JP7204159B1 - Combinations and food compositions - Google Patents
Combinations and food compositions Download PDFInfo
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- JP7204159B1 JP7204159B1 JP2022104090A JP2022104090A JP7204159B1 JP 7204159 B1 JP7204159 B1 JP 7204159B1 JP 2022104090 A JP2022104090 A JP 2022104090A JP 2022104090 A JP2022104090 A JP 2022104090A JP 7204159 B1 JP7204159 B1 JP 7204159B1
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Abstract
【課題】BDNF発現増加、BDNF-TrkBシグナル伝達系活性化等のための組合せ剤及び食品組成物の提供。【解決手段】プラズマローゲンと、クロレラ・ピレノイドサの細胞壁破砕物とを含む、BDNF発現増加、BDNF-TrkBシグナル伝達系活性化、又はBDNF-TrkB-CREBシグナル伝達系活性化のための組合せ剤。プラズマローゲンと、クロレラ・ピレノイドサの細胞壁破砕物とを含む、BDNF発現増加、BDNF-TrkBシグナル伝達系活性化、又はBDNF-TrkB-CREBシグナル伝達系活性化用の食品組成物。【選択図】なしAn object of the present invention is to provide a combination agent and a food composition for increasing BDNF expression, activating the BDNF-TrkB signal transduction system, and the like. A combination agent for increasing BDNF expression, activating the BDNF-TrkB signaling system, or activating the BDNF-TrkB-CREB signaling system, comprising plasmalogen and Chlorella pyrenoidosa cell wall debris. A food composition for increasing BDNF expression, activating the BDNF-TrkB signaling system, or activating the BDNF-TrkB-CREB signaling system, comprising a plasmalogen and a cell wall fragment of Chlorella pyrenoidosa. [Selection figure] None
Description
本発明は、BDNF発現増加、BDNF-TrkBシグナル伝達系活性化、又はBDNF-TrkB-CREBシグナル伝達系活性化のための組合せ剤及び食品組成物に関する。 The present invention relates to combinations and food compositions for increasing BDNF expression, activating the BDNF-TrkB signaling system, or activating the BDNF-TrkB-CREB signaling system.
脳由来神経栄養因子(brain-derived neurotrophic factor; BDNF)は、神経細胞の発生や成長、維持、修復に働き、さらに学習や記憶、情動、摂食、糖代謝などにおいても重要な働きをする分泌タンパク質である。 Brain-derived neurotrophic factor (BDNF) plays an important role in the development, growth, maintenance, and repair of nerve cells, as well as in learning, memory, emotion, food intake, and glucose metabolism. is protein.
近年、BDNFが、うつ病やアルツハイマー病患者の脳、主として海馬、大脳皮質で減少していることが確認されている。 In recent years, it has been confirmed that BDNF is reduced in the brains of patients with depression and Alzheimer's disease, mainly in the hippocampus and cerebral cortex.
BDNFと記憶・学習との関連をみても、空間認知の記憶・学習能力を評価する水迷路課題試験において、ラットでこの水迷路の成績と海馬におけるBDNF mRNA発現量との間に正の相関関係が報告されている。 Looking at the relationship between BDNF and memory and learning, in the water maze task test that evaluates memory and learning ability of spatial cognition, there is a positive correlation between the water maze performance and the amount of BDNF mRNA expression in the hippocampus in rats. has been reported.
また、BDNF発現を増加、あるいはBDNF特異的受容体を活性化させる化合物が、アルツハイマー病やうつ病等の精神疾患により低下した脳機能を改善させる可能性を示す結果が報告されている。 In addition, results have been reported indicating the possibility that compounds that increase BDNF expression or activate BDNF-specific receptors may improve brain function that has been reduced due to mental disorders such as Alzheimer's disease and depression.
このように、BDNFは、脳・神経系の疾患のバイオマーカーや創薬ターゲットとして着目されている。 Thus, BDNF is attracting attention as a biomarker and drug discovery target for diseases of the brain and nervous system.
また、BDNFは、神経障害性疼痛の発症、摂食抑制、肥満時の糖代謝の改善、心拍数や血圧の調節にも関わっている。更には、BDNFについては、身体運動による脳BDNF mRNAの誘導も知られている。 BDNF is also involved in the development of neuropathic pain, food intake suppression, improvement of glucose metabolism in obesity, and regulation of heart rate and blood pressure. Furthermore, for BDNF, the induction of brain BDNF mRNA by physical exercise is also known.
短期投与において海馬における脳由来神経栄養因子(BDNF)を増加させる作用があり、短期投与による抗うつ作用が期待される物質として、特開2012-102059号公報には、トゥルーラベンダー(Lavandula angustifolia)、スパイクラベンダー(Lavandula latifolia)、ラバンジン(Lavandula ×intermedia)及びオレガノ(Origanum vulgare)の抽出物並びに該植物抽出物を分画して得られる分画物が開示されている。 Japanese Patent Application Laid-Open No. 2012-102059 describes true lavender (Lavandula angustifolia) as a substance that has the effect of increasing brain-derived neurotrophic factor (BDNF) in the hippocampus in short-term administration and is expected to have an antidepressant effect by short-term administration. Extracts of spike lavender (Lavandula latifolia), lavandin (Lavandula x intermedia) and oregano (Origanum vulgare) and fractions obtained by fractionating said plant extracts are disclosed.
また特許6890810号公報には、シリンガレジノール-di-O-β-D-グルコシドを有効成分とするBDNF-TrkBシグナル伝達系活性化剤が、脳におけるBDNF産生の増加によりTrkBシグナル伝達系を活性化し、それにより神経細胞の発生、成長、維持、修復を高めることができることが開示されている。 Japanese Patent No. 6890810 discloses that a BDNF-TrkB signal transduction system activator containing syringaresinol-di-O-β-D-glucoside as an active ingredient activates the TrkB signal transduction system by increasing BDNF production in the brain. It is disclosed that it can activate, thereby enhancing neuronal cell development, growth, maintenance and repair.
