JP6145181B2 - MFAP-4 production promoter - Google Patents

Description

  The present invention relates to a substance capable of promoting MFAP-4 production.

Skin stretch streaks (striae distensae) are skin streaks that are caused by rapid overextension of the skin, and occur regardless of gender due to pregnancy, adolescent growth, rapid obesity, or the like. In the initial stage, it is reddish purple, and it is a streak without a clear depression, but gradually becomes more whitish and has a scar-like appearance. Skin stretch striations are often found in places such as the popliteal, lumbar region, buttocks, and thighs.
Skin-stretching streaks that occur during pregnancy are called striae gravidarum and are found in most pregnant women. Gestational lines occur in the abdomen, breast, and crotch from about half a year to the end of childbirth. After childbirth, it will become less noticeable over time, but many will not return completely.

  The skin consists of the epidermis, dermis, and subcutaneous tissue. Of these, the epidermis is highly extensible, while the dermis and subcutaneous tissue are difficult to stretch. It is thought that when the skin grows rapidly due to pregnancy, growth, obesity, etc., the elastic fiber structure of the dermis breaks down, and as a result, the skin extension filament is caused. It has been reported that elastin fibers are reduced in the skin that has developed a pregnancy line (Non-patent Document 1).

  Elastic fibers and collagen fibers existing in the extracellular matrix (ECM) play an important role in maintaining the elasticity of the dermis. Elastic fibers are constructed by polymerizing aggregates of tropoelastin molecules on microfibrils. Microfibrils that form the basis of elastic fibers are mainly composed of fibrillin-1, which is a 350 kDa glycoprotein, and are constructed by the action of various proteins on fibrillin-1 polymerized in series and in parallel.

  microfibrillar-associated protein 4 (MFAP-4) was first identified as a 255 amino acid protein containing an Arg-Gly-Asp (RGD) sequence and a fibrinogen-like domain. MFAP-4 is known as a causative gene of Smith-Magenis syndrome in which abnormalities (such as square faces, deeply indented eyes, swollen cheeks, and low protruding jaws) are recognized in the face shape (Non-patent Document 2). In addition, in a disease related to elastin, pseudoxanthoma elasticum, it has been reported that findings are observed at the site of accumulation of decomposed elastic fibers (Non-patent Document 3). However, the function and mechanism of action of MFAP-4 have not yet been fully clarified.

  MFAP-4 may play a role in the construction of ECM and elastic fibers in the dermis of the skin. Furthermore, it is expected that by controlling the expression of MFAP-4, it is possible to reduce elastic fibers and prevent cracks in the dermis when the skin grows rapidly due to pregnancy or obesity.

Himezu (hime bun) is also called tenshi (Tenshizu) and dragonfly, and refers to Semiaquilegia adoxoides belonging to the genus Ranunculaceae. This is a plant conventionally used for antipyretic and diuresis. Rosemary is also referred to as rosinus officinalis, which is also referred to as lava marinade or rosmarin, and belongs to the genus Rosemary or Mannenrou. The branches and leaves of this plant are used as perfumes. The linden tree (Western tree) is also referred to as the Boletus tree (Western linden tree), and refers to Tilia europaea of the genus Lindenaceae . It was previously known that this flower has a sweating effect.

  However, it has not been known so far that these plants play some role in MFAP-4 expression, ECM and elastic fiber construction.

Watson et al, 1998, British J Dermatol 138: 931-937 Zhao et al, 1995, Hum Mol Genet 4: 589-597 Hirano et al, 2002, J Dermatol Sci 28: 60-67

  The present invention relates to the provision of a naturally-derived material that can promote MFAP-4 production and promote the construction of ECM and elastic fibers.

