JP6969041B2 - Vascular Endothelial Nitric Oxide Synthetic Enzyme Production Promoter and Oral Composition - Google Patents
Vascular Endothelial Nitric Oxide Synthetic Enzyme Production Promoter and Oral Composition Download PDFInfo
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Description
本発明は、Ｔｉｅ２活性化剤、血管新生抑制剤、血管の成熟化剤、血管の正常化剤、血管の安定化剤、クローディン−５産生促進剤、リンパ管の安定化剤、ｅＮＯＳ産生促進剤、血管拡張剤、及び血小板凝集抑制剤、並びに医薬品組成物に関する。 The present invention relates to a Tie2 activator, an angiogenesis inhibitor, a blood vessel maturation agent, a blood vessel normalizing agent, a blood vessel stabilizer, a claudin-5 production promoter, a lymphatic vessel stabilizer, and an eNOS production promoter. Agents, vasodilators, platelet aggregation inhibitors, and pharmaceutical compositions.
血管は、血管内皮細胞と血管壁細胞（血管平滑筋細胞やペリサイト）とが、細胞外マトリックスを介して、間接的に又は直接的に接着する構造を有しており、酸素及び栄養素を生体組織に供給して生体組織から老廃物を除去する機能を有する。 Blood vessels have a structure in which vascular endothelial cells and vascular wall cells (vascular smooth muscle cells and pericite) adhere indirectly or directly via an extracellular matrix, and oxygen and nutrients are living in the living body. It has the function of supplying to tissues and removing waste products from living tissues.
一般に、血管の形成は、新たに血管が形成される血管発生（ｖａｓｃｕｌｏｇｅｎｅｓｉｓ）と、形成された既存の血管が伸長して分岐することにより、新たな血管のネットワークが形成される血管新生（ａｎｇｉｏｇｅｎｅｓｉｓ）との２段階に分けられる。前者は、血管内皮増殖因子（ＶＥＧＦ）が作用し、脈管形成とよばれる血管の初期発生からその後の血管新生に至るまで非常に広い範囲の血管形成に関与するものであり、後者は、アンジオポエチン（Ａｎｇ）が作用し、血管内皮細胞と血管壁細胞との接着の制御、血管の構造的安定化に関与するものである。 In general, the formation of blood vessels is angiogenesis (vasculogenesis) in which new blood vessels are formed and angiogenesis (angiogenesis) in which a network of new blood vessels is formed by the extension and branching of existing blood vessels that have been formed. It can be divided into two stages: In the former, vascular endothelial growth factor (VEGF) acts and is involved in a very wide range of angiogenesis from the initial development of blood vessels called angiogenesis to subsequent angiogenesis, and the latter is angiopoetin. (Ang) acts and is involved in the control of adhesion between vascular endothelial cells and vascular wall cells and the structural stabilization of blood vessels.
血管は通常の酸素状況では、血管内皮細胞とその周囲を裏打ちする血管壁細胞とが強固に接着しており、血管構造が安定に保たれているが、組織で低酸素が生じると血管壁細胞が血管内皮細胞から脱離し、無秩序な血管が増生することがある。このような現象は、血管新生と呼ばれており、腫瘍、慢性関節リウマチ、糖尿病網膜症、高脂血症、高血圧などの血管病変を主体とした疾患において、しばしば観察される。 Under normal oxygen conditions, blood vessels have strong adhesion between vascular endothelial cells and the vascular wall cells that line them, and the vascular structure is kept stable. However, when hypoxia occurs in tissues, vascular wall cells May desorb from vascular endothelial cells, resulting in the growth of disordered blood vessels. Such a phenomenon is called angiogenesis and is often observed in diseases mainly composed of vascular lesions such as tumors, rheumatoid arthritis, diabetic retinopathy, hyperlipidemia, and hypertension.
これらの血管新生は、血管内皮細胞に発現する受容体型チロシンキナーゼＴｉｅ２（Ｔｙｒｏｓｉｎｅ ｋｉｎａｓｅ ｗｉｔｈ Ｉｇ ａｎｄ ＥＧＦ ｈｏｍｏｌｏｇｙ ｄｏｍａｉｎ２）を活性化させることにより、抑制されることが知られている（例えば、特許文献１参照）。血管狭小化あるいは血管拡大化の抑制が原因となって生じる虚血性疾患は、Ｔｉｅ２の活性化により、血管腔が拡大化されることが報告されている（例えば、非特許文献１参照）。また、Ｔｉｅ２の活性化により、血管内皮細胞の細胞死を抑制することが報告されている（例えば、非特許文献２参照）。 These angiogenesis are known to be suppressed by activating the receptor tyrosine kinase Tie2 (Tyrosine kinase with Ig and EGF homology domain2) expressed in vascular endothelial cells (see, for example, Patent Document 1). ). It has been reported that in ischemic diseases caused by suppression of vascular narrowing or vascular enlargement, the vascular lumen is enlarged by activation of Tie2 (see, for example, Non-Patent Document 1). Further, it has been reported that activation of Tie2 suppresses cell death of vascular endothelial cells (see, for example, Non-Patent Document 2).
