JP2013234177A - Cell death-inhibiting composition - Google Patents
Cell death-inhibiting composition Download PDFInfo
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- JP2013234177A JP2013234177A JP2013083449A JP2013083449A JP2013234177A JP 2013234177 A JP2013234177 A JP 2013234177A JP 2013083449 A JP2013083449 A JP 2013083449A JP 2013083449 A JP2013083449 A JP 2013083449A JP 2013234177 A JP2013234177 A JP 2013234177A
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- cell death
- disease
- atp
- ischemic
- cell
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Abstract
Description
本発明は、オイデスマン型セスキテルペノイド誘導体を有効成分とする高カリウム環境下での細胞死抑制組成物に関する。 The present invention relates to a composition for inhibiting cell death in a high potassium environment, which comprises an Eudesman sesquiterpenoid derivative as an active ingredient.
日本をはじめ、北米、ヨーロッパ諸国では生活環境の変化と社会の高齢化によって脳・神経疾患ならびに循環器障害の多発が顕在化している。なかでも壮年期に発症する認知症のような神経疾患や虚血性心疾患の増加は大きな社会問題になっている。また、筋萎縮性側索硬化症(ALS)・パーキンソン病・加齢による大脳機能障害(アルツハイマー、老年性記憶障害等)における脳神経細胞死、脳梗塞などの虚血性脳機能障害の予後に起きる脳神経細胞死、さらに心筋梗塞や狭心症の治療予後に派生する心筋細胞死には、細胞死が大きく関わっていることが明らかになってきた。 In Japan, North America, and European countries, changes in the living environment and the aging of society have caused many brain and neurological diseases and cardiovascular disorders. In particular, an increase in neurological diseases such as dementia and ischemic heart disease that develop in middle age has become a major social problem. Also, cranial nerves that occur in the prognosis of ischemic cerebral dysfunction such as cerebral nerve cell death in cerebral dysfunction due to amyotrophic lateral sclerosis (ALS), Parkinson's disease, aging (Alzheimer, senile memory disorder, etc.) It has been clarified that cell death is greatly involved in cell death, and further, myocardial cell death derived from the prognosis of treatment for myocardial infarction and angina.
認知症には、アルツハイマー型認知症と脳血管認知症、レビー小体認知症、前頭側頭型認知症などがある。脳血管性の認知症は脳の血管にコレステロールがたまり、血管が硬くなる動脈硬化によって引き起こされる。認知症の治療に優れた治療薬はなく、回想法や運動療法などと組み合せて、症状の進行を抑制し、日常生活を維持していくのが現状である。 Dementia includes Alzheimer type dementia and cerebrovascular dementia, Lewy body dementia, frontotemporal dementia and the like. Cerebrovascular dementia is caused by arteriosclerosis, where cholesterol accumulates in the blood vessels of the brain and hardens the blood vessels. There is no excellent therapeutic agent for the treatment of dementia, and the current situation is to suppress the progression of symptoms and maintain daily life in combination with reminiscence and exercise therapy.
アルツハイマー病の神経病理学的特徴は、大脳皮質や海馬での神経原線維変性、老人斑と大量の神経細胞脱落である。神経原線維変性は微小管結合タンパクの１つであるタウタンパクが過剰にリン酸化され線維封入体となったもので、老人斑はアミロイドβタンパクの細胞外蓄積である。アルツハイマー病ではこのアミロイドβタンパクの蓄積が神経原線維変性を加速し、繊維化したタウタンパクは細胞内輸送を阻害する。さらにはアミロイドβタンパク自体がシナプスの機能障害などの細胞毒性を有する。 The neuropathological features of Alzheimer's disease are neurofibrillary degeneration in the cerebral cortex and hippocampus, senile plaques and massive neuronal loss. In neurofibrillary degeneration, tau protein, which is one of microtubule-binding proteins, is excessively phosphorylated to form fiber inclusions, and senile plaques are extracellular accumulation of amyloid β protein. In Alzheimer's disease, this accumulation of amyloid β protein accelerates neurofibrillary degeneration, and fibrotic tau protein inhibits intracellular transport. Furthermore, amyloid β protein itself has cytotoxicity such as synaptic dysfunction.
神経細胞（ニューロン）は、細胞体（脳細胞）、樹状突起、軸策、シナプスで構成されている。ニューロンはシナプス結合を介して信号伝達しており、ニューロンを同時刺激することにより、2つのニューロン間の信号伝達が持続的に向上する。このことを、神経科学の分野において、長期増強（LTP）という。記憶はこのシナプスに貯えられているとみられているので、長期増強は記憶の主要なメカニズムであると広く考えられている。特に、海馬LTPの機構解明が進んでおり、シナプス後細胞にカルシウムイオンが流入すること、このカルシウムの流入がグルタミン酸受容体の一種のNMDA受容体を介して行なわれることが認められている。 A neuron (neuron) is composed of a cell body (brain cell), a dendrite, an axon, and a synapse. Neurons transmit signals through synaptic connections, and by simultaneously stimulating neurons, signal transmission between the two neurons is continuously improved. This is called long-term potentiation (LTP) in the field of neuroscience. Since memory is thought to be stored at this synapse, long-term potentiation is widely considered to be the primary mechanism of memory. In particular, elucidation of the mechanism of hippocampal LTP is progressing, and it is recognized that calcium ions flow into post-synaptic cells, and that this calcium influx is performed through a kind of NMDA receptor of glutamate receptors.
このNMDA受容体は静止膜電位時にマグネシウムによってブロックされ、シナプス後細胞へのカルシウムの流入が阻害されているが、脱分極によりマグネシウムのブロックから開放され、受容体にグルタミン酸が結合した際に、カルシウムの細胞内への流入が起きてLTPが成立する（記憶の成立）。そして、長期記憶は、数分以内に成立するLTPに引き続いて伝達効率の増大状態を長期維持する相であり、タンパクの新合成を伴い、ニューロンの形態変化を介して、シナプスの新生が起こるのだろうと考えられている。 This NMDA receptor is blocked by magnesium at resting membrane potential, and the influx of calcium into post-synaptic cells is inhibited, but when released from magnesium block by depolarization and glutamate binds to the receptor, calcium is bound Flows into the cell and LTP is established (establishment of memory). And long-term memory is a phase in which the state of increased transmission efficiency is maintained for a long time following LTP that is established within a few minutes, accompanied by new protein synthesis, and the formation of synapses through neuronal morphological changes. It is thought that.
このように、記憶・学習行動などヒトの脳機能を実現するために、神経回路が物理的・生理的に、その性質を変化できることをシナプスの可塑性といい、アポトーシスによるニューロンの減少と発芽によるシナプス接合部の増加という物理的な変化とLTPにより信号の通りがよくなるという生理的な変化が含まれる。 Synaptic plasticity is the ability of a neural circuit to change its physical and physiological properties in order to realize human brain functions such as memory and learning behavior, and it is called synaptic plasticity. This includes physical changes such as an increase in joints and physiological changes in which signals are improved by LTP.
神経線維の電気信号の発生は、ニューロンを取り巻く膜の内側と外側に存在するイオンのアンバランスによるもので、細胞の内側にはカリウムイオンが多く、外側には主としてナトリウムイオンと塩素イオンが多く分布しており、細胞内外の濃度差で生じる電位差に起因するものである。このイオンの濃度勾配で生じ電位差が静止電位であり、この状態を膜が分極しているという。 The generation of electrical signals in nerve fibers is due to an imbalance of ions existing inside and outside the membrane surrounding the neuron, and there are many potassium ions inside the cell and mainly sodium ions and chloride ions on the outside. This is due to a potential difference caused by a difference in concentration inside and outside the cell. The potential difference generated by this ion concentration gradient is a static potential, and this state is said to be polarized.
この電位差が刺激によって一時的に逆転（脱分極）する現象が活動電位であり、インパルスやスパイクとも呼ばれる。発生した活動電位はシナプスに向かって軸索方向に伝播され、プレシナプスに達するとカルシウムチャネルが開いてカルシウムイオンが流入し、神経伝達物質がシナプス間隙に放出することにより情報が伝達される。以上のようにして電気インパルスは神経細胞間を伝達され、同様の過程が多数のニューロンで繰り返し行われる。これが神経細胞の情報伝達処理の概観である。 A phenomenon in which this potential difference is temporarily reversed (depolarized) by a stimulus is an action potential, which is also called an impulse or a spike. The generated action potential is propagated toward the synapse in the axon direction. When the presynapse is reached, the calcium channel opens, calcium ions flow in, and neurotransmitters are released into the synaptic gap to transmit information. As described above, electrical impulses are transmitted between nerve cells, and the same process is repeated in many neurons. This is an overview of information transmission processing of nerve cells.
神経細胞の活動が円滑に維持されるためには、静止膜電位を深く維持する必要がある。そのためには細胞外のカリウムイオンを低濃度に保つ必要がある。そして、細胞外のカリウムイオンを保つ要因として、(1)ATP生成とNaＫ-ATPアーゼ活性、(2)アストロサイトのカリウムイオンの取り込みが挙げられる。 In order to maintain the activity of nerve cells smoothly, it is necessary to maintain the resting membrane potential deeply. For this purpose, it is necessary to keep extracellular potassium ions at a low concentration. Factors that maintain extracellular potassium ions include (1) ATP production and NaK-ATPase activity, and (2) uptake of potassium ions in astrocytes.
ATPは生命活動におけるエネルギー源であり、基礎代謝におけるATP消費の40％がNaK-ATPアーゼで、その内訳は脳が13％、腎が10％を占めることが知られている。そして細胞内ATPが、例えば筋肉の収縮、生体物質の生合成、イオン輸送などを起こす際にエネルギーとして重要な働きを果たしている。 ATP is a source of energy in life activities, and 40% of ATP consumption in basal metabolism is NaK-ATPase, which is known to account for 13% of the brain and 10% of the kidney. Intracellular ATP plays an important role as energy, for example, in causing muscle contraction, biosynthesis of biological materials, ion transport, and the like.
また、虚血などの酸素欠乏によりATP産生不足が起こるとカリウムイオン濃度が上昇することになる。これは、ATP不足でNaK-ATPアーゼ機能が低下するためである。そして、細胞外にカリウムイオン濃度が上昇すると脱分極を誘導し、細胞内カルシウムイオン濃度が上昇してアポトーシスによる神経細胞死が生じることになる。 In addition, when ATP production deficiency occurs due to oxygen deficiency such as ischemia, the potassium ion concentration increases. This is because NaK-ATPase function decreases due to lack of ATP. And if potassium ion concentration rises outside a cell, depolarization will be induced, intracellular calcium ion concentration will rise, and the neuronal cell death by apoptosis will arise.
一方、認知機能低下ではNaK-ATPアーゼが低下することも知られており、細胞外にカリウムイオンが停滞することが認知機能低下の一因であることが示唆される。 On the other hand, it is also known that NaK-ATPase decreases in cognitive decline, suggesting that stagnation of potassium ions outside the cell is one cause of cognitive decline.
