JP2018145104A - Lipid-soluble extract for promoting migration and proliferation of vascular endothelial cell and extraction method therefor, and angiogenesis promoter and wound healing agent - Google Patents
Lipid-soluble extract for promoting migration and proliferation of vascular endothelial cell and extraction method therefor, and angiogenesis promoter and wound healing agent Download PDFInfo
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Abstract
Description
本発明は、血管内皮細胞の遊走と増殖を促進する脂溶性抽出物および当該脂溶性抽出物をマイタケから抽出する方法並びに当該脂溶性抽出物および当該脂溶性抽出物を有効成分として含有してなる血管新生促進剤又は創傷治癒剤に関する。 The present invention comprises a fat-soluble extract that promotes migration and proliferation of vascular endothelial cells, a method for extracting the fat-soluble extract from maitake, and the fat-soluble extract and the fat-soluble extract as active ingredients. The present invention relates to an angiogenesis promoter or a wound healing agent.
血管内皮細胞は、血管の恒常性維持に重要な役割を果たしている。血管内皮細胞が虚血状態の組織または細胞から放出される血管新生促進因子を認識すると、血管内皮細胞は遊走および増殖をして管腔形成を誘導し血管が新生される。新生した血管により組織や細胞を維持するために必要な酸素や栄養が虚血状態の組織または細胞に供給され、炎症の修復や創傷治癒がされる。 Vascular endothelial cells play an important role in maintaining blood vessel homeostasis. When a vascular endothelial cell recognizes a pro-angiogenic factor released from an ischemic tissue or cell, the vascular endothelial cell migrates and proliferates to induce tube formation and a blood vessel is regenerated. Oxygen and nutrients necessary for maintaining tissues and cells are supplied to the ischemic tissues or cells by the newly formed blood vessels, and inflammation is repaired and wound healing is performed.
また、運動不足や高脂肪食の摂取などの生活習慣によってもたらされる動脈硬化症は、心筋梗塞、狭心症、脳梗塞、血管性認知症および末梢動脈疾患などの虚血性疾患を引き起こす原因になっているが、その治療法として血管新生促進剤が利用されることも多い。 Arteriosclerosis caused by lifestyle habits such as lack of exercise and high-fat diet causes ischemic diseases such as myocardial infarction, angina pectoris, cerebral infarction, vascular dementia and peripheral arterial disease. However, angiogenesis-promoting agents are often used as a treatment method.
本発明に関連性を有する血管新生促進剤の文献として特許文献1乃至４及び非特許文献１に開示されている。特許文献1には、クエルセチン配糖体を有効成分とする虚血後血管新生促進剤が開示されていて、特許文献２には、シクロブチルプリン誘導体を有効成分とする血管新生促進剤が開示されている。 Patent Literatures 1 to 4 and Non-Patent Literature 1 disclose angiogenesis promoting agents relevant to the present invention. Patent Document 1 discloses a post-ischemic angiogenesis promoter containing quercetin glycoside as an active ingredient, and Patent Document 2 discloses an angiogenesis promoter containing a cyclobutylpurine derivative as an active ingredient. ing.
ここで、クエルセチンは薬物代謝酵素CYP2C8の阻害作用が知られ一部の抗がん剤との併用で副作用が懸念され、シクロブチルプリン誘導体は、生体内に及ぼす影響について不明な点が多く、プリン塩基を含むため高尿酸血症の原因になる可能性が懸念されている。 Here, quercetin is known to have an inhibitory effect on the drug-metabolizing enzyme CYP2C8, and there are concerns about side effects when used in combination with some anticancer drugs. Cyclobutylpurine derivatives have many unclear points regarding their effects on the body. There is concern that it may cause hyperuricemia because it contains a base.
さらに非特許文献１には、現在行われている血管新生治療は薬剤あるいは増殖因子などの血管新生促進剤の患部への注射や遺伝子治療に限定されることが開示されている。 Further, Non-Patent Document 1 discloses that currently performed angiogenesis treatment is limited to injection of an angiogenesis-promoting agent such as a drug or a growth factor into an affected area or gene therapy.
これらは、血管新生促進の効果は優れていると考えられるが、副作用や開胸及び開頭などにより患者への精神的および肉体的負担が大きい。そのため、患者に大きな負担を追わせることなく血流を改善できるような血管新生促進剤が待望されている。 These are considered to be excellent in the effect of promoting angiogenesis, but have a great mental and physical burden on the patient due to side effects, thoracotomy, and craniotomy. Therefore, an anti-angiogenic agent that can improve blood flow without causing a large burden on the patient is desired.
