JP5618395B2 - Composition for regulating autonomic nerve activity and method for regulating autonomic nerve - Google Patents
Composition for regulating autonomic nerve activity and method for regulating autonomic nerve Download PDFInfo
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Description
本発明は、自律神経活動を調節するための組成物および自律神経活動を調節する方法に関する。特に、本発明は、交感神経活動の亢進を抑制するための組成物、および/または、副交感神経活動を促進するための組成物に関する。また、本発明は交感神経活動の亢進を抑制する方法および副交感神経活動を促進する方法に関する。 The present invention relates to a composition for regulating autonomic nerve activity and a method for regulating autonomic nerve activity. In particular, the present invention relates to a composition for suppressing an increase in sympathetic nerve activity and / or a composition for promoting parasympathetic nerve activity. The present invention also relates to a method for suppressing an increase in sympathetic nerve activity and a method for promoting parasympathetic nerve activity.
自律神経系は、意識によって調節されない神経系の一部を構成している神経系であり、主として骨格筋を制御している体性運動神経系とは異なり心筋、平滑筋や腺組織を支配している。また、自律神経系は構造的にも運動神経系とは異なり節前神経と節後神経と呼ばれる2つの神経細胞から構成されている。自律神経系は交感神経系と副交感神経系の2つに大別され、それぞれ機能が異なっている。交感神経の活動により一般に心拍数の増大、血圧上昇、瞳孔拡大、血中グルコースの遊離などが起こり、副交感神経の活動により一般に消化管運動の増加や消化液の分泌などが刺激され、心拍数は減少することが知られている。両者は協同して働き生体の恒常性の維持に重要な役割を果たしている。
ペプチドまたはペプチド誘導体に関して自律神経活動への影響を直接測定した報告として、L-カルノシンの自律神経調節作用が報告されている。低用量のL-カルノシンの経口投与が、交感神経の活動を抑制し、副交感神経の活動を促進させる自律神経調節作用を示すこと、さらには交感神経活動の亢進に起因する高血圧および高血糖症状を改善する作用も示すことが報告されている(特許文献1)。しかし、この作用は静脈内投与により測定されており、経口投与による作用は不明であった。さらに、トリペプチドVal-Pro-Pro(VPP)およびその類縁体については、自律神経活動への影響を直接測定した報告はない。
また、ペプチドまたはペプチド誘導体に関して健忘改善効果を示した報告として、VPDPR、PDPR、CPR、シクロDPが0.5〜2.3mg/kgの脳室内または経口投与により、スコポラミン誘発健忘に対する改善効果が得られることが知られており、メカニズムの一つとしてμオピオイドレセプターを介する可能性が示唆されている(特許文献2)。また、これらのペプチドは経口投与により血清コレステロールの低下作用も有することが示されている(特許文献2)。さらに、XPLPR(XはL、I、M、F、W)が300mg/kgの側脳室内投与または経口投与によりスコポラミン誘発健忘に対する改善効果を示すことが報告されており、そのメカニズムの一つとして脳内C3aレセプターによるアセチルコリンの放出が示唆されている(特許文献3)。しかし、これらの作用はいずれも自律神経活動調節を介する作用を示したものではなく、トリペプチドVal-Pro-Pro(VPP)およびその類縁体については、健忘予防効果への影響を直接測定した報告はない。
The autonomic nervous system is a part of the nervous system that is not regulated by consciousness. Unlike the somatic motor nervous system that mainly controls skeletal muscle, the autonomic nervous system controls the heart muscle, smooth muscle, and glandular tissue. ing. Also, the autonomic nervous system is structurally different from the motor nervous system and is composed of two nerve cells called the prenodal nerve and the retronodal nerve. The autonomic nervous system is roughly divided into two types, the sympathetic nervous system and the parasympathetic nervous system, and their functions are different. Sympathetic activity generally causes an increase in heart rate, increased blood pressure, pupil enlargement, blood glucose release, etc., and parasympathetic activity generally stimulates an increase in gastrointestinal motility and secretion of digestive juices. It is known to decrease. The two work together to play an important role in maintaining homeostasis.
As a report of directly measuring the effect on autonomic nerve activity with respect to peptides or peptide derivatives, the autonomic nerve regulating action of L-carnosine has been reported. Oral administration of low-dose L-carnosine suppresses sympathetic nerve activity and exhibits parasympathetic nerve activity, and also exhibits hypertension and hyperglycemia due to increased sympathetic nerve activity. It has been reported that the effect of improving is also shown (Patent Document 1). However, this effect was measured by intravenous administration, and the effect by oral administration was unknown. Furthermore, there is no report that directly measured the effect on autonomic nerve activity for the tripeptide Val-Pro-Pro (VPP) and its analogs.
In addition, as a report showing amnesia-improving effects on peptides or peptide derivatives, it is possible to obtain an improvement effect on scopolamine-induced amnesia by intraventricular or oral administration of 0.5 to 2.3 mg / kg of VPDPR, PDPR, CPR, and cyclo DP. It has been known that the possibility of via a mu opioid receptor is suggested as one of the mechanisms (Patent Document 2). Moreover, it has been shown that these peptides also have an effect of lowering serum cholesterol by oral administration (Patent Document 2). Furthermore, it has been reported that XPLPR (X is L, I, M, F, W) shows an improvement effect on scopolamine-induced amnesia by intraventricular or oral administration of 300 mg / kg. It has been suggested that acetylcholine is released by C3a receptors in the brain (Patent Document 3). However, none of these actions showed an action through regulation of autonomic nervous activity, and the tripeptide Val-Pro-Pro (VPP) and its analogs were directly measured for their effects on amnesia prevention effects. There is no.
トリペプチドVal-Pro-Pro(VPP)およびその類縁体の機能についてもいくつかの報告がある。例えば、Val-Pro-Proを有効成分とするアンジオテンシン変換酵素阻害剤が知られており、Val-Pro-ProがACE阻害活性を示し、経口投与によるVal-Pro-Proの血圧降下作用が知られており、Val-Pro-Proを含有する発酵乳も血圧降下作用を有することが報告されている(特許文献4)。また、Val-Pro-ProおよびIle-Pro-Pro(IPP)を経口投与することにより、寒冷ストレスによる血圧変動が抑えられ、同時に、血中アミノ酸のバランス(Fischer比)の変化、胸腺や脾臓の萎縮が見られ、脾臓細胞反応性の低下等の免疫機能指標の低下に対して抑制効果を示すことが知られている(特許文献5)。さらに、Val-Pro-Proを皮膚に塗布することにより皮膚のクスミが改善されることが知られており、そのメカニズムとして、アンジオテンシン変換酵素阻害活性を持つ物質(VPP等)は平滑筋拡張作用のあるブラジキニンの分解を抑えるので血行が促進されることが示唆されている(特許文献6)。しかし、これらの報告において何れもペプチドの自律神経活動への影響を直接測定した例はなく、その調節作用に着目した具体的な報告はなされていない。 There are also several reports on the function of the tripeptide Val-Pro-Pro (VPP) and its analogs. For example, an angiotensin converting enzyme inhibitor containing Val-Pro-Pro as an active ingredient is known, Val-Pro-Pro shows ACE inhibitory activity, and the blood pressure-lowering action of Val-Pro-Pro by oral administration is known. It has been reported that fermented milk containing Val-Pro-Pro also has a blood pressure lowering effect (Patent Document 4). Moreover, oral administration of Val-Pro-Pro and Ile-Pro-Pro (IPP) suppresses blood pressure fluctuations due to cold stress, and at the same time, changes in blood amino acid balance (Fischer ratio), thymus and spleen It is known that atrophy is observed and an inhibitory effect is exhibited against a decrease in immune function index such as a decrease in spleen cell reactivity (Patent Document 5). In addition, it is known that Val-Pro-Pro is applied to the skin to improve skin smears. As a mechanism, substances with angiotensin-converting enzyme inhibitory activity (such as VPP) have an effect of smooth muscle expansion. It has been suggested that blood circulation is promoted by suppressing the degradation of some bradykinin (Patent Document 6). However, none of these reports have directly measured the effect of peptides on autonomic nerve activity, and no specific reports have focused on their regulatory effects.
