JP2010202574A - Production-enhancing agent for active oxygen species - Google Patents
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Description
本発明は活性酸素種産生亢進剤に関する。 The present invention relates to a reactive oxygen species production enhancer.
活性酸素種は、体内の様々な酸化還元反応に関わる。例えば、ステロイド合成カスケードの一員として働いたり、転写調節因子ＮＦ−κＢの活性化等を行なったりするなど、重要な働きを担っている（非特許文献１）。 Reactive oxygen species are involved in various redox reactions in the body. For example, it plays an important role such as working as a member of a steroid synthesis cascade or activating the transcriptional regulatory factor NF-κB (Non-patent Document 1).
ヒスチジンやヒスチジン誘導体は、元来体内に存在する物質であり、抵抗力が弱っている高齢者や小児へ適用しても安全である。ヒスチジンやヒスチジン誘導体である、ホモカルノシン、カルノシン、アンセリン等は、抗酸化能を有していることが知られており、この抗酸化能を利用してヒスチジンやヒスチジン誘導体を含有する食品、医薬品、化粧品が開発されている（特許文献１−３）。また、ヒスチジンやヒスチジン誘導体が赤血球減少を抑制することから、ヒスチジンやヒスチジン誘導体を含有した、赤血球減少を予防、改善する医薬品及び食品が開発されている（特許文献４）。 Histidine and histidine derivatives are substances originally present in the body, and are safe to apply to elderly people and children with weak resistance. Histidine and histidine derivatives, such as homocarnosine, carnosine, anserine, etc. are known to have antioxidant ability, and using this antioxidant ability, foods, pharmaceuticals containing histidine and histidine derivatives, Cosmetics have been developed (Patent Documents 1-3). In addition, since histidine and histidine derivatives inhibit red blood cell reduction, pharmaceuticals and foods containing histidine and histidine derivatives that prevent and improve red blood cell reduction have been developed (Patent Document 4).
活性酸素種の産生に関し、細胞の活性酸素種産生システムとして以下のシステムが知られている。細胞が刺激を受容すると、細胞外からカルシウムイオンが流入するため、また、小胞体からカルシウムイオンが細胞内に流出するため、細胞内のカルシウムイオン濃度が上昇する。細胞内カルシウムイオン濃度上昇は、ＰＫＣ（プロテインキナーゼＣ）を活性化する。活性化されたＰＫＣは細胞表面のＮＡＤＰＨ（ニコチンアミドアデニンジヌクレオチドリン酸）酸化酵素を活性化し、その結果、細胞外の酸素が還元され、活性酸素種の一種であるスーパーオキシドが産生される。スーパーオキシドは、過酸化水素やヒドロキシルラジカル等の各種活性酸素種へと変化する。 Regarding the production of reactive oxygen species, the following systems are known as reactive oxygen species production systems for cells. When cells receive a stimulus, calcium ions flow from the outside of the cells, and calcium ions flow from the endoplasmic reticulum into the cells, so that the intracellular calcium ion concentration increases. Increased intracellular calcium ion concentration activates PKC (protein kinase C). The activated PKC activates NADPH (nicotinamide adenine dinucleotide phosphate) oxidase on the cell surface. As a result, extracellular oxygen is reduced and superoxide, which is a kind of reactive oxygen species, is produced. Superoxide changes into various active oxygen species such as hydrogen peroxide and hydroxyl radical.
細胞が刺激に際して産生するスーパーオキシドの産生量を高めることは、体内で重要な働きをする活性酸素種の産生を亢進させるために有効である。したがって、本発明の目的は、食品や医薬品に安全に適用でき、細胞外刺激に際しての細胞のスーパーオキシド産生量を高める活性酸素種産生亢進剤を提供することにある。 Increasing the amount of superoxide produced by the cells upon stimulation is effective for enhancing the production of reactive oxygen species that play an important role in the body. Accordingly, an object of the present invention is to provide a reactive oxygen species production enhancer that can be safely applied to foods and pharmaceuticals and increases the amount of superoxide production of cells upon extracellular stimulation.
本発明者らは、ヒスチジンやヒスチジン誘導体が、刺激を与えられた細胞のスーパーオキシド産生亢進に関与していることを見出し、本発明を完成させた。 The present inventors have found that histidine and histidine derivatives are involved in enhancing superoxide production in stimulated cells, and have completed the present invention.
