JP7337341B2 - Method for producing (4E,8E,2S,3R,2'R)-N2'-hydroxypalmityl-l-O-β-D-glucopyranosyl-9-methyl-4,8-sphingadienine and use thereof - Google Patents

Description

The present invention provides a method for producing (4E,8E,2S,3R,2'R)-N2'-hydroxypalmityl-lO-β-D-glucopyranosyl-9-methyl-4,8-sphingadienine and Regarding use .

The stratum corneum of the skin suppresses evaporation of water from the body and has a barrier function to prevent stimulation from the outside.

It is known that when this barrier function is impaired by ultraviolet rays, surfactants, dryness, mechanical irritation, active oxygen, residual chlorine in tap water, etc., dry skin, atopic dermatitis, rough skin, etc. are induced. there is

Various formulations have been studied so far to prevent dryness of the skin and moisturize the skin.

For example, petrolatum formulations, heparin formulations, and urea formulations are expected to have the effect of suppressing water evaporation and moisturizing the skin when applied to the target area (see, for example, Patent Document 1).

Makoto Kawashima, 2 others, ``Usefulness of moisturizing agents for skin physiological dysfunction in patients with atopic dermatitis'', Journal of the Japanese Dermatological Association 117(6), 969-977, 2007

As described above, non-polar oils such as liquid paraffin and petrolatum, and various topical preparations are thought to have the effect of suppressing moisture evaporation from the skin and moisturizing it, and have been widely used as the core ingredients of ointment bases. Ta.

However, while skin care using topical preparations is important, it would be even more desirable if the skin could be moisturized by actions from within the body through eating and drinking in daily eating habits.

The present invention has been made in view of such circumstances, and includes (4E,8E,2S,3R,2'R)-N2'-hydroxypalmityl-lO-β-D-glucopyranosyl contained in enokitake mushrooms. A method for purifying -9-methyl-4,8-sphingadienine is provided .

Furthermore, in the present invention , (4E,8E,2S,3R,2'R)-N2'-hydroxypalmityl-lO-β-D-glucopyranosyl-9-methyl-4,8 is used as a cosmetic component. - Uses of sphingadienin are also provided.

(4E,8E,2S,3R,2'R)-N2'-hydroxypalmityl-lO-β-D-glucopyranosyl-9-methyl-4,8-sphingadie contained in Enokitake according to the present invention. In the method for purifying nin , a step of dissolving and/or suspending a chloroform-extracted dried product of dried enokitake mushroom in 3 parts by volume of a 50 ± 1 v/v% methanol aqueous solution to obtain a crudely purified concentrate, and equilibrating 1 volume A step of applying the crude purified concentrate to a first column with a reversed-phase solid phase as a packing material, and applying 2 to 4 parts by volume of an 80 ± 5 v / v% methanol aqueous solution to the first column and rinsing the packing material of the first column with 1 to 2 parts by volume of a 95-100v/v% methanol aqueous solution or methanol, adding water to the resulting rinse solution and methanol A step of obtaining 3 parts by volume of the primary purified liquid with a final concentration of 50 ± 1 v / v%, and a second column packed with 1 part by volume of the equilibrated reversed-phase solid phase , the primary purified liquid and rinsing the packing material of the second column with 1 ± 0.25 parts by volume of 83 ± 1 v / v% methanol aqueous solution, and 2 to 4 parts by volume of propanol. (4E,8E,2S,3R,2'R)-N2'-hydroxypalmityl-lO-β-D-glucopyranosyl-9-methyl-4,8- and a step of obtaining a semi-purified product having an increased sphingadienin content.

In the present invention , (4E,8E,2S,3R,2'R)-N2'-hydroxypalmityl-lO-β-D-glucopyranosyl-9-methyl-4,8- It was decided to use sphingadienine.

In addition , the component is characterized by being an active ingredient for the purpose of including moisturizing of the skin as an action or effect.

(4E,8E,2S,3R,2'R)-N2'-hydroxypalmityl-lO-β-D-glucopyranosyl-9-methyl-4,8-sphingadienin contained in Enokitake according to the present invention In the purification method of , a step of dissolving and / or suspending the chloroform-extracted dry matter of dried enokitake in 3 parts by volume of a 50 ± 1 v / v% methanol aqueous solution to obtain a crude purified concentrate, and equilibrating 1 part by volume A step of applying the crude purified concentrate to a first column packed with a reversed-phase solid phase, and filling the first column with 2 to 4 volume parts of 80 ± 5 v / v% methanol aqueous solution. A step of rinsing the agent, rinsing the packing material of the first column with 1 to 2 parts by volume of a 95 to 100v/v% methanol aqueous solution or methanol, and adding water to the resulting rinse solution to complete the methanol A step of obtaining 3 parts by volume of a primary purified liquid with a concentration of 50 ± 1 v / v%, and applying the primary purified liquid to a second column packed with 1 part by volume of an equilibrated reversed-phase solid phase. rinsing the packing material of the second column with 1 ± 0.25 parts by volume of 83 ± 1 v/v% methanol aqueous solution; and packing the second column with 2 to 4 parts by volume of propanol. (4E,8E,2S,3R,2'R)-N2'-hydroxypalmityl-lO-β-D-glucopyranosyl-9-methyl-4,8-sphinga and a step of producing a semi-purified product with an increased dienine content ratio, so that the yield is good and the contamination of contaminant components is suppressed as much as possible. 2S,3R,2'R)-N2'-Hydroxypalmityl-lO-β-D-glucopyranosyl-9-methyl-4,8-sphingadienine enriched semipurified product can be obtained. can.

Further, in the present invention, (4E,8E,2S,3R,2'R)-N2'-hydroxypalmityl-l-O-β-D-glucopyranosyl-9-methyl-4,8-su Since fingadienin is used, it is possible to impart a skin moisturizing effect to the cosmetic.

In addition, if the constituent component is an active ingredient for the purpose of including moisturizing of the skin as an action or effect, the cosmetic can achieve a moisturizing action or effect of the skin.