このように、Ｔｉｅ２の活性化は、血管新生を抑制することができるだけでなく、血管を成熟化、正常化、及び安定化させることも知られている。
例えば、血管再生医療では、Ｔｉｅ２の活性化により、血管における血管内皮細胞と血管壁細胞との接着を誘導して、血管を成熟化させることが知られている。
例えば、腫瘍、糖尿病性網膜症等で観察される血管壁細胞が血管内皮細胞に接着しないことにより無秩序な血管が増生する疾患では、Ｔｉｅ２の活性化により、血管壁細胞を内皮細胞に接着させ、血管を正常化させることが知られている。
例えば、種々の細胞内外の血管構造を破綻させる環境因子に対しては、Ｔｉｅ２の活性化により、血管の不安定化を抑制し、血管を安定化させることが知られている。
Thus, it is known that activation of Tie2 not only suppresses angiogenesis, but also matures, normalizes, and stabilizes blood vessels.
For example, in vascular regenerative medicine, it is known that activation of Tie2 induces adhesion between vascular endothelial cells and vascular parietal cells in blood vessels to mature blood vessels.
For example, in a disease in which vascular parietal cells observed in tumors, diabetic retinopathy, etc. do not adhere to vascular endothelial cells and disordered blood vessels grow, activation of Tie2 causes the vascular parietal cells to adhere to the endothelial cells. It is known to normalize blood vessels.
For example, it is known that activation of Tie2 suppresses destabilization of blood vessels and stabilizes blood vessels for environmental factors that disrupt various internal and external vascular structures.
このようなＴｉｅ２の活性化により血管新生を抑制する天然物としては、桂皮の抽出物などが提案されている（例えば、特許文献１参照）。しかしながら、これらの活性が不十分であるという問題がある。また、血管新生を抑制する物質としては、スラミン（ポリスルホン化ナフチルウレア化合物）が知られているが（例えば、特許文献２参照）、安全性に優れないという問題がある。 As a natural product that suppresses angiogenesis by activating Tie2, an extract of cinnamon bark has been proposed (see, for example, Patent Document 1). However, there is a problem that these activities are insufficient. Further, although suramin (polysulfone naphthylurea compound) is known as a substance that suppresses angiogenesis (see, for example, Patent Document 2), there is a problem that it is not excellent in safety.
血管やリンパ管を構成する内皮細胞には、クローディン−５（ｃｌａｕｄｉｎ−５、ＣＬＤ５）と呼ばれるタイトジャンクション構成タンパク質の一つが存在し、細胞同士の密着結合に関与している。特に、リンパ管内皮細胞では、加齢とともにクローディン−５の発現が低下することが報告され、クローディン−５とリンパ管の安定化との関連が示唆されている。また、リンパ管内皮細胞にもＴｉｅ２が発現し、リガンドであるアンジオポエチン−１（Ａｎｇ１）により刺激されたリンパ管内皮細胞は、Ａｎｇ１／Ｔｉｅ２シグナルを介してクローディン−５の発現を亢進することで、リンパ管の安定化に寄与することが明らかとなった（例えば、非特許文献３参照）。 Endothelial cells that make up blood vessels and lymph vessels contain one of the tight junction-constituting proteins called claudin-5 (CLD5), which is involved in tight junctions between cells. In particular, it has been reported that the expression of claudin-5 decreases with aging in lymphatic endothelial cells, suggesting a relationship between claudin-5 and lymphatic vessel stabilization. In addition, Tie2 is also expressed in lymphatic endothelial cells, and lymphatic endothelial cells stimulated by the ligand angiopoietin-1 (Ang1) enhance the expression of claudin-5 via the Ang1 / Tie2 signal. , It has been clarified that it contributes to the stabilization of lymphatic vessels (see, for example, Non-Patent Document 3).
しかしながら、クローディン−５の産生を促進する物質については、未だ報告がなく、そのような活性を有する物質の同定が望まれている。 However, there have been no reports on substances that promote the production of claudin-5, and identification of substances having such activity is desired.
