JP2023162090A - BACE1 INHIBITOR, AMYLOID β AGGREGATE FORMATION INHIBITOR, CYTOPROTECTANT FOR AMYLOID β AND ACETYLCHOLINESTERASE INHIBITOR - Google Patents
BACE1 INHIBITOR, AMYLOID β AGGREGATE FORMATION INHIBITOR, CYTOPROTECTANT FOR AMYLOID β AND ACETYLCHOLINESTERASE INHIBITOR Download PDFInfo
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Abstract
Description
本発明は、ＢＡＣＥ１阻害剤、アミロイドβ凝集体形成阻害剤、アミロイドβに対する細胞保護剤及びアセチルコリンエステラーゼ阻害剤に関する。さらに、本発明は、認知機能改善剤、記憶力の維持又は改善剤並びに認知症及び／又はMCIの治療及び／又は予防用の医薬組成物に関する。 The present invention relates to a BACE1 inhibitor, an amyloid β aggregate formation inhibitor, a cytoprotective agent against amyloid β, and an acetylcholinesterase inhibitor. Furthermore, the present invention relates to a cognitive function improving agent, a memory maintenance or improving agent, and a pharmaceutical composition for treating and/or preventing dementia and/or MCI.
認知症は、加齢に伴う脳機能障害であり、その有病率及び罹患率は、加齢に伴い著しく増大する。世界的な高齢化の進行に伴い、認知症の罹患者は増大傾向にあり、全世界の認知症罹患者数は、2020年には4000万人、2040年には8000万人に達するという推計もなされている。 Dementia is an age-related brain dysfunction, and its prevalence and morbidity increase significantly with age. As the global population ages, the number of people suffering from dementia is on the rise, and it is estimated that the number of people with dementia worldwide will reach 40 million in 2020 and 80 million in 2040. has also been done.
認知症の主なものとして、アルツハイマー型認知症(AD)、脳血管性認知症、レビー小体型認知症があるが、最も頻度が高いのはアルツハイマー病で、認知症全体の40～60%を占める。最近の疫学研究によると、脳血管性認知症の有病率や罹患率は治療法や予防法などの進歩に伴い年々減少する傾向にあるが、アルツハイマー型認知症の罹患者は確実に増加しているとも言われている(非特許文献１)。 The main types of dementia include Alzheimer's disease (AD), cerebrovascular dementia, and Lewy body dementia, but Alzheimer's disease is the most common, accounting for 40-60% of all dementia cases. occupy According to recent epidemiological studies, the prevalence and incidence of cerebrovascular dementia tend to decrease year by year due to advances in treatment and prevention methods, but the number of people suffering from Alzheimer's disease is steadily increasing. It is also said that (Non-patent Document 1).
上述のとおり、アルツハイマー型認知症は、高齢化の進行する現在社会において深刻な社会問題の一つとなっており、その治療や予防を目的とした医薬品やサプリメント等の開発が急務となっている。アルツハイマー型認知症の要因として、脳内におけるアミロイドβ(アミロイドβタンパク質：Ａβ)の蓄積及び脳内に蓄積したアミロイドβの神経毒性に起因する神経細胞死が考えられている。現在、アルツハイマー型認知症の症状の軽減のための治療として、脳の機能低下に対する対症療法としてのコリンエステラーゼ阻害剤又はグルタミン酸NMDA受容体拮抗薬の投与、脳の神経細胞の細胞死を抑制する効果を有する薬剤として、ペリンドブリル等の投与が行われている。しかし、ペリンドブリルには血圧降下作用の存在が知られており、却って症状を悪化させる懸念がある。 As mentioned above, Alzheimer's dementia has become one of the serious social problems in today's aging society, and there is an urgent need to develop pharmaceuticals, supplements, etc. for the treatment and prevention of Alzheimer's disease. Accumulation of amyloid β (amyloid β protein: Aβ) in the brain and neuronal cell death due to the neurotoxicity of amyloid β accumulated in the brain are thought to be factors in Alzheimer's dementia. Currently, treatments for alleviating the symptoms of Alzheimer's disease include the administration of cholinesterase inhibitors or glutamate NMDA receptor antagonists as symptomatic treatments for brain function decline, and the effects of suppressing the cell death of brain nerve cells. Perindobril and other drugs have been administered as drugs that have this effect. However, perindobril is known to have a blood pressure lowering effect, and there is a concern that it may actually worsen symptoms.
アミロイドβは、「アミロイドβ生成」と「アミロイドβ凝集」の２つの段階を経て脳内に蓄積すると考えられている。したがって、これらの段階の一方又は双方を阻害する活性を有する化合物は、アルツハイマー型認知症の予防又は治療に有効であることが期待される。アミロイドβ凝集を阻害する活性を有する組成物として、例えば、シソ抽出物を有効成分として含むものが提案されている(特許文献１)。また、アミロイドβ生成に関与するタンパク質プロセシング酵素の１つであるγセクレターゼを阻害する活性を有する組成物として、ヒシュカ等に由来する植物エキスを有効成分として含むγセクレターゼ阻害組成物が提案されている(特許文献２)。 Amyloid β is thought to accumulate in the brain through two stages: “amyloid β generation” and “amyloid β aggregation.” Therefore, compounds having the activity of inhibiting one or both of these stages are expected to be effective in preventing or treating Alzheimer's dementia. As a composition having the activity of inhibiting amyloid β aggregation, for example, a composition containing perilla extract as an active ingredient has been proposed (Patent Document 1). Additionally, a γ-secretase inhibiting composition containing a plant extract derived from Hishuka etc. as an active ingredient has been proposed as a composition having the activity of inhibiting γ-secretase, which is one of the protein processing enzymes involved in amyloid β production. (Patent Document 2).
本発明は、安全で高い活性を有するＢＡＣＥ１阻害剤、Ａβ凝集体形成阻害剤、Ａβに対する細胞保護剤及びアセチルコリンエステラーゼ阻害剤を提供することを目的とする。さらに、本発明は、認知機能改善剤、記憶力の維持又は改善剤並びに認知症及び／又はMCIの治療及び／又は予防用の医薬組成物を提供することを目的とする。 An object of the present invention is to provide a BACE1 inhibitor, an Aβ aggregate formation inhibitor, a cytoprotective agent against Aβ, and an acetylcholinesterase inhibitor that are safe and have high activity. Furthermore, the present invention aims to provide a cognitive function improving agent, a memory maintenance or improving agent, and a pharmaceutical composition for treating and/or preventing dementia and/or MCI.
本発明者らは、上記目的を達成すべく鋭意研究を重ねた結果、ボケ、リンデン、サンカクトウ、及びジャクゼツソウの抽出物が、優れたＢＡＣＥ１阻害活性、アミロイドβ凝集阻害活性、アミロイドβ誘導性細胞死の抑制作用及びアセチルコリンエステラーゼ阻害活性を示すという知見を得た。さらにインビボでの試験により、これらが短期記憶障害の改善作用を有するという知見も得た。 As a result of extensive research to achieve the above-mentioned objectives, the present inventors have found that extracts of Boke, Linden, Sankakutou, and Jakusetsou have excellent BACE1 inhibitory activity, amyloid-β aggregation inhibitory activity, and amyloid-β-induced cell death. We obtained the knowledge that this compound exhibits an inhibitory effect on acetylcholinesterase and an acetylcholinesterase inhibitory activity. Furthermore, in vivo tests have revealed that these compounds have an ameliorating effect on short-term memory impairment.
本発明は、これら知見に基づき、更に検討を重ねて完成されたものであり、次のＢＡＣＥ１阻害剤、Ａβ凝集体形成阻害剤、Ａβに対する細胞保護剤、アセチルコリンエステラーゼ阻害剤等を提供するものである。 The present invention was completed based on these findings and further studies, and provides the following BACE1 inhibitors, Aβ aggregate formation inhibitors, Aβ cell protection agents, acetylcholinesterase inhibitors, etc. be.
項１．ボケ、リンデン、サンカクトウの外果皮、及びナデシコ科に属する植物からなる群から選択される少なくとも1種の抽出物を含むＢＡＣＥ１阻害剤。
項２．ボケ、リンデン、サンカクトウの外果皮、及びナデシコ科に属する植物からなる群から選択される少なくとも1種の抽出物を含むＡβ凝集体形成阻害剤。
項３．ボケ、リンデン、サンカクトウの外果皮、及びナデシコ科に属する植物からなる群から選択される少なくとも1種の抽出物を含むＡβに対する細胞保護剤。
項４．前記ナデシコ科に属する植物が、ジャクゼツソウである、項１～３のいずれか一項に記載の剤。
項５．ボケ、リンデン、サンカクトウの外果皮、及びナデシコ科に属する植物からなる群から選択される少なくとも1種の抽出物を含むアセチルコリンエステラーゼ阻害剤。
項６．前記ナデシコ科に属する植物が、ジャクゼツソウである、項５に記載のアセチルコリンエステラーゼ阻害剤。
項７．ボケ、リンデン、サンカクトウの外果皮、及びナデシコ科に属する植物からなる群から選択される少なくとも1種の抽出物を含む認知機能改善剤。
項８．ボケ、リンデン、サンカクトウの外果皮、及びナデシコ科に属する植物からなる群から選択される少なくとも1種の抽出物を含む、記憶力の維持又は改善剤。
項９．前記ナデシコ科に属する植物が、ジャクゼツソウである、項７又は８に記載の剤。
項１０．ボケ、リンデン、サンカクトウの外果皮、及びナデシコ科に属する植物からなる群から選択される少なくとも1種の抽出物を含む、認知症及び／又はMCIの治療及び／又は予防用の医薬組成物。
項１１．前記ナデシコ科に属する植物が、ジャクゼツソウである、項１０に記載の医薬組成物。
Item 1. A BACE1 inhibitor comprising at least one extract selected from the group consisting of the exocarp of Bokeh, linden, and Cinnamon, and plants belonging to the Caryophyllaceae family.
Item 2. An Aβ aggregate formation inhibitor comprising at least one extract selected from the group consisting of the exocarp of P. japonica, linden, and C. japonica, and plants belonging to the Caryophyllaceae family.
Item 3. A cytoprotective agent against Aβ, comprising an extract of at least one selected from the group consisting of the exocarp of P. japonica, linden, and C. japonica, and plants belonging to the Caryophyllaceae family.
