JP2021195361A - β-GLUCOCEREBROSIDASE ACTIVATOR, CERAMIDE SYNTHESIS ACCELERATOR, AND SKIN EXTERNAL PREPARATION - Google Patents

Abstract

To improve a rough skin surface by a skin external preparation for promoting a ceramide synthetic amount by activating a β-glucocerebrosidase being an important enzyme which generates acyl ceramide required for lamella structure formation, since, a horny cell layer ceramide amount is reduced due to an external environment such as exposure to solar light such as ultraviolet light, excessive washing of a skin, and dryness, and an internal environment such as aging, resulting in a rough skin surface.SOLUTION: Inventors have found that, extracts activate β-glucocerebrosidase on an epidermal cell, the extracts are acquired from: jojoba, Saxifraga, Japanese medlar, Angelica, Withania Somnifera, gingko biloba, sour orange, Thymus vulgaris, Prunus persica, tea leaf, hamamelis, sage, aloe, harrow, rosemary, echinacea, nettle, horse chestnut, gardenia, oats, Kaffir lime, Arnica, black tea, rosa canina, quince, perforated sae lettuce, linden, watercress, jasmine, Lithosperumum root, Lamium album, Arctic wrack, rice bran, soybean, and tear grass, and the inventors have developed a skin external preparation including the above-mentioned extracts which promote an epidermal cell ceramide synthetic amount.SELECTED DRAWING: None

Description

The present invention has a composition that increases the amount of ceramide synthesis by activating an external skin preparation, that is, epidermal β-glucocerebrosidase, which improves rough skin caused by a decrease in the amount of ceramide in the stratum corneum and a decrease in the moisturizing barrier function. Regarding things.

The intercellular lipids in the stratum corneum play an important role in maintaining the moisturizing barrier function of the skin together with the sebaceous membrane and low molecular weight water-soluble substances. Ceramide, which accounts for about 50% of the intercellular lipids in the stratum corneum, is essential for forming a moisturizing barrier function. However, it is known that the amount of ceramide in the stratum corneum decreases due to the influence of sunlight exposure such as ultraviolet rays, excessive skin washing, and the influence of the internal environment such as aging and the external environment such as dryness. In fact, it is known that under such an environment, the water retention function of the stratum corneum is reduced and rough skin occurs.

As one of the measures to reduce the amount of ceramide described above, a method of promoting ceramide synthesis in the epidermis has been conventionally taken. In ceramide biosynthesis, there are many processes starting from the reaction of condensation of serine and palmitoyl-CoA. Β-Glucocerebrosidase during ceramide biosynthesis is an important enzyme for producing acyl ceramide and performing a sufficient epidermal barrier function. In fact, in transgenic mice genetically deficient in β-glucocerebrosidase activity, an incomplete lamellar structure of intercellular lipids in the stratum corneum was observed, and it was reported that transepidermal water evaporation was significantly increased. (Non-Patent Document 1). Therefore, it was considered that activation of β-glucocerebrosidase is essential for preventing deterioration of the barrier function and improving rough skin.

Patent Document 1 proposes a skin external preparation containing a specific yeast extract that activates β-glucocerebrosidase, and the material can enhance β-glucocerebrosidase mRNA expression in artificial skin. It is shown. However, although there is generally a tendency that the higher the RNA expression, the higher the expression of the protein showing enzyme activity, it cannot be said that there is a correlation between the amount of RNA and the amount of protein. By also evaluating the activity, it is considered that a more useful β-glucocerebrosidase activator can be found.

JP-A-2015-120643

WM Holleran et al., J Clin Invest. 93 (4): 1756-64 (1994)

Problems to be solved by the invention

The present invention has been made to provide a β-glucocerebrosidase activator, a ceramide synthesis promoter, and a skin external preparation containing these.