また特開2016-210696号公報には、プラズマローゲンを含む、健常な哺乳動物に対して投与されるように用いられる、学習記憶能力増強剤として、プラズマローゲンの90質量%以上が、エタノールアミンプラズマローゲン及びコリンプラズマローゲンであり、鳥組織から抽出されたものであり、プラズマローゲンに含有される(エタノールアミンプラズマローゲン:コリンプラズマローゲン)の質量比が、(1:5)~(5:1)であるプラズマローゲンが好ましい学習記憶能力増強剤が開示されている。 Further, in Japanese Patent Laid-Open No. 2016-210696, as a learning and memory enhancement agent containing plasmalogen, which is used to be administered to healthy mammals, 90% by mass or more of plasmalogen is ethanolamine plasma. Rogen and choline plasmalogen, which are extracted from avian tissue, and the mass ratio of (ethanolamine plasmalogen:choline plasmalogen) contained in the plasmalogen is (1:5) to (5:1) A learning and memory enhancer is disclosed, preferably a plasmalogen that is:
更に、特開2019-162042号公報には、魚介類又は水産動物からの抽出物であって、プラズマローゲン型リン脂質を高純度に含有し、且つ、ヒ素の含量が低減された、プラズマローゲン型リン脂質高含有抽出物及びその製造方法に関する発明が開示されている。 Furthermore, Japanese Patent Application Laid-Open No. 2019-162042 discloses an extract from fish and shellfish or aquatic animals, which contains plasmalogen-type phospholipids in high purity and has a reduced arsenic content. An invention relating to a phospholipid-rich extract and a method for producing the same has been disclosed.
本発明は、上記背景技術の下、BDNF発現増加、BDNF-TrkBシグナル伝達系活性化、又はBDNF-TrkB-CREBシグナル伝達系活性化のための組合せ剤及び食品組成物を提供することを目的とする。 Under the above background art, the purpose of the present invention is to provide combinations and food compositions for increasing BDNF expression, activating the BDNF-TrkB signaling system, or activating the BDNF-TrkB-CREB signaling system. do.
本発明の組合せ剤及び食品組成物は、次のように表わすことができる。 The combinations and food compositions of the invention can be expressed as follows.
(1) プラズマローゲンと、クロレラ・ピレノイドサの細胞壁破砕物とを含む、組合せ剤。 (1) A combination comprising a plasmalogen and a cell wall fragment of Chlorella pyrenoidosa.
(2) 上記プラズマローゲンとクロレラ・ピレノイドサの細胞壁破砕物の両者を共に含むことにより当該両者が同時に使用されるか、又は、上記プラズマローゲンを含む剤Aと、上記クロレラ・ピレノイドサの細胞壁破砕物を含む剤Bが、時間をあけて又は同時に使用される、上記(1)記載の組合せ剤。 (2) containing both the above-mentioned plasmalogen and the cell wall fragment of Chlorella pyrenoidosa together so that the two are used at the same time, or agent A containing the above-mentioned plasmalogen and the above-mentioned cell wall fragment of Chlorella pyrenoidosa; The combination according to (1) above, wherein agent B is used separately or simultaneously.
(3) BDNF発現増加のための上記(1)又は(2)記載の組合せ剤。 (3) A combination according to (1) or (2) above for increasing BDNF expression.
(4) BDNF-TrkBシグナル伝達系活性化のための上記(1)又は(2)記載の組合せ剤。 (4) The combination according to (1) or (2) above for activating the BDNF-TrkB signaling system.
(5) BDNF-TrkB-CREBシグナル伝達系活性化のための上記(1)又は(2)記載の組合せ剤。 (5) The combination according to (1) or (2) above for activating the BDNF-TrkB-CREB signaling system.
(6) 上記プラズマローゲンがマボヤ又はその他のホヤ由来である上記(1)又は(2)記載の組合せ剤。 (6) The combination according to (1) or (2) above, wherein the plasmalogen is derived from Ascidian or other sea squirts.
(7) プラズマローゲンと、クロレラ・ピレノイドサの細胞壁破砕物とを含む、BDNF発現増加用の食品組成物。 (7) A food composition for increasing BDNF expression, comprising a plasmalogen and a cell wall fragment of Chlorella pyrenoidosa.
(8) プラズマローゲンと、クロレラ・ピレノイドサの細胞壁破砕物とを含む、BDNF-TrkBシグナル伝達系活性化用の食品組成物。 (8) A food composition for activating the BDNF-TrkB signal transduction system, comprising a plasmalogen and a cell wall fragment of Chlorella pyrenoidosa.
(9) プラズマローゲンと、クロレラ・ピレノイドサの細胞壁破砕物とを含む、BDNF-TrkB-CREBシグナル伝達系活性化用の食品組成物。 (9) A food composition for activating the BDNF-TrkB-CREB signaling system, comprising a plasmalogen and a cell wall fragment of Chlorella pyrenoidosa.
(10) 上記プラズマローゲンがマボヤ又はその他のホヤ由来である上記(7)乃至(9)の何れか1項に記載の食品組成物。 (10) The food composition according to any one of (7) to (9) above, wherein the plasmalogen is derived from a sea squirt or another sea squirt.
本発明の組合せ剤及び食品組成物は、BDNF発現増加、BDNF-TrkBシグナル伝達系活性化、又はBDNF-TrkB-CREBシグナル伝達系活性化の効果を有する。 The combination agent and food composition of the present invention have effects of increasing BDNF expression, activating the BDNF-TrkB signaling system, or activating the BDNF-TrkB-CREB signaling system.
本発明の実施の形態を説明する。 An embodiment of the present invention will be described.