  Recently, a study group that includes the present inventors has shown that MFAP-4 in fibroblasts is involved in the expression of matrix metalloproteinases (MMPs), which are ECM-degrading enzymes such as collagen fibers and elastic fibers. Was shown (FIG. 1). In addition, MFAP-4 contributes to the formation of a polymer composed of tropoelastin and fibrillin-1 (FIG. 2), and also directly interacts with fibrillin-1 constituting the microfibril that forms the basis of elastic fibers. It became clear that it worked (FIG. 3). As a result of these studies, MFAP-4 inhibits the degradation of ECM such as collagen fibers and elastic fibers via MMP, and also interacts with fibrillin-1 in the ECM to polymerize fibrillin-1 to microfibrils. It was shown that it promotes the formation of elastic fibers by promoting, and as a result, has the function of maintaining the structure of ECM (FIG. 4).

  Therefore, if the expression of MFAP-4 can be promoted, it promotes the construction of ECM and elastic fibers in the tissue, suppresses the degradation thereof, maintains the structure, and as a result, prevents the skin extending streaks such as pregnancy lines. Can be planned.

  Based on the above research results, the present inventors searched for naturally-occurring substances that promote the expression of MFAP-4. As a result, the expression of cellular MFAP-4 in certain plants or their extracts was determined. It has been found that it has a promoting action, and is useful as a material for promoting the formation of ECM and elastic fibers in tissues and suppressing the degradation thereof, and also for preventing skin stretch streaks such as pregnancy lines.

That is, this invention provides the MFAP-4 production promoter which uses as an active ingredient at least 1 selected from Himezu, rosemary, linden trees, and those extracts.
Moreover, this invention provides the microfibril formation promoter which uses at least 1 selected from the said plant and those extracts as an active ingredient.
Moreover, this invention provides the elastic fiber formation promoter which uses as an active ingredient at least 1 selected from the said plant and those extracts.
Moreover, this invention provides the elastic fiber degradation inhibitor which uses as an active ingredient at least 1 selected from the said plant and those extracts.
Moreover, this invention provides the collagen degradation inhibitor which uses as an active ingredient at least 1 selected from the said plant and those extracts.
The present invention also provides a skin stretch streak preventive agent comprising as an active ingredient at least one selected from the above-mentioned plants and extracts thereof.

  According to the present invention, MFAP-4 production of cells can be promoted. Further, according to the present invention, the structure of collagen and elastic fibers in the tissue can be maintained through the promotion of MFAP-4 production, and as a result, skin stretch filaments such as pregnancy lines can be prevented.

A: Effect of MFAP-4 on MMP-12 expression. B: Effect of MFAP-4 on MMP-1 expression. Error bar = SD, **: P <0.01. ***: P <0.001. Effect of MFAP-4 on extracellular matrix. Scale bar = 50 μm. Interaction of MFAP-4 with fibrillin-1. Schematic diagram of elastic fiber construction.

  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 therapy.

  In the present specification, “improvement” refers to improvement of a disease or symptom, prevention or delay of worsening of the disease or symptom, or reversal, prevention or delay of progression of the 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, “MFAP-4” (microfibrillar-associated protein 4) is a protein known as Gene ID: 4239 and MIM ID: 6000059.
In the present invention, “MFAP-4 production promotion” refers to an increase in the amount of MFAP-4 protein, an increase in the expression level of MFAP-4 protein, an increase in the expression level of mRNA encoding MFAP-4 protein, It can be measured based on an arbitrary parameter such as an increase in the activation level of the transcription control region of the gene to be encoded.

In the present specification, “Himeuzu” refers to Semiaquilegia adoxoides belonging to the genus Ranunculaceae, “Rosemary” refers to Rosmarinus officinalis belonging to the genus Rosemary, and “Linaceae” Point to Tilia europaea .

  In the present invention, the plant listed above is not limited to any part of the plant, for example, whole plant, whole tree, leaf, stem, bud, flower, bud, tree, wood part, bark, root, rhizome, A temporary corm, tuberous root, lichen, frond, seed, fruit, resin, or the like, or a combination thereof may be used. Preferred sites are tuberous roots for himeuzu, leaves for rosemary, and flowers or leaves for linden.