Means to solve problems

The present inventor conducted a test to see if β-glucocerebrosidase in epidermal cells was activated in about 150 samples, mainly using an extract of a natural material, and diligently investigated it.
As a result, hojoba, yukinoshita, biwa, angelica, ashwaganda, ginkgo, daidai, garden thyme, peach, cha, hamamelis, sage, aloe, magwa, rosemary, gardenia, irakusa, malonie, gardenia, karasumugi, kobumikan, arnica , Kaninabara, Marumero, Anaaosa, Shinanoki, Dutch ginkgo, Jasmine, Echinacea purpurea, White dead-nettle, Hibamata, Black tea, Soybean, and Hatomugi have been found to activate β-glucocerebrosidase in epidermal cells. It was also confirmed that an external skin preparation containing these β-glucocerebrosidase activators is useful for improving rough skin.

The invention's effect

Hereinafter, the present invention will be described in detail.
Since the present invention activates β-glucocerebrosidase in epidermal cells, daily skin care using an external skin agent containing β-glucocerebrosidase activator can be used to improve the internal environment such as aging. It is possible to improve the amount of ceramide in the stratum corneum, which has decreased due to the influence of the external environment such as ultraviolet rays and excessive washing, by promoting ceramide synthesis, and as a result, to improve rough skin.

The β-glucocerebrosidase activator in the present invention is specifically an extract derived from a natural material shown in Table 1 below. In addition, these extracts can be used alone or in combination of two or more as the β-glucocerebrosidase activator of the present invention, the ceramide synthesis promoter, and the external skin preparation containing them.

Parts of natural materials The parts of natural materials in the present invention are not limited to the parts shown in Table 1, and may be appropriately used for whole plants, above-ground parts, leaves, flowers, stems, rhizomes, seeds, roots, fruits and the like. can.

Extraction solvent In the present invention, when solvent extraction is performed, water, an appropriate organic solvent (alcohols, esters, acetones, alkanes, etc.) and a mixture of two or more thereof are used as the extraction solvent. For organic solvents, lower alcohols having 1 to 5 carbon atoms having a linear or branched chain, lower esters having 1 to 5 carbon atoms having a linear or branched chain, and 1 to 5 carbon atoms having a linear or branched chain. Lower acetone and alkane, which is a liquid at normal temperature and pressure, are used. Specific examples of the lower alcohol having a linear or branched chain and having 1 to 5 carbon atoms include methanol, ethanol, propanol, butanol, 1,3-butylene glycol, propylene glycol, glycerin and the like, and more preferably. , Methanol, ethanol. Specific examples of the lower ester having a linear or branched chain and having 1 to 5 carbon atoms include methyl acetate, ethyl acetate and the like, and more preferably ethyl acetate. Examples of the lower acetone having a linear or branched chain and having 1 to 5 carbon atoms include methyl ethyl ketone, acetone and the like, and more preferably acetone. Examples of alkanes that are liquid at normal temperature and pressure include hexane. In the present invention, when the solvent extract extracted with the solvent is used as it is, it is preferable and more preferable to select and use one of these solvents which is also used as a food in consideration of safety. Is water, ethanol and mixtures thereof.

The amount of the extraction solvent added can be appropriately determined in accordance with the amount of the plant substance, the extraction temperature, and the like. Further, the solvent extraction may be performed at normal temperature and under normal pressure, and the extraction temperature can be appropriately determined in consideration of the boiling point of each solvent and the like.

In the present invention, in the case of solvent extraction, the weight ratio of the drug substance to the solvent is 1:20 to 1: 1, preferably 1:15 to 1: 5, and more preferably 1:10. be. Further, the temperature at the time of heating at the time of extraction is about 100 ° C. or lower from normal temperature (15 to 25 ° C.).

In the present invention, it is not always necessary to carry out solvent extraction, and liquid juice or oil obtained by squeezing from a natural material may be used as the extract.

Concentration According to another aspect of the present invention, an extract obtained by removing the solvent from the solvent extract can be obtained. As a method for removing the solvent, generally, vacuum concentration, heat concentration, ventilation concentration, freezing concentration, spray concentration, and other concentration methods, or a mixing method thereof can be used. The pressure, temperature, wind power, spray, etc. at the time of concentration can be appropriately set according to the amount of the obtained extract and the intended use. In the present invention, vacuum concentration is preferably used. When concentrating under reduced pressure, the pressure and temperature can be appropriately set according to the amount of the obtained concentrate and the intended use.

