JP4123565B2 - Evaluation method of antipsychotic fatigue active substance - Google Patents
Evaluation method of antipsychotic fatigue active substance Download PDFInfo
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- JP4123565B2 JP4123565B2 JP11726698A JP11726698A JP4123565B2 JP 4123565 B2 JP4123565 B2 JP 4123565B2 JP 11726698 A JP11726698 A JP 11726698A JP 11726698 A JP11726698 A JP 11726698A JP 4123565 B2 JP4123565 B2 JP 4123565B2
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- fatigue
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Description
【０００１】
【産業上の利用分野】
本発明は、簡便でかつ効率の良い、被験物質の抗精神疲労活性または精神疲労改善活性の評価方法に関する。
【０００２】
【従来の技術】
一般に、生体は肉体的運動によって局部的な筋肉疲労もしくは全身的な疲労を知覚するが、いわゆるストレスが生体に負荷された場合、肉体的運動を伴わなくても疲労を知覚することが経験的に認められる。このようなストレス負荷に起因して知覚される疲労は、一般に精神疲労と呼ばれる。
【０００３】
即ち、精神疲労とは、肉体的運動時に知覚される疲労とは異なり、生体が異常環境下に置かれたり、精神的に抑圧されまたは刺激されたりする際に知覚する疲労を意味するものである。異常環境としては、不快な高温又は低温などの温度異常、大音量や不快な騒音、極度の明暗、身体の痛み、身体の拘束等が、また精神的な抑圧または刺激には不安、興奮、緊張の持続等が挙げられる。
【０００４】
最近では、このような精神疲労の蓄積を、種々の生理学的要因を測ることにより客観的に認識する方法が開発されてきている。
【０００５】
例えば、筋肉中の乳酸、３−メチルヒスチジン及びコルチコステロンの濃度を測る方法や、筋肉中のリン酸化合物の動態を測る方法、さらには、味覚の変化に着目した疲労度の測定方法などがある（特開平９−７７６８８号公報）。
【０００６】
また、精神疲労の予防あるいは解消に利用し得るものとして、動植物由来の生薬、ビタミン類、アミノ酸類等が、飲食品や医薬品として使用されまたは提唱されている。これら精神疲労の予防に利用し得るもの（これを抗精神疲労活性物質とする）、あるいは精神疲労の解消に利用し得るもの（これを精神疲労改善物質とする）の探索は、現在もなお広く行われている。
【０００７】
【発明が解決しようとする課題】
しかしながら、上記の公知の精神疲労の測定方法は、いずれも手段が煩雑であったり技術の熟練が要求されることが多く、かつ測定で得られたデータの解釈も難しいものであった。そのため、多数の被験物質を短時間で処理する必要がある抗精神疲労活性あるいは精神疲労改善活性の評価、ひいては抗精神疲労活性物質あるいは精神疲労改善活性物質の探索には、満足できるものではなかった。また、従来の方法では、精神疲労の蓄積度合いを定量的に表現することについても、十分なものではなかった。
【０００８】
【課題を解決するための手段】
本発明者らは、より簡便でかつ定量性に優れる精神疲労に関する測定方法の開発を試みた。その過程において、生体にストレスが負荷されると、アミノ酸の一種であるアラニンの血液中の濃度が有意に変化することを見出し、かかる知見をもとに本発明を完成した。 すなわち本発明は、被験物質を投与した非ヒト動物にストレスを負荷することにより生ずる、該動物の血液中のアラニン濃度の変化を測定することを特徴とした、被験物質の抗精神疲労活性の評価方法である。
【０００９】
またもう一つの本発明は、ストレスを負荷した後の非ヒト動物に被験試料を投与することにより生ずる、該動物の血液中のアラニン濃度の変化を測定することを特徴とした、被験物質の精神疲労改善活性の評価方法である。
【００１０】
本発明者らは、マウスをモデル動物として、生体に精神疲労をもたらすストレスを持続的に負荷させる過程で、血液中に含まれるアミノ酸濃度にどのような変化が現れるかを調べた。その結果、血液中のアラニン濃度はストレス負荷直後において有意に低下し、さらにストレスを持続的に負荷させると血液中のアラニン濃度は徐々に減少していくことが明らかとなった。