Item 4. Item 4. The agent according to any one of Items 1 to 3, wherein the plant belonging to the Caryophyllaceae family is Caryophyllaceae.
Item 5. 1. An acetylcholinesterase inhibitor comprising at least one extract selected from the group consisting of the exocarp of Bokeh, linden, and Cinnamon, and plants belonging to the Caryophyllaceae family.
Item 6. Item 6. The acetylcholinesterase inhibitor according to Item 5, wherein the plant belonging to the Caryophyllaceae family is Caryophyllaceae.
Section 7. A cognitive function improving agent comprising at least one extract selected from the group consisting of the exocarp of P. japonica, linden, and C. japonica, and plants belonging to the Caryophyllaceae family.
Section 8. An agent for maintaining or improving memory, comprising at least one extract selected from the group consisting of the exocarp of P. japonica, linden, and C. japonica, and plants belonging to the Caryophyllaceae family.
Item 9. Item 8. The agent according to Item 7 or 8, wherein the plant belonging to the family Caryophyllaceae is Caryophyllaceae.
Item 11. Item 11. The pharmaceutical composition according to
本発明によれば、優れたＢＡＣＥ１阻害活性、アミロイドβ凝集阻害活性、アミロイドβ誘導性細胞死の抑制作用、及びアセチルコリンエステラーゼ阻害活性を有する、ＢＡＣＥ１阻害剤、Ａβ凝集体形成阻害剤、Ａβに対する細胞保護剤、及びアセチルコリンエステラーゼ阻害剤を提供することができる。さらに、本発明によれば、認知機能改善剤、記憶力の維持又は改善剤並びに認知症及び／又はMCIの治療及び／又は予防用の医薬組成物を提供することができる。また、これらの有効成分は、食用又は生薬として使用されているものであるので高い安全性を有する。 According to the present invention, a BACE1 inhibitor, an Aβ aggregate formation inhibitor, and a cell against Aβ have excellent BACE1 inhibitory activity, amyloid β aggregation inhibitory activity, amyloid β-induced cell death suppressing effect, and acetylcholinesterase inhibitory activity. Protective agents and acetylcholinesterase inhibitors can be provided. Further, according to the present invention, it is possible to provide a cognitive function improving agent, a memory maintenance or improving agent, and a pharmaceutical composition for treating and/or preventing dementia and/or MCI. Furthermore, these active ingredients are highly safe because they are used as food or herbal medicines.
以下、本発明の実施の形態について詳細に説明する。 Embodiments of the present invention will be described in detail below.
なお、本明細書において「含有する、含む(comprise)」とは、「本質的にからなる(essentially consist of)」という意味と、「のみからなる(consist of)」という意味をも包含する。 Note that in this specification, the term "comprise" includes the meanings of "essentially consist of" and "consist of".
本発明のＢＡＣＥ１阻害剤、Ａβ凝集体形成阻害剤、Ａβに対する細胞保護剤、アセチルコリンエステラーゼ阻害剤、認知機能改善剤、記憶力の維持又は改善剤(以下、「本発明のＢＡＣＥ１阻害剤等」と称することもある)、並びに認知症及び／又はMCI (Mild Cognition Impairment：軽度認知障害)の治療及び／又は予防用の医薬組成物は、ボケ、リンデン、サンカクトウの外果皮、及びナデシコ科に属する植物からなる群から選択される少なくとも1種の抽出物を含むことを特徴とする。 BACE1 inhibitor of the present invention, Aβ aggregate formation inhibitor, cytoprotective agent against Aβ, acetylcholinesterase inhibitor, cognitive function improving agent, memory maintenance or improving agent (hereinafter referred to as "BACE1 inhibitor of the present invention, etc.") A pharmaceutical composition for the treatment and/or prevention of dementia and/or MCI (Mild Cognition Impairment), which may be prepared from the exocarp of Bokeh, linden, and Cinnamon, and from plants belonging to the Caryophyllaceae family. It is characterized by containing at least one extract selected from the group consisting of:
ボケ(木瓜)(学名：Chaenomeles speciosa)は、バラ科に属する植物であり、中国原産で日本にも広く分布している。果実部分が古くから食用とされている(果実酒、ジャムなど)。 Chaenomeles speciosa (scientific name: Chaenomeles speciosa) is a plant belonging to the Rosaceae family, and is native to China and widely distributed in Japan. The fruit part has been used as food since ancient times (fruit wine, jam, etc.).
リンデン(学名：Tilia europaea、Tilia plantyphillos, Tilia cordata, Tilia argentea)とは、シナノキ科に属する植物であり、高い木でヨーロッパ、北米に植栽されている。主に花や葉部がリンデンフラワーとして、ハーブティーや香り付け等に古くから利用されている。 Linden (scientific name: Tilia europaea, Tilia plantyphillos, Tilia cordata, Tilia argentea) is a tall tree that belongs to the Tiliaceae family and is planted in Europe and North America. The flowers and leaves, mainly known as linden flowers, have been used for a long time in herbal teas and flavorings.
サンカクトウ(山核桃)(学名：Carya cathayensis Sarg.)とは、クルミ科に属する植物であり、産地は中国である。中国では、種子がナッツとして食されている。 Carya cathayensis Sarg. (scientific name: Carya cathayensis Sarg.) is a plant belonging to the walnut family, and is produced in China. In China, the seeds are eaten as nuts.
ナデシコ科に属する植物としては、具体的には、ジャクゼツソウ、カーネーション、ナデシコ、カスミソウ、サポナリア、サボンソウ、ハコベ等などが挙げられ、特にジャクゼツソウが好ましい。 Specific examples of plants belonging to the family Caryophyllaceae include Jasperum japonica, carnation, Dianthus dianthus, Gypsophila, Saponaria, Saponaria, Chickweed, and the like, with Jasperum japonica being particularly preferred.
ジャクゼツソウ(雀舌草)(学名：Stellaria alsine Grimm)とは、ナデシコ科に属する植物であり、アジア温帯地域に広く分布している。別名：ノミノフスマである。温野菜として、若茎、若葉が食用とされている。 Stellaria alsine Grimm (scientific name: Stellaria alsine Grimm) is a plant belonging to the Caryophyllaceae family and is widely distributed in temperate regions of Asia. Another name: Chimney chinensis. The young stems and young leaves are eaten as a hot vegetable.
ボケ、リンデン、サンカクトウの外果皮、及びナデシコ科に属する植物(以下、これらの植物をまとめて「本発明の植物」と称することもある)の抽出物の製造には、これらの植物の一部若しくは全体を使用することもできる。植物の一部としては、花、花穂、果皮（内果皮、外果皮、中果皮）、果実、果肉、茎、葉、枝、枝葉、幹、樹皮、根茎、根皮、根、種子、虫えい、心材、地上部、地下部などが挙げられ、これらを単独又は複数部位を組み合わせて使用することができる。抽出物の製造に使用する植物の部位としては、好ましくはボケは果実であり、リンデンは花及び葉であり、ナデシコ科に属する植物は葉である。また、抽出物の製造には、生の物、乾燥した物、切断又は粉砕された物などいずれの状態の植物も使用することができる。 In the production of extracts from the exocarps of lindens, lindens, and cicadas, and plants belonging to the Caryophyllaceae family (hereinafter, these plants may be collectively referred to as "plants of the present invention"), some of these plants are used. Alternatively, the whole can be used. Plant parts include flowers, flower spikes, pericarp (endocarp, exocarp, mesocarp), fruit, pulp, stem, leaves, branches, foliage, trunk, bark, rhizome, root bark, roots, seeds, and insect base. , heartwood, above-ground parts, underground parts, etc., and these parts can be used singly or in combination. The parts of the plant used for producing the extract are preferably fruits, flowers and leaves for linden, and leaves for plants belonging to the Caryophyllaceae family. In addition, plants in any state such as fresh, dried, cut or crushed plants can be used for producing the extract.
本発明の植物の抽出物を製造する方法としては、特に制限されず、通常用いられる方法により行うことができる。そのような方法としては、例えば、上記植物の各部位をそのまま又は適当な大きさに切断し、搾汁又は溶媒で抽出することや超臨界二酸化炭素抽出により行うことが挙げられる。抽出方法としては、溶媒抽出が特に好ましい。そのような溶媒抽出には水、有機溶媒又は含水有機溶媒を使用することができ、有機溶媒としては、例えば、メタノール、エタノール、１－プロパノール、２－プロパノール、１－ブタノール、２－ブタノール、２－メチル－１－プロパノール、２－メチル－２－プロパノール、１－ペンタノール、２－ペンタノール、３－ペンタノール等の炭素数1～5の低級アルコール、ジエチルエーテル等のエーテル類、酢酸メチル、酢酸エチル等のエステル類、アセトン等のケトン類、酢酸、氷酢酸、プロピオン酸等の有機酸等が挙げられる。抽出溶媒としては、好ましくは、水、メタノール、エタノール及びこれらの任意の2種以上を任意の割合で混合した水性溶媒であり、特に好ましい抽出溶媒は、水、食品添加物として認められている有機溶媒であるエタノールと水とを任意の割合で混合した水性溶媒である。 The method for producing the plant extract of the present invention is not particularly limited, and any commonly used method can be used. Examples of such methods include, for example, extracting each part of the plant as it is or cutting it into appropriate sizes, and extracting the juice or using a solvent, or using supercritical carbon dioxide extraction. As the extraction method, solvent extraction is particularly preferred. Water, organic solvents or water-containing organic solvents can be used for such solvent extraction, such as methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, 2-butanol, - Lower alcohols with 1 to 5 carbon atoms such as methyl-1-propanol, 2-methyl-2-propanol, 1-pentanol, 2-pentanol, and 3-pentanol, ethers such as diethyl ether, methyl acetate, Examples include esters such as ethyl acetate, ketones such as acetone, and organic acids such as acetic acid, glacial acetic acid, and propionic acid. Preferably, the extraction solvent is water, methanol, ethanol, or an aqueous solvent obtained by mixing any two or more of these in any ratio. Particularly preferable extraction solvents are water and organic solvents that are recognized as food additives. It is an aqueous solvent made by mixing the solvent ethanol and water in an arbitrary ratio.