FIG. 4 is an explanatory diagram showing spots of each fraction on a TLC plate. FIG. 4 is an explanatory diagram showing spots of each fraction on a TLC plate. FIG. 4 is an explanatory diagram showing spots of each fraction on a TLC plate. FIG. 2 is an explanatory diagram showing the spots and structure of ENH-1. FIG. 4 is an explanatory diagram showing spots and structures of ENH-4 and ENH-5. FIG. 3 is an explanatory diagram showing spots and structures of ENH-6, ENH-7, and ENH-8. FIG. 3 is an explanatory diagram showing spots and structure of ENH-12. FIG. 4 is an explanatory diagram showing spots and structures of ENE-1 and ENE-2. FIG. 2 is an explanatory diagram showing changes in expression of the CERS2 gene. FIG. 3 is an explanatory diagram showing changes in expression of the CERS3 gene. FIG. 3 is an explanatory diagram showing changes in expression of the CERS4 gene. FIG. 4 is an explanatory diagram showing cell viability 72 hours after addition of HPGMS. FIG. 4 is an explanatory diagram showing the amount of collagen produced 72 hours after the addition of HPGMS. FIG. 4 is an explanatory diagram showing the amount of hyaluronic acid produced 72 hours after the addition of HPGMS. A chromatogram obtained by HPLC analysis of a semi-purified product.

TECHNICAL FIELD The present invention relates to a functional food for moisturizing the skin, and in particular, to provide a functional food for moisturizing the skin, which can exert an excellent moisturizing effect on the skin when taken orally.

で示される(4E,8E,2S,3R,2'R)-N2'-ヒドロキシパルミチル-l-O-β-D-グルコピラノシル-9-メチル-4,8-スフィンガジエニン（(4E,8E,2S,3R,2'R)-N2'-hydroxypalmityl-l-O-β-D-glucopyranosyl-9-methyl-4,8-sphingadienine：以下、HPGMSともいう。）を含有する点で特徴的である。 In particular, in the functional food for moisturizing skin according to this embodiment, the following structural formula (I):

(4E,8E,2S,3R,2'R)-N2'-hydroxypalmityl-lO-β-D-glucopyranosyl-9-methyl-4,8-sphingadienine ((4E,8E, 2S,3R,2'R)-N2'-hydroxypalmityl-lO-β-D-glucopyranosyl-9-methyl-4,8-sphingadienine: hereinafter also referred to as HPGMS).

The HPGMS contained in the skin-moisturizing functional food according to this embodiment may be artificially added to the skin-moisturizing functional food, or may be naturally occurring.

The HPGMS to be added to the functional food for moisturizing the skin may be, for example, a food material containing HPGMS that has been subjected to a predetermined process as necessary, or extracted from a food material containing HPGMS or a predetermined material that is not a food material. It may be an extract extracted through a process.

In addition, functional foods for skin moisturizing that naturally contain HPGMS should be interpreted as cases where part or all of living organisms such as fungi, plants, and animals that produce HPGMS are used as functional foods for skin moisturizing. can be done.

In addition, regardless of whether it is artificially added or naturally occurring, HPGMS contained in functional foods for moisturizing skin can be derived from enokitake, for example.

The present inventors have so far conducted intensive research focusing on bioactive components possessed by various plants and mushrooms, and have discovered for the first time that HPGMS is contained in enokitake mushrooms.

In addition, as will be described later with reference to data, etc., it was clarified that HPGMS derived from Enoki mushroom can exhibit excellent skin moisturizing effect by oral ingestion.

In addition, Enokitake, which is the origin of HPGMS, can be, more limitedly, Ohki Shirayuki No. 919. Oki Shirayuki No. 919 contains a large amount of HPGMS compared to conventional enokitake, and is suitable for processing and extraction. The amount of intake necessary for exhibiting a moisturizing effect on the skin can be set to a practically ingestible amount, for example, an amount of about 125 g or less in terms of the weight of fresh food. This does not preclude taking more than 125g of Oki Shirayuki No. 919 for the purpose of moisturizing the skin. , 200g, 300g, etc., even if you take more than the aforementioned 125g, you can naturally expect a moisturizing effect.

In addition, in order to exert the moisturizing effect of the skin, it is necessary to orally ingest HPGMS in an amount of approximately 1 mg or more, more effectively 1.2 mg or more per day. If No. 919 itself is a functional food for moisturizing the skin, it is sufficient to orally ingest about 90 g of fresh food weight (85% moisture) to ingest 1 mg of HPGMS. As a result, it is possible to easily adjust the condition of the skin within the range of reasonable intake.

Regarding the skin moisturizing functional food according to the present embodiment, other than when Shirayuki Oki No. 919 itself is used as a skin moisturizing functional food, examples thereof include, for example, supplements, beverages, puddings and jelly, and processing. It can be provided in various forms such as foods, cooked products, and so-called cooking forms. As an example, if the dish is lightly boiled in boiling water and then cooked and eaten like somen noodles in a dashi stock, it is delicious and goes down smoothly, and Oki Shirayuki 919 can be easily and deliciously ingested in an amount equivalent to 1 mg of HPGMS. .

In addition, if it is a supplement-like, the dosage form is not particularly limited, and any dosage form such as tablets, capsules, fine granules, pills, lozenges, liquids, and jelly-like can be selected. can be done.

In addition to HPGMS as an active ingredient, the skin-moisturizing functional food according to the present embodiment can also contain excipients, additives, auxiliary ingredients, and the like depending on the dosage form.

Examples of excipients for solid formulations include lactose, crystalline cellulose, and starch.

Examples of additives include stabilizers, surfactants, solubilizers, plasticizers, sweeteners, antioxidants, flavoring agents, coloring agents, preservatives, inorganic fillers, and the like.

Examples of auxiliary ingredients include ingredients that impart further functions to the functional food for moisturizing the skin and enhance the functions of the active ingredients. It can be a component included in the number.