ATP産生はミトコンドリアが担っており、神経細胞への酸素及び基質の供給が必要である。虚血によりこれらの供給が停止すると、嫌気的な反応が促進され、細胞内に乳酸と水素イオン濃度が上昇する。細胞内の水素イオンの増加は細胞膜上のNa-H交換系を介して細胞外へ水素イオンをくみ出すと同時に細胞内にナトリウムイオン流入を引き起こす。これによりナトリウムイオンの過負荷が誘発されると、Na-Ca交換系を介してカルシウムイオンの流入しカルシウムイオンの過負荷が誘導される。この過負荷により、膜タンパクやミトコンドリアの変性を引き起こすことになる。 ATP production is carried out by mitochondria, and it is necessary to supply oxygen and substrates to nerve cells. When these supplies are stopped by ischemia, an anaerobic reaction is promoted, and the concentration of lactic acid and hydrogen ions in the cells increases. The increase in intracellular hydrogen ions pumps out hydrogen ions through the Na-H exchange system on the cell membrane and simultaneously causes sodium ions to flow into the cell. As a result, when sodium ion overload is induced, calcium ions flow in via the Na-Ca exchange system, and calcium ion overload is induced. This overload causes degeneration of membrane proteins and mitochondria.
ミトコンドリアは細胞質のカルシウムイオン濃度を低下させようとカルシウムイオンを取り込むが、ミトコンドリア内に大量のカルシウムイオン蓄積の結果、ミトコンドリア膜電位の低下をまねく。ミトコンドリア内膜の膜電位は約-180mVで、この値は細胞膜の約-90mVよりも低値となる。ミトコンドリア内膜の電子伝達系はミトコンドリア内膜を境界にした電気的勾配を作り出し、それを起電力にしてF1/F0ATPアーゼでADPをATPへと変換する。この膜電位の低下はATP産生能の低下を意味するものであり、細胞内のカルシウムイオンやナトリウムイオンの上昇はミトコンドリアのエネルギー産生能の低下を引き起こすことになる。 Mitochondria take up calcium ions to reduce the cytoplasmic calcium ion concentration, but as a result of the accumulation of a large amount of calcium ions in the mitochondria, the mitochondrial membrane potential decreases. The membrane potential of the inner mitochondrial membrane is about -180 mV, which is lower than about -90 mV of the cell membrane. The electron transport system of the inner mitochondrial membrane creates an electrical gradient bounded by the inner mitochondrial membrane, which is used as an electromotive force to convert ADP to ATP by F1 / F0ATPase. This decrease in membrane potential means a decrease in ATP production ability, and an increase in intracellular calcium ions and sodium ions causes a decrease in mitochondrial energy production ability.
アデニル酸キナーゼ(AK)は、エネルギー代謝に必要不可欠の酵素で、マグネシウムイオンの存在下、ATPとADPから、ADPを2分子生成するリン酸転移反応を可逆的に触媒し、細菌
から哺乳動物まで広く存在する酵素である。哺乳動物には、AK1、AK2、AK3の3種類があり、AK1は細胞質に存在し、骨格筋、脳及び赤血球に見出される。AK2はミトコンドリア膜間スペースに、AK3はミトコンドリアマトリックスに存在し、肝臓や腎臓にみられる。
Adenylate kinase (AK) is an enzyme that is indispensable for energy metabolism, and in the presence of magnesium ions, reversibly catalyzes the phosphotransfer reaction that produces two molecules of ADP from ATP and ADP, from bacteria to mammals. It is a widely existing enzyme. There are three types of mammals, AK1, AK2, and AK3. AK1 is present in the cytoplasm and is found in skeletal muscle, brain and erythrocytes. AK2 is present in the mitochondrial intermembrane space and AK3 is present in the mitochondrial matrix and is found in the liver and kidney.
哺乳類の場合、通常細胞内のAMP濃度は、ATP濃度の約1/100、ADP濃度の約1/10程度でほぼ平衡に達しており、ATPが枯渇するより前にエネルギー産生を亢進させるネガティブフィードバック制御が、効果的に働くことになる。AKはATPが枯渇したときにATPを生成することで虚血による細胞死を抑制することが知られており、AK2虚血耐性遺伝子として低酸素／再酸素化ストレスにおいて細胞死抑制効果があることが分かってきている。 In mammals, the intracellular AMP concentration is almost equilibrated at approximately 1/100 of the ATP concentration and approximately 1/10 of the ADP concentration, and negative feedback that enhances energy production before ATP is depleted. Control will work effectively. AK is known to suppress cell death due to ischemia by producing ATP when ATP is depleted, and as an AK2 ischemic resistance gene, it has a cell death suppressing effect in hypoxia / reoxygenation stress I know.
また、血流が乏しく低グルコースのような飢餓条件に暴露されると酸化的リン酸化にスイッチングが起こり、より効果的にATPを作り出すためにAMPキナーゼ（AMP依存性タンパク質キナーゼ）の活性が高まり適応を図るようになるが、AMPキナーゼはLKB1によってリン酸化されて活性化することが知られている。 In addition, oxidative phosphorylation is switched when exposed to starvation conditions such as low glucose and poor blood flow, and the activity of AMP kinase (AMP-dependent protein kinase) is increased and adapted to produce ATP more effectively. However, AMP kinase is known to be phosphorylated and activated by LKB1.
ATPは膜輸送体のエネルギー源であり、イオン輸送性ATPアーゼやABC(ATP-Binding Cassette)輸送体により、イオン、アミノ酸やペプチドなどが輸送される。認知症との関連性が知られているアミロイドβの除去に関与する脳内HDL産生にはApoE及びABCA1が関与すること考えられており、ABCA1欠損によりアミロイドβの脳内沈着が加速することも報告されている（非特許文献１）ことから、アストロサイトで新生された未成熟なHDLにABC輸送体を介してコレステロールが取り込まれ成熟することにより、アミロイドβのクリアランス機能が向上すると考えられる。このように、アミロイドβもATP加水分解に依存して脳から排泄されるといえる。 ATP is an energy source for membrane transporters, and ions, amino acids, peptides, and the like are transported by ion transporting ATPases and ABC (ATP-Binding Cassette) transporters. ApoE and ABCA1 are thought to be involved in HDL production in the brain involved in the removal of amyloid β, which is known to be associated with dementia, and ABCA1 deficiency may accelerate the deposition of amyloid β in the brain It has been reported (Non-Patent Document 1) that it is considered that the clearance function of amyloid β is improved by incorporating cholesterol into immature HDL born in astrocytes via the ABC transporter and maturing. Thus, it can be said that amyloid β is also excreted from the brain depending on ATP hydrolysis.
それゆえに、神経細胞の活動が円滑に維持されるためには、細胞外のカリウムイオンを低濃度に保ち静止膜電位を深く維持する必要がある。そのためには、AKの賦活化或いはミトコンドリアにおけるATP生成低下を抑制することにより、細胞膜NaK-ATPアーゼなどのATPアーゼを活性化することが大切である。 Therefore, in order to maintain the activity of nerve cells smoothly, it is necessary to maintain a low concentration of extracellular potassium ions and a deep resting membrane potential. For this purpose, it is important to activate ATPases such as cell membrane NaK-ATPase by inhibiting AK activation or reducing ATP production in mitochondria.
一方、アルツハイマーのような認知症でみられる神経細胞死（アポトーシス）において、過剰なグルタミン酸によるNMDA受容体の過剰な活性化や細胞内カルシウムの急激な上昇が主要な原因であることがよく知られている。 On the other hand, neuronal cell death (apoptosis) seen in dementia such as Alzheimer is well known to be caused mainly by excessive activation of NMDA receptor and rapid increase of intracellular calcium by excessive glutamate. ing.
このような過剰なグルタミン酸は、認知症でみられるような虚血での低酸素と低グルコースにより起こり得ることが示唆される。ATPが枯渇すると、ナトリウムポンプ活性が低下し、細胞内ナトリウムイオンが増加、細胞外カリウムイオンが増加し、膜電位の脱分極が生ずる。 It is suggested that such excess glutamate can be caused by hypoxia and low glucose in ischemia as seen in dementia. When ATP is depleted, sodium pump activity decreases, intracellular sodium ions increase, extracellular potassium ions increase, and membrane potential depolarization occurs.
ニューロンやアストロサイトのグルタミン酸トランスポーターは負の膜電位（細胞外ナトリウム高濃度）により促進されるので、細胞外カリウムイオンが増加して脱分極することにより、グルタミン酸トランスポーターの機能が低下し、グルタミン酸が細胞外に貯留することになる。また、脱分極により、電位依存性カルシウムチャネルからカルシウムイオンが細胞内に流入して、細胞内のカルシウム依存性プロテアーゼが活性化され、アポトーシスが起きる。 Since glutamate transporters of neurons and astrocytes are promoted by negative membrane potential (high extracellular sodium concentration), the function of glutamate transporters decreases due to depolarization due to the increase of extracellular potassium ions, and glutamate Will accumulate outside the cell. In addition, due to depolarization, calcium ions flow from the voltage-dependent calcium channel into the cell, the intracellular calcium-dependent protease is activated, and apoptosis occurs.
一方、細胞内のカルシウムイオンは、形質膜や小胞体に存在するCa-ATPアーゼ（Caポンプ）やNa-Ca交換輸送系により、定常的に低く抑えられている。ニューロンのエネルギー代謝の低下は、これらの機能も低下させる。カルシウムイオンの上昇がアポトーシスの直接的な原因となる。 On the other hand, intracellular calcium ions are constantly kept low by the Ca-ATPase (Ca pump) and Na-Ca exchange transport system present in the plasma membrane and endoplasmic reticulum. Reduced neuronal energy metabolism also reduces these functions. Increased calcium ions are a direct cause of apoptosis.
それゆえに、カリウムイオンの恒常性維持は、認知症や総合失調症のような神経疾患の治療に重要である。
以上のように、正常な神経活動としてニューロンを維持し、記憶を成立させるべくニューロンの新生を行なうためには、脳内の神経細胞、毛細血管やアストロサイトからなる微小環境が重要ある。即ち、細胞外のカリウムイオンを低濃度に保ち静止膜電位を深く維持すること、そのためにはAKの賦活化或いはミトコンドリアにおけるATP生成低下を抑制することは、細胞外環境の恒常性を維持してアポトーシスを抑制する。即ち、ニューロンを保護することであり、認知症の予防や治療に有用であると考えられる。
Therefore, maintenance of potassium ion homeostasis is important for the treatment of neurological diseases such as dementia and schizophrenia.
As described above, in order to maintain neurons as normal nerve activity and to regenerate neurons in order to establish memory, a microenvironment composed of nerve cells, capillaries and astrocytes in the brain is important. That is, maintaining a low concentration of extracellular potassium ions and maintaining the resting membrane potential deeply, and therefore suppressing AK activation or reducing ATP production in mitochondria maintains the constancy of the extracellular environment. Inhibits apoptosis. That is, it protects neurons and is considered useful for prevention and treatment of dementia.