すなわち、本発明は、ヒスチジン又はヒスチジン誘導体を含有する、活性酸素種産生亢進剤である。このような活性酸素種産生亢進剤は、食品や医薬品に安全に適用でき、細胞外刺激に際しての細胞のスーパーオキシド産生量を高めることができる。 That is, the present invention is a reactive oxygen species production enhancer containing histidine or a histidine derivative. Such an active oxygen species production enhancer can be safely applied to foods and pharmaceuticals, and can increase the amount of superoxide produced by cells upon extracellular stimulation.
上記ヒスチジン誘導体が、カルノシン、ホモカルノシン、アンセリン、２−メチルヒスチジン、３−メチルヒスチジン及びバレニンからなる群から選ばれるヒスチジン誘導体であることが好ましい。本発明の活性酸素種産生亢進剤がヒスチジン誘導体を含む形態である場合、該ヒスチジン誘導体が上記の群から選ばれるヒスチジン誘導体であると、刺激を与えられた細胞のスーパーオキシド産生量がより高くなる。 The histidine derivative is preferably a histidine derivative selected from the group consisting of carnosine, homocarnosine, anserine, 2-methylhistidine, 3-methylhistidine, and valenine. When the active oxygen species production enhancer of the present invention is in a form containing a histidine derivative, if the histidine derivative is a histidine derivative selected from the above group, the amount of superoxide produced by the stimulated cell is higher. .
本発明のヒスチジン又はヒスチジン誘導体を含有する活性酸素種産生亢進剤は、食品や医薬品に安全に適用でき、細胞外刺激に際しての細胞のスーパーオキシド産生量を高めることができる。 The active oxygen species production enhancer containing the histidine or histidine derivative of the present invention can be safely applied to foods and pharmaceuticals, and can increase the amount of superoxide produced by cells upon extracellular stimulation.
以下、本発明の好適な実施形態について説明する。 Hereinafter, preferred embodiments of the present invention will be described.
本発明の活性酸素種産生亢進剤は、ヒスチジン又はヒスチジン誘導体を含む。該ヒスチジン誘導体としては、例えば、カルノシン、ホモカルノシン、アンセリン、２−メチルヒスチジン、３−メチルヒスチジン及びバレニンが挙げられる。本発明の活性酸素種産生亢進剤は、特に、ヒスチジン、ホモカルノシン又はカルノシンを含むことが好ましい。 The active oxygen species production enhancer of the present invention contains histidine or a histidine derivative. Examples of the histidine derivative include carnosine, homocarnosine, anserine, 2-methylhistidine, 3-methylhistidine, and valenin. The active oxygen species production enhancer of the present invention preferably contains histidine, homocarnosine or carnosine in particular.
本発明の活性酸素種産生亢進剤に含まれるヒスチジン又はヒスチジン誘導体は市販のものを用いることができる。ヒスチジン又はヒスチジン誘導体は、公知の無機又は有機の担体或いは医療用賦形剤を加えて、常法にしたがい、経口投与剤のほか、外用剤等の非経口投与剤に製剤化することができる。経口投与剤の場合、その投与形態としては、例えば錠剤、カプセル剤、顆粒剤、散剤、シロップ剤、うがい薬等が挙げられる。これらの各種製剤は、主薬に賦形剤、結合剤、崩壊剤、滑沢剤、矯味矯臭剤、溶解補助剤、懸濁剤、コーティング剤などの医薬の製剤技術分野において通常使用しうる既知の補助剤を用いて製剤化することができる。また、本発明の活性酸素種産生亢進剤を、常法にしたがい、調味料、畜肉加工品、水産加工品、農産加工品、調味食品、調理済食品、デザート類、菓子類、又は乳油製品等の食品、或いは、飼料、餌料等の形態にして提供することも可能である。本発明に用いられる、医薬担体又は賦形剤或いは食品、飼料、餌料等の各々の成分は特に限定されるものではなく、当該活性酸素種産生亢進剤の具体的用途に応じて当業者が適宜選択できる。また、活性酸素種産生亢進剤の形態も特に限定されず、具体的用途に応じて、種々の固体や半固体、液体の形態とすることができる。 As the histidine or histidine derivative contained in the active oxygen species production enhancer of the present invention, a commercially available product can be used. Histidine or a histidine derivative can be formulated into a parenteral preparation such as an external preparation in addition to an oral preparation according to a conventional method by adding a known inorganic or organic carrier or medical excipient. In the case of an oral administration agent, examples of the administration form include tablets, capsules, granules, powders, syrups, and mouthwashes. These various preparations are known as commonly used in the pharmaceutical preparation technical field such as excipients, binders, disintegrants, lubricants, flavoring agents, solubilizers, suspension agents, coating agents, etc. It can be formulated using adjuvants. In addition, the active oxygen species production enhancer of the present invention is a seasoning, processed meat product, processed fishery product, processed agricultural product, seasoned food, cooked food, dessert, confectionery, milk oil product, etc. It is also possible to provide it in the form of food, feed, feed or the like. Each component of the pharmaceutical carrier or excipient or the food, feed, feed, etc. used in the present invention is not particularly limited, and a person skilled in the art appropriately determines the active oxygen species production enhancer according to the specific use. You can choose. Further, the form of the active oxygen species production enhancer is not particularly limited, and can be various solids, semi-solids, and liquids according to specific applications.