一酸化窒素については、その生理学的作用又は薬理学的作用が注目されており、様々な研究が行われてきている。一酸化窒素は、生体内において、Ｌ−アルギニンと分子状酸素とを基質とし、還元型ニコチンアミドアデニンジヌクレオチドリン酸（ＮＡＤＰＨ）、フラビンモノヌクレオチド（ＦＭＮ）、フラビンアデニンジヌクレオチド（ＦＡＤ）、テトラヒドロビオプテリンを補酵素として、一酸化窒素合成酵素（ＮＯＳ）により生成される。ＮＯＳには、酵素学的にもタンパク質分子としても異なる３種のアイソフォームが存在し、それぞれ誘導型一酸化窒素合成酵素（ｉＮＯＳ）、血管内皮型一酸化窒素合成酵素（ｅＮＯＳ）、神経型一酸化窒素合成酵素（ｎＮＯＳ）が知られている。 Regarding nitric oxide, its physiological or pharmacological action has attracted attention, and various studies have been conducted. Nitric oxide uses L-arginine and molecular oxygen as substrates in the living body, and reduces nicotinamide adenine dinucleotide phosphate (NADPH), flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), and tetrahydro. It is produced by nitric oxide synthase (NOS) with biopterin as a coenzyme. There are three types of isoforms in NOS that are different both enzymatically and as protein molecules: inducible nitric oxide synthase (iNOS), vascular endothelial nitric oxide synthase (eNOS), and neurotype one. Nitric oxide synthase (nNOS) is known.
これらＮＯＳのうちｅＮＯＳは、血管内皮細胞に局在しており細胞膜に結合している。このｅＮＯＳが活性化されると、一酸化窒素が継続的に産生され、その一酸化窒素は直ちに血管平滑筋に取り込まれ、グアニル酸シクラーゼを活性化する。これによりサイクリックグアノシン３’，５’−一リン酸（ｃＧＭＰ）が産生され、ｃＧＭＰ依存性タンパク質リン酸化酵素の活性化を介して血管平滑筋が弛緩されることにより、血管拡張作用を有し、血圧が低く保たれる。また、ｅＮＯＳにより産生された一酸化窒素は、血液中の血小板に作用することにより、血小板の凝集を抑制するとともに、凝固した血液の血管壁への接着を阻害する（例えば、非特許文献４参照）。 Of these NOSs, eNOS is localized in vascular endothelial cells and binds to the cell membrane. When this eNOS is activated, nitric oxide is continuously produced, and the nitric oxide is immediately taken up by vascular smooth muscle to activate guanylate cyclase. This produces cyclic guanosine 3', 5'-monophosphate (cGMP), which has a vasodilatory effect by relaxing vascular smooth muscle via activation of cGMP-dependent protein kinase. , Blood vessels are kept low. In addition, nitric oxide produced by eNOS acts on platelets in blood to suppress platelet aggregation and inhibit adhesion of coagulated blood to the blood vessel wall (see, for example, Non-Patent Document 4). ).
このような血管内皮型一酸化窒素合成酵素の産生を促進する天然物としては、ヒハツの抽出物などが提案されている（例えば、特許文献３参照）。しかしながら、その有効成分に関しては同定されておらず、更に高い活性を有する物質が望まれている。 As a natural product that promotes the production of such vascular endothelial nitric oxide synthase, an extract of long pepper and the like have been proposed (see, for example, Patent Document 3). However, the active ingredient has not been identified, and a substance having higher activity is desired.
したがって、優れたＴｉｅ２活性化作用、血管新生抑制作用、血管の成熟化作用、血管の正常化作用、血管の安定化作用、クローディン−５産生促進作用、リンパ管の安定化作用、ｅＮＯＳ産生促進作用、血管拡張作用、及び血小板凝集抑制作用を有する安全性の高い物質について、速やかな開発が強く求められているのが現状である。 Therefore, excellent Tie2 activation action, angiogenesis inhibitory action, blood vessel maturation action, blood vessel normalization action, blood vessel stabilizing action, claudin-5 production promoting action, lymphatic vessel stabilizing action, eNOS production promotion At present, there is a strong demand for rapid development of highly safe substances having an action, a vasodilatory action, and a platelet aggregation inhibitory action.
