JP2007022966A - Cell death inhibitory substance and method for utilizing the same - Google Patents
Cell death inhibitory substance and method for utilizing the same Download PDFInfo
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Description
本発明はリゾホスファチジルエタノールアミンを含有することを特徴とする細胞死抑制物質にする。また、本発明の細胞死抑制物質は細胞の分化誘導作用、神経細胞の神経突起伸長作用を有し、さらに脳神経細胞の外傷性障害、代謝性要因による障害、β−アミロイド蛋白質による障害または脳虚血性障害を予防あるいは治癒する作用を有する。さらに、本発明の細胞死抑制物質を有効成分とする医薬組成物や食用組成物を調製することができる。 The present invention provides a cell death inhibitor comprising lysophosphatidylethanolamine. Further, the cell death inhibitor of the present invention has a cell differentiation-inducing action, a neuronal neurite outgrowth action, and further, a traumatic disorder of brain neurons, a disorder due to metabolic factors, a disorder due to β-amyloid protein, or a brain imagination. Has the effect of preventing or curing blood disorders. Furthermore, a pharmaceutical composition or an edible composition containing the cell death inhibitor of the present invention as an active ingredient can be prepared.
これまで、アルツハイマー病等の細胞死を引き起こす疾患の予防法として、クルクミン、コール酸、シムノール用いる技術（例えば特許文献１）、アラキドン酸や中鎖脂肪酸を用いる技術（例えば特許文献２）、ヨモギやクマザサを用いる技術（例えば特許文献３）、テアニンを用いる技術（例えば特許文献４）、L-カルニチンまたはアルカノイルを用いる技術（例えば特許文献５）、ドコサヘキサエン酸を用いる技術（例えば特許文献６，７）、イソフラボノイドを用いる技術（例えば特許文献８，９）が知られている。 Until now, as a method for preventing diseases that cause cell death such as Alzheimer's disease, techniques using curcumin, cholic acid, and simnole (for example, Patent Document 1), techniques using arachidonic acid and medium chain fatty acids (for example, Patent Document 2), mugwort, Technology using Kumazasa (for example, Patent Document 3), Technology using theanine (for example, Patent Document 4), Technology using L-carnitine or alkanoyl (for example, Patent Document 5), Technology using docosahexaenoic acid (for example, Patent Documents 6 and 7) Techniques using isoflavonoids (for example, Patent Documents 8 and 9) are known.
一方、リゾホスファチジルエタノールアミンの細胞死抑制作用に関する先行技術は知られていない。
しかし、上記のアルツハイマー病等の細胞死を引き起こす疾病の予防法は効果が不十分で、実用化が遅れている。また、リゾホスファチジルエタノールアミンを、細胞死抑制物質として利用する先行技術は知られていない。 However, the above-described methods for preventing diseases that cause cell death such as Alzheimer's disease are not effective enough, and their practical application is delayed. Moreover, the prior art which utilizes lysophosphatidylethanolamine as a cell death inhibitor is not known.
そこで、本発明は、ＮＧＦと同様な細胞死抑制作用を有し、細胞の分化誘導作用、神経細胞の神経突起伸長作用、脳神経細胞の外傷性障害、代謝性要因による障害、β−アミロイド蛋白質による障害または脳虚血性障害に対して予防あるいは治癒する作用を持つ素材を提供する。 Therefore, the present invention has a cell death inhibitory action similar to that of NGF, a cell differentiation inducing action, a neuronal neurite outgrowth action, a traumatic disorder of brain neurons, a disorder due to metabolic factors, and a β-amyloid protein Provided is a material having an effect of preventing or curing a disorder or cerebral ischemic disorder.