溶媒抽出としては、エタノール抽出及び熱水抽出が特に好ましい。エタノール抽出に使用する溶媒としては、例えば、エタノール濃度が10～60容量%又は45～55容量%の含水エタノールが挙げられる。 As the solvent extraction, ethanol extraction and hot water extraction are particularly preferred. Examples of the solvent used for ethanol extraction include aqueous ethanol with an ethanol concentration of 10 to 60% by volume or 45 to 55% by volume.
抽出溶媒の使用量は、用いる植物の部位や抽出溶媒等により異なるが、質量比で、例えば、1：2～1：30 (植物原料：抽出溶媒)であり、1：3～1：20の範囲内が好ましい。 The amount of extraction solvent used varies depending on the part of the plant used, the extraction solvent, etc., but the mass ratio is, for example, 1:2 to 1:30 (plant material: extraction solvent), and 1:3 to 1:20. Preferably within this range.
抽出溶媒の温度は、室温を超え抽出溶媒の沸点以下の任意の温度とすることができ、抽出効率、被抽出物の耐熱性、揮発性等を考慮して決定されることが望ましい。必要に応じて、抽出効率を向上させるために、加熱した抽出溶媒を用いることもできる。熱水抽出を行う場合の温度は、例えば70℃以上、好ましくは80℃以上、より好ましくは95℃以上である。抽出時間としては、30分～15日の範囲内が挙げられる。抽出溶媒として水及び水性溶媒を用いる場合には、抽出効率を向上させるために、必要に応じて、酸、塩基、塩等を適宜含ませることができる。抽出に用いる水のpHとしては、特に制限されず、生体への使用を考慮して中性付近が好ましく、pH4～9がより好ましく、pH6～8が更に好ましい。 The temperature of the extraction solvent can be any temperature above room temperature and below the boiling point of the extraction solvent, and is desirably determined in consideration of extraction efficiency, heat resistance, volatility, etc. of the material to be extracted. If necessary, a heated extraction solvent can also be used to improve extraction efficiency. The temperature when performing hot water extraction is, for example, 70°C or higher, preferably 80°C or higher, and more preferably 95°C or higher. The extraction time is within the range of 30 minutes to 15 days. When using water or an aqueous solvent as an extraction solvent, an acid, a base, a salt, etc. can be appropriately included as needed in order to improve extraction efficiency. The pH of the water used for extraction is not particularly limited, but in consideration of its use in living organisms, it is preferably around neutrality, more preferably pH 4 to 9, and even more preferably pH 6 to 8.
溶媒抽出は任意の公知の方法により行うことができ、例えば、本発明の植物を溶媒中で所定時間混合後、ろ過、遠心分離、デカンテーション等により固形分と分離する方法、ソックスレー抽出法等の連続抽出法等の方法を用いることができる。 Solvent extraction can be carried out by any known method, for example, a method in which the plant of the present invention is mixed in a solvent for a predetermined period of time and then separated from the solid content by filtration, centrifugation, decantation, etc., Soxhlet extraction method, etc. Methods such as continuous extraction methods can be used.
ろ過により不溶分等を除去する場合には、必要に応じて、不純物を除去するために活性炭、ベントナイト、セライト等の吸着剤やろ過助剤を添加することもできる。特に抽出液の状態で用いる場合には、メンブレンフィルター等による除菌ろ過を併せて行うことが好ましい。 When removing insoluble matters by filtration, an adsorbent or filter aid such as activated carbon, bentonite, or celite may be added to remove impurities, if necessary. Particularly when the extract is used in the form of an extract, it is preferable to perform sterilization filtration using a membrane filter or the like.
本発明の植物の溶媒抽出物は、そのままでも使用することができ、必要に応じて、限外濾過、分子篩クロマトグラフィー(ゲル濾過)、吸着クロマトグラフィー、イオン交換クロマトグラフィー、アフィニティクロマトグラフィー、高速液体クロマトグラフィー(HPLC)、透析法、これらの組合せなどによる精製を行うことができる。 The solvent extract of the plant of the present invention can be used as it is, and if necessary, ultrafiltration, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, high performance liquid Purification can be performed by chromatography (HPLC), dialysis, a combination thereof, and the like.
本発明の植物の溶媒抽出物としては、回収された抽出液(必要に応じて更に精製されたものも含む)、当該抽出液を濃縮した濃縮液、凍結乾燥、スプレードライ等により当該抽出液の溶媒が除去された固形物などが含まれる。ここで、抽出液の濃縮、凍結乾燥及びスプレードライは、常法に従って行うことができる。 The solvent extract of the plant of the present invention includes a recovered extract (including one further purified if necessary), a concentrated liquid obtained by concentrating the extract, a liquid extracted by freeze-drying, spray-drying, etc. This includes solid matter from which the solvent has been removed. Here, concentration, freeze drying, and spray drying of the extract can be performed according to conventional methods.
アルツハイマー型認知症に関連する諸症状の改善に関連すると思われる活性の一例として、アミロイドβの生成を阻害する活性が挙げられる。アルツハイマー型認知症は、脳内にアミロイドβからなるアミロイド線維が沈着して生成した老人斑が広範に認められる神経変性疾患であり、アミロイドβの蓄積がアルツハイマー型認知症の発症の引き金であるというアミロイドβ仮説が提唱されている。アミロイドβは、アミノ酸残基数約700の一回膜貫通型前駆体タンパク質(アミロイド前駆体タンパク質：APP)が2種類のタンパク質プロセシング酵素により切断されることにより産生される。アミロイドβは、APPの細胞外領域から膜貫通領域にかけて存在しており、βセクレターゼによりアミロイドβのN末部分が、次いでγセクレターゼによりC末部分が切断させる。 An example of an activity that is thought to be related to the improvement of symptoms associated with Alzheimer's disease is the activity of inhibiting the production of amyloid β. Alzheimer's disease is a neurodegenerative disease in which senile plaques formed by the deposition of amyloid-β fibrils in the brain are widely observed, and the accumulation of amyloid-β is said to be the trigger for the onset of Alzheimer's disease. The amyloid β hypothesis has been proposed. Amyloid β is produced by cleavage of a single transmembrane precursor protein (amyloid precursor protein: APP), which has approximately 700 amino acid residues, by two types of protein processing enzymes. Amyloid β exists from the extracellular region to the transmembrane region of APP, and the N-terminal portion of amyloid β is cleaved by β-secretase, and then the C-terminal portion is cleaved by γ-secretase.
ＢＡＣＥ１ (β site amyloid cleaving enzyme)は、βセクレターゼを有する膜結合型のアスパラギン酸プロテアーゼであり、1999年にクローニングされて以来、阻害剤に関する多くの研究がなされている。一方、γセクレターゼは複数のサブユニットからなる複合タンパク質であるが、未だ正体は不明であり、APP以外にも、細胞の発生や分化に関与するいくつかの膜貫通型タンパク質も基質とすることが知られている。γセクレターゼの触媒中心であるプレセニリン(PS)をノックアウトしたマウスは胎生致死であることが知られており、γセクレターゼの阻害剤には危険な副作用が存在する可能性が懸念されている。 BACE1 (β site amyloid cleaving enzyme) is a membrane-bound aspartic protease having β-secretase, and since it was cloned in 1999, much research has been conducted on inhibitors. On the other hand, γ-secretase is a complex protein consisting of multiple subunits, but its identity is still unknown, and in addition to APP, it also uses several transmembrane proteins involved in cell development and differentiation as substrates. Are known. It is known that mice in which presenilin (PS), the catalytic center of γ-secretase, is knocked out are embryonic lethal, and there are concerns that γ-secretase inhibitors may have dangerous side effects.
アミロイド線維の脳内への沈着には、アミロイドβの凝集が深く関与している。アミロイドβは、単量体として存在する場合には無害であるが、分子間会合してβシート構造を形成することにより凝集し、線維状の凝集体を形成する。アミロイドβのコンホメーション変化の原因は必ずしも明らかではないが、アミロイドβの凝集を阻害する活性を有する化合物は、アルツハイマー型認知症の有効な治療薬となる可能性を有している。 Aggregation of amyloid β is deeply involved in the deposition of amyloid fibrils in the brain. Although amyloid β is harmless when it exists as a monomer, it aggregates by intermolecular association to form a β sheet structure and forms fibrous aggregates. Although the cause of conformational changes in amyloid β is not necessarily clear, compounds that have the activity of inhibiting amyloid β aggregation have the potential to become effective therapeutics for Alzheimer's dementia.
また、アルツハイマー型認知症では、脳内のアセチルコリンが減少するため、アセチルコリンの減少に関与するアセチルコリンエステラーゼの阻害剤が治療薬(例えば、ドネペジル塩酸塩、ガランタミン、リバスチグミン)として用いられている。 Furthermore, in Alzheimer's disease, acetylcholine in the brain decreases, and therefore inhibitors of acetylcholinesterase, which is involved in the decrease of acetylcholine, are used as therapeutic drugs (eg, donepezil hydrochloride, galantamine, rivastigmine).
本発明の植物の抽出物、特に本発明の植物の熱水抽出物及びエタノール抽出物は、高いアミロイドβの凝集を阻害する活性、高いＢＡＣＥ１阻害活性、アミロイドβ誘導性細胞死の抑制作用及びアセチルコリンエステラーゼ阻害活性を有しており、ＢＡＣＥ１阻害剤等、並びに認知症及び／又はMCIの治療及び／又は予防用の医薬組成物の有効成分として有効である。 The extract of the plant of the present invention, particularly the hot water extract and the ethanol extract of the plant of the present invention, have high amyloid-β aggregation inhibiting activity, high BACE1 inhibitory activity, amyloid-β-induced cell death suppressing effect, and acetyl It has cholinesterase inhibitory activity and is effective as a BACE1 inhibitor, etc., and as an active ingredient of a pharmaceutical composition for treating and/or preventing dementia and/or MCI.
本発明のＢＡＣＥ１阻害剤等、並びに認知症及び／又はMCIの治療及び／又は予防用の医薬組成物に含まれる本発明の植物の抽出物の割合は、特に制限されず、例えば、0.01～99質量%の濃度を挙げることができ、下限値としては0.1質量%、1質量%、2質量%、3質量%、4質量%、5質量%、6質量%、7質量%、8質量%、9質量%、10質量%であってもよく、その上限値は90質量%、80質量%、70質量%、60質量%、50質量%、10質量%であってもよい。 The proportion of the plant extract of the present invention contained in the BACE1 inhibitor, etc. of the present invention, and the pharmaceutical composition for treating and/or preventing dementia and/or MCI is not particularly limited, and is, for example, 0.01 to 99% The concentration in mass% can be listed, and the lower limit values are 0.1% by mass, 1% by mass, 2% by mass, 3% by mass, 4% by mass, 5% by mass, 6% by mass, 7% by mass, 8% by mass, It may be 9% by mass or 10% by mass, and its upper limit may be 90% by mass, 80% by mass, 70% by mass, 60% by mass, 50% by mass, or 10% by mass.