にて示される4,4-ジメチル-5a-エルゴスタ-8,24(28)-ジエン-3β-オール（4,4-dimethyl-5a-ergosta-8,24(28)-dien-3β-o1）を添加した場合、抗菌作用や抗アレルギーの効果、丈夫な骨をつくる働きの効果などが期待される。 For example, structural formula (II):

4,4-dimethyl-5a-ergosta-8,24(28)-dien-3β-ol (4,4-dimethyl-5a-ergosta-8,24(28)-dien-3β-o1) represented by When added, it is expected to have antibacterial and anti-allergic effects, as well as the ability to build strong bones.

にて示されるエルゴステロール(ergosterol)を添加した場合、抗菌効果や丈夫な骨をつくる働きの効果が期待される。 and the following structural formula (III):

When ergosterol shown in is added, it is expected to have an antibacterial effect and an effect of working to create strong bones.

にて示されるエルゴステロールペルオキシド(ergosterol peroxide)を添加した場合、美白効果や丈夫な骨をつくる働きの効果が期待される。 In addition, the following structural formula (IV):

When ergosterol peroxide shown in is added, it is expected to have a whitening effect and a strong bone-building effect.

にて示される2-ヒドロキシ-テトラコサン酸(2,3-ジヒドロキシ-1-ヒドロキシメチル-ヘプタデック-7-エニル)-アミド(2-hydroxy-tetracosanoic acid(2,3-dihydroxy-1-hydroxymethyl-heptadec-7-enyl)-amide)、所謂Trolliamideを添加した場合、保湿効果が期待される。 In addition, the following structural formula (V):

2-Hydroxy-tetracosanoic acid (2,3-dihydroxy-1-hydroxymethyl-heptadec-7-enyl)-amide (2-hydroxy-tetracosanoic acid (2,3-dihydroxy-1-hydroxymethyl-heptadec- 7-enyl)-amide), so-called Trolliamide, is expected to have a moisturizing effect.

にて示されるセレビステロール(Cerevisterol)を添加した場合、抗菌の効果が期待される。 In addition, the following structural formula (VI):

When cerevisterol shown in is added, an antibacterial effect is expected.

にて示されるエルゴステロールペルオキシドグルコシド(ergosterol peroxide glucoside)を添加した場合、美白効果や丈夫な骨をつくる働きの効果が期待される。 In addition, the following structural formula (VII):

When ergosterol peroxide glucoside shown in is added, it is expected to have a whitening effect and a function to build strong bones.

にて示されるカタセレブロシドA(Catacerebroside A)を添加した場合、保湿効果が期待される。 Further, the following structural formula (VIII):

Moisturizing effect is expected when Catacerebroside A shown in is added.

にて示されるエルゴスト-7-エン-3,5,6-トリオール(Ergost-7-ene-3,5,6-triol)を添加した場合、抗菌効果や丈夫な骨をつくる働きの効果が期待される。 In addition, the following structural formula (IX):

When Ergost-7-ene-3,5,6-triol shown in is added, antibacterial effects and strong bone-building effects are expected. be done.

にて示されるケイ皮酸(cinnamic acid)を添加した場合、抗酸化効果が期待される。 In addition, the following structural formula (X):

When cinnamic acid shown in is added, an antioxidant effect is expected.

にて示されるパラヒドロキシ安息香酸(p-hydroxy benzoic acid)を添加した場合、抗菌・抗酸化効果が期待される。 In addition, the following structural formula (XI):

Antibacterial and antioxidant effects are expected when p-hydroxy benzoic acid shown in is added.

The present application also proposes the use of Enokitake mushroom HPGMS as a functional food for skin moisturizing. The skin-moisturizing functional food may be a fresh Enokitake mushroom containing HPGMS as described above, or may be a processed food obtained by drying and pulverizing such Enokitake, or containing HPGMS derived from Enokitake. Food may be used.

Although enokitake mushrooms are not particularly limited, enokitake mushrooms containing 60 µg/g or more of HPGMS in a dry state are extremely efficient in the production of skin-moisturizing functional foods. In particular, when the skin-moisturizing functional food is fresh enokitake mushrooms containing HPGMS or processed foods made by drying and powdering enokitake mushrooms, as mentioned above, excessive intake should be considered in consideration of the balance with other ingredients. It can be said that Oki Shirayuki No. 919 is particularly suitable for this application.

The present application also provides a method for purifying HPGMS from Enokitake. In the purification method according to the present embodiment, a step of dissolving and / or suspending the chloroform-extracted dry matter of dried enokitake mushroom in 3 parts by volume of a 50 ± 1 v / v% methanol aqueous solution to obtain a crudely purified concentrated liquid, and equilibrating A step of applying the crude purified concentrate to a first column packed with 1 part by volume of InertSep (registered trademark) Pharma, and applying 2 to 4 parts by volume of 80 ± 5 v / v% methanol aqueous solution to the first column. A step of rinsing the packing material of the column in 1, rinsing the packing material of the first column with 1 to 2 parts by volume of a 95-100v/v% methanol aqueous solution or methanol, and adding water to the resulting rinse solution. In addition, a step of obtaining 3 parts by volume of a primary purified liquid with a final methanol concentration of 50 ± 1 v / v%, and a second column packed with 1 part of equilibrated InertSep (registered trademark) Pharma, A step of applying the primary purified liquid, a step of rinsing the packing material of the second column with 1 ± 0.25 parts by volume of 83 ± 1 v / v% methanol aqueous solution, and a step of rinsing the packing material of the second column with 2 to 4 parts by volume of propanol. The step of rinsing the packing material of the second column and making the resulting rinse liquid into a semi-purified product with an increased HPGMS content ratio is performed.

According to such a purification method, it is possible to obtain a semi-purified product in which the content of HPGMS contained in enokitake mushrooms is increased while suppressing the contamination of contaminant components as much as possible with a good yield.

In addition, the semi-purified product obtained in this way has relatively good removal of most of the sugars contained in enokitake mushrooms, and is extremely advantageous for measuring the content of HPGMS and the like.

Specifically, enokitake mushrooms contain an extremely large variety of sugars, and when liquid chromatography (LC) techniques are used to measure the amount of HPGMS contained in enokitake mushrooms, the The signal-to-noise ratio was poor (small), and analysis was difficult without the use of an instrument capable of extremely high-precision detection, such as LC/MS/MS.

