JP7325779B1 - CD39 expression promoter - Google Patents

Abstract

An object of the present invention is to clarify an active ingredient contained in Dharma cypress that has an excellent effect of promoting CD39 expression, and to provide a CD39 expression promoting agent containing the active ingredient. A CD39 expression promoter in skin Langerhans cells of the present invention comprises a compound having a bicyclic diterpene skeleton, a salt thereof, or a solvate thereof as an active ingredient, and a compound having a bicyclic diterpene skeleton. is at least one compound selected from the group consisting of sclareol, sclareol glycosides and isomanool glycosides. [Selection figure] None

Description

The present invention relates to a CD39 expression promoter, in particular, CD39 that is expressed in Langerhans cells of the skin, is involved in skin immune response, and is capable of promoting the expression of CD39 that is involved in suppressing inflammation derived from extracellular ATP. It relates to an expression promoter.

CD39, also known as ectonucleoside triphosphate diphosphohydrolase 1, NTP Dase 1, is a membrane protein, an ectonucleotidase that has a catalytic site on the extracellular surface and hydrolyzes ATP and ADP to AMP. CD39 is expressed on B cells, T cells, natural killer cells, dendritic cells, vascular endothelial cells, etc., and is involved in suppressing inflammatory reactions and activating platelet function by degrading extracellular ATP/ADP. are doing.

It is known that when cells receive physical or chemical external stimuli, extracellular ATP is secreted and serves as an alarm signaling substance. The amount of extracellular ATP released increases with aging. When extracellular ATP stimulates senescent cells, a phenomenon called SASP (senescence-associated secretory phenotype) occurs, in which various secreted factors whose main components are inflammatory cytokines, chemokines, and proteases are highly expressed. Causes tissue inflammation. Some of the SASP factors have beneficial effects on the body, such as enhancing the aging cell cycle arrest, but other SASP factors are related to promoting inflammation and tumorigenesis, chronic inflammation, cancer, etc. has been reported to be involved in the onset of various diseases (Non-Patent Document 1). Therefore, the function of CD39 to degrade extracellular ATP to prevent accumulation of extracellular ATP and suppress SASP has attracted attention.

On the other hand, the skin consists of epidermis, dermis and subcutaneous tissue. Among them, the epidermis, which is the outermost layer of the skin responsible for the skin barrier function, is mainly composed of keratinocytes (keratinocytes) or cells that have changed from these keratinocytes. It is organized in order of layers and basal cell layers. The epidermis has the function of protecting the body from mechanical, physical or chemical external forces and invasion of foreign substances. By eliminating foreign substances invading from the outside, the existing immune cells ensure the barrier function. Typical skin immune cells include Langerhans cells present in the spinous cell layer of the epidermis. Langerhans cells are dendritic cells that capture foreign antigens that have passed through the stratum corneum, migrate to specific lymph nodes, present antigens to T cells, and activate T cells. Activated T cells migrate to the skin, and when they recognize antigens, they produce cytokines and kill foreign substances such as bacteria. On the other hand, Langerhans cells are also involved in the induction of immune tolerance in the steady state in which foreign antigens are not sensed.

The above-described CD39 is expressed in these epidermal Langerhans cells, and therefore the ATPase activity by this CD39 has been used as a marker for Langerhans cells. Keratinocytes release extracellular ATP by physical and chemical stimuli such as shear force and stretching, and the released extracellular ATP becomes a signal transducing substance, causing apoptosis and skin inflammation. Therefore, CD39-expressing Langerhans cells suppress inflammatory reactions and regulate immune response functions by degrading extracellular ATP by CD39 so that extracellular ATP does not accumulate (Non-Patent Document 2). . In addition, it has been reported that when the number of Langerhans cells decreases due to insufficient zinc intake, CD39 also decreases and extracellular ATP is not sufficiently degraded, resulting in accumulation of extracellular ATP and skin inflammation. (Non-Patent Document 3).

Therefore, in order to suppress inflammation and excessive immune response caused by SASP and the like, research is being conducted to increase the expression level of CD39, or to suppress the decrease or activation of Langerhans cells. In Patent Document 1, as a CD39 gene expression promoter, CD39 gene expression promoter containing three components of carboxymethyl beta-glucan sodium, polyoxyethylene (POE) / polyoxypropylene (POP) random copolymer dimethyl ether and rose water agents have been proposed. In addition, Patent Document 2 proposes a Langerhans cytoreduction inhibitor containing, as an active ingredient, a plant extract selected from ragweed, Japanese pagoda, Phellodendron bark, licorice, rhizome, tencha, or angelica root. Patent Document 3 proposes a neuramin derived from morel mushroom. It is described that acid derivatives exhibit Langerhans cell activating action.

Furthermore, in Patent Document 4 by the applicants of the present application, it is reported that an extract of Aster spathulifolius has an effect of promoting CD39 expression in skin Langerhans cells.

Japanese translation of PCT publication No. 2017-511790 JP-A-2000-239144 JP 2017-140000 Japanese Patent No. 6887649

Shin Yoshimoto et al., “Obesity-induced gut microbial metabolite promotes liver cancer through senescence secretome”, Nature, Vol. 499, 2013, p.97-101 Norikatsu Mizumoto et al., “CD39 is the dominant Langerhans cell-associated ecto-NTPDase: Modulatory roles in inflammation and immune responsiveness”, Nature Medicine, Vol. 8, No. 4, 2002, p.358-365 Tatsuyoshi Kawamura et al., “Severe dermatitis with loss of epidermal Langerhans cells in human and mouse zinc deficiency”, The Journal of Clinical Investigation, Vol. 122, No. 2, 2012, p.722-732

However, the CD39 gene expression promoter reported in Patent Document 1 contains components produced by chemical synthesis, and therefore, CD39 gene expression promoters composed of naturally derived components with high safety to the human body. Accelerators are still expected. In addition, although Patent Documents 2 and 3 describe specific plant extracts having an effect of inhibiting or activating the decrease of Langerhans cells, respectively, the effect of promoting the expression of CD39 has not been investigated.