  Although the extract of the plant mentioned above should just be the extract from the arbitrary site | parts or the combination of the said plant mentioned above, it is preferable that it is the extract from the preferable site | part of the said plant mentioned above. These plant parts may be subjected to the extraction process as they are, or may be subjected to the extraction process after being pulverized, cut or dried.

  As the extract, commercially available products may be used, or various solvent extracts obtained by a conventional method, or diluted solutions thereof, concentrated solutions thereof, dried powders thereof, pastes or activated carbon treatments thereof. There may be. As an example, the extract in the present invention can be obtained by extracting the plant part at room temperature or under heating, or by using an extraction device such as a Soxhlet extractor.

  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, ethyl acetate, and the like. 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 fats and waxes and other oils; and mixtures thereof. Preferable examples include water, alcohols and aqueous solutions thereof, and ethanol is preferable as the alcohols. More preferred solvents are water and an aqueous ethanol solution.

The blending ratio (volume ratio) of alcohols and water is preferably 99.999-0: 0.001-100, more preferably 95-5: 5-95, and still more preferably 80-20: 20-80. 70-30: 30-70 are still more preferable, and 60-40: 40-60 are still more preferable.
As a usage-amount of a solvent, 1-100 mL is preferable with respect to 1 g of said plants (dry mass conversion), As extraction time, 1 minute-2 months are preferable and 10 minutes-4 weeks are more preferable. The extraction temperature at this time is 0 ° C. to the solvent boiling point, more preferably 10 to 60 ° C., and further preferably 15 to 40 ° C.

  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. For example, as a suitable example of immersion, immersion for 1 hour to 4 weeks is mentioned at 0 degreeC-solvent boiling point (preferably 15-40 degreeC). Moreover, when shortening extraction time, solid-liquid extraction with stirring is desirable. 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 shown in Examples below, the plant extract has an effect of significantly promoting the expression of MFAP-4 in cells. Therefore, the said plant and / or those extracts can be used for MFAP-4 production promotion. In addition, the plant and / or the extract thereof can promote the formation and suppression of elastic fiber formation in the tissue, the suppression of collagen degradation, or the skin extension line such as a pregnancy line through the MFAP-4 production promoting action. Can be used for the prevention of. The use can be in humans or non-human animals, or specimens derived therefrom, and can be therapeutic or non-therapeutic.

Therefore, as one aspect, the present invention provides a MFAP-4 production promoter containing as an active ingredient at least one selected from himeuzu, rosemary, linden, and extracts thereof.
As another aspect, the present invention provides a microfibril formation promoter containing at least one selected from the above plants and their extracts as an active ingredient.
As another aspect, the present invention provides an elastic fiber formation promoter comprising as an active ingredient at least one selected from the above-mentioned plants and extracts thereof.
As another aspect, the present invention provides an elastic fiber degradation inhibitor comprising as an active ingredient at least one selected from the above plants and extracts thereof.
As another aspect, the present invention provides a collagen degradation inhibitor comprising as an active ingredient at least one selected from the above plants and extracts thereof.
As another aspect, the present invention provides a prophylactic agent for skin stretch streak comprising at least one selected from the above plants and extracts thereof as an active ingredient.
In the present invention, the agent is composed essentially of at least one selected from himeuzu, rosemary and linden, and extracts thereof.
In the present invention, these plants and extracts may be used alone or in any combination of two or more.

  The plant and / or extract thereof is a composition for promoting production of MFAP-4, promoting formation and inhibiting degradation of elastic fibers in a tissue, inhibiting collagen degradation, or preventing a skin stretch streak such as a pregnancy line. It can be used for the production of pharmaceuticals, quasi drugs, cosmetics and the like. Such compositions, pharmaceuticals, quasi drugs, cosmetics and the like are also within the scope of the present invention.