On the other hand, if HPGMS is purified by the above-described purification method, the SN ratio is dramatically improved, and analysis can be easily performed even with general HPLC.

That is, the above-described purification method is a method for purifying HPGMS from enokitake mushrooms to be added to the functional food for moisturizing skin according to the present embodiment, and is also a purification method for analyzing HPGMS derived from enokitake mushrooms. is also extremely useful.

The present application also provides an external preparation for skin. The external preparation for skin according to this embodiment is characteristic in that it contains HPGMS. In the present application, external preparations for skin refer to pharmaceuticals, quasi-drugs, and cosmetics (including medicated cosmetics). included.

In addition, the present application also provides a cosmetic, which is one aspect of this external preparation for skin. That is, the cosmetic according to this embodiment is characteristic in that it contains HPGMS.

In the present application, cosmetics include not only so-called makeup cosmetics, but also basic cosmetics, hair tonics, perfumes, toothpastes, shampoos, rinses, soaps and cleansers used for washing the body, and toiletry products such as bath agents. It also includes medicinal cosmetics claiming preventive effects.

Examples of makeup cosmetics include foundations, eyebrows, mascara, eye shadows, eyeliners, lipsticks, glosses, cheeks, face powders, nail polishes, etc. Basic cosmetics include lotions, milky lotions, Facial cleansers, cleansers, serums, creams and the like can be mentioned. It should be noted that the above descriptions of external preparations for skin, cosmetics, and the like are examples of corresponding articles, etc., in order to facilitate understanding of the present invention, and the interpretation of each term is based on the articles, etc., listed above. It is not limited. However, it does not preclude the applicant from limiting the present invention to these articles and the like in obtaining the right of the present application.

The present application also provides a method of using HPGMS as a cosmetic component. Various cosmetics having a moisturizing effect have been proposed, but no cosmetics containing HPGMS as a constituent have been proposed so far.

In addition, it is significant to propose a new ingredient capable of inducing a moisturizing action or effect to a person who is allergic to a given moisturizing ingredient.

In particular, when the above constituents are used as active ingredients for the purpose of moisturizing the skin as an action or effect, it is expected that daily skin care will improve the skin quality. can.

The present application also provides a method of using enokitake mushrooms containing 60 µg/g or more of HPGMS in a dry state as an HPGMS extraction material for producing a skin external preparation containing HPGMS as a component.

It has not been known until now that HPGMS is contained in enokitake mushrooms, and the present application proposes for the first time that enokitake mushrooms can be used as an HPGMS extraction material for the production of external preparations for skin.

However, the amount of HPGMS contained in ordinary enokitake mushrooms is very small, and a considerable amount of enokitake mushrooms is required in order to produce a skin preparation for external use containing HPGMS capable of exhibiting excellent skin moisturizing effects.

Of course, this does not preclude the use of ordinary enokitake mushrooms as an HPGMS extraction material, but in this regard, enokitake mushrooms containing 60 µg/g or more of HPGMS such as Ohki Shirayuki No. 919 can be used for the production of HPGMS-containing external skin preparations. When used as an extraction material for HPGMS, HPGMS can be efficiently extracted from safe ingredients that have been popular as foods for many years.

In addition, the concentration of HPGMS can be appropriately adjusted according to the use in the functional foods, cosmetics, and external skin preparations according to the present embodiment, and when used in these, for example, these dosage forms In the case of wet properties such as liquid, paste, and jelly with water or other solvents, the concentration of 0.01 μg/mL or more, more preferably 0.1 μg/mL or more, makes the effect of HPGMS more pronounced. can be enjoyed to In the case of dry properties such as granules and powders, it is preferable to mix HPGMS at a concentration of 0.01 μg or more, more preferably 0.1 μg or more per use.

Hereinafter, HPGMS ((4E,8E,2S,3R,2'R)-N2'-hydroxypalmityl-l-O-β -D-glucopyranosyl-9-methyl-4,8-sphingadienine) will be further described with reference to experimental processes and results.

[1] Search Test for Useful Ingredients First, useful ingredients contained in Enokitake were searched. Specifically, Oki Shirayuki No. 919 produced in Oki-machi, Fukuoka Prefecture was dried, and 1 kg of powder of the dried product was used as the test sample.

(1) Extraction using various solvents A powdered specimen was immersed in methanol to obtain 340 g of methanol extract. Then, hexane was added to this methanol solution for liquid-liquid partitioning to obtain 74 g of hexane extract. Further, ethyl acetate was added to the residual liquid after fractionating the hexane extract, and liquid-liquid partitioning was performed to obtain 43 g of the ethyl acetate extract. In addition, the remaining aqueous phase after fractionating the ethyl acetate extract was freeze-dried and pulverized.

(2) Analysis of Hexane Extract Next, an open column packed with normal-phase silica gel was constructed, and the hexane extract was fractionated while gradiently using a mixed solvent of hexane and ethyl acetate as a mobile phase. The obtained fractions (fractions 1 to 33) were mixed solvents of hexane and ethyl acetate (Hexane:EtOAc = 85:15, 80:20, 60:40, 55:45, 35:65, 25:75) In addition, methanol, a mixed solvent of dichloromethane (DCM) and methanol (DCM:MeOH = 90:10), and a mixed solvent of hexane and acetone (Hexane:Acetone = 50:50) were used as developing solvents for thin-layer chromatography. provided. Figures 1 to 3 show the spots after development of each fraction.

