JP7623826B2 - TIPARP production promoter - Google Patents
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Description
本発明はTIPARP産生促進剤を提供する。 The present invention provides a TIPARP production promoter.
TIPARPは、免疫、幹細胞の多能性、転写因子の調節等における重要な調節因子である。また、TIPARPは、薬物や環境中の危険因子による毒性作用等を媒介する転写因子であるアリール炭化水素受容体（AhR）等の転写抑制因子である。TIPARPの産生が促進できれば、TIPARP減少又はAhRの活性化に起因する各種疾患や状態を予防・改善することが期待されている。 TIPARP is an important regulator of immunity, stem cell pluripotency, and transcription factor regulation. TIPARP is also a transcription repressor of the aryl hydrocarbon receptor (AhR), a transcription factor that mediates the toxic effects of drugs and environmental hazards. It is hoped that promoting the production of TIPARP will help prevent and improve various diseases and conditions caused by a decrease in TIPARP or activation of AhR.
本発明の課題は、TIPARP産生促進剤の提供にある。 The objective of the present invention is to provide a TIPARP production promoter.
本発明者らは、鋭意研究の結果、アルテロモナス発酵エキスおよび金銀花エキスに高いTIPARP産生促進効果があることを見出した。以上の発見により、以下の発明を完成するに至った：
（１）アルテロモナス発酵エキス及び／又は金銀花エキスを有効成分として含有するTIPARP産生促進剤。
（２）（１）に記載のTIPARP産生促進剤を含有する組成物。
As a result of intensive research, the present inventors have found that Alteromonas fermentation extract and Golden Flower extract have a high TIPARP production promoting effect. Based on the above findings, the present inventors have completed the following invention:
(1) A TIPARP production promoter containing Alteromonas fermentation extract and/or Gold and Silver Flower extract as active ingredients.
(2) A composition containing the TIPARP production promoter described in (1).
本発明によれば、TIPARP産生促進剤を含有する組成物や内服剤を提供することができる。 According to the present invention, it is possible to provide a composition or oral preparation containing a TIPARP production promoter.
本発明はアルテロモナス発酵エキス及び／又は金銀花エキスを有効成分として含有するTIPARP産生促進剤を提供する。TIPARP（TCDD(2,3,7,8-tetrachlorodibenzo-p-dioxin)-inducible poly-ADP-ribose polymerase）は、PARP7、ARTD14としても知られ、モノADPリボシルトランスフェラーゼ（mono-ADP-ribosyltransferases）およびアリール炭化水素受容体（AhR：Aryl hydrocarbon Receptor）の転写抑制因子である。TIPARPは、免疫、幹細胞の多能性、転写因子の調節等における重要な調節因子であり、TIPARPノックダウンにより肝臓脂肪が蓄積することや、乳癌においてTIPARPのメチル化が起こりその発現が減少することが知られている（非特許文献１、２）。本発明によりTIPARPの産生が促進できれば、免疫の改善、TIPARP減少に起因する各種疾患や状態を予防・改善することが期待される。 The present invention provides a TIPARP production promoter containing Alteromonas fermentation extract and/or kinginka extract as an active ingredient. TIPARP (TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin)-inducible poly-ADP-ribose polymerase), also known as PARP7 and ARTD14, is a transcriptional repressor of mono-ADP-ribosyltransferases and aryl hydrocarbon receptor (AhR). TIPARP is an important regulator in immunity, stem cell pluripotency, transcription factor regulation, etc., and it is known that knockdown of TIPARP causes liver fat accumulation, and that in breast cancer, TIPARP expression is reduced due to methylation (Non-Patent Documents 1 and 2). If the production of TIPARP can be promoted by the present invention, it is expected that immunity will be improved and various diseases and conditions caused by TIPARP reduction will be prevented and improved.