一方、副作用が軽減されることや注射や遺伝子治療に代わって食生活において心筋梗塞、狭心症、脳梗塞、血管性認知症および末梢動脈疾患などの虚血性疾患の改善を促進することが期待され、日常的に摂食できる天然物中から血管新生促進作用のある物質の探索も進められている。 On the other hand, it is expected to reduce side effects and promote improvement of ischemic diseases such as myocardial infarction, angina pectoris, cerebral infarction, vascular dementia and peripheral arterial disease in the diet instead of injection and gene therapy In addition, a search for a substance having an angiogenesis-promoting action from natural products that can be eaten on a daily basis is also in progress.
特許文献３には、天然物のモズクなどから抽出されたフコステロールを主成分とする血管新生促進剤が開示され、特許文献4には、天然物の冬虫夏草からの抽出物を有効成分とした血管新生促進剤が開示されている。これらは、いずれも食経験のある天然物由来であって、副作用が低減されていると考えられる。 Patent Document 3 discloses an angiogenesis-promoting agent mainly composed of fucosterol extracted from natural products such as mozuku, and Patent Document 4 discloses blood vessels containing an extract of natural products Cordyceps as an active ingredient. Neoplasia agents are disclosed. These are all derived from natural products with food experience, and are considered to have reduced side effects.
しかしながら、モズクは季節により収穫量が変化し、安定した収穫および供給が困難であり、冬虫夏草は種類が様々であり栽培が可能な種もあるが、特許文献4にかかる冬虫夏草は人工栽培例がなく、また、他のすべての冬虫夏草に一律に血管新生促進作用があるとは限らないことや、冬虫夏草は虫に寄生して死に至らしめるため人への安全性が懸念されるところである。 However, the yield of mozuku varies depending on the season, and stable harvesting and supply are difficult. In addition, all other Cordyceps are not uniformly pro-angiogenic, and Cordyceps parasitize insects and cause death, which is a concern for human safety.
本発明が解決しようとする課題は、通年で安定的に原料を供給でき、かつ、食経験が豊かで安全性が確保されている生鮮食品のマイタケ（学名：Ｇｒｉｆｏｌａ ｆｒｏｎｄｏｓａ）から血管内皮細胞の遊走と増殖を促進する脂溶性抽出物を得る方法を提供し、当該脂溶性抽出物を得て、さらに当該脂溶性抽出物を有効成分として副作用が懸念されない血管新生促進剤又は創傷治癒剤を提供することにある。 The problem to be solved by the present invention is the migration of vascular endothelial cells from Maitake (scientific name: Grifola frontosa), a fresh food product that can supply raw materials stably throughout the year, has a rich dietary experience, and ensures safety. And a method for obtaining a fat-soluble extract that promotes growth, obtaining the fat-soluble extract, and further providing an angiogenesis-promoting agent or wound healing agent that is free from side effects with the fat-soluble extract as an active ingredient There is.
本発明者らは、上記課題を解決するために鋭意研究を重ねた結果、有機溶媒によりマイタケから一次抽出物を得て、当該一次抽出物を水と酢酸エチルとで分配することで酢酸エチル層から二次抽出物を得て、さらに当該二次抽出物をヘキサンと酢酸エチルとのグラジエント溶液で溶出し、ヘキサン：酢酸エチルが１：４の割合で溶出した画分を脂溶性抽出物として得た。 As a result of intensive studies to solve the above problems, the present inventors obtained a primary extract from maitake with an organic solvent, and distributed the primary extract between water and ethyl acetate to thereby obtain an ethyl acetate layer. A secondary extract is obtained from the mixture, and the secondary extract is eluted with a gradient solution of hexane and ethyl acetate to obtain a fraction eluted with a hexane: ethyl acetate ratio of 1: 4 as a fat-soluble extract. It was.
この方法により、得られた脂溶性抽出物は血管内皮細胞の遊走と増殖とを顕著に促進するものであって、血管新生促進剤又は創傷治癒剤の有効成分となり得る。本発明は上述のような知見に基づいて完成された。 By this method, the obtained fat-soluble extract significantly promotes the migration and proliferation of vascular endothelial cells, and can be an active ingredient of an angiogenesis promoter or a wound healing agent. The present invention has been completed based on the above findings.