本発明は、自律神経活動を調節するための経口摂取可能な組成物が提供される。また、本発明は、自律神経活動を調節する方法を提供する。
特に、本発明は、交感神経活動の亢進を抑制するための経口摂取可能な組成物、および/または、副交感神経活動を促進するための経口摂取可能な組成物を提供する。
The present invention provides an orally ingestible composition for modulating autonomic nerve activity. The present invention also provides a method for regulating autonomic nerve activity.
In particular, the present invention provides an orally ingestible composition for suppressing an increase in sympathetic nerve activity and / or an ingestible composition for promoting parasympathetic nerve activity.
(1)本発明は、Val-Pro-Proまたはその塩を有効成分とする、自律神経活動調節用組成物である。
(2)本発明は、Val-Pro-Proまたはその塩を有効成分とする、副交感神経活動の促進用組成物でもある。
(3)本発明は、Val-Pro-Proまたはその塩を有効成分とする、交感神経活動の亢進抑制用組成物である。
(4)また、本発明は、自律神経の乱れおよびそれに起因する症状を予防、改善または緩和することを特徴とする(1)〜(3)のいずれかに記載の組成物である。
(5)本発明は、Val-Pro-Proまたはその塩を有効成分とする、健忘予防作用、血流促進作用または抗不安作用を有する(2)または(3)記載の組成物である。
(6)本発明は、Val-Pro-Proまたはその塩を有効成分とする、健忘予防用組成物でもある。
(7)本発明は、Val-Pro-Proまたはその塩を有効成分とする、血流促進用組成物でもある。
(8)本発明は、Val-Pro-Proまたはその塩を有効成分とする、不安抑制用組成物でもある。
(9)本発明は、経口摂取用である、(1)〜(8)のいずれかに記載の組成物でもある。
(10)また、本発明は、非ヒト動物にVal-Pro-Proまたはその塩を投与することを含む、非ヒト動物における自律神経活動を調節する方法である。
(11)本発明は、非ヒト動物にVal-Pro-Proまたはその塩を投与することを含む、非ヒト動物における副交感神経活動を促進する方法でもある。
(12)本発明は、非ヒト動物にVal-Pro-Proまたはその塩を投与することを含む、非ヒト動物における交感神経活動の亢進を抑制する方法でもある。
(13)本発明は、非ヒト動物において自律神経の乱れおよびそれに起因する症状を予防、改善または緩和する、(10)〜(12)記載の方法でもある。
(14)また、本発明は、非ヒト動物において健忘を予防する、血流を促進する、または不安を抑制する、(11)または(12)記載の方法でもある。
(15)本発明は、非ヒト動物にVal-Pro-Proまたはその塩を投与することを含む、非ヒト動物における健忘を予防する方法でもある。
(16)本発明は、非ヒト動物にVal-Pro-Proまたはその塩を投与することを含む、非ヒト動物における血流を促進する方法でもある。
(17)本発明は、非ヒト動物にVal-Pro-Proまたはその塩を投与することを含む、非ヒト動物における不安を抑制する方法でもある。
(18)本発明は、投与が経口投与である、(10)〜(17)のいずれかに記載の方法でもある。
(1) The present invention is a composition for regulating autonomic nervous activity comprising Val-Pro-Pro or a salt thereof as an active ingredient.
(2) The present invention is also a composition for promoting parasympathetic nerve activity, comprising Val-Pro-Pro or a salt thereof as an active ingredient.
(3) The present invention is a composition for suppressing enhancement of sympathetic nerve activity, comprising Val-Pro-Pro or a salt thereof as an active ingredient.
(4) Moreover, this invention is a composition in any one of (1)-(3) characterized by preventing, improving or relieving the disorder of an autonomic nerve, and the symptom resulting from it.
(5) The present invention is the composition according to (2) or (3), which has an amnesia-preventing action, a blood flow promoting action or an anxiolytic action, comprising Val-Pro-Pro or a salt thereof as an active ingredient.
(6) The present invention is also an amnesia-preventing composition comprising Val-Pro-Pro or a salt thereof as an active ingredient.
(7) The present invention is also a composition for promoting blood flow comprising Val-Pro-Pro or a salt thereof as an active ingredient.
(8) The present invention is also an anxiety-suppressing composition comprising Val-Pro-Pro or a salt thereof as an active ingredient.
(9) The present invention is also the composition according to any one of (1) to (8), which is for oral intake.
(10) The present invention also relates to a method for regulating autonomic nerve activity in a non-human animal, comprising administering Val-Pro-Pro or a salt thereof to the non-human animal.
(11) The present invention is also a method for promoting parasympathetic nerve activity in a non-human animal, comprising administering Val-Pro-Pro or a salt thereof to the non-human animal.
(12) The present invention is also a method for suppressing an increase in sympathetic nerve activity in a non-human animal, comprising administering Val-Pro-Pro or a salt thereof to the non-human animal.
(13) The present invention is also the method according to (10) to (12), which prevents, ameliorates or alleviates autonomic nerve disturbance and symptoms caused by the disorder in a non-human animal.
(14) The present invention is also the method according to (11) or (12), which prevents amnesia, promotes blood flow, or suppresses anxiety in a non-human animal.
(15) The present invention is also a method for preventing amnesia in a non-human animal, comprising administering Val-Pro-Pro or a salt thereof to the non-human animal.
(16) The present invention is also a method for promoting blood flow in a non-human animal, comprising administering Val-Pro-Pro or a salt thereof to the non-human animal.
(17) The present invention is also a method for suppressing anxiety in a non-human animal, comprising administering Val-Pro-Pro or a salt thereof to the non-human animal.
(18) The present invention is also the method according to any one of (10) to (17), wherein the administration is oral administration.