本発明のTIPARP産生促進剤の一態様では、TIPARP産生を促進することによりAhRの活性化を抑制する。AhRは、薬物や環境中の危険因子による毒性作用等を媒介する転写因子である。AhRは、芳香族炭化水素類やダイオキシン類と結合し異物代謝を誘導すること、成長因子、抗酸化因子、炎症物質などを誘導すること等が報告されている(非特許文献３)。皮膚では、AhRの活性化により、活性酸素の発生、くすみ、メラニン産生といった悪影響を及ぼすことが知られており、これらの悪影響は紫外線の蓄積により悪化する。本発明のようなTIPARP産生促進を利用することにより、AhRの活性化を抑制し、AhRの活性化に起因する上述のような各種疾患や状態を予防・改善することが期待される。 In one embodiment of the TIPARP production promoter of the present invention, the activation of AhR is suppressed by promoting TIPARP production. AhR is a transcription factor that mediates the toxic effects of drugs and environmental hazards. It has been reported that AhR binds to aromatic hydrocarbons and dioxins to induce xenobiotic metabolism, and induces growth factors, antioxidant factors, inflammatory substances, and the like (Non-Patent Document 3). It is known that activation of AhR in the skin causes adverse effects such as the generation of reactive oxygen species, dullness, and melanin production, and these adverse effects are exacerbated by the accumulation of ultraviolet rays. It is expected that the use of TIPARP production promoters as in the present invention will suppress AhR activation and prevent and improve the various diseases and conditions caused by AhR activation as described above.
TIPARPの産生促進とは、例えばTIPARP遺伝子の発現はTIPARPタンパク質量を増加させること、例えば何も付与していない状態（コントロール）に比べて、TIPARP産生促進剤を付与した場合にTIPARP遺伝子の発現又はTIPARPタンパク質量が増加していることを意味し得る。本発明におけるTIPARPの産生促進とは、例えば何も付与していない状態（コントロール）に比べて、TIPARP産生促進剤を付与した場合に、TIPARP遺伝子の発現又はTIPARPタンパク質量が、例えば5％以上、10％以上、20％以上、30％以上、40％以上、50％以上、60％以、70％以上、80％以上、90％以上、100％以上、200％以上、300％以上、400％以上、又は500％以上亢進していることを意味し得る。TIPARP遺伝子の発現量は、任意の公知技術により求めることができる。 Promoting the production of TIPARP means, for example, that the expression of the TIPARP gene increases the amount of TIPARP protein, for example, that the expression of the TIPARP gene or the amount of TIPARP protein increases when a TIPARP production promoter is administered compared to a state in which nothing is administered (control). Promoting the production of TIPARP in the present invention means, for example, that the expression of the TIPARP gene or the amount of TIPARP protein increases by 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 when a TIPARP production promoter is administered compared to a state in which nothing is administered (control). The expression amount of the TIPARP gene can be determined by any known technique.
このようなTIPARP産生促進作用は、in vivo、in vitro、ex vivo等を含む各種方法で測定できる。例えば、被験物質を肝細胞、乳腺細胞、脂肪細胞、皮膚細胞、といった細胞に投与し、その細胞におけるTIPARPのmRNA発現量をqPCRなどの方法により求める、あるいはタンパク量をウエスタンブロッティング等の方法により求める等により決定できる。しかしながら測定方法は上記方法に限定されず、他の任意の方法を採用してもよい。例えば、ヒト等の動物に投与した後に肝臓、乳房、脂肪、皮膚組織／モデルなどの試料におけるTIPARPのmRNA発現量又はTIPARPタンパク質量を測定するといったin vivoやex vivoの方法を採用してもよい。 Such TIPARP production promoting activity can be measured by various methods including in vivo, in vitro, ex vivo, etc. For example, the test substance can be administered to cells such as liver cells, breast cells, fat cells, skin cells, etc., and the amount of TIPARP mRNA expression in the cells can be determined by a method such as qPCR, or the amount of protein can be determined by a method such as Western blotting. However, the measurement method is not limited to the above-mentioned method, and any other method may be adopted. For example, an in vivo or ex vivo method may be adopted in which the amount of TIPARP mRNA expression or the amount of TIPARP protein is measured in samples such as liver, breast, fat, and skin tissue/models after administration to an animal such as a human.