(1) 本発明の組合せ剤及び食品組成物におけるプラズマローゲンは、グリセロール分子のsn-1(α)位にビニルエーテル結合を有する1-アルケニル-2-アシル-sn-グリセロ-3-燐酸同族体であって、一般に下記式[1]で表されるアルケニルエーテル型燐脂質である。 (1) The plasmalogen in the combination and food composition of the present invention is a 1-alkenyl-2-acyl-sn-glycero-3-phosphate analogue having a vinyl ether bond at the sn-1(α) position of the glycerol molecule. It is an alkenyl ether-type phospholipid generally represented by the following formula [1].
式[1]中、R1は一般には炭素数1乃至20のアルキル基(例えば、ドデシル基、テトラデシル基、ヘキサデシル基、オクタデシル基、イコサニル基)であり、R2は一般には多価不飽和脂肪酸(DHA、EPA、アラキドン酸などを含む)又はオレイン酸などの一価の不飽和脂肪酸の残基に相当する脂肪族炭化水素基(例えば、R2CO-基として、オクタデカジエノイル基、オクタデカトリエノイル基、イコサテトラエノイル基、イコサペンタエノイル基、ドコサテトラエノイル基、ドコサペンタエノイル基、ドコサヘキサエノイル基が挙げられる。好ましくはイコサペンタエノイル基又はドコサヘキサエノイル基である。)、Xは例えばエタノールアミン(下記式[2])、コリン(下記式[3])、セリン、イノシトール、又はグリセロール(好ましくはエタノールアミン又はコリン)である。 In formula [1], R 1 is generally an alkyl group having 1 to 20 carbon atoms (e.g., dodecyl group, tetradecyl group, hexadecyl group, octadecyl group, icosanyl group), and R 2 is generally a polyunsaturated fatty acid. (including DHA, EPA, arachidonic acid, etc.) or an aliphatic hydrocarbon group corresponding to the residue of a monovalent unsaturated fatty acid such as oleic acid (e.g. decatrienoyl group, icosatetraenoyl group, icosapentaenoyl group, docosatetraenoyl group, docosapentaenoyl group and docosahexaenoyl group, preferably icosapentaenoyl group or docosahexaenoyl group. enoyl group), X is, for example, ethanolamine (formula [2] below), choline (formula [3] below), serine, inositol, or glycerol (preferably ethanolamine or choline).
本発明におけるプラズマローゲンとしては、例えば、魚介類若しくは水産動物(例えば、ホヤなどの原索動物、ホタテ、レイシガイ、ムラサキイガイ、ミドリイガイ、マガキ、ヒザラガイ、チヂミボラ、クボガイ、アワビ、ムラサキインコガイなどの軟体動物、ナマコ、ヒトデ、ウニなどの棘皮動物門、イソギンチャクなどの腔腸動物、サケ、サンマ、カツオ、マグロ、クジラなどの脊椎動物)、哺乳類(例えば、牛、豚、馬、羊など)、鳥類(例えば、鶏、家鴨、鴨、七面鳥など)、嫌気性細菌など(好ましい例としてはマボヤ[Halocynthia roretzi]又はその他のホヤ)に含まれる各種プラズマローゲン(混合物又は単体)を用いることができるほか、それらのプラズマローゲンと同一の立体配置を有し、公知手段で化学合成されたものも排除されない。一般的には、前記魚介類若しくは水産動物その他から公知手段により抽出した、プラズマローゲンを含有する抽出物又は抽出物を処理したもの(抽出物の乾燥粉末、プラズマローゲンを濃縮したもの、プラズマローゲンを希釈若しくは増量したもの、抽出物にその他の公知手段による処理を施したものを含む、処理抽出物)を用いることができる。或いは、前記魚介類若しくは水産動物その他における一部についての公知手段による処理物(例えばホヤ可食部のフリーズドライ粉末)を用いることもできる。このような抽出物及び処理抽出物或いは処理物中のプラズマローゲン含有量は、例えば、0.05乃至99.9質量%である。好ましくは0.1乃至50質量%、より好ましくは0.2乃至40質量%、更に好ましくは0.3乃至30質量%である。 The plasmalogen in the present invention includes, for example, fish and shellfish or aquatic animals (e.g., protochordates such as sea squirts, scallops, mussels, mussels, green mussels, Pacific oysters, mussels, blue mullet, cucumbers, abalones, molluscs such as parakeets). , sea cucumbers, starfish, sea urchins, and other echinoderms; coelenterates, such as sea anemones; For example, chickens, domestic ducks, ducks, turkeys, etc.), anaerobic bacteria, etc. (a preferable example is Halocynthia roretzi or other sea squirts). which have the same configuration as the plasmalogen of , and chemically synthesized by known means are not excluded. In general, an extract containing plasmalogen extracted by known means from the fish, shellfish or aquatic animals, or a product obtained by processing the extract (dried extract powder, concentrated plasmalogen, plasmalogen-containing Processed extracts, including those that have been diluted or expanded, and those that have undergone processing of the extract by other known means) can be used. Alternatively, a part of the fish, shellfish or aquatic animals processed by known means (for example, freeze-dried powder of edible parts of sea squirt) can also be used. The plasmalogen content in such extracts and treated extracts or treated products is, for example, 0.05 to 99.9% by mass. It is preferably 0.1 to 50% by mass, more preferably 0.2 to 40% by mass, still more preferably 0.3 to 30% by mass.
(2) 本発明におけるクロレラ・ピレノイドサ(Chlorella pyrenoidosa)の細胞壁破砕物は、例えば次のようにして得ることができる。 (2) The crushed cell wall of Chlorella pyrenoidosa in the present invention can be obtained, for example, as follows.