The extract used in the present invention may be used as it is, or may be filtered and concentrated as needed. Further, the extract can be fractionated and purified by a column chromatography method, a countercurrent distribution method, or the like. Further, the above-mentioned products may be dried under reduced pressure or freeze-dried, then prepared into powder or paste, and appropriately formulated and used.

Drying / Drying According to another aspect of the present invention, the extract or the extract from which the solvent has been removed can be dried to obtain a dried product. In the present invention, the drying method is sun drying, (hot) air drying, vacuum drying, air drying, fluid drying, spray drying, freeze drying, vacuum drying, infrared drying, high frequency drying, and other drying methods, or these. Mixing method is used. Drying may be performed at the same time as the above-mentioned concentration.

External skin preparation The composition according to the present invention is suitable for blending in external skin preparations because it is excellent in usability and safety when applied to the skin. This external skin preparation is specifically a skin external preparation containing an extract derived from a natural material centered on a plant. Examples of the type of external skin preparation include various ones such as cream, lotion, milky lotion, lotion, cleansing gel, facial mask, and ointment. In the present invention, the amount of the extract derived from a general natural material added is about 0.0001% by weight or more and 20% by weight or less with respect to the total weight of the external skin preparation. When the extract is liquid juice or oil, it is about 0.0001% by weight or more and 100% by weight (the extract as it is) with respect to the total weight of the external skin preparation, while considering safety and the like. Use) The following.
When the extract composition according to the present invention is used as it is, the concentration of the extract is not limited as long as the content of the dry solid content as a solute is within the above range.

The external preparation for skin of the present invention can be prepared by blending with various forms of bases known as ordinary external preparations for skin, and the form of the external preparation is not particularly limited, for example, milky lotion, cream, and lotion. Any dosage form such as can be selected. Further, the present invention is not limited to general cosmetics, but includes pharmaceuticals, quasi-drugs, and medicated cosmetics.

Among the skin external preparations of the present invention, those that can be used as constituents together with the extract derived from the natural material described in [0007] are, for example, moisturizers, ultraviolet absorbers, complex lipids, substances having an active oxygen scavenging effect, and the like. Anti-inflammatory agents, vitamins and their derivatives, oily ingredients, surfactants, preservatives, powder ingredients, purified water, polymer compounds, gelling agents, antioxidants, cholesterols, plant sterols, lipoproteins, microorganisms Derived ingredients, algae extracts, blood circulation promoters, antilipid leaking agents, thickeners, coloring agents, beauty ingredients and the like can be mentioned, and can be appropriately selected and used as long as the effects of the present invention are not impaired.

Next, the present invention will be described in more detail with reference to Examples and Comparative Examples, but the present invention is not limited thereto. Further, the extraction method for the extract derived from the natural material used is not limited in any way.

<Production example 1 of an extract derived from a natural material> Production method of jojoba seed oil Extract 1 (vegetable oil) is obtained by squeezing crude jojoba oil from dried jojoba seeds by a pressing method, decolorizing it with active white clay, and then distilling and deodorizing it. Got

<Production example 2 of extract derived from natural material> Production method of hydrous ethanol extract Add 200 g of 50% (w / w) ethanol, which is 20 times the amount, to 10 g of natural material in the part shown in Table 1, and extract at 50 ° C. for 5 hours. After filtration and concentration, the mixture was freeze-dried and pulverized to obtain an extract (extracts 2 to 24, 27 to 29, 31 to 36).

<Production example 3 of extract derived from natural material> Production method of water extract Add 200 g of purified water in an amount of 20 times to 10 g of the natural material in the part shown in Table 1, extract at 50 ° C. for 5 hours, filter and concentrate, freeze-dry, and powder. The extract was obtained (extracts 25 to 26).