本発明の活性酸素種産生亢進剤に含まれるヒスチジン又はヒスチジン誘導体の量は、ヒスチジン誘導体の種類、症状、年令、体重、投与方法および剤形等によって異なるが、通常、経口投与される量が成人１日当り、例えばヒスチジンでは０．１８〜６．２ｇ、ホモカルノシンでは０．４８〜１．９２ｇ、カルノシンでは０．９０４〜１．９２ｇであると、細胞外刺激に際して細胞から産生されるスーパーオキシドの量がさらに高くなり、活性酸素種産生亢進剤としての機能がさらに高くなる。 The amount of histidine or histidine derivative contained in the active oxygen species production enhancer of the present invention varies depending on the type, symptom, age, body weight, administration method and dosage form of the histidine derivative. Superoxide produced from cells during extracellular stimulation, for example, 0.18 to 6.2 g for histidine, 0.48 to 1.92 g for homocarnosine, and 0.904 to 1.92 g for carnosine per day for an adult And the function as a reactive oxygen species production enhancer is further enhanced.
本発明の活性酸素種産生亢進剤に含まれるヒスチジン又はヒスチジン誘導体は、元来体内に含まれているため、毒性については格別の問題はなく、したがって、必要であれば上記範囲よりも多量に使用してもさしつかえない。 Since the histidine or histidine derivative contained in the active oxygen species production enhancer of the present invention is originally contained in the body, there is no particular problem with respect to toxicity. Therefore, if necessary, it is used in a larger amount than the above range. It's okay.
本発明の活性酸素種産生亢進剤は、ヒトのほか、イヌ、ネコ、ウシ、ウマ、ヤギ、ヒツジといった各種の哺乳動物に対して適用することができ、また、ウサギ、ラット、マウスといった実験動物に対しても適用することができる。 The active oxygen species production enhancer of the present invention can be applied to various mammals such as dogs, cats, cows, horses, goats and sheep, as well as humans, and laboratory animals such as rabbits, rats and mice. It can also be applied to.
本発明を以下の実施例に基づいてさらに詳細に説明するが、本発明はこれらの実施例に限定されるものではない。 The present invention will be described in more detail based on the following examples, but the present invention is not limited to these examples.
以下のようにして、細胞を刺激した際の細胞内カルシウムイオン濃度の上昇及びスーパーオキシドの産生量を、ヒスチジン又はヒスチジン誘導体存在下と非存在下とで比較した。細胞を刺激した際の細胞内カルシウムイオン濃度の上昇及びスーパーオキシド産生の評価には、化学発光及び蛍光変化を同時に計測する方法（特許第３１８３８６３号明細書（特許文献６）に記載の方法）を用いた。実験に用いる純水として、水道水をミリＱ(ミリポワ社製)により処理した超純水を使用した。 As described below, the increase in intracellular calcium ion concentration and the amount of superoxide produced when cells were stimulated were compared in the presence and absence of histidine or a histidine derivative. For the evaluation of the increase in intracellular calcium ion concentration and superoxide production when cells are stimulated, a method of simultaneously measuring chemiluminescence and fluorescence change (the method described in Japanese Patent No. 3183863 (Patent Document 6)) is used. Using. As pure water used in the experiment, ultrapure water obtained by treating tap water with MilliQ (Millipois) was used.