本発明の組成物はトリペプチドVal-Pro-Proを有効成分とする。有効成分であるペプチドVal-Pro-Proは有機化学的に合成したペプチドであってもよく、また天然物由来のペプチドであってもよい。これらのペプチドの有機化学的合成法としては、固相法(Boc法、Fmoc法)や液相法といった一般的な方法を用いることができ、例えば、島津製作所製のペプチド合成装置(PSSM-8型)といったペプチド自動合成装置を用いて合成したものであってもよい。ペプチド合成の反応条件等については、当業者の技術常識に基づき、選択する合成方法や適切な反応条件等を任意で設定することができる。化学的に合成されたペプチドの精製方法も当業者にはよく知られたものである。
本明細書において、ペプチドVal-Pro-Proについて言及する場合、特に明示した場合および文脈上除外されることが明らかである場合を除き、「Val-Pro-Pro」および「ペプチドVal-Pro-Pro」にはその塩が含まれる。そのような塩には、例えばナトリウム塩、カリウム塩などの生理的条件下で存在しえる塩が含まれる。また、本発明の組成物は、本発明の組成物の有効成分であるペプチド、Val-Pro-Pro以外に他のペプチドおよび遊離アミノ酸またはその塩を含んでいても良い。なお、本発明との関連において、アミノ酸の三文字表記および一文字表記ならびにペプチドの表記は当業者によく知られた一般的規則に従う。
The composition of the present invention contains the tripeptide Val-Pro-Pro as an active ingredient. The peptide Val-Pro-Pro which is an active ingredient may be a peptide synthesized organically or may be a peptide derived from a natural product. As an organic chemical synthesis method for these peptides, a general method such as a solid phase method (Boc method, Fmoc method) or a liquid phase method can be used. For example, a peptide synthesizer manufactured by Shimadzu Corporation (PSSM-8) Type) and a peptide synthesized using an automatic peptide synthesizer. About the reaction conditions of peptide synthesis, based on the common general knowledge of those skilled in the art, the synthesis method to be selected, appropriate reaction conditions, and the like can be arbitrarily set. Methods for purifying chemically synthesized peptides are also well known to those skilled in the art.
In this specification, when referring to the peptide Val-Pro-Pro, “Val-Pro-Pro” and “Peptide Val-Pro-Pro” unless otherwise stated and apparently excluded from context. "Includes salts thereof. Such salts include salts that may exist under physiological conditions such as sodium salts, potassium salts and the like. The composition of the present invention may contain other peptides and free amino acids or salts thereof in addition to the peptide, Val-Pro-Pro, which is an active ingredient of the composition of the present invention. In the context of the present invention, the three-letter code and single-letter code for amino acids and the code for peptides follow general rules well known to those skilled in the art.
本発明のある態様においては、ペプチドVal-Pro-Proは、Val-Pro-Pro配列を含有するタンパク質を含む原料(例えば、牛乳、馬乳、山羊乳または羊乳、およびそれらの脱脂粉乳や大豆、小麦など)をラクトバチルス・ヘルベティカス(Lactobacillus helveticus)種に属する菌などにより醗酵させて得られる醗酵物に由来するものであってもよい。醗酵の際のタンパク質原料含有量は特に限定されないが、一般には1〜19重量%が好ましいであろう。このような醗酵物をそのまま、あるいは遠心分離、ろ過、クロマトグラム、乾燥等の適切な処理をして本発明の組成物とすることもできる。 In some embodiments of the present invention, the peptide Val-Pro-Pro is a raw material comprising a protein containing the Val-Pro-Pro sequence (eg, milk, horse milk, goat milk or sheep milk, and their nonfat dry milk or soy , Wheat, etc.) may be derived from a fermented product obtained by fermenting with bacteria belonging to the species Lactobacillus helveticus. The content of the protein raw material during fermentation is not particularly limited, but generally 1 to 19% by weight will be preferable. Such a fermented product can be used as it is or after appropriate treatment such as centrifugation, filtration, chromatogram, and drying to obtain the composition of the present invention.
本発明の別の態様においては、ペプチドVal-Pro-ProはVal-Pro-Proを含有するタンパク質の加水分解物に由来しても良い。そのようなタンパク質には、例えば牛乳、馬乳、山羊乳、羊乳等のカゼインのような獣乳カゼイン又はその濃縮物が含まれる。また、獣乳カゼインを含む食品原料を麹菌や乳酸菌といった菌類によって醗酵させて得られる醗酵物に由来するものであってもよい。獣乳カゼインを加水分解又は醗酵する際のカゼイン濃度は、特に限定されないが、一般には1〜19重量%が好ましい。市販の酵素群を用いる場合には、通常、至適条件が設定されているが、前記カゼイン加水分解物が得られるように条件、例えば、使用酵素量や反応時間等を、用いる酵素群に応じて適宜変更して行なうことができる。例えば、獣乳カゼインを溶解した水溶液に、酵素群/獣乳カゼインが重量比で1/1000以上、好ましくは1/100〜1/10、特に好ましくは1/40〜1/10の割合でタンパク質分解酵素を添加することができる。反応条件は、使用する酵素群に応じて適宜選択できるが、通常25〜60℃、好ましくは45〜55℃、pH3〜11、好ましくは、5〜9とすることができる。また、反応時間については、2〜48時間、好ましくは7〜15時間にて行なうことができる。このようなタンパク質加水分解物、特にカゼイン加水分解物をそのまま、あるいは遠心分離、ろ過、クロマトグラム、乾燥等の処理をして本発明の組成物としてもよい。 In another embodiment of the present invention, the peptide Val-Pro-Pro may be derived from a hydrolyzate of a protein containing Val-Pro-Pro. Such proteins include animal milk casein, such as casein such as cow's milk, horse milk, goat milk, sheep milk, or concentrates thereof. Moreover, you may originate in the fermented material obtained by fermenting the foodstuff raw material containing animal milk casein with fungi, such as a gonococcus and a lactic acid bacterium. Although the casein concentration at the time of hydrolyzing or fermenting animal milk casein is not particularly limited, it is generally preferably 1 to 19% by weight. In the case of using a commercially available enzyme group, the optimum conditions are usually set. However, depending on the enzyme group used, the conditions such as the amount of enzyme used and the reaction time are used so that the casein hydrolyzate can be obtained. Can be changed as appropriate. For example, in an aqueous solution in which animal milk casein is dissolved, the enzyme group / animal milk casein has a weight ratio of 1/1000 or more, preferably 1/100 to 1/10, particularly preferably 1/40 to 1/10. Degrading enzymes can be added. Although reaction conditions can be suitably selected according to the enzyme group to be used, it is usually 25 to 60 ° C., preferably 45 to 55 ° C., pH 3 to 11, and preferably 5 to 9. The reaction time can be 2 to 48 hours, preferably 7 to 15 hours. Such a protein hydrolyzate, in particular casein hydrolyzate, may be used as it is, or may be subjected to treatment such as centrifugation, filtration, chromatogram, and drying to obtain the composition of the present invention.
