Type vii collagen secretagogue

Abstract

To provide a novel type VII collagen secretagogue.SOLUTION: The present invention provides a type VII collagen secretagogue containing as an active ingredient, one or more crude drugs selected from the group consisting of cassis, sweet tea, and cherry. In addition, the present invention provides a type VII collagen secretagogue containing one or more crude drugs selected from the group consisting of cassis, sweet tea, and cherry as active ingredients, that promotes type VII collagen secretion through promotion of TANGO1 expression and / or activation.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a type VII collagen secretion promoter. [Background technology]

[0002] Type VII collagen is known to be involved in the binding of the skin's basement membrane to the dermis, and abnormalities or deficiencies of type VII collagen are known to cause skin fragility disorders such as dystrophic epidermolysis bullosa (DEB / RDEB), as well as diseases and disorders such as erosion, joint contractures, hair loss, localized blisters on the dermal side of the basement membrane at the dermal-epidermal junction, in the limbs, and in the esophagus, generalized blisters, squamous cell carcinoma, decreased skin elasticity, and nail deformation and loss (Patent Documents 1 to 4, Non-Patent Documents 1 to 4).

[0003] To prevent and treat the above-mentioned diseases and disorders, various substances have been explored as agents for promoting type VII collagen expression / production, and efforts have been made to create antisense oligomers and vectors for increasing type VII collagen gene expression (Patent Documents 1 to 4). However, collagen is a large molecule, and when synthesized in the endoplasmic reticulum, it already forms a linear structure of about 300 nm, which poses a problem of not being secreted by normal transport vesicles with diameters of 60 to 90 nm (Non-Patent Documents 5 and 6). Therefore, there is a need to explore agents that not only promote type VII collagen gene expression and protein synthesis, but also promote the secretion of synthesized type VII collagen. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Laid-Open No. 2014-221739 [Patent Document 2] Japanese Patent Application Laid-Open No. 2006-206571 [Patent Document 3] Special Publication No. 2018-518167 [Patent Document 4] Special Publication No. 2019-508454 [Patent Document 5] Japanese Patent Application Laid-Open No. 2007-320891 [Patent Document 6] Japanese Patent Application Laid-Open No. 2005-255527 [Patent Document 7] Patent No. 6217038 [Patent Document 8] Japanese Patent Application Laid-Open No. 2012-6905 [Patent Document 9] Japanese Patent Application Publication No. 2019-59698 [Non-patent literature]

[0005] [Non-Patent Document 1] Experimental Dermatology, 2008, Volume 17, Issue 7, p. 553-568 https: / / doi.org / 10.1111 / j.1600-0625.2008.00723.x [Non-patent document 2] J Invest Dermatol. 2013 Jul;133(7):1910-3. doi: 10.1038 / jid.2013.10. Epub 2013 Jan 15. [Non-patent document 3] https: / / www.nanbyou.or.jp / entry / 5338 [Non-patent document 4] https: / / www.dermatol.or.jp / qa / qa31 / q02.html [Non-Patent Document 5] Kenta Saito, Department of Physiology, Graduate School of Pharmaceutical Sciences, University of Tokyo, Functional analysis of a novel endoplasmic reticulum membrane protein that controls the secretion of the giant molecule type VII collagen, https: / / www.astellas-foundation.or.jp / pdf / research / 21 / h21_25_saitou.pdf [Non-patent document 6] Kenta Saito, Department of Physiology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Analysis of the Secretion Mechanism of Type VII Collagen, the Causative Gene of Dystrophic Epidermolysis Bullosa, Cosmetology Research Report Vol. 20, 2012, https: / / www.kose-cosmetology.or.jp / research_report / archives / 2012 / fullVersion / Cosmetology%20Vol20%202012%20p62-65%20Saito_K.pdf Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide a novel type VII collagen secretion promoter. [Means for solving the problem]