Then, Patent Document 4 describes the use of an extract of Dermatophyllum japonicum as a CD39 expression promoting agent in skin Langerhans cells, but confirmation of the effect on the CD39 expression promoting action is generally performed under reaction conditions compared to in vivo. Under controlled conditions, CD39 expression-promoting effect was observed when 0.1 to 0.2% (that is, 1000 to 2000 ppm) of the antler extract was added to the cell culture medium. It has been reported that Therefore, it is presumed that, when using this Dharmacillus extract as a CD39 expression promoter, it is necessary to use it in percent order.

In addition, the extract of Paparia chinensis disclosed in Patent Document 4 is a mixture containing a plurality of components, and the specific active component as a CD39 expression promoting agent is not indicated.

Therefore, the present invention has been made in view of the above points, and an object of the present invention is to identify an active ingredient contained in Dermatophyllum japonicum, which has an excellent CD39 expression promoting effect, and to provide a CD39 expression promoting agent containing the active ingredient. to provide.

Another object of the present invention is to provide external skin preparations, cosmetics, and foods and drinks containing the novel CD39 expression promoter as an active ingredient.

Another object of the present invention is to provide an anti-inflammatory agent produced by extracellular ATP containing a novel CD39 expression promoter.

In order to solve the above problems, the present inventors performed various fractionation treatments on an extract of damselfly, and examined the obtained fractions. identified. Based on this knowledge, the present invention has been completed.

That is, the CD39 expression promoter in skin Langerhans cells of the present invention contains a compound having a bicyclic diterpene skeleton, a salt thereof, or a solvate thereof as an active ingredient, and the compound having a bicyclic diterpene skeleton is , sclareol, sclareol glycosides and isomanool glycosides. By administering sclareol, sclareol glycoside or isomanool glycoside, which are compounds having a bicyclic diterpene skeleton, CD39 expression in target cells can be promoted and CD39 can be increased. Therefore, the degradation of extracellular ATP released by external stimuli, aging, etc. is promoted, and excessive immune responses due to inflammation, SASP and the like are suppressed.

Further, in the CD39 expression promoter in skin Langerhans cells of the present invention, the sugar constituting the sclareol glycoside is preferably 6-deoxydose, fucose, or derivatives thereof. Thereby, sugars constituting suitable sclareol glycosides are selected.

In addition, the CD39 expression promoter in skin Langerhans cells of the present invention has the above-described sclareol glycoside represented by the following formula (I) (wherein R ¹ represents a hydrogen atom or an acetyl group) or formula (II) (formula wherein R ² represents a hydrogen atom or an acetyl group) is also preferred. Thereby, a sclareol glycoside suitable as a CD39 expression promoter is selected.

In the skin Langerhans cell CD39 expression-promoting agent of the present invention, the sugar constituting the isomanool glycoside is preferably 6-deoxydose, fucose, or a derivative thereof. Thereby, sugars constituting suitable isomanool glycosides are selected.

In addition, the CD39 expression promoter in skin Langerhans cells of the present invention has the above-described isomanool glycoside represented by the following formula (III) (wherein R ³ represents a hydrogen atom or an acetyl group) or formula (IV) ( In the formula, R ⁴ represents a hydrogen atom or an acetyl group) is also preferred. Thus, an isomanool glycoside suitable as a CD39 expression promoter is selected.

In addition, the external skin preparation for promoting CD39 expression in skin Langerhans cells, the cosmetic for promoting CD39 expression, and the food and drink for promoting CD39 expression of the present invention contain a CD39 expression promoter comprising a compound having a bicyclic diterpene skeleton described above. Contains as an active ingredient. As a result, an external preparation for skin, a cosmetic, and a food or drink that can suppress inflammation of the skin or the like and suppress excessive immune response can be obtained.

In addition, the anti-inflammatory agent produced by extracellular ATP of the present invention contains a CD39 expression promoter consisting of the aforementioned compound having a bicyclic diterpene skeleton. By administering a compound having a bicyclic diterpene skeleton described above, CD39 expression in target cells is promoted and CD39 is increased, so that extracellular ATP is sufficiently degraded and inflammation caused by extracellular ATP is suppressed. can do.

Moreover, this invention is the compound represented by following formula (V), its salt, or those solvates. The compound represented by the formula (V) is a novel sclareol glycoside isolated and purified from an extract of Dermatophyllum japonicum, and has an excellent CD39 expression-enhancing effect.

INDUSTRIAL APPLICABILITY According to the present invention, CD39 expression promoters, external preparations for skin, cosmetics, foods and drinks, and inhibitors of inflammation caused by extracellular ATP having the following excellent effects can be provided.
(1) It can enhance CD39 expression in target cells at a low dose.
(2) Since extracellular ATP is sufficiently degraded by CD39 whose expression is promoted, the inflammatory reaction caused by extracellular ATP is suppressed.
(3) It is highly safe to the human body because the active ingredient is an ingredient separated and refined from an edible Dharmacillus extract.
(4) An external preparation for skin, a cosmetic, and a food or drink having an effect of suppressing skin inflammation and an excessive immune response can be obtained.

In Example 1, the fraction Fr. 1 is a flow chart outlining a method for preparing 1-6. Fr. 2 is a flow chart outlining a method for isolating compounds c and b from 2. FIG. Fr. 4 is a flow chart outlining a method for isolating compounds e, a and d from 4. FIG. Fr. 1 is a flow chart outlining a method for isolating compounds f, i and g from 5. FIG. Fr. 6 is a flow chart outlining a method for isolating compound h from 6. FIG. 1 shows (a) ¹ H-NMR spectrum and (b) ¹³ C-NMR spectrum of compound a. FIG. FIG. 2 shows (a) ¹ H-NMR spectrum and (b) ¹³ C-NMR spectrum of compound b. FIG. 2 shows (a) ¹ H-NMR spectrum and (b) ¹³ C-NMR spectrum of compound c. FIG. 2 shows (a) ¹ H-NMR spectrum and (b) ¹³ C-NMR spectrum of compound d. FIG. 2 shows (a) ¹ H-NMR spectrum and (b) ¹³ C-NMR spectrum of compound e. FIG. 2 shows (a) ¹ H-NMR spectrum and (b) ¹³ C-NMR spectrum of compound f. 1 shows (a) ¹ H-NMR spectrum and (b) ¹³ C-NMR spectrum of compound g. FIG. FIG. 2 shows (a) ¹ H-NMR spectrum and (b) ¹³ C-NMR spectrum of compound h. 1 shows (a) ¹ H-NMR spectrum and (b) ¹³ C-NMR spectrum of compound i. FIG. 4 is a graph showing the CD39 expression level of THP-1 cells to which compounds a to c were added, respectively, in Example 4. FIG. 10 is a graph showing the CD39 expression level of THP-1 cells to which compounds d to f were added, respectively, in Example 4. FIG. 4 is a graph showing the CD39 expression level of THP-1 cells to which compounds g to i were added, respectively, in Example 4. FIG.

