JP6002510B2 - Estrogen receptor β activator - Google Patents

Description

  The present invention relates to an estrogen β receptor activator having high selectivity for estrogen receptor β.

It is known that when women reach menopause, the concentration of estrogen in the blood decreases significantly, and the function of the entire skin changes. In particular, collagen fibers are remarkably reduced in the dermis, and as a result, skin aging phenomena such as wrinkles and sagging become noticeable (Non-patent Documents 1 and 2).
It has been reported that female hormone replacement therapy (HRT) is performed on postmenopausal women, blood circulation is improved, and when estrogen is applied, wrinkles, elasticity, and water content are improved (Non-patent Document 3). 4). In addition, it has been reported that the application of estrogen suppresses the secretion of sebum from the sebaceous glands (Non-patent Document 5).

Here, it is known that estrogen binds to two types of estrogen receptors (ERα, ERβ) and exerts its action by working in concert. Estrogen first binds to the ligand binding domain (LBD) of ERα and ERβ and changes its conformation, followed by dimer (three combinations of ERα / α, ERβ / β and ERα / β). It forms (Nonpatent literature 6, 7). The ER that formed the dimer moves into the nucleus, binds to an estrogen response element existing on the genome, and after recruiting various coupling factors there, the transcriptional activity of the target gene is controlled (Non-Patent Document 8, 9).
It has been reported that the target genes of ERα and ERβ are mostly the same (Non-patent Document 10). However, recently, it has been suggested that ERα and ERβ have different physiological functions due to several different combinations of target genes (Non-patent Document 11). For example, it has been reported that ERα activates proliferation of breast cancer cells, while ERβ plays the opposite role of suppressing its proliferation (Non-patent Document 12). In addition, ERβ has been reported to have a function of protecting the skin from photoaging (Non-patent Document 13).
For these reasons, ERβ is an ideal molecule that can safely exert the action of estrogen. If ERβ can be selectively activated, it is useful for prevention and improvement of skin aging symptoms caused by estrogen reduction. Conceivable.

On the other hand, Heterosmilax erythrantha Bail is a plant of the Lily family, and the rhizomes are used to treat rheumatism, hemorrhoids, erythematosis, impetigo, syphilis, and to relieve tendon and bone pain, edema It is known to be useful for calming. Oroxylum indicum (L) Vent is a plant belonging to the family Cranaceae, and the seeds are useful as antitussive, expectorant, analgesic for sore throat, dry bronchitis, sound loss, vocalization, abdominal pain, etc. Inflammation, tonic, and astringent action are known to be useful in preventing diarrhea as well as infectious hepatitis, cystitis, swelling of the throat and painful eczema. Maclura cochinchinensis (Lour.) Corner is a mulberry plant with leaves useful for treating wounds, burns, neoplastic cervicitis, and dermatosis, and roots for improving blood circulation and treating cuts. It is known to be useful.
However, the relationship between these plants and estrogen receptor activation is not known.

Nicholas et al (2003) Am J Clin Dermatol 4: 371-378 Hall et al (2005) J Am Acad Dermatol 53: 555-68 Sator et al (2001) Maturitas 39: 43-55 PiCrard-Franchimon et al (1995) Maturitas 22: 151-154 Peter et al (1974) J Invest Dermatol 62: 191-201 Mukherjee et al (2010) Pharm Res 27: 1439-1468 Powell et al (2008) Proc Natl Acad Sci U S A 105: 19012-19017 Kulakosky et al (2002) J Mol Endocrinol 29: 137-152 Michael et al (2006) Genes Dev 2006 20: 1405–1428 Liu et al (2008) Proc Natl Acad Sci USA 105: 2604-2609 Liu et al (2008) Proc Natl Acad Sci U S A 105: 2604-2609 Helguero et al (2005) Oncogene 24: 6605-6616 Chang et al (2010) Mol Pharmacol 77: 744-750

  The present invention relates to providing an estrogen receptor β activator that is highly selective for estrogen receptor β.

  The present inventor constructed an evaluation system using a ligand binding domain (LBD) in the molecular structure of ERβ and a transcription activation ability evaluation system using an estrogen responsive element present on DNA, and among the estrogen receptors As a result of diligent research on substances that selectively enhance the activity of ERβ, it was found that there is an excellent estrogen receptor β-activating effect on Karasukibasankirai, Soriyayanoki and Kakatsugayu.