[0007] As a result of extensive research into the effects of various ingredients as type VII collagen secretion promoters, the inventors have found that black currant, sweet tea, and cherry blossom are particularly effective as type VII collagen secretion promoters, leading to the completion of the following invention: (1) A type VII collagen secretion promoter comprising one or more herbal medicines selected from the group consisting of black currant, sweet tea, and cherry blossom. (2) A type VII collagen secretion promoter according to (1), which promotes type VII collagen secretion by promoting the expression and / or activity of TANGO1. (3) A composition comprising the type VII collagen secretion promoter according to (1) or (2). [Effects of the Invention]

[0008] Administration of the type VII collagen secretion promoter of the present invention can promote the secretion of type VII collagen. According to the present invention, a composition containing the type VII collagen secretion promoter can be provided. Promotion of type VII collagen secretion is expected to be effective in treating / preventing skin fragility disorders, erosions, joint contractures, hair loss, localized blisters on the dermal side of the basement membrane at the dermal-epidermal junction, in the limbs, in the esophagus, etc., generalized blisters, squamous cell carcinoma, and nail deformation / loss, as well as maintaining / improving skin elasticity. [Brief explanation of the drawings]

[0009] [Figure 1] Figure 1 shows the results of Example 3, and is a graph comparing the TANGO1 expression-promoting effects of black currant, sweet tea, and cherry blossom with those of a control (control) without these herbal medicines. The results are shown as relative values ​​(%), with the TANGO1 expression level of the control set at 100 (Dunnett's test, #: P<0.1, *: P<0.05, **: P<0.01). [Figure 2] Figure 2 shows the results of Comparative Example 1, a graph comparing the type I collagen production-promoting effects of black currant, sweet tea, and cherry blossom with a control (Ctrl) containing no added herbal medicines. The results are shown as relative values ​​(%), with the amount of type I collagen produced in the control set at 100 (Dunnett's test, #: P<0.1, *: P<0.05, **: P<0.01). DETAILED DESCRIPTION OF THE INVENTION

[0010] The present invention provides a type VII collagen secretion promoter containing, as an active ingredient, one or more herbal medicines selected from the group consisting of black currant, sweet tea, and cherry blossom.

[0011] Type VII collagen is a type of collagen expressed by the COL7A1 (Collagen Type VII Alpha 1 Chain) gene. Mutations in the COL7A1 gene are known to cause disorders such as dystrophic epidermolysis bullosa (DEB / RDEB). Abnormalities or deficiencies of type VII collagen are also known to cause diseases and disorders such as various skin fragility disorders, erosions, joint contractures, hair loss, localized blisters on the dermal side of the dermal-epidermal junction basement membrane, the limbs, the esophagus, etc., generalized blisters, squamous cell carcinoma, and nail deformities and loss. Type VII collagen is also known to strengthen the bond between the skin's basement membrane and the dermis by lifting collagens such as type I collagen and type III collagen to the skin's basement membrane, thereby contributing to maintaining skin elasticity and preventing sagging (Patent Documents 1-4, Non-Patent Documents 1-4).

[0012] As mentioned above, type VII collagen is too large to be secreted by conventional transport vesicles. However, the existence of a specific cargo receptor complex (cTAGE / TANGO1 / Sec12) for secreting such large type VII collagen has been reported. For example, TANGO1 (transport and Golgi organization 1), a component of the cargo receptor complex, is known to bind to type VII collagen on the luminal side of the endoplasmic reticulum, thereby promoting the budding of type VII collagen molecules from the endoplasmic reticulum and promoting secretion. Therefore, promoting type VII collagen secretion by activating type VII collagen-specific secretory cofactors such as TANGO1 is effective. In fact, it has been reported that low expression of the TANGO1 gene results in the accumulation of type VII collagen in the endoplasmic reticulum and suppresses its secretion (Non-Patent Documents 5 and 6). Therefore, if the secretion of type VII collagen can be promoted by black currant, sweet tea, and / or cherry blossom, which the inventors have discovered to have high TANGO1 expression and / or activity-promoting effects, it is believed that the effects of type VII collagen in preventing / treating the various diseases and disorders mentioned above and maintaining / improving skin elasticity will be enhanced.

