JP6091112B2 - Retinoic acid-like agent - Google Patents

Description

  The present invention relates to a retinoic acid-like agent.

It is known that retinoic acid and its related compound, retinoid (retinoid), exert morphogenesis control action, cell differentiation growth control action and the like in vivo. Retinoic acid has stereoisomers such as all-trans retinoic acid (tretinoin), 9-cis retinoic acid (aritretinoin), and 13-cis retinoic acid (isotretinoin).
Retinoic acid is a natural ligand of the nuclear receptor retinoic acid receptor (RAR) and retinoid X receptor (RXR). When retinoic acid binds to RAR and RXR in vivo, RAR and RXR form a heterodimer. This binds to the promoter of the target gene group as a ligand-inducible transcription factor, thereby controlling the expression of the target gene group at the transcription level.

There are three subtypes (α, β, γ) in RAR and RXR, and RARγ and RXRα are mainly expressed in the skin (Non-patent Documents 1 and 2). RARγ is involved in suppression of differentiation of keratinocytes, and adapalene, which is one of RARγ selective agonists, specifically binds to RARγ and suppresses differentiation of epidermal keratinocytes. It is said that it can treat the face that is the first rash of acne.
In addition, when retinoic acid and retinoid are used externally, it is said that sebum secretion from the sebaceous gland can be suppressed by controlling PPAR involved in the proliferation / differentiation of sebaceous cells and the production of IL-1 and TNF-α. .

Furthermore, when retinoic acid is applied externally, epidermal turnover is accelerated, severe desquamation is observed, and at the same time, melanin is discharged around the epidermal basal layer, and dramatic epidermal melanin is released in a short period of about 2 weeks. It is reported that a decrease is observed (Non-Patent Documents 3 and 4), and retinoic acid suppresses dermis thinning (skin aging) due to photoaging by promoting collagen production in dermal fibroblasts and suppressing MMP. (Non-Patent Documents 5, 6, and 7).
Therefore, if RARγ is activated in the skin and can exhibit a retinoic acid-like action, it is considered useful for suppressing sebum secretion, acne, skin aging symptoms, epidermal pigmentation, and the like.

On the other hand, Polygonum hydropiper (Polygonum hydropiper) is a plant belonging to the polygonaceae (Polygonaceae), an anti-inflammatory, detoxification, diuretic, stop diarrhea, fever, insect bites, for the treatment of such meal, Sina forget-me-not (Cynoglossum amabile Staf et Drum.) Is a plant belonging to the family Boraginaceae and is known to be useful for the treatment of cough, vomiting, and convulsions, respectively.
However, the relationship between these plants and retinoic acid-like activity is not known.

Elder et al. J Invest Dermatol. 1992; 98 (6 Suppl): 36S-41S Mangelsdorf et al. Genes Dev. 1992; 6 (3): 329-44. Xiao, J .; H et al. , EMBO J 18 1999: 1539-1548. Yoshimura, K .; et al. , Plast Reconstr Surg 2000; 105: 1097-1108 Griffiths CE et al. , Skin Pharmacol. 1993; 6 (suppl 1): 70-77. Kligman AM et al. , Skin Pharmacol. 1993; 6 (Suppl 1): 78-82. Kligman, A et al. , J .; Am. Acad. Dermatol. 1986; 15: 836-859

  The present invention relates to providing a retinoic acid-like agent.

  The present inventor uses a ligand binding domain (LBD) in the molecular structure of RARγ to construct a system in which luciferase is emitted from a cell only when a substance (ligand) is bound to the LBD of RARγ. As a result of diligent research on substances that increase their activity by binding to, that is, exhibit a retinoic acid-like action, they have found that Yanagitade and Shinawasulenagus have excellent retinoic acid-like activity.

That is, the present invention relates to the following (1) to (5).
(1) A retinoic acid-like agent comprising as an active ingredient at least one selected from the group consisting of Yanagita, Shinawasunagus, and extracts thereof.
(2) The sebum secretion inhibitor which uses as an active ingredient at least 1 sort (s) selected from the group which consists of Yanagita, Shinawasunagusa, and those extracts.
(3) An acne preventive or ameliorating agent comprising at least one selected from the group consisting of Willow grove, Shinawasunagusa and extracts thereof as an active ingredient.
(4) A skin aging preventive or ameliorating agent comprising as an active ingredient at least one selected from the group consisting of Yanagita, Shinawasunagusa, and extracts thereof.
(5) An external preparation for skin containing cinnabiaceae or an extract thereof.