That is, the present invention relates to the following (1) to (4).
(1) Selected from the group consisting of plants selected from Heterosmilax erythrantha Bail, Oroxylum indicum (L) Vent, and Kakatsugayu (Maclura cochinchinensis (Lour.) Corner) and their extracts An estrogen receptor β activator comprising at least one active ingredient.
(2) A skin external preparation containing at least one selected from the group consisting of plants selected from Heterosmilax erythrantha Bail and Katsutsugayu (Maclura cochinchinensis (Lour.) Corner) and extracts thereof.
(3) Selected from the group consisting of plants selected from Heterosmilax erythrantha Bail, Oroxylum indicum (L) Vent, and Kakatsugayu (Maclura cochinchinensis (Lour.) Corner) and their extracts An agent for preventing or improving skin aging comprising at least one active ingredient.
(4) Selected from the group consisting of plants selected from Heterosmilax erythrantha Bail, Oroxylum indicum (L) Vent, and Kakatsugayu (Maclura cochinchinensis (Lour.) Corner) and their extracts A sebum secretion inhibitor comprising at least one active ingredient.

  The estrogen receptor β activator of the present invention selectively activates estrogen receptor β, and is safe without the risk of developing or worsening breast cancer. It is useful as a pharmaceutical, a quasi-drug, a cosmetic that can exert an effect of suppressing sebum secretion from the sebaceous gland, or as a material or preparation for blending into these.

It is a figure which shows the oil red dyeing | staining of a hamster auricle part tissue. It is a figure which shows the change of the sebaceous gland size by estradiol, PPT, or DPN application.

  In the present specification, “non-therapeutic” is a concept that does not include a medical act, that is, a treatment act on the human body by treatment.

  As used herein, “improvement” refers to improvement of a disease, symptom or condition, prevention or delay of worsening of the disease, symptom or condition, or reversal, prevention or delay of progression of a disease or symptom.

  As used herein, “prevention” refers to preventing or delaying the onset of a disease or symptom in an individual, or reducing the risk of developing an individual's disease or symptom.

  In this specification, Karasukisanshirai refers to Heterosmilax erythrantha Bail of the genus Karasibasankirai, Soriyayanoki refers to Oroxylum indicum (L) Vent of the genus Sorusayanoki, and Katsugayu refers to the genus Hari Point to Maclura cochinchinensis (Lour.) Corner.

Such a plant can be any part, for example, whole grass, leaves, stems, buds, flowers, buds, wood parts, bark, lichens, roots, rhizomes, corms, corms, tubers, seeds, fruits, mycorrhiza or Resin or the like, or a combination thereof can be used, but it is preferable to use a rhizome for Karasobasankirai, a bark or a seed for Sorusayanoki, and a root or a leaf for bonito.
Such a plant can be used as it is or after being dried, ground or the like. Moreover, the said site | part may be attached | subjected to an extraction process as it is, or may be attached | subjected to an extraction process, after grind | pulverizing, cut | disconnecting, or drying. The extract is derived from natural ingredients and has high safety.

Specific examples of the extraction means for obtaining the extract include solid-liquid extraction, liquid-liquid extraction, immersion, decoction, leaching, reflux extraction, ultrasonic extraction, microwave extraction, and stirring. In order to shorten the extraction time, solid-liquid extraction with stirring is desirable. An example of suitable conditions for this solid-liquid extraction is stirring at 100 to 400 rpm / min for 1 to 30 minutes.
In order to prevent the oxidation of the extract, a means for extracting under a so-called non-oxidizing atmosphere while removing dissolved oxygen by bubbling degassing or inert gas such as nitrogen gas may be used in combination.

  As the solvent for extraction, either a polar solvent or a nonpolar solvent can be used. Specific examples of the solvent include water; alcohols such as methanol, ethanol, propanol and butanol; polyhydric alcohols such as propylene glycol and butylene glycol; ketones such as acetone and methyl ethyl ketone; methyl acetate and ethyl acetate Esters; linear and cyclic ethers such as tetrahydrofuran and diethyl ether; polyethers such as polyethylene glycol; hydrocarbons such as squalane, hexane, cyclohexane and petroleum ether; aromatic hydrocarbons such as toluene; dichloromethane and chloroform And halogenated hydrocarbons such as dichloroethane; and supercritical carbon dioxide; pyridines; organic solvents such as oils and fats, other oils such as wax; and mixtures thereof. Preferable examples include water, alcohols, alcohol-water mixtures and hydrocarbons, with alcohol-water mixtures and hydrocarbons being more preferred. As the alcohol, an alcohol having 1 to 5 carbon atoms is preferable, and ethanol is more preferable. Hexane is preferred as the hydrocarbon.