本発明者等は、リゾホスファチジルエタノールアミンを含む抽出物が細胞死を抑制することを見いだし、本発明を完成するに至った。すなわち、本発明は次の［１］〜［１０］である。
［１］リゾホスファチジルエタノールアミンを含有することを特徴とする細胞死抑制物質。
［２］細胞死抑制作用、細胞の分化誘導作用、神経細胞の神経突起伸長作用、さらには脳神経細胞の外傷性障害、代謝性要因による障害、β−アミロイド蛋白質による障害または脳虚血性障害を予防あるいは治癒する作用を有する［１］に記載の細胞死抑制物質。
［３］シグナル伝達系タンパク質であるＡｋｔのリン酸化を促進する作用を有する［１］〜［２］に記載の細胞死抑制物質。
［４］シグナル伝達系タンパク質であるマイトジェン活性化キナーゼのリン酸化を促進する作用を有する［１］〜［２］に記載の細胞死抑制物質。
［５］［１］〜［４］に記載の細胞死抑制物質を有効成分として配合してなる医薬用組成物。
［６］［１］〜［４］に記載の細胞死抑制物質を有効成分として配合してなる食用組成物。
The present inventors have found that an extract containing lysophosphatidylethanolamine suppresses cell death, and has completed the present invention. That is, the present invention includes the following [1] to [10].
[1] A cell death inhibitor comprising lysophosphatidylethanolamine.
[2] Cell death inhibitory action, cell differentiation inducing action, nerve cell neurite outgrowth action, further prevention of traumatic injury of brain neurons, damage due to metabolic factors, damage caused by β-amyloid protein or cerebral ischemic damage Alternatively, the cell death inhibitor according to [1], which has a healing action.
[3] The cell death inhibitor according to [1] to [2], which has an action of promoting phosphorylation of Akt, which is a signal transduction protein.
[4] The cell death inhibitor according to [1] to [2], which has an action of promoting phosphorylation of a mitogen-activated kinase that is a signal transduction protein.
[5] A pharmaceutical composition comprising the cell death inhibitor according to [1] to [4] as an active ingredient.
[6] An edible composition comprising the cell death inhibitor according to [1] to [4] as an active ingredient.
すなわち、本発明は、リゾホスファチジルエタノールアミンを含有することを特徴とする細胞死抑制物質とその利用法に関する。 That is, the present invention relates to a cell death inhibitor comprising lysophosphatidylethanolamine and a method for using the same.
本発明によれば、リゾホスファチジルエタノールアミンを含有することを特徴とする、細胞死抑制作用、細胞の分化誘導作用、神経細胞の神経突起伸長作用、さらには脳神経細胞の外傷性障害、代謝性要因による障害、β−アミロイド蛋白質による障害または脳虚血性障害を予防あるいは治癒する作用のある細胞死抑制物質を提供できる。 According to the present invention, it is characterized by containing lysophosphatidylethanolamine, cell death inhibitory action, cell differentiation inducing action, nerve cell neurite outgrowth action, brain neuron traumatic disorder, metabolic factor It is possible to provide a cell death inhibitor having an action to prevent or cure a disorder caused by amyloidosis, a disorder caused by β-amyloid protein or a brain ischemic disorder.
以下、本発明について詳細に説明する。本発明で使用するリゾホスファチジルエタノールアミン(lysophosphatidylethanolamine)は、動植物の細胞中に存在し、特に卵黄や脳細胞に多く含有されている。リゾホスファチジルエタノールアミンは、細胞膜に存在するリン脂質の一種であるホスファチジルエタノールアミンから生合成される。ホスファチジルエタノールアミンは、卵黄や大豆レシチンに含まれるリン脂質の一種で、２つの脂肪酸を分子内に含有している。生体内では、ホスファチジルエタノールアミンがリン脂質加水分解酵素であるホスホリパーゼ(Phospholipase)Ａ２作用を受けて、ｓｎ−２位置にある１つの脂肪酸が除去されることによって、リゾホスファチジルエタノールアミンに変換される。
また、動植物中にもっとも多く見いだされるホスファチジルコリンを出発物質としてホスフォリパーゼＤを用いた塩基交換反応でホスファチジルエタノールアミンとし、次にホスフォリパーゼＡ２によりリゾホスファチジルエタノールアミンを調製することができる。本発明に用いるリゾホスファチジルエタノールアミンは上記の天然物中に含まれるものやホスフォリパーゼＡ２を使用して調製したものを単独または適宜組み合わせたものである。
Hereinafter, the present invention will be described in detail. The lysophosphatidylethanolamine used in the present invention is present in animal and plant cells, and is particularly abundant in egg yolk and brain cells. Lysophosphatidylethanolamine is biosynthesized from phosphatidylethanolamine, which is a kind of phospholipid present in cell membranes. Phosphatidylethanolamine is a kind of phospholipid contained in egg yolk and soybean lecithin and contains two fatty acids in the molecule. In vivo, phosphatidylethanolamine is converted to lysophosphatidylethanolamine by receiving phospholipase A2 which is a phospholipid hydrolase and removing one fatty acid at the sn-2 position.