本発明の組成物は、自律神経活動の調節作用を有する。自律神経の活動には交感神経の活動および副交感神経の活動が含まれるが、本発明の組成物は、交感神経活動の亢進を抑制する作用および副交感神経活動を促進する作用を有する。特に、本発明の組成物は交感神経の活動の亢進を抑制する、あるいは副交感神経活動を促進する結果、健忘予防作用、血流促進、抗不安作用を有する。また、本発明の組成物は血流促進作用を有し、特に皮膚動脈交感神経活動を抑制することによる皮膚血流促進作用を有する。さらに、本発明の組成物は自律神経の調節作用を有するため、広く、心拍数調節作用、血糖調節作用、胃液分泌調節作用、体温調節作用、血圧調節作用およびストレス緩和作用等を有する。
本発明の組成物およびその有効成分であるペプチドVal-Pro-Proの自律神経活動調節作用、特に交感神経活動の亢進を抑制する作用および副交感神経活動を促進する作用は、例えば以下のように確認することができる。
The composition of the present invention has an action of regulating autonomic nerve activity. Although autonomic nerve activity includes sympathetic nerve activity and parasympathetic nerve activity, the composition of the present invention has an action of suppressing an increase in sympathetic nerve activity and an action of promoting parasympathetic nerve activity. In particular, the composition of the present invention has the effect of preventing amnesia, promoting blood flow, and anxiolytic effects as a result of suppressing the increase in sympathetic nerve activity or promoting parasympathetic nerve activity. Further, the composition of the present invention has a blood flow promoting action, and particularly has a skin blood flow promoting action by suppressing cutaneous arterial sympathetic nerve activity. Furthermore, since the composition of the present invention has an autonomic nerve regulating action, it has broadly a heart rate regulating action, a blood sugar regulating action, a gastric secretion regulating action, a body temperature regulating action, a blood pressure regulating action, a stress relieving action and the like.
The action of the composition of the present invention and its active ingredient peptide Val-Pro-Pro to regulate the autonomic nervous activity, particularly the action to suppress the increase of sympathetic nerve activity and the action to promote parasympathetic nerve activity are confirmed as follows, for example. can do.
本発明の組成物の胃副交感神経活動促進作用は、例えば、ラットまたはマウス等の実験動物に本組成物を投与し、胃を支配する副交感(迷走)神経の活動電位を経時的に測定し、活動電位が上昇することによって確認することができる。また、ラットまたはマウス等に本発明の組成物を投与し、一定時間経過後(例えば15分〜30分後)色素、たとえばフェノールレッドをラットまたはマウス等の胃に投入し、一定時間経過後(たとえば20分後)そのラットまたはマウスの胃を摘出し残存色素の量を測定することによって胃排出能の増強、すなわち胃の活動が活発化することを指標として本組成物の副交感神経活動促進作用を確認することもできる。
本発明の組成物またはペプチドVal-Pro-Proの副腎交感神経活動に対する効果は、ラットまたはマウス等に本組成物を投与し、副腎を支配する副交感神経の活動電位を経時的に測定することによって確認することができる。活動電位が低下すれば副腎交感神経活動が抑制されたことが確認できる。
The gastric parasympathetic nerve activity promoting action of the composition of the present invention is, for example, by administering this composition to a laboratory animal such as a rat or a mouse, and measuring the action potential of the parasympathetic (vagus) nerve governing the stomach over time, This can be confirmed by an increase in action potential. In addition, the composition of the present invention is administered to rats or mice, etc., and after a certain period of time (for example, after 15 to 30 minutes), a dye such as phenol red is introduced into the stomach of rats or mice, etc. For example, after 20 minutes, the rat or mouse stomach is removed and the amount of residual pigment is measured to enhance gastric emptying ability, that is, the activity of the stomach is activated. Can also be confirmed.
The effect of the composition of the present invention or the peptide Val-Pro-Pro on the adrenal sympathetic nerve activity is obtained by administering the composition to rats or mice and measuring the action potential of the parasympathetic nerve governing the adrenal gland over time. Can be confirmed. If the action potential decreases, it can be confirmed that the adrenal sympathetic nerve activity is suppressed.
本発明の組成物またはペプチドVal-Pro-Proの皮膚動脈交感神経活動に対する効果は、例えば、本発明の組成物またはVal-Pro-Proをマウスまたはラット等に経口投与し、尾部皮膚動脈交感神経の活動電位を経時的に測定することによって確認することができる。また、発明の組成物またはペプチドVal-Pro-Proの血流促進作用は、マウスまたはラット等に本発明の組成物またはVal-Pro-Proを投与し、接触型レーザードップラー血流計などを用いて背部の正中線上の皮膚血流量を測定することによって確認することができる。血流量の測定はたとえば背部の正中線上でおこなうことができる。
本発明の組成物またはペプチドVal-Pro-Proの健忘予防作用は、例えばY字型迷路を用いた、アルツハイマー病治療薬の評価系に準じた系を用いて確認することができる。具体的には、ラットまたはマウスにスコポラミンのような健忘症を誘発する薬剤単独または本発明の組成物またはVal-Pro-Proをそのような薬剤と同時に投与、またはそのような薬剤の投与に先立って本発明の組成物またはVal-Pro-Proを投与し、Y字型迷路を用いた試験において異なるアームへの自発的交替行動変化率や迷路への総進入回数を指標として本発明の組成物の健忘予防作用を確認することができる。
これらの試験において陰性対照としてはたとえば水のみを投与した動物を用いることができる。薬剤によって誘導した健忘に対するVal-Pro-Proの予防作用を確認する実験の際には、スコポラミンのような健忘症を誘発する薬剤のみを投与した動物も対照として加えることができる。
The effect of the composition of the present invention or the peptide Val-Pro-Pro on the cutaneous arterial sympathetic nerve activity is obtained by, for example, orally administering the composition of the present invention or Val-Pro-Pro to a mouse or a rat, etc. This can be confirmed by measuring the action potential over time. In addition, the blood flow promoting action of the composition of the invention or the peptide Val-Pro-Pro can be achieved by administering the composition of the present invention or Val-Pro-Pro to a mouse or rat, etc., and using a contact laser Doppler blood flow meter or the like. This can be confirmed by measuring the skin blood flow on the midline of the back. The blood flow can be measured, for example, on the back midline.
The amnesia-preventing action of the composition of the present invention or the peptide Val-Pro-Pro can be confirmed using, for example, a system according to an evaluation system for Alzheimer's disease therapeutic drugs using a Y-shaped maze. Specifically, an agent that induces amnesia such as scopolamine or a composition of the present invention or Val-Pro-Pro is administered to a rat or mouse simultaneously with such an agent, or prior to administration of such an agent. The composition of the present invention was administered using the composition of the present invention or Val-Pro-Pro and the rate of spontaneous alternation behavior to different arms and the total number of times of entering the maze in the test using the Y-shaped maze. Can prevent amnesia.
In these tests, as a negative control, for example, animals administered with water alone can be used. In the experiment for confirming the preventive action of Val-Pro-Pro against amnesia induced by a drug, an animal administered only with a drug that induces amnesia such as scopolamine can be added as a control.