  The above composition, medicine, quasi-drug, cosmetic and the like can be produced or used for human or non-human animals. The plant and / or extract thereof is blended as a material in the composition, medicine, quasi-drug, cosmetics, etc., and promotes MFAP-4 production, promotes formation of elastic fibers and suppresses degradation of tissue, or collagen It may be an active ingredient for inhibiting the degradation of the skin or preventing the skin extending line such as the pregnancy line.

The said pharmaceutical or quasi-drug contains the said plant and / or those extracts as an active ingredient. The pharmaceutical or quasi drug can be administered in any dosage form. The administration form may be oral administration or parenteral administration such as external preparation. Dosage forms for oral administration include, for example, solid dosage forms such as tablets, coated tablets, granules, powders, capsules, and liquid dosage forms such as elixirol, syrups and suspensions, Examples of the dosage form for parenteral administration include injection, infusion, topical, external preparation, transdermal, transmucosal, nasal, enteral, inhalation, suppository, bolus, patch and the like.
As an example, the medicine or quasi drug may be in the form of a skin external preparation in the form of a lotion, cream, gel, ointment, patch or the like.

  The said pharmaceutical and quasi-drug may contain the said plant or those extracts alone, or may contain it in combination with a pharmaceutically acceptable carrier. Examples of such carriers include excipients, coating agents, binders, extenders, disintegrants, surfactants, lubricants, diluents, dispersants, buffers, osmotic pressure adjusting agents, pH adjusting agents, Dispersants, emulsifiers, preservatives, stabilizers, antioxidants, colorants, UV absorbers, moisturizers, thickeners, activity enhancers, anti-inflammatory agents, bactericides, fragrances, flavoring agents, flavoring agents, etc. It is done. Moreover, the said pharmaceutical and quasi-drug may contain the other active ingredient and pharmacological component, unless the MFAP-4 production promotion effect of the said plant or those extracts is lost.

  The above-mentioned medicine or quasi-drug can be produced from the above-mentioned plant and / or an extract thereof or, if necessary, by combining the above-mentioned carrier and / or other active ingredient or pharmacological ingredient by a conventional method. . Content of the said plant and / or those extracts in the said medicine or quasi-drug is 0.001-99.999 mass% normally in conversion of the dry weight of the said extract, for example. It is preferable to set it as 01-20 mass%.

The said cosmetics contain the said plant and / or those extracts as an active ingredient. The cosmetic may contain the plant or an extract thereof alone or in combination with a carrier acceptable as a cosmetic.
Examples of such carriers include excipients, coating agents, binders, extenders, disintegrants, surfactants, lubricants, diluents, dispersants, buffers, osmotic pressure adjusting agents, pH adjusting agents, Dispersants, emulsifiers, preservatives, stabilizers, antioxidants, colorants, UV absorbers, moisturizers, thickeners, activity enhancers, anti-inflammatory agents, bactericides, fragrances, flavoring agents, flavoring agents, etc. It is done. In addition, as long as the MFAP-4 production promoting action of the above plants or their extracts is not lost, the cosmetics are other active ingredients and cosmetic ingredients such as moisturizers, whitening agents, UV protection agents, cell activators. , Cleaning agents, keratolytic agents, make-up ingredients (for example, makeup bases, foundations, funny, powder, teak, lipstick, eye makeup, eyebrow, mascara, etc.) and the like.
Examples of cosmetic forms include creams, emulsions, lotions, suspensions, foams, gels, powders, packs, sheets, patches, sticks, cakes, and any other form that can be used for cosmetics.
As an example, the cosmetic may be a cosmetic for preventing pregnancy lines.

  The cosmetic can be produced by a conventional method from the plant and / or an extract thereof, or in combination with the carrier and / or other active ingredients and cosmetic ingredients as necessary. Content of the said plant and / or those extracts in the said cosmetics is 0.0001-99.999 mass% normally in conversion of the dry weight of the said extract, and 0.001-10 mass% It is preferable to do this.