先ずクロレラ濃度10乃至25重量%のクロレラ粉体・水懸濁液を10℃以下に調整する。次にこの懸濁液を、下記のような連続湿式微粉砕機に送入し、破砕直後のスラリーが40℃以下になるよう微粉砕する。次いで、このようにして得られたクロレラスラリーを、直ちに10℃以下に冷却することにより、細胞壁が破砕されたクロレラを、品質劣化を生じさせることなく得ることができる。 First, a chlorella powder/water suspension having a chlorella concentration of 10 to 25% by weight is adjusted to 10° C. or lower. Next, this suspension is fed into a continuous wet pulverizer as described below and pulverized so that the slurry immediately after pulverization has a temperature of 40° C. or lower. Then, the chlorella slurry thus obtained is immediately cooled to 10° C. or less, whereby chlorella with crushed cell walls can be obtained without deterioration in quality.
上記連続湿式微粉砕機としては、冷却外套を持つ密閉シリンダー中に、当該密閉シリンダー容量の80乃至85%の多数の直径0.5乃至1.5mmのグラスビーズが封入されたものを好ましく用いることができる。そのグラスビーズを流入液体と混和・回転させることにより、流入液体中の物質を効果的に摩砕し得る。 As the continuous wet pulverizer, a closed cylinder having a cooling jacket and a large number of glass beads with a diameter of 0.5 to 1.5 mm enclosed in 80 to 85% of the capacity of the closed cylinder is preferably used. can be done. By mixing and rotating the glass beads with the inflow liquid, substances in the inflow liquid can be effectively milled.
このようにして得られたクロレラ・ピレノイドサの細胞壁破砕物は、そのまま用いることもできるが、例えば、真空乾燥後粉砕を行う等の適宜の処理を施した後に使用してもよい。 The crushed cell wall of Chlorella pyrenoidosa obtained in this way can be used as it is, but it may be used after being subjected to an appropriate treatment such as vacuum drying followed by pulverization.
(3) 本発明の組合せ剤は、経口投与又は非経口投与により適用することができる。 (3) The combination of the invention can be applied by oral or parenteral administration.
本発明の組合せ剤は、例えば、プラズマローゲンと、クロレラ・ピレノイドサの細胞壁破砕物の両者を共に含むことにより、前記両者が同時に使用されるものとすることができる。 For example, the combination agent of the present invention can contain both the plasmalogen and the cell wall fragment of Chlorella pyrenoidosa, so that the two can be used at the same time.
或いは例えば、本発明の組合せ剤は、
プラズマローゲンを含む剤Aと、クロレラ・ピレノイドサの細胞壁破砕物を含む剤Bが、時間をあけて又は同時に使用されるものとすることができる。この場合、例えば、両方が経口投与剤であるもの、剤Aが非経口投与剤で剤Bが経口投与剤であるものなどのように、両者の投与経路が同じであるものとすることができ、異なるものとすることもできる。
Alternatively, for example, the combinations of the invention are
The agent A containing the plasmalogen and the agent B containing the cell wall fragment of Chlorella pyrenoidosa can be used at intervals of time or simultaneously. In this case, both administration routes can be the same, for example, both are orally administered agents, or agent A is a parenterally administered agent and agent B is an orally administered agent. , can be different.
(4) 本発明の組合せ剤及び食品組成物の経口投与の形態に特に限定はないが、例えば、粉末、錠剤、硬カプセル剤、軟カプセル剤、液剤とすることができる。本発明の組合せ剤が、プラズマローゲンを含む剤Aと、クロレラ・ピレノイドサの細胞壁破砕物を含む剤Bからなるものである場合は、剤Aと剤Bの形態が同じであっても異なっていてもよい。 (4) The form of oral administration of the combination and food composition of the present invention is not particularly limited, but may be, for example, powder, tablet, hard capsule, soft capsule, or liquid. When the combination of the present invention comprises agent A containing plasmalogen and agent B containing cell wall fragment of Chlorella pyrenoidosa, agent A and agent B may have the same or different forms. good too.
また種々の形態を形成する上で、各種賦形剤、結合剤、崩壊剤、滑沢剤、コーティング剤、着色剤、矯味剤、矯臭剤、可塑剤等を適宜用いることができる。 Various excipients, binders, disintegrants, lubricants, coating agents, coloring agents, corrigents, corrigents, plasticizers and the like can be appropriately used to form various forms.
賦形剤の例としては、糖類(乳糖、白糖、ブドウ糖、マンニトール)、マルチトール、デンプン(バレイショ、コムギ、トウモロコシ)、無機物(炭酸カルシウム、硫酸カルシウム、炭酸水素ナトリウム、塩化ナトリウム)、デキストリン、結晶セルロース、植物末(カンゾウ末、ゲンチアナ末)等を挙げることができる。 Examples of excipients include sugars (lactose, sucrose, glucose, mannitol), maltitol, starches (potato, wheat, corn), minerals (calcium carbonate, calcium sulfate, sodium bicarbonate, sodium chloride), dextrin, crystals. Cellulose, plant powder (licorice powder, gentian powder) and the like can be mentioned.
結合剤の例としては、デンプンのり液、アラビアゴム、ゼラチン、二酸化ケイ素、アルギン酸ナトリウム、メチ/レセルロース(MC)、エチルセルロース(EC)、ポリビニルピロリドン(PVP)、ポリビニルアルコール(PVA)、ヒドロキシプロピルセルロース(HPC)、カルポキシメチルセルロース(CMC)等を挙げることができる。 Examples of binders include starch paste, gum arabic, gelatin, silicon dioxide, sodium alginate, methy/recellulose (MC), ethyl cellulose (EC), polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), hydroxypropylcellulose. (HPC), carboxymethyl cellulose (CMC), and the like.
崩壊剤の例としては、デンプン、寒天、ゼラチン末、結晶セルロース、CMC・Na、CMC・Ca、炭酸カルシウム、炭酸水素ナトリウム、アルギン酸ナトリウム等を挙げることができる。 Examples of disintegrants include starch, agar, gelatin powder, crystalline cellulose, CMC·Na, CMC·Ca, calcium carbonate, sodium hydrogencarbonate, sodium alginate and the like.