  Since the retinoic acid-like agent of the present invention has an action of activating RARγ and suppressing sebum secretion from the sebaceous glands, skin aging symptoms such as acne, wrinkles, and reduction in skin, melanin pigments such as dullness and spots It is useful as a pharmaceutical, a quasi-drug, or a cosmetic that can exert a preventive or ameliorating effect on a disease or the like, or as a material or preparation for blending into these.

The figure which shows the sebum secretion inhibitory effect by a retinoic acid or a RARγ selective agonist. The figure which shows the sebum secretion suppression effect of this invention plant extract. The figure which shows the MMPs activity inhibitory effect of this invention plant extract.

  In the present specification, “retinoic acid-like” or “retinoic acid-like activity” means physiological activities similar to those of retinoic acid, such as binding to RAR and RXR, particularly RARγ, and activation thereof.

  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 the present specification, the Polygonum hydropiper, refers to Polygonum hydropiper of polygonaceae Polygonum, and Sina Myosotis, Cynoglossum amabile Staf et Drum of boraginaceae cynoglossum. Point to. As a willow tree, a herbal medicine, water buffalo obtained from a willow tree as a base plant may be used. Moreover, you may use the herbal medicine and a deer obtained as a base plant as a Shinawasunagusa.

Any of such plants can be used in any part, for example, whole grass, leaves, stems, buds, flowers, buds, roots, rhizomes, calluses, corms, tubers, seeds, etc., or combinations thereof. It is preferable to use the above-ground parts such as leaves and stems.
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. Examples of suitable conditions for this solid-liquid extraction include stirring at 100 to 400 r / 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.

In the case of using an alcohol-water mixture as a solvent for extraction, the alcohol concentration is not particularly limited, but is preferably 0.001% by volume or more, more preferably 5% by volume or more, more preferably 20% by volume. Or more, more preferably 30% by volume or more, more preferably 40% by volume or more, and preferably 95% by volume or less, more preferably 80% by volume or less, more preferably 70% by volume or less, more preferably 60% by volume. It is as follows. For example, 5-95 volume%, 20-80 volume%, 30-70 volume%, 40-60 volume% is mentioned.

  As a usage-amount of a solvent, 1-100 mL with respect to 1 g of each plant (dry mass conversion), Furthermore, 8-50 mL is preferable, As extraction time, 1 minute-100 days are preferable, and 1 minute-3 days are more preferable. The extraction temperature at this time is 0 ° C. to the boiling point of the solvent, more preferably 20 to 100 ° C., and further preferably 40 to 90 ° C.

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 of the present invention may be a crude product as long as it conforms to food and pharmaceutical acceptable standards and exhibits the effects of the present invention, and the obtained crude product is publicly known. These separation and purification methods may be combined as appropriate to increase their purity. 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 plant of the present invention or an extract thereof showed excellent retinoic acid-like activity in an in vitro evaluation system using LBD in the molecular structure of RARγ.
The evaluation system enables the evaluation of retinoic acid-like activity by discriminating the binding of a ligand to LBD, that is, the activation of RARγ, by the luciferase activity generated when the ligand binds to LBD. .
Moreover, the plant of this invention or those extracts showed the effect | action which suppresses the secretion of sebum in sebaceous gland cells.
And it is thought that the plant of this invention or those extracts is effective in the prevention, improvement, or treatment of various symptom considered that it binds and activates RARγ. For example, the plant of the present invention or an extract thereof is effective for the prevention or improvement of acne, the prevention or improvement of skin aging symptoms such as wrinkles and reduction of elasticity, and the prevention or improvement of melanin pigment diseases such as dullness and spots. It is believed that there is. “Wrinkles” are those that have deep grooves that are difficult to recover due to prolonged UV exposure, drying, aging, etc., that is, “deep wrinkles” that can be improved with moisturizers, etc. It is a concept including "

  Therefore, the plant of the present invention or an extract thereof has retinoic acid-like activity, inhibition of sebum secretion, prevention or improvement of acne, prevention or improvement of skin aging symptoms such as wrinkles and reduction of skin, dullness, melanin such as spots, etc. It can be used for prevention or amelioration of pigmented diseases. The use can be in humans or non-human animals, or specimens derived therefrom, and can be therapeutic or non-therapeutic.