例えば、認知症の治療薬としては、コリンエステラーゼ阻害薬、NMDA受容体拮抗薬やアミロイドβの産生・代謝に関与する酵素阻害薬及び免疫療法などが開発されてきている。例えば、軽度から中程度のアルツハイマー病に対しての第一選択薬はコリンエステラーゼ阻害薬の単剤使用であるが、副作用等により使用継続が困難であるか臨床効果がみられない場合は、別のコリンエステラーゼ阻害薬に変更したり、あるいはNMDA阻害薬の使用を考慮することが推奨されているが、これらは対症療法的な治療手段であるため、薬理効果と副作用のモニタリングを行いながら治療継続する必要がある。この治療反応性は、認知、ADL、行動など多角的な見地から評価されるべきものとされている。 For example, as therapeutic agents for dementia, cholinesterase inhibitors, NMDA receptor antagonists, enzyme inhibitors involved in amyloid β production / metabolism, immunotherapy, and the like have been developed. For example, the first-line drug for mild to moderate Alzheimer's disease is the use of a single cholinesterase inhibitor, but if it is difficult to continue using due to side effects or if there is no clinical effect, It is recommended to switch to cholinesterase inhibitors or consider using NMDA inhibitors, but these are symptomatic treatment measures, so it is necessary to continue treatment while monitoring pharmacological effects and side effects There is. This treatment responsiveness should be evaluated from various viewpoints such as cognition, ADL, and behavior.
一方、認知症の改善の目的で、脳血流を改善する方法が種々提案されており、例えば、脳血流を促進する物質として、イフェンプロジル、シンナリジン、ニセルゴリン、ビンポセチン、ビンカミン、エストロゲン、イチョウ葉、丹参などが知られているが、満足できる効果は得られていない。 On the other hand, various methods for improving cerebral blood flow have been proposed for the purpose of improving dementia.For example, as substances that promote cerebral blood flow, ifenprodil, cinnarizine, nicergoline, vinpocetine, vincamine, estrogen, ginkgo leaves, Tansan etc. are known, but a satisfactory effect has not been obtained.
また、脳血流を改善する手段として、人尿性キニナーゼを有効成分とする脳機能改善剤（特許文献１）、エンドセリン変換酵素阻害剤（特許文献２）、ムラサキイモ由来の血液循環改善剤（特許文献３）、イソロイシン、ロイシン及びバリンを有効成分とする医薬組成物（特許文献４）、コリンエステラーゼ阻害剤と加味温胆湯との併用（特許文献５）、アンジオテンシン２受容体阻害剤を有効成分とするアルツハイマー病の予防又は治療のための医薬（特許文献６）が提案されている。 In addition, as means for improving cerebral blood flow, a brain function improving agent containing human urinary kininase as an active ingredient (Patent Document 1), an endothelin converting enzyme inhibitor (Patent Document 2), a blood circulation improving agent derived from Murasaki potato ( Patent document 3), pharmaceutical composition containing isoleucine, leucine and valine as active ingredients (patent document 4), combined use of cholinesterase inhibitor and Kamisento (patent document 5), angiotensin 2 receptor inhibitor as active ingredient A pharmaceutical (Patent Document 6) for preventing or treating Alzheimer's disease is proposed.
また、脳虚血疾患の改善として、システインプロテアーゼインヒビター（特許文献７）、Ｌ−ブチルフタリド（特許文献８）が提案されている。また、神経伝達機能改善剤としてセレノシステイン含有タンパク質（特許文献９）、神経成長刺激としてＲｈｏ−キナーゼ阻害剤（特許文献１０）、細胞増殖因子（特許文献１１）、ＮＭＤＡ受容体活性化のためグリシントランスポーター阻害剤（特許文献１２）やシンナミド化合物の併用（特許文献１３）、アミロイドβ抑制のための多価不飽和脂肪酸（特許文献１４）、丹参由来成分（特許文献１５）やリポタンパク質関連ホスホリパーゼＡ２阻害剤（特許文献１６）が提案されている。しかし、これらの提案はいずれも根本的な治療手段ではないので、満足する効果が得られていないのが現状である。 As an improvement of cerebral ischemic disease, cysteine protease inhibitor (Patent Document 7) and L-butylphthalide (Patent Document 8) have been proposed. In addition, a selenocysteine-containing protein (Patent Document 9) as a neurotransmitter function improving agent, a Rho-kinase inhibitor (Patent Document 10), a cell growth factor (Patent Document 11) as a nerve growth stimulus, and a glycine for NMDA receptor activation Combined use of a transporter inhibitor (Patent Document 12) and a cinnamide compound (Patent Document 13), a polyunsaturated fatty acid for suppressing amyloid β (Patent Document 14), a component derived from Tansan (Patent Document 15) and a lipoprotein-related phospholipase An A2 inhibitor (Patent Document 16) has been proposed. However, since none of these proposals is a fundamental therapeutic measure, the present situation is that a satisfactory effect has not been obtained.
AK賦活化は、神経細胞の細胞死を原因とする疾患、例えばアルツハイマー、認知症、筋萎縮性側索硬化症、パーキンソン病等に有用と考えられるが、AK賦活化によりATP生成を促進し、細胞外カリウムイオンが生理的な状態より高濃度の条件で生じる神経細胞死を抑制する物質は知られていなかった。更に、このようなAK活性化やAK酵素タンパク質を増加させるAK賦活作用を有する物質は、神経細胞死を原因とする疾患以外にも、脳梗塞などの虚血性脳機能障害の予後に起きる脳神経細胞死、狭心症や心筋梗塞のような虚血性心疾患、虚血性大腸炎や虚血性視神経症などの虚血性疾患の治療や予防に有用であると考えられる。 AK activation is thought to be useful for diseases caused by neuronal cell death, such as Alzheimer, dementia, amyotrophic lateral sclerosis, Parkinson's disease, etc., but AK activation promotes ATP production, There has been no known substance that suppresses neuronal cell death caused by extracellular potassium ions at a higher concentration than the physiological state. Furthermore, such substances having AK activation action that increases AK activation and AK enzyme protein are not only diseases caused by neuronal cell death, but also cranial nerve cells that occur in prognosis of ischemic brain dysfunction such as cerebral infarction. It is considered useful for the treatment and prevention of ischemic heart diseases such as death, angina pectoris and myocardial infarction, ischemic diseases such as ischemic colitis and ischemic optic neuropathy.
また、AMPキナーゼは、蛋白合成、ミトコンドリア生合成、アポトーシス、オートファ
ジーといった多彩な機能に関わり、認知症、アルツハイマーに代表される神経変性疾患、糖尿病のような代謝疾患、癌、心疾患、脳卒中など多くの疾患との関わりも指摘されている（非特許文献２、３）。
AMP kinase is involved in various functions such as protein synthesis, mitochondrial biogenesis, apoptosis, and autophagy. Dementia, neurodegenerative diseases represented by Alzheimer, metabolic diseases such as diabetes, cancer, heart disease, stroke, etc. The relationship with many diseases has also been pointed out (Non-patent Documents 2 and 3).
本発明は、細胞外カリウムイオンが滞留して生じる細胞死に対して、細胞内ATP不足を補い細胞死を抑制することにより、（１）神経細胞死を原因とするアルツハイマー、認知症、筋萎縮性側索硬化症（ALS）等や、（２）脳梗塞などの虚血性脳機能障害の予後に起きる脳神経細胞死、（３）狭心症、心筋梗塞のような虚血性疾患、（４）糖尿病のような代謝疾患、（５）癌、（６）脳卒中の予防・治療に有効な物質を提供することを目的とする。 In the present invention, cell death caused by retention of extracellular potassium ions is compensated for by lack of intracellular ATP, thereby suppressing cell death. (1) Alzheimer, neuropathy caused by neuronal cell death, dementia, muscle atrophy Lateral sclerosis (ALS), etc. (2) Cerebral nerve cell death that occurs in the prognosis of ischemic cerebral dysfunction such as cerebral infarction, (3) Ischemic disease such as angina pectoris and myocardial infarction, (4) Diabetes It is an object of the present invention to provide a substance effective for the prevention and treatment of metabolic diseases such as (5) cancer and (6) stroke.
神経細胞の活動が円滑に維持されるためには、細胞外のカリウムイオンを低濃度に保ち静止膜電位を深く維持する必要がある。しかし、細胞外カリウムイオンが滞留して生じる細胞死に対して、細胞内ATP不足を補い細胞死を抑制する物質は知られていなかった。 In order to maintain the activity of nerve cells smoothly, it is necessary to maintain a low concentration of extracellular potassium ions and to maintain a deep resting membrane potential. However, a substance that compensates for the lack of intracellular ATP and suppresses cell death against cell death caused by retention of extracellular potassium ions has not been known.
そこで本発明者らは上記課題を解決するため鋭意研究を行ったところ、オイデスマン型セスキテルペノイド誘導体に、細胞外カリウムイオン濃度が上昇して生じる神経細胞死に対する抑制作用があることを見出した。 Thus, the present inventors conducted extensive research to solve the above problems, and found that the Eudesman sesquiterpenoid derivative has an inhibitory effect on neuronal cell death caused by an increase in extracellular potassium ion concentration.
前記オイデスマン型セスキテルペノイド誘導体は、細胞外カリウムイオン濃度の上昇で生じる神経細胞死に対する抑制作用を有し、細胞外カリウムイオン濃度の上昇で起こるAT
P産生低下を抑制することが確認された。即ち、細胞外カリウムイオン濃度が上昇して起こるATP産生低下に対して、ATP不足を補い、それにより細胞外カリウムイオンを低濃度に保ち、細胞外にカリウムイオンが停滞して生じる細胞死を抑制する。これにより、神経細胞死を原因とするアルツハイマー、認知症、筋萎縮性側索硬化症や、脳梗塞などの虚血性脳機能障害の予後に起きる脳神経細胞死、狭心症、心筋梗塞のような虚血性心疾患、虚血性大腸炎や虚血性視神経症などの虚血性疾患、糖尿病のような代謝疾患、癌、脳卒中の予防・治療に有効であることが示唆された。
The Eudesman-type sesquiterpenoid derivative has an inhibitory effect on neuronal cell death caused by an increase in extracellular potassium ion concentration, and is caused by an increase in extracellular potassium ion concentration.
It was confirmed that the decrease in P production was suppressed. In other words, ATP deficiency caused by an increase in extracellular potassium ion concentration compensates for ATP deficiency, thereby maintaining a low concentration of extracellular potassium ion and suppressing cell death caused by stagnation of potassium ion outside the cell. To do. As a result, neuronal cell death caused by Alzheimer, dementia, amyotrophic lateral sclerosis, and the prognosis of ischemic brain dysfunction such as cerebral infarction It was suggested that it is effective for the prevention and treatment of ischemic heart disease, ischemic diseases such as ischemic colitis and ischemic optic neuropathy, metabolic diseases such as diabetes, cancer, and stroke.