本発明にかかる血管新生促進剤は、日常の食生活における食品素材であるマイタケから抽出された脂溶性抽出物を有効成分としているため、副作用が生じるおそれが無く、また、当該脂溶性抽出物は天然物質のため合成などの手間も不要である。さらに、マイタケは通年で安定して大量生産が可能なため材料の提供も安定的に行うことができる。 The angiogenesis-promoting agent according to the present invention uses a fat-soluble extract extracted from maitake, which is a food material in daily eating habits, as an active ingredient, so there is no possibility of causing side effects, and the fat-soluble extract is Since it is a natural substance, there is no need for labor such as synthesis. Furthermore, since Maitake can be mass-produced stably throughout the year, materials can be provided stably.
発明の効果をより詳述すると、請求項１の発明にかかる方法を用いれば、通年で安定して栽培が可能なマイタケから、血管内皮細胞の遊走と増殖とを顕著に促進する脂溶性抽出物を容易に得る事ができ、安定的な供給もできる。 The effect of the invention will be described in more detail. A fat-soluble extract that remarkably promotes migration and proliferation of vascular endothelial cells from maitake, which can be cultivated stably throughout the year, by using the method according to the invention of claim 1. Can be easily obtained, and a stable supply can be achieved.
請求項２にかかる発明は、マイタケから抽出された血管内皮細胞の遊走と増殖とを顕著に促進する脂溶性抽出物であって、当該脂溶性抽出物を乾燥粉末化あるいは液状化などすることで血管新生促進剤または創傷治癒剤の有効成分として利用することができる。 The invention according to claim 2 is a fat-soluble extract that remarkably promotes the migration and proliferation of vascular endothelial cells extracted from maitake, and the fat-soluble extract is made into a dry powder or liquefied. It can be used as an active ingredient of an angiogenesis promoter or wound healing agent.
請求項３にかかる発明は、マイタケから抽出された血管内皮細胞の遊走と増殖とを顕著に促進する脂溶性抽出物を有効成分として錠剤、顆粒、粉末、カプセルへの封入、内服液への添加など様々な形態にして投与をできるようにすることで、副作用が生じることの無い血管新生促進剤として利用をすることができる。 The invention according to claim 3 uses a fat-soluble extract that remarkably promotes the migration and proliferation of vascular endothelial cells extracted from maitake as an active ingredient, and is added to tablets, granules, powders, capsules, and added to internal liquids. By making it possible to administer in various forms such as, it can be used as an angiogenesis promoter without causing side effects.
請求項４にかかる発明は、マイタケから抽出された血管内皮細胞の遊走と増殖とを顕著に促進する脂溶性抽出物を有効成分として錠剤、顆粒、粉末、カプセルへの封入、内服液への添加など様々な形態にして投与をできるようにすることで、副作用が生じることの無い創傷治癒剤として利用をすることができる。 The invention according to claim 4 uses a fat-soluble extract that remarkably promotes the migration and proliferation of vascular endothelial cells extracted from maitake as an active ingredient, and is added to tablets, granules, powders, capsules, and added to internal liquids. By making it possible to administer in various forms such as, it can be used as a wound healing agent with no side effects.
本発明に係る血管新生促進剤及び創傷治癒剤は、マイタケから抽出した血管内皮細胞の遊走と増殖とを促進する脂溶性抽出物を有効成分として構成される。 The angiogenesis-promoting agent and wound healing agent according to the present invention are composed of a fat-soluble extract that promotes migration and proliferation of vascular endothelial cells extracted from maitake as an active ingredient.
マイタケは担子菌類の食用きのこであって、日本国内では、２０１５年の農林水産省の統計によれば国内で年間約４８８００トンの生産量があって、通年で栽培がされている。本発明の材料であるマイタケの入手については、タンク培養などにより菌糸体を集める事もできるが、市販されている子実体を入手する方が容易である。 Maitake mushrooms are basidiomycetous edible mushrooms. In Japan, according to statistics from the Ministry of Agriculture, Forestry and Fisheries in 2015, the annual production is about 48,800 tons and is cultivated throughout the year. Regarding the acquisition of maitake, which is a material of the present invention, mycelium can be collected by tank culture or the like, but it is easier to obtain commercially available fruiting bodies.
また、本願発明の効果を得るためには、菌糸体の培養物を利用する事もできるが、本発明の効果を十分に得るためには、マイタケの子実体からマイタケ脂溶性抽出物を抽出することが好ましい。 In order to obtain the effect of the present invention, a mycelium culture can be used. However, in order to sufficiently obtain the effect of the present invention, a maitake fat-soluble extract is extracted from the fruit body of Maitake. It is preferable.
