JP2009511466A - Factors for inhibiting lipases and / or phospholipases in body fluids, cells and tissues and compositions containing them - Google Patents
Factors for inhibiting lipases and / or phospholipases in body fluids, cells and tissues and compositions containing them Download PDFInfo
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- JP2009511466A JP2009511466A JP2008534146A JP2008534146A JP2009511466A JP 2009511466 A JP2009511466 A JP 2009511466A JP 2008534146 A JP2008534146 A JP 2008534146A JP 2008534146 A JP2008534146 A JP 2008534146A JP 2009511466 A JP2009511466 A JP 2009511466A
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Abstract
本発明は、代謝症候群、心臓血管障害及び炎症性疾患の予防及び治療のための体液、細胞及び組織中のリパーゼ及び／又はホスホリパーゼを阻害するタンパク質、及び前記タンパク質を含む組成物に関する。前記タンパク質は、植物種から分離されるか或いは組換えＤＮＡ技術により合成又は生成される。最も好ましい実施形態において、本発明のタンパク質は、５〜１００個のアミノ酸残基を含有し、分子量が０．５〜１０ｋＤであり、グリコシル化を有するかまたは有さない。The present invention relates to a protein that inhibits lipase and / or phospholipase in body fluids, cells and tissues for the prevention and treatment of metabolic syndrome, cardiovascular disorders and inflammatory diseases, and a composition comprising said protein. The protein can be isolated from plant species or synthesized or produced by recombinant DNA techniques. In the most preferred embodiment, the proteins of the invention contain 5-100 amino acid residues, have a molecular weight of 0.5-10 kD and have or do not have glycosylation.
Description
（発明の分野）
本発明は、体液、細胞及び組織中のリパーゼ及び／又はホスホリパーゼ酵素を阻害又は軽減するタンパク質及びその組成物に関する。本発明に記載のタンパク質及びその組成物は、体液、細胞及び組織中のリパーゼ及びホスホリパーゼ酵素活性がもたらす臨床的な症状発現及び疾患の予防又は治療に有用である。本発明は、体液、細胞及び組織中のリパーゼ及び／又はホスホリパーゼ酵素の阻害剤であるタンパク質の分離、精製、特徴付けに関する。本発明は、トリプシン不活化に対して精製タンパク質の安定な組成物にも関する。
(Field of Invention)
The present invention relates to proteins and compositions thereof that inhibit or reduce lipase and / or phospholipase enzymes in body fluids, cells and tissues. The protein and composition thereof according to the present invention are useful for clinical manifestation and prevention or treatment of diseases caused by lipase and phospholipase enzyme activities in body fluids, cells and tissues. The present invention relates to the separation, purification, and characterization of proteins that are inhibitors of lipase and / or phospholipase enzymes in body fluids, cells and tissues. The invention also relates to a stable composition of purified protein against trypsin inactivation.
（先行技術の説明）
リパーゼ及びホスホリパーゼは、哺乳動物の代謝の重要な制御要素である。これらは他の代謝酵素クラスとは区別される共通の特徴を数多く共有しており、中でも最も重要なのが、これらの「二次元」基質、即ち、脂肪の小滴、リポタンパク質、リン脂質層、生体膜との結合であり、結果として開裂機序及び調節に影響を及ぼしている。
(Description of prior art)
Lipases and phospholipases are important regulatory elements of mammalian metabolism. They share many common features that distinguish them from other metabolic enzyme classes, most importantly these “two-dimensional” substrates: fat droplets, lipoproteins, phospholipid layers, It is a binding to biological membranes and consequently affects the cleavage mechanism and regulation.
膵リパーゼ（ＰＬ）は、トリグリセリドを部分的に加水分解し、胆汁酸と共に複合体を形成し、腸粘膜を経て吸収される脂肪酸とモノグリセリドとを得るのに効果的であると考えられている。肝リパーゼ（ＨＬ）及びリポタンパク質リパーゼ（ＬＰＬ）の２つは、循環血漿リポタンパク質中に存在するトリグリセリド及びリン脂質の加水分解を担う主な脂肪分解酵素である。何れのリパーゼも細胞表面プロテオグリカンを介して血管内皮に付着する。ＨＬは主にカイロミクロンレムナント、中間密度リポタンパク質及び高密度リポタンパク質の代謝に関与しているのに対して、ＬＰＬはカイロミクロン及び超低密度リポタンパク質からのトリグリセリドの加水分解を触媒する。こうした脂肪分解酵素としての従来の機能に加えて、ＨＬ及びＬＰＬは、細胞表面受容体及び／又はプロテオグリカンへのリポタンパク質の相互作用を媒介するリガンドとして働くようである。 Pancreatic lipase (PL) is believed to be effective in partially hydrolyzing triglycerides to form complexes with bile acids to obtain fatty acids and monoglycerides that are absorbed through the intestinal mucosa. Liver lipase (HL) and lipoprotein lipase (LPL) are the two main lipolytic enzymes responsible for the hydrolysis of triglycerides and phospholipids present in circulating plasma lipoproteins. Both lipases attach to the vascular endothelium via cell surface proteoglycans. HL is primarily involved in the metabolism of chylomicron remnants, intermediate density lipoproteins and high density lipoproteins, whereas LPL catalyzes the hydrolysis of triglycerides from chylomicrons and very low density lipoproteins. In addition to these conventional functions as lipolytic enzymes, HL and LPL appear to act as ligands that mediate the interaction of lipoproteins with cell surface receptors and / or proteoglycans.
肝臓外の部位でのトリグリセリドリッチリポタンパク質結合脂肪酸の蓄積及び分布はＬＰＬにより促進される。この酵素は又、全リポタンパク質粒子及び親油性ビタミンの細胞の取り込みを含めた幾つかの非脂肪分解に関連する機能に関与している。種々の脂質障害、肥満及びアテローム硬化症の病因には、ＬＰＬ発現の組織特異的な変異が関わっている。血管壁の細胞、特にマクロファージにより発現したＬＰＬには、泡沫細胞形成及びアテローム硬化症の促進に寄与する酵素の作用が更に確認された。コレステロールエステル転移タンパク質及びリポタンパク質リパーゼ系に特異的に作用する薬剤の開発が進められている。 Accumulation and distribution of triglyceride-rich lipoprotein-bound fatty acids at sites outside the liver is facilitated by LPL. This enzyme is also involved in several non-lipolysis-related functions including cellular uptake of total lipoprotein particles and lipophilic vitamins. The pathogenesis of various lipid disorders, obesity and atherosclerosis involves tissue-specific mutations in LPL expression. LPL expressed by vascular wall cells, particularly macrophages, was further confirmed to have an action of an enzyme that contributes to foam cell formation and promotion of atherosclerosis. Development of drugs that specifically act on the cholesterol ester transfer protein and lipoprotein lipase systems is underway.
過去数年間にわたり、ＨＬ及びＬＰＬがリポタンパク質代謝及びｉｎ ｖｉｖｏのアテローム硬化症の発症を調節する新規の代替機序についての本発明者の理解において大きな進歩を遂げた。又、トリグリセリドリッチリポタンパク質の血管内代謝についての本発明者等の理解にも進歩が見られた。今では、トリグリセリドリッチリポタンパク質結合アポリポタンパク質Ｅ、リポタンパク質リパーゼ及び肝リパーゼと、ヘパラン硫酸プロテオグリカン及びリポタンパク質受容体との複雑な細胞外相互作用が、トリグリセリドリッチリポタンパク質の肝細胞内への取り込みを促進することが知られている。最近の研究では、取り込まれたトリグリセリドリッチリポタンパク質の細胞内運命が極めて複雑であることも明らかになった。細胞内エンドソーム区画内部のトリグリセリドリッチリポタンパク質成分の単離には、アポリポタンパク質Ｅの再循環が含まれるのに対して、アポリポタンパク質Ｂと結びついている残りの脂質コアは、リソソームによる分解の影響を受けやすい。 Over the past few years, great progress has been made in the inventor's understanding of novel alternative mechanisms by which HL and LPL regulate lipoprotein metabolism and the development of in vivo atherosclerosis. Progress has also been made in our understanding of the intravascular metabolism of triglyceride-rich lipoproteins. Now, the complex extracellular interaction of triglyceride rich lipoprotein-binding apolipoprotein E, lipoprotein lipase and hepatic lipase with heparan sulfate proteoglycan and lipoprotein receptor facilitates the uptake of triglyceride rich lipoprotein into hepatocytes. It is known to promote. Recent studies have also shown that the intracellular fate of incorporated triglyceride-rich lipoprotein is extremely complex. Isolation of the triglyceride-rich lipoprotein component within the intracellular endosomal compartment involves recycling of apolipoprotein E, whereas the remaining lipid core associated with apolipoprotein B has the effect of degradation by lysosomes. Easy to receive.
糖尿病患者におけるアテローム硬化症の高い発症率は、マクロファージのＬＰＬ活性と相関している。多くの証拠が集まったことにより、血管壁でマクロファージがＬＰＬを生成すると、病変内部では脂質の蓄積が促進され、アテローム硬化症の発症が助長される場合があることが示唆されている。 The high incidence of atherosclerosis in diabetic patients correlates with macrophage LPL activity. A lot of evidence suggests that when macrophages produce LPL in the vascular wall, lipid accumulation is promoted inside the lesion, which may promote the development of atherosclerosis.
