WO2021241428A1 - エラスチン産生促進剤及び皮膚化粧料 - Google Patents

エラスチン産生促進剤及び皮膚化粧料 Download PDF

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WO2021241428A1
WO2021241428A1 PCT/JP2021/019322 JP2021019322W WO2021241428A1 WO 2021241428 A1 WO2021241428 A1 WO 2021241428A1 JP 2021019322 W JP2021019322 W JP 2021019322W WO 2021241428 A1 WO2021241428 A1 WO 2021241428A1
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Prior art keywords
sialidase
elastin
skin
neu2
cage
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English (en)
French (fr)
Japanese (ja)
Inventor
彰 南
隆 鈴木
康範 岩尾
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Nikken Foods Co Ltd
University of Shizuoka
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Nikken Foods Co Ltd
University of Shizuoka
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Priority to CN202180033344.0A priority Critical patent/CN115515558B/zh
Priority to JP2022526975A priority patent/JP7312993B2/ja
Priority to EP21813509.3A priority patent/EP4159284A4/en
Priority to US17/926,665 priority patent/US20230233437A1/en
Publication of WO2021241428A1 publication Critical patent/WO2021241428A1/ja
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A61K8/66—Enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations

Definitions

  • the present invention relates to an elastin production promoter, and more particularly to an elastin production promoter capable of increasing dermal elastin that has decreased with aging.
  • the skin is composed of epidermis, dermis and subcutaneous tissue. Of these, collagen fibers that impart strength to the skin and elastin that supports these collagen fibers are distributed in the dermis below the epidermis. Elastin is an elastic fiber having elasticity and elasticity, and has an action of bringing elasticity and firmness to the skin. However, it is known that the abundance of elastin decreases with aging and is one of the causes of wrinkles and sagging of the skin.
  • Patent Document 1 describes an elastin production-promoting agent containing lipocalin 2, a protein belonging to the lipocalin family, or a decomposition product thereof as an active ingredient
  • Patent Document 2 describes elastin containing a specific dipeptide or tripeptide as an active ingredient. Production promoters are described.
  • sialidase is a hydrolase that desorbs sialic acid from sugar chains.
  • Sialic acid mainly modifies the ends of sugar chains and is involved in a wide range of biological phenomena such as physiological functions, diseases, and viral infections. Therefore, it is known that sialidase plays a role in controlling cell functions such as cell differentiation / proliferation and apoptosis. Therefore, in order to deepen the knowledge about sialidase, which plays an important role in the living body, the present inventors have developed a fluorescence imaging probe capable of highly sensitively visualizing the enzymatic activity of sialidase on the tissue (Non-Patent Document 1).
  • Patent Document 3 newly found that sialidase activity is detected in pancreatic islands, and reports that a compound having sialidase inhibitory activity has an effect of suppressing an increase in blood glucose level and an effect of increasing insulin secretion. ing.
  • Cialidases are roughly classified into three categories: animal (mammalian) sialidases, viral sialidases, and bacterial sialidases. Among them, it has been reported that sialidase NEU1, which is a kind of animal sialidase, forms a molecular complex with elastin-binding protein (EBP) and cathepsin A, and is involved in the formation of elastin fiber assembly, that is, elastic fibers (they are involved in the formation of elastin fiber assembly, that is, elastic fibers).
  • EBP elastin-binding protein
  • Non-patent document 2 Non-patent document 2
  • Non-Patent Document 1 there has been no report of visualizing the distribution of sialidase activity in skin tissue using a fluorescence imaging probe as reported in Non-Patent Document 1. In addition, there is no report on the relationship between the phenomenon that the abundance of elastin in the dermis decreases with aging and sialidase.
  • the present invention has been made in view of the above points, and an object thereof is to provide a novel elastin production promoter.
  • Another object of the present invention is to provide an elastin production promoter capable of improving or preventing a decrease in dermal elastin due to aging.
  • Non-Patent Document 1 In the process of elucidating the involvement of sialidase in dementia using the fluorescent imaging probe described in Non-Patent Document 1, the present inventors happened to significantly reduce the sialidase activity of skin tissue with aging. I found out to do. Based on this finding, the present invention has been completed.
  • the elastin production promoter of the present invention contains sialidase as an active ingredient.
  • sialidase By administering the sialidase to the target tissue, the production of elastin is promoted, and the amount of elastin in the target tissue can be increased.
  • sialidase NEU2 sialidase NEU2. This selects a type of sialidase suitable as an elastin production promoter.