滑沢剤の例としては、ステアリン酸マグネシウム、ショ糖脂肪酸エステル、タルク、水素添加植物油、マクロゴール、シリコーン油等を挙げることができる。 Examples of lubricants include magnesium stearate, sucrose fatty acid ester, talc, hydrogenated vegetable oil, macrogol, and silicone oil.
コーティング剤の例としては、糖衣(白糖、HPC、セラック)、膠衣(ゼラチン、グリセリン、ソルビトール)、フイルムコーティング〔ヒドロキシプロピルメチルセルロース(HPMC)、EC、HPC、PVP〕、腸溶性コーティング〔ヒドロキシプロビルメチルセルロースフタレート(HPMCP)、セルロースアセテートフタレート(CAP)〕等を挙げることができる。 Examples of coating agents include sugar coating (white sugar, HPC, shellac), glue coating (gelatin, glycerin, sorbitol), film coating [hydroxypropyl methylcellulose (HPMC), EC, HPC, PVP], enteric coating [hydroxypropyl methyl cellulose phthalate (HPMCP), cellulose acetate phthalate (CAP)] and the like.
着色剤の例としては、水溶性食用色素、レーキ色素等を挙げることができる。矯味剤の例としては、乳糖、白糖、ブドウ糖、マンニトール、マルチトール等を挙げることができる。矯臭剤の例としては、芳香性精油類、光線遮断剤(酸化チタン)等を挙げることができる。可塑剤の例としては、フタル酸エステル類、植物油、ポリエチレングリコール)等を挙げることができる。 Examples of coloring agents include water-soluble food dyes and lake dyes. Examples of flavoring agents include lactose, sucrose, glucose, mannitol, maltitol and the like. Examples of flavoring agents include aromatic essential oils and light shielding agents (titanium oxide). Examples of plasticizers include phthalates, vegetable oils, polyethylene glycol) and the like.
(5) 本発明の組合せ剤及び食品組成物のヒトに対する経口投与量は、プラズマローゲンの含有量において例えば0.1mg乃至1000mg/日程度が好ましく、クロレラ・ピレノイドサの細胞壁破砕物の含有量において例えば1000mg乃至20000mg/日程度が好ましい。 (5) The oral dose of the combination and food composition of the present invention to humans is preferably, for example, about 0.1 mg to 1000 mg/day in terms of plasmalogen content, and in terms of content of cell wall fragments of Chlorella pyrenoidosa, for example, About 1000 mg to 20000 mg/day is preferable.
非経口剤としては、例えば、プラズマローゲンについて、皮下注射剤、静脈内注射剤、筋肉内注射剤、腹腔内注射剤、点滴剤等の注射剤として液剤、或いは、溶液、懸濁液、乳濁液、用時溶剤に溶解、懸濁又は乳化させて用いる固形の注射剤が挙げられるが、これらに限るものではない。 Parenteral agents include, for example, plasmalogen, as injections such as subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections, and drip infusions, liquids, solutions, suspensions, and emulsions. Examples include, but are not limited to, liquids and solid injections that are used by dissolving, suspending or emulsifying them in a solvent when used.
(6) 本発明の組合せ剤及び食品組成物は、例えば栄養食品、栄養補助食品又は飲料用液体等を構成する飲食組成物中に含有した形態とすることができ、本発明の効果を損なわない各種成分を含むものとすることができる。 (6) The combination and food composition of the present invention can be in the form of being contained in a food or drink composition that constitutes, for example, a nutritional food, a dietary supplement, or a liquid for drinking, without impairing the effects of the present invention. Various components can be included.
海馬における、脳由来神経栄養因子(BDNF)、高親和性BDNF受容体(TrkB)、及び核内転写因子cAMP-response element binding protein(CREB)それぞれの発現量、並びに、TrkBのリン酸化(TrkBの活性化指標)及びCREBのリン酸化(CREBの活性化指標)に対する、プラズマローゲンとクロレラ・ピレノイドサの細胞壁破砕物の投与の効果について、試験を行った。 Expression levels of brain-derived neurotrophic factor (BDNF), high-affinity BDNF receptor (TrkB), and nuclear transcription factor cAMP-response element binding protein (CREB) in hippocampus, and TrkB phosphorylation (TrkB The effect of administration of plasmalogen and cell wall debris of Chlorella pyrenoidosa on CREB activation index) and CREB phosphorylation (CREB activation index) was tested.
1.被験物質 1. test substance
ホヤ由来のプラズマローゲンと、クロレラ・ピレノイドサの細胞壁破砕物を用いた。 A plasmalogen derived from sea squirt and a cell wall fragment of Chlorella pyrenoidosa were used.
(1) ホヤ由来プラズマローゲン (1) Ascidian-derived plasmalogen
(1-1) ホヤから下記のように抽出された焼津水産化学工業株式会社製のホヤプラズマローゲンエキス(Lot.No.191109)に含まれるホヤ由来プラズマローゲンを披験物質とした。 (1-1) Ascidian-derived plasmalogen contained in sea squirt plasmalogen extract (Lot. No. 191109) manufactured by Yaizu Suisan Kagaku Kogyo Co., Ltd., which was extracted from sea squirt as follows, was used as a test substance.
前記ホヤプラズマローゲンエキス100gに主に含まれるホヤ由来プラズマローゲンは、グリセロールの2位のアシル基により分類した次の4種類であった。その4種類の合計量は5540mg、括弧内は4種類の合計量に占める各種類の質量%である。
EPA型 3120mg(56.3質量%)
DHA型 1810mg(32.7質量%)
アラキドン酸型 450mg(8.1質量%)
オレイン酸型 160mg(2.9質量%)
The sea squirt-derived plasmalogen mainly contained in 100 g of the sea squirt plasmalogen extract was classified into the following four types according to the acyl group at the 2-position of glycerol. The total amount of the four types is 5540 mg, and the mass % of each type in the total amount of the four types is shown in parentheses.