（実施例１）
スーパーオキシドを産生させる細胞として、ヒト前骨髄球系細胞であるＨＬ−６０細胞をジメチルスルホキシド（ＤＭＳＯ、分化誘導剤）により好中球様細胞に分化させた細胞を使用した。好中球様細胞に分化誘導した細胞を回収後、ＲＨバッファー（１０ｍＭ ＨＥＰＥＳ、１５４ｍＭ ＮａＣｌ、５．６ｍＭ ＫＣｌ、ｐＨ７．４）で１回洗浄し、１０％ウシ胎児血清入りＲＰＭＩ１６４０ｍｅｄｉｕｍ又はＧＩＴ培地（日本製薬株式会社製）に懸濁した。この細胞懸濁液にカルシウム検出用指示薬として３μＭのｆｌｕｏ３−ＡＭ（１−［２−アミノ−５−（２，７−ジクロロ−６−ヒドロキシ−３−オキシ−９−ザンテニル）フェノキシ］−２−（２−アミノ−５−メチルフェノキシ）エタン−Ｎ，Ｎ，Ｎ’，Ｎ’−四酢酸、蛍光性指示薬）を加えて３７℃、５％ＣＯ２存在下で４５分間インキュベートし、細胞内にｆｌｕｏ３−ＡＭを取り込ませた。この細胞を、ＲＨバッファーで１回洗浄し、同バッファーに懸濁し、終濃度が１．０×１０５ｃｅｌｌｓ／ｍｌとなるように体積を調製した。この細胞懸濁液を測定用セル（ポリメタクリル酸メチル樹脂製、カルテル社製）に入れ、ＣａＣｌ２とスーパーオキシド検出用指示薬としてＣＬＡ（２−メチル−６−フェニル−３，７−ジヒドロイミダゾ［１，２−ａ］ピラジン−３−オン、化学発光性指示薬）をそれぞれ終濃度が１ｍＭ、１μＭとなるように加え、さらに、濃度を調製したヒスチジン又はヒスチジン誘導体の水溶液を１５μｌ添加してそれぞれ総液量を１．５ｍｌとした（以下、「測定試料液」という）。また、コントロールとして、ヒスチジン及びヒスチジン誘導体を含んでいない純水を添加した測定試料液も調製した。
Example 1
As cells for producing superoxide, HL-60 cells, which are human promyelocytic cells, were differentiated into neutrophil-like cells with dimethyl sulfoxide (DMSO, differentiation inducer). After collecting the cells induced to differentiate into neutrophil-like cells, the cells were washed once with RH buffer (10 mM HEPES, 154 mM NaCl, 5.6 mM KCl, pH 7.4), and RPMI 1640 medium containing 10% fetal bovine serum or GIT medium (Japan) Suspended in Pharmaceutical Co., Ltd. To this cell suspension, 3 μM fluo3-AM (1- [2-amino-5- (2,7-dichloro-6-hydroxy-3-oxy-9-xanthenyl) phenoxy] -2-yl as an indicator for calcium detection was used. (2-amino-5-methylphenoxy) ethane-N, N, N ′, N′-tetraacetic acid, fluorescent indicator) was added and incubated at 37 ° C. in the presence of 5% CO 2 for 45 minutes. Fluo3-AM was incorporated. The cells were washed once with RH buffer and suspended in the same buffer, and the volume was adjusted so that the final concentration was 1.0 × 10 5 cells / ml. The cell suspension for measurement cell (polymethyl methacrylate resin, manufactured by cartel Co.) was placed in, as CaCl 2 and superoxide detection indicator CLA (2-methyl-6-phenyl-3,7-dihydro-imidazo [ 1,2-a] pyrazin-3-one and a chemiluminescent indicator) are added to a final concentration of 1 mM and 1 μM, respectively, and 15 μl of an aqueous solution of histidine or a histidine derivative whose concentration is adjusted is added to each of the total concentrations. The liquid volume was 1.5 ml (hereinafter referred to as “measurement sample liquid”). As a control, a measurement sample solution to which pure water not containing histidine and histidine derivatives was added was also prepared.