[0013] "Promotion of TANGO1 expression and / or activity" can mean, for example, an increase in the expression level of the TANGO1 gene or the amount of TANGO1 protein when a type VII collagen secretion promoter is administered compared to a state in which the agent is not administered (control), e.g., an enhancement with a statistically significant difference (e.g., Student's t-test) at a significance level of 5%. Alternatively, "promotion of TANGO1 expression and / or activity" can mean, for example, an enhancement of the expression level of the TANGO1 gene or the amount of TANGO1 protein when a type VII collagen secretion promoter is administered compared to a state in which the agent is not administered (control), e.g., an enhancement of 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100% or more, 200% or more, 300% or more, 400% or more, or 500% or more.

[0014] Such promoting effect on TANGO1 expression and / or activity can be measured by various methods, including in vivo, in vitro, ex vivo, etc. For example, a test substance is administered to cells such as liver cells, mammary gland cells, adipocytes, or skin cells, and the level of TANGO1 mRNA expression in the cells is determined by methods such as qPCR, or the amount of TANGO1 protein is determined by methods such as Western blotting. However, the measurement method is not limited to the above methods, and any other method may be employed. For example, an in vivo or ex vivo method may be employed in which the level of TANGO1 mRNA expression or the amount of TANGO1 protein is measured in samples such as liver, breast, adipose, or skin tissue / models after administration to an animal such as a human.

[0015] The type VII collagen secretion-promoting activity can also be measured by various methods including in vivo, in vitro, ex vivo, etc. For example, as described above, it may be determined by measuring the expression and / or activity of TANGO1, a type VII collagen-specific secretory cofactor such as TANGO1, or by measuring the expression and / or activity of other factors known to be involved in type VII collagen secretion, such as cTAGE5 reported in Non-Patent Document 6, etc. Alternatively, a method such as measuring the amount of type VII collagen inside or outside the lumen of the endoplasmic reticulum is also possible.

[0016] Thus, the present invention provides a type VII collagen secretion promoter containing one or more herbal medicines selected from the group consisting of black currant, sweet tea, and cherry blossom as an active ingredient, which promotes type VII collagen secretion through promoting the expression and / or activity of TANGO1. The present invention also provides a composition containing a type VII collagen secretion promoter containing one or more herbal medicines selected from the group consisting of black currant, sweet tea, and cherry blossom as an active ingredient. The composition of the present invention may be a cosmetic composition or a food composition. Furthermore, the composition of the present invention may be a composition for enhancing one or more effects selected from the treatment / prevention of skin fragility disorders, erosions, joint contractures, hair loss, localized blisters on the dermal side of the basement membrane at the dermal-epidermal junction, in the limbs, in the esophagus, etc., generalized blisters, squamous cell carcinoma, nail deformity / loss, etc., and maintenance / improvement of skin elasticity by promoting type VII collagen secretion through promotion of TANGO1 expression and / or activity.

[0017] The blackcurrant (Ribes nigrum) used in the present invention is a shrub of the Ribes genus in the family Ribesaceae. It is preferable to use the fruit. Blackcurrant is known to have the effect of moisturizing and firming the skin and promoting biocollagen synthesis (Patent Documents 5, 6, etc.).

[0018] The sweet tea (Rubus suavissimus) used in the present invention is a shrub belonging to the genus Rubus in the family Rosaceae. It is preferable to use the leaves. Sweet tea is known to promote biocollagen synthesis and inhibit the production of CML (advanced glycation end products) from collagen (Patent Documents 6 and 7, etc.).