EPA type 3120 mg (56.3% by mass)
DHA type 1810 mg (32.7% by mass)
Arachidonic acid type 450 mg (8.1% by mass)
Oleic acid type 160 mg (2.9% by mass)
(1-2) 前記ホヤプラズマローゲンエキスは、特開2019-162042号公報に記載の下記方法に準じてホヤから抽出されたものである。 (1-2) The sea squirt plasmalogen extract is extracted from sea squirt according to the following method described in JP-A-2019-162042.
すなわち、ホヤ可食部のフリーズドライ粉末を50.8g秤量し、ヘキサン66mL、エタノール121mL、及び水11mLを仕込み、混合し、撹拌しながら常温で30分間抽出した後、定性濾紙(型式:No.2)にて濾過し、抽出液と残渣を分離し、残渣には、再度、ヘキサン66mL、エタノール121mL、及び水11mLを仕込み、混合し、撹拌しながら常温で30分間抽出した後、定性濾紙(型式:No.2)にて濾過し、抽出液と残渣を分離した。抽出液2回分を併せて真空乾燥による溶媒除去を行い、ホヤ抽出物を得た。その収量は7.9gであった。 That is, 50.8 g of freeze-dried powder of sea squirt edible part was weighed, 66 mL of hexane, 121 mL of ethanol, and 11 mL of water were charged, mixed, and extracted with stirring at room temperature for 30 minutes, followed by qualitative filter paper (model: No. 2) to separate the extract from the residue. To the residue, 66 mL of hexane, 121 mL of ethanol, and 11 mL of water were again charged, mixed, and extracted at room temperature for 30 minutes with stirring. Model: No. 2) to separate the extract and residue. Two extracts were combined and the solvent was removed by vacuum drying to obtain a sea squirt extract. The yield was 7.9 g.
得られたホヤ抽出物を1.97g秤量し、0.1Mクエン酸-HClバッファー(pH4.5)5mL、ホスホリパーゼA1(PLA1製剤、三菱ケミカルフーズ製)を240mg添加し、50℃にて4時間撹拌し酵素反応させた。酵素処理液にアセトン16.7mLとヘキサン16.7mLを添加し、よく撹拌した後、遠心分離(2500rpm, 4℃, 20min)し、ヘキサン層(上層)と水層(下層)に分離させた。上層を回収し、ロータリーエバポレーターで乾固し、アセトンを30mL添加した。遠心分離し、アセトンを除去し、沈殿物を回収した。再度、アセトンを30mL添加し、遠心分離し、アセトンを除去し、沈殿物を回収した。沈殿物を真空乾燥し、酵素処理物を得た。その収量は345.9mgであった。 1.97 g of the obtained sea squirt extract was weighed, 5 mL of 0.1 M citric acid-HCl buffer (pH 4.5), and 240 mg of phospholipase A1 (PLA1 formulation, manufactured by Mitsubishi Chemical Foods) were added and incubated at 50° C. for 4 hours. The mixture was stirred and enzymatically reacted. After adding 16.7 mL of acetone and 16.7 mL of hexane to the enzyme-treated solution and stirring well, the mixture was centrifuged (2500 rpm, 4°C, 20 min) to separate the hexane layer (upper layer) and aqueous layer (lower layer). The upper layer was recovered, dried on a rotary evaporator, and 30 mL of acetone was added. Centrifuge to remove acetone and collect precipitate. Again, 30 mL of acetone was added and centrifuged to remove acetone and collect the precipitate. The precipitate was vacuum-dried to obtain an enzyme-treated product. The yield was 345.9 mg.
得られた酵素処理物100.0mgを秤量し、ヘキサン10mL、アセトン10mL、及び水3mLを加え、よく撹拌した後、遠心分離(2500rpm, 4℃, 20min)し、液/液分配させた。下層を除去し、更に、アセトン5mLと水3mLを添加して、よく撹拌した後、遠心分離(2500rpm, 4℃, 20min)し、液/液分配させた。その上層を回収し、ロータリーエバポレーターで溶媒除去させたうえ、真空乾燥して、プラズマローゲン含有抽出物(ホヤプラズマローゲンエキス)を得た。その収量は68.1mgであった。 100.0 mg of the enzyme-treated product obtained was weighed, 10 mL of hexane, 10 mL of acetone, and 3 mL of water were added, and the mixture was stirred well and then centrifuged (2500 rpm, 4°C, 20 min) for liquid/liquid partitioning. The lower layer was removed, 5 mL of acetone and 3 mL of water were added, and the mixture was stirred well and then centrifuged (2500 rpm, 4°C, 20 min) for liquid/liquid partitioning. The upper layer was recovered, the solvent was removed by a rotary evaporator, and the extract was dried in a vacuum to obtain a plasmalogen-containing extract (sea squirt plasmalogen extract). The yield was 68.1 mg.
(2) クロレラ・ピレノイドサの細胞壁破砕物 (2) Cell wall fragment of Chlorella pyrenoidosa
冷却外套を持つ密閉シリンダー中にその密閉シリンダー容量の80乃至85%の多数の直径0.5乃至1.5mmのグラスビーズが封入されており、そのグラスビーズを流入液体と混和・回転させることにより流入液体中の物質を摩砕する連続湿式微粉砕機(商品名:ダイノーミル[KD型] WAB, Inc.製)に、10℃以下に調整されたクロレラ・ピレノイドサ濃度10乃至25重量%のクロレラ・ピレノイドサ粉体・水懸濁液を送入して、破砕直後のスラリーが40℃以下になるよう微粉砕した。 A large number of glass beads with a diameter of 0.5 to 1.5 mm, 80 to 85% of the volume of the closed cylinder are enclosed in a closed cylinder with a cooling mantle, and the glass beads are mixed with the influent liquid and rotated. A continuous wet fine pulverizer (trade name: Dyno Mill [KD type] manufactured by WAB, Inc.) that grinds substances in the inflow liquid is fed with chlorella pyrenoidosa concentration of 10 to 25% by weight adjusted to 10 ° C or less. The pyrenoid sa powder/water suspension was fed and pulverized so that the slurry immediately after pulverization had a temperature of 40° C. or lower.