本発明は、アルテロモナス発酵エキス及び／又は金銀花エキスを有効成分として含有するTIPARP産生促進剤を提供する。本発明のTIPARP産生促進剤は、経口剤、あるいは経皮剤、注射剤と言った非経口剤等、任意の形態であってもよい。 The present invention provides a TIPARP production promoter containing Alteromonas fermentation extract and/or gypsophila extract as active ingredients. The TIPARP production promoter of the present invention may be in any form, such as an oral agent, or a parenteral agent such as a transdermal agent or an injection.
本発明で用いられるアルテロモナス発酵エキス（CAS番号:267233-41-0）は、深海に生息する微生物であるアルテロモナス（Alteromonas macleodii）から得られた発酵物のエキスであり、多糖類グルコシドの高分子である。抗炎症、皮膚刺激、敏感肌の症状を軽減させる作用などが知られている（特許文献１，２）。公知の方法により容易に発酵、精製、抽出等ができ、またUNIPEXIN NOVATIONS社のAbyssine PFといった市販品を容易に入手可能である。 The Alteromonas fermented extract (CAS number: 267233-41-0) used in the present invention is an extract of a fermented product obtained from Alteromonas macleodii, a microorganism that lives in the deep sea, and is a polymer of polysaccharide glucoside. It is known to have anti-inflammatory properties, skin irritation properties, and effects of reducing symptoms of sensitive skin (Patent Documents 1 and 2). It can be easily fermented, purified, extracted, etc. by known methods, and is also readily available as a commercial product such as Abyssine PF from UNIPEXIN NOVATIONS.
本発明で用いられる金銀花エキスは、スイカズラ（Lonicera japonica）の花蕾の抽出物である。抗炎症、抗菌、鎮痙作用などが知られている（特許文献３，４）。公知の方法により容易に乾燥、精製、抽出等ができ、また市販品を容易に入手可能である。生のままでも乾燥したものでも使用することができるが、抽出物、乾燥物、乾燥粉末、原料の粉末物、搾汁液等として用いることもでき、いずれの形態を用いるかは適宜選択することができる。 The honeysuckle extract used in the present invention is an extract of the flower buds of honeysuckle (Lonicera japonica). It is known to have anti-inflammatory, antibacterial and antispasmodic effects (Patent Documents 3 and 4). It can be easily dried, purified, extracted, etc. by known methods, and is also readily available as a commercially available product. It can be used either raw or dried, but it can also be used as an extract, dried product, dried powder, raw material powder, squeezed juice, etc., and the form to be used can be selected appropriately.
上記エキスの抽出は例えば溶媒抽出により行うことができる。溶媒抽出の場合には、発酵物又は植物の全体又は部分を必要に応じて乾燥させ、更に必要に応じて細断又は粉砕した後、水性抽出剤、水、例えば冷水、温水、又は沸点若しくはそれより低温の熱水、あるいは含水有機溶媒、有機溶媒、例えばエタノール、メタノール、エーテル、１，３－ブチレングリコール等を原料の性質や組成物の用途等により好ましい溶媒を適宜選択して常温で又は加熱して用いることにより抽出される。しかしながら、抽出方法は溶媒抽出に限定されず、当業界で知られている常用の手法によってもよく、本発明で用いる抽出物の抽出方法や抽出物の形態は、本発明の効果を損なわない限り任意である。上記抽出物の形態は、抽出液自体だけでなく、常用の手法により適宜希釈又は濃縮したものであってもよく、更に、抽出液を乾燥することによって得られる粉状あるいは塊状の固体であってもよい。 The above extract can be extracted by, for example, solvent extraction. In the case of solvent extraction, the fermented product or plant is dried in whole or in part as necessary, and further chopped or crushed as necessary, and then extracted by using an aqueous extractant, water, for example, cold water, warm water, or hot water at or below the boiling point, or a water-containing organic solvent, an organic solvent, for example, ethanol, methanol, ether, 1,3-butylene glycol, etc., at room temperature or with heating, with a suitable solvent selected according to the properties of the raw material and the use of the composition. 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 form of the above extract may be not only the extract itself, but also one that has been appropriately diluted or concentrated by conventional methods, and may further be a powdered or lumpy solid obtained by drying the extract.
