JP2013032311A - Endothelin action inhibitor and whitening agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an endothelin action inhibitor, a whitening agent and a skin external preparation.SOLUTION: The endothelin action inhibitor, a whitening agent and a skin external preparation include a compound represented by general formula (1) or its salt as an active constituent; wherein, R denotes a hydrogen atom or a 1 to 4C alkyl group.

Description

  The present invention relates to an endothelin action inhibitor, a whitening agent, and a skin external preparation.

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 and skin external preparation which can suppress the effect | action of endothelin and can suppress melanin production.

  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 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.
Furthermore, this invention relates to the skin external preparation 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 and skin external preparation 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.

Hereinafter, the present invention will be described in detail.
The endothelin action inhibitor, whitening agent, and skin external preparation 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.

In general formula (1), R 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. Among these, R is preferably a hydrogen atom or a methyl group, and more preferably a methyl group.
The endothelin action inhibitor, whitening agent and skin external preparation 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 compounds represented by the general formula (1) include the following compounds. However, the present invention is not limited to these. 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.

As a method of chemically synthesizing the compound represented by the general formula (1), for example, the exemplary compound (1-a) can be synthesized with reference to the following documents 1) to 3). Specifically, using a condensing agent such as dicyclohexylcarbodiimide, 6-methoxy-2-methyl-4-hydroxybenzoic acid and hydroxyl group protected with a benzyl group are condensed with decanol acid, and then the benzyl group of the product is deprotected. Can be synthesized (see Document 2 below).

1) Synthesis of orcinol tridepsides and aphthosin, an orcinol tetradepside. Bryan, Anthony J .; Elix, John A .; Norfolk, Susan. Australian Journal of Chemistry (1976), 29 (5), 1079-86.
2) Synthesis of lichen tridepsides. Jayalakshmi, V .; Neelakantan, Sankara; Seshadri, Tiruvenkata R. Indian Journal of Chemistry (1969), 7 (1), 56-8.
3) Lichen substances. LXXV. Synthesis of gyrophoric acid. 2. Umbilicarinic and umbilicaric acid. Asahina, Yasuhiko; Yosioka, Itiro. Berichte der Deutschen Chemischen Gesellschaft [Abteilung] B: Abhandlungen (1937), 70B 200-6.

A method for extraction / isolation from a natural material such as a plant is not particularly limited. For example, a plant or the like is extracted using a suitable solvent, and the above general formula ( The method of isolating the compound represented by 1) is mentioned. As a natural material such as a plant, for example, lichen (liquor, sarcophagus, scientific name: Umbilicaria esculenta ) of lichen 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, the whitening agent and the external preparation for skin of the present invention, the compound represented by the general formula (1) may be used alone as an active ingredient, or two or more kinds may be used in combination.

  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 components may be added to the endothelin action inhibitor or 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 components, the amount of the compound represented by the general formula (1) contained in the agent is not particularly limited, but the compound represented by the general formula (1) is 0. It is preferably contained at 00001-5 mass%, more preferably at 0.0001-5 mass%, and particularly preferably at 0.0001-3 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 systems, ointments, creams, water-oil two-layer systems, water-oil-powder three-layer systems. 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 1
Illustrative compound (1-a): Isolation of umbilical acid To 5 g of iwatake (manufactured by Shinwa Bussan), 100 mL of 95% ethanol aqueous solution was added, extracted at 40 ° C. for 1 week, filtered, and extracted with 88 mL of iwatake extract (solid content 0 .25 g) was obtained. 32 mg of this extract was fractionated by preparative HPLC (column: Inertsil ODS3 10 * 500 mm) to obtain 2.00 mg of component A (yield from extract solids: 6.25%).
The structural analysis of the isolated component A was performed by NMR. The results of structural analysis by NMR are shown in Table 1. As a result, from the NMR spectrum data of isolated component A (isolated component A was measured after being dissolved in 0.50 mL of DMSO-d6), it was found that the component had the structure of exemplary compound (1-a). Moreover, since it almost coincided with the spectrum data of umbilical acid reported in the literature (Phytochemistry, Vol48., No5, P815-822), the isolated component was identified as umbilical acid.
In the table, Me represents a methyl group. The number of each hydrogen atom indicates the number of the bonded carbon atom. In the case of a hydrogen atom of a hydroxyl group, the number of the carbon atom to which the oxygen atom is bonded is shown.