本発明は、前記従来における諸問題を解決し、以下の目的を達成することを課題とする。即ち、本発明は、優れたＴｉｅ２活性化作用を有し、安全性の高いＴｉｅ２活性化剤を提供することを目的とする。
また、本発明は、優れた血管新生抑制作用を有し、安全性の高い血管新生抑制剤を提供することを目的とする。
また、本発明は、優れた血管の成熟化作用、血管の正常化作用又は血管の安定化作用を有し、安全性の高い血管の成熟化剤、血管の正常化剤又は血管の安定化剤を提供することを目的とする。
また、本発明は、優れたクローディン−５産生促進作用を有し、安全性の高いクローディン−５産生促進剤を提供することを目的とする。
また、本発明は、優れたリンパ管の安定化作用を有し、安全性の高いリンパ管の安定化剤を提供することを目的とする。
また、本発明は、優れたｅＮＯＳ産生促進作用を有し、安全性の高いｅＮＯＳ産生促進剤を提供することを目的とする。
また、本発明は、優れた血管拡張作用を有し、安全性の高い血管拡張剤を提供することを目的とする。
また、本発明は、優れた血小板凝集抑制作用を有し、安全性の高い血小板凝集抑制剤を提供することを目的とする。
また、本発明は、優れたＴｉｅ２活性化作用、血管新生抑制作用、血管の成熟化作用、血管の正常化作用、血管の安定化作用、クローディン−５産生促進作用、リンパ管の安定化作用、ｅＮＯＳ産生促進作用、血管拡張作用、及び血小板凝集抑制作用の少なくともいずれかの作用を有し、安全性の高い医薬品組成物を提供することを目的とする。
An object of the present invention is to solve the above-mentioned conventional problems and to achieve the following objects. That is, an object of the present invention is to provide a highly safe Tie2 activator having an excellent Tie2 activating action.
Another object of the present invention is to provide a highly safe angiogenesis inhibitor having an excellent angiogenesis inhibitory effect.
Further, the present invention has an excellent blood vessel maturation action, blood vessel normalization action or blood vessel stabilizing action, and is a highly safe blood vessel maturation agent, blood vessel normalizing agent or blood vessel stabilizing agent. The purpose is to provide.
Another object of the present invention is to provide a highly safe claudin-5 production-promoting agent having an excellent claudin-5 production-promoting action.
Another object of the present invention is to provide a highly safe lymphatic vessel stabilizer having an excellent lymphatic vessel stabilizing effect.
Another object of the present invention is to provide a highly safe eNOS production-promoting agent having an excellent eNOS production-promoting action.
Another object of the present invention is to provide a highly safe vasodilator having an excellent vasodilatory effect.
Another object of the present invention is to provide a highly safe platelet aggregation inhibitor having an excellent platelet aggregation inhibitory effect.
Further, the present invention has an excellent Tie2 activating effect, angiogenesis inhibitory effect, blood vessel maturation effect, blood vessel normalizing effect, blood vessel stabilizing effect, claudin-5 production promoting effect, and lymphatic vessel stabilizing effect. , An object of the present invention to provide a highly safe pharmaceutical composition having at least one of an action of promoting eNOS production, an action of dilating blood vessels, and an action of suppressing platelet aggregation.
前記課題を解決するため本発明者が鋭意検討を重ねた結果、ピペリジンアルカロイドの一種であるピペリンが、優れたＴｉｅ２活性化作用、血管新生抑制作用、血管の成熟化作用、血管の正常化作用、血管の安定化作用、クローディン−５産生促進作用、リンパ管の安定化作用、ｅＮＯＳ産生促進作用、血管拡張作用、及び血小板凝集抑制作用を有することを知見し、本発明を完成したものである。 As a result of diligent studies by the present inventor in order to solve the above-mentioned problems, piperine, which is a kind of piperidine alkaloid, has an excellent Tie2 activation effect, angiogenesis inhibitory effect, blood vessel maturation effect, and blood vessel normalization effect. The present invention has been completed by discovering that it has a blood vessel stabilizing action, a claudin-5 production promoting action, a lymphatic vessel stabilizing action, an eNOS production promoting action, a vasodilatory action, and a platelet aggregation inhibitory action. ..
本発明は、本発明者による前記知見に基づくものであり、前記課題を解決するための手段としては、以下の通りである。即ち、
＜１＞ ピペリンを含有することを特徴とするＴｉｅ２活性化剤である。
＜２＞ ピペリンを含有することを特徴とする血管新生抑制剤である。
＜３＞ ピペリンを含有することを特徴とする血管の成熟化剤、血管の正常化剤又は血管の安定化剤である。
＜４＞ ピペリンを含有することを特徴とするクローディン−５産生促進剤である。
＜５＞ ピペリンを含有することを特徴とするリンパ管の安定化剤である。
＜６＞ ピペリンを含有することを特徴とする血管内皮型一酸化窒素合成酵素（ｅＮＯＳ）産生促進剤である。
＜７＞ ピペリンを含有することを特徴とする血管拡張剤である。
＜８＞ ピペリンを含有することを特徴とする血小板凝集抑制剤である。
＜９＞ 前記＜１＞に記載のＴｉｅ２活性化剤、前記＜２＞に記載の血管新生抑制剤、前記＜３＞に記載の血管の成熟化剤、血管の正常化剤又は血管の安定化剤、前記＜４＞に記載のクローディン−５産生促進剤、前記＜５＞に記載のリンパ管の安定化剤、前記＜６＞に記載の血管内皮型一酸化窒素合成酵素（ｅＮＯＳ）産生促進剤、前記＜７＞に記載の血管拡張剤、及び前記＜８＞に記載の血小板凝集抑制剤の少なくともいずれかを含有することを特徴とする医薬品組成物である。
The present invention is based on the above-mentioned knowledge by the present inventor, and the means for solving the above-mentioned problems are as follows. That is,
<1> A Tie2 activator characterized by containing piperine.