Hereinafter, CD39 expression promoters containing sclareol, sclareol glycosides or isomanool glycosides, which are compounds having a bicyclic diterpene skeleton of the present invention, skin external preparations for promoting CD39 expression, cosmetics, and food and drink containing the same products, as well as anti-inflammatory agents generated by extracellular ATP.

CD39 in the present invention is called ectonucleoside triphosphate diphosphohydrolase 1 (Ectonucleoside triphosphate diphosphohydrolase 1), ENTPD1, NTPDase1, or the like, and is an ectonucleotidase that mainly degrades ATP/ADP to AMP as a substrate. CD39 is a membrane protein with transmembrane domains at its N and C termini and a catalytic site on the extracellular surface. CD39 is expressed in B cells, T cells, natural killer cells, dendritic cells, vascular endothelial cells, and the like, and is also expressed in melanocytes in addition to Langerhans cells in skin-constituting cells.

CD39 expression promotion in the present invention refers to CD39 gene expression promotion or CD39 (protein) expression promotion, compared with a control in which the CD39 expression promoting agent of the present invention is not added or administered, It means that the expression of CD39 (protein) is enhanced. More specifically, the CD39 gene expression level is preferably 1.2-fold or more, more preferably 1.4-fold or more, and further preferably 1.6-fold or more, 1.8 times or more is particularly preferable. The CD39 gene expression level can be measured by known methods such as real-time PCR (QPCR) and microarray, and the CD39 expression level can be measured by known methods such as immunostaining and Western blotting.

The CD39 expression promoter of the present invention contains a compound having a bicyclic diterpene skeleton, a salt thereof, or a solvate thereof as an active ingredient. Compounds having a bicyclic diterpene skeleton include sclareol represented by the following chemical formula, as well as sclareol glycosides and isomanool glycosides. The compound having a bicyclic diterpene skeleton according to the present invention may be a salt, preferably a pharmacologically acceptable salt. The pharmacologically acceptable salt of this compound is not particularly limited as long as it is a salt formed with an acid or a base. Moreover, this compound or a salt thereof may be a solvate, and although not particularly limited, examples thereof include hydrates and organic solvates such as ethanol.

Sclareol represented by the following chemical formula is a compound classified as a bicyclic diterpene alcohol, and is known to be contained in clary sage (Salvia sclarea), a plant of the labiatae family. This time, the present inventors have isolated this sclareol from a fraction fractionated from an extract of Dermatophyllum japonicum and found that this sclareol has a CD39 expression promoting effect.

Similarly, the present inventors isolated sclareol glycosides from a fraction fractionated from an extract of Dermatophyllum japonicum and found that sclareol glycosides have CD39 expression-enhancing activity. Sugar components constituting sclareol glycosides include 6-deoxyidose, fucose (6-deoxygalactose), and derivatives thereof. Among these, L-6-deoxidose represented by formula (I) and D-fucose represented by formula (II) are preferably used. Further, in the following formula (I), the atom represented by R ¹ is preferably a hydrogen atom, and the molecule is preferably an acetyl group. On the other hand, in the following formula (II), the atom represented by R ² is preferably a hydrogen atom, and the molecule is preferably an acetyl group.

Among the compounds represented by the formula (I), a sclareol glycoside represented by the following formula (V) was recently discovered as a novel compound.

Furthermore, the present inventors isolated isomanool glycosides from a fraction fractionated from an extract of Dermatophyllum japonicum and found that isomanool glycosides have CD39 expression-enhancing activity. In addition, as shown in Example 4 described later, isomanool itself, which is the aglycone of isomanool glycoside, was not confirmed to have a CD39 expression promoting effect. Sugar components constituting isomanool glycosides include 6-deoxyidose, fucose (6-deoxygalactose), and derivatives thereof. Among these, L-6-deoxidose represented by formula (III) and D-fucose represented by formula (IV) are preferably used. Further, in the following formula (III), the atom represented by ^R3 is preferably a hydrogen atom, and the molecule is preferably an acetyl group. On the other hand, in the following formula (IV), the atom represented by ^R4 is preferably a hydrogen atom, and the molecule is preferably an acetyl group.

The above-mentioned compounds, ie, sclareol, sclareol glycosides and isomanool glycosides, can all be obtained by isolating and purifying from the plant body of Dalmagnum. The Asteraceae used in the present invention, whose scientific name is Aster spathulifolius, is a plant belonging to the family Asteraceae and the genus Sion. It is found in coastal rocky areas, etc., and is distributed in the western part of Honshu, the Kyushu region, and the Korean Peninsula in Japan.It is also cultivated as a garden plant. In the present invention, the place of production and the environment of cultivation are not particularly limited, and Dharma daisy of all places of production and cultivation environment can be used.

A method for isolating the above-mentioned compound from the plant of Dalmagnum will be described. First, an extract is obtained from the plant body of Dalmagnum. As the plant body of Dermatophyllum japonicum, in addition to using the whole plant, it is also possible to select and use one or more parts of the flower part, leaf part, stem part, root part, germination, seed, etc. In particular, it is preferable to use flower parts, leaf parts or stem parts. Extraction treatment is performed on the harvested fresh plant body of Dalmagnum or the dried plant body that has been subjected to drying treatment, in order to improve the extraction efficiency or to facilitate handling. It is also possible to apply an extraction treatment to plants that have undergone various pretreatments. The pretreatment is not particularly limited, but includes drying treatment, crushing treatment, pulverization treatment, and the like, and the pretreated plant body of Dharmacillus may be subjected to extraction treatment to obtain an extract of Dharmacillus.