Furthermore, phosphatidylethanolamine can be prepared by phosphatidylethanolamine by phosphatidylethanolamine by phosphatidylcholine by a base exchange reaction using phospholipase D starting from phosphatidylcholine found most often in animals and plants. The lysophosphatidylethanolamine used for this invention is what was contained in said natural product, and what was prepared using phospholipase A2, individually or in combination.
本発明では、リゾホスファチジルエタノールアミンの含有量を上げる目的で、クロマト処理を行うことができる。また、得られた分画物は、そのまま、あるいは凍結乾燥法、スプレードライなどの方法を用いて、固体化、粉末化して用いることが出来る。 In the present invention, chromatographic treatment can be performed for the purpose of increasing the content of lysophosphatidylethanolamine. Further, the obtained fraction can be used as it is or after solidification or powderization using a method such as freeze drying or spray drying.
本発明の細胞死抑制物質の作用機作としては、リゾホスファチジルエタノールアミンが細胞膜のレセプターを活性化し、次いで細胞生存のシグナル伝達経路であるマイトジェン活性化キナーゼ経路、ＰＩ３Ｋ／Ａｋｔ経路を活性化し、ミトコンドリアからのチトクロームＣの漏洩抑制、カスパーゼの活性化抑制等を経由して最終的に細胞死を抑制する。 As a mechanism of action of the cell death inhibitor of the present invention, lysophosphatidylethanolamine activates a receptor on the cell membrane, and then activates a mitogen-activated kinase pathway, PI3K / Akt pathway, which is a signal transduction pathway of cell survival. Cell death is finally suppressed through suppression of leakage of cytochrome C from the cells, suppression of caspase activation, and the like.
本発明の細胞死抑制物質は、リゾホスファチジルエタノールアミンを０．０１から５０％、好ましくは０．１から３０％、より好ましくは１から１０％含有する。リゾホスファチジルエタノールアミンの含有量が０．０１％未満では細胞死抑制作用が認められない。また。リゾホスファチジルエタノールアミン含有量が５０％より多くしても、活性の顕著な増加は認められない。 The cell death inhibitor of the present invention contains lysophosphatidylethanolamine in an amount of 0.01 to 50%, preferably 0.1 to 30%, more preferably 1 to 10%. When the content of lysophosphatidylethanolamine is less than 0.01%, the cell death inhibitory effect is not observed. Also. Even if the lysophosphatidylethanolamine content exceeds 50%, no significant increase in activity is observed.
次に、本発明の細胞死抑制物質を配合してなる医薬用組成物および食用組成物について説明する。本発明の細胞死抑制物質を配合してなる製剤は、これをそのまま、あるいは慣用の医薬製剤担体とともに医薬用組成物となし、動物およびヒトに投与することができる。医薬用組成物の剤形としては特に制限されるものではなく、必要に応じて適宜に選択すればよいが、例えば、錠剤、カプセル剤、顆粒剤、細粒剤、散剤等の経口剤、注射剤、坐剤等の非経口剤があげられる。 Next, a pharmaceutical composition and an edible composition comprising the cell death inhibitor of the present invention will be described. A preparation comprising the cell death inhibiting substance of the present invention can be administered to animals and humans as it is or as a pharmaceutical composition together with a conventional pharmaceutical preparation carrier. The dosage form of the pharmaceutical composition is not particularly limited, and may be appropriately selected according to need. For example, tablets, capsules, granules, fine granules, powders and other oral preparations, injections And parenterals such as suppositories and suppositories.