  When an alcohol-water mixture is used as a solvent for extraction, the blending ratio (volume ratio) of alcohols and water is preferably 0.001 to 100: 99.999-0, preferably 5 to 95. : 95-5 is more preferable, 20-80: 80-20 are more preferable, 30-70: 70-30 are still more preferable, 40-60: 60-40 are still more preferable. In the case of an ethanol aqueous solution, the ethanol concentration is preferably 40% by volume or more, more preferably 45% by volume or more. And preferably it is 60 volume% or less, More preferably, it is 55 volume%. Moreover, Preferably it is 40-60 volume%, More preferably, it is 45-55 volume%.

  As a usage-amount of a solvent, Preferably it is 1 mL or more with respect to 1 g of each plant (dry mass conversion), More preferably, it is 8 mL or more. And preferably it is 100 mL or less, More preferably, it is 50 mL or less. Moreover, Preferably it is 1-100 mL, More preferably, it is 8-50 mL. The extraction time is preferably 1 minute or longer, more preferably 10 minutes or longer. And it is preferably 100 days or less, more preferably 10 days or less. Moreover, it is preferably 1 minute to 100 days, more preferably 10 minutes to 10 days. The extraction temperature at this time is preferably 0 ° C. or higher, more preferably 10 ° C. or higher, and further preferably 20 ° C. or higher. And preferably it is below the boiling point of a solvent, More preferably, it is 100 degrees C or less, More preferably, it is 90 degrees C or less. Moreover, Preferably it is 0 degreeC-solvent boiling point, More preferably, it is 10-100 degreeC, More preferably, it is 20-90 degreeC.

The plant extract thus obtained may be used as it is as the extract or fraction, may be used as a diluted solution diluted with an appropriate solvent, or may be a concentrated extract or dry powder, or prepared in a paste form. It may be a thing. Also, freeze-dry and dilute with a solvent usually used for extraction, such as water, ethanol, propylene glycol, butylene glycol, water / ethanol mixture, water / propylene glycol mixture, water / butylene glycol mixture, etc. It can also be used. It can also be used by encapsulating in vesicles such as liposomes or microcapsules.
Moreover, the plant extract can also use a commercial item.

  The plant extract may be a crude product as long as it conforms to food and pharmaceutical acceptable standards and exhibits the effects of the present invention. These purities may be increased by appropriately combining purification methods. Examples of the purification means include organic solvent precipitation, centrifugation, ultrafiltration membrane, high performance liquid chromatograph, column chromatograph and the like.

As shown in Examples below, the above plants or their extracts showed excellent estrogen receptor β activation in an in vitro evaluation system using a ligand binding domain (LBD) in the molecular structure of ERβ.
The evaluation system uses a difference in the sequence of ligand binding domain (LBD) of ERα and ERβ (J Endocrinol 163: 379-383 (1999)), which binds ligand to LBD and activates ER. This makes it possible to selectively evaluate ERβ activation.
Specifically, ERβ / ERα, which is a binding ratio between ERα and ERβ, is preferably 20 or more, more preferably 30 or more. And preferably it is 300 or less, More preferably, it is 200 or less. Moreover, Preferably it is 20-300, More preferably, it is the range of 30-200.

Moreover, the said plant or those extracts showed the effect | action which activates the transcription | transfer of a target gene in the transcriptional activation ability evaluation system via ER (beta) using an estrogen response element (Estrogen Response Element: ERE, AGGTCAnnnTGACCT).
And it is thought that the said plant or those extracts are effective in prevention, improvement, or treatment of various diseases and symptoms considered to be useful to activate ERβ. For example, since it has been reported that ERβ has a function of protecting the skin from photoaging (Non-patent Document 13), the above-mentioned plant or an extract thereof can be used for skin wrinkles, tarmi (relaxation), elasticity, It is considered to be effective for prevention, improvement or treatment of skin aging symptoms such as reduction of elasticity.
Moreover, as shown in the reference examples described later, since the size of the sebaceous glands in the hamster pinna decreases due to the activation of ERβ, the above plants or their extracts are considered to be effective in suppressing sebum secretion.

  Therefore, the plant or an extract thereof can be used for selective activation of ERβ, prevention, improvement or treatment of skin aging symptoms, and suppression of sebum secretion. The use can be in humans or non-human animals, or specimens derived therefrom, and can be therapeutic or non-therapeutic.

  In addition, the plant or an extract thereof can be used as an estrogen receptor β activator, an external preparation for skin, a skin aging preventive or ameliorating agent, a sebum secretion inhibitor (hereinafter referred to as estrogen receptor β, etc.) It can be used to produce these agents. At this time, for the estrogen receptor β activator, at least one selected from the group consisting of the above-mentioned plants and extracts thereof alone or in addition to this, a carrier appropriately selected as necessary Those which are acceptable in the below-mentioned objects to be blended may be used. In addition, the said formulation can be manufactured by a conventional method according to the target object which should be mix | blended.