用いたヒスチジン及びヒスチジン誘導体の水溶液は以下の通りである：終濃度が０．３、１、３、又は１０ｍＭとなるように調製したヒスチジン水溶液、或いは終濃度が０．００１、０．０１、０．１、又は１ｍＭとなるように調製したカルノシン水溶液。 The aqueous solutions of histidine and histidine derivatives used are as follows: an aqueous histidine solution prepared to have a final concentration of 0.3, 1, 3, or 10 mM, or a final concentration of 0.001, 0.01, 0. Carnosine aqueous solution prepared to be 1 or 1 mM.
化学発光及び蛍光の同時測定装置（特許文献６に記載の装置）の測定試料用セルホルダーに測定試料液を入れ、攪拌しながら３７℃で４分間インキュベートした。６２．５ｍｓｅｃ間隔（５００ｍｓｅｃの８分の１）でＬＥＤ（５００ｎｍ）を点滅させ、励起光としてセルホルダー内の測定試料液への照射を開始した。 A measurement sample solution was placed in a cell holder for a measurement sample of a simultaneous chemiluminescence and fluorescence measurement apparatus (the apparatus described in Patent Document 6), and incubated at 37 ° C. for 4 minutes with stirring. The LED (500 nm) was blinked at an interval of 62.5 msec (1/8 of 500 msec), and irradiation of the measurement sample solution in the cell holder was started as excitation light.
照射開始から２．５分後に、刺激誘導剤として１００μｍｏｌ／ｌのｆ−ＭＬＰ（好中球遊走性ペプチド、ホルミルメチオニルロイシルフェニルアラニン）１５μｌを測定試料液にサンプルディスペンサーより添加して細胞を刺激し、Ｆｌｕｏ−３の蛍光強度及びＣＬＡの化学発光強度の変化から、細胞内カルシウムイオン濃度及び細胞外に産生されたスーパーオキシド量の変化を同時にモニターした。結果を、図１及び６に示す。 2.5 minutes after the start of irradiation, 15 μl of 100 μmol / l f-MLP (neutrophil migratory peptide, formylmethionyl leucylphenylalanine) as a stimulation inducer was added to the measurement sample solution from the sample dispenser to stimulate the cells. Then, from the change in the fluorescence intensity of Fluo-3 and the chemiluminescence intensity of CLA, changes in the intracellular calcium ion concentration and the amount of superoxide produced extracellularly were simultaneously monitored. The results are shown in FIGS.
（実施例２）
以下のヒスチジン及びヒスチジン誘導体の水溶液を用いた：終濃度が０．３、１、３、又は１０ｍＭとなるように調製したヒスチジン水溶液、終濃度が０．５、１、又は２ｍＭとなるように調製したホモカルノシン水溶液、或いは終濃度が０．００１、０．０１、０．１、又は１ｍＭとなるように調製したカルノシン水溶液。
(Example 2)
The following aqueous solutions of histidine and histidine derivatives were used: histidine aqueous solutions prepared to a final concentration of 0.3, 1, 3, or 10 mM, prepared to a final concentration of 0.5, 1, or 2 mM. A homocarnosine aqueous solution, or a carnosine aqueous solution prepared so that the final concentration is 0.001, 0.01, 0.1, or 1 mM.
実施例１と同様にして、測定試料液の総液量を１．５ｍｌに調製した。また、コントロールとして、ヒスチジン及びヒスチジン誘導体を含んでいない測定試料液を用意した。測定試料液に刺激誘導剤として１０μｍｏｌ／ｌのＰＭＡ（ＰＫＣ活性化剤、ホルボールミリステートアセテート）１５μｌを測定試料液にサンプルディスペンサーより添加して細胞を刺激した。ＣＬＡの化学発光強度の変化から細胞外に産生されたスーパーオキシド量の変化をモニターした。なお、ＰＭＡ刺激の場合、ＰＭＡにより活性化されたＰＫＣが細胞表面のＮＡＤＰＨ酸化酵素を活性化し、その結果、細胞外の酸素が還元されスーパーオキシドが産生されるので、細胞内カルシウム濃度によらず、スーパーオキシド産生が生じる。したがって、ＰＭＡ刺激の場合、細胞内カルシウムイオン濃度は変化しないので、細胞内カルシウムイオン濃度は測定しなかった。結果を図２、４及び７に示す。 In the same manner as in Example 1, the total amount of the measurement sample solution was adjusted to 1.5 ml. Moreover, the measurement sample liquid which does not contain histidine and a histidine derivative was prepared as control. Cells were stimulated by adding 15 μl of 10 μmol / l PMA (PKC activator, phorbol myristate acetate) as a stimulation inducer to the measurement sample solution from the sample dispenser. Changes in the amount of superoxide produced extracellularly were monitored from changes in the chemiluminescence intensity of CLA. In the case of PMA stimulation, PKC activated by PMA activates NADPH oxidase on the cell surface, and as a result, extracellular oxygen is reduced and superoxide is produced. Superoxide production occurs. Therefore, in the case of PMA stimulation, since the intracellular calcium ion concentration did not change, the intracellular calcium ion concentration was not measured. The results are shown in FIGS.