Extraction solvents include water, lower monohydric alcohols such as methyl alcohol, ethyl alcohol, 1-propanol, 2-propanol, 1-butanol or 2-butanol, glycerin, propylene glycol, dipropylene glycol or 1,3-butylene. Examples include solvents containing liquid polyhydric alcohols such as glycol, etc., and it is also possible to use a mixed solvent in which one or more of these are combined. Among these, water-containing alcohol is preferably used as the extraction solvent, and ethyl alcohol is preferably selected as the alcohol, from the viewpoint of safety to the human body, extraction efficiency, and the like. When hydrous alcohol is selected as the extraction solvent, the alcohol concentration is preferably 1 to 95 w/w%, more preferably 20 to 95 w/w%, and particularly preferably 40 to 90 w/w%. In addition, the extraction solvent may contain other components as long as they do not interfere with the extraction of the useful components from the damselfly.

As for the method of extracting from Apocynaceae, the plant bodies of Apocynaceae are added to an extraction solvent and immersed in the extraction solvent for extraction. For example, in the case of drying and pulverizing the plant body of Dalmagnum with a moisture content of less than 10%, it is preferable to use 2 to 100 parts by weight of the extraction solvent, preferably 2 to 50 parts by weight, per 1 part by weight of the plant body. is more preferable, and it is particularly preferable to use 3 to 30 parts by weight. As the extraction method, any method such as extraction at room temperature (1 to 30° C.), extraction with heating, extraction with hot water, extraction with pressurized hot water, or subcritical extraction can be used. As an example, when hydrous alcohol is used, extraction can be performed at room temperature of 1 to 30°C, preferably 10 to 30°C, more preferably 20 to 30°C. In addition, the extraction time is variously set according to the extraction method, the state of the plant body, the type of extraction solvent, the extraction temperature, etc. When performing extraction at room temperature using hydrous alcohol, it is 1 to 2 hours. About a week is preferable, about 1 to 10 days is more preferable, and about 2 to 8 days is particularly preferable. After the above-described extraction treatment, the dalmagnus extract is obtained by removing the residue by decantation, centrifugation, filtration, or the like. The obtained extracts include those made into concentrates and solids by subjecting them to treatments such as distillation under reduced pressure.

The dalmagnus extract obtained as described above is partitioned into two layers with ethyl acetate and water. Multiple bilayer distributions are preferred. The number of times of two-layer distribution is preferably about 2 to 10 times, particularly preferably about 4 to 5 times. The ethyl acetate fraction recovered by this solvent extraction operation contains the compound of the present invention.

The compound of the present invention can be isolated from the ethyl acetate fraction obtained as described above by purification according to a conventional method. Examples of the purification method include normal phase chromatography, reverse phase chromatography, thin layer chromatography, gel filtration chromatography, high performance liquid chromatography, and the like, and purification may be performed by combining one or more of these. is possible. Carriers, elution solvents, and the like used in various chromatographies can be appropriately selected according to the various chromatographies.

Furthermore, the compounds related to sclareol, sclareol glycosides and isomanool glycosides of the present invention may be synthetic products synthesized by known methods.

The CD39 expression promoter of the present invention contains the aforementioned sclareol, sclareol glycoside or isomanool glycoside as an active ingredient, and promotes CD39 expression and increases the CD39 expression level in CD39-expressing cells. It has the effect of causing The promotion of CD39 expression increases CD39 in target cells, thereby promoting the degradation of extracellular ATP and suppressing the accumulation of extracellular ATP. As a result, inflammation caused by extracellular ATP can be suppressed, and generation of SASP can also be prevented, thus suppressing excessive immune response. In particular, CD39 is expressed in Langerhans cells, which are cutaneous immune cells, and has the effect of degrading extracellular ATP and inhibiting the transmission of inflammation-inducing signals. By applying the CD39 expression promoter of the present invention to Langerhans cells, the amount of CD39 expressed in Langerhans cells is increased, thereby suppressing or calming skin inflammation and suppressing excessive immune response in the skin.

The CD39 expression promoter of the present invention can be used as an external skin preparation for suppressing skin inflammation, suppressing excessive immune response, and keeping the skin in a stable state. When applied to the skin, the expression level of CD39 in Langerhans cells increases, thereby promoting the decomposition of extracellular ATP released by external stimuli, aging, and the like. Therefore, skin inflammation caused by extracellular ATP and excessive immune response caused by SASP and the like are suppressed, and the skin can be kept in a stable state.

In addition, the CD39 expression promoter of the present invention can be used as a cosmetic having an action of suppressing skin inflammation, an action of suppressing excessive immune response, and an action of keeping the skin healthy. When applied to the skin, the expression level of CD39 in Langerhans cells increases, thereby promoting the decomposition of extracellular ATP released by external stimuli, aging, and the like. Therefore, skin inflammation, rough skin, itching, rashes, etc. are suppressed, and the skin can be kept in a healthy state. The cosmetic of the present invention can also be preferably applied for cosmetic purposes to prevent or improve skin aging.

The dose or additive amount of the CD39 expression promoter, external skin preparation, and cosmetic of the present invention cannot be defined unconditionally because it varies depending on the desired anti-inflammatory effect, administration method, age, etc., but the sclareol according to the present invention. , as sclareol glycoside or isomanool glycoside, it is preferably 0.02 ng/cm ² ·day to 100 μg/cm ² ·day per epidermal unit area (1 cm ² ), and 0.02 ng/cm ² ·day It is more preferable to be up to 10 μg/cm ² ·day.

The dosage forms of the CD39 expression promoter, external skin preparation and cosmetic of the present invention are not particularly limited, and examples include low-viscosity liquids, liquids such as lotions, emulsions, gels, pastes, creams, foams, packs, ointments, and powders. , aerosols or patches, as well as tablets, granules, capsules or liquids for internal use. In addition, the CD39 expression promoter according to the present invention can be applied to any of cosmetics, quasi-drugs, and pharmaceuticals. Examples of specific products include, but are not limited to, lotions, creams, milky lotions, serums, packs, cleansers, soaps, hair care agents, bath agents, makeup cosmetics, and the like.