本発明のＢＡＣＥ１阻害剤等、並びに認知症及び／又はMCIの治療及び／又は予防用の医薬組成物には、本発明の効果を妨げない範囲で、本発明の植物の抽出物以外の公知の成分を適宜配合することができる。 The BACE1 inhibitor of the present invention, etc., and the pharmaceutical composition for treating and/or preventing dementia and/or MCI may contain known ingredients other than the extract of the plant of the present invention, to the extent that they do not impede the effects of the present invention. Components can be mixed as appropriate.
本発明のＢＡＣＥ１阻害剤等は、飲食品(特に、保健、健康維持、増進等を目的とする飲食品(例えば、健康食品、機能性食品、栄養補助食品、サプリメント、特定保健用食品、栄養機能食品、又は機能性表示食品))、医薬品(医薬部外品も含む)などとして使用することができる。また、本発明のＢＡＣＥ１阻害剤等は、それぞれＢＡＣＥ１阻害作用、アミロイドβ凝集阻害作用、アミロイドβ誘導性細胞死の抑制作用、アセチルコリンエステラーゼ阻害作用、認知機能改善作用、及び記憶力の維持又は改善作用を付与する添加剤についての意味も包含するものである。 The BACE1 inhibitor of the present invention can be used in foods and drinks (especially foods and drinks for the purpose of health, health maintenance, promotion, etc.) (e.g., health foods, functional foods, nutritional supplements, supplements, foods for specified health uses, nutritional functional foods, etc.). It can be used as food (or food with functional claims), medicine (including quasi-drugs), etc. In addition, the BACE1 inhibitors of the present invention each have a BACE1 inhibitory effect, an amyloid β aggregation inhibitory effect, an amyloid β-induced cell death inhibitory effect, an acetylcholinesterase inhibitory effect, a cognitive function improving effect, and a memory maintenance or improving effect. It also includes the meaning of additives to be added.
上記の飲食品には、本発明の植物の抽出物をそのまま使用することもできるが、必要に応じて、ビタミン類、フラボノイド類、ミネラル類、キノン類、ポリフェノール類、アミノ酸、核酸、必須脂肪酸、清涼剤、結合剤、甘味料、着色料、香料、安定化剤、防腐剤、崩壊剤、滑沢剤、徐放調整剤、界面活性剤、溶解剤、湿潤剤等を配合することができる。 Although the extract of the plant of the present invention can be used as it is in the above-mentioned food and drink products, vitamins, flavonoids, minerals, quinones, polyphenols, amino acids, nucleic acids, essential fatty acids, Cooling agents, binders, sweeteners, colorants, fragrances, stabilizers, preservatives, disintegrants, lubricants, sustained release modifiers, surfactants, solubilizers, wetting agents, and the like can be added.
飲食品には、動物(ヒトを含む)が摂取できるあらゆる飲食品が含まれる。飲食品の種類は、特に限定されず、例えば、乳製品；発酵食品(ヨーグルト、チーズ等)；飲料類(コーヒー、ジュース、茶飲料のような清涼飲料、乳飲料、乳酸菌飲料、乳酸菌入り飲料、ヨーグルト飲料、炭酸飲料、日本酒、洋酒、果実酒のような酒等)；スプレッド類(カスタードクリーム等)；ペースト類(フルーツペースト等)；洋菓子類(チョコレート、ドーナツ、パイ、シュークリーム、ガム、キャンデー、ゼリー、クッキー、ケーキ、プリン等)；和菓子類(大福、餅、饅頭、カステラ、あんみつ、羊羹等)；氷菓類(アイスクリーム、アイスキャンデー、シャーベット等)；食品類(カレー、牛丼、雑炊、味噌汁、スープ、ミートソース、パスタ、漬物、ジャム等)；調味料類(ドレッシング、ふりかけ、旨味調味料、スープの素等)などが挙げられる。 Food and beverages include all foods and beverages that can be ingested by animals (including humans). The types of food and beverages are not particularly limited, and include, for example, dairy products; fermented foods (yoghurt, cheese, etc.); beverages (coffee, juice, soft drinks such as tea drinks, milk drinks, lactic acid bacteria drinks, drinks containing lactic acid bacteria, Yogurt drinks, carbonated drinks, Japanese sake, Western liquor, alcoholic beverages such as fruit wine, etc.); Spreads (custard cream, etc.); Pastes (fruit paste, etc.); Western sweets (chocolate, donuts, pies, cream puffs, gum, candy, etc.) Jelly, cookies, cakes, pudding, etc.); Japanese sweets (daifuku, mochi, manju, castella, anmitsu, yokan, etc.); Frozen confections (ice cream, popsicles, sorbet, etc.); Foods (curry, gyudon, rice porridge, etc.) Examples include miso soup, soup, meat sauce, pasta, pickles, jam, etc.); seasonings (dressing, furikake, umami seasonings, soup stock, etc.).
飲食品の製法も特に限定されず、適宜公知の方法に従うことができる。 The method for producing the food or drink is not particularly limited, and any known method can be followed as appropriate.
飲食品をサプリメントとして使用する際の投与単位形態については特に限定されず適宜選択でき、例えば、錠剤、カプセル剤、顆粒剤、液剤、散剤等が挙げられる。 The dosage unit form when food and drink is used as a supplement is not particularly limited and can be selected as appropriate, and includes, for example, tablets, capsules, granules, liquids, powders, and the like.
飲食品の摂取量は、摂取者の体重、年齢、性別、症状などの種々の条件に応じて適宜設定することができる。 The intake amount of food and drink can be appropriately set according to various conditions such as the weight, age, sex, and symptoms of the person taking the food.
上記の医薬品(以下の医薬品には「認知症及び／又はMCIの治療及び／又は予防用の医薬組成物」も含まれる)には、本発明の植物の抽出物のみを使用することもでき、ビタミン、生薬など日本薬局方に記載の他の医薬成分と混合して使用することもできる。 For the above pharmaceuticals (the following pharmaceuticals also include "pharmaceutical compositions for the treatment and/or prevention of dementia and/or MCI"), only the extract of the plant of the present invention can be used, It can also be used in combination with other pharmaceutical ingredients listed in the Japanese Pharmacopoeia, such as vitamins and herbal medicines.
医薬品として調製する場合、本発明の植物の抽出物をそのまま使用するか、又は医薬品において許容される成分とともに、タブレット(素錠、糖衣錠、フィルムコート錠、発泡錠、チュアブル錠、トローチ剤などを含む)、カプセル剤、丸剤、粉末剤(散剤)、細粒剤、顆粒剤、液剤、懸濁液、乳濁液、シロップ、ペースト、注射剤(使用時に、蒸留水又はアミノ酸輸液や電解質輸液等の輸液に配合して液剤として調製する場合を含む)などの形態に調製して、医薬用の製剤にすることが可能である。 When preparing a pharmaceutical product, the extract of the plant of the present invention can be used as it is, or it can be prepared in the form of tablets (including plain tablets, sugar-coated tablets, film-coated tablets, effervescent tablets, chewable tablets, lozenges, etc.) together with ingredients that are acceptable for pharmaceuticals. ), capsules, pills, powders (powders), fine granules, granules, solutions, suspensions, emulsions, syrups, pastes, injections (when used, distilled water or amino acid infusions, electrolyte infusions, etc.) (including the case where it is mixed into an infusion solution and prepared as a liquid preparation), and can be prepared into a pharmaceutical preparation.
医薬品には、本発明の植物の抽出物以外にも、必要に応じて、賦形剤、結合剤、崩壊剤、滑沢剤、懸濁化剤、増粘剤、抗酸化剤、吸収促進剤、pH調節剤、着色剤、保存剤、防腐剤、界面活性剤、安定化剤、甘味剤、矯味剤、香料等の薬学的に許容される成分を適宜配合することができる。 In addition to the plant extract of the present invention, pharmaceuticals may contain excipients, binders, disintegrants, lubricants, suspending agents, thickeners, antioxidants, and absorption enhancers as necessary. , a pH adjuster, a coloring agent, a preservative, a preservative, a surfactant, a stabilizer, a sweetener, a flavoring agent, a fragrance, and other pharmaceutically acceptable ingredients may be appropriately blended.
医薬品の投与方法は特に限定されず、例えば、経口投与、動脈内投与、静脈内投与、口腔内投与、直腸投与、経腸投与、経皮投与などにより行うことができる。 The method of administering the drug is not particularly limited, and can be carried out, for example, by oral administration, intraarterial administration, intravenous administration, intraoral administration, rectal administration, enteral administration, transdermal administration, and the like.
医薬品の投与量は、患者の体重、年齢、性別、症状、投与方法、剤型の種類などの種々の条件に応じて適宜決定することができる。 The dosage of the drug can be appropriately determined depending on various conditions such as the patient's weight, age, sex, symptoms, administration method, and type of dosage form.
飲食品及び医薬品の投与量及び摂取量としては、ヒトの場合、一般には製剤中に含有される有効成分の量で、好ましくは成人1日当り0.1～2000 mg/日である。もちろん投与量及び摂取量は、種々の条件によって変動するので、上記投与量及び摂取量より少ない量で十分な場合もあるし、或いは上記範囲を超えて必要な場合もある。 For humans, the dosage and intake of food, drink, and pharmaceutical products is generally the amount of the active ingredient contained in the formulation, preferably 0.1 to 2000 mg/day for an adult. Of course, the dosage and intake amount vary depending on various conditions, so there are cases where it is sufficient to use an amount smaller than the above-mentioned dosage and intake amount, or there are cases where an amount exceeding the above-mentioned range is necessary.
以上説明した本発明のＢＡＣＥ１阻害剤等、並びに認知症及び／又はMCIの治療及び／又は予防用の医薬組成物は、ヒトを含む哺乳動物に対して適用されるものである。 The BACE1 inhibitor, etc. of the present invention and the pharmaceutical composition for treating and/or preventing dementia and/or MCI described above are applicable to mammals including humans.