すなわち、本発明は、下記の一般式（１）、（２）又は（３）で示されるオイデスマン型セスキテルペノイド誘導体を有効成分とする高カリウム環境下での細胞死抑制組成物を提供する。
That is, the present invention provides a composition for inhibiting cell death in a high potassium environment containing an Eudesman sesquiterpenoid derivative represented by the following general formula (1), (2) or (3) as an active ingredient.
また、本発明は、前記オイデスマン型セスキテルペノイド誘導体が、アトラクチレノリドＩＩＩ、アトラクチレノリドＩＩ、β−オイデスモール、アトラクチレノリドＩのいずれか一つ以上である細胞死抑制組成物を提供する。 The present invention also provides a cell death inhibiting composition, wherein the Eudesman sesquiterpenoid derivative is one or more of atractylenolide III, atractylenolide II, β-eudesmol, and atractylenolide I. provide.
また、本発明は、前記組成物が、生薬または生薬エキスを有効成分とする細胞死抑制組成物を提供する。 In addition, the present invention provides a cell death inhibiting composition, wherein the composition comprises a crude drug or a crude drug extract as an active ingredient.
また、本発明は、前記生薬が白朮又は蒼朮である細胞死抑制組成物を提供する。 In addition, the present invention provides a cell death suppressing composition in which the herbal medicine is white cocoon or cocoon.
また、本発明は、前記細胞死抑止組成物を有効成分とする細胞賦活剤を提供する。 Moreover, this invention provides the cell activator which uses the said cell death suppression composition as an active ingredient.
また、本発明は、前記細胞死抑止組成物を有効成分とする細胞死に起因する疾患の治療剤を提供する。 Moreover, this invention provides the therapeutic agent of the disease resulting from the cell death which uses the said cell death suppression composition as an active ingredient.
また、本発明は、前記細胞死に起因する疾患が、アルツハイマー、認知症、筋萎縮性側索硬化症、パーキンソン病、脳梗塞、脳虚血性疾患、狭心症、心筋梗塞、虚血性心疾患、虚血性大腸炎、虚血性視神経症、虚血性疾患、代謝疾患、癌、脳卒中のいずれか一つ以上である治療剤を提供する。 In the present invention, the disease caused by cell death is Alzheimer, dementia, amyotrophic lateral sclerosis, Parkinson's disease, cerebral infarction, cerebral ischemic disease, angina pectoris, myocardial infarction, ischemic heart disease, Provided is a therapeutic agent that is any one or more of ischemic colitis, ischemic optic neuropathy, ischemic disease, metabolic disease, cancer, and stroke.
本発明により神経細胞死を原因とするアルツハイマー、認知症、筋萎縮性側索硬化症や、脳梗塞などの虚血性脳機能障害の予後に起きる脳神経細胞死、狭心症、心筋梗塞のような虚血性心疾患、虚血性大腸炎や虚血性視神経症などの虚血性疾患、糖尿病のような代謝疾患、癌、脳卒中の予防及び治療に有効である細胞死抑制組成物を提供する。 According to the present invention, Alzheimer's disease caused by neuronal cell death, dementia, amyotrophic lateral sclerosis, brain neuronal cell death caused by ischemic brain dysfunction such as cerebral infarction, angina pectoris, myocardial infarction, etc. Disclosed is a cell death inhibiting composition that is effective for the prevention and treatment of ischemic heart disease, ischemic diseases such as ischemic colitis and ischemic optic neuropathy, metabolic diseases such as diabetes, cancer, and stroke.
以下、本発明についてさらに詳細に説明する。 Hereinafter, the present invention will be described in more detail.
本発明の細胞死抑制剤の有効成分としては、オイデスマン型セスキテルペノイド誘導体（１）、（２）又は（３）を含むものであれば特に制限はない。 The active ingredient of the cell death inhibitor of the present invention is not particularly limited as long as it contains Eudesman sesquiterpenoid derivative (1), (2) or (3).
具体的には、オイデスマン型セスキテルペノイド誘導体の一般式（１）でＲがＨのアトラクチレノリドＩＩ、ＲがＯＨのアトラクチレノリドＩＩＩ、一般式（２）のβ−オイデスモール、一般式（３）のアトラクチレノリドＩを挙げることができる。 Specifically, the formula (1) of the Eudesman-type sesquiterpenoid derivative in which R is H atractylenolide II, R is OH atractylenolide III, β-eudesmol of the general formula (2), general formula Atractylenolide I of (3) can be mentioned.
そして、オイデスマン型セスキテルペノイド誘導体のアトラクチレノリドＩ、アトラクチレノリドＩＩ、アトラクチレノリドＩＩＩは生薬の白朮、β−オイデスモールは生薬の蒼朮を、それぞれ粉砕して得られる生薬末として使用することができる。 The Eudesmann sesquiterpenoid derivatives atractylenolide I, atractylenolide II, atractylenolide III are used as a crude drug powder obtained by pulverizing a crude drug white cocoon, and β-eudesmol is used as a crude drug powder obtained by grinding. can do.
更に、原料生薬を極性または非極性溶媒を用いて常法によって抽出して得られる抽出液、濃縮液及び乾燥物のような生薬エキスとして使用することができる。抽出方法としては、加熱抽出、加温抽出、低温抽出などが挙げられる。また、抽出溶媒は、水の他に、エタノール、酢酸エチルやアセトンなどの有機溶媒が挙げられ、それらを単独で用いるか、または２種類以上を適宜混合して使用することができる。 Furthermore, raw material crude drugs can be used as crude drug extracts such as extracts, concentrated liquids, and dried products obtained by extraction by a conventional method using a polar or nonpolar solvent. Examples of extraction methods include heat extraction, warm extraction, and low temperature extraction. In addition to water, organic solvents such as ethanol, ethyl acetate, and acetone can be used as the extraction solvent, and these can be used alone or in combination of two or more.
具体的な生薬エキスの調製例としては、前記生薬をその重量に対して５〜２５倍量、好ましくは８〜２０倍量の水を加えて、通常８０〜１００℃で３０分間〜２時間加熱してエキスを煎出し、熱水抽出分離することにより得られる。この熱水抽出液は、必要に応じて減圧濃縮して濃縮エキスとし、さらに、噴霧乾燥、減圧濃縮乾燥、凍結乾燥等により乾燥エキス粉末とすることもできる。 As a specific preparation example of a crude drug extract, the above-mentioned crude drug is added in an amount of 5 to 25 times, preferably 8 to 20 times the amount of water, and heated usually at 80 to 100 ° C. for 30 minutes to 2 hours. The extract is then brewed and extracted by hot water extraction. This hot water extract is concentrated under reduced pressure to obtain a concentrated extract, if necessary, and can also be formed into a dry extract powder by spray drying, vacuum concentration drying, freeze drying, or the like.
また、得られた濃縮エキスを、種々カラムクロマトグラフィー、イオン交換クロマトグラフィー、高速液体クロマトグラフィー等の適当な分離精製手段を用いて本発明の化合物を単離することもできる。 In addition, the compound of the present invention can be isolated from the obtained concentrated extract using appropriate separation and purification means such as various column chromatography, ion exchange chromatography, and high performance liquid chromatography.
有効量は０．１ｐＭ以上であることが望ましい。経口剤としての有効量は、患者の年齢、体重、疾患の程度によって異なるが、通常成人で本発明の化合物として、０．１ｍｇ〜５００ｍｇ、好ましくは１ｍｇ〜１００ｍｇを、１日数回に分けての服用が適当である。 The effective amount is desirably 0.1 pM or more. The effective amount as an oral preparation varies depending on the age, weight, and degree of disease of the patient, but it is usually 0.1 mg to 500 mg, preferably 1 mg to 100 mg, divided into several times a day as a compound of the present invention in an adult. It is appropriate to take.
このようにして得られたエキス粉末や本発明の化合物はそのままの形で使用することもできるが、通常、食品及び／又は医薬品に使用される通常の賦形剤（例えば、結晶セルロース、ショ糖脂肪酸エステル、白糖等）を加え、例えば、乾式造粒法或は湿式造粒法により造粒して製造し、このようにした造粒物をそのまま使用することもできるが、それらをさらに打錠機を用いた圧縮成形物として使用することもできる。 The extract powder thus obtained and the compound of the present invention can be used as they are, but are usually used as usual excipients for food and / or pharmaceuticals (for example, crystalline cellulose, sucrose) Fatty acid ester, sucrose, etc.) and granulated by dry granulation method or wet granulation method, for example, and such granulated product can be used as it is. It can also be used as a compression molded product using a machine.
また、コンプライアンスの観点から服用性を改善するために被覆剤で被覆したフィルムコート剤とすることもできる。また、成分の安定性の点や簡単に摂取できる形態として、粉砕したものをそのまままた上記造粒物をハードカプセルやソフトカプセルに充填し摂取してもよい。 Moreover, it can also be set as the film coat agent coat | covered with the coating agent in order to improve taking property from a viewpoint of a compliance. In addition, as a component stability point and a form that can be easily ingested, the pulverized product may be used as it is or filled with the above granulated product in a hard capsule or soft capsule.
また、抽出したエキスや単離した本発明の化合物に、通常、液状の食品などに使用される甘味料、酸味料、乳化剤、フレーバー、分散助剤などの賦形剤を加えて溶解し、液体の形状として製造することもできる。 In addition, the extracted extract or the isolated compound of the present invention is dissolved by adding excipients such as sweeteners, acidulants, emulsifiers, flavors, and dispersion aids that are usually used in liquid foods, etc. It can also be manufactured as a shape.
以上のような、内服固形の顆粒、錠剤、散剤、液剤の形態だけではなく、半固形状の形態のもの及び、水や湯などに溶解し液状にして用いることができる粉末状の形態などに加工することもできる。 As described above, not only in the form of internal solid granules, tablets, powders, and liquids, but also in semi-solid forms and powdered forms that can be dissolved and used in water or hot water. It can also be processed.
以下、試験例を挙げて本発明をさらに詳細に説明する。 Hereinafter, the present invention will be described in more detail with reference to test examples.
（試験例１）細胞外電解質の神経細胞に対する影響について、次の試験により確認した。
すなわち、PC12細胞をKClによる脱分極後にグルタミン酸を添加したときのATP産生量及び細胞生存率の変化を調べた。
(Test Example 1) The effect of extracellular electrolytes on nerve cells was confirmed by the following test.
That is, changes in ATP production and cell viability when PC12 cells were depolarized with KCl and glutamic acid was added were examined.