代謝症候群及び心臓血管障害を治療する薬剤標的としてのリパーゼの可能性がますます認められている。現在、脂質の吸収及び代謝に関する疾患の最先端療法は、体液、細胞及び組織のリパーゼ活性を阻害又は軽減すべきであると考えられている。 The potential of lipase as a drug target to treat metabolic syndrome and cardiovascular disorders is increasingly recognized. Currently, it is believed that state-of-the-art therapy for diseases related to lipid absorption and metabolism should inhibit or reduce lipase activity in body fluids, cells and tissues.
リパーゼ阻害剤は、種々の天然産物、特に微生物供給源に由来するものが報告されている。このような阻害剤の例には、トリグリセリド及びコレステロールエステルの加水分解を阻害するストレプトマイセス種由来のリプスタチン及びパンクリシンＡ−Ｅ又はこれらの合成誘導体が含まれており（Ｈｏｃｈｕｌｉら、Ｌｉｐｓｔａｔｉｎ，ａｎｄ Ｉｎｈｉｂｉｔｏｒ ｏｆ Ｐａｎｃｒｅａｔｉｃ Ｌｉｐａｓｅ，Ｐｒｏｄｕｃｅｄ ｂｙ Ｓｔｒｅｐｔｏｍｙｃｅｓ Ｔｏｘｙｔｒｉｃｉｎｉ，ＩＩ．Ｃｈｅｍｉｓｔｒｙ ａｎｄ Ｓｔｒｕｃｔｕｒｅ Ｅｌｕｃｉｄａｔｉｏｎ，Ｊ．Ａｎｔｉｂｉｏｔ．（Ｔｏｋｙｏ），１９８７ Ａｕｇ．，４０（８）：１０８６−９１；Ｆｅｒｎａｎｄｅｚら、Ｅｆｆｅｃｔｓ ｏｆ Ｔｅｔｒａｈｙｄｒｏｌｉｐｓｔａｔｉｎ，ａ Ｌｉｐａｓｅ Ｉｎｈｉｂｉｔｏｒ，ｏｎ Ａｂｓｏｒｐｔｉｏｎ ｏｆ Ｆａｔ ｆｒｏｍ ｔｈｅ Ｉｎｔｅｓｔｉｎｅ ｏｆ ｔｈｅ Ｒａｔ，Ｂｉｏｃｈｉｍ．Ｂｉｏｐｈｙｓ．Ａｃｔａ．，１９８９ Ｆｅｂ．２０，１００１（３）：２４９−５５；Ｙｏｓｈｉｎａｒｉら、Ｐａｎｃｌｉｃｉｎｓ，Ｎｏｖｅｌ Ｐａｎｃｒｅａｔｉｃ Ｌｉｐａｓｅ Ｉｎｈｉｂｉｔｏｒｓ，ＩＩ．Ｓｔｒｕｃｔｕｒａｌ ｅｌｕｃｉｄａｔｉｏｎ，Ｊ．Ａｎｔｉｂｉｏｔ．（Ｔｏｋｙｏ），１９９４ Ｄｅｃ．，４７（１２）：１３７６−８４）、肥満の治療に使用されている（特許文献１）。しかも、幾つかの分子がＣａｓｓｉａ ｎｏｍａｍｅから分離したタンニンを含む植物供給源から同定されている（特許文献２）。ＬＰＬはアテローム硬化症の病因に関与していることが明らかにされている（Ｍｅａｄら、Ｌｉｐｏｐｒｏｔｅｉｎ Ｌｉｐａｓｅ，ａ Ｋｅｙ Ｒｏｌｅ ｉｎ Ａｔｈｅｒｏｓｃｌｅｒｏｓｉｓ？，ＦＥＢＳ Ｌｅｔｔ．，１９９９ Ｎｏｖ．２６，４６２（１−２）：１−６）。ＬＰＬの阻害はアテローム硬化の進行を予防すると考えられている（Ｚｉｍｍｅｒｍａｎら、Ｌｉｐｏｐｒｏｔｅｉｎ Ｌｉｐａｓｅ Ｍｅｄｉａｔｅｓ ｔｈｅ Ｕｐｔａｋｅ ｏｆ Ｇｌｙｃａｔｅｄ ＬＤＬ ｉｎ Ｆｉｂｒｏｂｌａｓｔｓ，Ｅｎｄｏｔｈｅｌｉａｌ Ｃｅｌｌｓ，ａｎｄ Ｍａｃｒｏｐｈａｇｅｓ，Ｄｉａｂｅｔｅｓ，５０，１６４３−１６５３，２００１）。 Lipase inhibitors have been reported from various natural products, particularly those from microbial sources. Examples of such inhibitors include Lipstatin and pancrisin A-E from Streptomyces species that inhibit hydrolysis of triglycerides and cholesterol esters, or synthetic derivatives thereof (Hochuli et al., Lipstatin, and Inhibitor). . of Pancreatic Lipase, Produced by Streptomyces Toxytricini, II.Chemistry and Structure Elucidation, J.Antibiot (Tokyo), 1987 Aug., 40 (8): 1086-91; Fernandez et al., Effects of Tetrahydrolipstatin, a Lipase Inhibitor, on Absorptio of Fat from the Intestine of the Rat, Biochim. Biophys. Acta., 1989 Feb. 20, 1001 (3): 249-55; Yoshinari et al., Panclinics, Novel Pacific Inhibit. Tokyo), 1994 Dec., 47 (12): 1376-84), used for the treatment of obesity (Patent Document 1). Moreover, several molecules have been identified from plant sources containing tannins isolated from Cassia nome (Patent Document 2). LPL has been shown to be involved in the pathogenesis of atherosclerosis (Mead et al., Lipoprotein Lipase, a Key Role in Atherosclerosis ?, FEBS Lett., 1999 Nov. 26, 462 (1-2): 1 -6). Inhibition of LPL is believed to prevent the progression of atherosclerosis (Zimmerman et al., Lipoprotein Lipase Mediates the Uptake of Glycated LDL in Fibroblasts, Endothelial Cells, 65, 43).
ホスホリパーゼは、膜リン脂質に特異的に作用して加水分解し、シグナル伝達及び炎症プロセスに関わるメディエーターを産生する。アラキドン酸の産生では、ホスホリパーゼＡ２（ＰＬＡ２）の役割は周知であり、ロイコトリエン及びプロスタグランジン合成を担っている。ＰＬＡ２阻害剤は、種々の炎症性疾患及び変性疾患の薬剤として提唱されている。リポタンパク質関連ホスホリパーゼＡ２がアテローム硬化症に関与することが明らかにされており、アテローム硬化症の治療のためにリポタンパク質関連ホスホリパーゼＡ２を阻害することが提唱されている（Ｌｅａｃｈら、Ｌｉｐｏｐｒｏｔｅｉｎ−Ａｓｓｏｃｉａｔｅｄ ＰＬＡ２ Ｉｎｈｉｂｉｔｉｏｎ−Ａ Ｎｏｖｅｌ，Ｎｏｎ−Ｌｉｐｉｄ Ｌｏｗｅｒｉｎｇ Ｓｔｒａｔｅｇｙ ｆｏｒ Ａｔｈｅｒｏｓｃｌｅｒｏｓｉｓ Ｔｈｅｒａｐｙ，Ｆａｒｍａｃｏ，２００１ Ｊａｎ−Ｆｅｂ，５６（ｌ−２）：４５−５０）。
（要旨）
本発明は、体液、細胞及び組織中のリパーゼ及び／又はホスホリパーゼ酵素を阻害又は軽減するタンパク質、及び前記タンパク質を含む組成物に関する。本発明に記載のタンパク質及び前記タンパク質を含む組成物は、体液、細胞及び組織中のリパーゼ及び／又はホスホリパーゼ酵素活性がもたらす臨床的な症状発現及び疾患の予防又は治療に有用である。
(Summary)
The present invention relates to a protein that inhibits or reduces lipase and / or phospholipase enzymes in body fluids, cells and tissues, and a composition comprising the protein. The protein according to the present invention and the composition containing the protein are useful for clinical manifestation and prevention or treatment of diseases caused by lipase and / or phospholipase enzyme activities in body fluids, cells and tissues.
好ましい実施形態において、リパーゼ及び／又はホスホリパーゼ阻害活性を有するタンパク質は、組換え技術により合成、生成するか又は天然の供給源から分離することができる。 In preferred embodiments, a protein having lipase and / or phospholipase inhibitory activity can be synthesized, produced by recombinant techniques, or isolated from natural sources.
最も好ましい実施形態において、タンパク質は、Ｍｏｒｉｎｇａ属に属する植物種の種子から分離される。 In the most preferred embodiment, the protein is isolated from the seeds of plant species belonging to the genus Moringa.
好ましい実施形態において、タンパク質は、簡潔で、効果的であり、費用効率が高いプロセスにより精製される。 In a preferred embodiment, the protein is purified by a simple, effective and cost effective process.
最も好ましい実施形態において、タンパク質は、トリプシン阻害剤と合わせることによりトリプシン不活化から保護される。 In the most preferred embodiment, the protein is protected from trypsin inactivation by combining with a trypsin inhibitor.