When the sclareol, sclareol glycoside, or isomanool glycoside according to the present invention is blended as an active ingredient in the CD39 expression promoter, external preparation for skin, and cosmetic of the present invention, the amount is preferably 0.001. It is mass ppm to 1 mass %, more preferably 0.01 mass ppm to 0.1 mass %. By setting the blending amount of these compounds within this range, the compounds can be stably blended, the safety to the skin is high, and high CD39 expression promoting effect, inflammation suppressing effect or cosmetic effect can be exhibited. can.

In addition, the CD39 expression promoter, topical skin preparation and cosmetic of the present invention contain various commonly used medicinal ingredients such as moisturizing agents, whitening agents, anti-inflammatory agents, It can be used in combination with one or two or more medicinal agents selected from cell activators, UV protection agents, blood circulation promoters, antioxidants, and the like. Thereby, it is possible to further enhance the effect of the present invention.

Examples of moisturizing agents include aspartic acid, aspartate, alanine, arginine, alginic acid, sodium alginate, xylitol, glycine, glucose, cystine, cysteine, serine, polyethylene glycol, propylene glycol, dipropylene glycol, glycerin, polyglycerin, 1,3-butylene glycol, 1,2-pentanediol, hexylene glycol, sorbitol, POE methyl glucoside, maltitol, maltose, mannitol, lysine, honey, royal jelly, chondroitin sulfate, sodium chondroitin sulfate, hyaluronic acid, sodium hyaluronate , sodium acetyl hyaluronate, mucoitin sulfate, caronic acid, tranexamic acid, betaine, trehalose, chitosan, urea, ceramide, atelocollagen, cholesteryl-12-hydroxystearate, sodium lactate, bile salts, dl-pyrrolidone carboxylate, soluble One selected from collagen, diglycerin (EO) PO adduct, Angelica keiskei leaf extract, asparagus extract, Rose rose extract, quince seed extract, guava leaf extract, Yarrow extract and Melilot extract, etc. or Combinations of two or more can be mentioned.

Examples of whitening agents include L-ascorbic acid, L-ascorbate, L-ethyl L-ascorbate, L-ascorbic acid dipalmitate, L-ascorbic acid monopalmitate, and ascorbyl tetra-2-hexyldecanoate. , L-ascorbic acid-2-sodium sulfate, L-ascorbic acid phosphate magnesium salt, L-ascorbic acid phosphate sodium salt, L-ascorbic acid stearate, L-ascorbic acid-2-glucoside, DL- Vitamin C such as α-tocopherol-L-ascorbic acid diester dipotassium phosphate, placenta extract, kojic acid, ellagic acid, chamomile extract, fire thorn extract, rensch extract, tocotrienol, glutathione, arbutin, tranexamic acid, walnut extract, saxifrage extract, acerola extract, ageitsu extract, ferulic acid, disodium adenosine phosphate, linoleic acid, 4-n-butylresorcinol, 4-(4-hydroxyphenyl)-2-butanol, 5,5 1 or 2 selected from '-dipropyl-biphenyl-2,2'-diol, 4-methoxysalicylic acid potassium salt, hydroquinone, hydroquinone monobenzyl ether, pantetheine-s-sulfonic acid calcium, oil-soluble licorice extract, and the like Combinations of the above can be mentioned.

Examples of anti-inflammatory agents include aminocaproic acid, allantoin, indomethacin, bisabolol, saponin, lysotium chloride, azulene, guaiazulene, guaiazulene sulfonate, glycyrrhizic acid or derivatives thereof, glycyrrhetinic acid or derivatives thereof, salicylic acid or derivatives thereof, hinokitiol, One or a combination of two or more selected from photosensitizers, tranexamic acid or derivatives thereof, zinc oxide, turmeric extracts, Chinese ginger extracts, button extracts, litchi extracts and burnet extracts can be used.

Cell activators include, for example, aminobutyric acid, ginkgo biloba extract, fennel extract, mustard extract, carrot extract, clara extract, chlorella extract, saffron extract, soybean extract, turmeric extract, nicotinic acid , nicotinamide, pantothenic acid or derivatives thereof, biotin, retinol, leucine, photosensitizer, riboflavin or derivatives thereof, pyridoxine or derivatives thereof, or a combination of two or more thereof.

Examples of UV protection agents include oxybenzone, oxybenzosulfonic acid, tetrahydroxybenzophenone, dihydroxybenzophenone, dihydroxydimethoxybenzophenone, sodium dihydroxydimethoxybenzophenone sulfonate, 2-hydroxy-4-methoxybenzophenone, 2-hydroxy-4-methoxybenzophenone- 5-sulfonate, cinoxate, glyceryl mono-2-ethylhexanoate di-paramethoxycinnamate, methyl diisopropylcinnamate, methyl methoxycinnamate, octyl paramethoxycinnamate, isopropyl/diisopropyl paramethoxycinnamate Cinnamate mixture, glyceryl para-aminobenzoate, amyl para-dimethylaminobenzoate, octyl para-dimethylbenzoate, para-aminobenzoic acid (PABA), ethyl para-aminobenzoate, ethylene glycol salicylate, octyl salicylate, phenyl salicylate, homomenthyl salicylate, urocanine selected from acid, ethyl urocanate, ethylhexyl triazone, drometrizol, drometrizol trisiloxane, 4-methoxy-4'-tert-butyldibenzoylmethane, titanium oxide, talc, carmine, bentonite, kaolin and zinc oxide, etc. or a combination of two or more.

Blood circulation promoters include, for example, Japanese pepper extract, ginger extract, cnidium extract, chimp extract, capsicum extract, angelica extract, botan extract, nonylic acid werenylamide, benzyl nicotinate, and β nicotinic acid. - One or more selected from butoxyethyl ester, capsaicin, cantharis tincture, ictamol, caffeine, tannic acid, α-borneol, tocopherol nicotinate, inositol hexanicotinate, acetylcholine, cepharanthine, γ-oryzanol, etc. A combination of

Examples of antioxidants include astaxanthin, β-carotene, γ-oryzanol, kinetin, tocopherol, dibutylhydroxytoluene, flavonoids, SOD, catalase, fullerene, phytic acid, ferulic acid, chlorogenic acid, propyl gallate, and green tea extract. , rosemary extract, rosehip extract, calamus extract, horsetail extract, hamamelis extract, parsley extract, loquat leaf extract, grapefruit extract, meadowsweet extract, lychee extract, mugwort extract, peach leaf One or a combination of two or more selected from extracts, mango extracts, Botanpi extracts, pine bark extracts, platinum, ubiquinone, α-lipoic acid and the like can be mentioned.