含水有機溶媒の例として、含水エタノール等の含水低級アルコールを用いてもよく、その場合の含水率は、例えば0～10v/v％、10～40v/v％、20～30v/v％、30～50v/v％、50～80v/v％、80～99.5v/v％等であってもよい。 As an example of the water-containing organic solvent, a water-containing lower alcohol such as water-containing ethanol may be used, in which case the water content may be, for example, 0-10 v/v%, 10-40 v/v%, 20-30 v/v%, 30-50 v/v%, 50-80 v/v%, 80-99.5 v/v%, etc.
本発明のTIPARP産生促進剤は、経皮、経口、局所注入等各種投与経路で投与でき、本発明の有効成分をTIPARP産生促進の効果が十分発揮されるような量で適用することが好ましい。アルテロモナス発酵エキス及び／又は金銀花エキスの配合量は、それらの種類、目的、形態、利用方法などに応じて、適宜決めることができる。 The TIPARP production promoter of the present invention can be administered via various routes, such as transdermal, oral, and local injection, and it is preferable to apply the active ingredient of the present invention in an amount that sufficiently exerts the effect of promoting TIPARP production. The amount of Alteromonas fermentation extract and/or cypress extract to be blended can be appropriately determined depending on their type, purpose, form, and method of use.
本発明のTIPARP産生促進剤を経皮剤又は経口剤として利用する場合には、経皮剤又は経口剤全量に対して、アルテロモナス発酵エキス及び／又は金銀花エキスの乾燥重量が、0.00001～1.0重量%程度になるように調製することが好ましく、0.001～0.8重量%程程度、0.10～0.5重量%程度がより好ましい。 When the TIPARP production promoter of the present invention is used as a transdermal or oral preparation, it is preferable to prepare the transdermal or oral preparation so that the dry weight of the Alteromonas fermentation extract and/or the rhododendron extract is about 0.00001 to 1.0% by weight, more preferably about 0.001 to 0.8% by weight, and more preferably about 0.10 to 0.5% by weight.
また、投与頻度は、限定されないものの、一例によれば、例えば２週間に１回、１週間に１回、３日に１回、２日に１回、１日１回、１日２回、１日３回、１日４回等の頻度で投与することができる。また、都度摂取するものであっても、継続的に摂取するものであっても、例えば数か月の間隔を空け断続的に投与するものであってもよい。 The frequency of administration is not limited, but according to one example, it can be administered once every two weeks, once a week, once every three days, once every two days, once a day, twice a day, three times a day, four times a day, etc. It can also be administered on an as-needed basis, continuously, or intermittently, for example, at intervals of several months.
また、本発明のTIPARP産生促進剤は、経皮又は経口摂取用の組成物、例えば、医薬、化粧品、食品組成物に含有できる。本発明のTIPARP産生促進剤及び組成物の形態としては、例えば、粉末状、液体状、錠剤等の固形、顆粒、粒状、ペースト状、ゲル状、クリーム状など任意に選択することができる。 The TIPARP production promoter of the present invention can be contained in a composition for transdermal or oral ingestion, such as a pharmaceutical, cosmetic, or food composition. The form of the TIPARP production promoter and composition of the present invention can be selected from any of a variety of forms, such as powder, liquid, solid such as a tablet, granules, particles, paste, gel, cream, and the like.
