JP2012020950A - Inhibitor of endothelin action and whitening agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an inhibitor of endothelin action and a bleaching agent.SOLUTION: The inhibitor of endothelin action and the bleaching agent contain as an effective component, at least one kind of extract selected from a group composed of Pinusyunnanensis Franch, Juglanscinerea, Parmelia tinctorum Despre., Homalomenaocculta (Lour.) Schott, and Aletrisfarinosa.

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 extract of shochu, batagurumi, sekika, sennenken and alletris farinosa effectively suppresses the increase in calcium (ion) concentration in melanocytes caused by the action of endothelin. The knowledge that it was useful as a whitening component was obtained. The present invention has been completed based on these findings.

That is, the present invention is made up of the group consisting of Pinus yunnanensis Franch., Bataguru ( Juglans cinerea ), Sekika ( Parmelia tinctorum Despre.), Sennenken ( Homalomena occulta (Lour.) Schott) and Aletris farinosa ( Aletris farinosa ). The present invention relates to an endothelin action inhibitor containing at least one selected extract as an active ingredient.
The present invention also includes the group consisting of Pinus yunnanensis Franch., Batuguru ( Juglans cinerea ), Sekika ( Parmelia tinctorum Despre.), Sennenken ( Homalomena occulta (Lour.) Schott) and Aletris farinosa ( Aletris farinosa ). The present invention relates to a whitening agent containing at least one selected extract 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 | seat which added and extracted the extract of Shoko, Batagurumi, Sekika, and Aletris farinosa. In Example 2, it is a figure which shows the amount of melanin (relative value%) in the skin sheet which added each extract.

Hereinafter, the present invention will be described in detail.
Endothelin action inhibitors and whitening agents of the present invention include: Pinus yunnanensis Franch., Batagumi ( Juglans cinerea ), Sekika ( Parmelia tinctorum Despre.), Sennenken ( Homalomena occulta (Lour.) Schott), and Aletris Farinosa ( Aletris ). farinosa ) at least one extract selected from the group consisting of: As demonstrated in the examples described later, these extracts have an excellent endothelin action inhibitory effect and melanin production inhibitory effect.

First, the plant and lichen used in the present invention will be described.
In the present invention, shochu is a plant belonging to the genus Pinaceae, the scientific name is Pinus yunnanensis Franch., And the Japanese name is Yunnanmatsu (Unnansho, Unnanmatsu, Yunnanmatsu).
Bataguru is a plant belonging to the genus Juglans in the walnut family, and its scientific name is Juglans cinerea , also known as sylogrumi, butternut or white walnut.
Sekika is a lichen belonging to the genus Umenochoke, whose scientific name is Parmelia tinctorum Despr., And its Japanese name is stone flower, Umenogoke.
Sennenken is a plant belonging to the genus Homalomena , and its scientific name is Homalomena occulta (Lour.) Schott.
Aletris farinosa is a plant belonging to the genus Lilyaceae, and its scientific name is Aletris farinosa .
In the present invention, related plants and lichens belonging to the above-mentioned genera can also be used.

  As for the present invention, shochu, batagurumi, sekika, sennenken, and aretris farinosa can be used in any arbitrary part of those plants or lichens. For example, whole plant, whole plant, or any part of the above plant (root, rhizome, trunk, branch, stem, leaf, bark, sap, resin, flower, fruit, seed, peel, bud, bud, spike, heartwood, etc. ), The above-mentioned lichen leaf-like body, fruiting body and the like can be used. Moreover, you may use combining each site | part in multiple numbers.