  The estrogen receptor β activator, etc. is a human or veterinary drug, quasi-drug, makeup, which exhibits selective activation of ERβ, prevention, improvement or treatment of skin aging symptoms, and sebum secretion suppression effect. It may be an active ingredient of a food, food or feed, or may be a material or lumber used in combination with the pharmaceutical, quasi-drug, cosmetic, food or feed. As food, food and drinks with the concept of physiological functions such as activation of estrogen β receptor or prevention, improvement or treatment of skin aging symptoms, suppression of sebum secretion, etc., if necessary, functionality Includes food and drink, food and drink for the sick, and food for specified health use.

Here, dosage forms of the above-mentioned pharmaceuticals (including quasi-drugs) are tablets, capsules, granules, powders, syrups, intravenous injections, intramuscular injections, suppositories, inhalants, and transdermal absorption agents. , Eye drops, nasal drops, poultices, poultices, ointments, lotions, creams, oral preparations (dentifrices, liquid dentifrices, mouthwashes, gum massage creams, oral ointments, gargle tablets, troches, throats Or the like. The administration form may be either oral administration (internal use) or parenteral administration (external use, injection).
In order to prepare pharmaceutical formulations of such various dosage forms, the estrogen receptor β activator or the like alone or other pharmaceutically acceptable excipients, binders, extenders, disintegrants, Surfactants, lubricants, dispersants, buffers, preservatives, flavoring agents, fragrances, coating agents, carriers, diluents and the like can be used in appropriate combinations.

  Among these dosage forms, the preferred form is oral administration, and the content of the plant or the extract thereof in the preparation (in terms of the dry matter of the extract) is generally preferably 0.00001% by mass or more. More preferably, it is 0.0001 mass% or more. And preferably it is 10 mass% or less, More preferably, it is 1 mass% or less. Moreover, Preferably it is 0.00001-10 mass%, More preferably, it is 0.0001-1 mass%.

The food may be solid, semi-solid or liquid, for example, breads, cakes, noodles, confectionery, jelly, frozen foods, ice creams, dairy products, beverages and other various foods as described above. Examples include the same forms (tablets, capsules, syrups, etc.) as oral preparations.
To prepare various forms of foods, the estrogen receptor β activator alone or other food materials, solvents, softeners, oils, emulsifiers, preservatives, fragrances, stabilizers, colorants , Antioxidants, humectants, thickeners, and the like can be used in appropriate combinations. The content of the plant or the extract thereof in the food (in terms of a dry product of the extract) is generally preferably 0.01% by mass or more, more preferably 0.1% by mass or more, and further preferably 1 % By mass. And preferably it is 100 mass% or less, More preferably, it is 90 mass% or less, More preferably, it is 80 mass% or less. Moreover, Preferably it is 0.01-100 mass%, More preferably, it is 0.1-90 mass%, More preferably, it is 1-80 mass%.

Examples of the above feed include livestock feed used for cattle, pigs, chickens, sheep, horses, etc., feed for small animals used for rabbits, rats, mice, etc., feed for seafood used for tuna, eel, Thailand, Hamachi, etc. Pet foods used for dogs, cats, small birds, squirrels and the like.
In addition, when producing feed, in addition to the estrogen receptor β activator of the present invention, meat such as cattle, pigs and sheep, proteins, grains, bran, potatoes, sugars, vegetables, vitamins In addition, feed materials generally used for minerals and the like, and gelling agents, shape-preserving agents, pH adjusters, seasonings, preservatives, nutrient reinforcements and the like generally used for feed can be used in combination.
Further, the content of the plant or the extract thereof in the feed varies depending on the use form, but is usually preferably 0.0001% by mass or more, more preferably 0.001% by mass or more, in terms of dry matter, More preferably, it is 0.01 mass% or more. And preferably it is 20 mass% or less, More preferably, it is 10 mass% or less, More preferably, it is 5 mass% or less. Moreover, Preferably it is 0.0001-20 mass%, More preferably, it is 0.001-10 mass%, More preferably, it is 0.01-5 mass%.

  The dose or intake of the above-mentioned pharmaceuticals and the like may vary according to the condition, weight, sex, age or other factors of the subject. (In terms of product) is preferably 0.01 mg / kg or more, particularly preferably 0.1 mg / kg or more per day. And it is preferably 100 mg / kg or less, particularly preferably 10 mg / kg or less. Moreover, Preferably it is 0.01-100 mg / kg, Most preferably, it is 0.1-10 mg / kg.