Moreover, this invention provides the MFAP-4 production promotion method characterized by administering at least 1 selected from the said plant and those extracts. In this method, at least one selected from the above plants and their extracts is administered in an effective amount to a subject in need thereof to promote MFAP-4 production.
In addition, the present invention provides at least one selected from the above plants and their extracts, promotes formation and suppression of elastic fiber formation in tissues, suppresses collagen degradation, pregnancy lines, and the like Provide a method for preventing skin stretch streaking. In the method, at least one selected from the above plants and their extracts is used for promoting the formation and suppression of elastic fiber formation in tissues, inhibiting collagen degradation, or preventing skin stretch lines such as pregnancy lines. In an effective amount to a subject in need thereof.

  Subjects to be administered include animals, preferably humans or non-human mammals, more preferably humans. Preferably, the administration can be administered non-therapeutically for cosmetic purposes. Alternatively, the subject to be administered can be an animal-derived tissue, organ, cell, or fraction thereof. The tissue, organ, cell, or fraction thereof is preferably a naturally-derived or biologically or bioengineered tissue, organ, cell, or fraction thereof having the ability to express MFAP-4. More preferably, it is skin tissue, cells derived from skin tissue, or cultures thereof.

  The preferred dosage may vary according to the subject's species, weight, sex, age, condition or other factors. The dose, route, interval, and amount and interval of intake can be determined appropriately by those skilled in the art. For example, it is preferably 0.01 to 1000 mg / day, more preferably 0.1 to 100 mg / day per adult in terms of dry matter of the plant extract.

  EXAMPLES Hereinafter, an Example is shown and this invention is demonstrated more concretely.

Production Example Preparation of Plant Extract Production Example 1 Preparation of Himeuzu Extract 40 mL of 50% ethanol aqueous solution was added to 40 g of Himezu tuberous root (Shinwa product) and immersed at room temperature for 22 days. This was filtered to obtain a Himeuzu extract. When this extract was concentrated, the solid content was 14.89 g. This extract was diluted with 50% ethanol to give a 1% (w / v) solution.

Production Example 2 Preparation of Rosemary Extract 400 ml of 50% ethanol aqueous solution was added to 40 g of rosemary leaves (produced by Shinwa), and immersed for 28 days at room temperature. This was filtered to obtain a rosemary extract. When this extract was concentrated, the solid content was 6.53 g. This extract was diluted with 50% ethanol to give a 1% (w / v) solution.

Production Example 3 Preparation of linden leaves extract To 40 g of linden leaves (produced by Shinwa), 400 mL of a 50% ethanol aqueous solution was added and immersed for 19 days at room temperature. This was filtered to obtain a linden extract. When this extract was concentrated, the solid content was 5.89 g. This extract was diluted with 50% ethanol to give a 1% (w / v) solution.

Example 1 Promotion of MFAP-4 Production by Plant Extract Normal human dermal fibroblasts (Kurashikibo Co., Ltd.) were added to a 24-well plate at 3.0 × 10 ⁵ cells / well and cultured for 24 hours. The medium was changed, and then each well was extracted with any of the plant extracts prepared in Production Examples 1 to 3 as a test substance (final concentration 0.1% (v / v); 0.001% (w / v)), or the same amount of 50% ethanol was added as a control. Three days later, the culture supernatant was collected, and a MFAP-4 protein band was detected by Western blotting using the culture supernatant as a sample to quantify the expression level of MFAP-4 protein, and the relative expression level relative to the control was determined.
The results are shown in Table 1. All plant extracts listed in Table 1 promoted MFAP-4 expression.