本発明の組成物またはペプチドVal-Pro-Proの抗不安作用は、例えば、マウスの高架式十字迷路試験によって確認することができる。具体的には、マウスに水または本発明の組成物若しくはペプチドVal-Pro-Proを投与し、マウスを高所にある迷路の中心のプラットフォームに置き、一定時間内のオープンアーム(壁のないアーム)とクローズドアーム(壁のあるアーム)への侵入回数、及び滞在していた時間を測定し、水のみを投与した対照群に比較してオープンアームへの侵入回数比率、若しくは滞在時間比率が増加すれば抗不安作用が認められたとして確認することができる。 The anxiolytic effect of the composition of the present invention or the peptide Val-Pro-Pro can be confirmed, for example, by an elevated plus maze test in mice. Specifically, mice are administered water or a composition of the invention or peptide Val-Pro-Pro, the mice are placed on the platform at the center of the maze at a high altitude, and open arms (arms without walls) within a certain time. ) And closed arms (arms with walls), and the time spent staying was measured, and the ratio of the number of times of intrusion into the open arm or the staying time ratio increased compared to the control group administered with water alone. This can be confirmed as an anxiolytic effect.
本発明の組成物はその有効成分がVal-Pro-Proであり、経口投与または経口摂取することにより上述の所望の効果を達成することができる。本発明の組成物の投与又は摂取期間は、投与又は摂取する対象、たとえばヒトや非ヒト動物の年齢やその対象の健康状態等を考慮して種々調整することができる。非ヒト動物には非ヒト高等脊椎動物、特に非ヒト哺乳類が含まれ、イヌ、ネコ等の愛玩動物、ウシ、ウマ、ブタ、ヒツジ等の家畜が含まれるがこれらに限られない。本発明の組成物は1回の投与でも効果が見られるが、1日1回以上、継続摂取することで継続した効果が期待できる。本発明の組成物を医薬として用いる場合の形態は、経口投与用の製剤の形態とすることができる。例えば、錠剤、丸剤、硬カプセル剤、軟カプセル剤、マイクロカプセル、散剤、顆粒剤、液剤等が挙げられる。医薬として製造する場合は、例えば、適宜必要に応じて、製薬的に許容される担体、賦形剤、補形剤、防腐剤、安定化剤、結合剤、pH調節剤、緩衝剤、増粘剤、ゲル化剤、保存剤、抗酸化剤等を用い、一般に認められた製剤投与に要求される単位用量形態で製造することができる。
本発明の組成物は飲食品用素材または動物用飼料素材として用いることもでき、例えば、本発明の組成物または本発明の組成物の有効成分であるペプチドVal-Pro-Proを消化管運動促進作用、抗不安作用、血流促進作用、健忘予防、心拍数調節作用、血糖調節作用、胃液分泌調節作用、体温調節作用、またはストレス緩和作用等の効能を有する特定保健用食品等の機能性食品とすることができる。
所望の効果を得るための本組成物またはペプチドVal-Pro-Proの投与または摂取量は、1回あたり有効成分であるペプチドVal-Pro-Proの量として一般に0.001mg/kg体重〜100mg/kg体重程度である。特に、副交感神経活動促進が主として望まれる場合、本発明の組成物の摂取量は、Val-Pro-Proの量として0.09mg/kg体重〜15mg/kg体重が好ましい。交感神経活動の抑制効果が望まれる場合は、Val-Pro-Proの量として0.15mg/kg体重〜20mg/kg体重が好ましく、特に皮膚動脈交感神経活動に対する効果が主として望まれる場合は0.15mg/kg体重〜1.5mg/kg体重が好ましい。血流促進作用が主として望まれる場合は、約20mg/kg程度が好ましい。また主として抗不安作用が望まれる場合は、Val-Pro-Proの量として0.001mg/kg体重〜100mg/kg体重が特に好ましく、主として健忘予防作用が望まれる場合はVal-Pro-Proの量として0.64mg/kg体重〜10mg/kg体重が好ましい。1日あたりの摂取回数に応じて、食品、例えば機能性食品における1回あたりの摂取量を前記量より更に低くすることも可能である。適切な摂取量は、上述の通り種々の要因を考慮して更に調整することができる。
The active ingredient of the composition of the present invention is Val-Pro-Pro, and the above-mentioned desired effect can be achieved by oral administration or ingestion. The administration or ingestion period of the composition of the present invention can be variously adjusted in consideration of the age of the subject to be administered or ingested, for example, the age of the human or non-human animal, the health condition of the subject, and the like. Non-human animals include non-human higher vertebrates, particularly non-human mammals, including but not limited to pets such as dogs and cats, and domestic animals such as cows, horses, pigs and sheep. The effect of the composition of the present invention can be seen even after one administration, but a continuous effect can be expected by continuously ingesting once or more a day. When the composition of the present invention is used as a medicine, it can be in the form of a preparation for oral administration. Examples include tablets, pills, hard capsules, soft capsules, microcapsules, powders, granules, liquids and the like. In the case of producing as a pharmaceutical, for example, a pharmaceutically acceptable carrier, excipient, excipient, preservative, stabilizer, binder, pH adjuster, buffer, thickener, if necessary. Can be manufactured in unit dosage forms required for generally accepted formulation administration, using agents, gelling agents, preservatives, antioxidants, and the like.
The composition of the present invention can also be used as a material for food and drink or an animal feed material. For example, the peptide of the present invention or the peptide Val-Pro-Pro, which is an active ingredient of the composition of the present invention, promotes gastrointestinal motility. Functional foods such as foods for specified health use that have effects such as action, anti-anxiety action, blood flow promotion action, amnesia prevention, heart rate regulation action, blood glucose regulation action, gastric secretion regulation action, body temperature regulation action, stress relaxation action, etc. It can be.
The administration or intake of the present composition or peptide Val-Pro-Pro for obtaining a desired effect is generally 0.001 mg / kg body weight to 100 mg / kg as the amount of peptide Val-Pro-Pro which is an active ingredient per time. About weight. In particular, when promotion of parasympathetic nerve activity is mainly desired, the intake of the composition of the present invention is preferably 0.09 mg / kg body weight to 15 mg / kg body weight as the amount of Val-Pro-Pro. When an inhibitory effect on sympathetic nerve activity is desired, the amount of Val-Pro-Pro is preferably 0.15 mg / kg body weight to 20 mg / kg body weight, particularly 0.15 mg / kg when an effect on cutaneous artery sympathetic nerve activity is mainly desired. kg body weight to 1.5 mg / kg body weight is preferred. When blood flow promoting action is mainly desired, about 20 mg / kg is preferable. When an anxiolytic effect is mainly desired, the amount of Val-Pro-Pro is particularly preferably 0.001 mg / kg body weight to 100 mg / kg body weight, and when an amnesia-preventing effect is mainly desired, the amount of Val-Pro-Pro is 0.64 mg / kg body weight to 10 mg / kg body weight is preferred. Depending on the number of intakes per day, it is possible to further reduce the intake amount per time in foods such as functional foods. The appropriate intake can be further adjusted taking into account various factors as described above.