[0019] The cherry blossom (Cerasus Mill.) used in the present invention is a deciduous broadleaf tree of the genus Prunus or Prunus in the family Rosaceae. The cherry blossom species is not particularly limited, and various species may be used, such as Somei-Yoshino (Cerasus yedoensis), Yamazakura (Cerasus jamasakura), Oshimazakura (Cerasus speciosa), and Oyamazakura (Cerasus sargentii). It is preferable to use flowers and / or leaves. Cherry blossoms are known to promote the expression of type I collagen-producing genes, inhibit collagenase gene expression, suppress stratum corneum moisture content, inhibit increases in melanin levels, and improve wrinkles and skin elasticity (Patent Documents 8, 9, etc.).

[0020] However, there have been no reports of any of the above herbal medicines promoting type VII collagen secretion or TANGO1 expression / activity.

[0021] The type VII collagen secretion promoter of the present invention may contain, as an active ingredient, one or more herbal medicines selected from the group consisting of black currant, sweet tea, and cherry blossom, for example, at a concentration of 10% by mass or more, 20% by mass or more, 30% by mass or more, 40% by mass or more, 50% by mass or more, 60% by mass or more, 70% by mass or more, 80% by mass or more, 90% by mass or more, 95% by mass or more, or 99% by mass or more. In one embodiment, the type VII collagen secretion promoter of the present invention may consist of one or more herbal medicines selected from the group consisting of black currant, sweet tea, and cherry blossom.

[0022] The above-mentioned herbal medicines are known substances, can be easily squeezed, dried, purified, extracted, etc. by known methods, and are readily available as commercial products. They can be used in either raw or dried form, but from the standpoint of usability, formulation, etc., they can also be used as extracts, dried products, dried powders, powdered raw materials, squeezed juices, etc. The form to be used can be appropriately selected depending on the raw material, and further, treatments such as sterilization may be performed as necessary.

[0023] When used as an extract, the extract can be extracted, for example, by solvent extraction. In solvent extraction, the whole plant or various parts (fruits, leaves, flowers, roots, etc.) of the plant are dried as needed, and further shredded or crushed as needed. The extract is then extracted using an aqueous extractant, water (e.g., cold water, warm water, or hot water at or below boiling point), or a hydrous organic solvent, or an organic solvent (e.g., ethanol, methanol, ether, 1,3-butylene glycol, etc.), selected appropriately depending on the properties of the raw materials and the intended use of the composition, at room temperature or with heating. However, the extraction method is not limited to solvent extraction and may be any conventional method known in the art. The extraction method and form of the extract used in the present invention are arbitrary as long as they do not impair the effects of the present invention. The extract may be in the form of a liquid extract itself, or a liquid obtained by diluting or concentrating it as appropriate using conventional methods. It may also be in the form of a powder or lumpy solid obtained by drying the extract, or a squeezed juice obtained by diluting or concentrating it as appropriate using conventional methods.

[0024] Examples of the aqueous organic solvent include aqueous lower alcohols (e.g., C1 to C4) such as aqueous ethanol, and in this case the water content may be, for example, 0 to 10 v / v%, 10 to 15 v / v%, 10 to 20 v / v%, 20 to 30 v / v%, 30 to 50 v / v%, 50 to 80 v / v%, 60 to 85 v / v%, 70 to 90 v / v%, 85 to 99.5 v / v%, etc.

[0025] Methods for obtaining dry powder include shredding or crushing the whole plant or various parts (leaves, flowers, roots, etc.) and then drying, or drying the plant and then shredding or crushing it to obtain dry powder. Alternatively, a method can be used where the plant is shredded or crushed, fermented or enzymatically treated, dried, and then crushed to a predetermined particle size, as needed.

[0026] The type VII collagen secretion promoter or composition of the present invention may be a pharmaceutical product, a quasi-drug, a food composition, a cosmetic composition, or the like. It can be administered topically or orally. The form of topical administration can be selected arbitrarily, for example, from cream, emulsion, liquid, sheet, spray, gel, etc. The form of oral administration can be selected arbitrarily, for example, from tablets, supplements, beverages, powder, etc.