本発明のTIPARP産生促進剤及び組成物には、必要に応じて添加剤を任意に選択し併用することができる。添加剤としては賦形剤、着色剤、保存剤、増粘剤、結合剤、崩壊剤、分散剤、安定化剤、ゲル化剤、酸化防止剤、界面活性剤、保存剤、ｐＨ調整剤等については、公知のものを適宜選択して使用できる。 Additives can be selected arbitrarily and used in combination with the TIPARP production promoter and composition of the present invention as necessary. Additives such as excipients, colorants, preservatives, thickeners, binders, disintegrants, dispersants, stabilizers, gelling agents, antioxidants, surfactants, preservatives, and pH adjusters can be appropriately selected from known additives and used.
本発明は、アルテロモナス発酵エキス及び／又は金銀花エキスを例えば経皮、経口、局所注入経路で投与することにより肝細胞を含む肝臓組織、乳腺を含む乳房の組織、表皮又は真皮細胞を含む皮膚組織のTIPARP産生促進、TIPARP産生促進によるAhRの活性化を抑制を介する脂肪肝、乳癌、皮膚のくすみやメラニン生成といったTIPARPの抑制あるいはAhRの活性化により発症する疾患・状態を予防又は改善するための方法も提供する。本発明の方法は、美容を目的とする方法であり、医師や医療従事者による治療ではないことがある。 The present invention also provides a method for preventing or improving diseases or conditions caused by inhibition of TIPARP or activation of AhR, such as fatty liver, breast cancer, skin dullness, and melanin production, through the promotion of TIPARP production in liver tissue including hepatocytes, breast tissue including mammary glands, and skin tissue including epidermal or dermal cells, and the inhibition of AhR activation due to the promotion of TIPARP production, by administering Alteromonas fermentation extract and/or Golden Ginseng extract, for example, via a transdermal, oral, or local injection route. The method of the present invention is a method for cosmetic purposes and may not be a treatment performed by a doctor or medical professional.
さらに、本発明は、脂肪肝、乳癌、皮膚老化といったTIPARPの抑制あるいはAhRの活性化により発症する疾患・状態を予防又は改善するための経口剤又は経腸剤といった医薬の製造におけるアルテロモナス発酵エキス及び／又は金銀花エキスの使用も提供する。本発明は、例えば経口又は経皮投与により、肝臓、脂肪、皮膚組織のTIPARP産生促進することによる、脂肪肝、乳癌、皮膚のくすみやメラニン生成といったTIPARPが抑制されることにより発症する疾患・状態を予防又は改善するための方法に使用するためのアルテロモナス発酵エキス及び／又は金銀花エキスも提供する。 The present invention further provides the use of Alteromonas fermentation extract and/or Golden Flower extract in the manufacture of a pharmaceutical, such as an oral or enteral preparation, for preventing or ameliorating diseases and conditions caused by inhibition of TIPARP or activation of AhR, such as fatty liver, breast cancer, and skin aging. The present invention also provides Alteromonas fermentation extract and/or Golden Flower extract for use in a method for preventing or ameliorating diseases and conditions caused by inhibition of TIPARP, such as fatty liver, breast cancer, skin dullness, and melanin production, by promoting TIPARP production in liver, fat, and skin tissue, for example, by oral or transdermal administration.
次に実施例によって本発明をさらに詳細に説明する。なお、本発明はこれにより限定されるものではない。 Next, the present invention will be described in more detail with reference to examples. Note that the present invention is not limited to these examples.
実験１：試料の調製
TIPARP産生促進剤の候補試料として、アルテロモナス発酵エキス及び／又は金銀花エキスを以下の表のように調製した。
Experiment 1: Sample preparation
As candidate samples for TIPARP production promoters, Alteromonas fermentation extract and/or Golden Flower extract were prepared as shown in the table below.