本発明の組成物またはその有効成分であるペプチドVal-Pro-Proを含む食品、例えば機能性食品は、上述のようにして得たVal-Pro-Proを含有するタンパク質の分解物又はその濃縮物やVal-Pro-Proを含有するタンパク質を含む原料の醗酵物そのもの、あるいは醗酵物を粉末状や顆粒状にして各種食品に添加して製造することができる。また必要に応じて、食品に用いる他の成分、例えば、糖類、タンパク質、脂質、ビタミン、ミネラル、フレーバー、例えば、各種炭水化物、脂質、ビタミン類、ミネラル類、甘味料、香料、色素、テクスチュア改善剤等又はこれらの混合物等の添加物を添加し、栄養的バランスや風味等を改善してもよい。本発明の組成物またはその有効成分であるペプチドVal-Pro-Proを含む動物用飼料もヒト用食品と同様にして調製することができる。
例えば前述の機能性食品は、固形物、ゲル状物、液状物の何れの形態とすることができ、例えば、乳酸菌飲料等の醗酵乳製品、各種加工飲食品、乾燥粉末、錠剤、カプセル剤、顆粒剤等が挙げられ、更には各種飲料、ヨーグルト、流動食、ゼリー、キャンディ、レトルト食品、錠菓、クッキー、カステラ、パン、ビスケット、チョコレート等とすることができる。
本発明の組成物を含む特定保健用食品等の機能性食品を製造する場合、添加形態や製品形態によるが、最終製品に対して、含有される有効成分であるペプチドVal-Pro-Proの含有量が0.00001質量%〜10質量%、好ましくは0.00003質量%〜3質量%、さらに好ましくは0.0001質量%〜1質量%となるように調製する。
以下、実施例により本発明を具体的に説明するが、本発明の範囲は実施例に限られない。
A food comprising the composition of the present invention or its active ingredient peptide Val-Pro-Pro, for example, a functional food, is a protein degradation product containing Val-Pro-Pro obtained as described above or a concentrate thereof. It can be produced by adding a raw material fermented product containing a protein containing Val-Pro-Pro, or a fermented product in powder form or granular form to various foods. If necessary, other ingredients used in foods, such as sugars, proteins, lipids, vitamins, minerals, flavors, such as various carbohydrates, lipids, vitamins, minerals, sweeteners, fragrances, pigments, texture improvers Etc. or a mixture thereof may be added to improve nutritional balance, flavor, and the like. Animal feed containing the composition of the present invention or the peptide Val-Pro-Pro which is an active ingredient thereof can also be prepared in the same manner as human food.
For example, the functional food described above can be in any form of solid, gel, and liquid, for example, fermented milk products such as lactic acid bacteria beverages, various processed foods and drinks, dry powders, tablets, capsules, Examples thereof include granules, and various beverages, yogurt, liquid food, jelly, candy, retort food, tablet confectionery, cookies, castella, bread, biscuits, chocolate and the like.
When producing a functional food such as a food for specified health use containing the composition of the present invention, depending on the form of addition or product, the inclusion of the peptide Val-Pro-Pro which is an active ingredient contained in the final product The amount is adjusted to 0.00001% by mass to 10% by mass, preferably 0.00003% by mass to 3% by mass, and more preferably 0.0001% by mass to 1% by mass.
EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, the scope of the present invention is not restricted to an Example.
Val-Pro-Proの胃副交感神経活動に対する効果
体重約300gのWistar系雄ラット(約9週齢)(n=3)を用い、餌及び水は自由摂取させた。自律神経の活動を検討するために、3時間絶食後、明期の中間期にウレタン麻酔下に経口投与用のポリエチレンチューブを口腔内に挿入した。その後、胃を支配する副交感(迷走)神経を銀電極で吊り上げて、神経の電気活動を測定した。尚、手術開始から測定終了までチューブを気管に挿入して気道を確保し、保温装置にて体温(ラット直腸温)を35.0±0.5℃に保つようにした。神経の活動が落着いた時期に各Val-Pro-Proペプチド水溶液サンプルを1mlポリエチレンチューブにて経口投与し、神経活動の変化を測定した。サンプルは、Val-Pro-Pro 0.09mg/kg体重、0.15mg/kg体重または15mg/kg体重とした。対照として溶媒の水1mlを経口投与した。胃副交感神経の活動のデータは5分間毎の5秒あたりの発火頻度(pulse/5 s)の平均値にて解析し、刺激開始前の値(0分値)を100%として百分率で表し、その平均値±標準誤差で示した。対照群との有意差検定は、投与後5〜60分における相対活動電位の値を群として、分散分析法(ANOVA)によって行った。結果を図1に示す。
水投与群に比較し、Val-Pro-Pro 0.09mg/kg体重、0.15mg/kg体重および15mg/kg体重投与群のいずれにおいても投与後の胃副交感神経活動が有意に上昇した(図1)。Val-Pro-Proの経口投与は0.09mg/kg体重〜15mg/kg体重の範囲で胃副交感神経活動を促進することが示された。
Effect of Val-Pro-Pro on gastric parasympathetic nerve activity Wistar male rats (about 9 weeks old) (n = 3) weighing about 300 g were used, and food and water were ad libitum. In order to examine autonomic nerve activity, a polyethylene tube for oral administration was inserted into the oral cavity under urethane anesthesia in the middle of the light period after fasting for 3 hours. Thereafter, the parasympathetic (vagus) nerve that controls the stomach was lifted with a silver electrode, and the electrical activity of the nerve was measured. The tube was inserted into the trachea from the start of surgery to the end of measurement to secure the airway, and the body temperature (rat rectal temperature) was maintained at 35.0 ± 0.5 ° C. with a heat retaining device. When the nerve activity settled, each Val-Pro-Pro peptide aqueous solution sample was orally administered in a 1 ml polyethylene tube, and the change in nerve activity was measured. Samples were Val-Pro-Pro 0.09 mg / kg body weight, 0.15 mg / kg body weight or 15 mg / kg body weight. As a control, 1 ml of solvent water was orally administered. The data of gastric parasympathetic nerve activity is analyzed by the average value of the firing frequency (pulse / 5 s) per 5 seconds every 5 minutes, and the value before the start of stimulation (0 minute value) is expressed as a percentage. The average value ± standard error is shown. The significant difference test with the control group was carried out by analysis of variance (ANOVA) with the relative action potential value at 5 to 60 minutes after administration as a group. The results are shown in FIG.
Compared to the water-administered group, the gastric parasympathetic nerve activity after administration was significantly increased in any of the Val-Pro-Pro 0.09 mg / kg body weight, 0.15 mg / kg body weight, and 15 mg / kg body weight groups (FIG. 1). . It was shown that oral administration of Val-Pro-Pro promotes gastric parasympathetic nerve activity in the range of 0.09 mg / kg body weight to 15 mg / kg body weight.