  • the above-mentioned sialidase is a bacterial sialidase.
  • This selects a type of sialidase suitable as an elastin production promoter.
  • bacterial sialidase has high sialidase activity and excellent stability, so that it is easy to handle.
  • the above-mentioned bacterial sialidase is a sialidase derived from Arthrobacter sp.
  • a more suitable type of sialidase having high sialidase activity and excellent safety and stability is selected.
  • the elastin production promoter of the present invention is dermis elastin in which the above-mentioned elastin constitutes the fibrous tissue of the dermis. Thereby, a suitable target for promoting the production of elastin is selected.
  • the elastin production promoter of the present invention is for preventing or improving skin aging.
  • the amount of elastin present in the dermis decreases with age, causing wrinkles and sagging of the skin.
  • administration of sialidase promotes the production of elastin and increases the amount of elastin. Therefore, the firmness and elasticity of the skin can be maintained or restored, and the aging of the skin can be prevented or ameliorated.
  • the skin cosmetic of the present invention contains the above-mentioned elastin production promoter. As a result, a skin cosmetic having an effect of enhancing the firmness and elasticity of the skin can be obtained.
  • an elastin production promoter and a skin cosmetic having the following excellent effects. (1) It is possible to improve the production of elastin and increase the amount of elastin in the target tissue. (2) The abundance of dermal elastin can be increased, and the firmness and elasticity of the skin can be enhanced. (3) It is possible to prevent / improve the decrease of dermal elastin due to aging.
  • FIG. 3 is a fluorescence imaging image showing the sialidase activity of a rat skin tissue section and an H & E stained image of the same section in Example 1. It is a graph which shows the relationship between the age of a rat and the sialidase activity in a rat skin tissue in Example 2.
  • 3 is a graph showing the expression ratio of each isozyme of animal sialidase of dermis and muscle tissue (Dermis + Muscle) to rat subcutaneous adipose tissue (Fat) in Example 3.
  • 3 is a graph showing the mRNA expression level of the elastin gene by application of sialidase NEU2 to rat skin in Example 4.
  • FIG. 6 (c) a graph showing an elastin production promoting effect by applying bacterial sialidase to rat skin in Example 5.
  • FIG. 6 (a) is a graph showing the sialidase activity when bacterial sialidase is mixed with PBS or ionic liquid coringeranoic acid (CAGE), respectively. * In the graph: P ⁇ 0.05 vs. CAGE, **: P ⁇ 0.01 vs. Shows CAGE.
  • 6 is a fluorescence imaging image showing the distribution of sialidase activity in skin sections of each test group in Example 5.
  • 6 is an immunostaining image showing the distribution of elastin in dermis tissue sections of each test group in Example 5.
  • FIG. 6 is an autofluorescent elastin image showing the distribution of elastin in dermis tissue sections of each test group in Example 5.
  • the scale bar indicates 50 ⁇ m.
  • FIG. 10 (a) shows the data of the test group using SAMP1 as the aging-accelerated model mouse
  • FIG. 10 (b) shows the data of the test group using SAMP8 as the aging-accelerated model mouse.
  • ⁇ in the graph: P ⁇ 0.001 is shown.
  • the sialidase (sometimes called neuraminidase) in the present invention is a hydrolase that liberates sialic acid from a sugar chain
  • the sialidase activity refers to an activity that can liberate sialic acid from a sugar chain.
  • the sialidase activity is not particularly limited, but can be detected using, for example, 4MU-Neu5Ac or BTP-Neu5Ac developed by the present inventors.
  • the sialidase used in the present invention is not particularly limited as long as it has an elastin production promoting effect, but an animal (mammalian) sialidase or a bacterial sialidase is preferably used from the viewpoint of excellent elastin production promoting effect.
  • an animal (mammalian) sialidase or a bacterial sialidase is preferably used from the viewpoint of excellent elastin production promoting effect.
  • there are four types of isozymes NEU1, NEU2, NEU3 and NEU4 in animal sialidase and it is possible to use only one type of isozyme or a combination of a plurality of types.
  • sialidase NEU2 which has been clarified to be highly expressed in the dermis (see Examples described later), is particularly preferably used.
  • Cialidase NEU2 is a protein encoded by amino acids of 380 residues, has a molecular weight of about 42 kDa, and has an optimum pH near neutral. In addition, Cialidase NEU2 has excellent durability and stable enzyme activity, so that it is easy to handle. It should be noted that the animal sialidase can be used alone or in combination of a plurality of species.