本発明において錠剤、カプセル剤、顆粒剤、細粒剤、散剤としての経口剤は、例えば、デンプン、乳糖、白糖、マンニット、カルボキシメチルセルロース、コーンスターチ、無機塩類等を用いて常法に従って製造される。これらの製剤中の本発明の細胞死抑制物質の配合量は０．０１から５０％、好ましくは０．１から３０％、より好ましくは１から１０％含有する。細胞死抑制物質の含有量が０．０１％未満では細胞死抑制活性が認められない。また。細胞死抑制物質の含有量が５０％より多くしても、活性の顕著な増加は認められない。この種の製剤には本発明の細胞死抑制物質の他に、結合剤、崩壊剤、界面活性剤、滑沢剤、流動性促進剤、矯味剤、着色剤、香料等を適宜に使用することができる。 In the present invention, oral preparations such as tablets, capsules, granules, fine granules, and powders are produced according to a conventional method using, for example, starch, lactose, sucrose, mannitol, carboxymethylcellulose, corn starch, inorganic salts, and the like. . The compounding amount of the cell death inhibitor of the present invention in these preparations is 0.01 to 50%, preferably 0.1 to 30%, more preferably 1 to 10%. When the content of the cell death inhibitor is less than 0.01%, the cell death inhibitory activity is not observed. Also. Even if the content of the cell death inhibitor is more than 50%, no significant increase in activity is observed. In addition to the cell death inhibitor of the present invention, a binder, a disintegrant, a surfactant, a lubricant, a fluidity promoter, a corrigent, a colorant, a fragrance, and the like are appropriately used for this type of preparation. Can do.
上記の細胞死抑制物質を含有する医薬用組成物は懸濁液、エマルション剤、シロップ剤、エリキシル剤としても投与することができ、これらの各種剤形には、矯味矯臭剤、着色剤を含有させてもよい。 The above-mentioned pharmaceutical composition containing the cell death inhibitor can be administered as a suspension, emulsion, syrup, or elixir, and these various dosage forms contain a flavoring agent and a coloring agent. You may let them.
本発明の細胞死抑制物質は食用組成物としても利用可能である。すなわち、前述のようにして得られるリゾホスファチジルエタノールアミンを有効成分としてなる細胞死抑制物質は、これをそのまま液状、ゲル状あるいは固形状の食品、例えばジュース、清涼飲料、茶、スープ、豆乳、サラダ油、ドレッシング、ヨーグルト、ゼリー、プリン、ふりかけ、育児用粉乳、ケーキミックス、粉末状または液状の乳製品、パン、クッキー等に添加したり、必要に応じてデキストリン、乳糖、澱粉等の賦形剤や香料、色素等とともにペレット、錠剤、顆粒等に加工したり、またゼラチン等で被覆してカプセルに成形加工して健康食品や栄養補助食品等として利用できる。 The cell death inhibitor of the present invention can also be used as an edible composition. That is, the cell death inhibitor comprising lysophosphatidylethanolamine obtained as described above as an active ingredient is a liquid, gel or solid food such as juice, soft drink, tea, soup, soy milk, salad oil. , Dressing, yogurt, jelly, pudding, sprinkles, infant formula, cake mix, powdered or liquid dairy products, bread, cookies, etc. It can be processed into pellets, tablets, granules, etc. together with fragrances, pigments, etc., or coated with gelatin and formed into capsules for use as health foods, nutritional supplements and the like.
これらの食品類あるいは食用組成物における本発明の細胞死抑制物質の配合量は、当該食品や組成物の種類や状態等により一律に規定しがたいが、約０．０１〜５０重量％、より好ましくは０．１〜３０重量％である。配合量が０．０１重量％未満では経口摂取による所望の効果が小さく、５０重量％を超えると食品の種類によっては風味を損なったり当該食品を調製できなくなる場合がある。なお、本発明の細胞死抑制物質は、原料が食品であれば、これをそのまま食用に供してもさしつかえない。 The compounding amount of the cell death inhibitor of the present invention in these foods or edible compositions is difficult to define uniformly depending on the type and state of the food or composition, but is about 0.01 to 50% by weight, more Preferably it is 0.1 to 30 weight%. If the blending amount is less than 0.01% by weight, the desired effect by oral intake is small, and if it exceeds 50% by weight, the flavor may be impaired or the food may not be prepared depending on the type of food. In addition, if the raw material is a foodstuff, the cell death inhibitor of the present invention can be used for food as it is.