Val-Pro-Proの副腎交感神経活動に対する効果
体重約300gのWistar系雄ラット(約9週齢)(n=3)を用い、餌及び水は自由摂取させた。自律神経の活動を検討するために、3時間絶食後、明期の中間期にウレタン麻酔下に経口投与用のポリエチレンチューブを口腔内に挿入した。その後、副腎を支配する交感神経を銀電極で吊り上げて、神経の電気活動を測定した。尚、手術開始から測定終了までチューブを気管に挿入して気道を確保し、保温装置にて体温(ラット直腸温)を35.0±0.5℃に保つようにした。神経の活動が落着いた時期にVal-Pro-Proペプチド水溶液サンプル1mlをポリエチレンチューブにて経口投与し、神経活動の変化を測定した。サンプルは、Val-Pro-Pro 15mg/kg体重とした。対照として溶媒の水1mlを経口投与した。副腎交感神経の活動のデータは5分間毎の5秒あたりの発火頻度(pulse/5 s)の平均値にて解析し刺激開始前の値(0分値)を100%として百分率で表し、その平均値±標準誤差で示した。対照群との有意差検定は、投与後5〜60分における相対活動電位の値を群として、分散分析法(ANOVA)によって行った。結果を図2に示す。
水投与群に比較し、Val-Pro-Pro 15mg/kg体重投与により副腎交感神経活動が有意に低下した(図2)。Val-Pro-Proは15mg/kg体重の投与で副腎交感神経活動を抑制することが示された。
Effect of Val-Pro-Pro on adrenal sympathetic nerve activity Wistar male rats (about 9 weeks old) (n = 3) weighing about 300 g were used, and food and water were ad libitum. In order to examine autonomic nerve activity, a polyethylene tube for oral administration was inserted into the oral cavity under urethane anesthesia in the middle of the light period after fasting for 3 hours. Thereafter, the sympathetic nerve that controls the adrenal gland was lifted with a silver electrode, and the electrical activity of the nerve was measured. The tube was inserted into the trachea from the start of surgery to the end of measurement to secure the airway, and the body temperature (rat rectal temperature) was maintained at 35.0 ± 0.5 ° C. with a heat retaining device. When the nerve activity settled, 1 ml of Val-Pro-Pro peptide aqueous solution sample was orally administered in a polyethylene tube, and the change in nerve activity was measured. The sample was Val-
Compared to the water-administered group, administration of Val-
Val-Pro-Proの皮膚動脈交感神経活動に対する効果
体重約300gのWistar系雄ラット(約9週齢)(n=3)を用い、餌及び水は自由摂取させた。自律神経の活動を検討するために、3時間絶食後、明期の中間期にウレタン麻酔下に経口投与用のポリエチレンチューブを口腔内に挿入した。その後、尻尾の皮膚動脈交感神経を銀電極で吊り上げて、神経の電気活動を測定した。尚、手術開始から測定終了までチューブを気管に挿入して気道を確保し、保温装置にて体温(ラット直腸温)を35.0±0.5℃に保つようにした。これらの神経の活動が落着いた時期に各Val-Pro-Proペプチド水溶液サンプル1mlをポリエチレンチューブにて経口投与し、これらの神経活動の変化を測定した。サンプルは、Val-Pro-Pro 0.15mg/kg体重または1.5mg/kg体重とした。対照として溶媒の水1mlを経口投与した。皮膚動脈交感神経の活動のデータは、5分間毎の5秒あたりの発火頻度(pulse/5 s)の平均値にて解析し、刺激開始前の値(0分値)を100%として百分率で表し、その平均値±標準誤差で示した。対照群との有意差検定は、投与後5〜60分における相対活動電位の値を群として、分散分析法(ANOVA)によって行った。結果を図3に示す。
水投与群に比較し、Val-Pro-Pro 1.5mg/kg体重および0.15mg/kg体重群において投与後の皮膚動脈交感神経活動が有意に低下した(図3)。Val-Pro-Proの経口投与は0.15mg/kg体重〜1.5mg/kg体重の範囲で皮膚動脈交感神経活動を抑制することが示された。
Effect of Val-Pro-Pro on cutaneous arterial sympathetic nerve activity Wistar male rats (about 9 weeks old) (n = 3) weighing about 300 g were used, and food and water were ingested freely. In order to examine autonomic nerve activity, a polyethylene tube for oral administration was inserted into the oral cavity under urethane anesthesia in the middle of the light period after fasting for 3 hours. Then, the tail cutaneous artery sympathetic nerve was lifted with a silver electrode, and the electrical activity of the nerve was measured. The tube was inserted into the trachea from the start of surgery to the end of measurement to secure the airway, and the body temperature (rat rectal temperature) was maintained at 35.0 ± 0.5 ° C. with a heat retaining device. When these nerve activities settled, 1 ml of each Val-Pro-Pro peptide aqueous solution sample was orally administered through a polyethylene tube, and changes in these nerve activities were measured. The sample was Val-Pro-Pro 0.15 mg / kg body weight or 1.5 mg / kg body weight. As a control, 1 ml of solvent water was orally administered. Cutaneous arterial sympathetic nerve activity data is analyzed with the average value of the firing frequency (pulse / 5 s) per 5 seconds every 5 minutes, and the percentage before the start of stimulation (0 minute value) is 100%. Expressed as an average value ± standard error. The significant difference test with the control group was carried out by analysis of variance (ANOVA) with the relative action potential value at 5 to 60 minutes after administration as a group. The results are shown in FIG.
Cutaneous arterial sympathetic nerve activity after administration was significantly reduced in the Val-Pro-Pro 1.5 mg / kg body weight and 0.15 mg / kg body weight groups as compared to the water administration group (FIG. 3). Oral administration of Val-Pro-Pro was shown to suppress cutaneous arterial sympathetic nerve activity in the range of 0.15 mg / kg body weight to 1.5 mg / kg body weight.