その他動植物の抽出物といった天然由来成分や合成成分を含め、合計29種類の候補試料を調製した。試料は各エキスの溶媒(エタノール又はは1,3-BG(1,3-ブチレングリコール))を用いて培地中の終濃度が0.10重量%～0.5重量%となるように調整した。対照として何も含まない各エキスの溶媒を用いた。 A total of 29 types of candidate samples were prepared, including natural ingredients such as animal and plant extracts, as well as synthetic ingredients. The samples were adjusted using the solvent for each extract (ethanol or 1,3-BG (1,3-butylene glycol)) so that the final concentration in the medium was 0.10% to 0.5% by weight. As a control, the solvent for each extract that did not contain anything was used.
実験２：qPCRによるTIPARP発現解析
本研究では、正常ヒト表皮角化細胞（クラボウ:KK-4009）を用いた。表皮細胞は、EpiLife Medium M-EPI-500-CAにHuman Keratinocyte Growth Supplement(HKGS)を添加した培地(Thermo Fisher Scientific)を用いて、細胞培養ディッシュにて、37℃、5% CO2条件下で培養した。3.12x105 cells/cm2の密度で細胞を播種し細胞が定着したのち、過酸化水素処理を行い老化誘導した。老化状態はSA-β-gal(Dojindo:SG03 Cellular Senescence Detection Kit - SPiDER-βGal)の検出で確認した。さらに紫外線(UVB 400mJ/cm2,UVA 5J/cm2)を照射し6h培養した。各エキスおよび溶媒を添加し一晩培養後10nM FICZ(Aryl Hydrocarbon Receptor Agonist)を添加しAhRを活性化させた。24h培養後のtotal RNAを回収した。
Experiment 2: TIPARP expression analysis by qPCR Normal human epidermal keratinocytes (Kurabo: KK-4009) were used in this study. Epidermal cells were cultured in a cell culture dish at 37℃ under 5% CO2 conditions using EpiLife Medium M-EPI-500-CA supplemented with Human Keratinocyte Growth Supplement (HKGS) (Thermo Fisher Scientific). Cells were seeded at a density of 3.12x105 cells/ cm2 , and after the cells had settled, they were treated with hydrogen peroxide to induce senescence. The senescence state was confirmed by detection with SA-β-gal (Dojindo: SG03 Cellular Senescence Detection Kit - SPiDER-βGal). The cells were then exposed to ultraviolet light (UVB 400mJ/ cm2 , UVA 5J/ cm2 ) and cultured for 6 hours. After incubation overnight with each extract or solvent, 10 nM FICZ (Aryl Hydrocarbon Receptor Agonist) was added to activate AhR. Total RNA was collected after 24 hours of incubation.
上記方法で培養した正常ヒト表皮角化細胞におけるtotal RNAよりcDNAを合成した(Takara: PrimeScript RT reagent kit)。本cDNAを用いSYBR Green(KAPA SYBR Fast qPCRキット（UniversalqPCRキット）)によりqPCRを行った。Internal controlはβ-actinとしTIPARPのmRNA産生増加能を評価した。また同時にAhRの下流にある活性酸素産生遺伝子であるCYPA1AおよびCYP1B1の遺伝子発現も評価し、これらについては産生増加させないことを確認した。各遺伝子に対応するプライマの具体的な配列を下記表２に示す。
結果を図２に示す。図２に示すように、アルテロモナス発酵エキス及び金銀花エキスを投与すると、対照と比べて濃度依存的にTIPARPの発現量が有意に増加した。したがって、本発明のエキスは、優れたTIPARP産生促進効果があることがわかる。 The results are shown in Figure 2. As shown in Figure 2, when Alteromonas fermentation extract and Golden Flower extract were administered, the expression level of TIPARP increased significantly in a concentration-dependent manner compared to the control. Therefore, it can be seen that the extract of the present invention has an excellent effect of promoting TIPARP production.
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| JP2004083476A (en) | 2002-08-27 | 2004-03-18 | Horin:Kk | Tyrosinase activity inhibitor, melanin production inhibitor, fibroblast proliferation agent and estrogen-like agent, and skin cosmetic |
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