本発明の認知機能改善剤は、認知機能の改善を目的として使用される。ここで、「認知機能」とは、認知症において障害が見られる機能のことを意味する。「認知機能」としては、記憶力、注意力、言語機能、身につけた一連の動作を行う機能、五感を通じてまわりの状況を把握する機能、遂行機能、見当識などが挙げられる。「認知症」とは、後天的な脳の器質的障害により、一旦正常に発達した知能が不可逆的に低下した状態である。ここで、「改善」には、更なる症状の悪化の防止、すなわち維持、認知機能の悪化を遅延させる作用、一度悪化した認知機能を改善させる作用も包含される。 The cognitive function improving agent of the present invention is used for the purpose of improving cognitive function. Here, "cognitive function" means a function that is impaired in dementia. "Cognitive functions" include memory, attention, language functions, the ability to perform a series of learned movements, the ability to grasp the surrounding situation through the five senses, executive functions, and orientation. "Dementia" is a state in which intelligence, once normally developed, is irreversibly reduced due to acquired organic brain damage. Here, "improvement" includes prevention of further deterioration of symptoms, that is, maintenance, action to delay deterioration of cognitive function, and action to improve cognitive function once deteriorated.
本発明の認知機能改善剤を飲食品として使用する場合、「健常な中高年者の加齢によって低下する脳の活動性を改善し、認知機能の一部である記憶(言葉、数字、図形などを覚え、思い出すこと)の精度や判断の正確さを向上させる」、「認知機能の一部である記憶力(日常生活で見聞きした情報を覚え、思い出す力)を維持する機能がある」、「認知機能の一部である記憶力(数・ことば・状況などの情報を記憶し、思い出す力)を維持する機能がある」、「認知機能の一部である記憶力(見たり聞いたりした内容を記憶し、思い出す力)を維持する機能がある」、「認知機能の一部である記憶力(日常生活で見聞きした情報、言葉・物のイメージ、位置情報を覚え、思い出す力)をサポートする機能がある」、「認知機能の一部である記憶力(言葉・物のイメージ・位置情報を思い出す力)を維持する機能」、「認知機能の一部である、数・ことば・図形・状況などの情報の記憶をサポートする機能がある」、「認知機能の一部である記憶力(日常生活で生じる行動や判断を記憶し、思い出す力)を維持する機能がある」、「認知機能の一部である記憶(言葉や数字、図形などを覚え、思い出すこと)の精度や判断の正確さを向上させる」、「認知機能の一部である記憶(知覚・認識した物事の想起)の精度を高める」、「認知機能の一部である記憶力(少し前に見聞きしたことを思い出す力)を維持する」、「中高年の方の加齢に伴い低下する、認知機能の一部である記憶力、注意力、判断力、空間認識力を維持する」、「中高年の方の、認知機能の一部である記憶力(言葉や図形などを覚え、思い出す能力)を維持する」、「中高年の方の加齢に伴い低下する認知機能の一部(記憶力、注意力、判断力、空間認識力)を維持する作用」、「中高年の方の、認知機能の一部である数・ことば・図形・状況などの情報の記憶をサポートする機能がある」、「中高年の方の、認知機能の一部である数・ことば・図形・状況などの情報の記憶をサポートする機能がある」、「健常な中高年の方の加齢に伴い低下する認知機能の一部である記憶力(言葉や図形などを覚え、思い出す能力)を維持する」、「加齢により認知機能の一部である記憶機能(情報を記憶し、これをスムーズに思い出して判断する機能)が低下することを緩和する働きがある」、「脳の認知機能の一部である記憶力(少し前に見聞きしたことを思い出す力)を維持する」、「加齢に伴う記憶力の低下が気になる方に適した機能(記憶の保持・検索・再生に役立つ)がある」、「認知機能の一部である記憶(知覚・認識した物事の想起)の精度を高める」、「認知機能の一部である記憶(認知した言葉・物のイメージ・体験を思い出すこと)の精度や判断の正確さを向上させる」、「認知機能の一部である、記憶(言葉、人の顔)を維持する」、「中高年の方の加齢に伴い低下する、認知機能の一部である、数字・文字・図形・空間など情報の記憶をサポートする機能がある」、「認知機能の一部である記憶力(言葉や図形などを覚え、思い出す能力)を維持する」、「認知機能の一部である記憶力(日常生活で見たり聞いたりした情報を記憶し、思い出す力)を維持する機能がある」、「認知機能の一部である、数・ことば・図形・物語・色・状況などの情報の記憶をサポートする機能(物を覚えて記憶にとどめる力)」、「日常生活で疲労を感じる方の疲労感を軽減し、頭が冴えない・注意力低下といった疲労に伴う感覚の緩和、単純な記憶や判断を必要とする作業の効率向上に役立つ機能がある」等と表示してもよく、又はこれらに類似する表示を行ってもよい。 When the cognitive function improving agent of the present invention is used as a food or drink, it can improve brain activity that decreases with age in healthy middle-aged and elderly people, and improve memory, which is a part of cognitive function (words, numbers, shapes, etc.). ``Improve the accuracy of memory (remembering and recalling) and the accuracy of judgment'', ``It has the function of maintaining memory (the ability to remember and recall information seen and heard in daily life), which is a part of cognitive function'', ``Cognitive function It has the function of maintaining memory (the ability to memorize and recall information such as numbers, words, situations, etc.), which is part of the cognitive function. "It has a function that supports memory (the ability to remember and recall information seen and heard in daily life, images of words and objects, and location information), which is a part of cognitive function." ``The ability to maintain memory (the ability to remember words, images of objects, and location information), which is part of cognitive functions.'' ``It has a function to support memory (the ability to remember and recall actions and decisions that occur in daily life), which is a part of cognitive function.'' ``It has a function to maintain memory, which is a part of cognitive function. ``Improve the accuracy of memory (memorizing and recalling objects, numbers, shapes, etc.) and judgment,'' ``Improving the accuracy of memory (recalling things that have been perceived and recognized), which is a part of cognitive function,'' ``Cognitive function ``Maintaining memory (the ability to remember what you saw or heard a while ago)'', ``Maintaining memory, which is a part of cognitive functions that decline with age in middle-aged and older people, such as memory, attention, judgment, and spatial ability.'' ``Maintaining cognitive ability'', ``Maintaining memory (the ability to memorize and recall words, figures, etc.), which is a part of the cognitive function of middle-aged and elderly people'', ``Cognitive function that declines with age in middle-aged and elderly people'' ``Action to maintain some of the cognitive functions (memory, attention, judgment, spatial awareness)'' and ``Support the memory of information such as numbers, words, shapes, situations, etc., which are part of the cognitive functions of middle-aged and elderly people.'' "It has a function that supports the memory of information such as numbers, words, shapes, and situations, which is part of the cognitive functions of middle-aged and elderly people." Maintaining memory (the ability to memorize and recall words, figures, etc.), which is a part of cognitive function, and ``maintaining memory (ability to memorize information and recall it smoothly), which is part of cognitive function as we age. ``It works to alleviate the decline in memory (the ability to make decisions)'', ``maintains memory (the ability to remember what you saw and heard a while ago)'', which is a part of the brain's cognitive function'', and ``helps reduce the decline in memory that comes with aging.'' It has functions suitable for people who are concerned about its decline (useful for memory retention, retrieval, and replay)," "Improves the accuracy of memory (recall of things perceived and recognized), which is part of cognitive function." Improving the accuracy of memory (recalling recognized words, images of objects, experiences) and the accuracy of judgment, which are part of cognitive function, ), "It has a function that supports the memory of information such as numbers, letters, shapes, and spaces, which is part of the cognitive function that declines with age in middle-aged and elderly people." The ability to maintain memory (the ability to memorize and recall words, figures, etc.), which is part of the cognitive function, and the ability to maintain memory (the ability to remember and recall information seen and heard in daily life), which is part of the cognitive function. "There is a function that supports the memory of information such as numbers, words, shapes, stories, colors, situations, etc., which is part of the cognitive function (ability to remember and retain things in memory)", "Fatigue in daily life" It has functions that help reduce the feeling of fatigue in people who experience fatigue, alleviate feelings associated with fatigue such as dullness of mind and decreased attention, and improve efficiency in tasks that require simple memory and judgment.'' or a display similar to these may be used.
本発明のＢＡＣＥ１阻害剤等の適用対象者としては、認知症(特に、アルツハイマー病)の患者、MCIの患者、pre-MCIの患者などが挙げられるが、認知機能の維持を目的として、認知機能に問題がない高齢者も適用対象者に含まれる。 The BACE1 inhibitor of the present invention is applicable to patients with dementia (particularly Alzheimer's disease), MCI patients, pre-MCI patients, etc. This also includes elderly people who do not have any health problems.
後述する実施例で示すように、本発明者らは、本発明の植物の抽出物が優れたＢＡＣＥ１阻害活性、アミロイドβ凝集阻害活性、アミロイドβ誘導性細胞死の抑制作用及びアセチルコリンエステラーゼ阻害活性を示すことを見出した。そのため、本発明の植物の抽出物は、優れたＢＡＣＥ１阻害活性、アミロイドβ凝集阻害活性、アミロイドβ誘導性細胞死の抑制作用及びアセチルコリンエステラーゼ阻害活性を有するので、ＢＡＣＥ１阻害剤、Ａβ凝集体形成阻害剤、Ａβに対する細胞保護剤、アセチルコリンエステラーゼ阻害剤の有効成分として好適に使用することができる。さらに、後述する実施例で示すように、本発明者らは、本発明の植物の抽出物が優れた短期記憶障害の改善作用を示すことを見出した。本発明の植物の抽出物は、上記活性を有することから、認知機能改善剤、記憶力の維持又は改善剤並びに認知症及び／又はMCIの治療及び／又は予防用の医薬組成物の有効成分としても好適に使用することができる。 As shown in the Examples below, the present inventors have demonstrated that the plant extract of the present invention exhibits excellent BACE1 inhibitory activity, amyloid β aggregation inhibitory activity, amyloid β-induced cell death inhibitory effect, and acetylcholinesterase inhibitory activity. I found out that it shows. Therefore, the plant extract of the present invention has excellent BACE1 inhibitory activity, amyloid β aggregation inhibitory activity, amyloid β-induced cell death suppressing effect, and acetylcholinesterase inhibitory activity, and therefore can be used as a BACE1 inhibitor and Aβ aggregate formation inhibitor. It can be suitably used as an active ingredient of a drug, a cytoprotective agent against Aβ, and an acetylcholinesterase inhibitor. Furthermore, as shown in the Examples below, the present inventors have discovered that the extract of the plant of the present invention exhibits an excellent effect on improving short-term memory impairment. Since the plant extract of the present invention has the above-mentioned activity, it can also be used as an active ingredient of a cognitive function improving agent, a memory maintenance or improving agent, and a pharmaceutical composition for treating and/or preventing dementia and/or MCI. It can be suitably used.
