JP2017085923A - Agent for maintaining undifferentiated state of stem cell with alaria praelonga kjellman and agent for promoting proliferation thereof - Google Patents
Agent for maintaining undifferentiated state of stem cell with alaria praelonga kjellman and agent for promoting proliferation thereof Download PDFInfo
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- JP2017085923A JP2017085923A JP2015217429A JP2015217429A JP2017085923A JP 2017085923 A JP2017085923 A JP 2017085923A JP 2015217429 A JP2015217429 A JP 2015217429A JP 2015217429 A JP2015217429 A JP 2015217429A JP 2017085923 A JP2017085923 A JP 2017085923A
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- stem cells
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Abstract
Description
本発明は、幹細胞の未分化状態維持剤及び増殖促進剤並びに幹細胞の未分化状態維持方法及び増殖促進方法に関する。 The present invention relates to an agent for maintaining an undifferentiated state of a stem cell and an agent for promoting proliferation, a method for maintaining an undifferentiated state of a stem cell, and a method for promoting proliferation.
脊椎動物(特に哺乳動物)の組織は、傷害若しくは疾患、又は加齢等に伴い細胞・臓器の損傷が起こった場合、再生系が働き、細胞・臓器の損傷を回復しようとする。この作用に、当該組織に備わる幹細胞(組織幹細胞、体性幹細胞)が大きな役割を果たしている。幹細胞は、あらゆる細胞・臓器に分化する多能性を有しており、この性質により細胞・臓器の損傷部を補うことで回復に導くと考えられている。このような幹細胞を応用した、次世代の医療である再生医療に期待が集まっている。 When tissue of vertebrates (especially mammals) is damaged due to injury or disease, or with aging, etc., the regeneration system works and tries to recover the damage of cells / organs. Stem cells (tissue stem cells, somatic stem cells) provided in the tissue play a major role in this action. Stem cells have the pluripotency to differentiate into all cells and organs, and this property is thought to lead to recovery by compensating for damaged parts of cells and organs. Expectations are gathered for regenerative medicine, which is the next generation of medicine using such stem cells.
哺乳動物における幹細胞研究で最も進んでいる組織は骨髄である。骨髄には生体の造血幹細胞が存在しており、全ての血液細胞再生の源であることが明らかにされた。さらに骨髄には、造血幹細胞とは別に、その他の臓器・組織(例えば、骨、軟骨、筋肉、脂肪等)へ分化可能な幹細胞が包含されていることが報告されている(非特許文献1参照)。 The most advanced tissue in stem cell research in mammals is the bone marrow. The bone marrow contains living hematopoietic stem cells, which have been shown to be the source of all blood cell regeneration. Furthermore, it has been reported that the bone marrow contains stem cells that can be differentiated into other organs / tissues (eg, bone, cartilage, muscle, fat, etc.) in addition to hematopoietic stem cells (see Non-Patent Document 1). ).
さらに、近年、骨髄以外にも、皮膚、肝臓、膵臓、脂肪等、あらゆる臓器・組織に幹細胞が存在することが明らかにされ、各臓器・組織の再生及び恒常性維持を司っていることがわかってきた(非特許文献2〜5参照)。また、各組織に存在する幹細胞は可塑性に優れており、今まで自己複製が不可能であった臓器や組織の再生にも利用できる可能性がある。 Furthermore, in recent years, it has been clarified that stem cells exist in all organs / tissues other than bone marrow, such as skin, liver, pancreas, fat, etc., and is responsible for regeneration and homeostasis of each organ / tissue. It has been understood (see Non-Patent Documents 2 to 5). In addition, stem cells present in each tissue are excellent in plasticity and may be used for regeneration of organs and tissues that have been impossible to self-replicate until now.
一方で、これらの幹細胞のうちのいくつかは、加齢とともに減少することが知られており、各組織の恒常性維持のために幹細胞の減少を防ぐ技術の研究が積極的になされている(非特許文献6)。また、近年、幹細胞の能力(多能性)を、臓器や組織の再生へ応用するため、細胞移植治療や組織工学(再生医療や再生美容)の分野において幹細胞を生体組織から分離した後に培養し増殖させる技術の開発が進められている(非特許文献7、8)。 On the other hand, some of these stem cells are known to decrease with aging, and research on techniques to prevent the decrease of stem cells is actively conducted to maintain the homeostasis of each tissue ( Non-patent document 6). In recent years, stem cells have been isolated from living tissues in the field of cell transplantation and tissue engineering (regenerative medicine and regenerative beauty) in order to apply the ability (pluripotency) of stem cells to regeneration of organs and tissues. Development of techniques for propagation is in progress (Non-Patent Documents 7 and 8).
特に、幹細胞を生体外で培養する場合、幹細胞の能力である多能性を維持した状態、すなわち、未分化な状態を維持させたまま増殖させることが極めて重要である。もし、この培養時に幹細胞の未分化状態が維持できず分化誘導が進んでしまった場合、最終的に調製された幹細胞の能力(多能性)は失われていることになり、目的の効果(臓器や組織の再生等)を発揮できない。 In particular, when culturing stem cells in vitro, it is extremely important to proliferate while maintaining the pluripotency that is the ability of stem cells, that is, maintaining an undifferentiated state. If the undifferentiated state of the stem cells cannot be maintained during this culture and differentiation induction has progressed, the ability (pluripotency) of the finally prepared stem cells has been lost, and the desired effect ( Can not reproduce organs and tissues.
以上より、幹細胞を細胞移植治療や組織工学(再生医療や再生美容)に利用し、臓器や組織の再生を望む場合、幹細胞を、未分化状態を維持させたまま培養できなければならない。 As described above, when stem cells are used for cell transplantation treatment or tissue engineering (regenerative medicine or regenerative beauty) and regeneration of organs or tissues is desired, the stem cells must be able to be cultured while maintaining an undifferentiated state.
現在までに、幹細胞を、未分化状態を維持させたまま増殖させる技術について、幾つか報告があるが、未だ発展途上である。例えば、胚性幹細胞(ES細胞)や造血幹細胞は、支持細胞(ストローマ細胞、又はフィーダー細胞)と共培養することで未分化を維持することができる(特許文献1及び非特許文献9〜11参照)。しかしながら、最近になってフィーダー細胞由来の内在性ウイルスによる異種動物間の感染例が報告されており(非特許文献12参照)、支持細胞を使用した幹細胞の培養は、医療用途を目的とした幹細胞の培養には適していない。 To date, there have been several reports on techniques for proliferating stem cells while maintaining an undifferentiated state, but they are still under development. For example, embryonic stem cells (ES cells) and hematopoietic stem cells can be maintained undifferentiated by co-culture with supporting cells (stromal cells or feeder cells) (see Patent Literature 1 and Non-Patent Literatures 9 to 11). ). However, recently, there have been reports of cases of infection between different animals caused by feeder cells-derived endogenous viruses (see Non-Patent Document 12), and stem cell culture using feeder cells is a stem cell for medical purposes. It is not suitable for culturing.
その他の方法に、サイトカインを複雑に組合せることによって幹細胞の未分化状態を維持させる方法がある。例えば、マウスES細胞は、LIF(白血病抑制因子(Leukemia Inhibitory Factor))を培地に添加することによって、未分化性が維持される(特許文献2及び非特許文献13参照)。その他、初期作用性サイトカイントロンボポイエチン(TPO)、インターロイキン6(IL−6)、FLT−3リガンド、及び幹細胞因子(SCF)の存在下で、未分化性を維持させることが胚性幹細胞、体性幹細胞等で報告されている(特許文献3及び非特許文献14参照)。 As another method, there is a method of maintaining an undifferentiated state of a stem cell by complexly combining cytokines. For example, mouse ES cells are maintained undifferentiated by adding LIF (Leukemia Inhibitory Factor) to the medium (see Patent Document 2 and Non-Patent Document 13). In addition, maintaining embryonic stem cells in the presence of early-acting cytokines thrombopoietin (TPO), interleukin 6 (IL-6), FLT-3 ligand, and stem cell factor (SCF) It has been reported for somatic stem cells (see Patent Literature 3 and Non-Patent Literature 14).
しかしながら、サイトカインは、高価であり、採取原料や保存性等の問題があり、容易な使用は難しい。加えて、LIFの効果は極めて特定の細胞系統に限定的であり、特に霊長類のES細胞や体性幹細胞においては、LIFの添加のみでは未分化状態を維持することができないことが明らかにされている(非特許文献10参照)。 However, cytokines are expensive and have problems such as collection raw materials and storage stability, and easy use is difficult. In addition, the effect of LIF is limited to a very specific cell lineage. In particular, in primate ES cells and somatic stem cells, it has been clarified that the addition of LIF alone cannot maintain an undifferentiated state. (See Non-Patent Document 10).
現在報告されている幹細胞の未分化状態の維持方法はいずれも、煩雑な操作を必要とし、また未分化状態の維持効果が低い。従って、幹細胞を再生医療に利用するために、幹細胞を、未分化状態を維持したまま増殖させる技術が求められていた。つまり、安全且つ簡便で効率的に、幹細胞を、未分化状態を維持させたまま増殖させることができる技術が求められていた。 Any of the currently reported methods for maintaining the undifferentiated state of stem cells requires complicated operations and has a low effect of maintaining the undifferentiated state. Therefore, in order to use stem cells for regenerative medicine, a technique for proliferating stem cells while maintaining an undifferentiated state has been demanded. That is, there has been a demand for a technique that can proliferate stem cells while maintaining an undifferentiated state in a safe, simple and efficient manner.
本発明は、上述した実情に鑑み、幹細胞を、未分化状態を維持させたまま、効率良く増殖させる方法を提供することを目的とする。 In view of the above-described circumstances, an object of the present invention is to provide a method for efficiently proliferating stem cells while maintaining an undifferentiated state.
本発明者らは、上記課題を解決するため鋭意研究を行った結果、アイヌワカメの亜臨界水処理物又はその抽出物が、幹細胞に対する優れた未分化状態維持効果と増殖促進効果を有することを見出し、本発明を完成するに至った。 As a result of intensive studies to solve the above problems, the present inventors have found that a subcritical water treated product of Ainu sea turtle or an extract thereof has an excellent undifferentiated state maintenance effect and proliferation promoting effect on stem cells. The headline and the present invention were completed.
すなわち、本発明は、以下を包含する。
(1)温度が100〜374℃、且つ圧力が飽和蒸気圧以上の亜臨界状態にある水で処理したアイヌワカメの亜臨界水処理物又はその抽出物を有効成分として含有する幹細胞の未分化状態維持剤。
(2)100〜374℃、且つ圧力が飽和蒸気圧以上の亜臨界状態にある水で処理したアイヌワカメの亜臨界水処理物又はその抽出物を有効成分として含有する幹細胞の増殖促進剤。
(3)幹細胞を、100〜374℃、且つ圧力が飽和蒸気圧以上の亜臨界状態にある水で処理したアイヌワカメの亜臨界水処理物又はその抽出物を含有する培地で培養する工程を含む、幹細胞の製造方法。
(4)幹細胞を、100〜374℃、且つ圧力が飽和蒸気圧以上の亜臨界状態にある水で処理したアイヌワカメの亜臨界水処理物又はその抽出物を含有する培地で培養する工程を含む、幹細胞の未分化状態維持方法。
(5)幹細胞を、100〜374℃、且つ圧力が飽和蒸気圧以上の亜臨界状態にある水で処理したアイヌワカメの亜臨界水処理物又はその抽出物を含有する培地で培養する工程を含む、幹細胞の増殖促進方法。
(6)請求項1又は2に記載の剤を含む、化粧品、医薬品、又は医薬部外品。
(7)請求項1又は2に記載の剤を含む、飲食品。
That is, the present invention includes the following.
(1) An undifferentiated state of a stem cell containing, as an active ingredient, a subcritical water treated product of Ainu seaweed treated with water in a subcritical state at a temperature of 100 to 374 ° C. and a pressure equal to or higher than a saturated vapor pressure or an extract thereof Maintenance agent.
(2) A stem cell growth promoter containing, as an active ingredient, a subcritical water treated product of Ainu sea turtle treated with water in a subcritical state at a pressure of 100 to 374 ° C. or higher and a saturated vapor pressure or an extract thereof.