（試験方法）
1)神経前駆細胞及び培養
ラット副腎髄質由来褐色細胞腫PC12（独立行政法人理化学研究所 バイオリソースセンターより購入）は、１型コラーゲンでコートした100ｍｍディッシュを用いて、1×106個／ディッシュとなるように播種し、10％熱不活性化ウマ血清及び10％熱不活性化ウシ胎児血清（FBS）を有し、抗生物質（100IU／mlのペニシリン及び100mg／mlのストレプトマイシン）を補足した10mlのDMEM（ダルベッコ変法イーグル培地）中で、5％CO2を含む湿気のある雰囲気中37℃に調整したインキュベーターにより維持した。約7日間でコンフルエントに達した。その間、2回の培地交換を行なった。
(Test method)
1) Neural progenitor cells and culture Rat adrenal medulla-derived pheochromocytoma PC12 (purchased from RIKEN BioResource Center) is 1 × 10 6 cells / dish using a 100 mm dish coated with type 1 collagen 10 ml with 10% heat inactivated horse serum and 10% heat inactivated fetal bovine serum (FBS) supplemented with antibiotics (100 IU / ml penicillin and 100 mg / ml streptomycin) Maintained in DMEM (Dulbecco's Modified Eagle Medium) with an incubator adjusted to 37 ° C. in a humid atmosphere containing 5% CO 2 . It reached confluence in about 7 days. Meanwhile, the medium was changed twice.
2）PC12細胞に対する脱分極剤KCl及び細胞死誘導剤グルタミン酸の添加の影響
PC12細胞をKClの添加により細胞外のカリウムイオン濃度を高めて脱分極をさせた後に、神経細胞興奮因子であるグルタミン酸（L-グルタミン酸）を添加することで細胞死を誘導させることが可能なKCl及びグルタミン酸の添加量の検討を行った。1型コラーゲンでコートした96ウェルプレートに、PC12細胞を1×104個／ウェルで播種し、一晩培養した。その後、各ウェルの培地を吸引し、血清無添加のDMEM培地で希釈した0〜70mMKClを100μl／ウェルずつ添加した。24時間後に、0〜30mMグルタミン酸を100μl／ウェルずつ添加し、ウェルの総量200μl／ウェルとした。そして、インキュベーター内にプレートを静置し、24時間後に細胞内ATP量と細胞生存率の測定を行った。
2) Effects of addition of depolarizing agent KCl and cell death inducer glutamic acid on PC12 cells
KCl is capable of inducing cell death by adding neuronal excitatory factor glutamate (L-glutamate) after depolarizing PC12 cells by increasing extracellular potassium ion concentration by adding KCl And the addition amount of glutamic acid was examined. PC12 cells were seeded at 1 × 10 4 cells / well in a 96-well plate coated with type 1 collagen and cultured overnight. Thereafter, the medium in each well was aspirated, and 0 to 70 mM KCl diluted with serum-free DMEM medium was added at 100 μl / well. After 24 hours, 0-30 mM glutamic acid was added in an amount of 100 μl / well to make a total well volume of 200 μl / well. Then, the plate was allowed to stand in the incubator, and after 24 hours, the amount of intracellular ATP and the cell viability were measured.
3)細胞内ATP量の測定
各ウェルから培養上清を150μlずつ抜き、50μl／ウェルずつATP反応試薬（Cell Titer-GloTM LuminescentCell ViabilityAssay、Promega社製）を添加した。添加後、遮光したプレートを2分間振盪させた後、10分間常温で静置した。その後、プレートをホワイトプレートに移し、ルミノメーター（Centro／CentroXS3 LB960、Berthold Technologys社製
）でルシフェラーゼ発光をATP量として測定した。尚、ATP量は培地のみを添加し試験を行った場合の発光率100％とした。
3) Measurement of intracellular ATP amount 150 μl of the culture supernatant was extracted from each well, and 50 μl / well of ATP reaction reagent (Cell Titer-Glo ™ Luminescent Cell Viability Assay, manufactured by Promega) was added. After the addition, the light-shielded plate was shaken for 2 minutes and then allowed to stand at room temperature for 10 minutes. Thereafter, the plate was transferred to a white plate, and luciferase luminescence was measured as the amount of ATP using a luminometer (Centro / CentroXS3 LB960, manufactured by Berthold Technologies). In addition, the amount of ATP was set to 100% of the luminescence rate when the test was performed with only the medium added.
4)細胞生存率の測定
各ウェルから培養上清をアスピレーターで全て吸引し、DMEM培地で10倍に希釈したCCK8試薬を100μl／ウェル添加し、インキュベーターで4時間反応させた後、吸光度計（Multiskan Spectrum 、Thermo Fisher Scientific社製）で吸光度を測定した。尚、細胞生存率は培地のみを添加し試験を行った場合の細胞内ATP量を100％とした。
4) Measurement of cell viability All culture supernatants were aspirated from each well, CCK8 reagent diluted 10-fold with DMEM medium was added at 100 μl / well, reacted in an incubator for 4 hours, and then absorbance meter (Multiskan Absorbance was measured with Spectrum, manufactured by Thermo Fisher Scientific. The cell survival rate was defined as the amount of intracellular ATP when the test was performed with only the medium added.
（試験結果）
結果を図１及び２に示す。細胞内ATP量は、10mMグルタミン酸のみの刺激に比べて、50mM以上の KClで処理した後にグルタミン酸刺激した方が、ATPの生成を強く抑制することが確認された。一方、細胞生存率は、10mM以上のグルタミン酸のみの刺激に比べて、50mM以上のKCl添加24時間後に10mM以上のグルタミン酸の添加で更に細胞生存率が低下した。以上から、50mMKClにて脱分極させた後に10mMグルタミン酸の添加でATP生成の抑制及び細胞死を誘導することが確認できた。尚、グルタミン酸添加により、培地のpHが低下するが、グルタミン酸20mM以下では細胞生存率に影響がないことを確認した。
(Test results)
The results are shown in FIGS. It was confirmed that the intracellular ATP level strongly suppressed the production of ATP when stimulated with glutamate after treatment with 50 mM or more of KCl, compared to stimulation with 10 mM glutamate alone. On the other hand, the cell viability was further decreased by adding 10 mM or more glutamic acid 24 hours after addition of 50 mM or more of KCl, compared to stimulation with 10 mM or more of glutamic acid alone. From the above, it was confirmed that addition of 10 mM glutamic acid after depolarization with 50 mM KCl induces inhibition of ATP production and cell death. In addition, although the pH of the culture medium was lowered by addition of glutamic acid, it was confirmed that cell viability was not affected at glutamic acid of 20 mM or less.
以上の結果より、PC12細胞をKClによる脱分極後にグルタミン酸を添加したときのATP生成量及び細胞生存率の低下に対する効果の確認する試験方法として、PC12細胞を1×104個／ウェルで播種し、一晩培養後に、血清無添加DMEM培地を用い、55mMKClを100μl／ウェルずつ添加し、24時間後に10mMグルタミン酸を100μl／ウェルずつ添加し、その24時間後に細胞内ATP量及び細胞生存率の測定を行うこととした。 Based on the above results, PC12 cells were seeded at 1 × 10 4 cells / well as a test method to confirm the effects on the decrease in ATP production and cell viability when glutamic acid was added after depolarization with KCl. After overnight culture, use serum-free DMEM medium, add 55 mM KCl 100 μl / well, add 24 mM 10 mM glutamic acid 100 μl / well 24 hours later, and measure intracellular ATP and cell viability 24 hours later It was decided to do.
（試験例２）PC12細胞をKClによる脱分極後にグルタミン酸を添加したときのATP産生量及び細胞生存率の低下に対する本発明の各有効成分の効果を次の試験により確認した。 (Test Example 2) The effect of each active ingredient of the present invention on the decrease in ATP production and cell viability when PC12 cells were depolarized with KCl and glutamic acid was added was confirmed by the following test.
（試験方法）
1)神経前駆細胞及び培養
ラット副腎髄質由来褐色細胞腫PC12（独立行政法人理化学研究所 バイオリソースセンターより購入）は、１型コラーゲンでコートした100ｍｍディッシュを用いて、1×106個／ディッシュとなるように播種し、10％熱不活性化ウマ血清及び10％熱不活性化ウシ胎児血清（FBS）を有し、抗生物質（100IU／mlのペニシリン及び100mg／mlのストレプトマイシン）を補足した10mlのDMEM（ダルベッコ変法イーグル培地）中で、5％CO2を含む湿気のある雰囲気中37℃に調整したインキュベーターにより維持した。約7日間でコンフルエントに達した。その間、2回の培地交換を行なった。
(Test method)
1) Neural progenitor cells and culture Rat adrenal medulla-derived pheochromocytoma PC12 (purchased from RIKEN BioResource Center) is 1 × 10 6 cells / dish using a 100 mm dish coated with type 1 collagen 10 ml with 10% heat inactivated horse serum and 10% heat inactivated fetal bovine serum (FBS) supplemented with antibiotics (100 IU / ml penicillin and 100 mg / ml streptomycin) Maintained in DMEM (Dulbecco's Modified Eagle Medium) with an incubator adjusted to 37 ° C. in a humid atmosphere containing 5% CO 2 . It reached confluence in about 7 days. Meanwhile, the medium was changed twice.
2）PC12細胞に対する脱分極剤KCl及び細胞死誘導剤グルタミン酸の添加の影響
1型コラーゲンでコートした96ウェルプレートに、PC12細胞を1×104個／ウェルで播種し、一晩培養した。その後、各ウェルの培地を吸引し、血清無添加のDMEM培地で希釈した110mMKClを50μl／ウェルと、同様の培地で希釈した本発明のるアトラクチレノリドＩＩＩ（実施例１）、β−オイデスモール（実施例２）、パキマ酸（参考例３）、デヒドロパキマ酸（参考例３）の試料溶液を50μl／ウェルずつ添加した（培養液中カリウムイオン濃度は55mM）。
24時間後に、20mMグルタミン酸を100μl／ウェルずつ添加し、ウェルの総量200μl／ウェルとした（培養液中グルタミン酸濃度は10mM）。そして、インキュベーター内にプレートを静置し、24時間後に細胞内ATP量と細胞生存率、上清中のアデニル酸キナーゼ活性の測定を行った。
2) Effects of addition of depolarizing agent KCl and cell death inducer glutamic acid on PC12 cells
PC12 cells were seeded at 1 × 10 4 cells / well in a 96-well plate coated with type 1 collagen and cultured overnight. Thereafter, the medium in each well was aspirated and 110 mM KCl diluted with serum-free DMEM medium was 50 μl / well, and the actinylolide III according to the present invention diluted with the same medium (Example 1), β-eude Sample solutions of small (Example 2), pachymic acid (Reference Example 3), and dehydropachymic acid (Reference Example 3) were added at 50 μl / well (potassium ion concentration in the culture solution was 55 mM).
After 24 hours, 20 mM glutamic acid was added in an amount of 100 μl / well to make a total well volume of 200 μl / well (the concentration of glutamic acid in the culture medium was 10 mM). Then, the plate was allowed to stand in an incubator, and after 24 hours, the amount of intracellular ATP, cell viability, and adenylate kinase activity in the supernatant were measured.