次にマイタケからの脂溶性抽出物の抽出方法を説明する。 Next, a method for extracting a fat-soluble extract from maitake is described.
材料のマイタケの子実体又は菌糸体は、乾燥粉末化したものも使用できる。抽出収率を考慮するとマイタケを乾燥した後微粉末化する事が望ましいが、乾燥工程及び微粉末化の工程を省略すればコストを低減できるため、新鮮なマイタケを使用する事がより好ましい。 The fruit body or mycelium of the maitake material may be a dry powder. Considering the extraction yield, it is desirable to pulverize the maitake after drying, but it is more preferable to use fresh maitake because the cost can be reduced if the drying step and the pulverization step are omitted.
また、マイタケの子実体または菌糸体の乾燥は、天日干し、温風乾燥、熱風乾燥又は凍結乾燥などの方法を適用することができる。 Further, for drying the fruit bodies or mycelium of Maitake, methods such as sun-drying, hot air drying, hot air drying or freeze-drying can be applied.
次に、一次抽出について説明する。 Next, primary extraction will be described.
新鮮なマイタケまたはマイタケ乾燥微粉末は、溶媒中に浸漬し、その後当該溶媒を吸引濾過して抽出液を回収する。回収した抽出液は減圧下で溶媒を除去し、残渣を一次抽出物として得ることができる。 The fresh maitake or dried maitake powder is immersed in a solvent, and then the solvent is suction filtered to recover the extract. The collected extract is freed from the solvent under reduced pressure, and the residue can be obtained as a primary extract.
ここで、溶媒は、極性有機溶媒を用いる事ができ、詳しくは１−ブタノール、１−プロパノール、２−プロパノール、エタノール、アセトン、アセトニトリルまたはエタノールを単独または２以上の混合溶媒とすることができ、より好ましくはエタノールを使用する。さらに、水分の無い粉末状のマイタケから抽出する場合は、酢酸エチル、ヘキサン、トルエンなどの無極性有機溶媒の使用もできる。 Here, a polar organic solvent can be used as the solvent. Specifically, 1-butanol, 1-propanol, 2-propanol, ethanol, acetone, acetonitrile, or ethanol can be used alone or as a mixed solvent of two or more. More preferably ethanol is used. Furthermore, when extracting from the powdered maitake without a water | moisture content, nonpolar organic solvents, such as ethyl acetate, hexane, and toluene, can also be used.
また、浸漬期間や有機溶媒の量等は、抽出効率を考慮して適宜変更する事ができる。 Further, the immersion period, the amount of the organic solvent, and the like can be appropriately changed in consideration of the extraction efficiency.
次に二次抽出について説明する。一次抽出物は血管内皮細胞の遊走と増殖とを促進する作用をもつが、更に分画を行うことで血管内皮細胞の遊走と増殖とを促進する作用をより強力にすることができる。 Next, secondary extraction will be described. The primary extract has an action of promoting the migration and proliferation of vascular endothelial cells, but further fractionation can further enhance the action of promoting the migration and proliferation of vascular endothelial cells.
一次抽出物に蒸留水と低極性の有機溶媒から選択される１または２以上の有機溶媒とを添加して混合し、静置した後混合液が分離したら、蒸留水層を除いて有機溶媒層のみを残す。次に当該有機溶媒層を無水硫酸ナトリウムで脱水した後、減圧濃縮して二次抽出物を得ることができる。 When the primary extract is mixed with distilled water and one or more organic solvents selected from low-polarity organic solvents, mixed and separated after standing, the distilled water layer is removed and the organic solvent layer Leave only. Next, the organic solvent layer can be dehydrated with anhydrous sodium sulfate and then concentrated under reduced pressure to obtain a secondary extract.
ここで、上記低極性の有機溶媒には、塩化メチレン、酢酸エチル、クロロホルム、ジエチルエーテル、トルエン、ベンゼン、ヘキサン等があり、好ましくは酢酸エチル、クロロホルム、ヘキサンの何れかを単独または２以上の混合溶媒として用いる。より好ましくは酢酸エチルを用いる。 Here, the low-polar organic solvent includes methylene chloride, ethyl acetate, chloroform, diethyl ether, toluene, benzene, hexane, etc., and preferably any one of ethyl acetate, chloroform, hexane, or a mixture of two or more. Used as a solvent. More preferably, ethyl acetate is used.