（実施例３）
以下のヒスチジン及びヒスチジン誘導体の水溶液を用いた：終濃度が０．３、１、３、又は１０ｍＭとなるように調製したヒスチジン水溶液、或いは終濃度が０．００１、０．０１、０．１、又は１ｍＭとなるように調製したカルノシン水溶液。
(Example 3)
The following aqueous solutions of histidine and histidine derivatives were used: aqueous histidine solutions prepared to a final concentration of 0.3, 1, 3, or 10 mM, or final concentrations of 0.001, 0.01, 0.1, Alternatively, a carnosine aqueous solution prepared to 1 mM.
実施例１と同様にして、測定試料液の総液量を１．５ｍｌに調製した。また、コントロールとして、ヒスチジン及びヒスチジン誘導体を含んでいない測定試料液を用意した。測定試料液に刺激誘導剤として１０μｍｏｌ／ｌのカルシウムイオノフォアＡ２３１８７（細胞膜に直接作用し、カルシウムイオンの細胞膜における透過性を亢進して、細胞内にカルシウムイオンを取り込ませる作用を持つ刺激誘導剤）１５μｌを測定試料液にサンプルディスペンサーより添加して細胞を刺激した。実施例１と同様にＦｌｕｏ−３の蛍光強度及びＣＬＡの化学発光強度の変化から、細胞内カルシウムイオン濃度及び細胞外に産生されたスーパーオキシド量の変化を同時にモニターした。結果を図３及び８に示す。 In the same manner as in Example 1, the total amount of the measurement sample solution was adjusted to 1.5 ml. Moreover, the measurement sample liquid which does not contain histidine and a histidine derivative was prepared as control. 15 μl of 10 μmol / l calcium ionophore A23187 (stimulus inducer that acts directly on the cell membrane, enhances the permeability of calcium ions through the cell membrane, and incorporates calcium ions into the cells) as a stimulation inducer in the measurement sample solution Was added to the measurement sample solution from a sample dispenser to stimulate the cells. As in Example 1, changes in the intracellular calcium ion concentration and the amount of superoxide produced extracellularly were simultaneously monitored from changes in the fluorescence intensity of Fluo-3 and the chemiluminescence intensity of CLA. The results are shown in FIGS.
（実施例４）
ヒスチジン誘導体としてホモカルノシンを用いた。ホモカルノシン水溶液の濃度は、終濃度が０．５、１、又は２ｍＭとなるように調製した。実施例１と同様にして、測定試料液の総液量を１．５ｍｌに調製した。また、コントロールとして、ホモカルノシンを含んでいない測定試料液を用意した。測定試料液に終濃度５ｍＭとなるようにＥＧＴＡ（グリコールエーテルジアミン四酢酸、キレート剤）を添加して、細胞外のカルシウムイオンをキレートさせた。実施例１と同様にしてｆ−ＭＬＰ刺激を行った。実施例１と同様にＦｌｕｏ−３の蛍光強度及びＣＬＡの化学発光強度の変化から、細胞内カルシウムイオン濃度及び細胞外に産生されたスーパーオキシド量の変化を同時にモニターした。結果を図５に示す。
Example 4
Homocarnosine was used as the histidine derivative. The concentration of the homocarnosine aqueous solution was adjusted so that the final concentration was 0.5, 1, or 2 mM. In the same manner as in Example 1, the total amount of the measurement sample solution was adjusted to 1.5 ml. As a control, a measurement sample solution containing no homocarnosine was prepared. EGTA (glycol ether diamine tetraacetic acid, chelating agent) was added to the measurement sample solution to a final concentration of 5 mM to chelate extracellular calcium ions. In the same manner as in Example 1, f-MLP stimulation was performed. As in Example 1, changes in the intracellular calcium ion concentration and the amount of superoxide produced extracellularly were simultaneously monitored from changes in the fluorescence intensity of Fluo-3 and the chemiluminescence intensity of CLA. The results are shown in FIG.