<2> An angiogenesis inhibitor characterized by containing piperine.
<3> A blood vessel maturating agent, a blood vessel normalizing agent, or a blood vessel stabilizing agent, which is characterized by containing piperine.
<4> A claudin-5 production promoter characterized by containing piperine.
<5> A lymphatic vessel stabilizer containing piperine.
<6> A vascular endothelial nitric oxide synthase (eNOS) production promoter characterized by containing piperine.
<7> A vasodilator characterized by containing piperine.
<8> A platelet aggregation inhibitor characterized by containing piperine.
<9> The Tie2 activator according to <1>, the angiogenesis inhibitor according to <2>, the blood vessel maturation agent, the blood vessel normalizing agent, or the blood vessel stabilization according to <3>. Agent, claudin-5 production promoter according to <4>, vascular stabilizer according to <5>, vascular endothelial type nitrogen monoxide synthase (eNOS) production according to <6>. A pharmaceutical composition comprising at least one of an accelerator, the vasodilator according to <7>, and the platelet aggregation inhibitor according to <8>.
本発明のＴｉｅ２活性化剤によると、従来における前記諸問題を解決し、優れたＴｉｅ２活性化作用を有し、安全性の高いＴｉｅ２活性化剤を提供することができる。
本発明の血管新生抑制剤によると、従来における前記諸問題を解決し、優れた血管新生抑制作用を有し、安全性の高い血管新生抑制剤を提供することができる。
本発明の血管の成熟化剤、血管の正常化剤又は血管の安定化剤によると、従来における前記諸問題を解決し、優れた血管の成熟化作用、血管の正常化作用又は血管の安定化作用を有し、安全性の高い血管の成熟化剤、血管の正常化剤又は血管の安定化剤を提供することができる。
本発明のクローディン−５産生促進剤によると、従来における前記諸問題を解決し、優れたクローディン−５産生促進作用を有し、安全性の高いクローディン−５産生促進剤を提供することができる。
本発明のリンパ管の安定化剤によると、従来における前記諸問題を解決し、優れたリンパ管の安定化作用を有し、安全性の高いリンパ管の安定化剤を提供することができる。
本発明のｅＮＯＳ産生促進剤によると、従来における前記諸問題を解決し、優れたｅＮＯＳ産生促進作用を有し、安全性の高いｅＮＯＳ産生促進剤を提供することができる。
本発明の血管拡張剤によると、従来における前記諸問題を解決し、優れた血管拡張作用を有し、安全性の高い血管拡張剤を提供することができる。
本発明の血小板凝集抑制剤によると、従来における前記諸問題を解決し、優れた血小板凝集抑制作用を有し、安全性の高い血小板凝集抑制剤を提供することができる。
本発明の医薬品組成物によると、従来における前記諸問題を解決し、優れたＴｉｅ２活性化作用、血管新生抑制作用、血管の成熟化作用、血管の正常化作用、血管の安定化作用、クローディン−５産生促進作用、リンパ管の安定化作用、ｅＮＯＳ産生促進作用、血管拡張作用、及び血小板凝集抑制作用の少なくともいずれかの作用を有し、安全性の高い医薬品組成物を提供することができる。
According to the Tie2 activator of the present invention, it is possible to solve the above-mentioned problems in the past and provide a Tie2 activator having an excellent Tie2 activating action and high safety.
According to the angiogenesis inhibitor of the present invention, it is possible to solve the above-mentioned problems in the past and provide an angiogenesis inhibitor having an excellent angiogenesis inhibitory action and high safety.
According to the blood vessel maturation agent, blood vessel normalizing agent, or blood vessel stabilizing agent of the present invention, the above-mentioned problems in the prior art can be solved, and excellent blood vessel maturation action, blood vessel normalization action, or blood vessel stabilization can be achieved. It is possible to provide a blood vessel maturating agent, a blood vessel normalizing agent, or a blood vessel stabilizing agent having an action and high safety.
According to the claudin-5 production promoter of the present invention, it is possible to solve the above-mentioned problems in the past and provide a highly safe claudin-5 production promoter having an excellent claudin-5 production promoting action. Can be done.
According to the lymphatic vessel stabilizer of the present invention, it is possible to solve the above-mentioned problems in the prior art, provide an excellent lymphatic vessel stabilizing action, and provide a highly safe lymphatic vessel stabilizer.
According to the eNOS production promoter of the present invention, it is possible to solve the above-mentioned problems in the past and provide a highly safe eNOS production promoter having an excellent eNOS production promoting action.
