JP2012126653A - Inhibitor of activity of endothelin action and skin whitening agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an inhibitor for endothelin, and a skin whitening agent.SOLUTION: There are provided the inhibitor of activity of endothelin and the skin whitening agent, containing a compound represented by general formula (1) or a salt thereof as an active ingredient. (In the formula, Rrepresents a formyl group or a 1-4C alkyl; Rrepresents H or a 1-4C alkyl group).

Description

  The present invention relates to an endothelin action inhibitor and a whitening agent.

Endothelin is a peptide hormone derived from endothelial cells and acts on various cells and tissues through receptors. For example, it is known to cause an increase in intracellular calcium concentration in vascular smooth muscle cells and the like (Non-Patent Document 1).
In recent years, endothelin has promoted an increase in intracellular calcium concentration in epidermal melanocytes (melanocytes), promoted cell proliferation via intracellular signal transduction system, and enhanced the activity of tyrosinase, the rate-limiting enzyme of melanin synthesis. Has been reported (see Non-Patent Document 2). In addition, endothelin is one of the melanocyte activators produced by epidermal keratinocytes (keratinocytes) (Non-patent Document 3), and is an important factor in ultraviolet-induced pigmentation and senile pigment spot formation. It has been reported (Non-Patent Documents 4 and 5).
From the biological action of endothelin, it is expected that a substance capable of suppressing the action of endothelin is useful for improvement and prevention of melanin production and pigmentation.

Hirata Y. et al. (1988) Biochem. Biophys. Res. Commun. 154, 868-875 Yada et al. (1991) J. Biol. Chem. 266, 18352-18357 Imokawa et al. (1992) J. Biol. Chem. 267, 24675-24680 Imokawa et al. (1995) J. Invest. Dermatol. 105, 32-37 Kadono et al. (2001) J. Invest. Dermatol. 116, 571-577

  An object of the present invention is to provide an endothelin action inhibitor that effectively suppresses the action of endothelin. Moreover, this invention makes it a subject to provide the whitening agent which can suppress the effect | action of endothelin and can suppress a melanin production | generation.

  In view of the above problems, the present inventors have intensively studied to search for new substances that suppress the action of endothelin. As a result, it has been found that the compound represented by the following general formula (1) effectively suppresses the increase in calcium (ion) concentration in melanocytes caused by the action of endothelin, and the compound is useful as a novel whitening component. The knowledge that it is. The present invention has been completed based on these findings.

  That is, the present invention relates to an endothelin action inhibitor containing a compound represented by the following general formula (1) or a salt thereof as an active ingredient.

(In the formula, R ₁ represents a formyl group or an alkyl group having 1 to 4 carbon atoms. R ₂ represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms.)

  Moreover, this invention relates to the whitening agent which contains the compound or its salt represented by the said General formula (1) as an active ingredient.

  The endothelin action inhibitor of the present invention has an excellent endothelin action inhibitory effect, and can effectively suppress an increase in calcium concentration in melanocytes caused by endothelin. Moreover, the whitening agent of this invention can suppress the effect | action of endothelin with respect to a melanocyte, and can suppress a melanin production | generation.

In the reference example of Example 1, it is a figure which shows the increase rate (relative value%) of the intracellular calcium concentration in the system which added the German chamomile extract. In Example 2, it is a photograph of the skin sheet which added and cultivated beomices acid. In Example 2, it is a figure which shows the amount of melanin (relative value%) in the skin sheet which added the beomices acid.

Hereinafter, the present invention will be described in detail.
The endothelin action inhibitor and the whitening agent of the present invention contain a compound represented by the following general formula (1) or a salt thereof as an active ingredient. As demonstrated in the examples described later, the compound has an excellent endothelin action inhibitory effect and melanin production inhibitory effect.