[0027] The amounts of black currant, sweet tea, and cherry blossom in the type VII collagen secretion promoter or composition of the present invention can be appropriately determined depending on their type, purpose, form, method of use, etc. For example, in the case of topical administration, the amounts of black currant, sweet tea, and cherry blossom can be 0.0001 to 50% by weight, 0.001 to 50% by weight, 0.01 to 50% by weight, 0.01 to 5% by weight, 0.01 to 1% by weight, 0.01 to 0.1% by weight, etc., based on the total weight of the type VII collagen secretion promoter or composition of the present invention.

[0028] The administration frequency can be selected arbitrarily, such as once every 4 weeks, once every 2 weeks, once a week, once every 3 days, once every 2 days, once a day, twice a day, three times a day, four times a day, five times a day, or administration as needed, but is not limited to these.

[0029] The type VII collagen secretion promoter or composition of the present invention can be used in combination with any additive selected as needed. Additives that can be used include excipients and the like.

[0030] The excipient may be any one that is normally used when preparing the desired dosage form, and examples thereof include starches such as wheat starch, rice starch, corn starch, potato starch, dextrin, and cyclodextrin, crystalline celluloses, sugars such as lactose, glucose, sugar, reduced maltose, starch syrup, fructooligosaccharides, and emulsified oligosaccharides, and sugar alcohols such as sorbitol, erythritol, xylitol, lactitol, and mannitol. These excipients can be used alone or in combination of two or more.

[0031] Other additives that can be used include colorants, preservatives, thickeners, binders, disintegrants, dispersants, stabilizers, gelling agents, antioxidants, surfactants, preservatives, pH adjusters, oils, water, alcohols, chelating agents, silicones, ultraviolet absorbers, moisturizers, fragrances, various medicinal ingredients, antiseptics, and neutralizing agents, and can be appropriately selected and used.

[0032] The present invention also provides a method for promoting type VII collagen secretion through the promotion of TANGO1 expression and / or activity by administering one or more herbal medicines selected from the group consisting of black currant, sweet tea, and cherry blossom. The present invention also provides a method for enhancing one or more effects selected from the treatment / prevention of skin fragility disorders, erosions, joint contractures, hair loss, localized blisters on the dermal side of the basement membrane at the dermal-epidermal junction, the limbs, the esophagus, etc., generalized blisters, squamous cell carcinoma, nail deformity / loss, etc., and maintenance / improvement of skin elasticity, by promoting type VII collagen secretion through the promotion of TANGO1 expression and / or activity by administering one or more herbal medicines selected from the group consisting of black currant, sweet tea, and cherry blossom. The methods of the present invention are for cosmetic purposes and may not be performed by a physician or medical professional.

[0033] The present invention also provides use of one or more herbal medicines selected from the group consisting of black currant, sweet tea, and cherry blossom in the manufacture of a medicament for enhancing one or more effects selected from the treatment / prevention of skin fragility disorders, erosion, joint contracture, hair loss, localized blisters on the dermal side of the basement membrane at the dermal-epidermal junction, limbs, esophagus, etc., generalized blisters, squamous cell carcinoma, nail deformity / loss, etc., and maintaining / improving skin elasticity.The present invention also provides one or more herbal medicines selected from the group consisting of black currant, sweet tea, and cherry blossom for use in a method for enhancing one or more effects selected from the treatment / prevention of skin fragility disorders, erosion, joint contracture, hair loss, localized blisters on the dermal side of the basement membrane at the dermal-epidermal junction, limbs, esophagus, etc., generalized blisters, squamous cell carcinoma, nail deformity / loss, etc., and maintaining / improving skin elasticity, by promoting the secretion of type VII collagen through the promotion of TANGO1 expression and / or activity. [Example]

[0034] The present invention will now be described in more detail with reference to examples, although the present invention is not limited thereto.