  • Bacterial sialidase has the advantage of being easier to handle, as it has higher sialidase activity and enzyme stability than animal sialidase, and can be expressed in large quantities in Escherichia coli.
  • Examples of the bacterial sialidase include Arthrobacter sp., Bifidobacterium sp., Streptococcus sp., Vibrio cholerae, Salmonella Typhimurium, and Clostridium perfringens.
  • Cialidase derived from Arthrobacter ureafaciens commercially available: Nakaraitesk Co., Ltd., model number 24229) and Bifidobacterium bacterium from the viewpoint of promoting elastin production and safety.
  • Bifidobacterium bifidum-derived sialidase and the like are particularly preferably used.
  • examples of usable virus sialidase include neuraminidase (NA) of influenza A virus, neuraminidase (NA) of influenza B virus, and bacteriophage. It is possible to use only one type of sialidase or a combination of a plurality of types.
  • the sialidase in the present invention can be obtained by gene expression by gene recombination using a gene encoding sialidase according to a conventional method.
  • a recombinant vector can be obtained by inserting a DNA fragment encoding sialidase as an insert downstream of the promoter of an appropriate expression vector.
  • the promoter is a DNA sequence that exhibits transcriptional activity in a host cell, and can be appropriately selected depending on the type of host.
  • a transformant producing a protein having sialidase activity can be obtained.
  • sialidase can also be obtained by extraction and purification from cells or tissues containing sialidase.
  • the elastin production promoter of the present invention contains the above-mentioned sialidase as an active ingredient, and has an action of increasing the amount of elastin in a tissue containing elastin.
  • tissue containing elastin include the dermis of the skin, the blood vessel wall, the ligaments, and the lung tissue.
  • the dermis elastin contained in the dermis of the skin and constituting the fibrous tissue is increased. Has an effect. The amount of elastin present in the dermis decreases with age, causing wrinkles and sagging of the skin.
  • the elastin production promoter of the present invention can maintain or restore the firmness and elasticity of the skin, and can prevent or improve the aging of the skin.
  • the dose of the elastin production-promoting agent of the present invention cannot be unconditionally specified because it varies depending on the target improvement or preventive effect, age, administration method, etc., but the daily dose is usually used as the active ingredient sialidase. , 0.0001U to 10U, more preferably 0.001U to 1U, and even more preferably 0.005U to 0.5U.
  • the transdermal dose, 2 per coating area 1cm As an example, a sialidase as an active ingredient, it is preferable that about 0.00005U ⁇ 5U, the order of 0.0005 ⁇ 0.5 U It is more preferable, and it is more preferable that it is about 0.0025U to 0.25U.
  • a substance having a transdermal drug delivery action As the substance having a transdermal drug delivery action, a known TDDS (Transdermal Drug Delivery System) can be used as long as it does not interfere with the action and effect of the present invention. More specifically, it is preferable to use a so-called "ionic liquid", which is an organic salt that exhibits a liquid at normal temperature and pressure, for transdermal drug delivery.
  • TDDS Transdermal Drug Delivery System
  • choline geranic acid As the ionic liquid, those using choline as a cation are preferable, and examples thereof include choline geranic acid, choline oleic acid, choline malonic acid, etc. Among them, choline geranic acid (CAGE) represented by the following chemical formula 1 is particularly preferable. Can be used.
  • the substance having the transdermal drug delivery action as described above can be contained in the elastin production promoter as a compounding ingredient together with the active ingredient sialidase. This gives a one-agent type elastin accelerator.
  • the substance having a transdermal drug delivery action as described above can be administered separately from the active ingredient sialidase.
  • a substance having a transdermal drug delivery action such as CAGE is applied after the sialidase is applied.
  • CAGE a substance having a transdermal drug delivery action
  • TDDS Transdermal Drug Delivery System
  • microneedles needleless injectors
  • iontophoresis iontophoresis
  • ultrasonic introduction sonophoresis
  • the elastin production promoter of the present invention can be used as a skin cosmetic having an anti-aging effect. Thereby, the firmness and elasticity of the skin can be maintained or restored, and the wrinkles and sagging of the skin can be prevented or improved.
  • the elastin production promoter or skin cosmetic of the present invention may contain various components usually blended in an external preparation as long as the action and effect of the present invention are not impaired.
  • examples thereof include moisturizers, emollients, plant extracts, vegetable oils and fats, pH regulators, surfactants, thickeners, vitamins, amino acids, preservatives, antibacterial agents, fragrances and pigments.