  The cosmetics (including quasi-drugs) can be used as external preparations for skin, cleaning agents, makeup cosmetics, etc., and depending on the method of use, lotions, emulsions, gels, creams, ointments, powders, It can be provided in various dosage forms such as granules. Such cosmetics in various dosage forms include at least one selected from the group consisting of the above-mentioned plants and extracts thereof, and oily ingredients, humectants, powders, pigments, which can be blended with skin cosmetics. Emulsifiers, solubilizers, detergents, UV absorbers, thickeners, drugs (eg anti-inflammatory agents, bactericides, antioxidants, vitamins, fat metabolism promoting action or uncoupling protein expression promoting action are known Or a natural product), a fragrance, a resin, an antifungal agent, a plant extract, an alcohol or the like.

  The above-mentioned plants or their extracts in the total amount of the cosmetic are usually preferably 0.0001% by mass or more, more preferably 0.001% by mass or more, and still more preferably 0.001% by mass or more in terms of dry matter of the extract. 01% by mass or more. And preferably it is 20 mass% or less, More preferably, it is 10 mass% or less, More preferably, it is 5 mass% or less. Moreover, Preferably it is 0.0001-20 mass%, More preferably, it is 0.001-10 mass%, More preferably, it is 0.01-5 mass%.

With respect to the above-described embodiment, the following aspects are disclosed in the present invention.
<1> A plant selected from Heterosmilax erythrantha Bail, Oroxylum indicum (L) Vent, and Kakatsugayu (Maclura cochinchinensis (Lour.) Corner), and selected from the group consisting of these extracts An estrogen receptor β activator comprising at least one active ingredient.
<2> An external preparation for skin containing at least one selected from the group consisting of plants selected from Heterosmilax erythrantha Bail and bonito (Maclura cochinchinensis (Lour.) Corner) and extracts thereof.
<3> Selected from the group consisting of plants selected from Heterosmilax erythrantha Bail, Oroxylum indicum (L) Vent, and Kakatsugayu (Maclura cochinchinensis (Lour.) Corner) and their extracts An agent for preventing or improving skin aging comprising at least one active ingredient.
<4> Selected from the group consisting of plants selected from Heterosmilax erythrantha Bail, Oroxylum indicum (L) Vent, and Kakatsugayu (Maclura cochinchinensis (Lour.) Corner) and their extracts A sebum secretion inhibitor comprising at least one active ingredient.
<5> A plant selected from Heterosmilax erythrantha Bail, Oroxylum indicum (L) Vent, and Kakatsugayu (Maclura cochinchinensis (Lour.) Corner) for producing an estrogen receptor β activator And at least one use selected from the group consisting of extracts thereof.
<6> A plant selected from Heterosmilax erythrantha Bail and Kakatsugayu (Maclura cochinchinensis (Lour.) Corner) for producing a skin external preparation, and at least selected from the group consisting of extracts thereof One use.
<7> Plants selected from Heterosmilax erythrantha Bail, Oroxylum indicum (L) Vent, and Kakatsugayu (Maclura cochinchinensis (Lour.) Corner) for producing an agent for preventing or improving skin aging, And at least one use selected from the group consisting of those extracts.
<8> Plants selected from Heterosmilax erythrantha Bail, Oroxylum indicum (L) Vent), and Kakagaya (Maclura cochinchinensis (Lour.) Corner) for producing sebum secretion inhibitors, and those Use of at least one selected from the group consisting of:
<9> Plants selected from Heterosmilax erythrantha Bail, Oroxylum indicum (L) Vent, and Kakatsugayu (Maclura cochinchinensis (Lour.) Corner) for use as an estrogen receptor β activator And at least one selected from the group consisting of extracts thereof.
<10> At least selected from the group consisting of a plant selected from Heterosmilax erythrantha Bail and katsugayu (Maclura cochinchinensis (Lour.) Corner) and an extract thereof for use in a skin external preparation One kind.
<11> A plant selected from Heterosmilax erythrantha Bail, Oroxylum indicum (L) Vent, and Kakatsugayu (Maclura cochinchinensis (Lour.) Corner) for use in an agent for preventing or improving skin aging, And at least one selected from the group consisting of those extracts.
<12> Plants selected from Heterosmilax erythrantha Bail, Oroxylum indicum (L) Vent, and Katsugayu (Maclura cochinchinensis (Lour.) Corner) for use in sebum secretion inhibitors, and those At least one selected from the group consisting of
<13> selected from the group consisting of plants selected from Heterosmilax erythrantha Bail, Oroxylum indicum (L) Vent, and Kakatsugayu (Maclura cochinchinensis (Lour.) Corner) and their extracts A method for activating estrogen receptor β, comprising administering or ingesting at least one species to humans or animals.
<14> Selected from the group consisting of plants selected from Heterosmilax erythrantha Bail, Oroxylum indicum (L) Vent, and Kakatsugayu (Maclura cochinchinensis (Lour.) Corner) and their extracts A method for preventing or improving skin aging, wherein at least one kind is administered or ingested to a human or an animal.
<15> A plant selected from Heterosmilax erythrantha Bail, Oroxylum indicum (L) Vent, and Kakatsugayu (Maclura cochinchinensis (Lour.) Corner), and selected from the group consisting of these extracts A method for inhibiting sebum secretion, wherein at least one kind is administered or ingested to a human or an animal.
<16> The method according to any one of <13> to <15>, which is a non-therapeutic method.
<17> In the above items <1> to <16>, the plant is preferably used in the form of rhizome for Heterosmilax erythrantha Bail, bark or seed for Oroxylum indicum (L) Vent, (Maclura cochinchinensis (Lour.) Corner) preferably uses roots or leaves, respectively.
<18> In the above items <1> to <17>, the extraction solvent for obtaining the plant extract is preferably water, alcohols, alcohol-water mixtures, hydrocarbons, alcohol-water mixtures, hydrocarbons. Are more preferred. As the alcohol, an alcohol having 1 to 5 carbon atoms is preferable, and ethanol is more preferable. As the hydrocarbon, hexane is more preferable.
<19> In the above items <1> to <18>, the extraction solvent for obtaining the plant extract is preferably an alcohol-water mixture such as an ethanol aqueous solution, and in the case of an ethanol aqueous solution, the ethanol concentration is preferably Is 40% by volume or more, more preferably 45% by volume or more. And preferably it is 60 volume% or less, More preferably, it is 55 volume%. Further, it is preferably 40% by volume or more and 60% by volume or less, more preferably 45% by volume or more and 55% by volume or less.
<20> In the above <1> to <19>, the plant extract preferably uses an extraction solvent of 8 to 50 parts by mass with respect to 1 part by mass of the plant, and is 20 to 90 ° C. In the following, it is extracted for 10 minutes to 10 days.