次に脂溶性抽出物について説明する。二次抽出物は、一次抽出物よりも強い血管内皮細胞の遊走と増殖とを促進する作用を示すが、さらに純度を向上させるために分画することができる。すなわち、二次抽出物をシリカゲルに吸着させ、その後当該シリカゲル吸着物を有機溶媒で溶出させることで、血管内皮細胞の遊走と増殖とをさらに強く促進する作用を得る事ができる。 Next, the fat-soluble extract will be described. The secondary extract exhibits a stronger vascular endothelial cell migration and proliferation action than the primary extract, but can be fractionated to further improve the purity. That is, by adsorbing the secondary extract onto silica gel and then eluting the silica gel adsorbate with an organic solvent, it is possible to obtain an action that further promotes migration and proliferation of vascular endothelial cells.
すなわち、オープンカラムに充填されたシリカゲルに二次抽出物を吸着させ、ヘキサンと酢酸エチルとの混合有機溶媒を使用してグラジエント溶出法にしたがって分画操作を行い、ヘキサン：酢酸エチルが１：４の割合で溶出した分画を脂溶性抽出物として得ることができる。当該脂溶性抽出物は血管内皮細胞の遊走と増殖とをより強く促進する作用を有する。 That is, the secondary extract is adsorbed on silica gel packed in an open column, and fractionation operation is performed according to the gradient elution method using a mixed organic solvent of hexane and ethyl acetate, and hexane: ethyl acetate is 1: 4. The fraction eluted at a ratio of 1 can be obtained as a fat-soluble extract. The fat-soluble extract has an action of more strongly promoting the migration and proliferation of vascular endothelial cells.
ここで、上記シリカゲル吸着物を溶出させる有機溶媒は、無極性有機溶媒と極性有機溶媒との全ての中から選択される２以上の有機溶媒を混合して得られる有機溶媒を使用する事ができるが好ましくは、ヘキサンと酢酸エチルとを混合した有機溶媒を使用することができる。 Here, as the organic solvent for eluting the silica gel adsorbate, an organic solvent obtained by mixing two or more organic solvents selected from all of a nonpolar organic solvent and a polar organic solvent can be used. Preferably, an organic solvent in which hexane and ethyl acetate are mixed can be used.
また、オープンカラムに充填されるシリカゲルは順相カラムクロマトグラフィー充填用の各種シリカゲルが使用でき、シリカゲルの粒径は４０〜２００μｍが好ましい。 Moreover, the silica gel with which an open column is packed can use the various silica gel for normal phase column chromatography packing, and the particle size of a silica gel has preferable 40-200 micrometers.
なお、脂溶性抽出物を血管内皮細胞の細胞培養液に添加してＣＯ２インキュベーター内に所定時間放置することで、血管内皮細胞の遊走や増殖が促進された事を観察でき、さらに血管内皮細胞の管腔様形成も確認する事ができる。 In addition, it was possible to observe that the migration and proliferation of vascular endothelial cells were promoted by adding the fat-soluble extract to the cell culture solution of vascular endothelial cells and leaving it in a CO 2 incubator for a predetermined time. It is also possible to confirm the formation of a lumen.
血管内皮細胞の管腔様形成は、血管が新生されたことを示す現象である。従って、上記した方法により三次抽出された血管内皮細胞の遊走と増殖とを促進する脂溶性抽出物を、賦形剤、結合剤、崩壊剤、潤滑剤、乳化剤、着色剤、香料などに有効成分として添加して、食品に配合し、錠剤に打錠し、顆粒化し又はカプセルに内包した状態にして血管新生促進剤または創傷治癒剤とすることができる。 The lumen-like formation of vascular endothelial cells is a phenomenon indicating that a blood vessel has been renewed. Therefore, the fat-soluble extract that promotes the migration and proliferation of vascular endothelial cells tertiary-extracted by the above-described method is used as an active ingredient in excipients, binders, disintegrants, lubricants, emulsifiers, colorants, fragrances, etc. Can be added to the food, blended into foods, compressed into tablets, granulated, or encapsulated into an angiogenesis promoter or wound healing agent.