Reference Example 1 Effect of MFAP-4 on MMP Expression Normal human dermal fibroblasts were added to a 24-well plate at 1.0 × 10 ⁵ cells / well in the same procedure as in Example 1 and cultured for 24 hours. The medium was changed and the cells were divided into 3 groups and cultured for 8 days. The two groups were cultured in the presence or absence of 10 nM recombinant MFAP-4 (rMFAP-4) and twice in culture, a control siRNA sequence with no specificity for MFAP-4 (3. The siRNA was introduced into the cells by adding 75 μl) to the medium. The remaining one group was cultured in the absence of rMFAP-4, and twice during the culture, the MFAP-4 siRNA sequence (3.75 μl at 20 μM) was added to the medium and introduced into the cells.
After culture, total RNA was extracted from the cells, MMP-12 expression was measured by RT-PCR using TaqMan (registered trademark) gene expression assays (Applied Biosystems), and quantified as a relative value to the internal standard (GAPDH) did. MMP-1 expression was examined by the same procedure.
The results are shown in FIG. When MFAP-4 expression was suppressed with siRNA, the expression of MMP-12 (FIG. 1A) and MMP-1 (FIG. 1B) increased. Since MMP-12 and MMP-1 are enzymes involved in ECM degradation, these results indicate that MFAP-4 is involved in the degradation of ECM such as collagen fibers and elastic fibers.

Reference Example 2 Effect of inhibition of MFAP-4 expression on fibrillin-1 polymerization After preparing cells in the same procedure as in Reference Example 1, the cells were fixed with methanol, and tropoelastin antibody and fibrillin-1 antibody were used. Then, immunostaining was performed, nuclear staining was performed using DAPI, and formation of elastic fibers was observed under a fluorescence microscope.
The results are shown in FIG. When MFAP-4 expression was suppressed with siRNA, the formation of a polymer composed of tropoelastin and fibrillin-1 was impaired, indicating that MFAP-4 contributes to the polymerization of fibrillin-1. It was.

Reference Example 3 Interaction between MFAP-4 and fibrillin-1 In the same procedure as in Example 1, normal human dermal fibroblasts were added to a 24-well plate at 1.0 × 10 ⁵ cells / well and cultured for 24 hours. did. The medium was changed and the cells were cultured for 8 days. The culture supernatant is collected, filtered through a 0.45 μm membrane (Millipore), concentrated 10-fold using a 3 kDa cut-off membrane (Millipore), and 500 μL of the concentrated solution is incubated with 10 μg of MFAP-4 antibody or normal rabbit IgG. did. 20 μL of Protein G Sepharose beads (GE Healthcare) was added thereto and incubated, and then the beads were collected by centrifugation. Proteins were collected from the collected beads, suspended in 1 × SDS sample buffer (Thermo Fisher Scientific), and subjected to Western blotting using fibrillin-1 antibody.
The results are shown in FIG. Since fibrillin-1 was detected in the fraction collected by the MFAP-4 antibody, it was suggested that MFAP-4 and fibrillin-1 coexist in the ECM. Considering this result together with the influence of MFAP-4 on the polymerization of fibrillin-1 shown in Reference Example 2, MFAP-4 interacts with fibrillin-1 in the ECM, so It is suggested that it promotes polymerization to fibrils and is involved in elastic fiber formation (FIG. 4).

Summary of Reference Examples As shown in Reference Examples 1 to 3, MFAP-4 inhibits the degradation of ECM such as collagen fibers and elastic fibers via MMP and interacts with fibrillin-1 in the ECM to produce fibrillin- It promotes the polymerization of 1 to microfibrils to promote the formation of elastic fibers and consequently functions to maintain the structure of the ECM.
Therefore, by promoting the expression of MFAP-4, the elasticity of the tissue can be improved through the function of the MFAP-4, and the elasticity of the dermis due to rapid overextension of the skin caused by pregnancy, growth, obesity, etc. It can prevent the collapse of the fiber structure and the decrease of elastic fibers, and can prevent the skin extending line such as pregnancy line.

Claims (1)

Skin extensibility streak prophylactic agent comprising as an active ingredient, at least one selected from the human Meuzu and its extracts.