(3) including a step of culturing stem cells in a medium containing a subcritical water treated product of Ainu seaweed or an extract thereof treated with water in a subcritical state at 100 to 374 ° C. and a pressure equal to or higher than a saturated vapor pressure A method for producing stem cells.
(4) including a step of culturing a stem cell in a medium containing a subcritical water treated product of Ainu seaweed or an extract thereof treated with water in a subcritical state having a pressure equal to or higher than a saturated vapor pressure at 100 to 374 ° C. A method for maintaining the undifferentiated state of stem cells.
(5) including a step of culturing a stem cell in a medium containing a subcritical water treated product of Ainu seaweed or an extract thereof treated with water in a subcritical state having a pressure equal to or higher than a saturated vapor pressure at 100 to 374 ° C. A method for promoting the proliferation of stem cells.
(6) Cosmetics, pharmaceuticals, or quasi drugs containing the agent according to claim 1 or 2.
(7) Food / beverage products containing the agent of Claim 1 or 2.
本発明によれば、幹細胞を、未分化状態を維持したまま、効率的に増殖させることができる。従って、本発明は、再生医療や再生美容の分野において大きく貢献できるものである。 According to the present invention, stem cells can be efficiently proliferated while maintaining an undifferentiated state. Therefore, the present invention can greatly contribute to the fields of regenerative medicine and regenerative beauty.
以下、本発明を詳細に説明する。
本発明に係る幹細胞の未分化状態維持剤又は増殖促進剤は、アイヌワカメの亜臨界水処理物又はその抽出物を有効成分として含有する。
Hereinafter, the present invention will be described in detail.
The stem cell undifferentiated state maintenance agent or growth promoter according to the present invention contains a subcritical water treated product of Ainu seaweed or an extract thereof as an active ingredient.
本発明に用いるアイヌワカメとは、北海道東部等で生育するアイヌワカメ(Alaria praelonga Kjellman)のことであり、アイヌワカメ本体をそのまま使用してもよく、あるいは、乾燥、粉砕、細切等の処理を行ったものを使用することもできる。 The Ainu wakame used in the present invention is an Ainu wakame (Alaria prellalonga Kjellman) that grows in eastern Hokkaido, etc., and the Ainu wakame main body may be used as it is, or treatment such as drying, crushing, shredding, etc. You can also use what you did.
本発明に用いられる亜臨界水処理とは、所定温度及び圧力の条件下で亜臨界状態にした水と処理対象(本発明ではアイヌワカメ)とを接触させることである。例えば、水は、圧力22.12MPa、温度374.15℃まで上げると液体でも気体でもない状態を示す。この点を水の臨界点といい、臨界点より低い温度及び圧力の熱水を亜臨界水という。この亜臨界水は、誘電率低下とイオン積の向上により、優れた成分抽出作用と加水分解作用を有する。なお、亜臨界水処理に供給する水は、液体状態でも気体状態でも利用することができる。即ち、亜臨界水処理の処理槽へは、水蒸気を供給してもよく、水を供給してもよく、あるいはその両者を供給してもよい。亜臨界水処理時において、望まれる反応場としては気体よりも液体状態の方が反応は進みやすいので、密閉容器で強制的に液体の状態にした状態の水の使用が好ましい。より具体的には、金属やセラミックス等の耐圧容器にアイヌワカメと処理剤である水を入れて、密閉状態にし、水の亜臨界状態(温度:100℃以上、圧力:飽和蒸気圧以上)で、両者の接触を一定時間以上行うことで得られる処理物を亜臨界水処理物とすることができる。 The subcritical water treatment used in the present invention is to bring water in a subcritical state under the conditions of a predetermined temperature and pressure and the object to be treated (in the present invention, Ainu Wakame). For example, when water is raised to a pressure of 22.12 MPa and a temperature of 374.15 ° C., it shows a state where it is neither liquid nor gas. This point is called the critical point of water, and hot water at a temperature and pressure lower than the critical point is called subcritical water. This subcritical water has an excellent component extraction action and hydrolysis action due to a decrease in dielectric constant and an improvement in ionic product. The water supplied to the subcritical water treatment can be used in a liquid state or a gas state. That is, water vapor may be supplied to the treatment tank for subcritical water treatment, water may be supplied, or both of them may be supplied. At the time of subcritical water treatment, as a desired reaction field, the reaction is more likely to proceed in the liquid state than in the gas. Therefore, it is preferable to use water in a liquid state forcibly in a sealed container. More specifically, Ainu sea turtle and water as a treatment agent are put in a pressure vessel such as metal or ceramics, sealed, and in a subcritical state of water (temperature: 100 ° C. or higher, pressure: saturated vapor pressure or higher). The treated product obtained by performing the contact between them for a certain time or longer can be a subcritical water treated product.
本発明に用いられるアイヌワカメ亜臨界水処理物とは、アイヌワカメを亜臨界状態にある水によって処理したものであり、アイヌワカメと水溶物(水溶液等)との混合物又はその乾燥物を含む。また、前記の混合物をろ過したろ液、又はその乾燥物を含む。 The Ainu seaweed subcritical water treated product used in the present invention is a product of Ainu seaweed treated with water in a subcritical state, and includes a mixture of Ainu seaweed and an aqueous solution (such as an aqueous solution) or a dried product thereof. Moreover, the filtrate which filtered the said mixture, or its dried material is included.
アイヌワカメの亜臨界水処理温度は、アイヌワカメの亜臨界水処理物又はその抽出物中に含まれる有効成分が効率的に得られやすくなるという理由から100〜300℃の間が好ましく、120〜200℃の間がより好ましく、130〜150℃の間がさらに好ましい。100℃未満の場合は、有効成分の量が少なくなる傾向にある。また、亜臨界水処理の温度が300℃を超える場合は、有効成分の過分解を引き起こしやすくなる。 The subcritical water treatment temperature for Ainu sea turtle is preferably between 100 and 300 ° C., because the active ingredient contained in the subcritical water treated product of Ainu sea turtle or its extract is easily obtained, preferably 120 to More preferably, the temperature is between 200 ° C and more preferably between 130 and 150 ° C. When the temperature is less than 100 ° C., the amount of the active ingredient tends to decrease. Moreover, when the temperature of subcritical water treatment exceeds 300 degreeC, it will become easy to cause the excessive decomposition | disassembly of an active ingredient.
アイヌワカメの亜臨界水処理圧力は、各温度での飽和蒸気圧以上であれば良く、圧力0.1〜3.0MPaの間がより好ましく、0.26〜1.56MPaの間がさらに好ましく、0.28〜0.48MPaの間が最も好ましい。圧力0.1MPa未満の場合は、有効成分の量が少なくなる傾向にある。また、亜臨界水処理の圧力が、飽和蒸気圧を大きく上回るような過度の加圧には、別途加圧装置の追加及び設備の耐圧向上が必要となり、抽出の経済性が悪化する。 The subcritical water treatment pressure of Ainu Wakame may be equal to or higher than the saturated vapor pressure at each temperature, more preferably between 0.1 and 3.0 MPa, more preferably between 0.26 and 1.56 MPa, Most preferably between 0.28 and 0.48 MPa. When the pressure is less than 0.1 MPa, the amount of the active ingredient tends to decrease. In addition, excessive pressurization in which the pressure of the subcritical water treatment greatly exceeds the saturated vapor pressure requires the addition of a separate pressurization device and improvement of the pressure resistance of the equipment, which deteriorates the economics of extraction.
アイヌワカメの亜臨界水処理時間は、5〜90分の間で行うことが好ましく、10〜30分の間で行うことがより好ましい。5分未満の場合は、有効成分の量が少なくなる傾向にある。また、亜臨界水処理の時間が90分を超える場合は、有効成分の過分解を引き起こしやすくなる。 The subcritical water treatment time for Ainu Wakame is preferably 5 to 90 minutes, more preferably 10 to 30 minutes. If it is less than 5 minutes, the amount of the active ingredient tends to decrease. Moreover, when the time of subcritical water treatment exceeds 90 minutes, it becomes easy to cause the excessive decomposition | disassembly of an active ingredient.
すなわち、アイヌワカメの亜臨界水処理条件としては、温度は100〜300℃、圧力は0.1〜3.0MPa、時間は5〜90分で行うことが好ましい。この条件で行うことにより、アイヌワカメの亜臨界水処理物の有効成分を向上させることができるのである。
さらに、アイヌワカメの亜臨界水処理条件としては、温度は130〜150℃、圧力は0.28〜0.48MPa、時間は10〜30分で行うことが最も好ましい。この条件で行うことにより、アイヌワカメの亜臨界水処理物の有効成分を容易に向上させることができるのである。
That is, as subcritical water treatment conditions for Ainu Wakame, the temperature is preferably 100 to 300 ° C., the pressure is 0.1 to 3.0 MPa, and the time is preferably 5 to 90 minutes. By carrying out under these conditions, the active ingredient of the subcritical water treated product of Ainu Wakame can be improved.
Furthermore, as subcritical water treatment conditions for Ainu Wakame, it is most preferable that the temperature is 130 to 150 ° C., the pressure is 0.28 to 0.48 MPa, and the time is 10 to 30 minutes. By carrying out under these conditions, the active ingredient of the subcritical water treated product of Ainu Wakame can be easily improved.
本発明に用いられるアイヌワカメ亜臨界水処理物の抽出物とは、前記の亜臨界水処理物に溶媒を加えて、抽出した抽出液又はその乾燥物を含む。 The extract of Ainu Wakame subcritical water treated product used in the present invention includes an extract extracted by adding a solvent to the subcritical water treated product or a dried product thereof.
抽出に使用する溶媒としては、例えば、水、低級アルコール類(メタノール、エタノール、1−プロパノール、2−プロパノール、1−ブタノール、2−ブタノール等)、液状多価アルコール(1,3−ブチレングリコール、プロピレングリコール、グリセリン等)、ケトン類(アセトン、メチルエチルケトン等)、アセトニトリル、エステル類(酢酸エチル、酢酸ブチル等)、炭化水素類(ヘキサン、ヘプタン、流動パラフィン等)、エーテル類(エチルエーテル、テトラヒドロフラン、プロピルエーテル等)等が挙げられる。好ましくは、水、低級アルコール及び液状多価アルコールが良く、特に好ましくは、水、エタノールが良い。これらの溶媒は1種でも2種以上を混合して用いても良く、例えば30〜70v/v%のエタノール水溶液を使用することもできる。また、上記抽出溶媒に酸やアルカリを添加して、pH調整した溶媒を使用することもできる。また、抽出方法としては、連続抽出、浸漬抽出、超臨界抽出、亜臨界水抽出等が挙げられる。亜臨界抽出条件としては、上記の亜臨界水処理と同一又は別条件にて更に亜臨界水抽出を行うことができる。 Examples of the solvent used for extraction include water, lower alcohols (methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, etc.), liquid polyhydric alcohols (1,3-butylene glycol, Propylene glycol, glycerin, etc.), ketones (acetone, methyl ethyl ketone, etc.), acetonitrile, esters (ethyl acetate, butyl acetate, etc.), hydrocarbons (hexane, heptane, liquid paraffin, etc.), ethers (ethyl ether, tetrahydrofuran, Propyl ether, etc.). Preferably, water, lower alcohol and liquid polyhydric alcohol are preferable, and water and ethanol are particularly preferable. These solvents may be used alone or in combination of two or more. For example, an ethanol aqueous solution of 30 to 70 v / v% can also be used. Moreover, the acid which adjusted the pH by adding an acid or an alkali to the said extraction solvent can also be used. Examples of the extraction method include continuous extraction, immersion extraction, supercritical extraction, and subcritical water extraction. As subcritical extraction conditions, subcritical water extraction can be further performed under the same or different conditions as the above subcritical water treatment.