本発明の医薬用組成物および食用組成物は、細胞死を予防あるいは治癒をねらいとして利用するものであれば、それを使用する上で何ら制限を受けることなく適用される。 The pharmaceutical composition and the edible composition of the present invention can be applied without any limitation on the use of the pharmaceutical composition and edible composition as long as they are used for the purpose of preventing or curing cell death.
以下、本発明を実施例を用いて具体的に説明するが、本発明はこれに限定されるものではない。 EXAMPLES Hereinafter, although this invention is demonstrated concretely using an Example, this invention is not limited to this.
実験例１ 加水分解反応によるリゾホスファチジルエタノールアミンの製造例
大豆リン脂質（ツルーレシチン製、ホスファチジルコリン７５％、ホスファチジルエタノールアミン１４％、その他１１％）８０ｇを８０ｍＬのアセトンに溶かした。シリカゲル（Ｍｅｒｃｋ、７０−２３０ｍｅｓｈ）４５０ｇを１，０００ｍｌのアセトンに加えた後、７０ｍｍｘ７００ｍｍのガラスカラムに充填した。このカラムに、上記で溶解したリン脂質溶液を投入した後、アセトン５００ｍｌ、アセトン：エタノール混合溶液（９５：５）１０００ｍｌ、アセトン：エタノール混合溶液（９０：１０）１，５００ｍｌ、アセトン：エタノール混合溶液（８５：１５）２，０００ｍｌで溶出しながら、溶出されるリゾホスファチジルエタノールアミンを薄層クロマトグラフィーで調べた。ホスファチジルエタノールアミンが溶出される分画だけを集めて、ロータリーエバポレータで濃縮した。得られたホスファチジルエタノールアミンをＨＰＬＣ(カラム：シリカゲル６０ 野村化学製、溶離液 クロロホルム：メタノール＝９０：１０)で分析した結果、純度６０％のホスファチジルエタノールアミン（ホスファチジルコリン２２％含有）２０ｇが得られた。
Experimental Example 1 Production Example of Lysophosphatidylethanolamine by Hydrolysis Reaction 80 g of soybean phospholipid (manufactured by True Lecithin, 75% phosphatidylcholine, 14% phosphatidylethanolamine, 11% other) was dissolved in 80 mL of acetone. After adding 450 g of silica gel (Merck, 70-230 mesh) to 1,000 ml of acetone, it was packed into a 70 mm × 700 mm glass column. After the above-dissolved phospholipid solution is put into this column, 500 ml of acetone, 1000 ml of an acetone: ethanol mixed solution (95: 5), 1,500 ml of an acetone: ethanol mixed solution (90:10), an acetone: ethanol mixed solution (85:15) While eluting with 2,000 ml, the eluted lysophosphatidylethanolamine was examined by thin layer chromatography. Only fractions from which phosphatidylethanolamine was eluted were collected and concentrated on a rotary evaporator. The obtained phosphatidylethanolamine was analyzed by HPLC (column: silica gel 60, manufactured by Nomura Chemical, eluent chloroform: methanol = 90: 10). As a result, 20 g of phosphatidylethanolamine (containing 22% phosphatidylcholine) with a purity of 60% was obtained. .