また、本発明の植物は、食用又は生薬として使用されているものであるので高い安全性を有する。 Furthermore, the plants of the present invention are highly safe because they are used as food or herbal medicines.
以下、本発明を更に詳しく説明するため実施例を挙げる。しかし、本発明はこれら実施例等になんら限定されるものではない。 Examples are given below to explain the present invention in more detail. However, the present invention is not limited to these Examples in any way.
調製例
＜測定試料について＞
以下に示す植物(乾燥生薬、乾燥ハーブ)は全て国内の生薬卸より購入した。
・サンカクトウ(山核桃) 学名：Carya cathayensis Sarg. 抽出部位：外果皮
・ボケ(木瓜、別名：皺皮木瓜） 学名：Chaenomeles speciosa 抽出部位：果実
・ジャクゼツソウ(雀舌草) 学名：Stellaria alsine Grimm 抽出部位：葉
・リンデンフラワー(別名：セイヨウシナノキ) 学名 Tilia europaea 抽出部位：花部・葉部
・イチョウ 抽出部位：葉
・シソ 抽出部位：葉
Preparation example <About the measurement sample>
All of the plants shown below (dried crude drugs, dried herbs) were purchased from domestic crude drug wholesalers.
・Stellaria cathayensis Sarg. Scientific name: Carya cathayensis Sarg. Extracted part: Exocarp, Boke (also known as rugose quince) Scientific name: Chaenomeles speciosa Extracted part: Fruit, Stellaria alsine Grimm Scientific name: Stellaria alsine Grimm Extracted part : Leaves/linden flower (also known as linden flower) Scientific name: Tilia europaea Extracted parts: flowers/leaves/Ginkgo Extracted parts: leaves/perilla Extracted parts: leaves
＜抽出方法＞
(試験例１～４)
・50%エタノール抽出
フードプロセッサーを使用して各試料を破砕し、20倍量の50%エタノールを添加し、常温で2時間撹拌(抽出)を行った。撹拌後、ろ過して沈殿物を除去した。次いで、ロータリーエバポレーターでエタノールを除去し、凍結乾燥後、各抽出物を得た。なお、各抽出物はジメチルスルホキシド(DMSO)に溶解して測定に用いた。
<Extraction method>
(Test examples 1 to 4)
- 50% ethanol extraction Each sample was crushed using a food processor, 20 times the amount of 50% ethanol was added, and stirring (extraction) was performed at room temperature for 2 hours. After stirring, the mixture was filtered to remove the precipitate. Next, ethanol was removed using a rotary evaporator, and each extract was obtained after freeze-drying. Note that each extract was dissolved in dimethyl sulfoxide (DMSO) and used for measurement.
(試験例５)
・50%エタノール抽出
フードプロセッサーを使用して各試料を破砕し、10倍量の50%エタノールを添加し、常温で2時間撹拌(抽出)を行った。撹拌後、ろ過して沈殿物を除去した。次いで、ロータリーエバポレーターでエタノールを除去、濃縮し、凍結乾燥後、各抽出物を得た。
(Test example 5)
- 50% ethanol extraction Each sample was crushed using a food processor, 10 times the amount of 50% ethanol was added, and stirring (extraction) was performed at room temperature for 2 hours. After stirring, the mixture was filtered to remove the precipitate. Next, ethanol was removed and concentrated using a rotary evaporator, and each extract was obtained after freeze-drying.
・熱水抽出
フードプロセッサーを使用して試料を破砕し、10倍量の蒸留水を添加し、90℃で30分間加熱抽出を行った。抽出後、ろ過して沈殿物を除去した。次いで、ロータリーエバポレーターで濃縮し、凍結乾燥後、抽出物を得た。
・Hot water extraction The sample was crushed using a food processor, 10 times the volume of distilled water was added, and extraction was performed by heating at 90°C for 30 minutes. After extraction, the precipitate was removed by filtration. The extract was then concentrated using a rotary evaporator and freeze-dried to obtain an extract.
試験例１：ＢＡＣＥ１阻害活性の測定
ＢＡＣＥ１阻害活性の評価は、BACE1 FRETアッセイキット(Thermo Fisher Scientificより入手)を用いて行った。測定試料は最終濃度の30倍濃度となるようにDMSOに溶解し、キットに付属のバッファーで10倍希釈したものを使用した。測定方法は、96ウェルブラックプレート(ハーフエリア)に測定試料溶液(コントロールは10%DMSO)及び基質溶液を各ウェルに10μlずつ添加し、混和した。次いで、BACE1酵素溶液(ブランクはバッファー)を10μl添加して混和後、恒温槽内(28℃)で2時間静置した。次いで、反応停止液10μlを添加して混和後、プレートリーダーで蛍光強度(励起波長：545 nm、蛍光波長：585 nm)を測定した。ＢＡＣＥ１阻害率の算出は下式を用いて行った。
ＢＡＣＥ１阻害率（％）＝［（Ｃ－ＣＢ）－（Ｓ－ＳＢ）］／（Ｃ－ＣＢ）×１００
式中、Ｃは阻害剤非存在下における蛍光強度、ＣＢは阻害剤非存在下におけるバックグラウンド(コントロールバックグラウンド)、Ｓは阻害剤存在下における蛍光強度、ＳＢは阻害剤存在下におけるバックグラウンド(サンプルバックグラウンド)である。
Test Example 1: Measurement of BACE1 inhibitory activity Evaluation of BACE1 inhibitory activity was performed using a BACE1 FRET assay kit (obtained from Thermo Fisher Scientific). The measurement sample was dissolved in DMSO to a
BACE1 inhibition rate (%) = [(C-CB)-(S-SB)]/(C-CB) x 100
In the formula, C is the fluorescence intensity in the absence of the inhibitor, CB is the background in the absence of the inhibitor (control background), S is the fluorescence intensity in the presence of the inhibitor, and SB is the background in the presence of the inhibitor ( sample background).
結果を図１に示す。図１に示す通り、山核桃皮抽出物、ボケ抽出物、ジャクゼツソウ抽出物、リンデン抽出物は終濃度50μg/mlにおいて、ＢＡＣＥ１阻害活性を示すことが明らかとなった。 The results are shown in Figure 1. As shown in FIG. 1, it was revealed that the mountain kernel peach peel extract, the porphyry extract, the japonicum extract, and the linden extract exhibited BACE1 inhibitory activity at a final concentration of 50 μg/ml.
試験例２：Aβ凝集阻害活性の測定
アミロイドβ凝集阻害活性の評価には、in vitro評価法であるチオフラビンT法を用いた。チオフラビンTは凝集したAβと反応し、特異的な蛍光波長を示す試薬である。そのため、チオフラビンTを用いて、測定試料のAβ凝集阻害活性を簡便に測定することができる。測定は、Okudaらの方法(Journal of Alzheimer’s Disease 59, 313-328, 2017)に準拠して行った。具体的な手順は下記のとおりである。
Test Example 2: Measurement of Aβ aggregation inhibitory activity The Thioflavin T method, which is an in vitro evaluation method, was used to evaluate the amyloid β aggregation inhibitory activity. Thioflavin T is a reagent that reacts with aggregated Aβ and exhibits a specific fluorescence wavelength. Therefore, using Thioflavin T, the Aβ aggregation inhibitory activity of a measurement sample can be easily measured. Measurements were performed according to the method of Okuda et al. (Journal of Alzheimer's Disease 59, 313-328, 2017). The specific steps are as follows.
Aβ1-42 (株式会社ペプチド研究所より入手)を、濃度が0.2 mMとなるように0.1%アンモニア水に溶解した後、PBSでアミロイドβ濃度が20μMになるよう希釈した(Aβ溶液)。測定試料は、濃度が最終濃度の100倍になるようにDMSOに溶解した後、PBSで50倍希釈した(測定試料溶液)。Aβ溶液と測定試料溶液を、96ウェルブラックプレート(ハーフエリア)に25μlずつ添加して、ピペッティング撹拌した。プレートシールでウェルを密封して、37℃で24時間インキュベートした。次いで、100 mMトリス－グリシン緩衝液(pH：8.5)で6μMに調整したチオフラビンT (富士フイルム和光純薬株式会社製)を50μl加え、ピペッティング後、プレートリーダーで蛍光強度を測定した(励起波長：440 nm、蛍光波長：486 nm）。陽性対照として、アミロイドβ凝集阻害剤として知られるイチョウ葉抽出物、シソ葉抽出物を使用した。アミロイドβ(Aβ)凝集阻害率(%)は、下式を用いて求めた。
Ａβ凝集阻害率（％）＝［（Ｉ０－Ｂ０）－（Ｉ－Ｂ）］／（Ｉ０－Ｂ０）×１００
式中、Ｉ０は阻害剤非存在下における蛍光強度、Ｂ０は阻害剤非存在下におけるバックグラウンド、Ｉは阻害剤存在下における蛍光強度、Ｂは阻害剤存在下におけるバックグラウンドである。
Aβ1-42 (obtained from Peptide Institute Co., Ltd.) was dissolved in 0.1% ammonia water to a concentration of 0.2 mM, and then diluted with PBS to a concentration of amyloid β of 20 μM (Aβ solution). The measurement sample was dissolved in DMSO to a concentration of 100 times the final concentration, and then diluted 50 times with PBS (measurement sample solution). 25 μl each of the Aβ solution and measurement sample solution were added to a 96-well black plate (half area) and stirred by pipetting. Wells were sealed with plate seal and incubated at 37°C for 24 hours. Next, 50 μl of Thioflavin T (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) adjusted to 6 μM with 100 mM Tris-glycine buffer (pH: 8.5) was added, and after pipetting, the fluorescence intensity was measured with a plate reader (excitation wavelength : 440 nm, fluorescence wavelength: 486 nm). As a positive control, ginkgo biloba extract and perilla leaf extract, which are known as amyloid β aggregation inhibitors, were used. Amyloid β (Aβ) aggregation inhibition rate (%) was determined using the following formula.