In general formula (1), R ₁ represents a formyl group or an alkyl group having 1 to 4 carbon atoms. R ₂ represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. Examples of the alkyl group having 1 to 4 carbon atoms include methyl group, ethyl group, propyl group, isopropyl group, n-butyl group, i-butyl group, sec-butyl group, and t-butyl group.
R ₁ is preferably a formyl group or an alkyl group having 1 to 2 carbon atoms (methyl group, ethyl group), more preferably a formyl group or a methyl group.
R ₂ is preferably a hydrogen atom or an alkyl group having 1 to 2 carbon atoms (methyl group or ethyl group), more preferably a hydrogen atom or a methyl group.
The endothelin action inhibitor and the whitening agent of the present invention may contain a salt of the compound represented by the general formula (1) as an active ingredient. Examples of the salt include, but are not limited to, alkali metal salts such as lithium salt, sodium salt and potassium salt, alkaline earth metal salts such as calcium salt and magnesium salt, alkylamine salts such as trimethylamine and triethylamine, and quaternary ammonium salts. , Alkanolamine salts such as triethanolamine, diethanolamine and monoethanolamine, or amino acid salts such as lysine, histidine and arginine, and the like, preferably alkali metal salts, alkanolamine salts and amino acid salts. (Hereinafter, “the compound represented by the general formula (1) or a salt thereof” is collectively referred to as a compound represented by the general formula (1).)

  Specific examples of the compound represented by the general formula (1) are given below, but the present invention is not limited thereto. In the following exemplary compounds, Me represents a methyl group.

  The compound represented by the general formula (1) can be produced by chemical synthesis or can be obtained by extraction / purification from a natural product-derived material. It is also available as a commercially available reagent.

As a method of chemically synthesizing the compound represented by the general formula (1), for example, Australian Journal of Chemistry (1975), 28 (5), 1113-1124, Pharmaceutical Journal (1936), 56, 237-258, Examples include the methods described in Pharmaceutical Journal (1932), 52, 991-993 and the like.
When obtaining from a commercially available reagent etc., it can obtain from Namiki Shoji Co., Ltd., for example.

A method for extraction / isolation from a natural material such as a plant is not particularly limited. For example, a plant is extracted using a suitable solvent, and the above general formula ( The method of isolating the compound represented by 1) is mentioned. As the plant, for example, a setcha (snow tea, scientific name: Thamnolia vermicularis ) can be used. As the extraction solvent, those commonly used for extraction of plant components can be used, for example, water; alcohols such as methanol, ethanol, propanol, butanol; polyhydric alcohols such as ethylene glycol, propylene glycol, glycerin, butylene glycol, etc. Ketones such as acetone and methyl ethyl ketone; esters such as methyl acetate and ethyl acetate; chain and cyclic ethers such as tetrahydrofuran and diethyl ether; polyethers such as polyethylene glycol; halogens such as dichloromethane, chloroform and carbon tetrachloride; Hydrocarbons such as hexane, cyclohexane and petroleum ether; aromatic hydrocarbons such as benzene and toluene; pyridines; supercritical carbon dioxide; fats and oils, waxes and other oils, etc. Or it can be used in combination of two or more. Moreover, normal conditions can also be applied as extraction conditions. As an example, the plant is immersed or heated to reflux at 0 to 100 ° C. for several minutes to several weeks.

  In the endothelin action inhibitor or whitening agent of the present invention, the compound represented by the general formula (1) may be used alone as an active ingredient, or a mixture of two or more thereof may be used.

  As demonstrated in the examples described later, the compound represented by the general formula (1) can effectively suppress an increase in calcium concentration of melanocytes caused by endothelin. Furthermore, since the said compound can suppress the production | generation of melanin by suppressing the effect | action of endothelin with respect to a melanocyte, there exists a whitening effect. In the present invention, “whitening (action)” refers to suppressing the formation of melanin, returning to the original transparent skin color without excess melanin, or preventing pigmentation such as blackening of the skin or spots and freckles. Mean to suppress.

  In the present invention, the compound represented by the general formula (1) may be used as it is as an endothelin action inhibitor or a whitening agent. In addition, an appropriate liquid or solid excipient or extender such as titanium oxide, calcium carbonate, distilled water, lactose, starch or the like may be added to the compound. In this case, the amount of the compound represented by the general formula (1) contained in the agent is not particularly limited, but 0.00001 to 5% by mass of the compound represented by the general formula (1) is included. Is preferable, it is more preferable that it is contained 0.00001-3 mass%, and it is especially preferable that 0.0001-3 mass% is contained.

  In addition to the compound represented by the general formula (1), other medicinal ingredients may be added to the whitening agent of the present invention. For example, other whitening agents, moisturizers, antioxidants, ultraviolet absorbers, surfactants, thickeners, colorant types, and the like can be added. When used as a composition together with other medicinal ingredients, the amount of the compound represented by the general formula (1) contained in the whitening agent is not particularly limited, but the compound represented by the general formula (1) is 0. The content is preferably 0.0001 to 5% by mass, more preferably 0.0001 to 5% by mass, and particularly preferably 0.0001 to 3% by mass.