  That is, the plant of the present invention or an extract thereof is used as a retinoic acid-like agent, a sebum secretion inhibitor, an acne prevention or improvement agent, a skin aging prevention or improvement agent, and a skin external preparation (hereinafter referred to as a retinoic acid-like agent). And can be used to produce these agents. At this time, the retinoic acid-like agent or the like includes at least one selected from the group consisting of the plant of the present invention and an extract thereof alone, or in addition to this, a carrier or the like appropriately selected as necessary. In addition, what is permitted in the below-mentioned target object 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 retinoic acid-like agent itself has retinoic acid-like activity, inhibition of sebum secretion, prevention or improvement of acne, prevention or improvement of skin aging symptoms such as wrinkles and reduction of skin tension, melanin pigment disease such as spots, etc. It may be a drug for humans or animals, quasi-drugs, cosmetics or foods that exerts an effect of preventing or improving the above, or is used in combination with such pharmaceuticals, quasi-drugs, cosmetics, etc. It may be a raw material or a preparation.
As food, retinoic acid-like activity, suppression of sebum secretion, prevention or improvement of acne, prevention or improvement of skin aging symptoms such as reduction of wrinkles and tension, prevention or improvement of melanin pigment diseases such as dullness and spots, etc. It includes food and drink, functional food and drink, food and drink for the sick, food for specified health use, etc., which are based on the concept of physiology and display that effect as necessary.

The above pharmaceutical products (including quasi-drugs) include tablets, capsules, granules, powders, syrups, intravenous injections, intramuscular injections, suppositories, inhalants, transdermal absorption agents, eye drops, It may be any of nasal drops, poultices, poultices, ointments, lotions, creams, oral preparations, etc. The administration form may be either oral (internal) or parenteral (external, injection). Good.
In order to prepare such pharmaceutical preparations of various dosage forms, at least one selected from the group consisting of the plant of the present invention and an extract thereof is used alone, or other pharmaceutically acceptable excipients. Agents, 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 for external use, and the content of the plant of the present invention or the extract thereof in the preparation (in terms of the dry matter of the extract) is generally 0.00001% by mass or more, preferably Is 0.0001% by mass or more and 10% by mass or less, preferably 1% by mass or less. For example, 0.00001-10 mass% and 0.0001-1 mass% are mentioned.

The form of the food may be solid, semi-solid or liquid, for example, various foods such as breads, cakes, noodles, confectionery, jelly, frozen foods, ice creams, dairy products, beverages, Examples include the same forms (tablets, capsules, syrups, etc.) as the above-mentioned oral administration preparations.
In order to prepare various forms of food, at least one selected from the group consisting of the plant of the present invention and an extract thereof is used 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 of the present invention or the extract thereof in the food (in terms of dry matter of the extract) is generally 0.01% by mass or more, preferably 0.1% by mass or more, more preferably 1% by mass. % And 100% by mass, 90% by mass and 80% by mass. For example, 0.01-100 mass%, More preferably, 0.1-100 mass%, More preferably, 1-100 mass% is mentioned.

  The dose or intake of the above-mentioned pharmaceutical may vary according to the condition, weight, sex, age or other factors of the subject, but in the case of oral administration or intake, the plant of the present invention or an extract thereof per adult ( In terms of dry matter), it is 0.01 mg / kg or more per day, preferably 0.1 mg / kg or more, and 100 mg / kg or less, preferably 10 mg / kg or less. For example, 0.01 mg-100 mg / kg, Preferably 0.1 mg-10 mg / kg is mentioned.