以下、実施例を挙げて本発明を更に詳細に説明する。ただし、本発明の構成は、下記実施例に記載された内容に限られるものではなく、また、実施例の記述については、特許請求の範囲を限定し、あるいは特許請求の範囲を減縮するように解すべきものでもない。そして、本発明の構成は、特許請求の範囲に記載された技術的範囲内においての種々の変形が可能である。 Hereinafter, the present invention will be described in more detail with reference to examples. However, the configuration of the present invention is not limited to the contents described in the following embodiments, and the description of the embodiments limits the scope of claims or reduces the scope of claims. It is not something to be understood. The configuration of the present invention can be variously modified within the technical scope described in the claims.
マイタケ脂溶性抽出物の抽出工程の概略を図１に示すとともに、以下に新鮮なマイタケ子実体から脂溶性抽出物を抽出する実施例を詳説する。 The outline of the extraction process of the maitake fat-soluble extract is shown in FIG. 1, and an example of extracting the fat-soluble extract from fresh maitake fruit bodies will be described in detail below.
新鮮なマイタケ子実体１２.５ｋｇを、エタノール２１Ｌ中に浸漬して３日間常温で放置した。その後当該新鮮なマイタケが浸清しているエタノール溶液を吸引濾過して抽出液を回収して１回目の抽出液を得た。当該濾過の残渣を再び、エタノール２１Ｌ中に浸清し同様に３日間放置後吸引濾過をして２回目の抽出液を得た。１回目の抽出液と２回目の抽出液とを混合し、エバポレーターを使用して当該混合した抽出液から溶媒を蒸発させ、残留物を一次抽出物として得た。 12.5 kg of fresh maitake fruit bodies were immersed in 21 L of ethanol and left at room temperature for 3 days. Thereafter, the ethanol solution in which the fresh maitake was soaked was filtered by suction to collect the extract to obtain a first extract. The filtration residue was again soaked in 21 L of ethanol and left to stand for 3 days, followed by suction filtration to obtain a second extract. The first extract and the second extract were mixed, and the solvent was evaporated from the mixed extract using an evaporator to obtain a residue as a primary extract.
次に一次抽出物を酢酸エチルと水とを１：１に混合した溶媒に添加した後、攪拌し放置した。しばらくして酢酸エチル層と水層に分かれた時に、酢酸エチル層を回収した。回収した酢酸エチル層に無水硫酸ナトリウムを加えて脱水操作を行った。その後、エバポレーターを使用して酢酸エチル層から溶媒を蒸発させ、残留物を二次抽出物として得た。 Next, the primary extract was added to a solvent in which ethyl acetate and water were mixed at a ratio of 1: 1, and then stirred and left standing. After a while, when the ethyl acetate layer and the aqueous layer were separated, the ethyl acetate layer was recovered. The collected ethyl acetate layer was dehydrated by adding anhydrous sodium sulfate. Then, the solvent was evaporated from the ethyl acetate layer using an evaporator, and the residue was obtained as a secondary extract.
次に、酢酸エチルに溶解した二次抽出物をセライトに吸着させて完全に乾燥させた後、オープンカラムに充填したシリカゲル上にのせ、酢酸エチルとｎ−ヘキサンとの濃度比率を調整した混合溶媒を用いて濃度勾配溶出を行った。そして、ｎ−ヘキサンと酢酸エチルとの混合比が１：４で溶出した画分を脂溶性抽出物とした。本実施例では、脂溶性抽出物は２６．９ｍｇ得る事ができた。
なお、本実施例で得られた一次抽出物、二次抽出物および脂溶性抽出物はそれぞれ、血管内皮細胞の遊走と増殖とを促進することを実験的に確認をした。
Next, after the secondary extract dissolved in ethyl acetate was adsorbed on celite and completely dried, it was placed on silica gel packed in an open column, and a mixed solvent in which the concentration ratio of ethyl acetate and n-hexane was adjusted. Was used to perform gradient elution. And the fraction which eluted with the mixing ratio of n-hexane and ethyl acetate 1: 4 was made into the fat-soluble extract. In this example, 26.9 mg of fat-soluble extract could be obtained.
In addition, it was experimentally confirmed that the primary extract, the secondary extract and the fat-soluble extract obtained in this example promote the migration and proliferation of vascular endothelial cells, respectively.
次に実施例1で得られた脂溶性抽出物を用いた細胞遊走試験について実施例を説明するとともに、その結果として得られた血管内皮細胞の遊走率を図２に示した。 Next, while explaining an Example about the cell migration test using the fat-soluble extract obtained in Example 1, the migration rate of the vascular endothelial cell obtained as a result was shown in FIG.