In this invention, it is preferable to obtain an extract from the following site | parts especially among each site | part of the said plant or lichen.
In order to obtain an extract of pepper, it is preferable to use a resin of the above-mentioned plant, and a herbal medicine, matsuka, obtained from pepper as a base plant can also be used.
In order to obtain an extract of batagurumi, it is preferable to use the bark of the plant.
In order to obtain an extract of sekika, it is preferable to use the leaf-like body and fruit body of the lichen.
In order to obtain an extract of Sennenken, it is preferable to use the rhizome of the plant, and it is also possible to use the herbal medicine, Sennen Ken, obtained from Sennenken as a base plant.
In order to obtain an extract of Aretris falinosa, it is preferable to use the rhizome of the plant.
In the endothelin action inhibitor or whitening agent of the present invention, each of the above extracts may be used alone, or two or more of them may be used in combination.

There is no particular limitation on the method for producing the extract of shrimp, batagurumi, sekika, sennenken, and alletris farinosa used in the present invention, and the extract can be obtained by extracting the plant or lichen by a usual method. . Specifically, a dried product obtained by drying the plant or lichen, a squeezed juice obtained by compressing and extracting the pulverized product, a steam distillate, a crude extract using various extraction solvents, or a crude extract is distributed or distributed. An extract fraction obtained by purification by various chromatographies such as column chromatography can be used as the extract in the present invention.
The plant or lichen can be subjected to extraction as it is, but it is also preferable to add steps such as drying, shredding, and pulverization in order to further increase the extraction efficiency. Moreover, in this invention, you may use said extract, steam distillate, pressing material, etc. individually or in combination of 2 or more types.

In the present invention, it is more preferable to use an extract obtained by using an extraction solvent from a dried product obtained by drying the plant or lichen or a pulverized product thereof.
As the extraction solvent, any of a polar solvent and a nonpolar solvent can be used, and these can be mixed and used. For example, water; alcohols such as methanol, ethanol, propanol and butanol; polyhydric alcohols such as ethylene glycol, propylene glycol, glycerin and butylene glycol; 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; halogenated hydrocarbons such as dichloromethane, chloroform and carbon tetrachloride; hydrocarbons such as hexane, cyclohexane and petroleum ether; benzene Aromatic hydrocarbons such as toluene, pyridines; supercritical carbon dioxide; fats and oils, waxes, and other oils. Or the mixture which combined 2 or more types of the said solvent can be used as an extraction solvent. Among these, water, alcohols, water-alcohol mixed liquids, propylene glycol, glycerin, butylene glycol, and water-polyhydric alcohol mixed liquids are preferably used, and ethanol aqueous solutions are more preferably used.

  The extraction conditions for obtaining the extract used in the present invention differ depending on the solvent used and are not particularly limited. For example, when extracting with water, alcohols or a water-alcohol mixed solution, propylene glycol, butylene glycol, it is preferable. Uses 1 to 50 parts by volume of solvent with respect to 1 part by mass of plant or lichen, preferably 3 to 100 ° C, more preferably 20 to 80 ° C, still more preferably 20 to 40 ° C, preferably 1 It is preferable to immerse or heat reflux for a time to several weeks, more preferably about 1 day to 30 days. Moreover, in order to raise extraction efficiency, you may stir together or may perform a homogenization process in a solvent.

  The extract obtained by extraction with the above solvent may be used as it is, but it is also possible to fractionate and use a fraction having high activity by an appropriate separation means such as gel filtration, chromatography, precision distillation, etc. it can. In the present invention, the extract includes the various extracts thus obtained, a diluted solution thereof, a concentrated solution thereof, a purified product thereof or a dry powder thereof.

  As demonstrated in the below-mentioned Examples, the extract of shochu, batagurumi, sekika, sennenken and alltris farinosa can effectively suppress the increase in calcium concentration of melanocytes by endothelin. Furthermore, since these extracts can suppress the production of melanin by suppressing the action of endothelin on melanocytes, they have 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.