  The form of the cosmetic (including quasi-drugs) can be a skin external preparation, a cleaning agent, a makeup cosmetic, and the like. Depending on the method of use, lotion, emulsion, gel, cream, ointment, It can be provided in various dosage forms such as powder and granules. Such cosmetics in various dosage forms include at least one selected from the group consisting of the plant of the present invention and extracts thereof, and oily ingredients, moisturizers, powders, which can be blended in skin cosmetics. Pigments, emulsifiers, solubilizers, detergents, UV absorbers, thickeners, drugs (for example, anti-inflammatory agents, bactericides, antioxidants, vitamins, fat metabolism promoting action or uncoupling protein expression promoting action are known Prepared drugs or natural products), fragrances, resins, antibacterial and antifungal agents, plant extracts, alcohols, and the like.

  The content of the plant of the present invention or the extract thereof in the total amount of the cosmetic is usually 0.0001% by mass, preferably 0.001% by mass or more, more preferably 0, in terms of the dry matter of the extract. 0.01 mass% or more, and 20 mass% or less, preferably 10 mass% or less, more preferably 5 mass% or less. For example, 0.0001-20 mass%, Preferably it is 0.001-10 mass%, More preferably, 0.0001-5 mass% is mentioned.

In relation to the above-described embodiment, the present invention further discloses the following applications.
<1> A retinoic acid-like agent comprising as an active ingredient at least one selected from the group consisting of Yanagita, Shinagawa renagus and extracts thereof.
<2> A sebum secretion inhibitor comprising as an active ingredient at least one selected from the group consisting of Willow grove, Shinawasunagusa and extracts thereof.
<3> An acne preventive or ameliorating agent comprising at least one selected from the group consisting of Willow grove, Shinawasunagusa and extracts thereof as an active ingredient.
<4> A preventive or ameliorating agent for skin aging comprising as an active ingredient at least one selected from the group consisting of Yanagita, Shinawarenagususa and extracts thereof.
<5> An MMPs inhibitor comprising, as an active ingredient, at least one selected from the group consisting of Yanagita, Shinawasunagusa, and extracts thereof.
<6> An external preparation for skin containing Sinawasunagusa or an extract thereof.

<7> Use of at least one selected from the group consisting of Yanagita, Shinagawa renagus and extracts thereof for producing a retinoic acid-like agent.
<8> Use of at least one selected from the group consisting of Yanagita, Shinawarenagusa, and extracts thereof for producing a sebum secretion inhibitor.
<9> Use of at least one selected from the group consisting of Yanagita, Shinawasunagusa and extracts thereof for producing an acne prevention or amelioration agent.
<10> Use of at least one selected from the group consisting of Yanagita, Shinawasunagus, and extracts thereof for producing an agent for preventing or improving skin aging.
<11> Use of at least one selected from the group consisting of Yanagita, Shinawasunagusa and extracts thereof for producing an MMPs inhibitor.
<12> Use of Shinawasunagusa or an extract thereof for producing a skin external preparation.
<13> At least one selected from the group consisting of Yanagitade, Shinawasu renagusa and extracts thereof for use as a retinoic acid-like substance.
<14> At least one selected from the group consisting of Yanagita, Shinawasunagus, and extracts thereof for use in suppressing sebum secretion.
<15> At least one selected from the group consisting of Yanagita, Shinawasunagusa, and extracts thereof for use in preventing or improving acne.
<16> At least one selected from the group consisting of Yanagita, Shinagawa renagus and extracts thereof for use in preventing or improving skin aging.
<17> At least one selected from the group consisting of Yanagita, Shinawasunagusa, and extracts thereof for use in inhibiting MMPs.
<18> Shinawasunagusu or its extract for external use on the skin.
<19> A method for imparting retinoic acid-like activity, comprising administering or ingesting at least one selected from the group consisting of willow tree, Shinawasunagusa and extracts thereof to a subject in need thereof in an effective amount.
<20> A method for suppressing sebum secretion, comprising administering or ingesting an effective amount of at least one selected from the group consisting of Yanagita, Shinawarenagusa and extracts thereof to a subject in need thereof.
<21> A method for preventing or improving acne, comprising administering or ingesting at least one selected from the group consisting of Willow grove, Shinawarenagusa and extracts thereof to a subject in need thereof in an effective amount.
<22> A method for preventing or ameliorating skin aging, comprising administering or ingesting at least one selected from the group consisting of Willow grove, Shinawasunagusa and extracts thereof to a subject in need thereof in an effective amount.
<23> A method for inhibiting MMPs, comprising administering or ingesting an effective amount of at least one selected from the group consisting of Willow grove, Shinawasunagusa and extracts thereof to a subject in need thereof.
<24> The method <19> to <23>, which is a non-therapeutic method.
<25> In the above items <1> to <24>, the plant use site is preferably the above-ground part such as a leaf or a stem.
<26> In the above <1> to <25>, the extraction solvent for obtaining the plant extract preferably includes water, alcohols, alcohol-water mixed liquids, hydrocarbons, and alcohol-water mixing. Liquids and hydrocarbons are 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.
<27> In the above <1> to <26>, 0.001% by volume or more is preferable, more preferably 5% by volume or more, more preferably 20% by volume or more, more preferably 30% by volume or more, more preferably 40%. It is at least 95% by volume, preferably 95% by volume or less, more preferably 80% by volume or less, more preferably 70% by volume or less, more preferably 60% by volume or less. For example, 5-95 volume%, 20-80 volume%, 30-70 volume%, 40-60 volume% is mentioned.