上述の溶媒を用いて、アイヌワカメ亜臨界水処理物を溶媒抽出に供する。溶媒に対するアイヌワカメの亜臨界水処理物の割合は、固形分に換算して、例えば1〜50%(w/w)、好ましくは5〜25%(w/w)が挙げられる。例えば、アイヌワカメ亜臨界水処理物の乾燥物に水を加え、95〜100℃における熱水抽出を行うことで、アイヌワカメ亜臨界水処理物の抽出物を得ることができる。あるいは、アイヌワカメ亜臨界水処理物の乾燥物に低級アルコール(例えば、エタノール等)又は液状多価アルコール(例えば、プロピレングリコール、1,3−ブチレングリコール等)を添加し、常温(例えば5〜35℃)で抽出を行うことで、アイヌワカメ亜臨界水処理物の抽出物を得ることができる。 Ainu Wakame subcritical water treated product is subjected to solvent extraction using the above-mentioned solvent. The ratio of the processed subcritical water of Ainu Wakame to the solvent is, for example, 1 to 50% (w / w), preferably 5 to 25% (w / w) in terms of solid content. For example, an extract of Ainu Wakame subcritical water treatment product can be obtained by adding water to a dried product of Ainu Wakame subcritical water treatment product and performing hot water extraction at 95 to 100 ° C. Or lower alcohol (for example, ethanol etc.) or liquid polyhydric alcohol (for example, propylene glycol, 1,3-butylene glycol, etc.) is added to the dried product of Ainu Wakame subcritical water treated product, and room temperature (for example, 5-35). The extract of Ainu Wakame subcritical water treatment product can be obtained by performing the extraction at (° C.).
アイヌワカメ亜臨界水処理物又はその溶媒抽出物は、得られた溶液自体又は溶媒相自体をアイヌワカメ亜臨界水処理物の抽出物とすることができる。あるいは、必要に応じて、得られた溶液自体又は溶媒相を、濃縮(減圧濃縮、膜濃縮等による濃縮)、希釈、濾過、乾燥等の処理及び活性炭等による脱色、脱臭処理等に供して、得られた生成物をアイヌワカメ亜臨界水処理物の抽出物とすることができる。特に、抽出した溶液を濃縮乾固、噴霧乾燥、凍結乾燥等の処理に供し、得られた乾燥物をアイヌワカメ亜臨界水処理物の抽出物として用いることが好ましい。 In the Ainu Wakame subcritical water treatment product or the solvent extract thereof, the obtained solution itself or the solvent phase itself can be used as an extract of the Ainu Wakame subcritical water treatment product. Alternatively, if necessary, the obtained solution itself or the solvent phase is subjected to concentration (vacuum concentration, concentration by membrane concentration, etc.), dilution, filtration, drying, etc., decolorization with activated carbon, deodorization treatment, etc. The obtained product can be used as an extract of a processed Ainu Wakame subcritical water. In particular, it is preferable that the extracted solution is subjected to a treatment such as concentration to dryness, spray drying, freeze drying, and the like, and the obtained dried product is used as an extract of a processed Ainu Wakame subcritical water.
このようにして得られたアイヌワカメ亜臨界水処理物又はその抽出物は、幹細胞の増殖時に分化が進行せず、生体レベルで又は培養レベルで未分化状態を維持させつつ幹細胞を効率的に増殖させる作用を有するので、幹細胞の未分化状態維持剤又は増殖促進剤として使用できる。さらに、本発明の幹細胞の未分化状態維持剤又は増殖促進剤は、幹細胞の未分化状態を維持しつつ効率的に増殖させるための細胞培養用添加剤、研究用試薬としても使用することができる。 The Ainu Wakame subcritical water treatment product or the extract thus obtained does not proceed with differentiation when stem cells proliferate, and efficiently proliferates stem cells while maintaining an undifferentiated state at the biological level or culture level. Therefore, it can be used as an agent for maintaining an undifferentiated state of stem cells or a growth promoter. Furthermore, the stem cell undifferentiated state maintenance agent or growth promoter of the present invention can also be used as an additive for cell culture and a research reagent for efficiently proliferating while maintaining the undifferentiated state of stem cells. .
本発明に係る幹細胞の未分化状態維持剤又は増殖促進剤におけるアイヌワカメ亜臨界水処理物又はその抽出物の配合量は、特に限定されないが、例えば、当該薬剤全量に対し、乾燥物に換算して0.00001〜10重量%であることが好ましく、0.0001〜1重量%とすることがより好ましい。0.00001重量%未満であると効果が十分に発揮されにくい場合がある。 The compounding amount of the Ainu Wakame subcritical water treated product or extract thereof in the undifferentiated state maintaining agent or growth promoting agent for stem cells according to the present invention is not particularly limited. For example, the total amount of the drug is converted to a dry product. It is preferably 0.00001 to 10% by weight, and more preferably 0.0001 to 1% by weight. If it is less than 0.00001% by weight, the effect may not be sufficiently exhibited.
本発明に係る幹細胞の未分化状態維持剤又は増殖促進剤を、ヒトを含めた哺乳動物の幹細胞に適用することで、幹細胞の未分化状態を維持し、また幹細胞の増殖を促進することができる。本発明に係る幹細胞の未分化状態維持剤又は増殖促進剤を適用する幹細胞としては、本発明の目的に沿うものであれば特に限定されず、例えば胚性の幹細胞(ES細胞);骨髄、血液、皮膚(表皮、真皮、皮下組織)、脂肪、毛包、脳、神経、肝臓、膵臓、腎臓、筋肉やその他の組織に存在する体性の幹細胞;遺伝子導入等により人工的に作製された幹細胞(人工多能性幹細胞:iPS細胞)が挙げられる。好ましくは、本発明に係る幹細胞の未分化状態維持剤又は増殖促進剤は、骨髄、血液、皮膚又は脂肪組織由来の幹細胞に対してより効果を発揮する。ES細胞としては、例えば、着床以前の初期胚を培養することによって樹立されたES細胞、体細胞の核を核移植することによって作製された初期胚を培養することによって樹立されたES細胞、及びそれらのES細胞の染色体上の遺伝子を遺伝子工学の手法を用いて改変したES細胞が挙げられる。このようなES細胞は、例えば、自体公知の方法によって作製することができるが、所定の機関より入手でき、さらには市販品を購入することもできる。また、これらの幹細胞は、初代培養細胞、継代培養細胞又は凍結細胞のいずれであってもよい。 By applying the stem cell undifferentiated state maintenance agent or growth promoter according to the present invention to stem cells of mammals including humans, the undifferentiated state of stem cells can be maintained and the proliferation of stem cells can be promoted. . Stem cells to which the stem cell undifferentiated state maintaining agent or growth promoting agent according to the present invention is applied are not particularly limited as long as they meet the object of the present invention. For example, embryonic stem cells (ES cells); bone marrow, blood , Somatic stem cells present in the skin (epidermis, dermis, subcutaneous tissue), fat, hair follicle, brain, nerve, liver, pancreas, kidney, muscle and other tissues; stem cells artificially prepared by gene transfer etc. (Artificial pluripotent stem cells: iPS cells). Preferably, the stem cell undifferentiated state maintenance agent or growth promoter according to the present invention is more effective for stem cells derived from bone marrow, blood, skin, or adipose tissue. Examples of ES cells include ES cells established by culturing early embryos before implantation, ES cells established by culturing early embryos produced by nuclear transfer of somatic cell nuclei, And ES cells obtained by modifying genes on the chromosomes of those ES cells using genetic engineering techniques. Such ES cells can be prepared, for example, by a method known per se, but can be obtained from a predetermined institution, and further commercially available products can be purchased. In addition, these stem cells may be primary cultured cells, subcultured cells, or frozen cells.
さらに、本発明に係る幹細胞の未分化状態維持剤又は増殖促進剤は、幹細胞の分化の方向性及び分化の過程等について同等の特性を持っていれば、全ての哺乳動物由来の幹細胞に応用が可能である。例えば、本発明に係る幹細胞の未分化状態維持剤又は増殖促進剤は、ヒト、サル、マウス、ラット、モルモット、ウサギ、ネコ、イヌ、ウマ、ウシ、ヒツジ、ヤギ、ブタ等の哺乳動物の幹細胞に対して効果を発揮することができる。 Furthermore, the stem cell undifferentiated state maintenance agent or growth promoter according to the present invention can be applied to stem cells derived from all mammals as long as it has equivalent characteristics with respect to the direction of differentiation of the stem cells and the process of differentiation. Is possible. For example, the stem cell undifferentiated state maintaining agent or the growth promoting agent according to the present invention is a stem cell of a mammal such as a human, monkey, mouse, rat, guinea pig, rabbit, cat, dog, horse, cow, sheep, goat or pig. Can be effective.
本発明に係る幹細胞の未分化状態維持剤又は増殖促進剤の幹細胞への適用は、生体外であっても生体内であってもよく、いずれの場合もその作用を発揮できる。従って、本発明に係る幹細胞の未分化状態維持剤又は増殖促進剤は、その有効量を添加した幹細胞培養用培地にて幹細胞を培養することによって、又は、ヒトを含む哺乳動物に投与することによって、幹細胞の未分化状態を維持し、増殖を促進することができる。 The application of the stem cell undifferentiated state maintenance agent or growth promoter according to the present invention to the stem cell may be in vitro or in vivo, and in any case, the action can be exerted. Therefore, the stem cell undifferentiated state maintaining agent or growth promoting agent according to the present invention is obtained by culturing stem cells in a medium for culturing stem cells to which an effective amount thereof is added, or by administering to a mammal including a human. , Can maintain the undifferentiated state of stem cells and promote proliferation.
本発明に係る幹細胞の未分化状態維持剤又は増殖促進剤を生体内に投与する場合は、そのまま投与することも可能であるが、本発明の効果を損なわない範囲で適当な添加物とともに化粧品、医薬部外品、医薬品、飲食品等の各種組成物に配合して提供することができる。なお、本発明の医薬品には、動物に用いる薬剤、即ち獣医薬も包含されるものとする。 In the case where the stem cell undifferentiated state maintenance agent or growth promoter according to the present invention is administered in vivo, it can be administered as it is, but it is a cosmetic product with appropriate additives within a range not impairing the effects of the present invention, It can be provided by blending in various compositions such as quasi-drugs, pharmaceuticals, and foods and drinks. The pharmaceutical of the present invention includes drugs used for animals, that is, veterinary medicine.
本発明に係る幹細胞の未分化状態維持剤又は増殖促進剤は、有効成分であるアイヌワカメ亜臨界水処理物又はその抽出物が優れた幹細胞の未分化状態維持作用及び増殖促進作用を有するので、皮膚、骨芽、軟骨、筋肉、神経、脂肪、肝臓等の生体内の組織又は臓器の幹細胞に作用して当該組織又は臓器の障害又は損傷を治療、改善、及び予防するのにも有効である。また、幹細胞は、加齢等に伴い減少又は機能低下することから、本発明に係る幹細胞の未分化状態維持剤又は増殖促進剤は、上記生体内の組織又は臓器の幹細胞の減少や機能低下に関連する疾患を治療、改善、及び予防するのに有効である。ここで、組織又は臓器の障害又は損傷、幹細胞の減少や機能低下に関連する疾患としては、例えば、皮膚関連では、シワ、タルミ、シミ、くすみ、肌荒れ、皮膚の肥厚、毛穴のひらき、ニキビ痕、創傷、褥瘡、熱傷、瘢痕、ケロイド等が挙げられ、薄毛や脱毛等の頭皮や毛髪の損傷も含まれる。また、骨関連では、骨粗しょう症、骨折(脊椎圧迫骨折、大腿骨頚部骨折等)等、軟骨疾患では、変形性関節症、関節リウマチ、椎間板ヘルニア等、神経関連では、脊髄損傷、顔面神経麻痺、アルツハイマー病、筋萎縮性側索硬化症、パーキンソン病、加齢に伴う記憶低下等、血液関連では、再生不良性貧血、白血病等、心血管関連では心筋梗塞、閉塞性動脈硬化症等、歯科関連では歯周病、歯槽膿漏による歯槽骨損傷等、眼科関連では、網膜色素変性症、加齢黄斑変性症、緑内障等、肝臓・膵臓関連では肝炎、肝硬変、糖尿病等が挙げられるが、これらに限定されない。 Stem cell undifferentiated state maintenance agent or growth promoter according to the present invention is an active ingredient Ainu Wakame subcritical water treatment product or extract thereof has an excellent stem cell undifferentiated state maintenance action and growth promotion action, It is also effective in treating, improving, and preventing damage or damage to tissues or organs by acting on stem cells of tissues or organs in the body such as skin, osteoblast, cartilage, muscle, nerve, fat, liver, etc. . In addition, since stem cells decrease or decrease in function with aging or the like, the stem cell undifferentiated state maintaining agent or growth promoting agent according to the present invention reduces the stem cells or functions of organs or organs in the living body. It is effective in treating, ameliorating and preventing related diseases. Here, as diseases related to tissue or organ damage or damage, stem cell decrease or functional decline, for example, in the case of skin, wrinkles, tarmi, spots, dullness, rough skin, thickened skin, open pores, acne scars , Wounds, pressure ulcers, burns, scars, keloids, and the like, including scalp and hair damage such as thinning and hair loss. For bone-related, osteoporosis, fracture (vertebral compression fracture, femoral neck fracture, etc.), etc. For cartilage disease, osteoarthritis, rheumatoid arthritis, intervertebral disc herniation, etc., for nerve-related, spinal cord injury, facial paralysis , Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, memory decline with age, blood related, aplastic anemia, leukemia, etc., cardiovascular related, myocardial infarction, obstructive arteriosclerosis, etc. Related to periodontal disease, alveolar bone damage due to alveolar pyorrhea, ophthalmic related, retinitis pigmentosa, age-related macular degeneration, glaucoma, etc., liver / pancreas related include hepatitis, cirrhosis, diabetes, etc. It is not limited to.