  • the elastin production promoter or skin cosmetic of the present invention can be applied to various external preparation dosage forms by a conventionally used method as a dosage form usually applied to the skin, for example, lotion.
  • a dosage form usually applied to the skin for example, lotion.
  • Such as liquids, emulsions, gels, powders such as dry water, creams and the like can be mentioned.
  • It can also be applied to patches, facial masks, makeup products, cleansing agents, soaps, hair care agents, hair growth agents and the like.
  • the active ingredient sialidase and the substance having a transdermal drug delivery effect are separately formulated, the separately formulated ones can be mixed and administered at the time of use, or they are separately prepared. It is also possible to administer the isomers at the same time or at different times.
  • Example 1 Examination of sialidase activity in skin tissue (1) The presence or absence and distribution of sialidase activity in rat skin tissue was investigated using the fluorescence imaging probe (Non-Patent Document 1) developed by the present inventors.
  • This fluorescent imaging probe has a phenolic hydroxyl group of BTP3 [(2-benzothiazole-2-yl) -4-bromophenol], which is a fluorescent substance, and Neu5Ac (N-acetylneuramine), which is a molecular species of sialic acid recognized by sialidase. It is BTP3-Neu5Ac to which acid) is bound.
  • BTP3-Neu5Ac is soluble in water and its fluorescence is also controlled off, but when it reacts with sialidase, a water-insoluble fluorescent substance BTB3 is generated, deposited on the tissue, and fluorescently stained. Therefore, the site of sialidase activity can be histochemically fluorescently imaged.
  • the back skin of a Wistar male rat (12 weeks old, Nippon SLC Co., Ltd.) was used as an embedding agent for preparing frozen tissue sections (Tisshoe Tech (registered trademark) OCT Compound, Sakura Finetech Japan Co., Ltd.). ) was embedded.
  • a frozen tissue section having a thickness of 100 to 300 ⁇ m was prepared by cryostat (Leica Microsystems, Inc.).
  • the sections after observation were stained with hematoxylin and eosin (H & E staining) for observation.
  • the results are shown in FIG.
  • Example 2 2. Examination of sialidase activity in skin tissue (2) The relationship between sialidase activity in rat skin tissue and aging was investigated. Cialidase activity in each skin of Wistar male rats (Nippon SLC Co., Ltd.) on the 19th day of pregnancy (embryonic) (E19), 1-week-old, 6-week-old, 12-week-old, and over 21-month-old was measured. Twice the amount of 0.32 M sucrose was added to the skin of each rat collected, and homogenized at 200 rpm while cooling with ice.
  • Example 3 Examination of isozymes of sialidase present in the dermis
  • isozymes of animal sialidases There are four types of isozymes of animal sialidases: NEU1, NEU2, NEU3 and NEU4. The four isozymes differ in their localization and substrate specificity, and therefore their functions and roles are also different. Since strong sialidase activity was detected in the dermis subcutaneous layer of rat skin tissue in Example 1, an isozyme of sialidase highly expressed in the dermis was investigated.
  • TRIzol registered trademark
  • the mixture was centrifuged at 4 ° C. and 15,000 rpm for 20 minutes to separate the aqueous layer.
  • 250 ⁇ L of 2-propanol was added to 250 ⁇ L of the aqueous layer, and after inversion mixing, the mixture was allowed to stand at room temperature for 10 minutes. After standing, it was centrifuged at 4 ° C. and 15,000 rpm for 10 minutes, and after removing the supernatant, 500 ⁇ L of 75% ethanol was added and centrifuged at 4 ° C. and 15,000 rpm for 20 minutes.
  • RNA separated by a spectrophotometer (NanoDrop ND-1000, Thermo Fisher Scientific Co., Ltd.) was quantified, and the concentration of total RNA was adjusted to 100 ng / ⁇ L with UltraPure DNase / RNase-Free Distilled Water.
  • RNA sample isolated from the excised tissues (A) and (B) were measured by real-time RT-PCR method. Quantified. Specifically, using OneStep TB Green PrimeScript PLUS RT-PCR Kit (Takara Bio Inc.), measurements were taken according to the protocol, and the expression levels of each mRNA were compared. Thermal Cycler Dice (registered trademark) Real Time System Lite (Takara Bio Inc.) was used as the measuring device.
  • FIG. 3 (a) shows the mRNA expression level of sialidase Neu1
  • FIG. 3 (b) shows the mRNA expression level of sialidase Neu2
  • FIG. 3 (c) shows the mRNA expression level of sialidase Neu3
  • FIG. 3 (d) shows the mRNA expression of sialidase Neu4.
