaluation of the preparation over time were tested.

(1) Preparation of HPA-containing BB cream B components listed in Table 9 below were pulverized and mixed in a container to make it uniform. The A component was weighed into a production kettle where the entire amount was charged, and heated and mixed to make it uniform. Next, the B component, the C component, and the D component were sequentially added to the A component to make it uniform, and then the E component was added for emulsification. The homomixer was activated to make the whole uniform, and then cooled under reduced pressure. When the bulk temperature became 30 ° C. or lower, cooling and stirring were stopped, the pressure was returned to normal pressure, and then the container was filled.

The concentration of HPA in the prepared BB cream is about 300 μM.

(2) Evaluation of stability of formulation and usability evaluation In the same manner as in Example 2, the stability of the obtained face cream formulation was evaluated. In addition, usability evaluation by female panelists (10 persons) was performed in the same manner as in Example 2.

(3) Results
The BB cream of the present invention containing HPA has a good formulation stability, and even when stored at 40 ° C. for 1 month, no separation or precipitation of the emulsion is observed, and there is no discoloration or off-flavor, and a stable formulation It was in a state.

  Moreover, the BB cream of the present invention had no stickiness during use and no irritation during use. Furthermore, the skin after one month using the BB cream of the present invention had a slight whitening, improved skin gloss, and reduced wrinkles.

  The present invention provides a melanin production inhibitor and a whitening agent having a high inhibitory effect on melanin production, safety to living bodies, and stability over time, and using this, skin blackening, spots, buckwheat etc. Can contribute to the prevention, treatment or improvement of pigmentation such as buckwheat.

Claims (4)

A melanin production inhibitor comprising 4-((E) -3-hydroxy-1-propenyl) phenyl acetate as an active ingredient. A whitening agent containing 4-((E) -3-hydroxy-1-propenyl) phenyl acetate as an active ingredient. A method for inhibiting melanin production, comprising applying 4-((E) -3-hydroxy-1-propenyl) phenyl acetate to the skin. A whitening method characterized by applying 4-((E) -3-hydroxy-1-propenyl) phenyl acetate to the skin.