本発明に係る幹細胞の未分化状態維持剤又は増殖促進剤を化粧品や医薬部外品に配合する場合は、その剤形は、水溶液系、可溶化系、乳化系、粉末系、粉末分散系、油液系、ゲル系、軟膏系、エアゾール系、水−油二層系、又は水−油−粉末三層系等のいずれでもよい。また、当該化粧品や医薬部外品は、幹細胞の未分化状態維持剤又は増殖促進剤とともに、皮膚外用組成物において通常使用されている各種成分、添加剤、基剤等をその種類に応じて選択し、適宜配合し、当分野で公知の手法に従って製造することができる。その形態は、液状、乳液状、クリーム状、ゲル状、ペースト状、スプレー状等のいずれであってもよい。配合成分としては、例えば、油脂類(オリーブ油、ヤシ油、月見草油、ホホバ油、ヒマシ油、硬化ヒマシ油等)、ロウ類(ラノリン、ミツロウ、カルナウバロウ等)、炭化水素類(流動パラフィン、スクワレン、スクワラン、ワセリン等)、脂肪酸類(ラウリン酸、ミリスチン酸、パルミチン酸、ステアリン酸、ベヘニン酸等)、高級アルコール類(ミリスチルアルコール、セタノール、セトステアリルアルコール、ステアリルアルコール、ベヘニルアルコール等)、エステル類(ミリスチン酸イソプロピル、パルミチン酸イソプロピル、オクタン酸セチル、トリオクタン酸グリセリン、ミリスチン酸オクチルドデシル、ステアリン酸オクチル、ステアリン酸ステアリル等)、有機酸類(クエン酸、乳酸、α-ヒドロキシ酢酸、ピロリドンカルボン酸等)、糖類(マルチトール、ソルビトール、キシロビオース、N-アセチル-D-グルコサミン等)、蛋白質及び蛋白質の加水分解物、アミノ酸類及びその塩、ビタミン類、植物・動物抽出成分、種々の界面活性剤、保湿剤、紫外線吸収剤、抗酸化剤、安定化剤、防腐剤、殺菌剤、香料等が挙げられる。 In the case where the stem cell undifferentiated state maintenance agent or growth promoter according to the present invention is added to cosmetics or quasi drugs, the dosage form is an aqueous solution, a solubilization system, an emulsion system, a powder system, a powder dispersion system, Any of an oil liquid system, a gel system, an ointment system, an aerosol system, a water-oil two-layer system, or a water-oil-powder three-layer system may be used. In addition, the cosmetics and quasi-drugs are selected according to the type of various components, additives, bases, etc. that are usually used in the composition for external use of skin, together with an undifferentiated state maintenance agent or growth promoter for stem cells. And it mix | blends suitably and can manufacture according to a well-known method in this field | area. The form may be liquid, emulsion, cream, gel, paste, spray or the like. Examples of the ingredients include oils and fats (olive oil, coconut oil, evening primrose oil, jojoba oil, castor oil, hydrogenated castor oil, etc.), waxes (lanolin, beeswax, carnauba wax, etc.), hydrocarbons (liquid paraffin, squalene, Squalane, petrolatum, etc.), fatty acids (lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid etc.), higher alcohols (myristyl alcohol, cetanol, cetostearyl alcohol, stearyl alcohol, behenyl alcohol etc.), esters (myristin) Isopropyl acid, isopropyl palmitate, cetyl octanoate, glyceryl trioctanoate, octyldodecyl myristate, octyl stearate, stearyl stearate, etc., organic acids (citric acid, lactic acid, α-hydroxyacetic acid, pyrrolidone cal Acid, etc.), sugars (maltitol, sorbitol, xylobiose, N-acetyl-D-glucosamine, etc.), proteins and protein hydrolysates, amino acids and salts thereof, vitamins, plant / animal extracts, various interfaces Examples include activators, humectants, ultraviolet absorbers, antioxidants, stabilizers, preservatives, bactericides, and fragrances.
化粧品や医薬部外品の種類としては、例えば、化粧水、乳液、ジェル、美容液、一般クリーム、日焼け止めクリーム、パック、マスク、洗顔料、化粧石鹸、ファンデーション、おしろい、浴用剤、ボディローション、ボディシャンプー、ヘアシャンプー、ヘアコンディショナー、育毛剤等が挙げられる。 As types of cosmetics and quasi-drugs, for example, lotion, milky lotion, gel, beauty essence, general cream, sunscreen cream, pack, mask, face wash, cosmetic soap, foundation, funny, bath preparation, body lotion, Examples include body shampoos, hair shampoos, hair conditioners, hair restorers and the like.
本発明に係る幹細胞の未分化状態維持剤又は増殖促進剤を医薬品に配合する場合は、薬理学的及び製剤学的に許容しうる添加物と混合し、患部に適用するのに適した製剤形態の各種製剤に製剤化することができる。薬理学的及び製剤学的に許容しうる添加物としては、その剤形、用途に応じて、適宜選択した製剤用基材や担体、賦形剤、希釈剤、結合剤、滑沢剤、コーティング剤、崩壊剤又は崩壊補助剤、安定化剤、保存剤、防腐剤、増量剤、分散剤、湿潤化剤、緩衝剤、溶解剤又は溶解補助剤、等張化剤、pH調節剤、噴射剤、着色剤、甘味剤、矯味剤、香料等を適宜添加し、公知の種々の方法にて経口又は非経口的に全身又は局所投与することができる各種製剤形態に調製すればよい。本発明の医薬品を上記の各形態で提供する場合、通常当業者に用いられる製法、たとえば日本薬局方の製剤総則[2]製剤各条に示された製法等により製造することができる。 When the stem cell undifferentiated state maintenance agent or growth promoter according to the present invention is added to a pharmaceutical, it is mixed with a pharmacologically and pharmaceutically acceptable additive and is suitable for application to an affected area. Can be formulated into various preparations. The pharmacologically and pharmaceutically acceptable additives include pharmaceutical bases and carriers, excipients, diluents, binders, lubricants, and coatings that are appropriately selected according to the dosage form and application. Agents, disintegrants or disintegration aids, stabilizers, preservatives, preservatives, extenders, dispersants, wetting agents, buffers, solubilizers or solubilizers, isotonic agents, pH adjusters, propellants A coloring agent, a sweetening agent, a corrigent, a fragrance, and the like may be added as appropriate, and various preparations that can be administered systemically or locally orally or parenterally by various known methods may be used. When the pharmaceutical product of the present invention is provided in each of the above-described forms, it can be produced by a method usually used by those skilled in the art, for example, the method shown in the General Formulation [2] Formulation of the Japanese Pharmacopoeia.
本発明の医薬品の形態としては、特に制限されるものではないが、例えば錠剤、糖衣錠剤、カプセル剤、トローチ剤、顆粒剤、散剤、液剤、丸剤、乳剤、シロップ剤、懸濁剤、エリキシル剤等の経口剤、注射剤(例えば、皮下注射剤、静脈内注射剤、筋肉内注射剤、腹腔内注射剤)、点滴剤、座剤、軟膏剤、ローション剤、点眼剤、噴霧剤、経皮吸収剤、経粘膜吸収剤、貼付剤等の非経口剤等が挙げられる。また、使用する際に再溶解させる乾燥生成物にしてもよく、注射用製剤の場合は単位投与量アンプル又は多投与量容器の状態で提供される。 The form of the pharmaceutical product of the present invention is not particularly limited. For example, tablets, sugar-coated tablets, capsules, troches, granules, powders, liquids, pills, emulsions, syrups, suspensions, elixirs Oral preparations such as pills, injections (eg, subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections), drops, suppositories, ointments, lotions, eye drops, sprays, transdermal Non-oral agents such as skin absorbents, transmucosal absorbents, patches and the like can be mentioned. Moreover, it may be a dry product which is redissolved when used, and in the case of an injectable preparation, it is provided in the form of a unit dose ampoule or a multi-dose container.
本発明に係る幹細胞の未分化状態維持剤又は増殖促進剤を、前記皮膚関連の損傷や疾患を治療、改善、及び予防するための医薬品として用いる場合に適した形態は外用製剤であり、例えば、軟膏剤、クリーム剤、ゲル剤、液剤、貼付剤等が挙げられる。軟膏剤は、均質な半固形状の外用製剤をいい、油脂性軟膏、乳剤性軟膏、水溶性軟膏を含む。ゲル剤は、水不溶性成分の抱水化合物を水性液に懸濁した外用製剤をいう。液剤は、液状の外用製剤をいい、ローション剤、懸濁剤、乳剤、リニメント剤等を含む。 A form suitable for using the undifferentiated state maintenance agent or growth promoter for stem cells according to the present invention as a pharmaceutical product for treating, ameliorating, and preventing the skin-related damage or disease is an external preparation, for example, Examples include ointments, creams, gels, solutions, patches and the like. The ointment refers to a homogeneous semi-solid external preparation, and includes an oily ointment, an emulsion ointment, and a water-soluble ointment. The gel is an external preparation in which a water-insoluble component hydrate compound is suspended in an aqueous liquid. The liquid preparation refers to a liquid external preparation and includes lotions, suspensions, emulsions, liniments and the like.
本発明の医薬品は、上記疾患の発症を抑制する予防薬として、及び/又は、正常な状態に改善する治療薬として機能する。本発明の医薬品の有効成分は、天然物由来であるため、非常に安全性が高く副作用がないため、前述の疾患の治療、改善、及び予防用医薬として用いる場合、ヒト、マウス、ラット、ウサギ、イヌ、ネコ等の哺乳動物に対して広い範囲の投与量で経口的に又は非経口的に投与することができる。 The pharmaceutical agent of the present invention functions as a prophylactic agent that suppresses the onset of the above diseases and / or a therapeutic agent that improves the normal state. Since the active ingredient of the pharmaceutical of the present invention is derived from natural products, it is very safe and has no side effects. Therefore, when used as a medicament for the treatment, amelioration, and prevention of the aforementioned diseases, humans, mice, rats, rabbits It can be administered orally or parenterally in a wide range of doses to mammals such as dogs and cats.
本発明の化粧品、医薬品、医薬部外品における幹細胞の未分化状態維持剤又は増殖促進剤の含有量は特に限定されないが、製剤(組成物)全重量に対して、アイヌワカメ亜臨界水処理物又はその抽出物の乾燥固形分に換算して、0.001〜30重量%が好ましく、0.01〜10重量%がより好ましい。0.001重量%以下では効果が低く、また30重量%を超えても効果に大きな増強はみられにくい。上記の量があくまで例示であって、組成物の種類や形態、一般的な使用量、効能・効果等を考慮して適宜設定・調整すればよい。また、製剤化における有効成分の添加法については、予め加えておいても、製造途中で添加してもよく、作業性を考えて適宜選択すればよい。 The content of the stem cell undifferentiated state maintaining agent or growth promoter in the cosmetics, pharmaceuticals, and quasi drugs of the present invention is not particularly limited, but the Ainu Wakame subcritical water treated product with respect to the total weight of the preparation (composition). Or 0.001-30 weight% is preferable and 0.01-10 weight% is more preferable in conversion into the dry solid content of the extract. If it is 0.001% by weight or less, the effect is low, and even if it exceeds 30% by weight, the effect is hardly increased. The above amounts are merely examples, and may be appropriately set and adjusted in consideration of the type and form of the composition, the general usage amount, efficacy and effects, and the like. Moreover, about the addition method of the active ingredient in formulation, it may add previously, may be added in the middle of manufacture, and should just select suitably in consideration of workability | operativity.