本実施例では、血管内皮細胞としてヒト臍帯静脈内皮細胞（Human Umbilical Vein Endothelial Cells：以下、ＨＵＶＥＣという。）を使用した。あらかじめ継代培養されたＨＵＶＥＣを培養培地であるHuMedia-EG２に懸濁して、ＨＵＶＥＣが２×１０４個/ ウェルになるように、RadiusTM ９６-well Cell Migration Assay （Cell Biolabs社製）に播種した。常法により２４時間培養後、脂溶性抽出物をジメチルスルホキシドに溶解して、当該培養後の培養培地に添加した。このときの脂溶性抽出物の濃度は０、１０μｇ／ｍＬになるように調整をした。また、ポジティブコントロールとして血管内皮増殖因子 (Vascular endothelial growth factor: 以下VEGF)を２０ｎｇ／ｍＬになるように加えた試験区を設けた。それぞれを添加した後１６時間経過後、RadiusTM Gelを除き、さらに８時間後、それぞれ細胞染色を行った。細胞染色を写真撮影してその画像から細胞遊走率（％）を画像処理ソフトImageJを用いて算出した。 In this example, human umbilical vein endothelial cells (hereinafter referred to as HUVEC) were used as vascular endothelial cells. HUVEC that had been subcultured in advance were suspended in HuMedia-EG2, which is a culture medium, and seeded in a Radius ™ 96-well Cell Migration Assay (manufactured by Cell Biolabs) so that HUVEC was 2 × 10 4 cells / well. . After culturing for 24 hours by a conventional method, the fat-soluble extract was dissolved in dimethyl sulfoxide and added to the culture medium after the culture. At this time, the concentration of the fat-soluble extract was adjusted to 0 or 10 μg / mL. Further, as a positive control, a test group was prepared in which vascular endothelial growth factor (hereinafter referred to as VEGF) was added to 20 ng / mL. After 16 hours from the addition of each, Radius ™ Gel was removed, and after 8 hours, cell staining was performed. The cell staining was photographed, and the cell migration rate (%) was calculated from the image using image processing software ImageJ.
本実施例の結果、ＨＵＶＥＣの細胞遊走率は図３に示したとおりであり、脂溶性抽出物はＨＵＶＥＣの遊走を活性化した。 As a result of the present example, the cell migration rate of HUVEC was as shown in FIG. 3, and the fat-soluble extract activated HUVEC migration.
次に実施例1で得られた脂溶性抽出物を用いた細胞増殖試験について実施例を説明するとともに、その結果得られた細胞増殖率を図３に示した。 Next, while explaining an Example about the cell growth test using the fat-soluble extract obtained in Example 1, the cell growth rate obtained as a result was shown in FIG.
あらかじめ継代培養されたＨＵＶＥＣを培養培地であるHuMedia-EG２に懸濁して、コラーゲンコート９６ウェルプレートにＨＵＶＥＣが１×１０４個/ ウェルになるように播種した。そして、ＨＵＶＥＣを常法により２４時間培養した後、ジメチルスルホキシドに溶解させた脂溶性抽出物を当該培養培地に添加した。このときの脂溶性抽出物の濃度はそれぞれ０、２．４、１２、６０ μｇ／ｍＬになるように調整した。当該脂溶性抽出物を添加して２４時間経過後にそれぞれに細胞増殖測定試薬を添加し、さらに３時間経過後、それぞれの吸光度を測定し、その測定値から細胞増殖率（％）を算出した。 HUVEC that had been subcultured in advance were suspended in HuMedia-EG2, which is a culture medium, and seeded on a collagen-coated 96-well plate at 1 × 10 4 HUVECs / well. And after culturing HUVEC for 24 hours by a conventional method, a fat-soluble extract dissolved in dimethyl sulfoxide was added to the culture medium. At this time, the concentration of the fat-soluble extract was adjusted to 0, 2.4, 12, and 60 μg / mL, respectively. After 24 hours have elapsed since the addition of the fat-soluble extract, a cell proliferation measurement reagent was added to each, and after another 3 hours, the absorbance was measured, and the cell proliferation rate (%) was calculated from the measured value.
本実施例の結果、ＨＵＶＥＣの細胞増殖率は図３に示したとおりとなった。脂溶性抽出物の濃度に依存してＨＵＶＥＣの細胞増殖率が上昇し、脂溶性抽出物がＨＵＶＥＣの増殖を促進させた結果が得られた。 As a result of this example, the cell proliferation rate of HUVEC was as shown in FIG. The cell growth rate of HUVEC increased depending on the concentration of the fat-soluble extract, and the result that the fat-soluble extract promoted the growth of HUVEC was obtained.