In addition to the components described above, the CD39 expression promoter, topical skin preparation, and cosmetic of the present invention include components that are commonly used in topical skin preparations and cosmetics, as long as they do not impair the effects of the present invention. Water, fats and oils, waxes, hydrocarbons, fatty acids, higher alcohols, esters, plant extracts, vitamins, water-soluble polymers, surfactants, metallic soaps, alcohols, polyhydric alcohols, pH adjustment Ingredients such as agents, preservatives, fragrances, powders, thickeners, pigments, chelating agents, etc. can be appropriately added.

Furthermore, the CD39 expression-enhancing food and drink of the present invention contain the above-described sclareol, sclareol glycoside, or isomanool glycoside as an active ingredient. The food and drink composition according to the present invention includes supplement forms such as tablets, capsules, granules, and syrups, beverages such as soft drinks, fruit juice drinks, and alcoholic beverages, sweets such as candy, gum, cookies, biscuits, and chocolate, It can be in any form such as bread, porridge, cereals, noodles, jellies, soups, dairy products, and seasonings. When used as a food or drink in this way, it is possible to combine various other active ingredients, nutrients such as vitamins, minerals, amino acids, etc. within the range that does not affect the efficacy of the active ingredients of the present invention. The food and drink products of the present invention include supplements, health foods, functional foods, foods for specified health uses, and the like. In addition, the daily intake of the food and drink of the present invention is preferably 5 mg to 5000 mg (60 kg body weight), preferably 10 mg to 2000 mg (60 kg body weight), as sclareol, sclareol glycoside or isomanool glycoside. body weight) is more preferable.

EXAMPLES The present invention will be described in more detail below with reference to Examples, but the present invention is not limited by these Examples.

[Example 1]
1. Preparation of Dharmacillus extract After harvesting, the flower part was recovered from the dried Dharmacillus plant body and pulverized with a pulverizer. After 300 g of this pulverized material was immersed in 2 L of 90 w/w % ethanol aqueous solution and extracted for 3 days, the resulting extract was filtered through a cotton plug and concentrated under reduced pressure using an evaporator to obtain an ethanol extract (Fig. 1). . This ethanol extract was partitioned between 200 mL of ethyl acetate and 200 mL of water. After vigorously mixing with a separatory funnel, the mixture was allowed to stand, and only the ethyl acetate layer was collected (Fig. 1). The bilayer distribution was repeated a total of 4 times. The ethyl acetate layer was dried by an evaporator to obtain an ethyl acetate fraction (16.3 g).

[Example 2]
2. Isolation and Purification of Dermatophyllum japonicum Extract As shown in FIG. (63-200 μm), Merck), 200 mL hexane:ethyl acetate=9:1, 200 mL hexane:ethyl acetate=8:2, 200 mL hexane:ethyl acetate=7:3, 200 mL hexane: Elution was performed in the order of ethyl acetate=5:5, 300 mL of ethyl acetate and 300 mL of methanol, and six elution fractions Fr. 1-6 were obtained. Each Fr. 1:71 mg, Fr. 2: 177.3 mg, Fr. 3: 36.7 mg, Fr. 4: 299.5 mg, Fr. 5: 384.7 mg, Fr. 6: Fractionated with 248 mg. These six elution fractions were examined for their CD39 expression promotion effect using the same material and method as in Example 4, which will be described later. 4, Fr. 5 and Fr. 6 was confirmed to have the effect of promoting CD39 expression. Also, when these six fractions were subjected to ¹ H-NMR profiling, Fr. 2, Fr. 4, Fr. 5 and Fr. A characteristic peak was detected in 6. Therefore, Fr. 2, Fr. 4, Fr. 5 and Fr. Attempts were made to isolate compounds contained in the four fractions of 6.

As shown in FIG. 2, Fr. 2 to 90 mg was separated and subjected to preparative thin layer chromatography (PTLC: PLC silica gel 60 F ₂₅₄ , product of Merck & Co.). Toluene (100%) was used as a developing solvent, and five fractions Fr. 2-1 to 2-5 were obtained. Fr. 2-3 was subjected to PTLC using hexane:ethyl acetate=9:1 as a developing solvent. Fr. 2-3-2 was subjected to PTLC with benzene 100% and Fr. Compound c (21.4 mg) was isolated from 2-3-2-3. On the other hand, Fr. 2-4 was also subjected to PTLC using hexane:ethyl acetate=9:1 as a developing solvent. As a result, the resulting fraction Fr. Compound b (12.3 mg) was obtained from 2-4-2.

As shown in FIG. 3, Fr. 4 to 225 mg was taken and subjected to PTLC. Using hexane:ethyl acetate=3:1 as a developing solvent, four fractions Fr. 4-1 to 4-4 were obtained. Fr. 4-4 was subjected to PTLC again using chloroform:methanol=98:2 as a developing solvent, and five fractions Fr. 4-4-1 to 4-4-5 were obtained. The resulting Fr. 4-4-3 was subjected to PTLC with a developing solvent of hexane:ethyl acetate=2:1, and Fr. Compound e (17.9 mg) was isolated from 4-4-3-3. On the other hand, Fr. 4-4-5 was also subjected to PTLC using a developing solvent of hexane:ethyl acetate=2:1. As a result, Fr. 4-4-5-3 and Fr. Compound a (94.3 mg) and compound d (23.3 mg) were obtained from 4-4-5-4, respectively.