また、本発明に係る幹細胞の未分化状態維持剤又は増殖促進剤は、飲食品にも配合できる。また、本発明において、飲食品とは、一般的な飲食品のほか、医薬品以外で健康の維持や増進を目的として摂取できる食品、例えば、健康食品、機能性食品、保健機能食品、又は特別用途食品を含む意味で用いられる。健康食品には、栄養補助食品、健康補助食品、サプリメント等の名称で提供される食品を含む。保健機能食品は食品衛生法又は健康増進法により定義され、特定の保健の効果や栄養成分の機能、疾病リスクの低減等を表示できる、特定保健用食品及び栄養機能食品が含まれる。 Moreover, the undifferentiated state maintenance agent or growth promoter of stem cells according to the present invention can also be added to food and drink. In addition, in the present invention, the food and drink is a general food and drink, a food that can be ingested for the purpose of maintaining and promoting health other than pharmaceuticals, for example, health food, functional food, health functional food, or special use Used to include food. The health food includes foods provided under the names of nutritional supplements, health supplements, supplements, and the like. Functional health foods are defined by the Food Sanitation Law or Health Promotion Law, and include specific health foods and nutritional functional foods that can display the effects of specific health, the function of nutritional components, the reduction of disease risk, and the like.
飲食品の形態は、食用に適した形態、例えば、固形状、液状、顆粒状、粒状、粉末状、カプセル状、クリーム状、ペースト状のいずれであってもよい。特に、上記の健康食品等の場合の形状としては、例えば、タブレット状、丸状、カプセル状、粉末状、顆粒状、細粒状、トローチ状、液状(シロップ状、乳状、懸濁状を含む)等が好ましい。 The form of the food or drink may be any form suitable for edible use, for example, solid, liquid, granular, granular, powder, capsule, cream, or paste. In particular, the shape in the case of the above health foods, for example, tablets, rounds, capsules, powders, granules, fine granules, troches, liquids (including syrups, milks, suspensions) Etc. are preferred.
飲食品の種類としては、茶飲料(緑茶、ウーロン茶、紅茶等)、清涼飲料(スポーツドリンク、ミネラルウォーター、ニア・ウォーター等)、炭酸飲料、乳飲料、コーヒー飲料、果汁・野菜汁入り飲料、栄養ドリンク、ゼリー飲料等の各種飲料、粉末スープ、菓子類(キャンディー、グミ、ガム、錠菓等)、麺類、パン類、乳製品、水産・畜産加工食品、油脂及び油脂加工食品等が挙げられるが、これらに限定はされない。 The types of food and drink include tea drinks (green tea, oolong tea, tea, etc.), soft drinks (sports drinks, mineral water, near water, etc.), carbonated drinks, milk drinks, coffee drinks, fruit / vegetable juice drinks, nutrition Various drinks such as drinks and jelly drinks, powdered soups, confectionery (candy, gummi, gum, tablet confectionery, etc.), noodles, breads, dairy products, processed fishery products, processed foods, fats and oils and processed foods, etc. However, it is not limited to these.
本発明の飲食品は、その種類に応じて通常使用される添加物を適宜配合してもよい。添加物としては、食品衛生法上許容されうる添加物であればいずれも使用できるが、例えば、ブドウ糖、ショ糖、果糖、異性化液糖、アスパルテーム、ステビア等の甘味料;クエン酸、リンゴ酸、酒石酸等の酸味料;デキストリン、デンプン等の賦形剤;結合剤、希釈剤、香料、着色料、緩衝剤、増粘剤、ゲル化剤、安定剤、保存剤、乳化剤、分散剤、懸濁化剤、防腐剤等が挙げられる。 The food / beverage products of the present invention may be appropriately blended with additives usually used depending on the type. Any additive that is acceptable under the Food Sanitation Law can be used as the additive. For example, sweeteners such as glucose, sucrose, fructose, isomerized liquid sugar, aspartame, stevia; citric acid, malic acid Acidulants such as tartaric acid; excipients such as dextrin and starch; binders, diluents, fragrances, colorants, buffers, thickeners, gelling agents, stabilizers, preservatives, emulsifiers, dispersants, suspensions Examples include turbidity agents and preservatives.
本発明の飲食品が一般的な飲食品の場合は、その飲食品の通常の製造工程においてアイヌワカメ亜臨界水処理物又はその抽出物を添加する工程を含めることによって製造することができる。また、健康食品の場合は、前記の医薬品の製造方法に準じればよく、例えば、タブレット状のサプリメントでは、アイヌワカメ亜臨界水処理物又はその抽出物に、賦形剤等の添加物を添加、混合し、打錠機等で圧力をかけて成形することにより製造することができる。また、必要に応じてその他の材料(例えば、ビタミンC、ビタミンB2、ビタミンB6等のビタミン類、カルシウム等のミネラル類、食物繊維等)を添加することもできる。 When the food / beverage products of the present invention are general food / beverage products, the food / beverage products can be produced by including a step of adding a processed Ainuwakame subcritical water product or an extract thereof in a normal production process of the food / beverage product. In addition, in the case of health foods, it may be according to the above-mentioned pharmaceutical production method. For example, in the case of tablet-like supplements, additives such as excipients are added to the processed Ainu Wakame subcritical water or the extract thereof. It can be manufactured by mixing and molding by applying pressure with a tableting machine or the like. Moreover, other materials (for example, vitamins such as vitamin C, vitamin B2, and vitamin B6, minerals such as calcium, dietary fiber, etc.) can be added as necessary.
本発明の飲食品におけるアイヌワカメ亜臨界水処理物又はその抽出物の配合量は、幹細胞の未分化状態維持効果と増殖促進効果を発揮できる量であればよいが、対象飲食品の一般的な摂取量、飲食品の形態、効能・効果、呈味性、嗜好性及びコスト等を考慮して適宜設定すればよい。 The amount of Ainu Wakame subcritical water-treated product or extract thereof in the food or drink of the present invention may be any amount that can exhibit the effect of maintaining the undifferentiated state of stem cells and the effect of promoting proliferation, What is necessary is just to set suitably in consideration of the amount of intake, the form of food and drink, efficacy and effect, taste, palatability and cost.
本発明はまた、幹細胞を、アイヌワカメ亜臨界水処理物又はその抽出物を含有する培地で培養することで、幹細胞の未分化状態を維持させたまま、幹細胞増殖を促進する方法に関する。換言すれば、本発明に係る方法は、幹細胞を、アイヌワカメ亜臨界水処理物又はその抽出物を含有する培地で培養する工程を含む、幹細胞の製造方法、幹細胞の未分化状態維持方法又は幹細胞の増殖促進方法ということができる。 The present invention also relates to a method of accelerating stem cell proliferation while maintaining the undifferentiated state of stem cells by culturing stem cells in a medium containing a processed Ainu Wakame subcritical water or an extract thereof. In other words, the method according to the present invention includes a step of culturing a stem cell in a medium containing a processed Ainu seaweed subcritical water or an extract thereof, a method for producing a stem cell, a method for maintaining an undifferentiated state of a stem cell, or a stem cell It can be said that it is a method for promoting the growth of
本発明に係る方法において、幹細胞の培養には、幹細胞の未分化状態維持及び増殖のために一般的に使用されている培地を用いればよい。例えば、幹細胞の生存及び増殖に必要な成分(無機塩、炭水化物、ホルモン、必須アミノ酸、非必須アミノ酸、ビタミン、脂肪酸)を含む基本培地、具体的には、Dulbecco’s Modified Eagle Medium(D−MEM)、Minimum Essential Medium(MEM)、RPMI 1640、Basal Medium Eagle(BME)、Dulbecco’s Modified Eagle Medium:Nutrient Mixture F−12(D−MEM/F−12)、Glasgow Minimum Essential Medium(Glasgow MEM)、ハンクス液(Hank’s balanced salt solution)等が挙げられる。また、培地に、増殖因子として塩基性線維芽細胞増殖因子(bFGF)及び/又は白血球遊走阻止因子(LIF)を添加してもよい。さらに、必要に応じて、培地は、上皮細胞増殖因子(EGF)、腫瘍壊死因子(TNF)、ビタミン類、インターロイキン類、インスリン、トランスフェリン、ヘパリン、ヘパラン硫酸、コラーゲン、フィブロネクチン、プロゲステロン、セレナイト、B27−サプリメント、N2−サプリメント、ITS−サプリメント、抗生物質等を含有してもよい。 In the method according to the present invention, a culture medium generally used for maintaining and proliferating the undifferentiated state of stem cells may be used for culturing stem cells. For example, a basic medium containing components (inorganic salts, carbohydrates, hormones, essential amino acids, non-essential amino acids, vitamins, fatty acids) necessary for stem cell survival and proliferation, specifically, Dulbecco's Modified Eagle Medium (D-MEM) ), Minimum Essential Medium (MEM), RPMI 1640, Basal Medium Eagle (BME), Dulbecco's Modified Eagle Medium (Nentient Mixture F-12 (D-MEM / F-M / G). Hanks' solution (Hank's balanced salt solution) etc. are mentioned. In addition, basic fibroblast growth factor (bFGF) and / or leukocyte migration inhibitory factor (LIF) may be added to the medium as growth factors. Further, if necessary, the medium may be epidermal growth factor (EGF), tumor necrosis factor (TNF), vitamins, interleukins, insulin, transferrin, heparin, heparan sulfate, collagen, fibronectin, progesterone, selenite, B27. -It may contain supplements, N2-supplements, ITS-supplements, antibiotics and the like.
また、上記以外には、1〜20%の含有率で血清が培地に含まれることが好ましい。しかしながら、血清はロットの違いにより成分が異なり、その効果にバラツキがあるため、ロットチェックを行った後に使用することが好ましい。 In addition to the above, it is preferable that serum is contained in the medium at a content of 1 to 20%. However, since serum has different components depending on the lot and there are variations in its effects, it is preferably used after lot check.
市販品の培地としては、インビトロジェン製の間葉系幹細胞基礎培地や、三光純薬製の間葉系幹細胞基礎培地、TOYOBO社製のMF培地、Sigma社製のハンクス液(Hank’s balanced salt solution)等を用いることができる。 Commercially available media include Mesenchymal stem cell basal medium manufactured by Invitrogen, Mesenchymal stem cell basal medium manufactured by Sanko Junyaku Co., Ltd., MF medium manufactured by TOYOBO, Hanks' solution manufactured by Sigma (Hank's balanced salt solution) ) Etc. can be used.
幹細胞の培養に用いる培養器は、幹細胞の培養が可能なものであれば特に限定されないが、例えば、フラスコ、シャーレ、ディッシュ、プレート、チャンバースライド、チューブ、トレイ、培養バッグ、ローラーボトル等が挙げられる。 The incubator used for stem cell culture is not particularly limited as long as stem cells can be cultured. Examples thereof include flasks, petri dishes, dishes, plates, chamber slides, tubes, trays, culture bags, and roller bottles. .
培養器は、細胞非接着性であっても接着性であってもよく、目的に応じて適宜選択される。細胞接着性の培養器は、細胞との接着性を向上させる目的で、細胞外マトリックス等による細胞支持用基質等で処理したものを用いてもよい。細胞外基質としては、例えば、コラーゲン、ゼラチン、ポリ−L−リジン、ポリ−D−リジン、ラミニン、フィブロネクチン等が挙げられる。 The incubator may be non-cell-adhesive or adhesive, and is appropriately selected according to the purpose. As the cell-adhesive incubator, for the purpose of improving the adhesion to cells, a cell-treated culture vessel treated with a cell support substrate using an extracellular matrix or the like may be used. Examples of the extracellular matrix include collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, fibronectin and the like.