  As a method for evaluating the physiological action of endothelin on melanocytes, that is, an increase in calcium (calcium ion) concentration in melanocytes, for example, intracellular calcium (Ca) labeling reagents Fura-2AM and Fluo-4AM were incorporated into cells. Later, there is a method of measuring a fluorescence intensity ratio indicating intracellular Ca concentration with a digital imaging microscope or the like. This is a technique that has been used conventionally, but it is difficult to handle a large number of evaluation samples simultaneously with a microscope. In the present invention, as will be described in detail in Examples below, FDSS (Functional Drug Screening System) is used as an evaluation method with high throughput in order to efficiently search for and identify endothelin inhibitory substances having higher effectiveness. The candidate substances were evaluated. As a result, the above five extracts were identified as particularly effective.

In the endothelin action inhibitor or whitening agent of the present invention, an extract of shochu, batagurumi, sekika, sennenken, or aletris farinosa may be used alone, or two or more of them may be used in combination.
In the present invention, the extract of shrimp, batagurumi, sekika, sennenken, or aretris farinosa 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 extract. In this case, the amount of the extract contained in the agent is not particularly limited, but the extract is preferably contained in an amount of 0.0001 to 50% by mass, more preferably 0.001 to 25% by mass. ,
It is particularly preferable that the content is 0.01 to 10% by mass.

In addition to the above extract, 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 extract contained in the whitening agent is not particularly limited, but the extract is preferably contained in an amount of 0.00001 to 5% by mass, 0.0001 to More preferably 5% by mass is contained,
It is particularly preferable that the content is 0.001 to 5% 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 above extract, components used in normal skin external preparations, such as surfactants, oily substances, polymer compounds, preservatives, other medicinal ingredients, powders UV absorbers, dyes, fragrances, emulsion stabilizers, pH adjusters, and the like can be appropriately blended. The amount of the external preparation for skin containing the extract varies depending on the content of the active ingredient. For example, in the case of cream or ointment, 0.1 to 5 μg per cm ^{2 of} skin surface, Similarly, it is preferable to use 0.1 to 10 μg.

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

(Reference Example 1) Preparation of German chamomile extract To 40 g of flower portion of German chamomile (Japanese name: Chamomile, Shinwa Bussan Co., Ltd.), add 400 mL of 50% ethanol-containing aqueous solution, and extract at room temperature for 14 days. Filtration gave a German chamomile extract (221 mL) (evaporation residue 2.63%).

(Production Example 1) Preparation of Shoko extract To 40 g of the resin part of Shoko (Japanese name: Unnansho, Shinwa Bussan Co., Ltd.), 400 mL of a 50% ethanol-containing aqueous solution was added and room temperature was 41 days. After extraction, it was filtered to obtain a pepper extract (365 mL) (evaporation residue 0.60%).

(Production Example 2) Preparation of batagurumi extract To 40 g of bark of batagurumi (scientific name: Juglans cineria , obtained from Moutain Rose Harbs), 400 mL of 95% ethanol-containing aqueous solution was added, extracted at room temperature for 27 days, filtered, and batagurumi An extract (333 mL) was obtained (evaporation residue 1.24%).

(Production Example 3) Preparation of Sekika Extract To Sekika (Japanese name: Ishihana, obtained from Shinwa Bussan Co., Ltd.) 40 g of leaf-like body part was added 400 mL of 95% ethanol-containing aqueous solution, extracted at room temperature for 27 days, filtered To obtain a sesame extract (325 mL) (evaporation residue 0.60%).

(Production Example 4) Preparation of Sennenken Extract 400 mL of 95% ethanol-containing aqueous solution was added to 40 g of rhizomes of Sennenken (Japanese name: Ken Sennen Ken, obtained from Shinwa Bussan Co., Ltd.), extracted for 21 days at room temperature, and filtered. Sennenken extract (367 mL) was obtained (evaporation residue 0.70%).

(Production Example 5) Preparation of Aletris farinosa extract 500 ml of 95% ethanol-containing aqueous solution was added to 50 g of rhizome part of Aletris farinosa (scientific name: Aletris farinosa , obtained from Monteagle Herbs), extracted at room temperature for 15 days, and filtered. As a result, an extract of Aretris farinosa (425 mL) was obtained (evaporation residue 0.23%).