好ましい実施形態において、リパーゼ及び／又はホスホリパーゼを阻害するための組成物は、本発明に記載のタンパク質の治療有効量及び薬学的に不活性なアジュバント、希釈剤又は担体を含む。 In a preferred embodiment, a composition for inhibiting lipase and / or phospholipase comprises a therapeutically effective amount of a protein according to the invention and a pharmaceutically inert adjuvant, diluent or carrier.
好ましい実施形態において、本発明に記載のタンパク質を含むリパーゼ及び／又はホスホリパーゼを阻害するための組成物は、他の有効成分と合わせられ得る。 In a preferred embodiment, a composition for inhibiting lipases and / or phospholipases comprising a protein according to the present invention can be combined with other active ingredients.
本発明に記載のタンパク質又は前記タンパク質を含む組成物は、生理的条件下でリパーゼ及び／又はホスホリパーゼを阻害する能力があると考えられており、それによって代謝症候群、心臓血管障害及び炎症性疾患の予防又は治療に即した有効性があると考えられる。 The protein according to the invention or a composition comprising said protein is considered to be capable of inhibiting lipase and / or phospholipase under physiological conditions, thereby preventing metabolic syndrome, cardiovascular disorders and inflammatory diseases. It is considered effective for prevention or treatment.
好ましい実施形態において、本発明に記載のタンパク質又はタンパク質を含む組成物は、代謝症候群、心臓血管障害及び炎症性疾患の予防及び治療のために、体液、細胞及び組織中のリパーゼ及び／又はホスホリパーゼを阻害するのに有用である。 In a preferred embodiment, the protein or composition comprising the protein according to the present invention comprises a lipase and / or phospholipase in body fluids, cells and tissues for the prevention and treatment of metabolic syndrome, cardiovascular disorders and inflammatory diseases. Useful to inhibit.
別の実施形態において、本発明に記載のタンパク質又はタンパク質を含む組成物は、肥満、糖尿病及びアテローム硬化症のような代謝障害の予防又は治療に使用することができる。 In another embodiment, the protein or protein-containing composition described in the present invention can be used for the prevention or treatment of metabolic disorders such as obesity, diabetes and atherosclerosis.
更に別の実施形態において、本発明に記載のタンパク質又はタンパク質を含む組成物は、単球系細胞、血管細胞、肝細胞及び脂肪組織に脂質が蓄積するのを阻害又は軽減するのに使用することができる。 In yet another embodiment, the protein or composition comprising the protein of the present invention is used to inhibit or reduce lipid accumulation in monocyte cells, vascular cells, hepatocytes and adipose tissue. Can do.
更に別の実施形態において、本発明に記載のタンパク質又はタンパク質を含む組成物は、ホスホリパーゼの活性化及び／又は作用によって引き起こされる関節炎、アテローム硬化症及び敗血症性ショック等の炎症性疾患の予防又は治療に使用することができる。 In yet another embodiment, the protein or composition comprising the protein of the present invention is used to prevent or treat inflammatory diseases such as arthritis, atherosclerosis and septic shock caused by activation and / or action of phospholipase. Can be used for
更に別の実施形態において、本発明に記載のタンパク質又はタンパク質を含む組成物は、皮膚及び頭髪の手入れ用及び美容調製物（ｃｏｓｍｅｔｉｃ ｐｒｅｐａｒａｔｉｏｎ）のために使用することができる。 In yet another embodiment, the protein or protein-containing composition according to the invention can be used for skin and hair care and cosmetic preparations.
更に別の実施形態において、本発明に記載のタンパク質又はタンパク質を含む組成物は、リパーゼ及び／又はホスホリパーゼを分泌する微生物病原体によって引き起こされる細胞及び組織の損傷の予防又は治療に使用することができる。 In yet another embodiment, the proteins or compositions comprising the proteins of the present invention can be used for the prevention or treatment of cell and tissue damage caused by microbial pathogens that secrete lipases and / or phospholipases.
リパーゼの阻害特性に基づく更に別の実施形態において、タンパク質を含む組成物は、体液、細胞及び組織中のリパーゼ及び／又はホスホリパーゼ活性によって引き起こされるか、または悪化させる疾患を治療及び予防する動物薬において使用することができる。 In yet another embodiment based on the inhibitory properties of lipase, the composition comprising the protein is in an animal drug that treats and prevents diseases caused or exacerbated by lipase and / or phospholipase activity in body fluids, cells and tissues. Can be used.
好ましい実施形態において、本発明は、代謝症候群、心臓血管障害及び炎症性疾患を予防及び治療するための体液、細胞及び組織中のリパーゼ及び／又はホスホリパーゼの阻害に有用な、タンパク質を単独でか、又は薬学的に許容される適切なアジュバントを含む薬学的処方物も提供する。 In a preferred embodiment, the present invention provides a protein useful for inhibition of lipase and / or phospholipase in body fluids, cells and tissues to prevent and treat metabolic syndrome, cardiovascular disorders and inflammatory diseases, Alternatively, a pharmaceutical formulation comprising a suitable pharmaceutically acceptable adjuvant is also provided.
好ましい実施形態において、本発明は、トリプシン阻害から保護するための安定したタンパク質の送達にも関する。好ましい実施形態において、本発明は、分離したタンパク質の経口送達も提供する。 In a preferred embodiment, the present invention also relates to the delivery of stable proteins to protect against trypsin inhibition. In preferred embodiments, the present invention also provides for oral delivery of separated proteins.
本発明は又、本発明に記載のタンパク質の治療有効量を単独でか、又は薬学的に許容されるアジュバントと組み合わせて医薬品を製造する方法も提供する。本発明に記載のタンパク質は、他の有効成分と合わせられ得る。 The present invention also provides a method for producing a pharmaceutical product, either alone or in combination with a pharmaceutically acceptable adjuvant, a therapeutically effective amount of the protein described in the present invention. The proteins described in the present invention can be combined with other active ingredients.
（本発明の説明）
本発明は、５〜１００個のアミノ酸残基を含有し、分子量が０．５〜１０ｋＤの範囲であり、グリコシル化を有するかまたは有さないタンパク質に関する。このタンパク質は、リパーゼ及び／又はホスホリパーゼの酵素活性に対する阻害作用又は軽減作用を有する。このタンパク質は組換えＤＮＡ技術により合成又は生成される場合もあれば、植物材料から分離される場合もある。
(Description of the present invention)
The present invention relates to proteins containing 5 to 100 amino acid residues, having a molecular weight in the range of 0.5 to 10 kD, and with or without glycosylation. This protein has an inhibitory action or a reducing action on the enzyme activity of lipase and / or phospholipase. This protein may be synthesized or produced by recombinant DNA technology or may be isolated from plant material.
本発明に開示されるタンパク質は、Ｍｏｒｉｎｇａ属に属する種から分離され、更に好ましくはＭｏｒｉｎｇａ植物の種子から分離される。このタンパク質は、本明細書の以下の実施例に開示する方法により分離することができる。次に、分離したタンパク質は、適切な技術により精製される。次に、精製タンパク質は、以下の配列について、ＳＤＳ ＰＡＧＥ及びＬＣ−ＭＳ技術によって特徴付けられる。 The proteins disclosed in the present invention are isolated from species belonging to the genus Moringa, more preferably from seeds of Moringa plants. This protein can be isolated by the methods disclosed in the Examples herein below. The separated protein is then purified by appropriate techniques. The purified protein is then characterized by SDS PAGE and LC-MS techniques for the following sequences:
上記タンパク質は、以下の配列番号１：ＣＧＱＱＬＲＮＩＳＰＰＱＲＣＰＳＬＲＱＡＶＱＬＡＨＱＱＱＧＱＧＰＱＱＶＲＱＭＹＲの部分的な配列番号を有する。 The protein has the following partial SEQ ID NO: CGQQLRRNISPPRCCPSLRQAVQLAHQQQGQGPQQVRQMYR.
更に、このタンパク質を、リパーゼ阻害活性について合成基質及び天然基質の存在下で試験した。 In addition, the protein was tested for lipase inhibitory activity in the presence of synthetic and natural substrates.
トリプシン存在下で、Ｍｏｒｉｎｇａ種子タンパク質の活性について前記タンパク質を更に解析した。 The protein was further analyzed for the activity of Moringa seed protein in the presence of trypsin.
Ｍｏｒｉｎｇａ種子タンパク質は、トリプシン存在下ではリパーゼ阻害活性を失うことが明らかにされた。そこで、トリプシン不活化に対するタンパク質保護に関する試験を実施した。トリプシン阻害剤の存在下では、このタンパク質がリパーゼ阻害剤として有効であることが明らかにされた。 Moringa seed protein was shown to lose lipase inhibitory activity in the presence of trypsin. Therefore, a test for protein protection against trypsin inactivation was performed. This protein has been shown to be effective as a lipase inhibitor in the presence of a trypsin inhibitor.