According to the vasodilator of the present invention, it is possible to solve the conventional problems and provide a highly safe vasodilator having an excellent vasodilatory action.
According to the platelet aggregation inhibitor of the present invention, it is possible to solve the conventional problems and provide a highly safe platelet aggregation inhibitor having an excellent platelet aggregation inhibitory action.
According to the pharmaceutical composition of the present invention, the above-mentioned problems in the prior art are solved, and excellent Tie2 activation action, angiogenesis inhibitory action, blood vessel maturation action, blood vessel normalization action, blood vessel stabilizing action, and platelets -5 It is possible to provide a highly safe pharmaceutical composition having at least one of an action of promoting production, a stabilizing action of lymphatic vessels, a promoting action of eNOS, a vasodilatory action, and an inhibitory action on platelet aggregation. ..
（Ｔｉｅ２活性化剤、血管新生抑制剤、血管の成熟化剤、血管の正常化剤、血管の安定化剤、クローディン−５産生促進剤、リンパ管の安定化剤、ｅＮＯＳ産生促進剤、血管拡張剤、及び血小板凝集抑制剤）
本発明のＴｉｅ２活性化剤、血管新生抑制剤、血管の成熟化剤、血管の正常化剤、血管の安定化剤、クローディン−５産生促進剤、リンパ管の安定化剤、ｅＮＯＳ産生促進剤、血管拡張剤、及び血小板凝集抑制剤は、ピペリンを含有し、更に必要に応じてその他の成分を含有してなる。
(Tie2 activator, angiogenesis inhibitor, blood vessel maturation agent, blood vessel normalizing agent, blood vessel stabilizer, claudin-5 production promoter, lymphatic vessel stabilizer, eNOS production promoter, blood vessel Dilator and platelet aggregation inhibitor)
Tie2 activator, angiogenesis inhibitor, blood vessel maturation agent, blood vessel normalizing agent, blood vessel stabilizer, claudin-5 production promoter, lymphatic vessel stabilizer, eNOS production promoter of the present invention. , Vasodilators, and platelet aggregation inhibitors contain piperin and, if necessary, other components.
前記Ｔｉｅ２活性化剤は、Ｔｉｅ２をリン酸化することで、その活性体（リン酸化Ｔｉｅ２）に変換するＴｉｅ２活性化作用を有する。前記Ｔｉｅ２の活性化により、細胞内チロシンキナーゼドメインの自己リン酸化が惹起され、血管内皮細胞と血管壁細胞との接着が誘導される。血管狭小化あるいは血管拡大化の抑制が原因となって生じる虚血性疾患では、Ｔｉｅ２の活性化により、血管腔が拡大化される。また、Ｔｉｅ２の活性化により、血管内皮細胞の細胞死を抑制することができる。 The Tie2 activator has a Tie2 activating action of phosphorylating Tie2 to convert it into an active substance (phosphorylated Tie2). The activation of Tie2 induces autophosphorylation of the intracellular tyrosine kinase domain and induces adhesion between vascular endothelial cells and vascular parietal cells. In ischemic diseases caused by suppression of vascular narrowing or vascular enlargement, activation of Tie2 enlarges the vascular lumen. In addition, activation of Tie2 can suppress cell death of vascular endothelial cells.
前記血管新生抑制剤は、既存の血管から形成される新たな血管のネットワークを抑制する血管新生抑制作用を有する。低酸素状態では、Ｔｉｅ２の活性化が一時的に抑制され、血管内皮細胞と血管壁細胞との接着が乖離し、接着が乖離された血管内皮細胞から新しい血管のネットワークが形成される。前記血管新生抑制剤は、このような血管壁細胞が内皮細胞に接着しないことによる無秩序な血管の増生を抑制することができる。 The angiogenesis inhibitor has an angiogenesis inhibitory action that suppresses a network of new blood vessels formed from existing blood vessels. In a low oxygen state, the activation of Tie2 is temporarily suppressed, the adhesion between the vascular endothelial cells and the vascular parietal cells is dissociated, and a new vascular network is formed from the vascular endothelial cells from which the adhesion is dissociated. The angiogenesis inhibitor can suppress the disordered growth of blood vessels due to the non-adhesion of such angiogenic parietal cells to the endothelial cells.
前記血管の成熟化剤は、血管内皮細胞と血管壁細胞との接着を誘導して、血管内環境因子（細胞及び液性因子）が容易に血管外に漏出しないように血管内皮細胞間の接着斑を形成する成熟化作用を有する。また、血管再生医療においては、Ｔｉｅ２の活性化により、血管における血管内皮細胞と血管壁細胞との接着を誘導して、血管を成熟化させることができる。 The vascular maturation agent induces adhesion between vascular endothelial cells and vascular wall cells, and adhesion between vascular endothelial cells so that intravascular environmental factors (cells and humoral factors) do not easily leak out of the blood vessel. It has a maturation effect that forms plaques. Further, in angiogenic medicine, activation of Tie2 can induce adhesion between vascular endothelial cells and vascular parietal cells in blood vessels to mature blood vessels.