  • FIG. 3 shows the level of mRNA expression level in the dermis and the muscle tissue (Dermis + Muscle) immediately below the dermis when the mRNA expression level of the subcutaneous adipose tissue (Fat) is 1. From this result, it was clarified that among the four isozymes, sialidase NEU2 was highly expressed in the dermis.
  • Example 4 Elastin production promoting effect by sialidase NEU2
  • sialidase NEU2 is present in a large amount in the dermis. Therefore, the elastin production promoting effect by sialidase NEU2 was investigated. Specifically, sialidase NEU2 was prepared by expressing recombinant protein in cultured cells, applied to rat skin for a predetermined period of time, and then the amount of elastin contained in rat skin tissue was measured.
  • a DNA fragment of a gene (2531bp) encoding rat sialidase NEU2 was prepared by the cloning method.
  • the DNA fragment of the prepared rat Neu2 gene was cloned into a vector as an insert to obtain a recombinant vector containing the Neu2 gene.
  • the constructed NEU2 expression vector was introduced into C6 glioma cells and transfected to obtain NEU2-producing cells.
  • the obtained NEU2-producing cells were cultured in MEM medium to express proteins.
  • the expressed NEU2 protein was recovered from the medium and concentrated by ultrafiltration. PBS (pH 7.3) containing 4MU-Neu5Ac was added to this concentrate, and the mixture was incubated at 27 ° C. for 1 hour.
  • the enzymatic activity of sialidase NEU2 obtained by expression of the recombinant protein was 21.3.4MU nmol / min / mL.
  • the stratum corneum which is the outermost cell of the skin, has the function of a physical barrier that prevents the invasion of foreign substances. Therefore, even if a protein such as sialidase NEU2 is applied to the skin surface, it is difficult to allow the stratum corneum to permeate and act inside the skin tissue. Therefore, in this example, cholinergic acid (CAGE), which is an ionic liquid, was used for transdermal administration of the sialidase NEU2 obtained as described above.
  • CAGE cholinergic acid
  • the structural formula of CAGE is shown in Chemical Formula 2 below. CAGE is known to have excellent drug solubility and a transdermal drug delivery action (see Special Table 2016-535781).
  • a sample solution (CAGE + Neu2) in which CAGE and Cialidase NEU2 were mixed was prepared, and a coating test was conducted.
  • a double application test (Neu2, CAGE) in which a certain amount of Cialidase NEU2 was applied and then CAGE was applied.
  • a test was also conducted in which only PBS (phosphate buffer) and CAGE were applied as sample solutions.
  • the skin tissue of the part where the sample solution was applied was collected. After homogenizing the collected skin tissue, the amount of elastin contained in the homogenate of the skin tissue was measured using an elastin colorimetric determination kit (Fastin Elastin Assay Kit, manufactured by Biocolor). The results are shown in the graph of FIG. According to this result, the amount of elastin in the skin tissue was applied both when Cialidase NEU2 and CAGE were applied as a mixed solution (CAGE + Neu2) and when Cialidase NEU2 was applied and then CAGE was applied (Neu2, CAGE). Was found to increase. From this, it was clarified that the transdermal administration of Cialidase NEU2 promotes the production of elastin in the skin tissue.
  • Cialidase NEU2 since the increase in the amount of elastin is large when Cialidase NEU2 is applied and then CAGE is applied (Neu2, CAGE), CAGE applies the previously applied Cialidase NEU2 to the skin. It was found that it can be quickly taken up above and delivered inside the skin.
  • RNA expression of the elastin gene was investigated. Specifically, one day after the last application of each sample solution shown in Table 1, tissues such as skin and subcutaneous fat on the flank of the rat were collected, and TRIzol (registered trademark) Reagent (Invitrogen Co., Ltd.) was used. Total RNA was separated from rat skin by the same method as in Example 3. Using the isolated RNA sample, the mRNA expression level of the elastin gene in rats was measured by the real-time RT-PCR method. Real-time RT-PCR was performed according to the protocol using the One-Step SYBR PrimeScript PLUS RT-PCR kit (Takara Bio Inc.) and primers.
  • Thermal Cycler Dice (registered trademark) Real Time System Lite (Takara Bio Inc.) was used as the measuring device.
  • the expression level of elastin mRNA was determined as a relative amount of elastin mRNA to the internal standard GAPDH (glyceraldehyde-3-phosphate dehydrogenase). The results are shown in the graph of FIG.