EXAMPLE 1 Verification of endothelin action inhibitory effect About each extract prepared by the said manufacture example, 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) apparatus, the extract having a predetermined concentration was pretreated for 1 minute, and then the ligand endothelin (ET-1, final concentration 100 nM) was added, and Fluo- Fluorescence of 4AM was measured using 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.
In the 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 extract is calculated, The endothelin 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 As a positive control, the German chamomile extract prepared in Reference Example 1 (evaporation residue 2.63%, 50% EtOH solvent) was used. German chamomile extract is known to suppress the increase in Ca concentration of melanocytes due to the action of endothelin and suppress melanin production.
German chamomile extract was pretreated at final concentrations of 0.5 v / v% and 1.0 v / v%, and used for 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 action 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 extract of the present invention Each of the extracts prepared in Production Examples 1 to 5 (evaporation residue 1 w / v%) was pretreated at a final concentration of 0.3 v / v% and evaluated as described above. It used for the system and the Ca concentration increase rate by endothelin stimulation was calculated (N = 6). The results are shown in Table 1.

  As is clear from Table 1, in all of the systems to which the extracts of shochu, batagurumi, sekika, sennenken, and aletris farinosa were added, an increase in intracellular Ca concentration by endothelin stimulation was suppressed by 20% or more. That is, the extract of Shochu, Batagurumi, Sekika, Sennenken, and Aletris farinosa used in the present invention has an excellent endothelin inhibitory action of suppressing an increase in intracellular Ca concentration by 20% or more, and the inhibitory effect is known. It was the same as or higher than the German chamomile extract, which is a whitening component.

Example 2 Verification of inhibitory action on melanin production Using the extract produced above, the inhibitory action on melanin production in 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 the culture, each extract (evaporation residue 1 w / v%) prepared in Production Examples 1 to 3 and 5 was added at a final concentration of 0.1 to 0.5 v / v% (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, in the system to which the extracts of ginger, batagurumi, sekika, and alletris farinosa were added, the skin color was brighter than that of the control system, and skin blackening due to melanin production was suppressed. . In addition, as shown in FIG. 3, the amount of melanin in the skin tissue was higher in the system to which the extract of shochu, batagurumi, sekika, and alletris farinosa was added, compared to the control system, at an added concentration that did not show cytotoxicity. Was reduced by more than 40%.
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, the extract of shrimp, batagurumi, sekika, sennenken, and aletris farinosa used in the present invention effectively suppresses the action of endothelin that promotes an increase in intracellular calcium concentration, thereby suppressing melanin production and whitening agent. As useful.

(Prescription example)
Using the extract obtained in the above production example as an active ingredient, a lotion having the composition shown below,
A milky lotion, a cosmetic liquid and a cream were each prepared by a conventional method.

1. Preparation of lotion (composition) (Formulation: mass%)
1,3-butylene glycol 8.0
Glycerin 5.0
Ethanol 3.0
Shoko extract 3.0
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%)
Bata Rumi Extract 5.0
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%)
Sekika Extract 5.0
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%)
Sennenken extract 3.0
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%)
Aletris farinosa extract 3.0
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 preservative Appropriate amount Perfume Appropriate amount

Claims (2)

At least one species selected from the group consisting of Pinus yunnanensis Franch., Bataguru ( Juglans cinerea ), Sekika ( Parmelia tinctorum Despre.), Sennenken ( Homalomena occulta (Lour.) Schott) and Aletris farinosa ( Aletris farinosa ) An endothelin action inhibitor containing an extract as an active ingredient. At least one species selected from the group consisting of Pinus yunnanensis Franch., Bataguru ( Juglans cinerea ), Sekika ( Parmelia tinctorum Despre.), Sennenken ( Homalomena occulta (Lour.) Schott) and Aletris farinosa ( Aletris farinosa ) A whitening agent containing an extract as an active ingredient.