JP2017078077A - Retinoic acid-like agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a retinoic acid-like agent that activates RARγ, is useful as pharmaceuticals, quasi drugs or cosmetics that can exhibit the effect of promoting the cure of skin wounds, shows bioactivity comparable to retinoic acids, and is derived from plant extract.SOLUTION: The retinoic acid-like agent has, as plant extract, at least one selected from the group consisting of Mimosa pudica L., Pteris multifida Poir. and extract thereof, as an active ingredient.SELECTED DRAWING: None

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.
RAR and RXR have three subtypes (α, β, γ), and it is known that RARγ and RXRα are mainly expressed in the skin (Non-patent Documents 1 and 2).

When retinoic acid is applied externally, a strong growth promoting effect of epidermal keratinocytes is observed in the epidermis, and the epidermis thickens. This is because keratinocytes immediately above the basal layer secrete HB-EGF (heparin-binding EGF-like growth factor) by the action of retinoic acid, and basal cells receive HB-EGF and are promoted by paracrine. It is thought that. In the dermis, angiogenesis induction is also observed in the dermal papillary layer, and it has been reported that it promotes wound healing of the skin along with the effect of promoting the proliferation of keratinocytes (Non-patent Document 3).
Therefore, if RARγ is activated in the skin and can exhibit a retinoic acid-like action, it is considered effective for promoting wound healing in the skin.

On the other hand, mimosa (Mimosa pudica L.), a plant belonging to mimosoideae (Mimosaceae), antipyretic, for the treatment of enteritis and gastritis, Inomotosou (Pteris multifida Poir.) Is a plant belonging to Inomotosou family (Pteridaceae), hemostatic Each is known to be useful for detoxification.
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. Kitano et al. , Arch Dermatol Res. 2001; 293 (10): 515-521.

  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 a substance that enhances its activity by binding to, that is, exhibits a retinoic acid-like action, it has excellent retinoic acid-like activity in Mimosa pudicica L. and Yomomotoso ( Pteris multifida Poir .). I found.

That is, the present invention relates to a retinoic acid-like agent containing as an active ingredient at least one selected from the group consisting of Mizou pudicica L. , Inomotoso ( Pteris multifida Poir. ) And extracts thereof . .

  Since the retinoic acid-like agent of the present invention activates RARγ, it is useful as a pharmaceutical, quasi-drug or cosmetic that can exert an effect of promoting skin wound healing, or as a material or preparation for blending into these. is there.

  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.

In the present specification, the term “swordfish” refers to Mimosa pudicica L. Inomotoso refers to Pteris multifida Poir. Of the genus Inomotosou . Point to. As the white-spotted grass, a herbal medicine obtained by using white-spotted grass as a base plant, garlic (Ganshu-sou) may be used. Moreover, you may use the herbal medicine obtained by using Inomotoso as a base plant, Kashio grass (Hobisou) as Inomotoso.

Such plants can use any arbitrary part, for example, whole grass, leaves, stems, buds, flowers, buds, roots, rhizomes, temporary corms, 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.

  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 to 60% by volume.

  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, activation of RARγ, using luciferase activity generated when the ligand binds to LBD as an index. It is.
And it is thought that the plant of these invention or those extracts is effective in the improvement of various symptom considered to bind | bond | couple to RAR (gamma) and to activate this. For example, the plant of the present invention or an extract thereof is considered effective for promoting wound healing of skin.

  Therefore, the plant of the present invention or an extract thereof can be used to promote wound healing of skin because of retinoic acid-like activity. The use can be in humans or non-human animals, or specimens derived therefrom, and can be therapeutic or non-therapeutic.

  Moreover, the plant of this invention or those extracts can be used as a retinoic acid-like agent, and can be used in order to manufacture this agent further. At this time, for the retinoic acid-like agent, 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, You may use what is accept | permitted in the below-mentioned target object which should be mix | blended. 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 may be a human or veterinary drug, quasi-drug, cosmetic, food or feed that exhibits retinoic acid-like activity, skin wound healing promoting effect, or It may be a material or a preparation used by blending with the pharmaceutical, quasi-drug, cosmetic or the like. As food, the concept of physiological functions such as retinoic acid-like activity, skin wound healing promoting effect, etc., food and drink, functional food and drink, food and drink for the sick, food for specified health, as necessary Etc.

The pharmaceuticals (including quasi drugs) can be administered in any dosage form. Examples of the dosage form include parenteral administration such as injections, suppositories, inhalants, transdermal absorption agents, external preparations, or oral administration such as tablets, capsules, granules, powders, syrups and the like.
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 oral administration, and the content of the plant of the present invention or the extract thereof in the preparation (in terms of the dried product of the extract) is generally 0.00001 to 10% by mass. And is more preferably 0.0001 to 1% by mass.

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 preferably 0.01 to 100% by mass, and 0.1 to 100% by mass. More preferably, it is more preferably 1 to 100% by 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 at least one selected from the group consisting of the plant of the present invention and extracts thereof, meat such as cattle, pigs, sheep, protein, grains, bran, Commonly used feed materials such as potatoes, sugars, vegetables, vitamins, minerals, etc., and gelling agents, shape-preserving agents, pH adjusters, seasonings, preservatives, nutritional supplements that are generally used in feeds, etc. Can be used in combination.
Moreover, although content of the plant of this invention or those extracts in feed changes with the usage forms, it is 0.0001-20 mass% normally in dry matter conversion, 0.001-10 mass% Is preferable, and 0.01-5 mass% is more preferable.

  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, but in the case of oral administration or intake, the plant of the present invention or an extract thereof per adult As dry matter, it is preferably 0.01 mg to 100 mg / kg, more preferably 0.1 mg to 10 mg / kg per day.

  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 to 20% by mass, preferably 0.001 to 10% by mass in terms of the dry matter of the extract. 0.01 to 5% by mass is more preferable.

Example 1 RARγ binding activity <Preparation of plant extract>
(Production Example 1) Preparation of extract of mimosa pudica L. 140 g of herbaceous herbaceous herb that uses cabbage grass as a base plant is shredded, and 1800 mL of 50 (v / v)% ethanol is added and extracted twice at 85 ° C. (First: 2 hours, Second: 1 hour) After filtration, the solvent was removed to obtain 16.0 g of a solid content. The solid content was diluted so that the evaporation residue was 1.0 (w / v)%, and a swordfish extract was prepared.

(Production Example 2) Preparation of extract of Inomotoso ( Pteris multifida Poir. ) 100 g of herbal medicine Bibiso using Inomotoso as a base plant, 2,000 mL of 50 (v / v)% ethanol was added and extracted twice at 85 ° C ( (First time: 2 hours, second time: 1 hour), followed by filtration to remove the solvent to obtain 7.1 g of a solid content. The solid content was diluted to an evaporation residue of 1.0 (w / v)% to prepare an Inomotoso extract.

<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 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.

Claims (1)

A retinoic acid-like agent comprising, as an active ingredient, at least one selected from the group consisting of Mizosa pudicica L. , Inomotoso ( Pteris multifida Poir .) And extracts thereof.