<Production Example 4 of Extract Derived from Natural Material> Method for Producing Arabian jasmine (Jasmine) Extract 5 L of hexane was added to 500 g of Arabian jasmine flowers, and the mixture was extracted at room temperature for 5 days. After solid-liquid separation, hexane was distilled off and ethanol was added to the concentrate. 1,3-Butylenglucol was added to the ethanol-soluble portion, and the mixture was allowed to stand in a refrigerator and then filtered to obtain an extract (extract 30).

<Production example 5 of extract derived from natural material> Method of producing Coix seed extract 1000 g of 1,3-butylene glycol and 1000 g of purified water are added to 100 g of crude seeds of Coix seeds excluding dried seed coat, and 2 at room temperature. Extraction was carried out with stirring for days, and the mixture was filtered to obtain 1500 g of an extract (extract 37).

(Test Example 1) Evaluation test of β-glucocerebrosidase activity of epidermal cells In this section, epidermal cells are used as an index of whether an extract derived from a natural material promotes β-glucocerebrosidase activation in human skin. The test was carried out. This test is a test on whether the β-glucocerebrosidase activity of epidermal cells is increased by culturing in a medium containing an extract derived from a natural material.
As the test sample, the extracts 1 to 36 prepared in Production Examples 1 to 4 were used. Human immortalized epidermal cells (HaCaT cells) were cultured and maintained in DMEM medium containing 5% fetal bovine serum (hereinafter abbreviated as FBS), and 1.5 × 10 ⁵ cells were placed in a 24-well plate using DMEM medium at the time of passage. / Holes were sown and cultured at 37 ° C. under 5% carbon dioxide for 24 hours.
The test sample was added as follows.
(Extract 1, positive control, enzyme inhibitor)
Galactosylceramide (Fujifilm Wako Pure Chemical Industries, Ltd.) was used as a positive control, and conduritol-B-epoxide (Sigma Aldrich) was used as a specific inhibitor of β-glucocerebrosidase. The sample was dissolved in DMEM medium and cultured under the same conditions for 48 hours in a filter-sterilized medium. The control cells containing no sample were cultured only in the medium under the same conditions.
(Extracts 2-36 excluding extract 30) The test sample was dissolved in purified water, replaced with DMEM medium supplemented with a filter-sterilized solution, and cultured under the same conditions for 48 hours. For the control cells containing no extract, purified water (sterilized water) sterilized by a filter was replaced with DMEM medium to which the same amount as above was added, and the cells were cultured under the same conditions.
(Extract 30)
The extract 30 was replaced with DMEM medium containing 0.1% (v / v), and the cells were cultured under the same conditions for 48 hours. For control cells containing no extract, replace with DMEM medium containing ethanol and 1,3-butylene glycol (ethanol / 1,3-butylene glycol = 1/5) in an amount equivalent to that of extract 30. It was cultured under the same conditions. After culturing for 48 hours, the cells were washed once with PBS (-), cells were lysed by adding PBS (-) containing 0.5% Triton X-100, and then the supernatant (crude enzyme solution) was placed on two 96-well plates. ) Was fractionated, and β-glucocerebrosidase activity was evaluated and protein was quantified. Protein quantification was performed using the BCA Protein Assay kit (Thermo Fisher Scientific). For the measurement of β-glucocerebrosidase activity, 4-methylumbelliferone-β-D-glucopyranoside (Sigma Aldrich) was reacted with the supernatant at 37 ° C. for 1 hour, and the concentration of 4-methylumbelliferone generated after the reaction was stopped was measured. Fluorescence intensity was measured and carried out. The 4-methylumbelliferone (nmol) / Protein (μg) value was calculated, and the activity (%) of the test sample when the control cell value was 100% was calculated and compared. The results are shown in Table 2.

As shown in Table 2, in epidermal cells cultured in a medium containing extracts derived from each natural material (extracts 1-36), β-glucocerebrosidase enzyme activity is the enzymatic activity of control cells containing no extract. It was confirmed that the increase was 5% or more.
In addition, in the positive control galactosylceramide, the β-glucocerebrosidase activity was increased, and the enzyme activity was inhibited by the enzyme inhibitor conduritol-B-epoxide. Therefore, the β-glucocerebrosidase activity was determined in this test. It was also confirmed that it can be evaluated appropriately.