前記血管の正常化剤は、血管内皮細胞同士の接着を高めて血管壁細胞の血管内皮細胞への裏打ちを促進することにより、血管透過性の破綻した血管や無秩序な血管の増生を招く異常な血管を、正常な状態にする正常化作用を有する。また、腫瘍、糖尿病性網膜症等で観察されるような血管壁細胞が血管内皮細胞に接着しないことによる無秩序な血管の増生を引き起こす疾患では、Ｔｉｅ２の活性化により、血管壁細胞を内皮細胞に接着させ、血管を正常化させることができる。 The vascular normalizing agent is an abnormal condition that increases the adhesion between vascular endothelial cells and promotes the lining of vascular wall cells to vascular endothelial cells, thereby leading to the proliferation of vascular permeability-impaired blood vessels and disordered blood vessels. It has a normalizing effect that restores blood vessels to a normal state. In addition, in diseases that cause disordered vascular growth due to the non-adhesion of vascular parietal cells to vascular endothelial cells, such as those observed in tumors and diabetic retinopathy, the activation of Tie2 turns the vascular parietal cells into endothelial cells. It can be adhered and the blood vessels can be normalized.
前記血管の安定化剤は、既存の血管に対する障害、血管内皮細胞同士の解離、及び血管内皮細胞と血管壁細胞との解離を抑制して、血管内皮細胞の細胞死を抑制する血管の安定化作用を有する。また、種々の細胞内外の血管構造を破綻させる環境因子に対しては、Ｔｉｅ２の活性化により、血管の不安定化を抑制し、血管を安定化させることができる。 The vascular stabilizer suppresses damage to existing blood vessels, dissociation between vascular endothelial cells, and dissociation between vascular endothelial cells and vascular parietal cells, and stabilizes blood vessels that suppress cell death of vascular endothelial cells. Has an effect. In addition, for environmental factors that disrupt various internal and external vascular structures, the activation of Tie2 can suppress the destabilization of blood vessels and stabilize the blood vessels.
前記クローディン−５産生促進剤は、血管やリンパ管を構成する内皮細胞においてクローディン−５の産生を促進する作用を有する。タイトジャンクション構成タンパク質の一つであるクローディン−５の産生促進により、内皮細胞同士の接着が安定化され、血管やリンパ管の構造を維持、安定化することができる。 The claudin-5 production promoter has an action of promoting the production of claudin-5 in the endothelial cells constituting blood vessels and lymphatic vessels. By promoting the production of claudin-5, which is one of the tight junction constituent proteins, the adhesion between endothelial cells is stabilized, and the structures of blood vessels and lymph vessels can be maintained and stabilized.
前記リンパ管の安定化剤は、リンパ管内皮細胞同士の接着を高めてリンパ管の構造を維持、安定化する作用を有する。前記クローディン−５産生の促進により、内皮細胞同士の接着が安定化され、リンパ管を安定化させることができる。また、リンパ管内皮細胞に発現するＴｉｅ２の活性化によっても、クローディン−５の産生を促進でき、リンパ管の不安定化を抑制し、リンパ管を安定化させることができる。 The lymphatic vessel stabilizer has an effect of enhancing adhesion between lymphatic vessel endothelial cells to maintain and stabilize the lymphatic vessel structure. By promoting the production of claudin-5, the adhesion between endothelial cells can be stabilized and the lymphatic vessels can be stabilized. In addition, activation of Tie2 expressed in lymphatic endothelial cells can also promote the production of claudin-5, suppress the destabilization of lymphatic vessels, and stabilize the lymphatic vessels.
前記ｅＮＯＳ産生促進剤は、血管内皮細胞に局在する血管内皮型一酸化窒素合成酵素（ｅＮＯＳ）の産生を促進する作用を有する。前記ｅＮＯＳ産生の促進により、血管内皮における一酸化炭素の産生が促進され、ｃＧＭＰ依存性タンパク質リン酸化酵素の活性化を介して血管平滑筋が弛緩されることにより、血管が拡張され、血圧が低く保たれる。また、ｅＮＯＳにより産生された一酸化窒素は、血液中の血小板に作用することにより、血小板の凝集を抑制するとともに、凝固した血液の血管壁への接着を阻害することができる。 The eNOS production promoter has an action of promoting the production of vascular endothelial nitric oxide synthase (eNOS) localized in vascular endothelial cells. The promotion of eNOS production promotes the production of carbon monoxide in the vascular endothelium, and the relaxation of vascular smooth muscle through the activation of cGMP-dependent protein kinase causes dilation of blood vessels and low blood pressure. Be kept. In addition, nitric oxide produced by eNOS can act on platelets in blood to suppress the aggregation of platelets and to inhibit the adhesion of the coagulated blood to the blood vessel wall.