【００１１】
このことは、ストレスが負荷されたモデル動物の血液中でのアラニン濃度を測定することにより、生体にストレスが負荷されたか否かまたは精神疲労がどの程度蓄積されているかを、客観的に調べることができることを意味する。
【００１２】
即ち、モデル動物の平常時の血液中に含まれるアラニン濃度を予めまたは同時に測定し、かかる平常時のアラニン濃度に比べてどの程度アラニン濃度が減じているかを確認することで、精神疲労の発生とその蓄積を調べることができる。
【００１３】
本発明は、上述の血液中のアラニン濃度の測定を利用したものである。本発明によれば、モデル動物に対して被験試料を投与した後にストレスを負荷し、その際のモデル動物の血液中のアラニン濃度の減少を測定することで、被験試料の有する抗精神疲労活性を評価し、または抗精神疲労活性物質を探索することができる。また、本発明によれば、モデル動物にストレスを負荷した後に被験試料を投与して、ストレスにより生じたアラニン濃度の減少が回復するか否かを調べることにより、被験物質の精神疲労改善活性を評価し、または精神疲労改善物質の探索を行うことができる。
【００１４】
【発明の実施の形態】
本発明で使用することができる非ヒト動物としては、哺乳類動物であれば特に制限はないが、特に実験の利便性から動物実験に汎用されるウイスター（wister）系ラット、ＳＤ（Sprague-Dawley）系ラット、ｄｄＹ系マウス、ＩＣＲ系マウスが特に好ましい。
【００１５】
本発明において非ヒト動物に対してストレスを負荷する方法としては、非ヒト動物の身体を拘束して水に浸す方法（拘束水浸ストレス）や、コミュニケーションボックス、宙づり、拘束、条件付けストレス等が挙げられる。
【００１６】
本発明における血液中のアラニンの濃度の測定は、生体から採取された血液を遠心分離して調製される血清を測定試料とすることが好ましい。測定は、この試料をもとに高速液体クロマトグラフ（ＨＰＬＣ）を用いて行うことができる。その際使用できるカラムは、アミノ酸の検出に使用できるカラムであればよく、例えば資生堂社製カプセルパックＵＧ１２０や、エイコム社製パックドカラムＭＡ−５０ＤＳ等が挙げられる。また、同様の測定は市販されているアミノ酸自動分析機を用いて行うこともできる。この測定方法は使用する機器の取扱説明書に従って処理すればよい。
【００１７】
その他、本発明においてはアミノ酸濃度を検出、測定できる方法であれば、その測定方法には特別な制限はない。
【００１８】
【発明の効果】
本発明により、再現性がよく、しかも、簡便な精神疲労の蓄積予防、解消または低減作用を有する抗精神疲労活性物質または精神疲労改善物質の評価、検出に使用できる。
【００１９】
【実施例】
以下、実施例を挙げて本発明をさらに詳細に説明する。
【００２０】
＜実施例＞
ウイスター系ラット（雄、６週齢）１４匹を用い、試験食として２０％カゼイン食を７日間投与して飼育した。その後ラットを２群に分け、一方の群にのみラットの体を網で縛り２６℃の水浴に１日当たり７時間浸水させる拘束水浸ストレスを負荷し、これ以外は両群のラットを同一の条件下でさらに７日間飼育して、拘束水浸ストレスを負荷する前、および負荷開始後１日後、３日後、７日後に各群のラットから血液を採取した。
【００２１】
採取した血液中のアミノ酸濃度は以下の操作により測定した。採取した血液から遠心分離操作（１，８００×ｇ、１０分間）で調製した血清に、３％スルホサリチル酸を血清と同量添加し、４℃で２４時間放置後、さらに遠心分離（１１，６００×ｇ、１０分間）し、得られた上澄を分取した。これを、０．４５μｍのメンブランフィルターで濾過し、濾液を日立製作所（株）製全自動アミノ酸自動分析機（Ｌ−８５００）を用いてアラニンの濃度を測定した。その結果を図１に示す。
【００２２】
拘束水浸ストレスを負荷しないラットでは、血液中のアラニン濃度に殆ど変化が認められない一方、拘束水浸ストレスを負荷した被験ラットでは、ストレス負荷開始直後から血液中のアラニン濃度は有意に低下し、このストレス負荷の回数に反比例して血液中のアラニン濃度が減少していくことが確認できた。
【図面の簡単な説明】
【図１】は、拘束水浸ストレスを負荷したラットと、同ストレスを負荷しないラットのそれぞれにおける、血液中のアラニン濃度の変化を示す。[0001]
[Industrial application fields]
The present invention relates to a simple and efficient method for evaluating antipsychotic fatigue activity or mental fatigue ameliorating activity of a test substance.