Aβ aggregation inhibition rate (%) = [(I 0 - B 0 ) - (IB)] / (I 0 - B 0 ) x 100
In the formula, I 0 is the fluorescence intensity in the absence of an inhibitor, B 0 is the background in the absence of the inhibitor, I is the fluorescence intensity in the presence of the inhibitor, and B is the background in the presence of the inhibitor.
結果を図２に示す。図２に示す通り、山核桃皮抽出物、ボケ抽出物、ジャクゼツソウ抽出物、リンデン抽出物は終濃度10μg/mlにおいて、Aβ凝集阻害活性を示した。また、その活性は陽性対照である、イチョウ葉やシソ葉の抽出物と同等、もしくはそれらよりも強いことが明らかとなった。 The results are shown in Figure 2. As shown in FIG. 2, the mountain kernel peach peel extract, the porphyry extract, the japonicum extract, and the linden extract exhibited Aβ aggregation inhibitory activity at a final concentration of 10 μg/ml. It was also revealed that its activity was equivalent to or stronger than the positive controls, extracts of ginkgo biloba and perilla leaves.
試験例３： Aβ誘導性細胞死抑制作用の評価
ヒト神経芽細胞種株SH-SY5Y (株式会社ケー・エー・シーより入手)をDMEM培地(3%FBS、抗生物質含む)で40×104 cells/mlとなるように懸濁し、24ウェルI型コラーゲンコートプレートに0.5 mlずつ播種し、24時間前培養した。次いで、測定サンプル(DMSOに溶解)を0.5%DMEM培地(0.5%FBS、抗生物質含む、以下同様)に溶解し、培地交換を行った。1時間後に、Aβ1-42 (株式会社ペプチド研究所より入手)をDMSOに溶解してフィルター滅菌し、10μMとなるように各ウェルに添加(DMSO終濃度1%)して、22時間後に生細胞数の評価を行った。生細胞数の評価にはセルカウンティングキット-8 (株式会社同仁化学研究所)を用いた。キット付属の基質溶液を50μlずつウェルに添加し、37℃で60分反応後、プレートリーダーで吸光度450 nmを測定した。サンプル及びAβ1-42を添加していないウェル(コントロール)の吸光度を1として、各ウェルの吸光度の値を相対値(生細胞数相対値)で算出した。なお、相対値の算出においてはブランクとしてDMEM培地又はサンプルを溶解したDMEM培地を設定し、各々のブランク吸光度を差し引いた値を用いた。
Test Example 3: Evaluation of Aβ-induced cell death suppression effect Human neuroblastoma line SH-SY5Y (obtained from KAC Corporation) was grown in DMEM medium (3% FBS, containing antibiotics) at 40×10 4 The cells were suspended at a concentration of cells/ml, seeded in 0.5 ml portions onto a 24-well type I collagen coated plate, and precultured for 24 hours. Next, the measurement sample (dissolved in DMSO) was dissolved in 0.5% DMEM medium (0.5% FBS, containing antibiotics, the same applies hereinafter), and the medium was replaced. After 1 hour, Aβ1-42 (obtained from Peptide Institute Co., Ltd.) was dissolved in DMSO, filter sterilized, added to each well at 10 μM (DMSO final concentration 1%), and 22 hours later, live cells were dissolved. A number of evaluations were conducted. Cell Counting Kit-8 (Dojindo Kagaku Kenkyusho Co., Ltd.) was used to evaluate the number of viable cells. 50 μl of the substrate solution provided with the kit was added to each well, and after reacting at 37°C for 60 minutes, absorbance at 450 nm was measured using a plate reader. The absorbance value of each well was calculated as a relative value (relative value of the number of living cells), with the absorbance of the sample and wells to which Aβ1-42 was not added (control) set as 1. In addition, in calculating the relative value, a DMEM medium or a DMEM medium in which the sample was dissolved was set as a blank, and the value obtained by subtracting each blank absorbance was used.
結果を図３に示す。図３に示す通り、アミロイドβを細胞に添加することで生細胞数が低下した。一方、山核桃皮抽出物、ボケ抽出物、ジャクゼツソウ抽出物、リンデン抽出物を終濃度20μg/mlで細胞に事前添加することにより、アミロイドβ添加による細胞死が抑制された。すなわち、各抽出物がAβ誘導性細胞死抑制作用を有することが明らかとなった。 The results are shown in Figure 3. As shown in FIG. 3, adding amyloid β to cells reduced the number of viable cells. On the other hand, cell death caused by the addition of amyloid-β was suppressed by adding in advance to the cells a mountain kernel peach extract, a bokeh extract, a japonicum extract, and a linden extract at a final concentration of 20 μg/ml. In other words, it was revealed that each extract had an inhibitory effect on Aβ-induced cell death.
試験例４：アセチルコリンエステラーゼ阻害活性の測定
アセチルコリンエステラーゼ(Sigma-Aldrich)を0.1 unit/mlとなるように0.01Mリン酸緩衝液(pH7.4)に溶解し酵素溶液とした。DTNB試薬(株式会社同仁化学研究所)を0.15 mMとなるように0.1M リン酸緩衝液(pH7.4)に溶解しDTNB溶液とした。測定試料はDMSOに溶解し、0.01Mリン酸緩衝液(pH7.4)で希釈して試料溶液を作製した。アセチルチオコリン(東京化成工業株式会社)は2.25 mMとなるように蒸留水に溶解し基質溶液とした。なお、本評価系ではリン酸緩衝液は既製品(富士フイルム和光純薬株式会社、生化学用)を使用した。次に、試料溶液20μl、DTNB溶液200μl、酵素溶液20μlを96ウェルプレートに添加、混合し、恒温槽内(24℃)で10分静置した。次に基質溶液30μlを添加、混合し、恒温槽内(24℃)で30分間反応させた。反応後、プレートリーダーで吸光度405 nmを測定し、アセチルコリンエステラーゼ阻害率を算出した。
アセチルコリンエステラーゼ阻害率（％）＝［（Ｃ－ＣＢ）－（Ｓ－ＳＢ）］／（Ｃ－ＣＢ）×１００
式中、Ｃは阻害剤非存在下における吸光度、ＣＢは阻害剤非存在下におけるバックグラウンド(コントロールバックグラウンド)、Ｓは阻害剤存在下における吸光度、ＳＢは阻害剤存在下におけるバックグラウンド(サンプルバックグラウンド)である。
Test Example 4: Measurement of acetylcholinesterase inhibitory activity Acetylcholinesterase (Sigma-Aldrich) was dissolved in 0.01M phosphate buffer (pH 7.4) to a concentration of 0.1 unit/ml to prepare an enzyme solution. DTNB reagent (Dojindo Kagaku Kenkyusho Co., Ltd.) was dissolved in 0.1M phosphate buffer (pH 7.4) to a concentration of 0.15 mM to obtain a DTNB solution. The measurement sample was dissolved in DMSO and diluted with 0.01M phosphate buffer (pH 7.4) to prepare a sample solution. Acetylthiocholine (Tokyo Kasei Kogyo Co., Ltd.) was dissolved in distilled water to a concentration of 2.25 mM to prepare a substrate solution. In this evaluation system, a ready-made phosphate buffer (Fujifilm Wako Pure Chemical Industries, Ltd., for biochemistry use) was used. Next, 20 μl of the sample solution, 200 μl of the DTNB solution, and 20 μl of the enzyme solution were added to a 96-well plate, mixed, and allowed to stand in a constant temperature bath (24° C.) for 10 minutes. Next, 30 μl of the substrate solution was added, mixed, and reacted in a constant temperature bath (24° C.) for 30 minutes. After the reaction, absorbance at 405 nm was measured using a plate reader, and the acetylcholinesterase inhibition rate was calculated.
Acetylcholinesterase inhibition rate (%) = [(C-CB)-(S-SB)]/(C-CB) x 100
In the formula, C is the absorbance in the absence of the inhibitor, CB is the background in the absence of the inhibitor (control background), S is the absorbance in the presence of the inhibitor, and SB is the background in the presence of the inhibitor (sample background). ground).
結果を図４に示す。図４に示す通り、山核桃皮抽出物、ボケ抽出物、ジャクゼツソウ抽出物、リンデン抽出物は終濃度37μg/mlにおいて、アセチルコリンエステラーゼ阻害活性を示した。 The results are shown in Figure 4. As shown in FIG. 4, the mountain kernel peach peel extract, the peach extract, the japonica extract, and the linden extract exhibited acetylcholinesterase inhibitory activity at a final concentration of 37 μg/ml.
試験例５：認知機能改善評価
βアミロイド1-42脳室内単回投与による学習記憶障害に対する被験物質の効果をY迷路試験で評価した。
Test Example 5: Evaluation of Cognitive Function Improvement The effect of the test substance on learning and memory impairment caused by a single intracerebroventricular administration of β-amyloid 1-42 was evaluated using a Y-maze test.
＜試験系＞
試験動物として、雄性Slc: ddYマウス(SPF、日本エスエルシー株式会社)を使用した。当該マウスとして、5週齢のものを入手した。入手1日後の体重範囲は24.4～32.1 gであった。入手した動物には5日間の検疫期間、その後1日の馴化期間を設けた。体重推移及び一般状態に異常の認められない動物を群分けに用いた。群分けはコンピュータプログラムを用いて、各群の平均体重がほぼ等しくなるように馴化終了日に行った。
<Test system>
Male Slc: ddY mice (SPF, Japan SLC Co., Ltd.) were used as test animals. The mice were obtained at the age of 5 weeks. The body weight range 1 day after acquisition was 24.4 to 32.1 g. The obtained animals underwent a 5-day quarantine period, followed by a 1-day acclimatization period. Animals with no abnormality observed in weight change or general condition were used for grouping. Grouping was performed using a computer program on the day at the end of acclimatization so that the average weight of each group was approximately equal.