Example 1 RARγ binding activity <Preparation of plant extract>
(Production Example 1) Preparation of extract of Polygonum hydropiper 50 g of crude herb varicella (water leopard) based on willow tide is added, and 2000 mL of 50 (v / v)% ethanol is added twice at 85 ° C. After extraction (first time: 2 hours, second time: 1 hour), the mixture was filtered and the solvent was removed to obtain 6.7 g of a solid content. The solid content was diluted to an evaporation residue of 1.0 (w / v)% to prepare a willow extract.

(Production Example 2) Basswood Myosotis (Cynoglossum amabile Staf et Drum.) Preparation Sina Myosotis extracts were minced herbal dog屎花(Kushika) 100 g to MotoHara plants, 50 (v / v)% ethanol 2000mL In addition, extraction was performed twice at 85 ° C. (first time: 2 hours, second time: 1 hour), followed by filtration to remove the solvent to obtain 16.1 g of a solid content. The solid content was diluted so that the evaporation residue was 1.0 (w / v)%, and a Sinawas lengus extract was prepared.

<Evaluation method>
pBIND-RARγ and pGL4.35 [luc2P / 9XGAL4UAS / Hygro] were purchased from Promega.
1) Preparation of pBIND-RARγ First, a region containing RBD of LARγ was amplified by PCR using cDNA derived from human skin tissue as a template and using primers shown in Table 1 containing restriction enzyme sites of PvuI and EcoRI. did.

  The PCR product amplified with the primers in Table 1 and pBIND-RARγ cleaved with PvuI and EcoRI were ligated using In Fusion Advantage PCR Cloning Kit (Takara Bio Inc.). Then, according to the protocol shown by Promega, JM109 Competent Cells (Promega) were transformed, and the plasmid was extracted from Escherichia coli into which the target plasmid was introduced, using EndoFree Plasmid Maxi Kit (QIAGEN).

2) Cell culture HEK293, which is a human kidney-derived cell line, was cultured in DMEM containing 10% Fetal Bovine Serum (FBS) (37 ° C, 5% CO ₂ ).

3) Introduction of plasmid into HEK293 and addition of RARγ agonist HEK293 was seeded on a 96-well plate to a cell density of 3.0 × 10 4 cells / well. After 12 hours, the culture solution (phenol red-free DMEM containing 5% charcoal-treated FBS) was replaced, and pBIND-RARγ and pGL4.35 [luc2P / 9XGAL4UAS / Hygro] were used using Lipofectamine 2000 (Invitrogen), The cells were introduced into the cells according to the protocol attached to the product. 24 hours after introduction, a control solvent (a culture solution containing 0.1% dimethylsulfoxide), adapalene (Tocris Bioscience) as a positive control (final concentration 10 nM), plant extracts prepared in the above Production Examples 1 and 2 ( The culture solution was replaced with a culture solution containing a final concentration of 0.1, 0.5, or 1 (v / v)%), and further cultured for 24 hours (n = 6).