本発明のリパーゼ／ホスホリパーゼ阻害剤との組み合わせのために、天然に存在するトリプシン阻害剤の内、ダイズ（Ｇｌｙｃｉｎｅ ｍａｘ）、ライマメ（Ｐｈａｓｅｏｌｕｓ ｌｉｍｅｎｓｉｓ）、Ｐｒｏｓｏｐｉｓ ｊｕｌｉｆｉｏｒａの種子、ヒマワリ種子（Ｈｅｌｉａｎｔｈｕｓ ａｎｎｕｓ）等及びこれらの組み合わせを選択した。ダイズタンパク質存在下で、リパーゼ阻害についてタンパク質を試験した。 Among the naturally occurring trypsin inhibitors for combination with the lipase / phospholipase inhibitors of the present invention, soybean (Glycine max), Lima bean (Phaseolus limensis), Prosopsi julifiora seed, sunflower seed (Helianthus annus) and the like These combinations were selected. The protein was tested for lipase inhibition in the presence of soy protein.
本発明は、本発明に記載のタンパク質の治療有効量及び薬学的に不活性なアジュバント、希釈剤又は担体を含む、リパーゼ及び／又はホスホリパーゼを阻害するための組成物にも関する。本発明に記載のタンパク質を含む、リパーゼ又はホスホリパーゼを阻害するための組成物は又、別の有効成分と合わせられ得る。 The invention also relates to a composition for inhibiting lipase and / or phospholipase comprising a therapeutically effective amount of a protein according to the invention and a pharmaceutically inert adjuvant, diluent or carrier. Compositions for inhibiting lipases or phospholipases comprising the proteins described in the present invention can also be combined with other active ingredients.
本発明に記載のタンパク質又は前記タンパク質を含む組成物は、生理的条件下でリパーゼ及び／又はホスホリパーゼを阻害する能力を有すると考えられており、それによって代謝症候群、心臓血管障害及び炎症性疾患の予防又は治療に即した有効性があると考えられる。 The protein according to the invention or a composition comprising said protein is considered to have the ability to inhibit lipase and / or phospholipase under physiological conditions, thereby preventing metabolic syndrome, cardiovascular disorders and inflammatory diseases. It is considered effective for prevention or treatment.
本発明に記載のタンパク質又はタンパク質を含む組成物は、代謝症候群、心臓血管障害及び炎症性疾患を予防及び治療するために、体液、細胞及び組織中のリパーゼ及び／又はホスホリパーゼを阻害するのに有用である。 A protein or composition comprising a protein according to the invention is useful for inhibiting lipases and / or phospholipases in body fluids, cells and tissues to prevent and treat metabolic syndrome, cardiovascular disorders and inflammatory diseases It is.
好ましい実施形態において、本発明に記載のタンパク質又はタンパク質を含む組成物は、肥満、糖尿病及びアテローム硬化症のような代謝障害の予防又は治療に使用することができる。 In a preferred embodiment, the protein or composition comprising the protein according to the present invention can be used for the prevention or treatment of metabolic disorders such as obesity, diabetes and atherosclerosis.
更なる態様において、本発明に記載のタンパク質又はタンパク質を含む組成物は、単球系細胞、血管細胞、肝細胞及び脂肪組織に脂質が蓄積するのを阻害又は軽減するのに使用することができる。 In a further aspect, the protein or composition comprising the protein of the present invention can be used to inhibit or reduce lipid accumulation in monocyte cells, vascular cells, hepatocytes and adipose tissue. .
更に他の態様において、本発明に記載のタンパク質又はタンパク質を含む組成物は、ホスホリパーゼの活性化及び／又は作用によって引き起こされる関節炎、アテローム硬化症及び敗血症性ショック等の炎症性疾患の予防及び治療に使用することができる。 In yet another aspect, the protein or composition comprising the protein of the present invention is used for the prevention and treatment of inflammatory diseases such as arthritis, atherosclerosis and septic shock caused by activation and / or action of phospholipase. Can be used.
更に別の態様において、本発明に記載のタンパク質又はタンパク質を含む組成物は、リパーゼ及び／又はホスホリパーゼを分泌する微生物病原体によって引き起こされる細胞及び組織の損傷を予防又は治療するために使用することができる。 In yet another aspect, the proteins or compositions comprising the proteins of the present invention can be used to prevent or treat cell and tissue damage caused by microbial pathogens that secrete lipases and / or phospholipases. .
更に別の態様において、本発明に記載のタンパク質又はタンパク質を含む組成物は、皮膚及び頭髪の手入れ用及び美容調製物のために使用することができる。 In yet another aspect, the proteins or compositions comprising the proteins according to the invention can be used for skin and hair care and cosmetic preparations.
本発明に記載のタンパク質又は前記タンパク質を含む組成物は、従来の任意の経口、頬側、鼻内、単位投薬形態の吸入スプレーによってか、非経口（例えば、静脈内、筋肉内、皮下、胸骨内（ｉｎｔｒａｓｔｅｍａｌ）又は注入技術による）、局所（例えば、粉末、軟膏又はドロップ）、経皮、槽内、膣内、腹腔内、膀胱内又は直腸に投与することができる。本発明の別の態様において、本発明の化合物及び少なくとも１つの他の薬学的に活性な因子を、個別に投与する場合もあれば、両方を含む薬学的組成物で投与する場合もある。このような投与には、一般的に経口が好まれる。しかし、治療されている患者が嚥下することができないか、経口投与が困難であるか望ましくなければ、非経口又は経皮投与が妥当な場合がある。 The protein according to the invention or a composition comprising said protein can be applied by any conventional oral, buccal, nasal, unit dose inhalation spray or parenterally (eg intravenous, intramuscular, subcutaneous, sternum) It can be administered intra- or intra-injection techniques), topically (eg, powder, ointment or drop), transdermal, intravaginal, intravaginal, intraperitoneal, intravesical or rectal. In another aspect of the present invention, the compound of the present invention and at least one other pharmaceutically active agent may be administered separately or in a pharmaceutical composition comprising both. For such administration, oral is generally preferred. However, parenteral or transdermal administration may be appropriate if the patient being treated cannot swallow, or oral administration is difficult or undesirable.
本発明に記載のタンパク質又は前記タンパク質を含む組成物は、任意の改変放出処方物、制御放出処方物又は持続放出処方物の形態で投与することができる。 The proteins according to the invention or compositions comprising said proteins can be administered in the form of any modified release formulation, controlled release formulation or sustained release formulation.
従って、体液、細胞及び組織中のリパーゼ及び／又はホスホリパーゼ活性を軽減するための本発明にしたがう処方物は、必須有効成分として、本発明のタンパク質を含む。 Accordingly, a formulation according to the present invention for reducing lipase and / or phospholipase activity in body fluids, cells and tissues comprises the protein of the present invention as an essential active ingredient.
好ましい実施形態において、本発明は、体液、細胞及び組織中のリパーゼ及び／又はホスホリパーゼ活性を軽減するための、本発明のタンパク質を含む処方物を提供し、このタンパク質は、単独でか、或いは既知の薬学的に許容される不活性なアジュバント、希釈剤又は担体と組み合わせて処方され得る。 In a preferred embodiment, the present invention provides a formulation comprising a protein of the present invention for reducing lipase and / or phospholipase activity in body fluids, cells and tissues, the protein alone or known In combination with a pharmaceutically acceptable inert adjuvant, diluent or carrier.
本発明によるタンパク質を含む処方物は、１つ以上の常用添加剤、担体、助剤等と共に配合することができる。これは、経口投与用に配合することができ、医薬分野で使用することができる。経口投与に適切な形態の例には、錠剤、カプセル剤、顆粒剤、細粒剤、球状体、シロップ剤及び飲料が挙げられる。好ましい実施形態において、これは球状体の形態で処方される。最も好ましい実施形態において、この球状体は腸溶性コーティングされる。 Formulations containing proteins according to the present invention can be formulated with one or more conventional additives, carriers, auxiliaries and the like. It can be formulated for oral administration and can be used in the pharmaceutical field. Examples of forms suitable for oral administration include tablets, capsules, granules, fine granules, spheroids, syrups and beverages. In a preferred embodiment, it is formulated in the form of spheres. In the most preferred embodiment, the spheroids are enteric coated.
適切な担体材料の例は、水、ゼラチン、アラビアゴム、ラクトース、デンプン、ステアリン酸マグネシウム、タルク、植物油、ポリアルキレングリコール、ワセリン等である。薬学的処方物は、固体形態（例えば、錠剤、糖衣剤、坐剤又はカプセル剤として）又は液体形態（例えば、溶液、懸濁物又はエマルジョンとして）で調合され得る。医薬調製物は滅菌される場合もあれば、保存剤、安定化剤、湿潤剤、乳化剤、浸透圧を変えるための塩又は緩衝剤等のアジュバントを含有する場合もある。これらは治療上価値がある物質を含有することができる。 Examples of suitable carrier materials are water, gelatin, gum arabic, lactose, starch, magnesium stearate, talc, vegetable oil, polyalkylene glycol, petrolatum and the like. The pharmaceutical formulations can be formulated in solid form (eg as tablets, dragees, suppositories or capsules) or in liquid form (eg as solutions, suspensions or emulsions). The pharmaceutical preparations may be sterilized or may contain preservatives, stabilizers, wetting agents, emulsifiers, adjuvants such as salts or buffers to alter osmotic pressure. They can contain therapeutically valuable substances.
本発明による処方物を調製するため、必須成分は、１つ以上の薬学的に許容されるビヒクル、担体、賦形剤、結合剤、防腐剤、酸化防止剤、安定化剤、矯味剤、緩衝剤等と混合され、続いて所望の単位投薬形態に形成される。 In order to prepare the formulation according to the invention, the essential ingredients are one or more pharmaceutically acceptable vehicles, carriers, excipients, binders, preservatives, antioxidants, stabilizers, flavoring agents, buffers. Mixed with an agent, etc., and then formed into the desired unit dosage form.