このようにして得られたクロレラ・ピレノイドサのスラリーを、直ちに10℃以下に冷却し、更に、真空乾燥させた後、粉砕することにより、被験物質であるクロレラ・ピレノイドサの細胞壁破砕物を、乾燥粉末として得た。 The slurry of Chlorella pyrenoidosa thus obtained is immediately cooled to 10° C. or less, further dried in a vacuum, and then pulverized to obtain a dry powder of the crushed cell wall of Chlorella pyrenoidosa, which is the test substance. obtained as
2.方法 2. Method
6週齢のSD系雄性ラットを1週間予備飼育後、Cont群(対照)、CHL群(クロレラ投与)、H-Pls群(ホヤ由来プラズマローゲン投与)、H-Pls & CHL群(ホヤ由来プラズマローゲン及びクロレラ投与)の4群に分けた(各群の個体数は5)。 After preliminarily feeding 6-week-old male SD rats for 1 week, Cont group (control), CHL group (chlorella administration), H-Pls group (ascidian-derived plasmalogen administration), H-Pls & CHL group (ascidian-derived plasma Rogen and chlorella administration) were divided into 4 groups (the number of individuals in each group was 5).
第1日から第7日までの7日間にわたり毎日、各群のラットに対し次の披験物質を投与した。 The following test substance was administered to the rats of each group every day for 7 days from the 1st day to the 7th day.
(1) Cont群
[a]サラダ油0.5mLを経口ゾンデにて1回胃内投与
[b]MF飼料(オリエンタル酵母工業株式会社製)25gを摂餌
(1) Cont group
[a] Administer 0.5 mL of salad oil once in the stomach using an oral probe
[b] 25 g of MF feed (manufactured by Oriental Yeast Co., Ltd.)
図1-5において、CHL: -、H-Pls: - In Figure 1-5, CHL: -, H-Pls: -
(2) CHL群
[a]サラダ油0.5mLを経口ゾンデにて1回胃内投与
[b]クロレラ・ピレノイドサ細胞壁破砕物の乾燥粉末を1質量%(600mg/kg体重)配合したMF飼料25gを摂餌
(2) CHL group
[a] Administer 0.5 mL of salad oil once in the stomach using an oral probe
[b] Feeding 25 g of MF feed containing 1% by mass (600 mg/kg body weight) of dry powder of Chlorella pyrenoidosa cell wall crushed product
図1-5において、CHL: +、H-Pls: - In Figure 1-5, CHL: +, H-Pls: -
(3) H-Pls群
[a]ホヤ由来プラズマローゲン(前記4種類)0.07mg(0.2mg/kg体重)を含む量のホヤプラズマローゲンエキスを含有するサラダ油0.5mLを経口ゾンデにて1回胃内投与
[b]MF飼料25gを摂餌
(3) H-Pls group
[a] 0.5 mL of salad oil containing sea squirt plasmalogen extract in an amount containing 0.07 mg (0.2 mg/kg body weight) of sea squirt-derived plasmalogens (the above four types) was intragastricly administered once using an oral probe.
[b] 25 g of MF feed
図1-5において、CHL: -、H-Pls: + In Figure 1-5, CHL: -, H-Pls: +
(4) H-Pls & CHL群
[a]ホヤ由来プラズマローゲン(前記4種類)0.07mg(0.2mg/kg体重)を含む量のホヤプラズマローゲンエキスを含有するサラダ油0.5mLを経口ゾンデにて1回胃内投与
[b]クロレラ・ピレノイドサ細胞壁破砕物の乾燥粉末を1質量%(600mg/kg体重)配合したMF飼料25gを摂餌
(4) H-Pls & CHL group
[a] 0.5 mL of salad oil containing sea squirt plasmalogen extract in an amount containing 0.07 mg (0.2 mg/kg body weight) of sea squirt-derived plasmalogens (the above four types) was intragastricly administered once using an oral probe.
[b] Feeding 25 g of MF feed containing 1% by mass (600 mg/kg body weight) of dry powder of Chlorella pyrenoidosa cell wall crushed product
図1-5において、CHL: +、H-Pls: + In Figure 1-5, CHL: +, H-Pls: +
投与を完了した翌日である第8日に、各群の各ラットより海馬を摘出し、ウエスタンブロット法にて、海馬における、脳由来神経栄養因子(BDNF)、高親和性BDNF受容体(TrkB)、及び核内転写因子cAMP-response element binding protein(CREB)それぞれの発現量、並びに、TrkBのリン酸化(TrkBの活性化指標)及びCREBのリン酸化(CREBの活性化指標)を解析した。 On the 8th day, the day after the administration was completed, the hippocampus was excised from each rat in each group, and the brain-derived neurotrophic factor (BDNF) and high-affinity BDNF receptor (TrkB) in the hippocampus were detected by Western blotting. , and nuclear transcription factor cAMP-response element binding protein (CREB) expression levels, TrkB phosphorylation (TrkB activation index) and CREB phosphorylation (CREB activation index) were analyzed.