  • Example 5 Elastin production promoting effect by bacterial sialidase
  • sialidase There are multiple types of sialidase, such as animal sialidase present in mammals, viral sialize present in viruses, and bacterial sialidase present in bacteria. Of these, the elastin production-promoting effect of bacterial sialidase, which has higher sialidase activity than animal sialidase, was investigated.
  • a sample solution (CAGE + AUSA) was prepared by mixing a certain amount of cholinergic acid (CAGE) and Cialidase (AUSA) derived from Arthrobacter ureafaciens in the same manner as in Example 4, and a coating test was conducted.
  • a test was also conducted in which only PBS (phosphate buffer) and CAGE were applied as sample solutions.
  • each sample solution was applied to the area of a circle having a diameter of 15 mm from which the hair was removed from the rat.
  • the application was performed twice a day for 5 days (10 times in total).
  • elastin in the skin tissue subjected to the application test was confirmed by immunohistochemical staining (Fig. 8) and autofluorescence of elastin (Fig. 9). Specifically, an extended section was prepared from the dermis of the collected skin tissue and fixed with 4% paraformaldehyde in PBS. 100 ⁇ L of normal goat serum (Goat Serum 2%) was added as a blocking agent for immunohistochemical staining, and the mixture was incubated at room temperature for 30 minutes. Subsequently, 100 ⁇ L of the primary antibody (Elastin Polyclonal antibody) was added, and the mixture was incubated overnight at 4 ° C.
  • the primary antibody Elastin Polyclonal antibody
  • elastin is an autofluorescent substance that emits autofluorescence
  • the autofluorescence of elastin was observed using a fluorescence microscope for dermis sections fixed with 4% paraformaldehyde in PBS. The results are shown in FIG.
  • a large amount of elastin autofluorescence was observed, and an increase in the amount of elastin was observed.
  • Example 6 Elastin production promoting effect on aging-promoting model mice
  • sialidase is transdermally administered to individuals in a state where the elastin amount is reduced due to the decrease in elastin activity due to aging using the aging-promoting model mice. It was investigated whether this could promote the production of elastin.
  • SAMP1 and SAMP8 which are SAMPs (Senescence-Accelerated Mouse Prone) showing accelerated aging and short life span, were selected (24 weeks old, male, Nippon SLC Co., Ltd.).
  • the hair on the left and right backs of the Society for Senescence Acceleration Model Mouse was removed, and a site to which the sample solution was applied was provided.
  • Table 3 With the contents shown in Table 3 below, each sample solution was applied to the area of a circle having a diameter of 10 mm from which the hair of the mouse was removed. The application was performed twice a day for 5 days (10 times in total).
  • the skin tissue of the part to which the sample solution was applied was collected, and a part of the collected skin tissue was homogenized.
  • the amount of elastin contained in the homogenate of the collected skin tissue was measured using an elastin colorimetric kit (Fastin Elastin Assay Kit, a product of Biocolor). The results are shown in the graph of FIG.
  • Example 7 Examination of Stability of Enzyme Activity of Siaridase NEU1-producing cells producing rat-derived sialidase NEU1 were obtained by the same method as in Example 4 and using C6 glioma cells.
  • the NEU1-producing cells were cultured in MEM medium to express sialidase NEU1.
  • the cells were collected and subjected to cytolysis treatment to obtain a solubilized solution containing the expressed sialidase NEU1.
  • PBS (pH 4.6) containing 4MU-Neu5Ac was added to this solubilized solution, and the mixture was reacted at 27 ° C. for a predetermined time.
  • sialidase NEU2 can be suitably used as an elastin production promoter as compared with sialidase NEU1 when applied to the skin.
  • the present invention provides an elastin production-promoting agent and a skin cosmetic that have an excellent elastin production-promoting effect and can prevent or improve the decrease of dermal elastin due to aging. It is widely useful in the industry of.

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PCT/JP2021/019322 2020-05-25 2021-05-21 エラスチン産生促進剤及び皮膚化粧料 Ceased WO2021241428A1 (ja)

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CN202180033344.0A CN115515558B (zh) 2020-05-25 2021-05-21 弹性蛋白产生促进剂以及皮肤化妆品
JP2022526975A JP7312993B2 (ja) 2020-05-25 2021-05-21 エラスチン産生促進剤及び皮膚化粧料
EP21813509.3A EP4159284A4 (en) 2020-05-25 2021-05-21 Elastin production promoter and cosmetic preparation for skin
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