得られた純度６０％のホスファチジルエタノールアミン（ホスファチジルコリン２２％含有）１５ｇを１５０ｍｌのジエルエーテルに溶かした後、ここにＣａＣｌ2が１００ｍＭ含有された酢酸ナトリウム緩衝液（ｐＨ５．６、１００ｍＭ）２０ｍＬを添加した。この溶液に５ｍｌのレシターゼ（１０，０００ＩＵ／ｍｌ、Ｎｏｖｏ
Ｎｏｒｄｉｓｋ社）を添加し、３０℃で１３時間激しく攪拌しながら反応した。溶液を静置させた後、上澄み液を除去した。すなわち、溶媒及び生成された脂肪酸を除去した。沈殿部分を常温で２００ｍＬのヘキサン：エタノール：水（１：１：０．３）の混合溶液で抽出した。下部の水層を除去し、ここに５０ｍｌのエタノールを添加して、常温でろ過した。ろ過物にさらにヘキサン：エタノール（１：１）溶液を１００ｍＬ処理し、ろ過し、乾燥した。得られた乾燥物について液体クロマトグラフィーでリゾホスファチジルエタノールアミンを定量した結果、純度は９０％、収量は５ｇであった。
15 g of the obtained 60% pure phosphatidylethanolamine (containing 22% phosphatidylcholine) was dissolved in 150 ml of diethyl ether, and 20 mL of sodium acetate buffer (pH 5.6, 100 mM) containing 100 mM CaCl 2 was added thereto. did. To this solution was added 5 ml of lecitase (10,000 IU / ml, Novo
Norddisk) was added and the reaction was carried out at 30 ° C. with vigorous stirring for 13 hours. After allowing the solution to stand, the supernatant was removed. That is, the solvent and the produced fatty acid were removed. The precipitated part was extracted at room temperature with 200 mL of a mixed solution of hexane: ethanol: water (1: 1: 0.3). The lower aqueous layer was removed, 50 ml of ethanol was added thereto, and the mixture was filtered at room temperature. The filtrate was further treated with 100 mL of a hexane: ethanol (1: 1) solution, filtered and dried. As a result of quantifying lysophosphatidylethanolamine by liquid chromatography on the obtained dried product, the purity was 90% and the yield was 5 g.
実験例２ 粗抽出物の調製例
乾燥全卵（キューピー製）１ｋｇを攪拌槽に仕込み、そこにエタノール２Ｌと水０．４Lを加え、常温で５時間撹拌した。その後、濾過により抽出液と残渣を分離した。抽出液をエバポレーターにより濃縮し、茶褐色の粗抽出物２５０ｇを得た。粗抽出物中のリゾホスファチジルエタノールアミン含量は１１％であった。
Experimental Example 2 Preparation Example of Crude Extract 1 kg of dried whole eggs (Kewpie) was placed in a stirring tank, 2 L of ethanol and 0.4 L of water were added thereto, and the mixture was stirred at room temperature for 5 hours. Thereafter, the extract and the residue were separated by filtration. The extract was concentrated by an evaporator to obtain 250 g of a brownish brown crude extract. The lysophosphatidylethanolamine content in the crude extract was 11%.
実験例３ 細胞死抑制物質の調製例
上記の粗抽出物１０ｇを、分取用高速液体クロマトグラフィー（ギルソン社製、モデル３０３）で分画した。カラムとしてデベロシル６０−１０ φ５０ｍｍ×５００ｍｍ（野村化学製）を用い、検出波長は２１０ｎｍ、溶媒はクロロホルム：メタノール＝９０：１０、溶媒流量を５０ｍL／分とした。分画に際し、５分ごとに溶離液を分取し、分取物に含まれる溶媒をロータリーエバポレーターで乾燥して、乾固した分画物を得た。画分各物中のリゾホスファチジルエタノールアミン含量を上記の方法で測定し、最も含量が高かった分画物すなわち９０％品を細胞死抑制物質とした。
Experimental Example 3 Preparation Example of Cell Death Inhibiting Substance 10 g of the above crude extract was fractionated by preparative high performance liquid chromatography (Gilson, model 303). Develosil 60-10 φ50 mm × 500 mm (manufactured by Nomura Chemical) was used as the column, the detection wavelength was 210 nm, the solvent was chloroform: methanol = 90: 10, and the solvent flow rate was 50 mL / min. In fractionation, the eluent was collected every 5 minutes, and the solvent contained in the fraction was dried by a rotary evaporator to obtain a solid fraction. The lysophosphatidylethanolamine content in each fraction was measured by the above method, and the fraction with the highest content, ie, 90% product, was used as a cell death inhibitor.