本発明にしたがう処方に際して、錠剤、カプセル剤等に混合することができるアジュバントの例には、アラビアゴム、コーンスターチ及びゼラチン等の結合剤、ステアリン酸マグネシウム等の滑沢剤、結晶セルロース等の賦形剤、α化デンプン及びアルギン酸等の膨潤剤、ショ糖、ラクトース及びサッカリン等の甘味剤及びペパーミント及びチェリー等の矯味剤が挙げられる。カプセル剤への処方に際して、油等の液体担体も上述のアジュバントと共に混合することができる。 Examples of adjuvants that can be mixed in tablets, capsules and the like in the formulation according to the present invention include binders such as gum arabic, corn starch and gelatin, lubricants such as magnesium stearate, and shaping such as crystalline cellulose. Agents, swelling agents such as pregelatinized starch and alginic acid, sweeteners such as sucrose, lactose and saccharin, and flavoring agents such as peppermint and cherry. In formulating into capsules, a liquid carrier such as oil can be mixed with the above-mentioned adjuvant.
更に、コーティング剤としてか、或いは調製物の物理的形態を変えるために、他の材料を加えることもできる。例えば、錠剤は、シュラック、糖又は任意の酸性ｐＨ耐性ポリマーでコーティングすることができる。シロップ剤及びエリキシル剤は、甘味剤としてショ糖を、防腐剤としてメチルパラベン又はプロピルパラベンを、及び／又は矯味剤としてペパーミント又はオレンジ風味を共に添加することができる。 In addition, other materials can be added as coating agents or to change the physical form of the preparation. For example, tablets can be coated with shellac, sugar or any acidic pH resistant polymer. For syrups and elixirs, sucrose can be added as a sweetening agent, methylparaben or propylparaben as a preservative, and / or peppermint or orange flavor as a flavoring agent.
本発明によれば、上記処方物は、総血清脂質コレステロールを下げる医薬として、そして肥満、虚血性心疾患、動脈硬化症、脳血管性痴呆、糖尿病、血管障害性パーキンソン病、炎症性疾患等の治療のために使用することができる。 According to the present invention, the above formulation is used as a medicine for lowering total serum lipid cholesterol, and for obesity, ischemic heart disease, arteriosclerosis, cerebrovascular dementia, diabetes, vascular disorder Parkinson's disease, inflammatory disease, etc. Can be used for treatment.
本発明に記載の処方物は、乾燥重量に換算して約１０〜２０００ｍｇ／日の量で１日１回又は数回投与してもよい。 The formulation described in the present invention may be administered once or several times a day in an amount of about 10 to 2000 mg / day in terms of dry weight.
本発明による処方物の有効成分は、血清脂質レベル又は血中総コレステロールレベル或いは組織中の脂肪の蓄積を軽減するために種々の食物に添加することができる。本発明による有効成分を添加することができる食物の例には、茶飲料、ジュース、コーヒー、飲料、炭酸飲料、チューイングガム、キャンディー、キャラメル、チョコレート及びアイスクリームが挙げられる。 The active ingredients of the formulations according to the invention can be added to various foods in order to reduce serum lipid levels or blood total cholesterol levels or fat accumulation in tissues. Examples of foods to which the active ingredient according to the present invention can be added include tea beverages, juices, coffees, beverages, carbonated beverages, chewing gum, candy, caramel, chocolate and ice cream.
更なる態様において、本発明に記載のタンパク質又はこのタンパク質を含む組成物は、動物の体液、細胞及び組織中のリパーゼ及び／又はホスホリパーゼ活性によって引き起こされるか、または悪化される疾患を治療及び予防するための動物薬として有用な場合がある。 In a further embodiment, the protein according to the invention or a composition comprising this protein treats and prevents diseases caused or exacerbated by lipase and / or phospholipase activity in animal body fluids, cells and tissues. It may be useful as a veterinary medicine.
本発明は更に、上記タンパク質を含む処方物の投与により体液、細胞及び組織中のリパーゼ及び／又はホスホリパーゼ活性を阻害又は軽減するための方法にも関する。本発明は、本明細書で以下に開示する特異的な酵素検定を使用した、このタンパク質がリパーゼ及び／又はホスホリパーゼの強力な阻害剤であるという本発明者等の発見に基づく。更に、本発明は、処方の間、このタンパク質が安定なままであり、それによってこのタンパク質の活性が維持されるという本発明者等の観察にも基づく。ｉｎ ｖｉｔｒｏの結果を確認した後、そのリパーゼ活性について動物試験を実施した。 The invention further relates to a method for inhibiting or reducing lipase and / or phospholipase activity in body fluids, cells and tissues by administration of a formulation comprising the protein. The present invention is based on the inventors' discovery that this protein is a potent inhibitor of lipase and / or phospholipase using the specific enzyme assay disclosed hereinbelow. Furthermore, the present invention is also based on our observation that the protein remains stable during formulation, thereby maintaining the activity of the protein. After confirming the in vitro results, an animal test was conducted for its lipase activity.
本発明の更なる態様及び特徴を、以下の非限定的な実施例に例示する。 Further aspects and features of the invention are illustrated in the following non-limiting examples.
（実施例１）
Ｍｏｒｉｎｇａ種子からのリパーゼ阻害タンパク質の分離
粉末状にした種子１００ｇｍを、ＭＱ水１リットルに室温にて４８時間浸漬した。その抽出液をＷｈａｔｍａｎ濾紙で濾過した。凍結乾燥器（−５０℃）を使用して濾液を２日間濃縮し、適切な技法で精製したタンパク質単離物を得た。Ｂｒａｄｆｏｒｄ法を使用してタンパク質を評価した。
Example 1
Separation of lipase-inhibiting protein from Moringa seeds 100 gm of powdered seeds were immersed in 1 liter of MQ water at room temperature for 48 hours. The extract was filtered through Whatman filter paper. The filtrate was concentrated for 2 days using a lyophilizer (−50 ° C.) to give a protein isolate purified by the appropriate technique. Proteins were evaluated using the Bradford method.
結果：粉末１０ｍｇには、タンパク質が４９４μｇ含まれていた。 Result: 10 mg of powder contained 494 μg of protein.
（実施例２）
分離したタンパク質の精製
Ｍｏｒｉｎｇａ種子の粉末試料を水に浸漬した。抽出液を濾過した後、清澄な抽出液を０．１Ｍリン酸緩衝液（１：１）と混合した。次に、その溶液を遠心分離し、上清を回収して、室温にて３０分間アセトン沈殿（冷アセトン）に供した。得られた溶液を遠心分離し、沈殿物を回収してｐＨ８．０の０．１Ｍリン酸緩衝液に溶解した。この溶液をよく混合してＴＣＡ沈殿に供した。その溶液を再び遠心分離した。最後に、粘着性のある沈殿物をｐＨ８．０の０．１Ｍリン酸緩衝液に撹拌することによって溶解した。次に、上のタンパク質溶液をゲル濾過クロマトグラフィーで精製し、５０ｍＭ、ｐＨ８．０のリン酸緩衝液で平衡化したＳｅｐｈａｄｅｘ Ｇ−５０カラムを使用して純粋なタンパク質を得た。Ｂｒａｄｆｏｒｄマイクロプレート検定により、回収した画分のタンパク質活性について検討し、タンパク質の純度を、クマシーブリリアントブルーで染色した１７％ＳＤＳ−ＰＡＧＥ（Ｌａｅｍｍｌｉ，１９７０）ゲルで泳動（ｒｕｎ）することによって確認した。
(Example 2)
Purification of separated protein A powder sample of Moringa seeds was immersed in water. After the extract was filtered, the clear extract was mixed with 0.1 M phosphate buffer (1: 1). The solution was then centrifuged and the supernatant was collected and subjected to acetone precipitation (cold acetone) at room temperature for 30 minutes. The resulting solution was centrifuged, and the precipitate was recovered and dissolved in 0.1 M phosphate buffer having a pH of 8.0. This solution was mixed well and subjected to TCA precipitation. The solution was centrifuged again. Finally, the sticky precipitate was dissolved by stirring in 0.1M phosphate buffer at pH 8.0. The above protein solution was then purified by gel filtration chromatography to obtain pure protein using a Sephadex G-50 column equilibrated with 50 mM, pH 8.0 phosphate buffer. The protein activity of the collected fractions was examined by Bradford microplate assay, and the purity of the protein was confirmed by running on a 17% SDS-PAGE (Laemmli, 1970) gel stained with Coomassie Brilliant Blue.