3)細胞内ATP量の測定
各ウェルから培養上清を150μlずつ抜き、50μl／ウェルずつATP反応試薬（Cell Titer-GloTM Luminescent Cell Viability Assay、Promega社製）を添加した。添加後、遮光したプレートを2分間振盪させた後、10分間常温で静置し、ルミノメーター（Centro／CentroXS3 LB960、Berthold Technologys社製）でルシフェラーゼ発光をATP量として測定した。尚、ATP量は培地のみを添加し試験を行った場合の発光率100％とした。
3) Measurement of intracellular ATP amount 150 μl of the culture supernatant was extracted from each well, and 50 μl / well of ATP reaction reagent (Cell Titer-Glo ™ Luminescent Cell Viability Assay, manufactured by Promega) was added. After the addition, the light-shielded plate was shaken for 2 minutes, then allowed to stand at room temperature for 10 minutes, and luciferase luminescence was measured as the amount of ATP with a luminometer (Centro / CentroXS3 LB960, manufactured by Berthold Technologies). In addition, the amount of ATP was set to 100% of the luminescence rate when the test was performed with only the medium added.
4)細胞生存率の測定
各ウェルから培養上清をアスピレーターで全て吸引し、DMEM培地で10倍に希釈したCCK8試薬を100μl／ウェル添加し、インキュベーターで4時間反応させた後、吸光度計（Multiskan Spectrum 、Thermo Fisher Scientific社製）で吸光度を測定した。尚、細胞生存率は培地のみを添加し試験を行った場合の細胞内ATP量を100％とした。
4) Measurement of cell viability All culture supernatants were aspirated from each well, CCK8 reagent diluted 10-fold with DMEM medium was added at 100 μl / well, reacted in an incubator for 4 hours, and then absorbance meter (Multiskan Absorbance was measured with Spectrum, manufactured by Thermo Fisher Scientific. The cell survival rate was defined as the amount of intracellular ATP when the test was performed with only the medium added.
5)AK活性の測定
各ウェルから培養上清を20μlずつホワイトプレートに移した後にルミノメーター（Centro／CentroXS3 LB 960、Berthold Technologies社製）に設置し、オートインジェクターで各ウェルに50μlずつ AK Detection Reagent（ToxiLight BioAssay Kit、LONZA社製）を添加した。5分間静置後、ルミノメーターでルシフェラーゼ発光をAK活性として測定した。尚、AK活性は培地のみを添加し試験を行った場合のAK活性を100％とした。
5) Measurement of AK activity Transfer 20 μl of the culture supernatant from each well to a white plate, place it on a luminometer (Centro / CentroXS3 LB 960, manufactured by Berthold Technologies), and use 50 μl of AK Detection Reagent in each well with an autoinjector. (ToxiLight BioAssay Kit, manufactured by LONZA) was added. After standing for 5 minutes, luciferase luminescence was measured as AK activity with a luminometer. The AK activity was 100% when the test was performed with only the medium added.
（試験結果）
結果を図３〜５に示す。PC12細胞は55mMKClによる脱分極下、10mMグルタミン酸の添加によりATPの産生が低下し細胞死が誘導されたが、10 nM〜100μMのアトラクチレノリドＩＩＩ、β-オイデスモール、パキマ酸及びデヒドロパキマ酸を添加することにより、ATP量の低下抑制作用が認められ、CCK8による細胞生存率が110〜120％増加した。
一方、培養上清中のAK活性については、濃度依存的に有意に増加し、特にデヒドロパキマ酸が顕著であった。このAK活性の増加は、各成分添加で細胞死が増えていないことから、AK酵素タンパクが増加したことによる結果であることが示唆される。
(Test results)
The results are shown in FIGS. PC12 cells were depolarized with 55 mM KCl and the addition of 10 mM glutamic acid reduced ATP production and induced cell death.However, 10 nM to 100 μM atractylenolide III, β-eudesmol, pachymic acid and dehydropachymic acid were added. By adding, the effect of suppressing the decrease in the amount of ATP was observed, and the cell survival rate by CCK8 increased by 110 to 120%.
On the other hand, the AK activity in the culture supernatant increased significantly depending on the concentration, and dehydropachymic acid was particularly remarkable. This increase in AK activity is due to the increase in AK enzyme protein since cell death did not increase with the addition of each component.
（試験例３）細胞外電解質の神経細胞に対する影響について、次の試験により確認した。
すなわち、PC12細胞にKClを添加したときのATP産生量及び細胞生存率、細胞外AK活性の変化を調べた。
(Test Example 3) The effect of extracellular electrolyte on nerve cells was confirmed by the following test.
That is, changes in ATP production, cell viability, and extracellular AK activity when KCl was added to PC12 cells were examined.
（試験方法）
1)神経前駆細胞及び培養
ラット副腎髄質由来褐色細胞腫PC12（独立行政法人理化学研究所 バイオリソースセンターより購入）は、１型コラーゲンでコートした100ｍｍディッシュを用いて、1×106個／ディッシュとなるように播種し、10％熱不活性化ウマ血清及び10％熱不活性化ウシ胎児血清（FBS）を有し、抗生物質（100IU／mlのペニシリン及び100mg／mlのストレプトマイシン）を補足した10mlのDMEM（ダルベッコ変法イーグル培地）中で、5％CO2を含む湿気のある雰囲気中37℃に調整したインキュベーターにより維持した。約7日間でコンフルエントに達した。その間、2回の培地交換を行なった。
(Test method)
1) Neural progenitor cells and culture Rat adrenal medulla-derived pheochromocytoma PC12 (purchased from RIKEN BioResource Center) is 1 × 10 6 cells / dish using a 100 mm dish coated with type 1 collagen 10 ml with 10% heat inactivated horse serum and 10% heat inactivated fetal bovine serum (FBS) supplemented with antibiotics (100 IU / ml penicillin and 100 mg / ml streptomycin) Maintained in DMEM (Dulbecco's Modified Eagle Medium) with an incubator adjusted to 37 ° C. in a humid atmosphere containing 5% CO 2 . It reached confluence in about 7 days. Meanwhile, the medium was changed twice.
2）PC12細胞に対するKClの添加の影響
PC12細胞に対して細胞死誘導に適したKCl添加量の検討を行った。1型コラーゲンでコートした96ウェルプレートに、PC12細胞を1×104個／ウェルで播種し、一晩培養した。その後、各ウェルの培地を吸引し、血清無添加のDMEM培地で希釈した0〜100mMKClを100μl／ウェルずつ添加した。そして、インキュベーター内にプレートを静置し、24時間後に細胞内ATP量と細胞生存率の測定を行った。
2) Effect of KCl addition on PC12 cells
The amount of KCl added to induce cell death was examined for PC12 cells. PC12 cells were seeded at 1 × 10 4 cells / well in a 96-well plate coated with type 1 collagen and cultured overnight. Thereafter, the medium in each well was aspirated, and 100 μl / well of 0-100 mM KCl diluted with serum-free DMEM medium was added. Then, the plate was allowed to stand in the incubator, and after 24 hours, the amount of intracellular ATP and the cell viability were measured.
3)細胞内ATP量の測定
各ウェルから培養上清を100μlずつ抜き、50μl／ウェルずつDMEM培地を加えた後、50μl／ウェルずつATP反応試薬（Cell Titer-GloTM LuminescentCell ViabilityAssay、Promega社製）を添加した。添加後、遮光したプレートを2分間振盪させた後、10分間常温で静置した。その後、ルミノメーター（Centro／CentroXS3 LB960、Berthold Technologys社製）でルシフェラーゼ発光をATP量として測定した。尚、ATP量は培地のみを添加し試験を行った場合の発光率100％とした。
3) Measurement of intracellular ATP amount Remove 100 μl of culture supernatant from each well, add 50 μl / well of DMEM medium, and then add 50 μl / well of ATP reaction reagent (Cell Titer-Glo ™ Luminescent Cell Viability Assay, Promega) Was added. After the addition, the light-shielded plate was shaken for 2 minutes and then allowed to stand at room temperature for 10 minutes. Thereafter, luciferase luminescence was measured as the amount of ATP with a luminometer (Centro / CentroXS3 LB960, manufactured by Berthold Technologies). In addition, the amount of ATP was set to 100% of the luminescence rate when the test was performed with only the medium added.
4)細胞生存率の測定
各ウェルから培養上清をアスピレーターで全て吸引し、DMEM培地で10倍に希釈したCCK8試薬を100μl／ウェル添加し、インキュベーターで4時間反応させた後、吸光度計（Multiskan Spectrum 、Thermo Fisher Scientific社製）で吸光度を測定した。尚、細胞生存率は培地のみを添加し試験を行った場合の細胞内ATP量を100％とした。
4) Measurement of cell viability All culture supernatants were aspirated from each well, CCK8 reagent diluted 10-fold with DMEM medium was added at 100 μl / well, reacted in an incubator for 4 hours, and then absorbance meter (Multiskan Absorbance was measured with Spectrum, manufactured by Thermo Fisher Scientific. The cell survival rate was defined as the amount of intracellular ATP when the test was performed with only the medium added.
5)AK活性の測定
各ウェルから培養上清を20μlずつホワイトプレートに移した後にルミノメーター（Centro／CentroXS3 LB 960、Berthold Technologies社製）に設置し、オートインジェクターで各ウェルに50μlずつ AK Detection Reagent（ToxiLight BioAssay Kit、LONZA社製）を添加した。5分間静置後、ルミノメーターでルシフェラーゼ発光をAK活性として測定した。尚、AK活性は培地のみを添加し試験を行った場合のAK活性を100％とした。
5) Measurement of AK activity Transfer 20 μl of the culture supernatant from each well to a white plate, place it on a luminometer (Centro / CentroXS3 LB 960, manufactured by Berthold Technologies), and use 50 μl of AK Detection Reagent in each well with an autoinjector. (ToxiLight BioAssay Kit, manufactured by LONZA) was added. After standing for 5 minutes, luciferase luminescence was measured as AK activity with a luminometer. The AK activity was 100% when the test was performed with only the medium added.
（試験結果）
結果を図６〜８に示す。細胞内ATP量は、55mM以上の KCl添加でATPの生成を顕著に低下した。また細胞生存率は、70mM以上のKCl添加で細胞生存率が顕著に低下した。一方、AK活性は90mM以上のKCl添加でAK活性が顕著に増加した。
以上から、70mMKCl添加でATP生成の抑制及び細胞死の誘導、AK活性の増加を確認できた。以上の結果より、PC12細胞に対してKClを添加したときのATP生成量及び細胞生存率の低下に対する効果を確認する試験方法として、PC12細胞を1×104個／ウェルで播種する。続いて、一晩培養後に、血清無添加DMEM培地を用い、70mMKClを100μl／ウェルずつ添加し、24時間後に被験試料を添加し細胞内ATP量、CCK8による細胞生存率及び上清中のAK活性の測定を行うこととした。
(Test results)
The results are shown in FIGS. The amount of ATP in the cell markedly decreased the production of ATP by adding 55 mM or more of KCl. In addition, the cell viability decreased remarkably when KCl of 70 mM or more was added. On the other hand, AK activity significantly increased when 90 mM or more of KCl was added.