幹細胞培養に使用される培地に対するアイヌワカメ亜臨界水処理物又はその抽出物の添加濃度は、上述の本発明に係る幹細胞の未分化状態維持剤又は増殖促進剤におけるアイヌワカメ亜臨界水処理物又はその抽出物の含有量に準じて適宜決定することができるが、固形分に換算して、例えば10〜100000μg/mL、好ましくは100〜10000μg/mLの濃度が挙げられる。また、幹細胞の培養期間中、アイヌワカメ亜臨界水処理物又はその抽出物を、定期的に培地に添加してもよい。 The concentration of the Ainu Wakame subcritical water treatment product or extract thereof added to the medium used for stem cell culture is the Ainu Wakame subcritical water treatment product in the above-described stem cell undifferentiated state maintenance agent or growth promoter, or Although it can determine suitably according to content of the extract, it is 10-100,000 microgram / mL in conversion to solid content, Preferably the density | concentration of 100-10000 microgram / mL is mentioned. Further, during the stem cell culture period, the Ainu Wakame subcritical water treated product or the extract thereof may be periodically added to the medium.
幹細胞の培養条件は、幹細胞の培養に用いられる通常の条件に従えばよく、特別な制御は必要ではない。例えば、培養温度は、特に限定されるものではないが約30〜40℃、好ましくは36〜37℃である。CO2ガス濃度は、例えば約1〜10%、好ましくは約2〜5%である。なお、培地の交換は2〜3日に1回行うことが好ましく、毎日行うことがより好ましい。前記培養条件は、幹細胞が生存及び増殖可能な範囲で適宜変動させて設定することもできる。 Stem cell culture conditions may be in accordance with normal conditions used for stem cell culture, and no special control is required. For example, the culture temperature is not particularly limited, but is about 30 to 40 ° C, preferably 36 to 37 ° C. The CO 2 gas concentration is, for example, about 1 to 10%, preferably about 2 to 5%. In addition, it is preferable to perform culture medium exchange once every 2 to 3 days, and it is more preferable to perform it every day. The culture conditions can also be set by varying as appropriate within the range in which the stem cells can survive and proliferate.
幹細胞の未分化状態維持は、例えば、本発明に係る幹細胞の未分化状態維持剤の非存在下で培養した幹細胞と比較して、本発明に係る幹細胞の未分化状態維持剤の存在下で培養した該幹細胞において幹細胞未分化マーカー遺伝子の発現レベルがmRNAレベル又はタンパク質レベルで培養開始時の発現レベルと同程度のレベルに有意に維持されているか否かで評価することができる。幹細胞未分化マーカー遺伝子としては、例えばCD44遺伝子(Mol Biol Cell. 2002 Dec;13(12):4279−95. Human adipose tissue is a source of multipotent stem cells.)等が挙げられる。 Stem cell undifferentiated state maintenance is performed, for example, in the presence of the stem cell undifferentiated state maintaining agent according to the present invention as compared to the stem cell cultured in the absence of the stem cell undifferentiated state maintaining agent according to the present invention. The stem cell undifferentiated marker gene expression level in the stem cells can be evaluated by whether or not it is maintained at the mRNA level or the protein level at the same level as the expression level at the start of culture. Examples of the stem cell undifferentiated marker gene include CD44 gene (Mol Biol Cell. 2002 Dec; 13 (12): 4279-95. Human adipose tissue of a source of multiple cells.) And the like.
幹細胞未分化マーカー遺伝子発現レベルの測定方法としては、mRNAレベルでは、例えば幹細胞未分化マーカー遺伝子に特異的なプライマーやプローブを用いたRT−PCR、定量PCRやノーザンブロッティング等の方法が挙げられ、また、タンパク質レベルでは、例えば幹細胞未分化マーカー遺伝子によりコードされるタンパク質に特異的な抗体を用いた免疫染色、ELISA、フローサイトメトリー、ウエスタンブロッティング等の免疫学的方法が挙げられる。 Examples of the method for measuring the level of expression of stem cell undifferentiated marker gene include, for example, RT-PCR using primers and probes specific to stem cell undifferentiated marker gene, quantitative PCR, Northern blotting and the like at the mRNA level. At the protein level, for example, immunological methods such as immunostaining using an antibody specific for a protein encoded by a stem cell undifferentiation marker gene, ELISA, flow cytometry, Western blotting and the like can be mentioned.
発現レベルの測定の結果、培養開始時(100%未分化状態)の幹細胞における幹細胞未分化マーカー遺伝子の発現レベルと本発明の幹細胞の未分化状態維持剤の存在下で所定時間培養後の幹細胞における幹細胞未分化マーカー遺伝子の発現レベルとの相対比が、本発明の幹細胞の未分化状態維持剤の非存在下で培養した場合の同相対比(コントロール)よりも大きい場合に幹細胞の未分化状態を維持できたと判定することができる。 As a result of measuring the expression level, the expression level of the stem cell undifferentiation marker gene in the stem cell at the start of culture (100% undifferentiated state) and the stem cell after culturing for a predetermined time in the presence of the stem cell undifferentiated state maintaining agent of the present invention When the relative ratio of the expression level of the stem cell undifferentiation marker gene is greater than the same relative ratio (control) when cultured in the absence of the stem cell undifferentiated state maintenance agent of the present invention, the stem cell undifferentiated state It can be determined that it was maintained.
また、幹細胞の増殖促進は、例えば、本発明に係る幹細胞の増殖促進剤の非存在下で培養した幹細胞と比較して、本発明に係る幹細胞の増殖促進剤の存在下で培養した該幹細胞の細胞数が有意に増加されているか否かで評価することができる。細胞数の測定は、例えば、MTT法やWST法等により、市販の細胞数測定キットを用いて行うことができる。測定の結果、培養開始時の幹細胞の細胞数と本発明の幹細胞の増殖促進剤の存在下で所定時間培養後の幹細胞の細胞数との相対比が、本発明の幹細胞の増殖促進剤の非存在下で培養した場合の同相対比(コントロール)よりも大きい場合に幹細胞の増殖を促進できたと判定することができる。 In addition, the proliferation promotion of the stem cell is achieved by, for example, the stem cell cultured in the presence of the stem cell proliferation promoter according to the present invention, as compared with the stem cell cultured in the absence of the stem cell proliferation promoter according to the present invention. It can be evaluated by whether or not the number of cells is significantly increased. The number of cells can be measured using, for example, a commercially available cell number measurement kit by the MTT method, the WST method, or the like. As a result of the measurement, the relative ratio between the number of stem cells at the start of culture and the number of stem cells cultured for a predetermined time in the presence of the stem cell proliferation promoter of the present invention is the non-existence of the stem cell proliferation promoter of the present invention. It can be determined that the proliferation of stem cells could be promoted when the relative ratio (control) was greater when cultured in the presence.
上記の本発明に係る方法により調製された幹細胞は移植材料(細胞移植剤)として用いることができ、従来の骨髄移植又は臍帯血移植と同一の方法で実施できる。 Stem cells prepared by the above-described method according to the present invention can be used as a transplant material (cell transplant agent), and can be performed by the same method as conventional bone marrow transplantation or umbilical cord blood transplantation.
上記の本発明に係る幹細胞の未分化状態維持剤又は増殖促進剤あるいは本発明に係る方法に準じて、アイヌワカメ亜臨界水処理物又はその抽出物を、単独で、あるいは培地と別々に又は培地と混合し、幹細胞の未分化状態維持又は増殖促進のための試薬キットとして提供することもできる。当該キットは、必要に応じて取扱い説明書等を含むことができる。あるいは、アイヌワカメ亜臨界水処理物又はその抽出物を、培地と混合し、幹細胞の未分化状態維持又は増殖促進用培地として提供することもできる。 According to the above-described stem cell undifferentiated state maintenance agent or growth promoter or the method according to the present invention, the Ainu Wakame subcritical water treated product or the extract thereof is used alone or separately from the medium or the medium. And a reagent kit for maintaining the undifferentiated state of stem cells or promoting proliferation thereof. The kit can include an instruction manual or the like as necessary. Alternatively, the Ainu Wakame subcritical water treatment product or an extract thereof can be mixed with a medium and provided as a medium for maintaining the undifferentiated state of stem cells or promoting proliferation.
以下、実施例を用いて本発明をより詳細に説明するが、本発明の技術的範囲はこれら実施例に限定されるものではない。 EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, the technical scope of this invention is not limited to these Examples.
[実施例1]アイヌワカメ亜臨界水処理物及びその抽出物
(1)アイヌワカメ亜臨界水処理物
製造例1
亜臨界水処理缶に、アイヌワカメ35gを入れ、処理温度:140℃、処理圧力:0.37MPa、処理時間:10分間で亜臨界水処理を行った(亜臨界水処理条件#1)。亜臨界水処理の終了後、処理缶内の処理物を凍結乾燥させることでアイヌワカメ亜臨界水処理物1を33.1g得た。
[Example 1] Ainu Wakame subcritical water treated product and extract thereof (1) Ainu Wakame subcritical water treated product Production Example 1
35 g of Ainu Wakame was put into a subcritical water treatment can, and a subcritical water treatment was performed at a treatment temperature of 140 ° C., a treatment pressure of 0.37 MPa, and a treatment time of 10 minutes (subcritical water treatment condition # 1). After the completion of the subcritical water treatment, the treated product in the treatment can was freeze-dried to obtain 33.1 g of Ainu Wakame subcritical water treated product 1.
製造例2〜製造例5(アイヌワカメ亜臨界水処理物2〜5)については、表1に示した製造例の条件一覧に示した処理温度、処理圧力、処理時間とした以外は、製造例1と同様に行った。また、製造例2〜製造例5の回収量については表1に示した。 Production Examples 2 to 5 (Ainu Wakame Subcritical Water Treatment Products 2 to 5) are production examples except that the treatment temperature, treatment pressure, and treatment time shown in the conditions list of the production examples shown in Table 1 are used. Same as 1. The recovered amounts of Production Examples 2 to 5 are shown in Table 1.
(2)アイヌワカメ亜臨界水処理物の抽出物
製造例6A 熱水抽出物
アイヌワカメ亜臨界水処理物1(製造例1)10gに、精製水200mLを加え、95〜100℃で2時間抽出した。濾過した後、その濾液を濃縮し、凍結乾燥して熱水抽出物を得た。
(2) Ainuwakame subcritical water extract
Production Example 6A To 10 g of the hot water extract Ainu Wakame subcritical water treated product 1 (Production Example 1), 200 mL of purified water was added and extracted at 95 to 100 ° C. for 2 hours. After filtration, the filtrate was concentrated and lyophilized to obtain a hot water extract.
製造例6B 50%エタノール抽出物
アイヌワカメ亜臨界水処理物1(製造例1)10gに、50%エタノール水溶液を200mL加え、一昼夜抽出した。濾過した後、その濾液を減圧濃縮し、凍結乾燥することにより、50%エタノール抽出物を得た。
Production Example 6B 50% ethanol extract 200 g of 50% ethanol aqueous solution was added to 10 g of Ainu Wakame subcritical water treated product 1 (Production Example 1), and extracted all day and night. After filtration, the filtrate was concentrated under reduced pressure and freeze-dried to obtain a 50% ethanol extract.
製造例6C エタノール抽出物
アイヌワカメ亜臨界水処理物1(製造例1)10gに、エタノールを200mL加え、一昼夜抽出した。濾過した後、その濾液を減圧濃縮し、凍結乾燥することにより、エタノール抽出物を得た。
Production Example 6C Ethanol extract To 10 g of Ainu Wakame subcritical water treated product 1 (Production Example 1), 200 mL of ethanol was added and extracted all day and night. After filtration, the filtrate was concentrated under reduced pressure and freeze-dried to obtain an ethanol extract.
製造例2〜5を用いて、上記の製造例6A〜6Cと同様にアイヌワカメ亜臨界水処理物の抽出物(製造例7A〜10C)を製造した(表2)。 Extracts (Production Examples 7A to 10C) of Ainu Wakame subcritical water treated products were produced in the same manner as in Production Examples 6A to 6C using Production Examples 2 to 5 (Table 2).