（実施例３）
タンパク質の特徴付け
Ｍｏｒｉｎｇａ種子から分離したタンパク質を用いて、ＳＤＳ−ＰＡＧＥ（１７％）を実行し、クマシーブルーで染色した。５＋／−１ｋＤバンドを切り出し、シリコン処理したチューブに移して５０％メタノール２００μＬで一晩洗浄、脱色した。ゲル片をアセトニトリル中で脱水し、０．１Ｍ炭酸水素アンモニウム中の１０ｍＭジチオールスレイトール３０μＬで再水和し、室温にて０．５時間還元した。ＤＴＴ溶液を除去し、０．１Ｍ炭酸水素アンモニウム中の５０ｍＭヨードアセトアミド３０μＬで、試料を室温にて０．５時間アルキル化した。試薬を除去し、アセトニトリル１００μＬ中でゲル片を脱水した。アセトニトリルを除去し、０．１Ｍ炭酸水素アンモニウム１００μＬ中でゲル片を再水和した。その断片をアセトニトリル１００μＬ中で脱水し、アセトニトリルを除去して、真空遠心分離により断片を完全に乾燥させた。氷上の５０ｍＭ炭酸水素アンモニウム中の２０ｎｇ／μＬトリプシンで、ゲル片を１０分間再水和した。余分なトリプシン溶液を除去し、５０ｍＭ炭酸水素アンモニウム２０μＬを添加した。試料を３７℃にて一晩消化し、ポリアクリルアミドから形成されたペプチドを５０％アセトニトリル／５％ギ酸の３０μＬアリコート２つで抽出した。これらの抽出液を合わせて、ＭＳ分析のために２５μＬまで蒸発させた。
(Example 3)
Protein Characterization SDS-PAGE (17%) was performed using proteins isolated from Moringa seeds and stained with Coomassie Blue. The 5 +/− 1 kD band was cut out, transferred to a siliconized tube, washed overnight with 200 μL of 50% methanol and decolorized. The gel pieces were dehydrated in acetonitrile, rehydrated with 30 μL of 10 mM dithiol threitol in 0.1 M ammonium bicarbonate and reduced at room temperature for 0.5 hours. The DTT solution was removed and the sample was alkylated with 30 μL of 50 mM iodoacetamide in 0.1 M ammonium bicarbonate at room temperature for 0.5 hours. The reagent was removed and the gel pieces were dehydrated in 100 μL of acetonitrile. Acetonitrile was removed and the gel pieces were rehydrated in 100 μL of 0.1M ammonium bicarbonate. The fragment was dehydrated in 100 μL of acetonitrile, the acetonitrile was removed, and the fragment was completely dried by vacuum centrifugation. Gel pieces were rehydrated with 20 ng / μL trypsin in 50 mM ammonium bicarbonate on ice for 10 minutes. Excess trypsin solution was removed and 20 μL of 50 mM ammonium bicarbonate was added. Samples were digested overnight at 37 ° C. and peptides formed from polyacrylamide were extracted with two 30 μL aliquots of 50% acetonitrile / 5% formic acid. These extracts were combined and evaporated to 25 μL for MS analysis.
Ｆｉｎｎｉｇａｎ ＬＣＱイオントラップ型質量分析計装置と、Ｐｒｏｔａｎａナノスプレーイオン源を、自己充填式８ｃｍ×内径７５μｍ Ｐｈｅｎｏｍｅｎｅｘ Ｊｕｐｉｔｅｒ １０μｍ Ｃ１８逆相キャピラリーカラムで接続して、ＬＣ−ＭＳ装置を構成した。抽出液を０．５〜５μＬ容量注入し、流速０．２５μＬ／分で、アセトニトリル／０．１Ｍ酢酸勾配によりペプチドをカラムから溶出した。ナノスプレーイオン源を２．８ｋＶで操作した。フルスキャン質量スペクトルを取得する機器の倍化動作（ｄｏｕｂｌｅ ｐｌａｙ）能力を使用して、消化液を分析してペプチド分子量及びプロダクトイオンスペクトルを決定し、連続的なスキャンでアミノ酸配列を決定した。 A Finnigan LCQ ion trap mass spectrometer apparatus and a Protana nanospray ion source were connected with a self-packing 8 cm × 75 μm inner diameter Phenomenex Jupiter 10 μm C18 reverse phase capillary column to constitute an LC-MS apparatus. The extract was injected at a volume of 0.5-5 μL, and the peptide was eluted from the column with an acetonitrile / 0.1 M acetic acid gradient at a flow rate of 0.25 μL / min. The nanospray ion source was operated at 2.8 kV. Using the instrument's double play capability to obtain full scan mass spectra, digestive fluid was analyzed to determine peptide molecular weight and product ion spectra, and amino acid sequences were determined in successive scans.
以下に示す通り、タンパク質の部分的な配列番号を確認した。
ＣＧＱＱＬＲＮＩＳＰＰＱＲＣＰＳＬＲＱＡＶＱＬＡＨＱＱＱＧＱＧＰＱＱＶＲＱＭＹＲ
（実施例３）
合成基質の存在下におけるＭｏｒｉｎｇａ種子タンパク質のリパーゼ阻害活性
リパーゼ検定
膵リパーゼを使用するという変更を伴って、Ｗｉｎｋｌｅｒ ａｎｄ Ｓｔｕｃｋｍａｎｎ，１９７９に記載の方法により酵素検定を実施した。９６ウェル形式を使用して検定を計画した。この検定で使用した基質はパルチミン酸ｐ−ニトロフェノール（Ｓｉｇｍａ、カタログ番号Ｎ−２７５２）であった。パルチミン酸ｐ−ニトロフェノール４．５ｍｇをＮ，Ｎ−ジメチルホルムアミド（Ｓｉｇｍａ、カタログ番号Ｄ−４５５１）２００μＬ中に溶解し、容積をＰｈ８．０の０．１Ｍリン酸緩衝液で１０ｍＬとした。０．１Ｍ−リン酸緩衝液中で、酵素を５ｍｇ／ｍＬ濃度で溶解することによりリパーゼ（Ｓｉｇｍａ、カタログ番号Ｌ−３１２６）試料を調製した。反応混合物は基質溶液−１５０μＬ、リン酸緩衝液（ｐＨ８．０、０．１Ｍ）−４０μＬ及びリパーゼ溶液−１０μＬからなった。反応混合物を３７℃にてインキュベートし、インキュベート後に光学密度を４０５ｎｍで測定した。酵素活性を国際単位（ＩＵ）の形で表した。標準的な検定条件下で、基質（パルチミン酸ｐ−ニトロフェノール）／ｍＬ／分から遊離フェノール１ｎｍを放出する量をリパーゼの１酵素単位と定義した（Ｗｉｎｋｌｅｒ Ｋ．Ｗ．ａｎｄ Ｓｔｕｃｋｍａｎ Ｍ，１９７９ Ｇｌｙｃｏｇｅｎ ｈｙａｌｕｒｏｎａｔｅ ａｎｄ ｓｏｍｅ ｏｔｈｅｒ ｐｏｌｙｓａｃｃｈａｒｉｄｅｓ ｇｒｅａｔｌｙ ｅｎｈａｎｃｅｓ ｔｈｅ ｆｏｒｍａｔｉｏｎ ｏｆ ｅｘｏｌｉｐａｓｅ ｂｙ Ｓｅｒｒａｔｉａ ｍａｒｃｅｓｃｅｎｓ，Ｊ．ｏｆ Ｂａｃｔｅｒｉｏｌｏｇｙ １３８：６６３−６７０）。これはｐ−ニトロフェノールの標準グラフから導き出したものである。
As shown below, the partial SEQ ID NO of the protein was confirmed.
CGQQLRNISPPQRCPSLRQAVQLAHQQQGQGPQQVRQMYR
(Example 3)
Lipase Inhibitory Activity of Moringa Seed Protein in the Presence of Synthetic Substrate Lipase Assay Enzyme assay was performed by the method described in Winkler and Stuckmann, 1979, with the modification that pancreatic lipase was used. The assay was planned using a 96 well format. The substrate used in this assay was p-nitrophenol palmitate (Sigma, catalog number N-2752). 4.5 mg of palmitic acid p-nitrophenol was dissolved in 200 μL of N, N-dimethylformamide (Sigma, catalog number D-4551), and the volume was adjusted to 10 mL with 0.1 M phosphate buffer of Ph 8.0. A lipase (Sigma, catalog number L-3126) sample was prepared by dissolving the enzyme at a concentration of 5 mg / mL in 0.1 M phosphate buffer. The reaction mixture consisted of -150 μL substrate solution, 40 μL phosphate buffer (pH 8.0, 0.1 M) and -10 μL lipase solution. The reaction mixture was incubated at 37 ° C. and the optical density was measured at 405 nm after incubation. Enzyme activity was expressed in the form of International Units (IU). The amount of 1 nm free phenol released from the substrate (p-nitrophenol palmitate) / mL / min under standard assay conditions was defined as one enzyme unit of lipase (Winkler KW and Stuckman M, 1979 Glycogen hyaluronate). and some other polysaccharides Greatly enhances the formation of exolative by Serratia marcescens, J. of Bacteriology 138: 663-670). This is derived from the standard graph of p-nitrophenol.
Ｍｏｒｉｎｇａ種子タンパク質によるリパーゼ阻害
阻害検定を用量依存的な様式で実施した。タンパク質の濃度は、４０μｇ〜０．１５６μｇ／ｍＬ反応混合物と照合した。検定は、対照のリン酸緩衝液の代わりに阻害剤溶液４０μＬを使用したことを除き、上に記載した検定とほぼ同じであった。放出したｐ−ニトロフェノールを４０５ｎｍで記録した。単純に国際単位（ＩＵ）の変化に基づいて、阻害率換算で酵素阻害を表したが、これは標準グラフから算出したものである。
Lipase inhibition by Moringa seed protein Inhibition assays were performed in a dose-dependent manner. The protein concentration was checked with a 40 μg to 0.156 μg / mL reaction mixture. The assay was similar to the assay described above, except that 40 μL of inhibitor solution was used instead of the control phosphate buffer. The released p-nitrophenol was recorded at 405 nm. Enzyme inhibition was simply expressed in terms of inhibition based on changes in international units (IU), which was calculated from a standard graph.