次に実施例1で得られた脂溶性抽出物を用いたＨＵＶＥＣの管腔様形成試験の実施例を説明するとともに、その結果得られた管腔様の全長について図４に示した。 Next, an example of the HUVEC lumen-like formation test using the fat-soluble extract obtained in Example 1 was described, and the resulting lumen-like full length was shown in FIG.
あらかじめ継代培養されたＨＵＶＥＣを培養培地であるHuMedia-EG２ （倉敷紡績株式会社製）に懸濁して、コラーゲンコート９６ウェルプレートに、ＨＵＶＥＣが１．５×１０４個/ ウェルになるように播種した。常法で２時間培養後、培養培地を除去し、コラーゲン溶液 （株式会社高研製）を各ウェルにそれぞれ５０μＬ加えてゲル化させた。その後、脂溶性抽出物をジメチルスルホキシドに溶解させてから上記培養培地に懸濁して、上記ゲル化させた各ウェルに添加した。このときの脂溶性抽出物の濃度は０、１０ μｇ／ｍＬになるようにそれぞれ調整した。また、ポジティブコントロールとしてVEGFを２０ｎｇ／ｍＬになるように加えた試験区も設けた。当該添加４８時間経過後、それぞれの試験区において管腔様の形成についての結果を写真撮影し、画像処理ソフトのImageJを用いて管腔様の全長（ｍｍ）を計測した。 Suspended HUVECs in advance are suspended in HuMedia-EG2 (Kurashiki Boseki Co., Ltd.), which is a culture medium, and seeded in a collagen-coated 96-well plate so that HUVECs are 1.5 × 10 4 cells / well. did. After culturing for 2 hours in a conventional manner, the culture medium was removed, and 50 μL of collagen solution (manufactured by Koken Co., Ltd.) was added to each well to cause gelation. Thereafter, the fat-soluble extract was dissolved in dimethyl sulfoxide, suspended in the culture medium, and added to each gelled well. The concentration of the fat-soluble extract at this time was adjusted to 0 and 10 μg / mL, respectively. Moreover, the test group which added VEGF so that it might become 20 ng / mL as a positive control was also provided. After 48 hours from the addition, the results of the formation of lumen-like were photographed in each test section, and the total length (mm) of the lumen-like was measured using ImageJ of image processing software.
本実施例の結果、ＨＵＶＥＣの管腔様の全長は図４に示したとおりとなった。脂溶性抽出物によってＨＵＶＥＣの管腔様が伸長し、血管新生が促進される事が示された。 As a result of the present example, the total length of the lumen-like HUVEC was as shown in FIG. It was shown that the lipid-soluble extract elongates the lumen-like shape of HUVEC and promotes angiogenesis.
本発明によれば、マイタケから得られた脂溶性抽出物は、ＨＵＶＥＣの遊走と増殖とを促進し、管腔様形成が顕著に見られた。このことにより、本発明にかかる脂溶性抽出物が血管内皮細胞の管腔様形成を促し、血管新生をさせることが明確となった。 According to the present invention, the fat-soluble extract obtained from maitake promoted the migration and proliferation of HUVEC, and a lumen-like formation was observed remarkably. Thus, it has been clarified that the fat-soluble extract according to the present invention promotes the formation of vascular endothelial cells in a lumen-like manner and causes angiogenesis.
本発明にかかる脂溶性抽出物の抽出方法は、マイタケから血管内皮細胞の遊走と増殖とを促進する脂溶性抽出物を得る事ができ、当該脂溶性抽出物は血管新生促進剤または創傷治癒剤の有効成分として使用する事が期待でき、さらに、当該脂溶性抽出物は食経験豊富な生鮮食品のマイタケから抽出した成分であるため、副作用が無く安全性を備えた医薬品や健康食品分野で利用することが可能である。 The method for extracting a fat-soluble extract according to the present invention can obtain a fat-soluble extract that promotes migration and proliferation of vascular endothelial cells from maitake, and the fat-soluble extract is an angiogenesis promoter or a wound healing agent. In addition, since the fat-soluble extract is an ingredient extracted from maitake, a fresh food with abundant experience, it has no side effects and is used in the field of pharmaceuticals and health foods with safety. Is possible.
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| JP2006232751A (en) * | 2005-02-25 | 2006-09-07 | Gunma Prefecture | Endothelial cell damage inhibitor and its use |
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