From the above, suppression of ATP production, induction of cell death, and increase in AK activity were confirmed by adding 70 mM KCl. From the above results, PC12 cells are seeded at 1 × 10 4 cells / well as a test method for confirming the effect on the decrease in ATP production and cell viability when KCl is added to PC12 cells. Subsequently, after overnight culture, using serum-free DMEM medium, add 70 mM KCl at 100 μl / well, add the test sample 24 hours later, add intracellular ATP, cell viability by CCK8, and AK activity in the supernatant It was decided to measure.
（試験例４）PC12細胞へKClを添加したときのATP産生量及び細胞生存率の低下に対する本発明の各有効成分の効果を次の試験により確認した。 Test Example 4 The effect of each active ingredient of the present invention on the decrease in ATP production and cell viability when KCl was added to PC12 cells was confirmed by the following test.
（試験方法）
1)神経前駆細胞及び培養
ラット副腎髄質由来褐色細胞腫PC12（独立行政法人理化学研究所 バイオリソースセンターより購入）は、１型コラーゲンでコートした100ｍｍディッシュを用いて、1×106個／ディッシュとなるように播種し、10％熱不活性化ウマ血清及び10％熱不活性化ウシ胎児血清（FBS）を有し、抗生物質（100IU／mlのペニシリン及び100mg／mlのストレプトマイシン）を補足した10mlのDMEM（ダルベッコ変法イーグル培地）中で、5％CO2を含む湿気のある雰囲気中37℃に調整したインキュベーターにより維持した。約7日間でコンフルエントに達した。その間、2回の培地交換を行なった。
(Test method)
1) Neuroprogenitor cells and cultured rat adrenal medullary pheochromocytoma PC12 (purchased from RIKEN BioResource Center) is 1 × 10 6 cells / dish using a 100 mm dish coated with type 1 collagen 10 ml with 10% heat inactivated horse serum and 10% heat inactivated fetal bovine serum (FBS) supplemented with antibiotics (100 IU / ml penicillin and 100 mg / ml streptomycin) Maintained in DMEM (Dulbecco's Modified Eagle Medium) with an incubator adjusted to 37 ° C. in a humid atmosphere containing 5% CO 2 . It reached confluence in about 7 days. Meanwhile, the medium was changed twice.
2）PC12細胞に対するKCl添加による細胞死に対する抑制効果
1型コラーゲンでコートした96ウェルプレートに、PC12細胞を1×104個／ウェルで播種し、一晩培養した。その後、各ウェルの培地を吸引し、血清無添加の70mMKCl 添加DMEM培地で希釈した本発明のアトラクチレノリドＩ（Stanford Materials company社より購入）、アトラクチレノリドＩＩ（Stanford Materials company社より購入）、アトラクチレノリドＩＩＩ（実施例１）、パキマ酸（参考例３）、デヒドロパキマ酸（参考例３）、β−オ
イデスモール（実施例２）、リグスチリド（参考例４）、アリソールA（和光純薬工業社より購入）、ギンセノシド Rg1（参考例５）、エルゴステロール（和光純薬工業社より購入）の試料溶液を100μl／ウェルずつ添加した（培養液中カリウムイオン濃度は70mM）。24時間後に細胞内ATP量と細胞生存率、上清中のアデニル酸キナーゼ活性の測定を行った。
2) Inhibitory effect on cell death by adding KCl to PC12 cells
PC12 cells were seeded at 1 × 10 4 cells / well in a 96-well plate coated with type 1 collagen and cultured overnight. Thereafter, the culture medium in each well is aspirated and diluted with serum-free 70 mM KCl-added DMEM medium. Atractylenolide I of the present invention (purchased from Stanford Materials company), atractylenolide II (purchased from Stanford Materials company) ), Atractylenolide III (Example 1), pachymic acid (Reference Example 3), dehydropachymic acid (Reference Example 3), β-eudesmol (Example 2), ligustilide (Reference Example 4), alisol A (sum) 100 μl / well of sample solutions of Ginsenoside Rg1 (Reference Example 5) and Ergosterol (purchased from Wako Pure Chemical Industries, Ltd.) were added each (purchased from Kojun Pharmaceutical Co., Ltd.) (potassium ion concentration in the culture solution was 70 mM). After 24 hours, intracellular ATP amount, cell viability, and adenylate kinase activity in the supernatant were measured.
3)細胞内ATP量の測定
各ウェルから培養上清を100μlずつ抜き、50μl／ウェルずつDMEM培地を加えた後、50μl／ウェルずつATP反応試薬（Cell Titer-GloTM Luminescent Cell Viability Assay、Promega社製）を添加した。添加後、遮光したプレートを2分間振盪させた後、10分間常温で静置し、ルミノメーター（Centro／CentroXS3 LB960、Berthold Technologys社製）でルシフェラーゼ発光をATP量として測定した。尚、ATP量は培地のみを添加し試験を行った場合の発光率100％とした。
3) Measurement of intracellular ATP amount Remove 100 μl of culture supernatant from each well, add 50 μl / well of DMEM medium, and then add 50 μl / well of ATP reaction reagent (Cell Titer-Glo ™ Luminescent Cell Viability Assay, Promega) Made) was added. After the addition, the light-shielded plate was shaken for 2 minutes, then allowed to stand at room temperature for 10 minutes, and luciferase luminescence was measured as the amount of ATP with a luminometer (Centro / CentroXS3 LB960, manufactured by Berthold Technologies). In addition, the amount of ATP was set to 100% of the luminescence rate when the test was performed with only the medium added.
4)細胞生存率の測定
各ウェルから培養上清をアスピレーターで全て吸引し、DMEM培地で10倍に希釈したCCK8試薬を100μl／ウェル添加し、インキュベーターで4時間反応させた後、吸光度計（Multiskan Spectrum 、Thermo Fisher Scientific社製）で吸光度を測定した。尚、細胞生存率は培地のみを添加し試験を行った場合の細胞内ATP量を100％とした。
4) Measurement of cell viability All culture supernatants were aspirated from each well, CCK8 reagent diluted 10-fold with DMEM medium was added at 100 μl / well, reacted in an incubator for 4 hours, and then absorbance meter (Multiskan Absorbance was measured with Spectrum, manufactured by Thermo Fisher Scientific. The cell survival rate was defined as the amount of intracellular ATP when the test was performed with only the medium added.
5)AK活性の測定
各ウェルから培養上清を20μlずつホワイトプレートに移した後にルミノメーター（Centro／CentroXS3 LB 960、Berthold Technologies社製）に設置し、オートインジェクターで各ウェルに50μlずつ AK Detection Reagent（ToxiLight BioAssay Kit、LONZA社製）を添加した。5分間静置後、ルミノメーターでルシフェラーゼ発光をAK活性として測定した。尚、AK活性は培地のみを添加し試験を行った場合のAK活性を100％とした。
5) Measurement of AK activity Transfer 20 μl of the culture supernatant from each well to a white plate, place it on a luminometer (Centro / CentroXS3 LB 960, manufactured by Berthold Technologies), and use 50 μl of AK Detection Reagent in each well with an autoinjector. (ToxiLight BioAssay Kit, manufactured by LONZA) was added. After standing for 5 minutes, luciferase luminescence was measured as AK activity with a luminometer. The AK activity was 100% when the test was performed with only the medium added.
（試験結果）
結果を図９〜２０に示す。PC12細胞は70mMKClの添加によりATPの産生が低下し細胞死が誘導されるが、0.1pM〜100nMのアトラクチレノリドＩ、アトラクチレノリドＩＩ、アトラクチレノリドＩＩＩ、パキマ酸、デヒドロパキマ酸、β−オイデスモール、リグスチリド、アリソールA、ギンセノシド Rg1、エルゴステロールを添加することにより、ATP量の有意な低下抑制作用が認められ、CCK8による細胞生存率も有意に増加した。一方、培養上清中のAK活性も増加したが、細胞賦活化による細胞増殖が促進した結果であると推察される。
(Test results)
The results are shown in FIGS. In PC12 cells, the addition of 70 mM KCl decreases ATP production and induces cell death. However, 0.1 pM to 100 nM atractylenolide I, atractylenolide II, atractylenolide III, pachymic acid, dehydropachymic acid, By adding β-eudesmol, ligustilide, allisole A, ginsenoside Rg1, and ergosterol, a significant decrease in ATP level was observed, and the cell survival rate by CCK8 also increased significantly. On the other hand, although the AK activity in the culture supernatant was also increased, it is presumed that this was the result of the promotion of cell proliferation by cell activation.
以下に、実施例および参考例を挙げて本発明をさらに具体的に説明する。 Hereinafter, the present invention will be described more specifically with reference to Examples and Reference Examples.
実施例１（アトラクチレノリドＩＩＩ）
粉末化した白朮１ｋｇにメタノールを５Ｌ加え、室温で２４時間抽出し、ろ過後抽出液を得た。更に、残渣にメタノール３Ｌを加え、同様に室温２４時間して抽出液を得た。これら抽出液を合わせて、減圧濃縮し、白朮抽出エキスを得た。このエキスをヘキサンで抽出し、ヘキサン画分約５０ｇを得た。この画分１０ｇをシリカゲルカラムを用い、ヘキサン/酢酸エチル（１００：０→０：１００）及びメタノールで順次溶出した。ＴＬＣによりアトラクチレノリドＩＩＩが確認された画分約２ｇについてヘキサンで再結晶すると、アトラクチレノリドＩＩＩ（３５０ｍｇ）を得た。
Example 1 (Atractylenolide III)
To 1 kg of powdered white rabbit, 5 L of methanol was added and extracted at room temperature for 24 hours. After filtration, an extract was obtained. Further, 3 L of methanol was added to the residue, and an extract was obtained in the same manner at room temperature for 24 hours. These extracts were combined and concentrated under reduced pressure to obtain a birch extract. This extract was extracted with hexane to obtain about 50 g of a hexane fraction. 10 g of this fraction was sequentially eluted with hexane / ethyl acetate (100: 0 → 0: 100) and methanol using a silica gel column. About 2 g of the fraction in which atractylenolide III was confirmed by TLC was recrystallized from hexane to obtain atractylenolide III (350 mg).