4) Measurement of luciferase activity For the activity measurement of firefly luciferase (an index of RBD binding activity of RARγ) and Renilla luciferase (an index of plasmid introduction efficiency), Dual Glo ™ Luciferase Assay System (Promega) was used according to the attached protocol. did. The activity of firefly luciferase was divided by the activity of Renilla luciferase, and the corrected value was used as the relative luminescence intensity (RLU).
Each activity value was shown as a relative value when the RLU in the positive control was 100. The evaluation of the binding activity was performed by comparison with the binding activity value when a control solvent as a negative control was added.

<Result>
The results are shown in Table 2. When the fluorescence intensity from HEK293 cells was measured, the fluorescence intensity increased in the cell group in which the plant extract was added to the culture solution compared to the group in which the control solvent was added. Since the fluorescence measured in the Examples reflects the ability of RARγ to bind to LBD, it was confirmed that the above two extracts have retinoic acid-like activity.

Reference Example 1 Sebum secretion inhibitory action of retinoic acid and RARγ selective agonist <Evaluation Method>
Hamster primary cultured sebaceous gland cells (Kurabo) were seeded on a 24-well plate at 1 × 10 ⁵ cells / well and cultured for 3 days in Humdia-BG (Kurabo), then Dimethylsulfoxide (control), all-trans In Humedia-BD (Kurabo) to which retinoic acid (atRA, Wako Pure Chemical Industries, Ltd.), adapalene (adapalene), BMS961 (Tocris Bioscience) was added to 0.1%, 100 μM, 10 nM, and 10 nM, respectively. And further cultured for 3 days (n = 6). After culturing, each well was washed twice with PBS, 10 μg / ml Nile Red staining solution was added dropwise, and the mixture was stained for 30 minutes at 37 ° C. and CO ₂ 5%. After staining, the plate was washed twice with PBS, applied with an excitation wavelength of 485 nm, and the fluorescence intensity at a wavelength of 565 nm was measured.

<Result>
When the fluorescence intensity from the hamster primary cultured sebaceous gland cells was measured, the fluorescence intensity was significantly decreased in the group to which atRA, adapalene, or BMS961 was added, compared to the control group. Statistical significance test was analyzed using Dunnett method (***: p <0.001). The results are shown in FIG.
From FIG. 1, it was confirmed that retinoic acid and a RARγ selective agonist are effective in suppressing sebum secretion. In addition, although the sebum secretion inhibitory action by adapalene has not been clarified so far, like the retinoic acid and other RARγ selective agonists, it was confirmed to significantly suppress the sebum secretion from the sebaceous glands.

Example 2 Sebum secretion inhibitory action of plant extract <Evaluation method>
Hamster primary cultured sebaceous gland cells (Kurabo) were seeded on a 24-well plate to 1 × 10 ⁵ cells / well, cultured for 3 days in Humdia-BG (Kurabo), then Dimethylsulfoxide (control) ), Adapalene as a positive control, or Humedia-BD (Kurabo Co., Ltd.) further supplemented with the plant extracts prepared in Production Examples 1 and 2 to 1 (v / v)%, respectively. Cultured for days (n = 3). After culturing, each well was washed twice with PBS, 10 μg / ml Nile Red staining solution was added dropwise, and the mixture was stained for 30 minutes at 37 ° C. and CO ₂ 5%. After staining, the plate was washed twice with PBS, applied with an excitation wavelength of 485 nm, and the fluorescence intensity at a wavelength of 565 nm was measured.

<Result>
When the fluorescence intensity from the hamster primary cultured sebaceous gland cells was measured, the fluorescence intensity was statistically significantly decreased in the group to which the plant extract prepared in Production Examples 1 and 2 was added, compared with the control group. It was. Statistical significance test was analyzed using Dunnett method (*: p <0.05, ***: p <0.001). The results are shown in FIG.
The fluorescence measured above is emitted from Nile Red bound to neutral lipids, and most of the lipids that make up sebum are neutral lipids, reflecting the amount of sebum in the sebaceous gland cells. Conceivable. Therefore, it was confirmed that the plant extract has a sebum secretion inhibitory action.