にて示される4,4-ジメチル-5a-エルゴスタ-8,24(28)-ジエン-3β-オール（4,4-dimethyl-5a-ergosta-8,24(28)-dien-3β-o1）であることが明らかとなった。 Of these plates, first, attention was paid to fraction 8 shown in FIG. The dry powder of this fraction 8 was washed with methanol, and the resulting precipitate was dissolved in dichloromethane and subjected to thin layer chromatography. Got a spot. The component constituting this spot was tentatively named ENH-1, and the same component was identified. ENH-1 has the following structural formula (II):

4,4-dimethyl-5a-ergosta-8,24(28)-dien-3β-ol (4,4-dimethyl-5a-ergosta-8,24(28)-dien-3β-o1) represented by It became clear that

にて示されるエルゴステロール(ergosterol)であることが明らかとなった。 Next, attention was paid to fractions 11 and 12 shown in FIG. Fractions 11 and 12 were subjected to crystallization treatment, and the obtained crystals were dissolved in a predetermined solvent and subjected to thin-layer chromatography. A single spot was obtained as shown in the Cry' lane. The component constituting this spot was tentatively named ENH-2, and the same component was identified. ENH-2 has the following structural formula (III):

It was found to be ergosterol shown in

にて示されるエルゴステロールペルオキシド(ergosterol peroxide)であることが明らかとなった。 Also, attention was paid to the fraction 15 shown in FIG. From the cochromatographic and NMR analysis of this fraction 15, it was named ENH-3, which constitutes the spot from which the arrow drawn in the "15" lane of the plate developed with Hexane:EtOAc = 60:40 solvent. The component has the following structural formula (IV):

It was found to be ergosterol peroxide shown in.

にて示される2-ヒドロキシ-テトラコサン酸(2,3-ジヒドロキシ-1-ヒドロキシメチル-ヘプタデック-7-エニル)-アミド(2-hydroxy-tetracosanoic acid(2,3-dihydroxy-1-hydroxymethyl-heptadec-7-enyl)-amide)、所謂Trolliamideであり、ENH-5と命名されたレーン1のスポットを構成する成分は、構造式(VI)：
にて示されるセレビステロール(Cerevisterol)であることが明らかとなった。 Also, attention was paid to the fraction 27 shown in FIGS. Using a reversed-phase plate and using methanol as a developing solvent, three spots from fraction 27 were isolated and identified. As shown in the right plate of FIG. has the structural formula (V):

2-Hydroxy-tetracosanoic acid (2,3-dihydroxy-1-hydroxymethyl-heptadec-7-enyl)-amide (2-hydroxy-tetracosanoic acid (2,3-dihydroxy-1-hydroxymethyl-heptadec- 7-enyl)-amide), so-called Trolliamide, and the component constituting the spot in lane 1 named ENH-5 has the structural formula (VI):
It was found to be Cerevisterol shown in.

にて示されるエルゴステロールペルオキシドグルコシド(ergosterol peroxide glucoside)であり、ENH-7と命名された「54・60」レーンのスポットを構成する成分は、構造式(I)：

で示される(4E,8E,2S,3R,2'R)-N2'-ヒドロキシパルミチル-l-O-β-D-グルコピラノシル-9-メチル-4,8-スフィンガジエニン（(4E,8E,2S,3R,2'R)-N2'-hydroxypalmityl-l-O-β-D-glucopyranosyl-9-methyl-4,8-sphingadienine）であり、ENH-8と命名された「5」レーンのスポットを構成する成分は、構造式(VIII)：

にて示されるカタセレブロシドA(Catacerebroside A)であることが明らかとなった。 Also, attention was paid to the fractions 32 and 33 shown in FIG. Gradient analysis was performed using a mixed solvent of water and methanol as the mobile phase by medium pressure liquid chromatography (MPLC) using a reversed phase column, and methanol was developed again for the fractions corresponding to the three peaks obtained. Subjected to reversed-phase TLC as a solvent, each obtained single spot was attempted to identify. As a result, as shown in FIG. , structural formula (VII):

is ergosterol peroxide glucoside represented by the structural formula (I):

(4E,8E,2S,3R,2'R)-N2'-hydroxypalmityl-lO-β-D-glucopyranosyl-9-methyl-4,8-sphingadienine ((4E,8E, 2S,3R,2'R)-N2'-hydroxypalmityl-lO-β-D-glucopyranosyl-9-methyl-4,8-sphingadienine) and constitutes the "5" lane spot designated ENH-8. The component has the structural formula (VIII):

It was found to be Catacerebroside A shown in.

にて示されるエルゴスト-7-エン-3,5,6-トリオール(Ergost-7-ene-3,5,6-triol)であることが明らかとなった。 Also, attention was paid to the fraction 28 shown in FIG. Gradient analysis was performed using a mixed solvent of water and methanol as the mobile phase by medium pressure liquid chromatography (MPLC) using a reversed-phase column. As a result of trying to identify each single spot obtained, as shown in FIG. IX):

Ergost-7-ene-3,5,6-triol (Ergost-7-ene-3,5,6-triol) represented by

In this way, each component corresponding to ENH-1, ENH-2, ENH-3, ENH-4, ENH-5, ENH-6, ENH-7, ENH-8, and ENH-12 is It was shown to be contained in a certain enokitake mushroom, Shirayuki Ohki No.919.

In addition, by isolating the ingredients separately, ENH-1 is 6 mg, ENH-4 is 3.5 mg, ENH-5 is 5.5 mg, ENH-6 is 15 mg, 170 mg of ENH-7, 13 mg of ENH-8 and 4 mg of ENH-12 have been isolated, specifically ENH-7 (4E,8E,2S,3R,2'R)-N2'-hydroxypalmityl- It was shown that the content of l-O-β-D-glucopyranosyl-9-methyl-4,8-sphingadienine (HPGMS) was remarkably high. It should be noted that since the values of each component are the isolated yields, they are estimated to be less than the actual content, so that the actual content is greater than the above content.

にて示されるケイ皮酸(cinnamic acid)であり、ENE-2と命名された「ENE-2」レーンのスポットを構成する成分は、構造式(XI)：

にて示されるパラヒドロキシ安息香酸(p-hydroxy benzoic acid)であることが明らかとなった。 (3) Analysis of Ethyl Acetate Extract Next, an open column packed with normal-phase silica gel was constructed, and a mixed solvent of hexane and ethyl acetate and a mixed solvent of ethyl acetate and methanol were used as mobile phases. Fractionation of the extract was performed. Among the obtained fractions (fractions 1 to 32), fractions 10 to 11 and fractions 15 to 22 are subjected to preparative TLC using a reversed-phase plate with 60% methanol aqueous solution as the developing solvent to form each spot. When an attempt was made to identify the components, as shown in FIG. , structural formula (X):

The component that constitutes the spot in the "ENE-2" lane, named ENE-2, is cinnamic acid represented by the structural formula (XI):

It was found to be p-hydroxy benzoic acid represented by

Thus, it was shown that each component corresponding to ENE-1 and ENE-2 was contained in Enokitake and Shirayuki Ohki No. 919, which are the test samples.