The endothelin action inhibitor and whitening agent of the present invention can be used in the form of a skin external preparation. “Skin external preparation” means those applied to the skin as skin cosmetics, external pharmaceuticals, quasi-drugs, etc., and their dosage forms are aqueous solutions, solubilization systems, emulsification systems, powder systems, gels It can take a wide variety of forms such as a system, an ointment system, a cream, a water-oil two-layer system, and a water-oil-powder three-layer system. Examples thereof include face wash, lotion, milky lotion, cream, gel, essence (beauty serum), pack, mask, foundation, ointment, sheet-like product, and the like.
When used in the form of a skin external preparation, in addition to the compound represented by the general formula (1), components used for normal skin external preparation, such as surfactants, oily substances, polymer compounds, preservatives Other than the above, medicinal components, powders, ultraviolet absorbers, dyes, fragrances, emulsion stabilizers, pH adjusters, and the like can be appropriately blended. The amount of the external preparation for skin containing the compound represented by the general formula (1) varies depending on the content of the active ingredient. For example, in the case of a cream or ointment, the amount is 0.1 per 1 cm ^{2 of} the skin surface. In the case of ˜5 μg and a liquid preparation, it is also preferable to use 0.1˜10 μg.

  EXAMPLES Hereinafter, although this invention is demonstrated further in detail based on an Example, this invention is not limited to this.

Production Example (Production Example 1) Beomices acid Setha (manufactured by Shinwa Bussan Co., Ltd.) 41.6 g was added 1 L of 95% ethanol aqueous solution, extracted at 30 ° C. for 30 days, filtered, and the setter on the filter paper was 200 mL of 95% ethanol aqueous solution In addition to the first filtrate, 1100 mL of setcha extract (solid content 2.98 g) was obtained. 56 mL of this extract (solid content: 151.7 mg) was fractionated by HPLC to obtain 28.00 mg of beomices acid (yield 18.5%).
The structural analysis of the isolated component was performed by NMR. The NMR spectrum data of the isolated component was in good agreement with the spectrum data of beomices acid reported in the literature (Japanese Journal of Food Chemistry (Eclipse Chemical Journal) Vol.14 (2), 2007). The identified component was identified as beomices acid. The results of structural analysis by NMR are shown in Table 1. In the table, Me represents a methyl group.

(Reference Example 1) Squamatoic acid 24 mL (solid content 65.04 mg) of the Setcha extract obtained in Production Example 1 was fractionated by HPLC to obtain 5.6 mg of squamatoic acid (yield 8.61%). . The structural analysis of the isolated component was performed by NMR. Since the NMR spectral data of the isolated component was almost consistent with the spectral data of squamatoic acid (Yunnan Zhiwu Yanjiu, 24 (4), 525-530 (2002)) reported in the literature, the isolated component was Identified as acid. The results of structural analysis by NMR are shown in Table 2. In the table, Me represents a methyl group.

(Production Example 2) Barbatic acid Barbatic acid (obtained from Namiki Shoji Co., Ltd.) commercially available as a reagent was used for activity evaluation described below.

(Production Example 3) Difractic acid Difractic acid commercially available as a reagent (obtained from Namiki Shoji Co., Ltd.) was used for activity evaluation described below.

(Reference Example 2) German chamomile extract To a flower portion of German chamomile (Japanese name: chamomile, Shinwa Bussan Co., Ltd.) 40 g was added 400 mL of a 50% ethanol-containing aqueous solution, extracted at room temperature for 14 days, and then filtered. German chamomile extract (221 mL) was obtained (2.63% evaporation residue).