(Test Example 2) Application test of external skin preparation An additional test was conducted on the Coix seed (Yokuinin) extract that showed the highest β-glucocerebrosidase enzyme activation in Test Example 1.
Twenty women aged 30 to 60 years who are aware of rough skin on a daily basis are used as a panel, and the number of external skin agents (Example 1) shown in Table 3 containing the extract 37 (Coix seed extract) is increased to 10 people. It was distributed, and the comparative skin external preparation was distributed to the remaining 10 people. In the winter season (January-March), I had my face applied with an appropriate amount of the external study agent after washing my face twice daily in the morning and at night for 12 weeks. The results of the questionnaire regarding items such as inflammation (improvement of rough skin) and dryness (improvement of moisturizing power) due to application were evaluated according to the following criteria, and are shown in Table 4.
Evaluation Criteria Evaluation ◎: Remarkable effect was observed (60% of all panelists exceeded)
Evaluation ○: A clear effect was observed (over 40% of all panelists and 60% or less).
Evaluation △: Some effect was observed (20% more than 40% or less of all panelists)
Evaluation ×: No effect was observed (0% to 20% of all panelists)

As shown in Table 4, it was confirmed that the external skin preparation of Example 1 containing the extract derived from the natural material alleviated the rough skin symptom by applying it for a long period of time.

An example of blending an extract derived from a natural material into a skin external preparation according to the present invention is shown below. In addition, the compounding example does not limit the present invention.

(Formulation Example 1) Cream The following components (1) to (10) and separately the following components (11) to (15) and (17) are heated and dissolved at 75 ° C., emulsified, and stirred while stirring 50. The cream was adjusted by cooling to ° C. and adding component (16).
(Ingredient) (% by weight)
(1) Extract 1 (jojoba seed oil) 3.0%
(2) Squalane 2.0%
(3) Methylpolysiloxane 0.5%
(4) Stearyl alcohol 0.5%
(5) Cetyl alcohol 0.5%
(6) Tri (caprylic capric acid) glyceryl 12.5%
(7) Glyceryl monostearate 5.0%
(8) Diglyceryl monostearate 1.5%
(9) Decaglyceryl monostearate 3.0%
(10) Propyl paraoxybenzoate 0.1%
(11) Xanthan gum 0.1%
(12) Extract 37 (Coix seed extract) 0.5%
(13) 1,3-butylene glycol 2.5%
(14) Methyl paraoxybenzoate 0.2%
(15) Fragrance 0.1%
(16) Remaining amount of purified water

(Mixing Example 2) Toner Mixing and dissolving the following components (7) to (9) to obtain liquid A, and separately mixing and dissolving the following components (1) to (6) and (10) to obtain liquid B. , Liquid A and liquid B were uniformly mixed to obtain a lotion.
(Ingredient) (% by weight)
(1) Glycerin 5.0%
(2) 1,3-butylene glycol 5.0%
(3) Melon placenta extract 0.5%
(4) Pomegranate flower extract 0.1%
(5) Extract 37 (Coix seed extract) 0.5%
(6) Polyoxyethylene sorbitan lauric acid ester 1.2%
(7) Ethyl alcohol 5.0%
(8) Phenoxyethanol 0.1%
(9) Fragrance 0.1%
(10) Remaining amount of purified water