Example 3 Matrix Metalloprotease (MMPs) Activity Inhibitory Action (1) Cell Culture Human dermal fibroblasts were cultured in DMEM containing 5% Fetal Bovine Serum (FBS) (37 ° C., 5% CO ₂ ).

(2) Collection of culture supernatant Human dermal fibroblasts were seeded on a 6-well plate to a cell density of 3.0 × 10 ⁵ cells / well. After 24 hours, the culture medium (FBS-free DMEM) was changed, and further cultured for 24 hours. After culturing, the culture medium was replaced with 50% ethanol as a control solvent or the plant extract prepared in Production Examples 1 and 2 (final concentration 1 (v / v)%), and the culture was continued for 24 hours. The culture supernatant was collected (n = 2).

(3) Measurement of MMPs activity Each culture supernatant prepared with a protein concentration as an enzyme source of MMPs in 96-well plate, or a culture solution containing GM6001 (Calbiochem), which is an inhibitor of MMPs, as a positive control (final concentration 10 nM) ) Was added (protein concentration was 100 ng / well), and 50 μl of Fluorescence Substitution IX (R & D Systems: final concentration 10 nM) was added and mixed as a substrate to be cleaved into a plurality of MMPs. Incidentally, TCNB buffer for dilution of the culture supernatant _{(50mM Tris, 10mM CaCl 2,} 150mM NaCl, 0.05% Brij-35: pH7.5) was used. After incubating the mixture at 37 ° C. for 30 minutes, the fluorescence intensity was measured with a fluorescence plate reader (Ex: 355 nm, Em: 430 nm). The substrate cleaving activity of each culture supernatant when the substrate cleaving activity of Control was 100% was expressed as a relative value to Control.

(4) Results Table 3 shows the results. When the substrate-cleaving activity of MMPs in the culture supernatant was measured by fluorescence intensity, it was found that each group cultured in a culture solution containing a willow extract or a Sinagus renusa extract was significantly more compared to the group to which a control solvent was added. The fluorescence intensity was decreased. (The statistical significance test was analyzed using Dunnett's method (**: p <0.01). Therefore, in the cell group to which the above plant extract was added, the substrate cleavage activity of MMPs was reduced, that is, the activity of MMPs. Was found to be inhibited.

  MMPs, a group of proteases that use extracellular matrix proteins as the main substrate, are greatly elevated by UV irradiation and cause a decrease or degeneration of the extracellular matrix, which is one of the major factors of photoaging. (Gary J. Fisher et al., Nature, Vol.379, No.6563, 335-339 (1996), Gary J. Fisher et al., ARCH DERMATOL, Vol.138, No.11 , 1462-1470 (2002)). It has also been reported that wrinkle formation can be prevented when an inhibitor of MMP-2, -9 is applied to the back of hairless mice and irradiated with UVB (Inomata et al, THE JOURNAL OF INVESTIGATIVE DERMATOLOGY, Vol. 120, No. 1, 128-134 (2003)). Therefore, the MMPs activity inhibitory substance is considered to be able to prevent and improve skin aging such as skin talmi and wrinkles. From the above results, the plant extract of the present invention is considered to have the effect.

Claims (6)

  A retinoic acid-like agent comprising at least one selected from the group consisting of Willow tide and its extract (except when used for acne prevention or improvement or anti-aging).   A sebum secretion inhibitor comprising as an active ingredient at least one selected from the group consisting of Yanagita and its extract (except when used for preventing or improving acne).   A retinoic acid-like agent comprising, as an active ingredient, at least one selected from the group consisting of Shinawarenagusa and its extract.   The sebum secretion inhibitor which uses as an active ingredient at least 1 sort (s) selected from the group which consists of Shinawarenagusa and its extract.   The acne prevention or improvement agent which uses as an active ingredient at least 1 sort (s) selected from the group which consists of Shinawarena rush and its extract.   A skin aging preventive or ameliorating agent comprising as an active ingredient at least one selected from the group consisting of Shinawarenagusa and an extract thereof.