図１〜３は、それぞれｆ−ＭＬＰ、ＰＭＡ又はカルシウムイオノフォアにて刺激した場合の、細胞内カルシウムイオン濃度の上昇及びスーパーオキシド産生量のコントロール（ヒスチジン非含有測定試料液）に対する割合と測定試料液中ヒスチジン濃度との関係を示す。縦軸には、コントロールの測定試料液の化学発光及び蛍光のピーク面積を１とし、ヒスチジンを含有するそれぞれの測定試料液の化学発光及び蛍光のピーク面積の、コントロールのピーク面積に対する割合を表示した。横軸にはヒスチジン濃度を対数目盛りで表示した。 FIGS. 1 to 3 show the ratio of the increase in intracellular calcium ion concentration and superoxide production control (histidine-free measurement sample solution) and the measurement sample solution when stimulated with f-MLP, PMA or calcium ionophore, respectively. The relationship with a medium histidine density | concentration is shown. On the vertical axis, the chemiluminescence and fluorescence peak areas of the control measurement sample solution are set to 1, and the ratio of the chemiluminescence and fluorescence peak areas of each measurement sample solution containing histidine to the control peak area is displayed. . The horizontal axis represents the histidine concentration on a logarithmic scale.
図４は、ＰＭＡで刺激した場合の、スーパーオキシド産生量のコントロール（ホモカルノシン非含有測定試料液）に対する割合と測定試料液中ホモカルノシン濃度との関係を示す。図５は、ＥＧＴＡで細胞外のカルシウムイオンをキレートし、ｆ−ＭＬＰで刺激した場合の、細胞内カルシウムイオン濃度の上昇及びスーパーオキシド産生量のコントロール（ホモカルノシン非含有測定試料液）に対する割合と測定試料液中ホモカルノシン濃度との関係を示す。縦軸には、コントロールの測定試料液の化学発光及び蛍光のピーク面積を１とし、ホモカルノシンを含有するそれぞれの測定試料液の化学発光及び蛍光のピーク面積の、コントロールのピーク面積に対する割合を表示した。横軸にはホモカルノシン濃度を対数目盛りで表示した。 FIG. 4 shows the relationship between the ratio of the superoxide production amount to the control (the measurement sample solution not containing homocarnosine) and the concentration of homocarnosine in the measurement sample solution when stimulated with PMA. FIG. 5 shows the ratio of the increase in intracellular calcium ion concentration and superoxide production amount (control sample solution containing no homocarnosine) when chelating extracellular calcium ions with EGTA and stimulating with f-MLP. The relationship with the homocarnosine density | concentration in a measurement sample liquid is shown. On the vertical axis, the chemiluminescence and fluorescence peak areas of the control measurement sample solution are set to 1, and the ratio of the chemiluminescence and fluorescence peak areas of each measurement sample solution containing homocarnosine to the control peak area is displayed. did. The horizontal axis represents the homocarnosine concentration on a logarithmic scale.
図６〜８は、それぞれｆ−ＭＬＰ、ＰＭＡ又はカルシウムイオノフォアで刺激した場合の、細胞内カルシウムイオン濃度の上昇及びスーパーオキシド産生量のコントロール（カルノシン非含有測定試料液）に対する割合と測定試料液中カルノシン濃度との関係を示す。縦軸には、コントロールの測定試料液の化学発光及び蛍光のピーク面積を１とし、カルノシンを含有するそれぞれの測定試料液の化学発光及び蛍光のピーク面積の、コントロールのピーク面積に対する割合を表示した。横軸にはカルノシン濃度を対数目盛りで表示した。 FIGS. 6 to 8 show the ratio of the increase in intracellular calcium ion concentration and control of superoxide production (measured sample solution containing no carnosine) and the measured sample solution when stimulated with f-MLP, PMA or calcium ionophore, respectively. The relationship with the carnosine concentration is shown. On the vertical axis, the peak area of chemiluminescence and fluorescence of the control measurement sample solution is 1, and the ratio of the peak area of chemiluminescence and fluorescence of each measurement sample solution containing carnosine to the control peak area is displayed. . The horizontal axis represents the carnosine concentration on a logarithmic scale.