(Formulation Example 3) Emulsion The following components (1) to (10) and separately the following components (11) to (14) and (16) are heated and dissolved at 75 ° C. to obtain solutions A and B, respectively. Liquid B was added to the liquid to emulsify, cooled to 50 ° C. with stirring, and component (15) was added to prepare a milky lotion.
(Ingredient) (% by weight)
(1) Extract 1 (jojoba seed oil) 1.0%
(2) Squalane 2.0%
(3) Behenyl alcohol 1.0%
(4) Tri (caprylic capric acid) glyceryl 2.0%
(5) Tetraglycerin condensed ricinoleic acid 0.1%
(6) Propylene glycol monooleate 0.5%
(7) Glyceryl monostearate 1.0%
(8) Hexaglyceryl monomyristate 1.0%
(9) Decaglyceryl monomyristate 0.5%
(10) Propyl paraoxybenzoate 0.1%
(11) Extract 30 (Arabian jasmine flower extract) 0.1%
(12) Pleurotus cornucopiae extract 0.2%
(13) 1,3-butylene glycol 3.0%
(14) Methyl paraoxybenzoate 0.1%
(15) Fragrance 0.1%
(16) Remaining amount of purified water

(Formulation Example 4) Cleansing Gel The following components (1) to (3) and separately the following components (4) to (7) and (9) are heated and dissolved at 70 ° C. to obtain solutions A and B, respectively. Add solution B to solution A and stir until uniform. The mixture was cooled to 50 ° C. with stirring, and the component (8) was added to prepare a cleansing gel.
(Ingredient) (% by weight)
(1) Hexaglyceryl monomyristate 20.0%
(2) Liquid paraffin 59.7%
(3) Paraoxybenzoic acid ester 0.3%
(4) Extract 30 (Arabian jasmine flower extract) 0.1%
(5) Jojoba leaf extract 0.1%
(6) Concentrated glycerin 5.0%
(7) Sorbitol 5.0%
(8) Fragrance 0.1%
(9) Remaining amount of purified water

(Formulation Example 5) Packing agent The following components (1) to (6) and (11) are heated and dissolved at 70 ° C. to prepare a liquid A, and (8) to (10) are mixed to obtain a liquid B. After the liquid B was mixed with the liquid A, it was cooled and (7) was uniformly dispersed to prepare a pack.
(Ingredient) (% by weight)
(1) Polyvinyl alcohol 20.0%
(2) Glycerin 5.0%
(3) Kaolin 6.0%
(4) Extract 37 (Coix seed extract) 0.5%
(5) Bergenia ligratha root extract 0.1%
(6) Rose-apple leaf extract 0.1%
(7) Benzalkonium chloride 20.0%
(8) Ethanol 20.0%
(9) Methyl paraoxybenzoate 0.2%
(10) Fragrance 0.1%
(11) Remaining amount of purified water

In the present invention, it was confirmed that the extracts derived from a plurality of natural materials are an activator of epidermal β-glucocerebrosidase and a ceramide synthesis promoter. It has been found that a skin external preparation containing these extracts has an effect of improving rough skin symptoms caused by a decrease in the amount of ceramide due to an internal environment such as aging and an external environment such as sunlight and dry conditions.

Claims (3)

Johova, Yukinoshita, Biwa, Angelica, Ashwaganda, Ginkgo, Daidai, Tachijakousou, Peach, Cha, Hamamelis, Sage, Aloe, Magwa, Rosemary, Echinacea, Nettle, Maronie, Gardenia, Karasumugi, Kobumikan, Arnica, Tea Β-Glucocerebros characterized by containing one or more extracts selected from the extracts obtained from ginkgo biloba, anaaosa, shinanoki, ginkgo biloba, jasmine, murasaki, white dead-nettle, hibamata, black tea, soybean, and sage. Celebrosis activator. Jojoba, Yukinoshita, Biwa, Angelica, Ashwaganda, Ginkgo, Daidai, Garden Thyme, Peach, Cha, Hamamelis, Sage, Aloe, Magwa, Rosemary, Gardenia, Nettle, Maronie, Gardenia, Karasumugi, Kobumikan, Arnica, Tea Promotion of ceramide synthesis, characterized by containing one or more extracts selected from the extracts obtained from Marumero, Anaaosa, Shinanoki, Dutch nettle, Jasmine, Murasaki, White dead-nettle, Jojoba, Black tea, Soybean, and Hatomugi. Agent. An external skin preparation comprising the β-glucocerebrosidase activator according to claim 1 or the ceramide synthesis promoter according to claim 2.