Reference Example 1 Suppression of sebum secretion
<Application of compound to hamster pinna>
Using a 6-week-old male Syrian hamster (manufactured by Japan SLC), 5 control groups to which only 10% glycerin-containing ethanol solvent was applied, and 100 nM estradiol (manufactured by Wako Pure Chemical Industries, Ltd., the same shall apply hereinafter) E2) 5 groups, PPT group to which 160 nM 1,3,5-tris (4-hydroxyphenyl) -4-propyl-1H-pyrazole (PPT, ERα selective agonist, manufactured by Tocris, the same applies below) 5 DPN groups to which 700 nM 2,3-bis (4-hydroxyphenyl) propionitrile (DPN, ERβ selective agonist, manufactured by Tocris, the same applies below) is prepared, and 100 μL of each solution is prepared. Was applied to the auricular region once a day for 2 consecutive weeks (PPT and DPN application concentrations were set based on the EC50 value of estradiol). Thereafter, the auricle was collected with reference to past reports, embedded in OCT compound, and stored at −80 ° C. until use.

<Oil red staining (neutral lipid staining method)>
From the frozen auricular tissue embedded in the OCT compound, a cryosection was prepared with a thickness of 6 μm using a cryostat and affixed on a glass slide (one slide for one animal). To fix the tissue, it was immersed in 10% neutral buffered formalin for 1 minute and then immersed in distilled water (DW) to remove the OCT compound. Next, after dipping in 60% isopropanol to remove excess isopropanol, 100 μL of oil red staining solution was mounted and staining was performed for 5 minutes. After completion of the staining, washing was performed in the order of 60% isopropanol and DW, and sealing was performed using a water-soluble mounting medium.

<Extraction of oil red staining positive area>
In order to extract oil red staining positive areas in the sebaceous glands, tissue images were taken under a microscope, and image analysis was performed using Image Pro Plus (Media Cybernetics), which is image analysis software. Five sebaceous glands including the duct portion were randomly selected from the captured image, and the number of pixels of the oil red staining positive region (red) in one sebaceous gland was calculated.

<Tissue image after oil red staining>
The tissue image after oil red staining is shown in FIG. The staining intensity in the vicinity of the basal cells (outermost layer cells) was weak, and the staining intensity tended to increase as the differentiation process of mature cells progressed (differentiation progressed toward the center of sebaceous glands). A decrease in sebaceous gland size was also visually confirmed in the estradiol group, the PPT group, and the DPN group compared to the control group.