ヒスチジンが測定試料液に存在した場合、いずれの刺激によっても、ヒスチジン非存在下のコントロールよりも細胞から放出されるスーパーオキシド産生量が高くなる傾向が観察された。また、ホモカルノシンが測定試料液に存在した場合も、ホモカルノシン非存在下のコントロールよりもスーパーオキシド産生量が高く、ホモカルノシン濃度依存的にスーパーオキシド産生量が高くなった。特に細胞外のカルシウムをキレートさせてｆ−ＭＬＰで刺激した場合、濃度依存的な産生量の上昇が顕著であった。また、カルノシンが測定試料液に存在した場合も、いずれの刺激によっても、カルノシン非存在下のコントロールよりもスーパーオキシド産生量が高くなる傾向が観察された。 When histidine was present in the measurement sample solution, a tendency was observed that the amount of superoxide released from the cells was higher than that of the control in the absence of histidine by any stimulation. In addition, when homocarnosine was present in the measurement sample solution, the amount of superoxide produced was higher than that of the control in the absence of homocarnosine, and the amount of superoxide produced was increased depending on the homocarnosine concentration. In particular, when extracellular calcium was chelated and stimulated with f-MLP, the concentration-dependent increase in production was significant. In addition, when carnosine was present in the measurement sample solution, a tendency was observed that the amount of superoxide produced was higher than that of the control in the absence of carnosine by any stimulus.
本発明のヒスチジン又はヒスチジン誘導体を含有する活性酸素種産生亢進剤は、細胞の活性酸素種産生を亢進する製剤として医薬品や食品に利用できる。また、本発明の活性酸素種産生亢進剤は、副作用の心配が少なく、抵抗力が弱っている高齢者や小児へも利用しやすいと思われる。 The active oxygen species production enhancer containing the histidine or histidine derivative of the present invention can be used in pharmaceuticals and foods as a preparation for enhancing the production of reactive oxygen species in cells. In addition, the active oxygen species production enhancer of the present invention is less likely to cause side effects and is likely to be used by elderly people and children who have weak resistance.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2000143696A (en) * | 1998-09-03 | 2000-05-26 | Eisai Co Ltd | Peptide having physiological activity |
| JP2001050953A (en) * | 1999-08-09 | 2001-02-23 | Natl Food Res Inst | Method for measuring produced amount of leukocyte active oxygen and oxidation stress |
| JP2005247700A (en) * | 2004-03-01 | 2005-09-15 | Pias Arise Kk | Composition for inhibiting active oxygen, and skin care preparation and cosmetic compounded with the composition |
| JP2006265161A (en) * | 2005-03-23 | 2006-10-05 | Japan Research & Development Association For New Functional Foods | Antioxidant composition |
| JP2006348035A (en) * | 2006-07-06 | 2006-12-28 | Kose Corp | Preparation suitable for external application |
-
2009
- 2009-03-03 JP JP2009049333A patent/JP2010202574A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2000143696A (en) * | 1998-09-03 | 2000-05-26 | Eisai Co Ltd | Peptide having physiological activity |
| JP2001050953A (en) * | 1999-08-09 | 2001-02-23 | Natl Food Res Inst | Method for measuring produced amount of leukocyte active oxygen and oxidation stress |
| JP2005247700A (en) * | 2004-03-01 | 2005-09-15 | Pias Arise Kk | Composition for inhibiting active oxygen, and skin care preparation and cosmetic compounded with the composition |
| JP2006265161A (en) * | 2005-03-23 | 2006-10-05 | Japan Research & Development Association For New Functional Foods | Antioxidant composition |
| JP2006348035A (en) * | 2006-07-06 | 2006-12-28 | Kose Corp | Preparation suitable for external application |
Non-Patent Citations (3)
| Title |
|---|
| JPN6013030870; Ｊ．Ｂｉｏｌ．Ｃｈｅｍ． Ｖｏｌ．２６５, 1990, ｐ．６６９３-６６９９ * |
| JPN6013030873; 臨床病理 Ｖｏｌ．５６, 2008, ｐ．９６７-９７２ * |
| JPN6013030875; Ｍｕｔａｔｉｏｎ Ｒｅｓｅａｒｃｈ Ｖｏｌ．２２８, 1990, ｐ．２２１-２２８ * |
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