In addition, according to a separate component isolation experiment, it was shown that ENE-1 was contained in 9.2 mg and ENE-2 was contained in 6 mg or more per 1 kg of dry powder of Enokitake and Oki Shirayuki No. 919, which were the test subjects.

[2] Expression Evaluation Test for Ceramide Synthase Gene The skin is composed of the epidermis and dermis, and the epidermis is classified into the stratum basale, the stratum spinosum, the stratum granulosum and the stratum corneum. A lamellar structure composed of lipids such as ceramides between stratum corneum cells located in the outermost layer of the skin plays a role as a physical barrier to retain water in the body and prevent invasion of bacteria and viruses.

In this study, (4E,8E,2S,3R,2'R)-N2'-hydroxypar To evaluate the effect of mytyl-l-O-β-D-glucopyranosyl-9-methyl-4,8-sphingadienine (HPGMS) on improvement of epidermal cells by examining changes in the expression level of ceramide synthase gene.

(1) Experimental method A specific experimental method will be described below.

(a) Cell culture Human epidermal keratinocytes (HaCaT cells) were grown to confluency using Dulbecco's Modified Eagle Medium (DMEM) (high glucose) (containing 1% penicillin-streptomycin and 10% fetal bovine serum (FBS)). It was pre-cultured in a φ10 cm dish until it became .

Then, the cells were washed with phosphate-buffered saline (PBS), resuspended in medium, seeded in a 24-well plate at a concentration of 1.0×10 ⁵ cells/well, and placed in a CO ₂ incubator (37°C, 5% CO ₂ ). was cultured overnight.

(b) Sample addition
24 hours after seeding HaCaT cells, 1 mL of serum-free DMEM (high glucose) containing HPGMS (containing 1% penicillin-streptomycin) was added to each well and cultured in a CO ₂ incubator (37°C, 5% CO ₂ ) for 48 hours. did.

(c) Changes in gene expression levels by real-time PCR
HaCaT cells were collected 48 hours after addition of HPGMS, and total RNA was extracted using RNeasy Mini kit (Qiagen).

cDNA was synthesized from the extracted total RNA using ReverTra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO).

Using the synthesized cDNA as a template, real-time PCR was performed using an AriaMX (Agilent) apparatus. THUNDERBIRD SYBR qPCR Mix (TOYOBO) was used for the real-time PCR reaction. For the real-time PCR reaction, 5'-CCGATTACCTGCTGGAGTCAG-3' and 5'-GGCGAAGACGATGAAGATGTTG-3' were used as primers for the ceramide synthase gene CERS2, and 5'-ACATTCCACAAGGCAACCATTG-3' and 5'- CTCTTGATTCCGCCGACTCC-3', 5'-GGAGGCCTGTAAGATGGTCA-3' and 5'-GAGGACCAGTCGGGTGTAGA-3' as primers for ceramide synthase gene CERS4, 5'-GGGTCAGAAGGACTCCTATG-3' and 5'- as primers for internal standard β-actin GTAACAATGCCATGTTCAAT-3' was used.

As real-time PCR reaction conditions, 40 cycles of a two-step PCR reaction consisting of initial denaturation at 95°C for 60 seconds, denaturation at 95°C for 15 seconds, and annealing/extension at 60°C for 60 seconds were performed.

(2) Results and discussion
We added HPGMS to HaCaT cells, one of human epidermal keratinocytes, and investigated the expression behavior of ceramide synthase CERS2, CERS3 and CERS4 genes using real-time PCR.

9 to 11 show changes in the expression of the ceramide synthase gene in HaCaT cells to which HPGMS was added. It has been reported that the ceramide synthase CERS2 gene is highly expressed in many cell tissues such as kidney, liver and intestine. On the other hand, the ceramide synthase CERS3 gene is mainly expressed in skin and keratinocytes. Furthermore, it is known that the ceramide synthase CERS4 gene is expressed not only in skin but also in leukocytes, heart and liver.

As a result of this test, it was confirmed that the expression of mainly the CERS3 gene and the CERS4 gene increased approximately 3.5 times compared to the control when HPGMS was added at 100 μg/mL. On the other hand, the CERS2 gene was found to be expressed at the same level as the control.

These results suggest that HPGMS has the effect of enhancing the expression of the ceramide synthase CERS3 and CERS4 genes. Therefore, HPGMS can be expected to have the effect of promoting ceramide synthesis, which plays an important role in the skin barrier function, and the associated water retention effect.

[3] Evaluation Test for Hyaluronic Acid and Collagen Production Fibroblasts in the skin dermis produce important components such as “hyaluronic acid” and “collagen” for constituting the skin. Hyaluronic acid is a major component of the extracellular matrix and participates in various intercellular interactions.

Hyaluronic acid is an essential element for retaining moisture in the skin, and it is believed that the skin produces and accumulates hyaluronic acid to prevent water loss in the body and protect against physical irritation. . Collagen is a structural protein that forms connective tissue such as animal skin. Collagen is important for keeping skin strong and youthful without wrinkles.

If the production of hyaluronic acid and collagen in fibroblasts can be promoted, it is believed that fresh and firm skin can be maintained. In this study, test substances are added to human fibroblasts, and changes in the production of hyaluronic acid and collagen are measured to evaluate the effects of HPGMS on skin moisture retention and wrinkle prevention.

(1) Experimental method A specific experimental method will be described below.

(a) Cell culture Adult human dermal fibroblasts (NHDF-Ad) were used as model cells. NHDF-Ad cells were precultured in a φ10 cm dish using Dulbecco's Modified Eagle Medium (DMEM) (high glucose) (containing 1% penicillin-streptomycin and 10% fetal bovine serum (FBS)) until confluent. After that, the cells were washed with phosphate buffered saline (PBS), resuspended in medium, seeded in a 96-well plate at a density of 0.5×10 ⁴ cells/well, and placed in a CO ₂ incubator (37°C, 5% CO ₂ ). was cultured overnight.