結果：
（表−１）
result:
(Table-1)
（実施例４）
天然基質の存在下におけるＭｏｒｉｎｇａ種子タンパク質の阻害活性
Ｉｓｈｉｉａ Ｃら、１９８８，Ｉｎｈｉｂｉｔｉｏｎ ｏｆ ｌｉｐａｓｅ ｂｙ ｐｒｏｔｅｉｎｓ ａｎｄ ｔｈｅｉｒ ｉｎｈｉｂｉｔｏｒｙ ｍｅｃｈａｎｉｓｍ，Ｎｉｐｐｏｎ Ｓｈｏｋｕｈｉｎ Ｋｏｇｙｏ Ｇａｋｋａｉｓｈｉ，３５（６），４３０−４３９の変法にしたがうことにより、反応混合物中に遊離した遊離脂肪酸を滴定することによりリパーゼ活性を測定した。試験管内で、３７℃にて３０分間振盪させて反応を実施した。反応混合物を、ｐＨ−７．０のＭｃｌｌｖａｉｎｅ緩衝液１．５ｍＬ、オリーブオイル０．２４ｍＬ及び阻害剤溶液０．５ｍＬで調製し、水を入れて最終容積を５．５ｍＬとした。５分間のプレインキュベーション後、酵素溶液０．５ｍＬ（５．０ｍｇ）を添加して反応を開始した。次に、ｎ−プロピルアルコール：石油エーテル（１：４）の混合溶液１０ｍＬを添加して反応を停止し、次に混合物を２分間勢いよく振盪した。上層を１ｍＬピペットで取り、指示薬としてフェノールフタレインを使用して０．０２Ｍのアルコール性ＫＯＨで滴定した。阻害剤溶液を緩衝液に置き換えたことを除き、標準反応混合物を、記載したように調製した。標準検定条件下でμＭ／分放出した脂肪酸を酵素の１国際単位（ＩＵ）とみなした。
(Example 4)
Moringa Seed Protein Inhibitory Activity in the Presence of Natural Substrate Ishiia C et al., 1988, Inhibition of Lipidy by Proteins and the Inhibitory Mechanism, Nippon Shokuhin Kogyo 4 Lipase activity was measured by titrating free fatty acids released into the mixture. The reaction was carried out in a test tube by shaking at 37 ° C. for 30 minutes. The reaction mixture was prepared with 1.5 mL of Mclvaine buffer at pH-7.0, 0.24 mL of olive oil and 0.5 mL of inhibitor solution, and water was added to a final volume of 5.5 mL. After 5 minutes of preincubation, 0.5 mL (5.0 mg) of enzyme solution was added to initiate the reaction. Next, 10 mL of a mixed solution of n-propyl alcohol: petroleum ether (1: 4) was added to stop the reaction, and then the mixture was shaken vigorously for 2 minutes. The upper layer was taken with a 1 mL pipette and titrated with 0.02 M alcoholic KOH using phenolphthalein as an indicator. A standard reaction mixture was prepared as described, except that the inhibitor solution was replaced with buffer. Fatty acids released at μM / min under standard assay conditions were considered as one international unit (IU) of enzyme.
結果：
（表−２）
result:
(Table-2)
（実施例５）
Ｍｏｒｉｎｇａ種子タンパク質のリパーゼ阻害活性に対するトリプシンの影響
Ｍｏｒｉｎｇａ種子タンパク質のトリプシン開裂後に、リパーゼ阻害活性を実施した。分離したタンパク質（５００μｇ／ｍＬ Ｂｒａｄｆｏｒｄ法により評価した）を、ｐＨ−９．０の５０ｍＭトリス緩衝液中のトリプシン（０．５％）及び試料緩衝液と共に１：１：２の割合で、３７℃にて２４時間インキュベートした。インキュベーション後、全試料を７０℃にて１０分間熱失活させた。対照におけるリン酸緩衝液の代わりに試験溶液（トリプシン処理済／未処理）４０μＬを使用したことを除き、先に記載の通りリパーゼ阻害検定を実施した。放出したｐ−ニトロフェノールを４０５ｎｍで記録した。単純に国際単位（ＩＵ）の変化に基づいて、阻害率換算でリパーゼ活性を表したが、これは標準グラフから算出したものである。
(Example 5)
Effect of trypsin on lipase inhibitory activity of Moringa seed protein Lipase inhibitory activity was performed after trypsin cleavage of Moringa seed protein. Separated protein (assessed by 500 μg / mL Bradford method) was added at a ratio of 1: 1: 2 with trypsin (0.5%) and sample buffer in 50 mM Tris buffer at pH-9.0 at 37 ° C. For 24 hours. After incubation, all samples were heat inactivated at 70 ° C. for 10 minutes. A lipase inhibition assay was performed as described above, except that 40 μL of test solution (trypsinized / untreated) was used instead of phosphate buffer in the control. The released p-nitrophenol was recorded at 405 nm. The lipase activity was simply expressed in terms of inhibition based on changes in the International Unit (IU), which was calculated from the standard graph.
結果：トリプシン開裂後に、Ｍｏｒｉｎｇａ種子タンパク質がリパーゼ阻害活性を失ったことが観察されたが、これはタンパク質にリパーゼ阻害活性が存在することを示唆するものである。 Results: It was observed that Moringa seed protein lost lipase inhibitory activity after trypsin cleavage, suggesting that the protein has lipase inhibitory activity.
（表−３） (Table-3)
ダイズタンパク質を用いての、トリプシンからのＭｏｒｉｎｇａ種子タンパク質の保護
安定した形態でＭｏｒｉｎｇａ種子タンパク質を存在させるためには、トリプシン阻害からこのタンパク質を保護することが必須であった。それゆえ、保護のためにダイズタンパク質を選択した。
Protection of Moringa seed protein from trypsin using soy protein In order for Moringa seed protein to be present in a stable form, it was essential to protect this protein from trypsin inhibition. Therefore, soy protein was selected for protection.
ダイズ種子からのダイズタンパク質の分離
２リットルのガラスビーカー内で、ダイズ種子５００ｇｍとＭｉｌｌｉ Ｑ水１．５リットルとを装填し、水浴でビーカーを６５℃にて（外部温度）９０分間加熱した。９０分後、ダイズ種子抽出液を濾過し室温に冷却した。凍結乾燥器（−５０℃）を使用して濾液を２日間濃縮した。粗タンパク質が丸底フラスコの壁面に張り付き、これを、スパチュラを使用して擦過し粉末を細かくした。得られた総固体粉末タンパク質は１２．５ｇｍであり、吸湿性を備えていた。Ｂｒａｄｆｏｒｄ法を使用してタンパク質を評価した。
Separation of Soybean Protein from Soybean Seeds In a 2 liter glass beaker, 500 gm of soybean seeds and 1.5 liter of Milli Q water were charged and the beaker was heated at 65 ° C. (external temperature) for 90 minutes in a water bath. After 90 minutes, the soybean seed extract was filtered and cooled to room temperature. The filtrate was concentrated for 2 days using a lyophilizer (−50 ° C.). The crude protein stuck to the wall of the round bottom flask and this was scraped using a spatula to make the powder fine. The total solid powder protein obtained was 12.5 gm and was hygroscopic. Proteins were evaluated using the Bradford method.
結果：固体１０ｍｇにはタンパク質が１８．５２μｇ含有されていた。 Result: 10 mg of the solid contained 18.52 μg of protein.
タンパク質保護検定
種々の濃度のダイズタンパク質を使用して、トリプシンからＭｏｒｉｎｇａ種子タンパク質を保護することを実施した。Ｍｏｒｉｎｇａ種子タンパク質（Ｂｒａｄｆｏｒｄ法による評価で４９．１μｇ／１００μＬ）を、トリプシン（０．５％）溶液１００μＬ及びｐＨ−９．０、５０ｍＭの緩衝液中の種々の濃度のダイズタンパク質１００μＬ及び試料緩衝液と共に１：１：１：１の比で、３７０℃にて２４時間インキュベートした。インキュベーション後、全試料を７０℃にて１０分間熱失活させた。対照におけるリン酸緩衝液の代わりに試験溶液（処理済／未処理）４０μＬを使用したことを除き、先に記載の通り検定を実施した。放出したｐ−ニトロフェノールを４０５ｎｍでのＯＤとして記録した。単純に国際単位（ＩＵ）の変化に基づいて、阻害率換算でリパーゼ活性を表したが、これは標準グラフから算出したものである。
Protein Protection Assay Protection of Moringa seed protein from trypsin was performed using various concentrations of soy protein. Moringa seed protein (49.1 μg / 100 μL as assessed by Bradford method), 100 μL of trypsin (0.5%) solution and 100 μL of soy protein at various concentrations in buffer of pH-9.0, 50 mM and sample buffer And incubated at 370 ° C. for 24 hours at a ratio of 1: 1: 1: 1. After incubation, all samples were heat inactivated at 70 ° C. for 10 minutes. The assay was performed as described above except that 40 μL of test solution (treated / untreated) was used instead of phosphate buffer in the control. The released p-nitrophenol was recorded as the OD at 405 nm. The lipase activity was simply expressed in terms of inhibition based on changes in the International Unit (IU), which was calculated from the standard graph.