馬血清１０容量％、牛胎児血清５容量％を含むＤＭＥＭ培地（日水製薬）にＰＣ−１２細胞（岐阜薬科大学から分与）を５万個／ｍＬ濃度で培養し、２４時間後に無血清のＤＭＥＭ培地に交換した。さらに２４時間後に本発明の実験例１の細胞死抑制物質を１００または５００μｇ／ｍL添加し、４日後の生細胞の数を倒立顕微鏡下で判定した。陽性対象として、ＮＧＦβ（シグマ製）を２５ｎｇ／ｍL添加し同じ操作を行った。結果を表１に示した。同表中の記号は、培養４日後の生細胞数を表し、−：生細胞なし、＋：若干死細胞あり、＋＋および＋＋＋：死細胞なしを意味する。 PC-12 cells (distributed from Gifu Pharmaceutical University) are cultured at a concentration of 50,000 cells / mL in DMEM medium (Nissui Pharmaceutical) containing 10% horse serum and 5% fetal bovine serum, and serum free 24 hours later The DMEM medium was replaced. Further, after 24 hours, 100 or 500 μg / mL of the cell death inhibitor of Experimental Example 1 of the present invention was added, and the number of viable cells after 4 days was determined under an inverted microscope. As a positive target, 25 ng / mL of NGFβ (manufactured by Sigma) was added and the same operation was performed. The results are shown in Table 1. The symbols in the table represent the number of viable cells after 4 days of culture, and mean-: no live cells, +: some dead cells, ++ and ++: no dead cells.
既往の方法で、１８日齢のラット胎児から大脳ニューロンを採取し、馬血清１０容量％、牛胎児血清５容量％を含むＤＭＥＭ培地（日水製薬）で２４時間培養した。２４時間後に無血清のＤＭＥＭ培地に交換し、さらに２４時間後に実験例３の細胞死抑制物質を１００または５００μｇ／ｍL添加した。３０秒後に大脳ニューロンの細胞質のタンパク質をリシスバッファーで採取した。採取したタンパク質を、ＳＤＳ−ＰＡＧＥで分離後、タンパク質をナイロンメンブレンにブロットした。ブロッキング後に抗リン酸化ＡｋｔラビットＩｇＧ抗体で反応後、アビジンービオチン化ホースラディッシュペルオキシダーゼコンプレックスで処理した。ペルオキシダーゼ活性をジアミノベンジディン−Ｈ２Ｏ２を用いてリン酸化Ａｋｔを可視化することにより、Ａｋｔのリン酸を測定した。陽性対象として、ＮＧＦβ（シグマ製）を２５ｎｇ／ｍL添加し同じ操作を行った。結果を表２に示した。同表中の記号は、Ａｋｔのリン酸化について−：なし、＋：あり、＋＋および＋＋＋：顕著にありを意味する。 Cerebral neurons were collected from 18-day-old rat fetuses by a conventional method, and cultured for 24 hours in DMEM medium (Nissui Pharmaceutical) containing 10% horse serum and 5% fetal bovine serum. After 24 hours, the medium was replaced with serum-free DMEM medium, and after 24 hours, 100 or 500 μg / mL of the cell death inhibitor of Experimental Example 3 was added. After 30 seconds, cytoplasmic proteins of cerebral neurons were collected with a lysis buffer. The collected proteins were separated by SDS-PAGE, and the proteins were blotted onto a nylon membrane. After blocking, the reaction with an anti-phosphorylated Akt rabbit IgG antibody was followed by treatment with an avidin-biotinylated horseradish peroxidase complex. The phosphorylation of Akt was measured by visualizing the phosphorylated Akt with peroxidase activity using diaminobenzidine-H2O2. As a positive target, 25 ng / mL of NGFβ (manufactured by Sigma) was added and the same operation was performed. The results are shown in Table 2. The symbols in the table mean-: none, +: yes, ++ and ++: notably about phosphorylation of Akt.