(b) Sample Addition After overnight culture, the medium was replaced with a serum-free medium (containing 1% penicillin-streptomycin) containing the sample, and cultured in a CO ₂ incubator for 72 hours. Samples were added at the following final concentrations.

(c) Hyaluronic Acid Production Test and Collagen Production Test Culture supernatants were collected 72 hours after sample addition, and the amount of produced hyaluronic acid or collagen was measured using a commercially available ELISA kit.

(d) Measurement of cell viability by MTT method After collecting the culture supernatant 72 hours after addition of the sample, 100 μL of sample-free serum-free medium (containing 1% penicillin-streptomycin) was added to each well of a 96-well plate. .

Next, 20 μL of MTT staining solution (5 mg/mL in PBS) was added to each well, and static culture was carried out in a CO ₂ incubator for 4 hours. The medium was removed, 100 μL of hydrochloric acid-isopropanol solution was added to each well, and formazan was completely dissolved by pipetting. Absorbance at 570 nm was measured with a microplate reader to calculate cell viability.

(2) Results and discussion The effects of HPGMS on the production of hyaluronic acid and collagen were investigated using adult human dermal fibroblasts (NHDF-Ad).

FIG. 12 shows the cell viability 72 hours after addition of HPGMS. It was confirmed that the treatment groups with HPGMS concentrations of 10, 100 and 200 μg/mL had significantly higher cell viability than the control (water). In the other HPGMS-treated plots, no significant change in cell viability was observed compared to the control (water).

FIG. 13 shows the amount of collagen produced per cell 72 hours after the addition of HPGMS. FIG. 14 shows the amount of hyaluronic acid produced per cell 72 hours after the addition of HPGMS. It was confirmed that the concentrations of collagen and hyaluronic acid per cell were increased in the treatment group with HPGMS added at concentrations of 0.01 and 0.1 μg/mL compared to the control.

Based on the above results, HPGMS is expected to promote the production of collagen and hyaluronic acid, and to retain moisture in the skin and prevent wrinkles.

[4] Purification Test of HPGMS Next, a purification test of HPGMS was conducted. As described above, the use of the semi-purified product obtained by the method for purifying HPGMS contained in enokitake mushrooms according to the present embodiment is not particularly limited. went.

First, 5.0 g of a dry powder sample of Ohki Shirayuki No. 919 was extracted with 250 mL of chloroform. After filtering the extract under reduced pressure, the solvent was dried using a vacuum evaporator.

Next, the resulting dried product was dissolved and/or suspended in about 3 parts by volume of a methanol/water mixture, such as 3 mL of a 50±1 v/v % methanol aqueous solution, to obtain a crude concentrate.

Processing was then performed using InertSep® Pharma, a reversed-phase solid-phase extraction column. That is, the resulting crude concentrate was applied (injected) to five InertSep Pharma columns (first columns). Fractions were pressure sedimented by centrifugation at 1200 xg for 1 minute. The liquid discharged from the lower end of the column during injection was removed.

Similarly, add 500 μL of 80% methanol aqueous solution (Fr.1) to each column, centrifuge and discard the effluent. Add 1 mL of 83% methanol aqueous solution, centrifuge and discard the effluent. The operation was performed twice (Fr,2,3). In addition, the packing material of the first column was rinsed with 2 to 4 volume parts of 80±5 v/v % methanol aqueous solution while centrifuging.

Next, 1 mL of methanol was added to each column and subjected to centrifugal separation, and the rinse solution (Fr.4) obtained from each column was collected and the solvent was dried to obtain a solid sample. This solid sample was again dissolved and/or suspended in about 3 parts by volume of a methanol/water mixture, for example, 3 mL of a 50±1 v/v % methanol aqueous solution to prepare a primary purified liquid for the second stage of purification.

Next, the first column was thoroughly washed and the primary purified liquid was injected into the second column obtained by equilibrating with water, and 1 ± 0.25 volume part of 83 ± 1 v / v% methanol aqueous solution The packing of the second column was rinsed by adding, for example, 1 mL of 83% aqueous methanol solution and centrifuging. A new column can also be used for the second column.

Then, 2 to 4 parts by volume of propanol, such as 3 mL of isopropanol, was added and subjected to centrifugation, and the obtained rinse was used as a semi-purified product with an increased HPGMS content. A semi-purified product can be obtained by drying the rinse liquid using a vacuum evaporator or the like. The obtained semi-purified product was used as it was or dissolved in a predetermined solvent and subjected to HPLC analysis as a concentrated fraction of HPGMS.

Agilent 1260 infinity II was used for HPLC analysis. Inertsil ODS-P (4.6×150 mm 5.0 μm) was used for component separation, and analysis was performed under isocratic conditions using a 92% aqueous methanol solution as the mobile phase. The flow rate is 1.0 mL/min.

A photodiode array (PDA) detector (210 nm, 214 nm, 230 nm, 250 nm, 280 nm) manufactured by Agilent and an evaporative high scattering detector (ELSD) were used as detectors. The temperature in the ELSD was 80°C, the nebulizer gas temperature was 30°C, the nebulizer gas flow rate was 1.0 SLM, and the injection volume was 5 μL.

In addition, for HPGMS quantification, a calibration curve was created using isolated HPGMS samples. A standard curve was prepared using HPGMS samples adjusted to 0.5, 1.0, 1.5, and 2.0 mg/mL.

From 6.45 mg of the semi-purified product obtained by drying, 1.7 mg was collected, dissolved in 100 μL of isopropanol, and subjected to HPLC analysis. As a result, a chromatogram as shown in FIG. 15(a) was obtained, and the peak of HPGMS indicated by a black triangle could be confirmed without being disturbed by the enokitake mushroom-derived saccharides.

Further, when the content was calculated based on the prepared calibration curve, it was found that 79.4 μg of HPGMS was contained per 1 g of dry powder of Shirayuki Ohki No.919.