[0002]
[Prior art]
In general, living bodies perceive local muscle fatigue or general fatigue due to physical exercise, but when so-called stress is applied to a living body, it is empirically found that perceives fatigue without physical exercise. Is recognized. Fatigue perceived due to such a stress load is generally called mental fatigue.
[0003]
In other words, mental fatigue means fatigue perceived when a living body is placed in an abnormal environment, mentally suppressed or stimulated, unlike fatigue perceived during physical exercise. . The abnormal environment includes uncomfortable temperature abnormalities such as high or low temperature, loud sound and unpleasant noise, extreme light and darkness, physical pain, physical restraint, etc., and mental depression or anxiety, tension For example.
[0004]
Recently, a method for objectively recognizing such accumulation of mental fatigue by measuring various physiological factors has been developed.
[0005]
For example, there are a method for measuring the concentration of lactic acid, 3-methylhistidine and corticosterone in the muscle, a method for measuring the kinetics of the phosphate compound in the muscle, and a method for measuring the degree of fatigue focusing on changes in taste. (Japanese Patent Laid-Open No. 9-77688).
[0006]
In addition, animal and plant-derived herbal medicines, vitamins, amino acids, and the like have been used or proposed as foods and drinks and medicines that can be used to prevent or eliminate mental fatigue. The search for those that can be used to prevent mental fatigue (this is an anti-mental fatigue active substance) or that can be used to eliminate mental fatigue (this is a substance that improves mental fatigue) is still widespread. Has been done.
[0007]
[Problems to be solved by the invention]
However, all of the above-mentioned known methods for measuring mental fatigue are complicated and often require skill, and it is difficult to interpret the data obtained by the measurement. Therefore, the evaluation of antipsychotic fatigue activity or mental fatigue ameliorating activity that requires treatment of a large number of test substances in a short time, and consequently the search for antipsychotic fatigue active substance or mental fatigue ameliorating active substance, was not satisfactory. . Further, the conventional method is not sufficient for quantitatively expressing the degree of accumulation of mental fatigue.
[0008]
[Means for Solving the Problems]
The present inventors have attempted to develop a measurement method related to mental fatigue that is simpler and more excellent in quantitativeness. In the process, when stress is applied to the living body, the concentration of alanine, which is a kind of amino acid, in blood is significantly changed, and the present invention has been completed based on such knowledge. That is, the present invention provides an evaluation of antipsychotic fatigue activity of a test substance, characterized by measuring a change in alanine concentration in the blood of the animal caused by stressing a non-human animal administered with the test substance. Is the method.
[0009]
Another aspect of the present invention relates to the spirit of a test substance characterized by measuring a change in alanine concentration in blood of a non-human animal that has been stressed and that is caused by administering the test sample. This is a method for evaluating fatigue improving activity.
[0010]
The present inventors investigated how changes in amino acid concentrations in blood appear in the process of continuously applying stress that causes mental fatigue to a living body using a mouse as a model animal. As a result, it was clarified that the alanine concentration in the blood decreased significantly immediately after the stress load, and that the alanine concentration in the blood gradually decreased when the stress was continuously applied.
[0011]
This can be done by objectively examining whether the body is stressed or how much mental fatigue is accumulated by measuring the alanine concentration in the blood of a model animal under stress. Means you can.
[0012]
That is, by measuring the alanine concentration in the normal blood of the model animal in advance or simultaneously and confirming how much the alanine concentration is reduced compared to the normal alanine concentration, The accumulation can be examined.
[0013]
The present invention utilizes the above-described measurement of alanine concentration in blood. According to the present invention, the stress is applied after the test sample is administered to the model animal, and the decrease in the alanine concentration in the blood of the model animal at that time is measured to thereby increase the antipsychotic fatigue activity of the test sample. You can evaluate or search for antipsychotic fatigue active substances. Further, according to the present invention, a test sample is administered after stress is applied to a model animal, and the test substance is tested for mental fatigue improvement activity by examining whether or not the decrease in the alanine concentration caused by the stress is recovered. Evaluate or search for substances that improve mental fatigue.
[0014]
DETAILED DESCRIPTION OF THE INVENTION
The non-human animal that can be used in the present invention is not particularly limited as long as it is a mammal, but in particular for convenience of experiment, a Wistar rat, SD (Sprague-Dawley), which is widely used for animal experiments. Strain rats, ddY mice, and ICR mice are particularly preferred.