As shown in FIG. 4, Fr. 5 to 220 mg was taken and subjected to PTLC. Using hexane:ethyl acetate=3:2 as a developing solvent, four fractions Fr. 5-1 to 5-4 were obtained. Of these, Fr. 5-3 was further subjected to PTLC using chloroform:methanol=98:2 as a developing solvent, and Fr. Compound f (6.1 mg) was obtained from 5-3-2. Fr. 5-4 was subjected to PTLC using hexane:ethyl acetate=1:1 as a developing solvent. The resulting Fr. 5-4-5 was subjected to PTLC using chloroform:methanol=99:1 as a developing solvent, and Fr. Compound g (30.3 mg) was obtained from 5-4-5-3. On the other hand, Fr. 5-4-4 was subjected to PTLC using chloroform:methanol=98:2 as a developing solvent, and Fr. 5-4-4-4 was obtained. Fr. 5-4-4-4 was subjected to reversed-phase PTLC (PLC silica gel 60 RP-18 F _254S , product of Merck) using acetonitrile:water=6:4 as a developing solvent, and the resulting Fr. 5-4-4-4-5 was subjected to reverse phase PTLC with methanol:water=6:4. As a result, Fr. New compound i (4.4 mg) was isolated from 5-4-4-4-5-2.

As shown in FIG. 5, Fr. 6 to 230 mg was taken and subjected to PTLC. Using chloroform:methanol=9:1 as a developing solvent, Fr. Got 6-3. Fr. PTLC was repeated on 6-3 using ethyl acetate (100%) and ethyl acetate:methanol=97:3 as a developing solvent. The resulting Fr. 6-3-3-2 was subjected to reversed-phase PTLC using acetonitrile:water=6:4 as a developing solvent, and the resulting Fr. 6-3-3-2-4 was subjected to reverse phase PTLC with methanol:water=6:4. As a result, Fr. Compound h (23.9 mg) was obtained from 6-3-3-2-4-2.

[Example 3]
3. Structure Determination of Compounds ai Structural analysis of the nine compounds isolated in Example 2 was performed. For structural analysis, each compound was analyzed by ¹ H-NMR and ¹³ C-NMR using an NMR apparatus (JNM-EX400, product of JEOL Ltd.). CDCl ₃ (chloroform-D1, 99.8 atom % D, product of Merck Co.) and heavy water (99.9 atom % D, product of Merck Co.) were used as deuterium solvents during measurement. Chemical shifts obtained from ¹ H-NMR and ¹³ C-NMR were compared with a compound database (SciFinder, Institute of Chemical Information) for structural analysis. When there was no corresponding compound in the database, 2D NMR (HSQC, HMBC, COZY) was measured and combined with spectral data such as HR-ESI-MS to determine the planar structure of the compound. Furthermore, NOESY was measured to examine the relative configuration. Further, in structural analysis, an HGS molecular model (manufactured by Maruzen Publishing Co., Ltd.) and ChemDraw (manufactured by Hulinks Co., Ltd.) of molecular structure editor software were used. Spectra obtained by ¹ H-NMR analysis (observation frequency: 400 MHz) and ¹³ C-NMR analysis (observation frequency: 100 MHz) of each compound are shown in FIGS.

As a result of analysis, Fr. Compound c (Fr.2-3-2-3) isolated from 2 was germacrone and compound b (Fr.2-4-2) was isomanool.

Also, as a result of the analysis, Fr. Compound e isolated from 4 (Fr.4-4-3-3) is isomanool glycoside (13R)-Labda-7,14-diene 13-Oa-L-(4'-O-acetyl )-6′-deoxyidopyranoside, compound a (Fr.4-4-5-3) is sclareol, compound d (Fr.4-4-5-4) is isomanool glycoside (13R)- Labda-7,14-diene 13-Oa-L-6'-deoxyidopyranoside.

Also, as a result of the analysis, Fr. Compound f (Fr. 5-3-2) isolated from 5 was nevadencin, a flavonoid compound. Compound i (Fr. 5-4-4-5-2) is a newly discovered sclareol glycoside and is a novel compound represented by the following formula (V), and compound g (Fr. 5-4 -5-3) was an isomanool glycoside (13R)-Labda-7,14-diene 13-O-β-D-fucopyranoside.

Also, as a result of the analysis, Fr. Compound h (Fr.6-3-3-2-4-2) isolated from 6 is a sclareol glycoside (13R)-Labda-14(15)-ene-8,13-diol 13-O- α-L-6'-deoxyidopyranoside.

[Example 4]
4. Investigation of CD39 expression promotion effect in Langerhans cell-like cells Langerhans cells are cells that are difficult to isolate from human skin tissue and culture for a long time in a state that can be used for testing. Therefore, THP-1 cells, which are Langerhans-like cells, are often used as an alternative to Langerhans cells in tests involving Langerhans cells such as skin sensitization assays. In addition, THP-1 cells are also used as an alternative to Langerhans cells in Patent Documents 1 and 4 mentioned above. Therefore, in the present invention, THP-1 cells, which are Langerhans cell-like cells and express CD39, were used to examine the effect of promoting CD39 expression.

First, each of the compounds a to i isolated in Example 2 was dissolved in absolute ethanol to prepare a 10 mg/mL sample solution. In addition, commercially available sclareol (compound a) reagent (product code: S0916, product of Tokyo Chemical Industry Co., Ltd.) and nevadensine (compound f) reagent (product code: HY-N1377, product of MedChemexpress) were obtained, and these were also It was dissolved in anhydrous ethanol to prepare a 10 mg/mL sample solution.

About 100,000 THP-1 cells (ATCC (registered trademark) number: TIB-202) were seeded in each well (3.34 mL volume/well) of a commercially available 24-well cell culture plate, and RPMI containing 10% FBS was added. 998 μL of −1640 medium was added and cultured for 24 hours. After that, compounds a to i or sample solutions of commercially available reagents (each with a concentration of 10 mg/mL) and absolute ethanol were mixed at the formulation amounts shown in Table 1 below, and 2 μL each was added to the wells. Addition concentrations ranged from 0 μg/mL (control) to 20 μg/mL. After addition, culture was performed for 24 hours. Testing was done with N=2 for each concentration.

After culturing, THP-1 cells were recovered from each well, and total RNA was obtained using a total RNA purification kit (FastGene RNA purification kit, product of Nippon Genetics Co., Ltd.). Next, cDNA was synthesized from each total RNA using a cDNA synthesis kit (ReverTra Ace, product of Toyobo Co., Ltd.). Using this cDNA, the expression level of CD39 was measured by real-time PCR (QPCR).