Production Example 2
Illustrative compound (1-b): Isolation of gyrophoric acid 32 mg of the extract obtained in Production Example 1 was fractionated by preparative HPLC, and 4.50 mg of component B (yield 14.06% from the solid content of the extract) was obtained. Obtained.
The structural analysis of the isolated component B was performed by NMR. The results of structural analysis by NMR are shown in Table 2. From the NMR spectrum data of isolated component B (isolated component B was measured after being dissolved in a mixed solvent of 0.45 mL of DMSO-d6 and 0.05 mL of 20% deuterated hydrochloric acid-containing heavy water), the component was exemplified by the compound (1- It was found to have the structure of b). Moreover, since it almost coincided with the spectrum data (Phytochemistry, Vol48., No5, P815-822) of gyrophoric acid reported in the literature, the isolated component was identified as gyrophoric acid.
In the table, Me represents a methyl group. The number of each hydrogen atom indicates the number of the bonded carbon atom. In the case of a hydrogen atom of a hydroxyl group, the number of the carbon atom to which the oxygen atom is bonded is shown.

Comparative Example 1
Isolation of ethyl orthoselate 32 mg of the extract obtained in Production Example 1 was fractionated by preparative HPLC to obtain 10.80 mg of component C (yield 33.75% from the solid content of the extract).
The structural analysis of the isolated component C was performed by NMR. The results of structural analysis by NMR are shown in Table 3. NMR data for isolated component C (isolated component C measured after dissolution in 0.50 mL of CDCl ₃ ) has been reported in the literature Spectral data of ethyl orthoserate (Chinese Chinese medicine magazine, Vol26., No9 , P608-610), the isolated component was identified as ethyl orthoselate.
In the table, Me represents a methyl group. The number of each hydrogen atom indicates the number of the bonded carbon atom. In the case of a hydrogen atom of a hydroxyl group, the number of the carbon atom to which the oxygen atom is bonded is shown.

Reference Example 1: German chamomile extract To a flower portion of German chamomile (Japanese name: chamomile, obtained from Shinwa Bussan Co., Ltd.), 40 mL of a 50% ethanol-containing aqueous solution was added, extracted at room temperature for 14 days, filtered, A German chamomile extract (221 mL) was obtained (evaporation residue 2.63%).

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) Evaluation of German chamomile extract As a positive control, the German chamomile extract prepared above (2.63% evaporation residue, 50% EtOH solvent) was used. 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 isolated compound Each compound isolated above was pretreated with the final concentrations shown in Table 4 and subjected to the evaluation system, and the Ca concentration increase rate by endothelin stimulation was calculated (N = 6). The results are shown in Table 4.

As is clear from Table 4, in the system to which exemplified compound (1-a): umbilical acid and (1-b): gyrophoric acid were added, the increase in intracellular Ca concentration by endothelin stimulation was 52 μM, respectively. It was confirmed that it was suppressed by 60% and 10%. That is, the compound represented by the general formula (1) has an excellent endothelin inhibitory action, and the inhibitory effect is equivalent to or higher than that of a German chamomile extract, which is a known whitening component. It was.
On the other hand, in the system to which ethyl orthoserate was added, the increase in intracellular Ca concentration was suppressed to 5% or less even at a concentration of 127.5 μM.

(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
Umbilical 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

2. Preparation of milky lotion (composition) (formulation: mass%)
Umbilical 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 (added moles of ethylene oxide: 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%)
Umbilical 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 antiseptic

4). Preparation of serum (composition) (Composition: mass%)
Umbilical 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%)
Umbilical 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 (addition moles of ethylene oxide: 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 antiseptic

Claims (3)

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 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 represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms.) A skin external preparation containing a compound represented by the following general formula (1) or a salt thereof as an active ingredient.

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

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