[0015]
Examples of the method of applying stress to a non-human animal in the present invention include a method of restraining the body of a non-human animal and immersing it in water (restraint water immersion stress), a communication box, hanging, restraining, and conditioning stress. It is done.
[0016]
In the measurement of the concentration of alanine in blood in the present invention, it is preferable to use serum prepared by centrifuging blood collected from a living body as a measurement sample. The measurement can be performed using a high performance liquid chromatograph (HPLC) based on this sample. The column that can be used in this case may be any column that can be used for amino acid detection, and examples thereof include Capsule Pack UG120 manufactured by Shiseido Co., Ltd., and packed column MA-50DS manufactured by Aicom. Moreover, the same measurement can also be performed using the commercially available amino acid automatic analyzer. This measurement method may be processed in accordance with the instruction manual of the equipment used.
[0017]
In addition, in the present invention, as long as the amino acid concentration can be detected and measured, the measuring method is not particularly limited.
[0018]
【The invention's effect】
According to the present invention, it can be used for evaluation and detection of an antipsychotic fatigue active substance or mental fatigue improving substance having a good reproducibility and having a simple effect of preventing, eliminating or reducing the accumulation of mental fatigue.
[0019]
【Example】
Hereinafter, the present invention will be described in more detail with reference to examples.
[0020]
<Example>
14 Wistar rats (male, 6 weeks old) were used and bred by administering a 20% casein diet as a test diet for 7 days. The rats were then divided into two groups, and only one of the groups was subjected to restraint immersion stress that tied the rats' body with a net and immersed in a 26 ° C water bath for 7 hours per day. Under the condition, the animals were reared for another 7 days, and blood was collected from each group of rats before loading with restraint water immersion stress, and after 1 day, 3 days, and 7 days after the start of loading.
[0021]
The amino acid concentration in the collected blood was measured by the following procedure. 3% sulfosalicylic acid is added to the serum prepared from the collected blood by centrifugation (1,800 × g, 10 minutes) in the same amount as the serum, left at 4 ° C. for 24 hours, and then centrifuged (11,600). Xg for 10 minutes), and the resulting supernatant was collected. This was filtered through a 0.45 μm membrane filter, and the concentration of alanine was measured using a fully automated amino acid automatic analyzer (L-8500) manufactured by Hitachi, Ltd. The result is shown in FIG.
[0022]
In rats that did not receive restraint water immersion stress, there was almost no change in the alanine concentration in the blood, whereas in test rats loaded with restraint water immersion stress, the alanine concentration in the blood decreased significantly immediately after the start of stress loading. It was confirmed that the alanine concentration in the blood decreased in inverse proportion to the number of stress loads.
[Brief description of the drawings]
BRIEF DESCRIPTION OF DRAWINGS FIG. 1 shows changes in blood alanine concentration in rats loaded with restraint water immersion stress and rats not loaded with the stress.
Claims (3)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP11726698A JP4123565B2 (en) | 1998-04-27 | 1998-04-27 | Evaluation method of antipsychotic fatigue active substance |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP11726698A JP4123565B2 (en) | 1998-04-27 | 1998-04-27 | Evaluation method of antipsychotic fatigue active substance |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH11304792A JPH11304792A (en) | 1999-11-05 |
| JP4123565B2 true JP4123565B2 (en) | 2008-07-23 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP11726698A Expired - Lifetime JP4123565B2 (en) | 1998-04-27 | 1998-04-27 | Evaluation method of antipsychotic fatigue active substance |
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| Country | Link |
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| JP (1) | JP4123565B2 (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005078448A1 (en) * | 2004-02-17 | 2005-08-25 | Soiken Inc. | Apparatus for assessing degree of fatigue, method of assessing degree of fatigue and use thereof |
| JP4218842B2 (en) | 2004-07-14 | 2009-02-04 | 株式会社ウイルス医科学研究所 | Fatigue degree evaluation method and use thereof |
| JP5295571B2 (en) * | 2005-12-28 | 2013-09-18 | ライオン株式会社 | Solution for internal use for fatigue recovery |
| JP7360807B2 (en) * | 2019-04-26 | 2023-10-13 | 株式会社東レリサーチセンター | Methods to assist in detecting fatigue conditions |
-
1998
- 1998-04-27 JP JP11726698A patent/JP4123565B2/en not_active Expired - Lifetime
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| Publication number | Publication date |
|---|---|
| JPH11304792A (en) | 1999-11-05 |
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