製造例11 アイヌワカメ亜臨界水処理物6
亜臨界水処理缶に、アイヌワカメ35gを入れ、処理温度:140℃、処理圧力:0.37MPa、処理時間10分間で亜臨界水処理を行った。亜臨界水処理の終了後、処理缶内の処理物をろ過した後、そのろ液を濃縮し、凍結乾燥してアイヌワカメ亜臨界水処理物6を11.5g得た。
Production Example 11 Ainu Wakame Subcritical Water Treatment Product 6
35 g of Ainu Wakame was put into a subcritical water treatment can, and the subcritical water treatment was performed at a treatment temperature of 140 ° C., a treatment pressure of 0.37 MPa, and a treatment time of 10 minutes. After completion of the subcritical water treatment, the treated product in the treatment can was filtered, and the filtrate was concentrated and freeze-dried to obtain 11.5 g of Ainu Wakame subcritical water treated product 6.
比較製造例1 アイヌワカメの熱水抽出物
アイヌワカメ10gに、精製水2Lを加え、95〜100℃で2時間抽出した。濾過した後、その濾液を濃縮し、凍結乾燥してアイヌワカメの熱水抽出物1.5gを得た。
Comparative Production Example 1 Ainu Wakame hot water extract 10 g of Ainu Wakame was added with 2 L of purified water and extracted at 95-100 ° C. for 2 hours. After filtration, the filtrate was concentrated and freeze-dried to obtain 1.5 g of a hot water extract of Ainu Wakame.
[実施例2]アイヌワカメ亜臨界水処理物の抽出物の幹細胞に対する未分化状態維持効果及び増殖促進効果の評価
以下に、実施例1において製造したアイヌワカメ亜臨界水処理物の抽出物を用いた、幹細胞に対する未分化状態維持効果及び増殖促進効果の実験例とその結果を示す。
[Example 2] Evaluation of undifferentiated state maintaining effect and growth promoting effect on stem cells of extract of Ainu Wakame subcritical water treatment product Extract of Ainu Wakame subcritical water treatment product produced in Example 1 is used below. The experimental example of the undifferentiated state maintenance effect with respect to the stem cell and the proliferation promotion effect and the result are shown.
(実験例1)幹細胞に対する未分化状態維持効果の評価
Dulbecco’s Modified Eagle Medium培養液(Sigma社製)にウシ胎児血清(FBS、1%、Sigma社製)、ITS−X(100倍希釈、GIBCO社製)、Hydorocortison(0.4μg/mL、SIGMA社製)を加えて調製した培地を用いて、ヒト成体幹細胞(DSファーマバイオメディカル社製)を24wellディッシュに5x104個播種し、各抽出物(比較製造例1、製造例6A、製造例7A)を最終濃度が625μg/mLになるように培地に添加し、3日間培養を続けた。なお、陽性対照としてFGF−2(10ng/mL、Pepro Teck社製)を用いた。
(Experimental example 1) Evaluation of an undifferentiated state maintenance effect with respect to a stem cell Dulbecco's Modified Eagle Medium culture solution (made by Sigma), fetal bovine serum (FBS, 1%, made by Sigma), ITS-X (100-fold dilution, 5 × 10 4 human adult stem cells (manufactured by DS Pharma Biomedical) were seeded in a 24 well dish using a medium prepared by adding GIBCO) and Hydrocortison (0.4 μg / mL, manufactured by SIGMA), and each extraction. The product (Comparative Production Example 1, Production Example 6A, Production Example 7A) was added to the medium so that the final concentration was 625 μg / mL, and the culture was continued for 3 days. As a positive control, FGF-2 (10 ng / mL, manufactured by Pepro Teck) was used.
培養3日後に、細胞をPBS(−)にて2回洗浄し、4%パラホルムアルデヒドを加え、室温で10分間インキュベーションして細胞を固定した。固定後、細胞をPBS(−)にて2回洗浄し、0.1%ウシ血清アルブミン(BSA、和光純薬工業社製)含有PBS(−)に抗CD44抗体(200倍希釈、Santa Cruz Biotechnology社製)を添加した一次抗体溶液を加え、37℃で1時間インキュベーションした。次に、2次抗体Alexa488(500倍希釈、Molecular Probes社製)で37℃60分間反応させた。細胞をPBS(−)にて2回洗浄し、蛍光顕微鏡(Olympus社製)を用いて観察し、CD44の発現を緑色の蛍光として画像を撮影した。取得した画像について、市販の解析ソフトを用いてCD44の相対発現量(100μm2あたりの蛍光強度)を算出した。試料を添加せずに培養した細胞におけるCD44の相対発現量を1とし、これに対し、試料を添加して培養した細胞におけるCD44の相対発現量の値を算出し、評価した。結果を表3に示す。 After 3 days of culture, the cells were washed twice with PBS (−), 4% paraformaldehyde was added, and the cells were fixed by incubation at room temperature for 10 minutes. After fixation, the cells were washed twice with PBS (−), and anti-CD44 antibody (diluted 200-fold, Santa Cruz Biotechnology) was added to PBS (−) containing 0.1% bovine serum albumin (BSA, manufactured by Wako Pure Chemical Industries, Ltd.). And a primary antibody solution added thereto was added and incubated at 37 ° C. for 1 hour. Next, the reaction was performed at 37 ° C. for 60 minutes with the secondary antibody Alexa 488 (diluted 500 times, manufactured by Molecular Probes). The cells were washed twice with PBS (−) and observed using a fluorescence microscope (manufactured by Olympus), and images were taken with CD44 expression as green fluorescence. For the acquired images, the relative expression level of CD44 (fluorescence intensity per 100 μm 2 ) was calculated using commercially available analysis software. The relative expression level of CD44 in cells cultured without adding the sample was set to 1, and the relative expression level of CD44 in cells cultured with the sample added was calculated and evaluated. The results are shown in Table 3.
表3に示すように、アイヌワカメ亜臨界水処理物の抽出物(製造例6A及び7A)に、顕著な幹細胞の未分化状態維持効果が認められた。以上より、アイヌワカメ亜臨界水処理物の抽出物の極めて優れた幹細胞の未分化状態維持効果を明らかにした。なお、本実験例で用いた幹細胞以外にも、胚性の幹細胞(ES細胞)についても同様な試験を行ったところ、顕著な幹細胞の未分化状態維持効果が認められた。また、アイヌワカメの亜臨界水処理物6(製造例11)についても同様に優れた幹細胞の未分化状態維持効果を示した。 As shown in Table 3, a remarkable effect of maintaining the undifferentiated state of stem cells was observed in the extract of Ainu Wakame subcritical water treated (Production Examples 6A and 7A). From the above, it was clarified that the extract of the Ainu Wakame subcritical water treated product was excellent in maintaining the undifferentiated state of stem cells. In addition to the stem cells used in this experimental example, a similar test was performed on embryonic stem cells (ES cells), and a remarkable effect of maintaining the undifferentiated state of stem cells was observed. In addition, Ainu Wakame's subcritical water treated product 6 (Production Example 11) also showed an excellent effect of maintaining the undifferentiated state of stem cells.
(実験例2)幹細胞に対する増殖促進効果の評価
ヒト幹細胞培養液(東洋紡社製)を用いて培養したヒト成体幹細胞(東洋紡社製)を、6cmディッシュに3×105個播種し、各抽出物(製造例6A、製造例7A及び比較製造例1)の最終濃度が625μg/mLになるように添加し、3日間培養を続けた。
(Experimental example 2) Evaluation of proliferation promoting effect on stem cells 3 × 10 5 adult human stem cells (manufactured by Toyobo Co., Ltd.) cultured using human stem cell culture solution (manufactured by Toyobo Co., Ltd.) are seeded in a 6 cm dish, and each extract is extracted. (Production Example 6A, Production Example 7A and Comparative Production Example 1) were added so that the final concentration was 625 μg / mL, and the culture was continued for 3 days.
3日間の培養後、細胞をPBS(−)にて3回洗浄した後、ラバーポリスマンにて集め、それぞれの細胞数をカウントした。 After culturing for 3 days, the cells were washed three times with PBS (−) and then collected by a rubber policeman, and the number of each cell was counted.
抽出物未添加時の総細胞数をコントロールとし、コントロールを100(%)とした場合の、各抽出物(製造例6A、製造例7A及び比較製造例1)添加時の細胞数の増減(%)を算出し、幹細胞増殖促進効果の評価を行った。これらの試験結果を以下の表4に示す。 Increase / decrease (%) in the number of cells when each extract (Production Example 6A, Production Example 7A and Comparative Production Example 1) was added, with the total number of cells when no extract was added as a control and the control as 100 (%) ) Was calculated, and the stem cell proliferation promoting effect was evaluated. The test results are shown in Table 4 below.
表4に示すように、アイヌワカメ亜臨界水処理物の抽出物(製造例6A及び7A)に、顕著な幹細胞増殖促進効果が認められた。なお、上述のコントロールの値を100%とした場合、培養開始時のヒト成体幹細胞数は、27%であった。以上より、アイヌワカメ亜臨界水処理物の抽出物の極めて優れた幹細胞増殖促進効果を明らかにした。なお、本実験例で用いた幹細胞以外にも、胚性の幹細胞(ES細胞)についても同様な試験を行ったところ、顕著な幹細胞増殖促進効果が認められた。また、アイヌワカメの亜臨界水処理物6(製造例11)についても同様に優れた幹細胞の増殖促進効果を示した。 As shown in Table 4, the extract of Ainu Wakame subcritical water treated product (Production Examples 6A and 7A) showed a significant stem cell proliferation promoting effect. When the control value described above was 100%, the number of adult human stem cells at the start of culture was 27%. From the above, the extremely excellent stem cell proliferation promoting effect of the extract of Ainu Wakame subcritical water treatment was clarified. In addition to the stem cells used in this experimental example, a similar test was performed on embryonic stem cells (ES cells), and a remarkable effect of promoting stem cell proliferation was observed. In addition, Ainu Wakame's subcritical water treated product 6 (Production Example 11) also showed an excellent stem cell proliferation promoting effect.
以上に示すように、アイヌワカメ亜臨界水処理物又はその抽出物を幹細胞に適用することで、従来の技術に比べて、簡便且つ効率的に、未分化状態を維持させたまま幹細胞の増殖を促進させることが可能になった。 As described above, by applying the Ainu Wakame subcritical water treatment product or its extract to stem cells, the proliferation of stem cells can be achieved while maintaining an undifferentiated state more easily and efficiently than conventional techniques. It became possible to promote.
[実施例3]製品の処方例
アイヌワカメ亜臨界水処理物の抽出物を配合した製品の処方例を以下に示す。
(処方例1)ローション
処方 配合量(重量%)
1.アイヌワカメ亜臨界水処理物の熱水抽出物(製造例6A) 0.1
2.1,3−ブチレングリコール 8.0
3.グリセリン 2.0
4.キサンタンガム 0.02
5.クエン酸 0.01
6.クエン酸ナトリウム 0.1
7.エタノール 5.0
8.パラオキシ安息香酸メチル 0.1
9.ポリオキシエチレン硬化ヒマシ油(40E.O.) 0.1
10.香料 0.1
11.精製水 残量
[Example 3] Formulation example of product A formulation example of a product containing an extract of Ainu Wakame subcritical water treated product is shown below.
(Formulation Example 1) Lotion Formulation Blending amount (% by weight)
1. Hot water extract of Ainu Wakame subcritical water treated product (Production Example 6A) 0.1
2. 1,3-butylene glycol 8.0
3. Glycerin 2.0
4). Xanthan gum 0.02
5. Citric acid 0.01
6). Sodium citrate 0.1
7). Ethanol 5.0
8). Methyl paraoxybenzoate 0.1
9. Polyoxyethylene hydrogenated castor oil (40E.O.) 0.1
10. Fragrance 0.1
11. Purified water remaining
成分1〜6及び11と、成分7〜10をそれぞれ均一に溶解した後、両者を混合し濾過しローションを調製する。 Components 1 to 6 and 11 and components 7 to 10 are uniformly dissolved, then both are mixed and filtered to prepare a lotion.