（表−４） (Table-4)
タンパク質の組成物
Ｍｏｒｉｎｇａ種子タンパク質（５０．０ｇｍｓ）、微結晶性セルロース（３３６．０ｇｍｓ）及びラクトース（８４．０ｇｍｓ）を個別に４０＃を通過させ、適切なミキサー内で１５分間幾何学的（ｇｅｏｍｅｔｒｉｃａｌ）に撹拌した。トウモロコシデンプン（３０．０ｇｍｓ）と必要量の水を使用して、１０％デンプンペーストを調製した。ミキサーにデンプンペーストを加え、押し出しに適切な粘稠度の生地が形成されるまで混合を続けた。０．８ｍｍ放射状スクリーンを装着したＦｕｊｉ Ｐａｕｄａｌ ＥＸＤＳ−６０押し出し機内に、生地混合物を装填した。乾燥オーブンを使用して、押し出された生地（ｅｘｔｒｕｄｅ）を４０℃にて８時間乾燥させた。Ｆｕｊｉ Ｐａｕｄａｌ Ｑ２３０を使用して球状体化し、これらを４０℃にて一晩乾燥させる。乾燥した球状体を篩いにかけ、２４番を通過し、メッシュｎ０．４０上に保持された球状体を更に加工した。
Protein Composition Moringa seed protein (50.0 gms), microcrystalline cellulose (336.0 gms) and lactose (84.0 gms) are individually passed through 40 # and geometrically (geometrical) for 15 minutes in a suitable mixer. ). A 10% starch paste was prepared using corn starch (30.0 gms) and the required amount of water. Starch paste was added to the mixer and mixing continued until a dough of suitable consistency for extrusion was formed. The dough mixture was loaded into a Fuji Paudal EXDS-60 extruder equipped with a 0.8 mm radial screen. The extruded dough was dried at 40 ° C. for 8 hours using a drying oven. Spheroidize using Fuji Paudal Q230 and dry them overnight at 40 ° C. The dried spheres were sieved and passed through No. 24 and the spheres retained on mesh n0.40 were further processed.
１）密封（ｓｅａｌ）コーティング、２）腸溶性コーティングの２つの工程でコーティングを行った。 Coating was performed in two steps: 1) seal coating and 2) enteric coating.
フィルム形成ポリマーとしてＨＰＭＣを使用することにより密封コーティングを行い、腸溶性ポリマーとしてＥｕｄｒａｇｉｔ Ｌ３０Ｄ５５を、着色剤として黄色酸化鉄を補って腸溶性コーティングを行った。サイズ０の透明な硬ゼラチンカプセルに、球状体を充填した。Ｂｒａｄｆｏｒｄ法を使用して、コーティングした球状体並びに充填カプセルに関するｉｎ−ｖｉｔｒｏタンパク質分析を行った。 Seal coating was performed by using HPMC as the film-forming polymer, and enteric coating was performed supplementing Eudragit L30D55 as the enteric polymer and yellow iron oxide as the colorant. Size 0 transparent hard gelatin capsules were filled with spheroids. In-vitro protein analysis was performed on coated spheroids as well as filled capsules using the Bradford method.
分析データ：
（表−５）
Analysis data:
(Table-5)
試料Ｂ＊＊＝充填カプセル試料
グラフには濃度に対する吸光度をプロットした
Ｒ＝０．９５０４５８
得られた方程式：Ｙ＝１．１３６１６×Ｘ＋０．０６８２６８１
式中、Ｙは吸光度であり、Ｘは濃度（μｇ／μＬ）である
計算結果：
試料Ａ＊＝０．３９９０μｇ／μＬ
試料Ｂ＊＊＝０．３６６８μｇ／μＬ
上述のデータから、処方プロセス中は、タンパク質は破壊されていないことが推測できる。
The resulting equation: Y = 1.13616 × X + 0.0682281
In the formula, Y is the absorbance, and X is the concentration (μg / μL).
Sample A * = 0.3990 μg / μL
Sample B ** = 0.3668 μg / μL
From the above data it can be inferred that the protein is not destroyed during the formulation process.
（実施例８）
動物試験
４群のＷｉｓｔａｒ雄ラットにおいて、Ｍｏｒｉｎｇａ種子タンパク質にダイズタンパク質（先に分離した）を添えたものと添えないものを用いて、短期動物実験を実施した。雄Ｗｉｓｔａｒを一晩絶食させ、脂質エマルジョン（水中ヒマワリ油１０ｍＬ）１．２ｍＬ、あるいは脂質エマルジョン１．２ｍＬに、群ごとに定めた種々の処方物（群：対照、群１：Ｍｏｒｉｎｇａ固体抽出物５０ｍｇ（タンパク質２．５ｍｇ）、群２：タンパク質９２．６μｇを含有するダイズ固体抽出物５０ｍｇ及び群３：Ｍｏｒｉｎｇａ固体抽出物５０ｍｇ（タンパク質２．５ｍｇ）及びダイズ固体抽出物５０ｍｇ（タンパク質９２．６μｇ））をあわせてラットに経口投与した。血液試料を採取し、血漿トリアシルグリセロール濃度を決定した。
トリグリセリド濃度（ｍｇ／ｄｌ）
平均値：
(Example 8)
Animal Tests Short-term animal experiments were conducted in 4 groups of Wistar male rats, with and without Moringa seed protein plus soy protein (previously separated). Male Wistar is fasted overnight and lipid formulations (10 mL sunflower oil in water) 1.2 mL, or lipid emulsion 1.2 mL, various formulations defined for each group (group: control, group 1: Moringa solid extract 50 mg (Protein 2.5 mg), Group 2: 50 mg soybean solid extract containing 92.6 μg protein and Group 3: 50 mg Moringa solid extract (2.5 mg protein) and 50 mg soybean solid extract (92.6 μg protein)) Were orally administered to rats. A blood sample was taken and the plasma triacylglycerol concentration was determined.
Triglyceride concentration (mg / dl)
Average value:
Claims (41)
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| Application Number | Priority Date | Filing Date | Title |
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| PCT/IN2005/000330 WO2007039911A1 (en) | 2005-10-05 | 2005-10-05 | Agent and compositions comprising the same for inhibiting lipases and/or phospholipases in body fluids, cells and tissues |
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| EP (1) | EP1931701A1 (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JP2023519026A (en) * | 2020-05-21 | 2023-05-09 | アジャンス フランセーズ プール ル デヴロプマン ダル ウラ | Extract of Moringa peregrina seed solids, process for obtaining same and use thereof in cosmetic or nutriceutical compositions |
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| EP2025242A1 (en) * | 2007-07-25 | 2009-02-18 | Universität Hohenheim | Feed and feed additive for herbivores, and method for manufacturing the same |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999048512A1 (en) * | 1998-03-24 | 1999-09-30 | Laboratoires Serobiologiques (Societe Anonyme) | Use of at least one protein extract of the moringa genus plant seeds and corresponding cosmetic and/or pharmacological composition |
| WO2003057230A1 (en) * | 2002-01-14 | 2003-07-17 | Philippe Jean Louis Thurot | Composition used for combating cholesterol and improving digestion |
| JP2005508304A (en) * | 2001-07-19 | 2005-03-31 | オプティマ アンビローヌマン ソシエテ アノニム | Moringa seed protein |
-
2005
- 2005-10-05 EP EP05823512A patent/EP1931701A1/en not_active Withdrawn
- 2005-10-05 JP JP2008534146A patent/JP2009511466A/en active Pending
- 2005-10-05 WO PCT/IN2005/000330 patent/WO2007039911A1/en active Application Filing
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999048512A1 (en) * | 1998-03-24 | 1999-09-30 | Laboratoires Serobiologiques (Societe Anonyme) | Use of at least one protein extract of the moringa genus plant seeds and corresponding cosmetic and/or pharmacological composition |
| JP2005508304A (en) * | 2001-07-19 | 2005-03-31 | オプティマ アンビローヌマン ソシエテ アノニム | Moringa seed protein |
| WO2003057230A1 (en) * | 2002-01-14 | 2003-07-17 | Philippe Jean Louis Thurot | Composition used for combating cholesterol and improving digestion |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2023519026A (en) * | 2020-05-21 | 2023-05-09 | アジャンス フランセーズ プール ル デヴロプマン ダル ウラ | Extract of Moringa peregrina seed solids, process for obtaining same and use thereof in cosmetic or nutriceutical compositions |
| JP7357983B2 (en) | 2020-05-21 | 2023-10-10 | アジャンス フランセーズ プール ル デヴロプマン ダル ウラ | Extract of Moringa peregrina seed solids, method for obtaining same and use thereof in cosmetic product compositions or nutricosmetic compositions |
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| Publication number | Publication date |
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| WO2007039911A1 (en) | 2007-04-12 |
| EP1931701A1 (en) | 2008-06-18 |
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