Example 1 Verification of endothelin action inhibitory effect About each compound and extract which were manufactured above, the endothelin action inhibitory effect was verified using the following evaluation system.
(1) Evaluation system Normal human newborn epidermis-derived melanocytes (NHEMs; Kurabo Industries Co., Ltd.) are seeded at 3 × 10 ⁴ cells / well (200 μL / well) in a 96-well plate for fluorescence detection, and under 5% CO ₂ . Cultured at 37 ° C. Medium 254 containing PMA (-) growth additive (HMGS) was used as the medium.
After 3 days of culture, the medium was removed by decantation from the culture plates and replaced in the assay buffer containing intracellular ^{Ca 2 ++} measuring reagent Fluo4-AM, and incubated for 1 hour at 37 ° C.. Next, 20 seconds after the start of measurement in an FDSS (Functional Drug Screening System) device, the compound at a predetermined concentration was pretreated for 1 minute, and then the ligand endothelin (ET-1, final concentration 100 nM) was added, and Fluo4-AM Fluorescence of Ex. 480 nm / Em. Detection was performed over time from the start of measurement at a measurement wavelength of 540 nm until 5 minutes later.
Relative value (%) when the fluorescence increase ratio of Fluo4-AM in the control (Max ratio-Min ratio) is 100, the rate of increase in intracellular calcium (calcium ion) concentration (%) of each compound is calculated, and endothelin The action inhibitory effect was evaluated. As a control, ethanol or a 10-95 v / v% aqueous ethanol solution added at a final concentration of 0.1-0.5 v / v% was used.

(2) Reference Example: Evaluation of German Chamomile Extract The German chamomile extract prepared above (2.63% evaporation residue, 50% EtOH solvent) was used as a positive control. It is known that a German chamomile extract suppresses an increase in Ca concentration of melanocytes by the action of endothelin and suppresses melanin production.
The German chamomile extract was pretreated at final concentrations of 0.5 v / v% and 1.0 v / v% and subjected to the above evaluation system, and the rate of increase in calcium (Ca) concentration by endothelin stimulation was calculated (N = 6). ). The results are shown in FIG.
As is clear from FIG. 1, the concentration-dependent Ca concentration increase inhibitory effect was confirmed in the system to which the German chamomile extract was added. In particular, in a system with a concentration of 1 v / v%, an increase in Ca concentration was suppressed by 20% or more, and an excellent endothelin inhibitory action was confirmed.

(3) Evaluation of the compound of the present invention The compound prepared above was pretreated with the final concentrations shown in Table 3 and subjected to the above evaluation system, and the Ca concentration increase rate due to endothelin stimulation was calculated (N = 6). The results are shown in Table 3.

As is apparent from Table 3, it was confirmed that the increase in intracellular Ca concentration by stimulation with endothelin was suppressed by 45% or more in all the systems to which beomices acid, barbatic acid and difractic acid were added at a concentration of 50 μM. That is, it was found that the compound represented by the general formula (1) has an excellent endothelin inhibitory action, and the inhibitory effect is more than that of the German chamomile extract, which is a known whitening component.
On the other hand, in the system to which squamatoic acid was added, the increase in intracellular Ca concentration was suppressed by about 4% at a concentration of 50 μM.

Example 2 Verification of inhibitory action on melanin production Using the above-produced beomices acid, the inhibitory action on melanin production in the skin was verified.
EPI-100-NMM113 in which a three-dimensional cultured skin model containing melanocytes (MEL300A; Kurabo Industries Co., Ltd.) was added with endothelin-1 (ET-1) and SCF (stem cell growth factor) which are melanocyte activators at a final concentration of 10 nM. Using the medium, the cells were cultured under the conditions of 37 ° C. and 5% CO ₂ . From the first day of culture, beomices acid was added at a final concentration of 50 μM (N = 2). The medium was changed once every 3 days. After 14 days, the skin model was washed with PBS and photographed. The results are shown in FIG. Thereafter, cell respiration activity was measured using Alamar Blue reagent, and it was confirmed that the final concentration was a concentration that did not show cytotoxicity (results not shown).
Subsequently, the skin model cup was washed with PBS, the skin sheet was peeled off with tweezers, transferred to a tube, and washed with PBS three times. After washing three times with 50% ethanol and twice with 100% ethanol, the mixture was allowed to stand overnight at room temperature and dried completely. 200 μL of 2M NaOH was added and dissolved at 100 ° C., and the absorbance of the supernatant obtained by centrifugation was measured at a measurement wavelength of 405 nm to calculate the amount of melanin. The results are shown in FIG. The amount of melanin was expressed as a relative value (%) when the amount of control melanin was 100. As a control, ethanol or a 10-95 v / v% aqueous ethanol solution added at a final concentration of 0.1-0.5 v / v% was used.