3.結果 3. result
(1) ホヤ由来プラズマローゲンとクロレラ・ピレノイドサの細胞壁破砕物の投与によるBDNF発現量への影響を表す図1[数値は平均±SEMを示す(n=5);*p<0.05 クロレラ投与群(CHL群)との比較、#p<0.05 ホヤプラズマローゲン投与群(H-Pls群)との比較]に示されるように、H-Pls & CHL群(ホヤ由来プラズマローゲン及びクロレラ投与群)においては、BDNF発現量が顕著に増加した。 (1) Fig. 1 showing the effect of administration of sea squirt-derived plasmalogen and Chlorella pyrenoidosa cell wall debris on BDNF expression level [Values indicate mean ± SEM (n = 5); CHL group), #p < 0.05 Comparison with sea squirt plasmalogen administration group (H-Pls group)], in the H-Pls & CHL group (ascidian-derived plasmalogen and chlorella administration group) , BDNF expression level increased significantly.
(2) ホヤ由来プラズマローゲンとクロレラ・ピレノイドサの細胞壁破砕物の投与によるTrkB発現量への影響を表す図2[数値は平均±SEMを示す(n=5);*p<0.05 対照群(Cont群)との比較、#p<0.05 クロレラ投与群(CHL群)との比較]に示されるように、H-Pls & CHL群(ホヤ由来プラズマローゲン及びクロレラ投与群)においては、TrkB発現量の顕著な低下が認められた。 (2) Fig. 2 showing the effect on TrkB expression level by administration of sea squirt-derived plasmalogen and cell wall debris of Chlorella pyrenoidosa [values indicate mean ± SEM (n = 5); group), #p < 0.05 Comparison with chlorella-administered group (CHL group)], in the H-Pls & CHL group (ascidian-derived plasmalogen and chlorella-administered group), TrkB expression level A significant decrease was observed.
(3) ホヤ由来プラズマローゲンとクロレラ・ピレノイドサの細胞壁破砕物の投与によるTrkBリン酸化への影響を表す図3[数値は平均±SEMを示す(n=5);*p<0.05 対照群(Cont群)との比較、#p<0.05 クロレラ投与群(CHL群)との比較]に示されるように、H-Pls & CHL群(ホヤ由来プラズマローゲン及びクロレラ投与群)においては、TrkBリン酸化が顕著に促進された。 (3) Fig. 3 showing the effect on TrkB phosphorylation by administration of sea squirt-derived plasmalogen and cell wall debris of Chlorella pyrenoidosa [values indicate mean ± SEM (n = 5); group), #p < 0.05 Comparison with chlorella-administered group (CHL group)], TrkB phosphorylation was significantly facilitated.
(4) ホヤ由来プラズマローゲンとクロレラ・ピレノイドサの細胞壁破砕物の投与によるCREB発現量への影響は図4[数値は平均±SEMを示す(n=5)]に示される。 (4) The effects of administration of sea squirt-derived plasmalogen and cell wall debris of Chlorella pyrenoidosa on CREB expression levels are shown in FIG.
(5) ホヤ由来プラズマローゲンとクロレラ・ピレノイドサの細胞壁破砕物の投与によるCREBリン酸化への影響を表す図5[数値は平均±SEMを示す(n=5);*p<0.05 対照群(Cont群)との比較、#p<0.05 クロレラ投与群(CHL群)との比較、\p<0.05 ホヤプラズマローゲン投与群(H-Pls群)との比較]に示されるように、H-Pls & CHL群(ホヤ由来プラズマローゲン及びクロレラ投与群)においては、CREBリン酸化が顕著に促進された。 (5) Fig. 5 showing the effect on CREB phosphorylation by administration of sea squirt-derived plasmalogen and cell wall debris of Chlorella pyrenoidosa [values indicate mean ± SEM (n = 5); group), #p < 0.05 comparison with chlorella administration group (CHL group), \p < 0.05 comparison with sea squirt plasmalogen administration group (H-Pls group)], H-Pls & In the CHL group (ascidian-derived plasmalogen and chlorella-administered group), CREB phosphorylation was significantly promoted.
以上のように、ホヤ由来プラズマローゲンとクロレラ・ピレノイドサ細胞壁破砕物を1週間投与したH-Pls & CHL群において、Cont群(対照)、CHL群(クロレラ・ピレノイドサ細胞壁破砕物の1週間単独投与)、H-Pls群(ホヤ由来プラズマローゲンの1週間単独投与)と比較して、顕著な、BDNF発現量の増加、TrkBリン酸化の促進、及び、CREBのリン酸化の促進が認められた。 As described above, in the H-Pls & CHL group in which sea squirt-derived plasmalogen and crushed chlorella pyrenoidosa cell wall were administered for one week, the Cont group (control) and the CHL group (one week administration of crushed chlorella pyrenoidosa cell wall) , Compared to the H-Pls group (single administration of sea squirt-derived plasmalogen for one week), significant increases in BDNF expression, promotion of TrkB phosphorylation, and promotion of CREB phosphorylation were observed.
すなわち、ホヤ由来プラズマローゲンとクロレラ・ピレノイドサ細胞壁破砕物を併せて投与することにより、海馬において、ホヤ由来プラズマローゲン単独投与又はクロレラ・ピレノイドサ細胞壁破砕物単独投与に比し効果的に、BDNF発現量の増加、TrkBリン酸化の促進(BDNF-TrkBシグナル伝達系活性化)、及び、CREBのリン酸化の促進(BDNF-TrkB-CREBシグナル伝達系活性化)を図り得る。 That is, by administering the sea squirt-derived plasmalogen and the chlorella pyrenoidosa cell wall fragment together, in the hippocampus, compared with the administration of the sea squirt-derived plasmalogen alone or the chlorella pyrenoidosa cell wall fragment alone, the expression level of BDNF was effectively reduced. increase, promotion of TrkB phosphorylation (BDNF-TrkB signaling system activation), and promotion of CREB phosphorylation (BDNF-TrkB-CREB signaling system activation).
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