(処方例2) クリーム
処方 配合量(重量%)
1.アイヌワカメ亜臨界水処理物の熱水抽出物(製造例7A) 0.1
2.スクワラン 5.5
3.オリーブ油 3.0
4.ステアリン酸 2.0
5.ミツロウ 2.0
6.ミリスチン酸オクチルドデシル 3.5
7.ポリオキシエチレンセチルエーテル(20E.O.) 3.0
8.ベヘニルアルコール 1.5
9.モノステアリン酸グリセリン 2.5
10.香料 0.1
11.パラオキシ安息香酸メチル 0.2
12.パラオキシ安息香酸エチル 0.05
13.1,3−ブチレングリコール 8.5
14.精製水 残量
(Formulation Example 2) Cream Formulation Formulation amount (% by weight)
1. Hot water extract of Ainu Wakame subcritical water treated product (Production Example 7A) 0.1
2. Squalane 5.5
3. Olive oil 3.0
4). Stearic acid 2.0
5. Beeswax 2.0
6). Octyldodecyl myristate 3.5
7). Polyoxyethylene cetyl ether (20E.O.) 3.0
8). Behenyl alcohol 1.5
9. Glycerol monostearate2.5
10. Fragrance 0.1
11. Methyl paraoxybenzoate 0.2
12 Ethyl paraoxybenzoate 0.05
13.1,3-Butylene glycol 8.5
14 Purified water remaining
成分2〜9を加熱溶解して混合し、70℃に保ち油相とする。成分1及び11〜14を加熱溶解して混合し、75℃に保ち水相とする。次いで、油相に水相を加えて乳化して、かき混ぜながら冷却し、45℃で成分10を加え、さらに30℃まで冷却して製品とする。 Ingredients 2-9 are dissolved by heating and mixed, and kept at 70 ° C. to obtain an oil phase. Ingredients 1 and 11 to 14 are dissolved by heating and mixed, and kept at 75 ° C. to form an aqueous phase. Next, the aqueous phase is added to the oil phase to emulsify, and the mixture is cooled while stirring. The component 10 is added at 45 ° C., and further cooled to 30 ° C. to obtain a product.
(処方例3)乳液
処方 配合量(重量%)
1.アイヌワカメ亜臨界水処理物の熱水抽出物(製造例8A) 0.1
2.スクワラン 5.0
3.オリーブ油 5.0
4.ホホバ油 5.0
5.セタノール 1.5
6.モノステアリン酸グリセリン 2.0
7.ポリオキシエチレンセチルエーテル(20E.O.) 3.0
8.ポリオキシエチレンソルビタンモノオレエート(20E.O.) 2.0
9.香料 0.1
10.プロピレングリコール 1.0
11.グリセリン 2.0
12.パラオキシ安息香酸メチル 0.2
13.精製水 残量
(Prescription Example 3) Emulsion
Formulation amount (% by weight)
1. Hot water extract of Ainu Wakame subcritical water treated product (Production Example 8A) 0.1
2. Squalane 5.0
3. Olive oil 5.0
4). Jojoba oil 5.0
5. Cetanol 1.5
6). Glycerol monostearate 2.0
7). Polyoxyethylene cetyl ether (20E.O.) 3.0
8). Polyoxyethylene sorbitan monooleate (20E.O.) 2.0
9. Fragrance 0.1
10. Propylene glycol 1.0
11. Glycerin 2.0
12 Methyl paraoxybenzoate 0.2
13. Purified water remaining
成分2〜8を加熱溶解して混合し、70℃に保ち油相とする。成分1及び10〜13を加熱溶解して混合し、75℃に保ち水相とする。油相に水相を加えて乳化して、かき混ぜながら冷却し、45℃で成分9を加え、さらに30℃まで冷却して製品とする。 Ingredients 2 to 8 are dissolved by heating and mixed, and kept at 70 ° C. to obtain an oil phase. Ingredients 1 and 10-13 are dissolved by heating and mixed, and kept at 75 ° C. to form an aqueous phase. The aqueous phase is added to the oil phase to emulsify, and the mixture is cooled while stirring.
(処方例4)ゲル剤
処方 配合量(重量%)
1.アイヌワカメ亜臨界水処理物の50%エタノール抽出物(製造例8B)0.1
2.エタノール 5.0
3.パラオキシ安息香酸メチル 0.1
4.ポリオキシエチレン硬化ヒマシ油(40E.O.) 0.1
5.香料 適量
6.1,3−ブチレングリコール 5.0
7.グリセリン 5.0
8.キサンタンガム 0.1
9.カルボキシビニルポリマー 0.2
10.水酸化カリウム 0.2
11.精製水 残量
(Formulation example 4) Gel formulation Formulation amount (% by weight)
1. 50% ethanol extract of Ainu Wakame subcritical water treated product (Production Example 8B) 0.1
2. Ethanol 5.0
3. Methyl paraoxybenzoate 0.1
4). Polyoxyethylene hydrogenated castor oil (40E.O.) 0.1
5. Perfume appropriate amount 6.1,3-butylene glycol 5.0
7). Glycerin 5.0
8). Xanthan gum 0.1
9. Carboxyvinyl polymer 0.2
10. Potassium hydroxide 0.2
11. Purified water remaining
成分2〜5と、成分1及び6〜11をそれぞれ均一に溶解し、両者を混合して製品とする。 Ingredients 2 to 5 and ingredients 1 and 6 to 11 are uniformly dissolved, and both are mixed to obtain a product.
(処方例5)軟膏
処方 配合量(重量%)
1.アイヌワカメ亜臨界水処理物の50%エタノール抽出物(製造例9B) 2.0
2.ポリオキシエチレンセチルエーテル(30E.O.) 2.0
3.モノステアリン酸グリセリン 10.0
4.流動パラフィン 5.0
5.セタノール 6.0
6.パラオキシ安息香酸メチル 0.1
7.プロピレングリコール 10.0
8.精製水 残量
(Formulation Example 5) Ointment Formulation Formulation amount (% by weight)
1. 50% ethanol extract of Ainu Wakame subcritical water treated product (Production Example 9B) 2.0
2. Polyoxyethylene cetyl ether (30E.O.) 2.0
3. Glycerol monostearate 10.0
4). Liquid paraffin 5.0
5. Cetanol 6.0
6). Methyl paraoxybenzoate 0.1
7). Propylene glycol 10.0
8). Purified water remaining
成分2〜5を加熱溶解して混合し、70℃に保ち油相とする。成分1及び6〜8を加熱溶解して混合し、75℃に保ち水相とする。油相に水相を加えて乳化し、かき混ぜながら30℃まで冷却して製品とする。 Ingredients 2 to 5 are dissolved by heating and mixed, and kept at 70 ° C. to obtain an oil phase. Ingredients 1 and 6 to 8 are dissolved by heating and mixed, and kept at 75 ° C. to obtain an aqueous phase. The aqueous phase is added to the oil phase to emulsify, and the product is cooled to 30 ° C. with stirring to obtain a product.
(処方例6)パック
処方 配合量(重量%)
1.アイヌワカメ亜臨界水処理物の50%エタノール抽出物(製造例10B)0.1
2.ポリビニルアルコール 12.0
3.エタノール 5.0
4.1,3−ブチレングリコール 8.0
5.パラオキシ安息香酸メチル 0.2
6.ポリオキシエチレン硬化ヒマシ油(20E.O.) 0.5
7.クエン酸 0.1
8.クエン酸ナトリウム 0.3
9.香料 適量
10.精製水 残量
(Formulation example 6) Pack
Formulation amount (% by weight)
1. 50% ethanol extract of Ainu Wakame subcritical water treated product (Production Example 10B) 0.1
2. Polyvinyl alcohol 12.0
3. Ethanol 5.0
4.1,3-Butylene glycol 8.0
5. Methyl paraoxybenzoate 0.2
6). Polyoxyethylene hydrogenated castor oil (20 EO) 0.5
7). Citric acid 0.1
8). Sodium citrate 0.3
9. Perfume appropriate amount10. Purified water remaining
成分1〜10を均一に溶解し製品とする。 Ingredients 1 to 10 are uniformly dissolved to obtain a product.
(処方例7)錠剤
処方 配合量(重量%)
1.アイヌワカメ亜臨界水処理物のエタノール抽出物(製造例6C) 1.0
2.乾燥コーンスターチ 25.0
3.カルボキシメチルセルロースカルシウム 20.0
4.微結晶セルロース 40.0
5.ポリビニルピロリドン 7.0
6.タルク 3.0
(Formulation Example 7) Tablet Formulation Formulation amount (% by weight)
1. Ethanol extract of Ainu Wakame subcritical water treated product (Production Example 6C) 1.0
2. Dried corn starch 25.0
3. Carboxymethylcellulose calcium 20.0
4). Microcrystalline cellulose 40.0
5. Polyvinylpyrrolidone 7.0
6). Talc 3.0
成分1〜5を混合し、次いで10%の水を結合剤として加えて、押出し造粒後乾燥する。成形した顆粒に成分6を加えて混合し打錠する。1錠0.52gとする。 Components 1-5 are mixed, then 10% water is added as a binder and dried after extrusion granulation. Ingredient 6 is added to the molded granules, mixed and compressed into tablets. One tablet is 0.52 g.
(処方例8)飲料
処方 配合量(重量%)
1.アイヌワカメ亜臨界水処理物のエタノール抽出物(製造例7C) 0.1
2.ステビア 0.05
3.リンゴ酸 5.0
4.香料 0.1
5.精製水 残量
(Prescription Example 8) Beverage Formulation Blending amount (% by weight)
1. Ethanol extract of Ainu Wakame subcritical water treated product (Production Example 7C) 0.1
2. Stevia 0.05
3. Malic acid 5.0
4). Fragrance 0.1
5. Purified water remaining
成分1〜4を成分5の一部の精製水に撹拌溶解する。次いで、成分5の残りの精製水を加えて混合し、90℃に加熱して50mLのガラス瓶に充填する。 Components 1 to 4 are dissolved in a part of purified water of component 5 with stirring. The remaining purified water of Component 5 is then added and mixed, heated to 90 ° C. and filled into a 50 mL glass bottle.
本発明の活用例として、再生医療や再生美容への応用が期待される。例えば、本発明を利用することで、再生医療や再生美容に用いる未分化状態の幹細胞を簡便に効率良く調製することが可能となる。さらに、幹細胞の移植後又は組織に存在する幹細胞に対して、本発明に係るアイヌワカメ亜臨界水処理物又はその抽出物を、直接的に注入するか又は経口投与、塗布、貼付等により適用することで、該幹細胞を、未分化状態を維持させたまま増殖させることが可能である。 As an application example of the present invention, application to regenerative medicine and regenerative beauty is expected. For example, by utilizing the present invention, it becomes possible to easily and efficiently prepare undifferentiated stem cells used for regenerative medicine and regenerative beauty. Furthermore, the Ainu Wakame subcritical water-treated product or extract thereof according to the present invention is applied directly to the stem cells after transplantation of stem cells or in tissues, or applied by oral administration, application, pasting, etc. Thus, the stem cells can be grown while maintaining an undifferentiated state.
すなわち、本発明は、再生医療や再生美容における、幹細胞の調製方法及び/又は幹細胞の未分化状態維持剤若しくは増殖促進剤としての利用が可能である。
That is, the present invention can be used as a preparation method of stem cells and / or an undifferentiated state maintaining agent or growth promoting agent for regenerative medicine and regenerative beauty.
Claims (7)
Food-drinks containing the agent of Claim 1 or 2.
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JP2020094003A (en) * | 2018-12-13 | 2020-06-18 | 日本メナード化粧品株式会社 | Agent for maintaining the undifferentiated state of stem cell and agent for promoting the proliferation of stem cell |
EP3995128A4 (en) * | 2019-07-04 | 2023-08-09 | Natura Cosméticos S.A. | Method for obtaining bioactive ingredients, use of subcritical-water extraction process, bioactive ingredient, use of bioactive ingredient and cosmetic composition |
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JP2020094003A (en) * | 2018-12-13 | 2020-06-18 | 日本メナード化粧品株式会社 | Agent for maintaining the undifferentiated state of stem cell and agent for promoting the proliferation of stem cell |
JP7202610B2 (en) | 2018-12-13 | 2023-01-12 | 日本メナード化粧品株式会社 | Stem cell undifferentiated state maintenance agent and growth promoter |
EP3995128A4 (en) * | 2019-07-04 | 2023-08-09 | Natura Cosméticos S.A. | Method for obtaining bioactive ingredients, use of subcritical-water extraction process, bioactive ingredient, use of bioactive ingredient and cosmetic composition |
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