[0035] Example 1: Sample preparation As candidate samples for type VII collagen secretion promoters, black currant, sweet tea, and cherry blossom were prepared as shown in the table below. [Table 1] A total of 15 candidate samples were prepared, including natural ingredients such as animal and plant extracts, as well as synthetic ingredients. The samples were prepared using DMSO and diluted appropriately with culture medium to a concentration of 10 μg / ml as the evaluation samples. The same amount of DMSO without the evaluation samples was used as a control.

[0036] Example 2: Cultivation of human skin fibroblasts Adult human dermal fibroblasts (Thermo Fisher Scientific) were cultured at 40,000 cells / cm 2The cells were seeded onto a cell culture dish at a density of 100 μg / ml and cultured overnight in Dulbecco's Modified Eagle Medium (DMEM) containing 10% FBS (Fetal Bovine Serum) at 37°C in a 5% CO2 atmosphere, and then subjected to Example 3 and Comparative Example 1.

[0037] Example 3: Evaluation of TANGO1 expression promoting effect Addition of sample The medium for the cells cultured overnight in Example 2 was replaced with DMEM containing 0.5% FBS, and after 24 hours, each sample was added to the cells at a concentration of 10 μg / mL, followed by further culture for 24 hours. As a control, the same amount of DMSO was added.

[0038] Extraction of RNA from cells 24 hours after the addition of the sample, the medium was removed, and cells were lysed and RNA was extracted using a commercially available RNA extraction reagent (RNeasy Mini Kit, Qiagen).

[0039] Evaluation of TANGO1 expression levels by qPCR Quantitative PCR was performed using the extracted RNA as a template using commercially available PCR reagents (TaqMan RNA-to-CT 1step Kit, Cat. No. 4392938) (Life Technologies) and a PCR device (LightCycler 480 System) (Roche) to measure the expression level of the TANGO1 gene. Expression levels of the β-actin gene were also measured simultaneously as an internal control. Gene-specific PCR primers (Thermo Fisher Scientific, Cat. No. Hs01558382_m1 (TANGO1) and Hs01060665_g1 (β-actin)) were used.

[0040] The results are shown in Figure 1. The TANGO1 gene expression level when each sample was added is shown as a comparison with the control TANGO1 gene expression level, which was set at 100. Figure 1 confirms that black currant, sweet tea, and cherry blossom significantly promoted TANGO1 expression compared to the control without these herbal medicines. This suggests that black currant, sweet tea, and cherry blossom have the effect of promoting type VII collagen secretion through the promotion of TANGO1 expression.

[0041] Comparative Example 1: Evaluation of type I collagen production promoting effect Addition of sample The medium for the cells cultured overnight in Example 2 was replaced with DMEM containing 0.5% FBS, and after 24 hours, each sample was added to the cells at a concentration of 10 μg / mL, followed by further culture for 72 hours. As a control, the same amount of DMSO was added.

[0042] Measurement of type I collagen levels by ELISA 72 hours after the addition of the sample, the culture supernatant was collected, and the amount of type I collagen was measured using a Procollagen Type I C-peptide (PIP) EIA Kit (Takara Bio Inc.).

[0043] The results are shown in Figure 2. As can be seen from Figure 2, when black currant, sweet tea, and cherry blossoms were added, the amount of type I collagen did not increase, and in fact tended to decrease compared to the control, indicating no promotion of type I collagen production. This suggests that the effects of black currant, sweet tea, and cherry blossoms may be specific to type VII collagen. [Industrial Applicability]

[0044] The present invention can promote type VII collagen secretion by administering a type VII collagen secretion promoter containing black currant, sweet tea, and cherry blossom as active ingredients.

Claims

1. A type VII collagen secretion promoter containing black currant.

2. The type VII collagen secretion promoter according to claim 1, which promotes type VII collagen secretion by promoting the expression and / or activity of TANGO1.

3. A composition comprising the type VII collagen secretion promoter according to claim 1 or 2.

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