実施例２（β−オイデスモール）
粉末化した蒼朮１ｋｇにエタノールを５Ｌ加え、室温で２４時間抽出し、ろ過後抽出液
を得た。更に、残渣にエタノール３Ｌを加え、同様に室温２４時間抽出後ろ過して抽出液を得た。これら抽出液を合わせて、減圧濃縮し、蒼朮抽出エキスを得た。このエキスを酢酸エチルで抽出し、酢酸エチル画分約３０ｇを得た。この画分１０ｇをシリカゲルカラムを用い、ジエチルエーテル/酢酸エチル（１００：２→１００：１００）で順次溶出し、ＴＬＣによりβ−オイデスモールが確認された画分を回収して、β−オイデスモール（１８０ｍｇ）を得た。
Example 2 (β-eudesmol)
5 kg of ethanol was added to 1 kg of powdered koji and extracted at room temperature for 24 hours. After filtration, an extract was obtained. Further, 3 L of ethanol was added to the residue. Similarly, extraction was performed at room temperature for 24 hours, followed by filtration to obtain an extract. These extracts were combined and concentrated under reduced pressure to obtain a koji extract. This extract was extracted with ethyl acetate to obtain about 30 g of an ethyl acetate fraction. 10 g of this fraction was eluted sequentially with diethyl ether / ethyl acetate (100: 2 → 100: 100) using a silica gel column, and the fraction in which β-eudesmol was confirmed by TLC was collected, and β-eudesmol was recovered. (180 mg) was obtained.
参考例３（デヒドロパキマ酸及びパキマ酸）
粉末化した茯苓５ｋｇにメタノールを加えて３時間加熱還流抽出し、ろ過後、抽出液を減圧濃縮し、茯苓抽出エキスを得た。このエキスをシリカゲルカラムを用い、クロロホルム/メタノール（１：０→１：１）で順次溶出した。次に、所望の画分を９０％メタノール溶液を溶媒として、逆相系分取高速液体クロマトグラフィーを繰り返し付し、デヒドロパキマ酸（５０ｍｇ）、パキマ酸（２００ｍｇ）を得た。
Reference example 3 (dehydropachymic acid and pachymic acid)
Methanol was added to 5 kg of powdered koji and heated under reflux for 3 hours. After filtration, the extract was concentrated under reduced pressure to obtain a koji extract. This extract was sequentially eluted with chloroform / methanol (1: 0 → 1: 1) using a silica gel column. Next, reverse phase preparative high performance liquid chromatography was repeatedly applied to the desired fraction using a 90% methanol solution as a solvent to obtain dehydropachymic acid (50 mg) and pachymic acid (200 mg).
参考例４（リグスチリド）
粉末化したセンキュウ５ｋｇをエタノール２５Ｌに５日間放置し、ろ過後、抽出液を減圧乾燥した。抽出物を水に懸濁し酢酸エチル３Ｌにて抽出し、酢酸エチル画分を減圧乾燥し、センキュウ抽出エキスを得た。このエキスをシリカゲルカラムを用い、ｎ−ヘキサン／酢酸エチル混液（３：１）を用い、ＴＬＣによりリグスチリドが確認された画分を回収して、リグスチリド（１５００ｍｇ）を得た。
Reference Example 4 (Ligustilide)
5 kg of powdered Senkyu was left in 25 L of ethanol for 5 days, filtered, and the extract was dried under reduced pressure. The extract was suspended in water and extracted with 3 L of ethyl acetate, and the ethyl acetate fraction was dried under reduced pressure to obtain a Senkyu extract. A fraction in which ligustilide was confirmed by TLC was collected using an n-hexane / ethyl acetate mixed solution (3: 1) using a silica gel column, and ligustilide (1500 mg) was obtained.
参考例５（ギンセノシドＲｇ１）
粉末化した人参５ｋｇをメタノール５Ｌで加熱抽出し、ろ過後、抽出液を減圧乾燥した。抽出物を水３Ｌに懸濁し、水飽和ブタノールＩＬで抽出し、減圧乾燥して、人参抽出エキスを得た。このエキスをシリカゲルカラムを用い、クロロホルム／メタノール／水混液（６５：３５：１０）下層を用い、ＴＬＣによりリグスチリドが確認された画分を回収して、ギンセノシドＲｇ１（１００ｍｇ）を得た。
Reference Example 5 (Ginsenoside Rg1)
5 kg of powdered carrots were heated and extracted with 5 L of methanol, and after filtration, the extract was dried under reduced pressure. The extract was suspended in 3 L of water, extracted with water-saturated butanol IL, and dried under reduced pressure to obtain a carrot extract. Using this silica gel column, the fraction in which ligustilide was confirmed by TLC was collected using a lower layer of a chloroform / methanol / water mixture (65:35:10), and ginsenoside Rg1 (100 mg) was obtained.
実施例６（アトラクチレノリドＩＩＩ含有の白朮抽出エキス）
白朮１００ｇに精製水を１Ｌ加え、１００℃で１時間加熱してエキスを煎出し、熱時抽出液を分離し、凍結乾燥したエキス４１．５ｇを得た。
Example 6 (White agate extract containing atractylenolide III)
1 L of purified water was added to 100 g of white birch, and the extract was decocted by heating at 100 ° C. for 1 hour, and the hot extract was separated to obtain 41.5 g of lyophilized extract.
実施例７（β−オイデスモール含有の蒼朮抽出エキス）
蒼朮１００ｇに精製水を１Ｌ加え、１００℃で１時間加熱してエキスを煎出し、熱時抽出液を分離し、凍結乾燥したエキス４１．４ｇを得た。
Example 7 (β-eudesmol-containing koji extract)
1 L of purified water was added to 100 g of koji, and the extract was decocted by heating at 100 ° C. for 1 hour. The hot extract was separated and 41.4 g of freeze-dried extract was obtained.
参考例８（デヒドロパキマ酸及びパキマ酸含有の茯苓抽出エキス）
茯苓１６０ｇに精製水を１．６Ｌ加え、１００℃で１時間加熱してエキスを煎出し、熱時抽出液を分離し、凍結乾燥したエキス３．６ｇを得た。
Reference Example 8 (Dehydropachymic acid and coconut extract containing pachymic acid)
1.6 L of purified water was added to 160 g of koji, and the extract was decocted by heating at 100 ° C. for 1 hour, and the hot extract was separated to obtain 3.6 g of freeze-dried extract.
実施例９（顆粒剤）
白朮抽出エキス（実施例６） １３８０ｇ
ヒドロキシプロピルセルロース ７０ｇ
乳糖 １０５０ｇ
合計 ２５００ｇ
Example 9 (granule)
White birch extract (Example 6) 1380 g
Hydroxypropylcellulose 70g
Lactose 1050g
Total 2500g
（製造方法）
上記の各成分を混合し、その混合物を常法により顆粒とし、２．５ｇずつに分包して実施例９の顆粒剤を得た。
(Production method)
The above components were mixed, and the mixture was granulated by a conventional method and divided into 2.5 g portions to obtain granules of Example 9.
実施例１０（顆粒剤）
アトラクチレノリドＩＩＩ（実施例１） ３０ｇ
ヒドロキシプロピルセルロース ７０ｇ
乳糖 ７３９７ｇ
合計 ７５００ｇ
Example 10 (granule)
Atractylenolide III (Example 1) 30 g
Hydroxypropylcellulose 70g
Lactose 7397g
Total 7500g
（製造方法）
上記の各成分を混合し、その混合物を常法により顆粒とし、２．５ｇずつに分封して実施例１０の顆粒剤を得た。
(Production method)
The above components were mixed, and the mixture was granulated by a conventional method and separated into 2.5 g portions to obtain granules of Example 10.
参考例１１（錠剤）
デヒドロパキマ酸（参考例３） ５０ｇ
結晶セルロース ２００ｇ
乳糖 ５９０ｇ
クロスカルメロースナトリウム １００ｇ
含水二酸化ケイ素 ５０ｇ
ステアリン酸マグネシウム １０ｇ
小 計 １０００ｇ
Reference Example 11 (tablet)
50 g of dehydropachymic acid (Reference Example 3)
200g of crystalline cellulose
Lactose 590g
Croscarmellose sodium 100g
Hydrous silicon dioxide 50g
Magnesium stearate 10g
Subtotal 1000g
（製造方法）
「日局」製剤総則、錠剤の項に準じて錠剤を製する。すなわち上表に記載の、デヒドロパキマ酸からステアリン酸マグネシウムまでの成分をとり、一錠重量２００ｍｇの参考例１１の錠剤を得た。
(Production method)
Prepare tablets according to “JP” General Rules for Preparations, Tablets. That is, the components of dehydropachymic acid to magnesium stearate described in the above table were taken to obtain tablets of Reference Example 11 having a tablet weight of 200 mg.
実施例１２（液剤）
蒼朮抽出エキス（実施例７） ４１００ｇ
白糖 ８０００ｇ
精製水 残 量
合計 ９０ｋｇ
Example 12 (Liquid)
Koji Extract (Example 7) 4100g
8000g of white sugar
Total amount of purified water remaining 90kg
（製造方法）
上記成分に精製水を加えて加熱溶解し、冷後、精製水を加えて全量９０ｋｇとする。この液を３０ｇずつ容器に分注し、締栓後、加熱殺菌し実施例１２の液剤を得た。
(Production method)
Purified water is added to the above components and dissolved by heating. After cooling, purified water is added to make a total of 90 kg. 30 g of this liquid was dispensed into a container, and after capping, heat sterilized to obtain a liquid preparation of Example 12.
参考例１３（エルゴステロール含有の猪苓末顆粒剤）
猪苓末 ４５００ｇ
ヒドロキシプロピルセルロース ２２５ｇ
乳糖 ２７７５ｇ
合計 ７５００ｇ
Reference Example 13 (powder granule containing ergosterol)
4500g of powder
225g of hydroxypropylcellulose
Lactose 2775g
Total 7500g
（製造方法）
上記の各成分を混合し、その混合物を常法により顆粒とし、２．５ｇずつに分封して参考例１３の顆粒剤を得た。
(Production method)
The above-mentioned components were mixed, and the mixture was granulated by a conventional method, and separated into 2.5 g portions to obtain granules of Reference Example 13.
参考例１４（アリソールA含有の沢瀉末顆粒剤）
沢瀉末 ３０００ｇ
ヒドロキシプロピルセルロース ２００ｇ
乳糖 ４３００ｇ
合計 ７５００ｇ
Reference Example 14 (Arisol A-containing granule powder)
3000g
Hydroxypropylcellulose 200g
Lactose 4300g
Total 7500g
（製造方法）
上記の各成分を混合し、その混合物を常法により顆粒とし、２．５ｇずつに分封して参考例１４の顆粒剤を得た。
(Production method)
The above-mentioned components were mixed, and the mixture was granulated by a conventional method.
Claims (7)
A cell death inhibitor composition comprising an Eudesman sesquiterpenoid derivative represented by the following general formula (1), (2) or (3) as an active ingredient.
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| CN104490869A (en) * | 2014-12-09 | 2015-04-08 | 上海交通大学医学院附属第三人民医院 | Application of atractylenolide-III derivative in preparation of platelet aggregation resisting drug and platelet aggregation resisting drug |
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| Publication number | Publication date |
|---|---|
| JP6108452B2 (en) | 2017-04-05 |
| JP2013234178A (en) | 2013-11-21 |
| JP2013234179A (en) | 2013-11-21 |
| JP6087706B2 (en) | 2017-03-01 |
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