<Measurement of sebaceous gland size>
The results of analyzing the sebaceous gland size of each group using Image Pro Plus are shown in FIG. Statistical significance test was analyzed using Dunnett's method. As a result, the sebaceous gland size was significantly reduced in the estradiol group, PPT group, and DPN group compared to the control group (p <0.01: control vs estradiol, control vs PPT, p <0.05: control vs DPN). . Although no significant difference was observed, the PPT group suppressed sebaceous gland size slightly more strongly than the DPN group.
This suggests that ERβ activation is effective in suppressing sebum secretion.

Reference Example 2 Construction of evaluation system
pBIND-ERα and pGL4.35 [luc2P / 9XGAL4UAS / Hygro] were purchased from Promega. pBIND-ERβ was constructed by cloning the LBD of ERβ (amino acid sequence: 222-531) into the pBIND vector.
In this evaluation system, the transcriptional activity of firefly luciferase encoded by pGL4.35 increases only when the test substance reacts with ER LBD (for details, see Pharm Res 27: 1439-1468 (2010)).

HEK293 cells are seeded on a 96-well plate at a cell density of 4.0 × 10 ⁴ cells / well, and 12 hours later, the culture medium (DMEM containing 5% charcoal-treated serum) is replaced with pBIND-ERα or pBIND-ERβ. Lipofectamine 2000 (Invtrogen) was used together with pGL4.35 [luc2P / 9XGAL4UAS / Hygro] and introduced into cells according to the protocol attached to the product. 24 hours after the introduction, 50 (v / v)% ethanol as a negative control and estradiol, PPT or DPN as a positive control were respectively added to the final concentrations shown in Table 1, and further cultured for 24 hours.
For the activity measurement of firefly luciferase (an index of ER activity) and Renilla luciferase (an index of efficiency of introducing plasmid), a Dual-Glo ™ Luciferase Assay System (Promega) was used, and the luciferase activity was measured according to the attached protocol. The luminescence intensity of firefly luciferase divided by the luminescence intensity of Renilla luciferase was calculated as a relative light unit (RLU). The value of each binding activity was shown as a relative value when RLU was 1 when estradiol as a positive control was added at a final concentration of 10 nM.
The results are shown in Table 1. The numerical values in the column for ERα and ERβ indicate the binding activity when each compound is added to the cell into which pBIND-ERα is introduced and the cell into which pBIND-ERβ is introduced. The evaluation of the binding activity enhancing effect was performed by comparison with the binding activity value when a 50 (v / v)% ethanol aqueous solution as a negative control was added.

  As a result, in the cells into which pBIND-ERα was introduced, the positive activity of estradiol and ERα selective agonist, PPT, enhanced the binding activity with ERα. On the other hand, in the cells into which pBIND-ERβ was introduced, estradiol and an ERβ selective agonist, DPN, enhanced the binding activity with ERβ, but PPT did not enhance the binding activity with ERβ. From this, it was confirmed that a substance that selectively enhances the binding activity with ERβ can be searched by the evaluation system.

Example 1
Selective enhancement of ERβ binding activity in each extract
<Preparation of plant extract>
(Production Example 1) Preparation of extract of Heterosmilax erythrantha Bail 1000 mL of 50 (v / v)% ethanol-water mixed solution was added to 100 g of Karasiba Sankirai rhizome and extracted at room temperature for 7 days. After extraction, insoluble matters were filtered to obtain an extract. The extract was concentrated under reduced pressure to obtain 9.23 g of a dried extract. The obtained dried extract was dissolved in a 50% (v / v)% ethanol-water mixed solution so as to have an evaporation residue of 1% to prepare a sample for evaluating the activity of Karasiba sankirai.

(Production Example 2) Preparation of extract of Oroxylum indicum (L) Vent To 100 g of bark and seeds of Sorusayanoki, 1000 mL of 50 (v / v)% ethanol-water mixed solution was added and extracted at room temperature for 7 days. After extraction, insoluble matters were filtered to obtain an extract. The extract was concentrated under reduced pressure to obtain 21.68 g of the dried extract. In the same manner as in the above (1), a sample for evaluating the activity of Sorusayanoki was prepared from the obtained extract.

(Production Example 3) Preparation of extract of Maclura cochinchinensis (Lour.) Corner To 100 g of roots and leaves of bonito, 1000 mL of a 50 (v / v)% ethanol-water mixed solution was added and extracted at room temperature for 7 days. After extraction, insoluble matters were filtered to obtain an extract. The extract was concentrated under reduced pressure to obtain 10.01 g of a dried extract. In the same manner as in the above (1), a bonito activity sample was prepared from the obtained extract.