QPCR is performed using a commercially available QPCR reagent kit (PrimeTime (registered trademark) Gene Expression Master Mix, product of Integrated DNA Technologies Co., Ltd.) and a QPCR measurement device (LightCycler (registered trademark) 96, product of Roche Diagnostics Co., Ltd.). went. As CD39 primers and probes for QPCR, the primers and probes of SEQ ID NOs: 1 to 3 shown in Table 2 below were used. On the other hand, human glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is a housekeeping gene, was selected as an internal standard, and the primers and probes of SEQ ID NOS: 4 to 6 shown in Table 2 below were used in place of the CD39 primers and probes described above. QPCR was performed on the All probes were added with IBFQ as a terminal quencher at the 3′ end and ZEN was added as an intermediate quencher between 9 and 10 bases from the 5′ end using a double quencher system probe (Integrated DNA Technologies Inc. product). The fluorescent dye added to the 5' end of the CD39 probe (SEQ ID NO: 3) was FAM, and the fluorescent dye added to the 5' end of the GAPDH probe (SEQ ID NO: 6) was HEX. The relative expression level was calculated from the expression level of GAPDH, which is an internal standard, and used as the expression level of CD39. The results are shown in FIGS. 15-17. The graphs of FIGS. 15 to 17 show the values when the CD39 expression level in the control THP-1 cells (concentration 0 μg/mL) to which no compound was added was taken as 1.00.

As shown in FIG. 15, when compound a (sclareol) was added to THP-1 cells, which are Langerhans-like cells, the expression level of CD39 increased by 1.2 times or more at an addition concentration of only 2 μg/mL (equivalent to 2 ppm). was found to increase to In the test using the sample isolated in Example 2, the expression level of CD39 reached 1.9 times at the addition concentration of 16 µg/mL (Fig. 15(a)). Moreover, when a commercially available sclareol reagent was used, a 2.55-fold increase in the CD39 expression level was observed at an addition concentration of 12 μg/mL (FIG. 15(b)).

On the other hand, compound b (isomanool), which has a bicyclic diterpene skeleton very similar to sclareol, interestingly did not increase the CD39 expression level (Fig. 15(c)). In addition, compound c (germacrone) having a sesquiterpene skeleton was not confirmed to have the effect of promoting CD39 expression similarly to isomanool (Fig. 15(d)). Furthermore, no CD39 expression-promoting effect was confirmed for compound f (nevadencin), which is a flavonoid compound (FIGS. 16(c) and (d)).

As described above, isomanool alone was not confirmed to have the effect of promoting CD39 expression, but compound d, compound e, and compound g, which are isomanool glycosides, were confirmed to have the effect of promoting CD39 expression (Fig. 16(a). and (b), FIG. 17(a)). Among them, compound g, which is a fucopyranoside, had the highest CD39 expression-promoting effect, and the CD39 expression level reached 1.95-fold at an addition concentration of 10 µg/mL (Fig. 17(a)). In addition, it was found that all isomanool glycosides showed peak effects at lower doses (about 8 to 12 μg/mL) than sclareol and sclareol glycosides.

In addition, compound h and compound i isolated as sclareol glycosides were also confirmed to have an excellent CD39 expression promoting effect (FIGS. 17(b) and (c)). Among them, compound h, which is 6-deoxide pyranoside, increased the expression level of CD39 by 2.11 times at a concentration of 20 μg/mL (FIG. 17(b)). In addition, compound i (4-O-acetyl-6-deoxyidopyranoside), which was discovered as a novel compound, was observed to increase the expression level of CD39 by 1.61 times at a concentration of 12 μg/mL ( FIG. 17(c)).

As described above, according to the present invention, it was clarified that by using sclareol, sclareol glycoside or isomanool glycoside, a high CD39 expression promoting effect can be obtained at a very low additive concentration on the order of ppm. .

The present invention is not limited to the above-described embodiments or examples, and includes various design changes within the scope of the invention within the scope of the invention described in the claims. .

The CD39 expression-promoting agent of the present invention is widely used in the medical and cosmetic fields because it has the effect of suppressing inflammation caused by external stimuli, aging, etc. and suppressing excessive immune response.

Claims (10)

Containing 2 mass ppm to 0.1 mass% of a compound having a bicyclic diterpene skeleton or a salt thereof, or a solvate thereof,
A CD39 expression promoter in skin Langerhans cells, wherein the compound having a bicyclic diterpene skeleton is at least one compound selected from the group consisting of sclareol, sclareol glycosides and isomanool glycosides. 2. The agent for promoting CD39 expression in skin Langerhans cells according to claim 1, wherein the sugar constituting said sclareol glycoside is 6-deoxydose or fucose . 2. The agent for promoting CD39 expression in skin Langerhans cells according to claim 1, wherein the sclareol glycoside is a compound represented by the following formula (I) or formula (II).

(In the formula, R ¹ represents a hydrogen atom or an acetyl group.)

(In the formula, ^R2 represents a hydrogen atom or an acetyl group.) 2. The agent for promoting CD39 expression in skin Langerhans cells according to claim 1, wherein the sugar constituting said isomanool glycoside is 6-deoxydose or fucose . The CD39 expression promoter in skin Langerhans cells according to claim 1, wherein the isomanool glycoside is a compound represented by the following formula (III) or formula (IV).

(In the formula, ^R3 represents a hydrogen atom or an acetyl group.)

(In the formula, ^R4 represents a hydrogen atom or an acetyl group.) An external skin preparation for promoting CD39 expression in skin Langerhans cells, comprising the CD39 expression promoter according to any one of claims 1 to 5 as an active ingredient. A cosmetic for promoting CD39 expression in skin Langerhans cells, comprising the CD39 expression promoting agent according to any one of claims 1 to 5 as an active ingredient. Food and drink for promoting CD39 expression in skin Langerhans cells, containing the CD39 expression promoting agent according to any one of claims 1 to 5. An inhibitor of inflammation caused by extracellular ATP, comprising the CD39 expression promoter according to any one of claims 1 to 5. A compound represented by the following formula (V), a salt thereof, or a solvate thereof.

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