Val-Pro-Proの健忘予防作用
ddY系雄性マウス(約7週齢)を用い(n=15)、餌及び水は自由摂取させた。被検物質として、Val-Pro-Pro 0.64mg/kg体重、6.4mg/kg体重、10mg/kg体重を用いた。各量のVal-Pro-Proは自発的交替行動を評価するY字迷路試験の実施60分前にマウスに単回経口投与した。また、Y字迷路試験の実施30分前には、マウスに記憶障害および/または認知障害を誘発するため、スコポラミンを1mg/kg体重となるよう背部に皮下投与した。Y字迷路試験では、一本のアームの長さが40cm、壁の高さが12cm、床の幅が3cm、上部の幅が10cmで3本のアームがそれぞれ120度の角度で接続されたY字迷路を実験装置として用いた。マウスをY字迷路のいずれかのアームの先端に置き、8分間にわたって迷路内を自由に探索させ、マウスが移動したアームを順に記録した。マウスが測定時間内に各アームに移動した回数をカウントし、これを総進入数とし、この中で連続して異なる三つのアームを選択した組み合わせ(例えば、3本のアームをそれぞれA、B、Cとした際に、進入したアームの順番がABCBACACBの場合は重複も含めて4とカウントする)を調べ、この数を自発的交替行動数とした。自発的交替行動数を総進入数から2を引いた数で割り、それに100を掛けて求めた値を自発的交替行動変化率とし、これを自発的交替行動の指標とした。測定値は群毎に平均値±標準誤差で表した。対照群またはスコポラミン対照群との有意差検定はスチューデント(Student)のt検定で行った。結果を図4に示す。Val-Pro-Proは0.64mg/kg体重〜10mg/kg体重の範囲で健忘予防作用を示すことが示された。
Amnesia preventive action of Val-Pro-Pro
ddY male mice (approximately 7 weeks of age) were used (n = 15), and food and water were ad libitum. As test substances, Val-Pro-Pro 0.64 mg / kg body weight, 6.4 mg / kg body weight, and 10 mg / kg body weight were used. Each dose of Val-Pro-Pro was administered orally once to
Val-Pro-Proの末梢血流促進作用
6週齢の雌性Hos:HR-1ヘアレスマウスを日本SLC株式会社より購入し、1週間の馴化飼育後に下記の試験に供した。試験時の週齢は7〜15週の範囲とした。体重を測定した後、イソフルラン(アボットジャパン社)によるガス麻酔を行なった。導入時麻酔は濃度4%、流速1L/minの条件で1分間行なった。維持麻酔はマウスピース使用下で、濃度1%、流速1L/分の条件とした。維持麻酔開始後、マウスは37℃に設定したホットプレート(オムロン社製)の上に置き保温した。プレートとマウスの間にはキムタオルを一枚挟んだ。麻酔導入後30分以降で、直前の血流量が10分間以上安定したところで、ゾンデを用いて各サンプルを単回経口投与した。サンプルは、投与されるVal-Pro-Pro が20mg/kgとなるよう10ml/kg体重で投与した。対照として溶媒の水10ml/kg体重を経口投与した。血流量測定にはアドバンス社製の接触型レーザードップラー血流計を用いた。接触型プローブは、背部の正中線上、尾根部から上部3cmの皮膚にビニールテープを用いて貼り付けた。血流量(Flow)を評価指標として、投与後1分間の間隔毎に平均値を算出し、投与前1分間の平均値を100とした相対値として表した。対照群との群間比較は投与後5〜60分の測定値を群としてANOVA解析によって行った。結果を図5に示す。Val-Pro-Proは20mg/kg体重で投与した場合に血流促進作用を有することが示された。
Peripheral blood flow promoting action of Val-Pro-Pro
Six-week-old female Hos: HR-1 hairless mice were purchased from Japan SLC Co., Ltd. and subjected to the following test after acclimation breeding for one week. The age at the time of the test was in the range of 7 to 15 weeks. After measuring the body weight, gas anesthesia with isoflurane (Abbott Japan) was performed. During introduction, anesthesia was performed for 1 minute under conditions of a concentration of 4% and a flow rate of 1 L / min. Maintenance anesthesia was performed using a mouthpiece under conditions of a concentration of 1% and a flow rate of 1 L / min. After the start of maintenance anesthesia, the mouse was placed on a hot plate (manufactured by OMRON) set at 37 ° C. and kept warm. A piece of Kim Towel was placed between the plate and the mouse. After 30 minutes after the induction of anesthesia, each sample was orally administered once using a sonde when the previous blood flow was stable for 10 minutes or more. Samples were administered at 10 ml / kg body weight such that the Val-Pro-Pro administered was 20 mg / kg. As a control, 10 ml / kg body weight of solvent was orally administered. A contact type laser Doppler blood flow meter manufactured by Advance was used for blood flow measurement. The contact type probe was attached to the skin 3 cm above the ridge on the midline of the back using vinyl tape. Using the blood flow rate (Flow) as an evaluation index, an average value was calculated at intervals of 1 minute after administration, and expressed as a relative value with the average value for 1 minute before administration being 100. Comparison between the control group and the control group was performed by ANOVA analysis with the measured value of 5 to 60 minutes after administration as a group. The results are shown in FIG. Val-Pro-Pro was shown to have a blood flow promoting effect when administered at 20 mg / kg body weight.
高架式十字迷路試験によるVal-Pro-Proの抗不安作用の評価
ddY 系雄性マウス(約6週齢)を用い(n=15)、予備飼育後、Val-Pro-Pro(0.001mg/kg体重, 0.1mg/kg体重, 10mg/kg体重または100mg/kg体重)をそれぞれ単回経口投与し、投与30分後に高架式十字迷路試験により抗不安作用を検討した。高架式十字迷路は、長さ65cm、幅5cmの塩化ビニル製の板を2本、十字型に交差させた形状をし、その1本の通路の周囲には中央の交差部分を除いて、高さ50cmの壁(黒色アクリル製樹脂)が取り付けられている。すなわち、十字迷路は壁のない2本のアーム(オープンアーム)と壁のある2本のアーム(クローズドアーム)、及び中央の交差部分で構成されている。高架式十字迷路試験では、迷路の中央の交差部に、一方の壁のあるアームに向けてマウスを置き、5分間迷路上を自由に探索させ、その行動を観察し、オープンアームへの侵入回数を測定した。測定値から5分間の試験中のオープンアーム侵入回数比率を求め、これを抗不安作用の指標とした。尚、対照群には蒸留水を単回経口投与した。各測定値は群毎に平均値±標準誤差で表した。結果を図6に示す。Val-Pro-Proは0.001mg/kg体重〜100mg/kg体重の範囲においてオープンアーム侵入回数比率の値が対照群に比べて高く、抗不安作用を有することが示された。
Evaluation of anxiolytic activity of Val-Pro-Pro by elevated plus maze test
Using ddY male mice (approximately 6 weeks old) (n = 15), after preliminary breeding, Val-Pro-Pro (0.001 mg / kg body weight, 0.1 mg / kg body weight, 10 mg / kg body weight or 100 mg / kg body weight) Were administered orally once, and 30 minutes after administration, the anxiolytic effect was examined by an elevated plus maze test. The elevated cross maze is formed by crossing two vinyl chloride plates with a length of 65 cm and a width of 5 cm in a cross shape, and around the single passage, except for the central intersection, A 50cm wall (black acrylic resin) is attached. That is, the cross maze is composed of two arms with no walls (open arms), two arms with walls (closed arms), and a central intersection. In the elevated cross maze test, place the mouse on the arm with one wall at the center of the maze, let it freely explore the maze for 5 minutes, observe its behavior, and enter the open arm Was measured. The ratio of the number of open arm intrusions during the 5-minute test was determined from the measured values, and this was used as an anxiolytic action index. In the control group, distilled water was orally administered once. Each measured value was expressed as an average value ± standard error for each group. The results are shown in FIG. Val-Pro-Pro had an anti-anxiety action in the range of 0.001 mg / kg body weight to 100 mg / kg body weight, with a higher value of the open arm invasion frequency ratio than the control group.
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