<Evaluation method>
HEK293 cells are seeded on a 96-well plate at a cell density of 4.0 × 10 ⁴ cells / well. After 12 hours, the culture medium (DMEM containing 5% charcoal-treated serum) is exchanged, and pBIND-ERα or pBIND-ERβ Was introduced into cells together with pGL4.35 [luc2P / 9XGAL4UAS / Hygro] using Lipofectamine 2000 (Invtrogen) according to the protocol attached to the product. 24 hours after introduction, 50 (v / v)% ethanol aqueous solution as a negative control, estradiol as a positive control (final concentration 10 nM), or the plant extract prepared in the above Production Examples 1 to 3 was used as the final concentration shown in Table 2. And further cultured for 24 hours.
Luciferase activity was measured in the same manner as described above, and the value obtained by dividing the emission intensity of firefly luciferase by the emission intensity of Renilla luciferase was calculated as a Relative light unit (RLU). Each activity value is shown as a relative value when RLU in the positive control is 1. The evaluation of the binding activity enhancing effect was performed by comparison with the binding activity value when a 50 (v / v)% ethanol aqueous solution as a negative control was added. The results are shown in Table 2.

<Result>
From Table 2, it was recognized that the plant extracts of the above Production Examples 1 to 3 have a significantly higher binding activity with ERβ than ERα.
From this, it was confirmed that the plant extract has an action of binding and activating ERβ with high selectivity.

Reference Example 3 Construction of an evaluation system for transcription activation ability via ERβ
It is known that ERβ binds to ERE (AGGTCAnnnTGACCT) existing on the genome and controls transcription of a target gene. Therefore, a system that can evaluate the presence of ERβ-mediated transcriptional activation using ERE was constructed.
pERE (firefly luciferase) was purchased from SABiosciences. pRL-CMV (Renilla luciferase) was purchased from Promega.

HEK293 cells are seeded on a 96-well plate at a cell density of 4.0 × 10 ⁴ cells / well. After 12 hours, the culture medium (DMEM containing 5% charcoal-treated serum) is replaced, and pERE, pRL-CMV and pcDNA3− ERβ was introduced into cells using Lipofectamine 2000 (Invtrogen) according to the protocol attached to the product. 24 hours after introduction, 50 (v / v)% ethanol as a negative control and estradiol, PPT, or DPN as positive controls were added to the final concentrations shown in Table 3, respectively, and further cultured for 24 hours. For the activity measurement of firefly luciferase (index of transcriptional activation ability) and Renilla luciferase (index of efficiency of introduction of plasmid), Dual-Glo ™ Luciferase Assay System (Promega) was used according to the attached protocol. The luminescence intensity of firefly luciferase divided by the luminescence intensity of Renilla luciferase was calculated as a relative light unit (RLU). The ability to activate transcription through ERβ was evaluated by comparison with RLU when a 50 (v / v)% aqueous ethanol solution as a negative control was added.

  As a result, when PPT or DPN was added, DPN had higher ERβ-mediated transcription activation ability than PPT. From this, it was confirmed that the said evaluation system is useful for evaluating the transcriptional activation ability via ERβ.

Example 2 Evaluation of transcription activation ability via ERβ in each extract
<Evaluation method>
HEK293 cells are seeded on a 96-well plate at a cell density of 4.0 × 10 ⁴ cells / well. After 12 hours, the culture medium (DMEM containing 5% charcoal-treated serum) is replaced, and pERE, pRL-CMV and pcDNA3− ERβ was introduced into cells using Lipofectamine 2000 (Invtrogen) according to the protocol attached to the product. 24 hours after introduction, 50 (v / v)% ethanol as a negative control, estradiol as a positive control, or each plant extract prepared in the above Production Examples 1 to 3 was added so as to have a final concentration described in Table 4, The culture was further continued for 24 hours. Luciferase activity was measured in the same manner as described above, and the value obtained by dividing the emission intensity of firefly luciferase by the emission intensity of Renilla luciferase was calculated as a Relative light unit (RLU). The ability to activate transcription through ERβ was evaluated by comparison with RLU when a 50 (v / v)% aqueous ethanol solution as a negative control was added.

<Result>
From Table 4, the plant extract of the above Production Examples 1 to 3 was recognized to have transcription activation ability via ERβ. From the above, it was confirmed that the plant extract binds to ERβ with high selectivity and has an action of activating transcription of the target gene.

Claims (2)

An estrogen receptor β activator comprising an extract of Karasukisan Sankirai as an active ingredient. Sebum secretion inhibitor as an active ingredient an extract of crow Kiba San hate.