As is clear from FIG. 2, the skin color added with beomices acid was brighter than the control system, and the skin darkening due to melanin production was suppressed. In addition, as shown in FIG. 3, in the system to which beomices acid was added, the amount of melanin in the skin tissue was reduced by 45% or more compared to the control system at an added concentration that did not show cytotoxicity.
As described above, endothelin is known to act on melanocytes to increase calcium concentration and increase melanin production, and substances that suppress the action of endothelin are useful as whitening ingredients. Also in the said Example of this invention, it was actually confirmed that the substance which suppresses the effect | action of endothelin with respect to a melanocyte can suppress melanin production effectively. Therefore, it can be seen that the compound represented by the general formula (1) can suppress melanin production by effectively suppressing the action of endothelin that promotes the increase of intracellular calcium concentration, and is useful as a whitening agent. It was.

(Prescription example)
Using the compound obtained in the above production example as an active ingredient, lotions, emulsions, serums and creams having the compositions shown below were prepared by conventional methods.

1. Preparation of lotion (composition) (Formulation: mass%)
1,3-butylene glycol 8.0
Glycerin 5.0
Ethanol 3.0
Veomices acid 0.0001
Chamomile extract 3.0
Kyokyo Extract 1.0
Clove extract 1.0
Xanthan gum 0.1
Hyaluronic acid 0.1
Disodium phosphate 0.1
Sodium dihydrogen phosphate 0.1
Ascorbic acid 2-glucoside 2.0
Purified water Remaining fragrance Appropriate amount Preservative Appropriate amount

2. Preparation of milky lotion (composition) (formulation: mass%)
Barbatic acid 0.001
Chamomile extract 1.0
Kyokyo Extract 1.0
Altea extract 2.0
Squalane 3.0
Olive oil 3.0
Glycerin 5.0
Polyoxyethylene hydrogenated castor oil (number of moles of ethylene oxide added: 40) 1.0
Carboxyvinyl polymer 0.2
Potassium hydroxide 0.1
Xanthan gum 0.2
Edetate disodium 0.02
Purified water Remaining antiseptic

3. Preparation of serum (composition) (Composition: mass%)
Difractic acid 0.001
Chamomile extract 1.0
Kyokyo Extract 1.0
Clove extract 1.0
Carboxyvinyl polymer 0.2
Acrylic acid / alkyl methacrylate copolymer 0.2
Potassium hydroxide 0.2
Xanthan gum 0.1
Hyaluronic acid 0.2
Sodium citrate 0.15
Citric acid 0.03
Glycerin 10.0
1,3-butylene glycol 5.0
Edetate dinatrim 0.05
Purified water Remaining preservative Appropriate amount Perfume Appropriate amount

4). Preparation of serum (composition) (Composition: mass%)
Veomices acid 0.001
Chamomile extract 1.0
Clove extract 1.0
Kyokyo Extract 1.0
Xanthan gum 0.2
Carboxymethylcellulose 0.2
Carboxyvinyl polymer 0.2
Potassium hydroxide 0.1
Citric acid 0.03
Sodium citrate 0.15
Glycerin 5.0
Propylene glycol 3.0
Polyethylene glycol (molecular weight 1500) 1.0
Polyethylene glycol monostearate 0.5
Purified water Remaining antiseptic

5. Preparation (composition) of cream (formulation: mass%)
Difractic acid 0.001
Chamomile extract 2.0
Kyokyo Extract 2.0
Clove extract 2.0
Methylpolysiloxane 3.0
Squalane 2.0
Neopentyl glycol dicaprate 3.0
Stearyl alcohol 1.5
Cetanol 1.0
Polyoxyethylene hydrogenated castor oil (number of moles of ethylene oxide added: 60) 0.5
Acrylic acid / alkyl methacrylate copolymer 0.3
Potassium hydroxide 0.15
Xanthan gum 0.1
Edetate disodium 0.05
Purified water Remaining preservative Appropriate amount Perfume Appropriate amount

Claims (2)

An endothelin action inhibitor containing a compound represented by the following general formula (1) or a salt thereof as an active ingredient.
(In the formula, R ₁ represents a formyl group or an alkyl group having 1 to 4 carbon atoms. R ₂ represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms.) A whitening agent containing a compound represented by the following general formula (1) or a salt thereof as an active ingredient.
(In the formula, R ₁ represents a formyl group or an alkyl group having 1 to 4 carbon atoms. R ₂ represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms.)