マウスは、管理温度：20.0～26.0℃、管理湿度：40.0～70.0%RH、明暗各12時間(照明：6時～18時)、換気回数：12回／時(フィルターを通した新鮮空気)に維持された飼育室で飼育した。マウスは、平床式プラスチック製ケージ(W：310×D：360×H：175 mm)を用いて1ケージあたり5匹まで、群分け後は1ケージあたり6匹までの群飼育をした。飼料としては、固型飼料(MF、オリエンタル酵母工業株式会社)を、飲料水としては、水道水をそれぞれ自由に摂取させた。 Mice were kept under controlled temperature: 20.0 to 26.0°C, controlled humidity: 40.0 to 70.0% RH, 12 hours each of light and darkness (lighting: 6:00 to 18:00), and ventilation frequency: 12 times/hour (fresh air through a filter). They were kept in a maintained breeding room. Mice were housed in groups of up to 5 mice per cage using flat-bed plastic cages (W: 310 x D: 360 x H: 175 mm), and up to 6 mice per cage after grouping. The animals were given free access to solid feed (MF, Oriental Yeast Co., Ltd.) as feed, and tap water as drinking water.
＜投与＞
投与経路は、経口投与とした。投与方法としては、ディスポーザブルマウス用経口ゾンデ(有限会社フチガミ器械)を取り付けたポリプロピレン製ディスポーザブル注射筒(テルモ株式会社)を用いて強制経口投与した。被験物質については投与操作時には、懸濁液をマグネチックスターラーで撹拌し、注射筒に吸引した。
<Administration>
The administration route was oral administration. The administration method was forcible oral administration using a polypropylene disposable syringe (Terumo Corporation) equipped with a disposable mouse oral probe (Fuchigami Kikai Co., Ltd.). For the test substance, during administration, the suspension was stirred with a magnetic stirrer and aspirated into a syringe.
＜実験スケジュール＞
投与液量は、投与日の体重を基準として、10 mL/kgで算出した。投与回数は、投与開始日を投与1日とし、1日1回、14日間の合計14回とした。投与8日にβアミロイドを注入し、投与14日にY迷路試験を行った。
<Experiment schedule>
The amount of liquid administered was calculated at 10 mL/kg based on the body weight on the day of administration. The number of administrations was once a day for 14 days, with the first day of administration being the first day of administration, for a total of 14 times. β-amyloid was injected on the 8th day of administration, and a Y-maze test was performed on the 14th day of administration.
＜群構成＞
群構成は、コントロールとして、生理食塩液を脳室内投与し媒体(0.5%MC)を経口投与する偽手術群、βアミロイドを脳室内投与し媒体(0.5%MC)を経口投与する媒体対照群を設け、ボケ熱水抽出物、ボケ50%エタノール抽出物、リンデン50%エタノール抽出物の500 mg/kg投与群を、それぞれ1群10例で設定した。0.5%MCはメチルセルロースを注射用水で0.5w/v%濃度となるように溶解したものである。被験物質は、それぞれ0.5%MCに懸濁させて用いた。
<Group composition>
The group composition was a sham surgery group in which physiological saline was administered intracerebroventricularly and vehicle (0.5% MC) was orally administered, and a vehicle control group in which β-amyloid was administered intracerebroventricularly and vehicle (0.5% MC) was orally administered. A group of 500 mg/kg of Bokeh hot water extract,
＜βアミロイドの注入＞
動物をペントバルビタールナトリウム(東京化成工業株式会社) 40 mg/kgで腹腔内投与(投与液量：10 mL/kg)し、麻酔した。麻酔後、頭皮にレボブピバカイン塩酸塩(ポプスカイン(登録商標) 0.25%注、丸石製薬株式会社)を皮下投与(0.1 mL)した。動物の頭頂部の毛を刈り、頭部を脳定位固定装置に固定した。頭頂部をイソジン(登録商標)で消毒後に切開して、頭蓋骨を露出させ、頭蓋骨上の結合組織を綿棒で取り除いたのち、ブロワーで乾燥させてブレグマの位置を見やすくした。歯科用ドリルを用いてブレグマより側方1 mm (右側)、後方0.2 mmの頭蓋骨にステンレス製パイプ刺入用の穴を開けた。骨表面から2.5 mmの深さまで外径0.5 mmのシリコンチューブ及びマイクロシリンジに接続されたステンレス製パイプを垂直に刺入した。脳室内にβアミロイド溶液(偽手術群は生理食塩液を3μL) 3μL (200 pmol/3μL)をマイクロシリンジポンプで3分間かけて注入した。注入後は、ステンレス製パイプを挿入したまま3分間静置し、ステンレス製パイプをゆっくりと抜去した。頭蓋穴を非吸収性骨髄止血剤(ネストップ(登録商標)、アルフレッサファーマ株式会社)で塞ぎ、頭皮を縫合した。動物を脳定位固定装置から外し、飼育ケージに戻した。なお、ステンレス製パイプ及びシリコンチューブは滅菌済みのものを使用した。
<Injection of β-amyloid>
The animals were anesthetized by intraperitoneal administration of pentobarbital sodium (Tokyo Kasei Kogyo Co., Ltd.) at 40 mg/kg (administration volume: 10 mL/kg). After anesthesia, levobupivacaine hydrochloride (Popskine (registered trademark) 0.25% injection, Maruishi Pharmaceutical Co., Ltd.) was subcutaneously administered (0.1 mL) to the scalp. The hair on the top of the animal's head was shaved, and the head was fixed in a stereotaxic apparatus. After disinfecting the top of the head with Isodine (registered trademark), an incision was made to expose the skull, and the connective tissue on the skull was removed with a cotton swab, and then dried with a blower to make it easier to see the location of bregma. A hole for inserting a stainless steel pipe was made in the skull 1 mm lateral (right side) and 0.2 mm posterior to bregma using a dental drill. A silicone tube with an outer diameter of 0.5 mm and a stainless steel pipe connected to a microsyringe were inserted vertically to a depth of 2.5 mm from the bone surface. 3 μL (200 pmol/3 μL) of β-amyloid solution (3 μL of physiological saline for the sham surgery group) was injected into the ventricles of the brain over 3 minutes using a microsyringe pump. After injection, the stainless steel pipe was left still inserted for 3 minutes, and then the stainless steel pipe was slowly removed. The cranial hole was closed with a non-absorbable bone marrow hemostatic agent (Nestop (registered trademark), Alfresa Pharma Co., Ltd.), and the scalp was sutured. Animals were removed from the stereotaxic apparatus and returned to their housing cages. Note that the stainless steel pipe and silicone tube used were sterilized.
＜Y迷路試験(自発的交替行動試験)＞
検査には、1本のアームの長さが39.5 cm、床の幅が4.5 cm、壁の高さが12 cmで、3アームがそれぞれ120度に分岐しているプラスチック製のY字型迷路(有限会社ユニコム)を用いた。測定前に、装置の床面の照度が10～40 lx (実測値：10.5～13.0 lx)になるように調節した。
<Y-maze test (spontaneous alternation behavior test)>
The test consisted of a plastic Y-maze with one arm length of 39.5 cm, floor width of 4.5 cm, wall height of 12 cm, and three arms branching at 120 degrees each ( Unicom Co., Ltd.) was used. Before measurement, the illuminance on the floor of the apparatus was adjusted to 10 to 40 lx (actual value: 10.5 to 13.0 lx).
動物をY字型迷路のいずれかのアームに置き、8分間迷路内を自由に探索させた。動物が測定時間内に移動したアームの番号(A～C)を記録し、アームに移動した回数を数え、総エントリー数とした。次に、この中で連続して異なる3つのアームを選択した組み合わせを調べ、この数を自発的交替行動数とした。さらに以下の式を用いて自発的交替行動率を算出した。
自発的交替行動率(%)=［自発的交替行動数／(総エントリー数－2)］×100
Animals were placed in either arm of the Y-maze and allowed to freely explore the maze for 8 min. The number of the arm (A to C) to which the animal moved within the measurement time was recorded, and the number of times the animal moved to the arm was counted, which was taken as the total number of entries. Next, we examined combinations in which three different arms were selected consecutively, and this number was taken as the number of spontaneous alternation behaviors. Furthermore, the spontaneous replacement behavior rate was calculated using the following formula.
Spontaneous replacement behavior rate (%) = [Number of spontaneous replacement actions / (total number of entries - 2)] × 100
例えば、Y字型迷路の各アームをA、B、Cとし、動物がACBABACBABの順で移動した場合の自発的交替行動数は、ACB、CBA、BAC、ACB、CBAの5となり、これを総エントリー数10から2を引いた8で割った値に100を掛けた数値62.5を自発的交替行動率とした。 For example, if the arms of a Y-shaped maze are A, B, and C, and the animal moves in the order ACBABACBAB, the number of spontaneous alternations will be 5, which is ACB, CBA, BAC, ACB, and CBA. The number of entries, 10 minus 2, divided by 8, multiplied by 100, 62.5, was taken as the spontaneous replacement behavior rate.
＜統計学的方法＞
有意差検定は以下の組み合わせで2群間比較を行った。2群間比較はF検定による等分散性の検定を行い、等分散の場合はStudentのt検定を、不等分散の場合はWelchのt検定を行った。有意水準は5%とした。なお、有意差検定には、市販の統計プログラム(SASシステム ver. 9.4、SAS Institute Japan株式会社)を使用した。
・偽手術群vs媒体対照群
・媒体対照群vsボケ熱水抽出物群、ボケ50%エタノール抽出物群、リンデン50%エタノール抽出物群
<Statistical method>
Significant difference tests were performed between two groups using the following combinations. For comparisons between two groups, equality of variance was tested using the F test; Student's t-test was used in the case of equal variance, and Welch's t-test was used in the case of unequal variance. The significance level was set at 5%. A commercially available statistical program (SAS system ver. 9.4, SAS Institute Japan Co., Ltd.) was used for the significance test.
・Sham surgery group vs. vehicle control group ・Vehicle control group vs. Bokeh hot water extract group,
Y迷路試験の結果(自発的交替行動率)を図５に示す。媒体対照群は、偽手術群と比較して自発的交替行動率の有意な低下が認められた(p<0.01)。ボケ熱水抽出物群、ボケ50%エタノール抽出物群及びリンデン50%エタノール抽出物群は、媒体対照群と比較して自発的交替行動率の有意な上昇が認められた(p<0.01)。
The results of the Y-maze test (spontaneous alternation behavior rate) are shown in Figure 5. A significant decrease in spontaneous alternation behavior rate was observed in the vehicle control group compared to the sham surgery group (p<0.01). A significant increase in spontaneous alternation behavior rate was observed in the Bokeh hot water extract group,
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