On the other hand, as a comparison target, a conventional cultivar Enokitake sample was also quantified in the same manner, and a chromatogram such as that shown in FIG. 15(b) was obtained. Met.

From the above results, Oki Shirayuki No. 919 was found to contain an amount of HPGMS that is necessary for the water retention effect to be achieved in a daily diet. In addition, the result that the content was higher than that of the conventional variety was obtained.

[5] Preparation of skin-moisturizing functional food and effect of the same cosmetic on skin Next, preparation of skin-moisturizing functional food containing HPGMS derived from Oki Shirayuki No. 919 obtained by the above-mentioned [4] HPGMS purification test was carried out. Ta.

A mixed powder containing HPGMS and the rest being lactose was tableted into a functional food for moisturizing the skin.

In the test, 10 subjects were divided into 5 test group subjects and 5 control group subjects. was performed by ingesting a control food that had the same appearance but did not contain HPGMS. The subjects in the test group took tablets in an amount equivalent to 0.1 µg of HPGMS per dose twice a day in the morning and evening for two weeks.

In addition, on the last day of ingestion, the subjects themselves were asked to evaluate the moist state of their skin. The evaluation is "-3: Worse", "0: No change", "1: Slightly improved", "2: Improved", "3: Significantly improved". Evaluation points were summed up and evaluated.

As a result, the total evaluation score of the control group was 4 points, while the total evaluation score of the test group was 7 points. It was shown that the moisturizing effect of

[6] Preparation of Cosmetics and Effect of Cosmetics on Skin Next, a lotion containing HPGMS derived from Shirayuki Ohki No. 919 obtained by the above-mentioned [4] HPGMS purification test was prepared.

A test lotion containing HPGMS as a constituent was prepared by adding HPGMS to a commercially available lotion so that the final concentration was 0.1 μg/ml. This test lotion is both an external preparation for skin according to the present embodiment and a cosmetic according to the present embodiment.

In the study, 10 subjects were divided into 5 test group subjects and 5 control group subjects. A control lotion was applied externally. The subjects in the test group applied 5 ml of the test lotion (equivalent to 0.5 µg of HPGMS) on their faces twice a day, morning and evening, for two weeks.

In addition, on the final day, the subjects themselves were asked to evaluate the moist state of their skin. The evaluation is "-3: Worse", "0: No change", "1: Slightly improved", "2: Improved", "3: Significantly improved". Evaluation points were summed up and evaluated.

As a result, the total evaluation score of the control group was 6 points, while the total evaluation score of the test group was 12 points. It was shown that excellent skin moisturizing effect can be exhibited.

で示される(4E,8E,2S,3R,2'R)-N2'-ヒドロキシパルミチル-l-O-β-D-グルコピラノシル-9-メチル-4,8-スフィンガジエニンを含有することとしたため、経口摂取により優れた皮膚の保湿効果を発揮できる皮膚保湿用機能食品を提供することができる。 As described above, according to the functional food for moisturizing skin according to the present embodiment, the following structural formula (I):

(4E,8E,2S,3R,2'R)-N2'-hydroxypalmityl-lO-β-D-glucopyranosyl-9-methyl-4,8-sphingadienine represented by It is possible to provide a skin-moisturizing functional food that exhibits an excellent skin-moisturizing effect by oral ingestion.

で示される(4E,8E,2S,3R,2'R)-N2'-ヒドロキシパルミチル-l-O-β-D-グルコピラノシル-9-メチル-4,8-スフィンガジエニンを含有することとしたため、外用により優れた皮膚の保湿効果を発揮できる皮膚外用剤や化粧料を提供することができる。 Further, according to the skin external preparation and the cosmetic according to the present embodiment, the following structural formula (I):

(4E,8E,2S,3R,2'R)-N2'-hydroxypalmityl-lO-β-D-glucopyranosyl-9-methyl-4,8-sphingadienine represented by Therefore, it is possible to provide an external preparation for skin and a cosmetic that can exert excellent moisturizing effect on the skin when applied externally.

Finally, the description of each embodiment mentioned above is an example of this invention, and this invention is not limited to the above-mentioned embodiment. Therefore, it goes without saying that various modifications other than the above-described embodiments can be made in accordance with the design and the like within the scope not departing from the technical idea of the present invention.

Claims (3)

a step of dissolving and/or suspending the chloroform-extracted dry matter of dried enokitake mushroom in 3 parts by volume of a 50±1 v/v% methanol aqueous solution to obtain a crude concentrate;
A step of applying the crude purified concentrate to a first column packed with 1 part by volume of an equilibrated reversed-phase solid phase;
rinsing the packing material of the first column with 2 to 4 parts by volume of an 80±5 v/v% methanol aqueous solution;
Rinse the packing material of the first column with 1 to 2 parts by volume of a 95 to 100 v/v% methanol aqueous solution or methanol, add water to the resulting rinse solution, and the final concentration of methanol is 50 ± 1 v / v. obtaining 3 parts by volume of the primary purified liquid at %;
a step of applying the primary purified liquid to a second column packed with 1 part by volume of an equilibrated reversed-phase solid phase;
rinsing the packing material of the second column with 1±0.25 parts by volume of 83±1 v/v% methanol aqueous solution;
The packing material of the second column was rinsed with 2 to 4 parts by volume of propanol, and the resulting rinse was treated with (4E,8E,2S,3R,2'R)-N2'-hydroxypalmityl-lO- and a step of producing a semi-purified product with an increased content of β-D-glucopyranosyl-9-methyl-4,8-sphingadienin (4E,8E,2S,3R,2 A method for purifying 'R)-N2'-hydroxypalmityl-lO-β-D-glucopyranosyl-9-methyl-4,8-sphingadienine. Use of (4E,8E,2S,3R,2'R)-N2'-hydroxypalmityl-l-O-β-D-glucopyranosyl-9-methyl-4,8-sphingadienine as a constituent of cosmetics . 3. Use according to claim 2 , characterized in that said constituent is an active ingredient for the purpose of including moisturizing of the skin as an action or effect.