96穴プレートに４×105個/ｍｌのＰＣ−１２細胞（岐阜薬科大学から分与）懸濁液を50μl、つまり２×104個/ウェルずつ播種した。37℃、５％炭酸ガス存在下で２日間培養後、培養液を除き、ＰＢＳでウェルの底に接着した細胞を洗い、試験例２の細胞死抑制物質を１００または５００μｇ／ｍL添加した無血清培地50μlを加えた。所定時間（０、24、48時間）培養した後、ＭＴＴの８ｍｇ/ｍｌ溶液を6.3μl添加し、37℃で２時間インキュベートした後リシスバッファーを50μｌ添加し、細胞を溶解した。37℃で一晩インキュベート後562nmの吸光度をマイクロプレートリーダー（アマシャム社製）を使用して測定した。
スタート時のＯＤ562−所定時間培養時のＯＤ562（染色度）を求めて、生細胞数の指標とした。結果を図１に示した。
A 96-well plate was seeded with 50 μl, that is, 2 × 10 4 cells / well, of 4 × 10 5 cells / ml of PC-12 cells (distributed from Gifu Pharmaceutical University). After culturing at 37 ° C. in the presence of 5% carbon dioxide gas for 2 days, the culture solution was removed, the cells adhering to the bottom of the well were washed with PBS, and serum-free serum supplemented with 100 or 500 μg / mL of the cell death inhibitor of Test Example 2 was added. 50 μl of medium was added. After culturing for a predetermined time (0, 24, 48 hours), 6.3 μl of an 8 mg / ml solution of MTT was added, incubated at 37 ° C. for 2 hours, and then 50 μl of lysis buffer was added to lyse the cells. After overnight incubation at 37 ° C., the absorbance at 562 nm was measured using a microplate reader (Amersham).
OD562 at the start-OD562 (staining degree) at the time of culturing for a predetermined time was determined and used as an index of the number of living cells. The results are shown in FIG.
ヨモギ抽出物（一丸ファルコス（株）製）、テアニン（太陽化学（株）製）、ドコサヘキサエン酸（日本油脂（株）製）に関して実施例１の方法で細胞死抑制作用を測定した。結果を表１に示した。 The cell death inhibitory effect was measured by the method of Example 1 for mugwort extract (Ichimaru Falcos Co., Ltd.), theanine (Taiyo Kagaku Co., Ltd.), and docosahexaenoic acid (Nippon Yushi Co., Ltd.). The results are shown in Table 1.
ヨモギ抽出物（一丸ファルコス（株）製）、テアニン（太陽化学（株）製）、ドコサヘキサエン酸（日本油脂（株）製）に関して実施例２の方法で細胞死抑制作用を測定した。結果を表２に示した。 The cell death inhibitory effect was measured by the method of Example 2 for mugwort extract (Ichimaru Falcos Co., Ltd.), theanine (Taiyo Kagaku Co., Ltd.), and docosahexaenoic acid (Nippon Yushi Co., Ltd.). The results are shown in Table 2.
ヨモギ抽出物（一丸ファルコス（株）製）、テアニン（太陽化学（株）製）、ドコサヘキサエン酸（日本油脂（株）製）に関して実施例４の方法で細胞死抑制作用を測定した。結果を図１に示した。 The cell death inhibitory effect was measured by the method of Example 4 with respect to mugwort extract (Ichimaru Falcos Co., Ltd.), theanine (Taiyo Kagaku Co., Ltd.), and docosahexaenoic acid (Nippon Yushi Co., Ltd.). The results are shown in FIG.
表１、２、図１に示されるように、本発明の細胞死抑制物質は、従来細胞死抑制作用があると言われていた、ヨモギ抽出物、テアニン、ドコサヘキサエン酸よりも格段に優れた細胞死抑制作用が認められる。また、陽性対象としたＮＧＦと同等の細胞死抑制作用を有していた。 As shown in Tables 1 and 2 and FIG. 1, the cell death inhibitor of the present invention is a cell far superior to mugwort extract, theanine, and docosahexaenoic acid, which has been conventionally said to have a cell death inhibitory effect. Death-inhibiting action is observed. Moreover, it had the cell death inhibitory effect equivalent to NGF made into the positive object.
本発明は人に対してばかりでなく、家畜等の動物にも使用することができる。 The present invention can be used not only for humans but also for animals such as livestock.
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