前記血管拡張剤は、前記ｅＮＯＳにより産生された一酸化窒素によって血管平滑筋が弛緩されることにより、血管を拡張し、血圧を低く保つ作用を有する。 The vasodilator has an effect of dilating blood vessels and keeping blood pressure low by relaxing vascular smooth muscle by nitric oxide produced by the eNOS.
前記血小板凝集抑制剤は、前記ｅＮＯＳにより産生された一酸化窒素によって血小板内のｃＧＭＰレベルを上昇させることにより血小板凝集を抑制する作用を有する。 The platelet aggregation inhibitor has an effect of suppressing platelet aggregation by increasing the cGMP level in platelets by nitric oxide produced by the eNOS.
＜ピペリン＞
前記ピペリンは、ピペリジンアルカロイドの一種であり、下記構造式（１）で表される化合物である。
前記ピペリンとしては、ピペリンを含む植物から製造したものであってもよく、市販品であってもよい。前記市販品としては、例えば、販売元コードＮｏ．１６２−１７２４１（和光純薬工業株式会社）などが挙げられる。
The piperine is a kind of piperidine alkaloid and is a compound represented by the following structural formula (1).
The piperine may be produced from a plant containing piperine or may be a commercially available product. Examples of the commercially available product include the distributor code No. 162-17241 (Wako Pure Chemical Industries, Ltd.) and the like can be mentioned.
＜＜ピペリンの製造方法＞＞
前記ピペリンの製造方法としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、植物抽出物調製工程、植物抽出物溶出工程、及び溶出物精製工程を含み、更に必要に応じてその他の工程を含む。
<< Manufacturing method of piperine >>
The method for producing the piperine is not particularly limited and may be appropriately selected depending on the intended purpose. For example, it includes a plant extract preparation step, a plant extract elution step, and an eluate purification step, and further if necessary. And other steps are included.
−植物抽出物調製工程−
前記植物抽出物調製工程は、植物の抽出物を調製する工程である。
前記植物としては、前記ピペリンを抽出できる植物であれば、特に制限はなく、目的に応じて適宜選択することができ、例えば、コショウ（Ｐｉｐｅｒ ｎｉｇｒｕｍ）、ヒハツ（Ｐｉｐｅｒ ｌｏｎｇｕｍ又はＰｉｐｅｒ ｒｅｔｒｏｆｒｕｃｔｕｍ）、西アフリカ産のコショウ（Ｐｉｐｅｒ ｇｕｉｎｅｅｎｓｅ）などが挙げられる。これらの中でも、ヒハツが好ましい。
-Plant extract preparation process-
The plant extract preparation step is a step of preparing a plant extract.
The plant is not particularly limited as long as it is a plant capable of extracting the piperine, and can be appropriately selected depending on the intended purpose. Pepper (Piper guineaense) and the like. Among these, long pepper is preferable.
前記植物抽出物の抽出原料であるヒハツは、コショウ科コショウ属の蔓性の常緑木本であり、学名は、Ｐｉｐｅｒ ｌｏｎｇｕｍ又はＰｉｐｅｒ ｒｅｔｒｏｆｒｕｃｔｕｍである。前記ヒハツは、インドや東南アジアの地域から容易に入手可能である。前記ヒハツの果穂は、多肉質の円筒状であり、乾燥物は、香辛料として広く用いられている。 The long pepper, which is the raw material for extracting the plant extract, is a vine-like evergreen tree of the genus Pepper in the family Piperaceae, and its scientific name is Piper longum or Piper retrofructum. The long peppers are readily available from regions of India and Southeast Asia. The fruit ears of the long pepper are fleshy and cylindrical, and the dried product is widely used as a spice.
前記植物抽出物は、植物の抽出に一般に用いられる方法を利用することによって、容易に得ることができる。なお、前記植物抽出物には、前記植物の抽出液、該抽出液の希釈液若しくは濃縮液、該抽出液の乾燥物、又はこれらの粗精製物若しくは精製物のいずれもが含まれる。 The plant extract can be easily obtained by using a method generally used for extracting plants. The plant extract includes an extract of the plant, a diluted solution or a concentrated solution of the extract, a dried product of the extract, or a crude product or a purified product thereof.
前記植物の抽出部位としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、前記植物がヒハツの場合、ヒハツの果穂部、葉部、茎部、花部、根部などが挙げられる。これらの中でも、果穂部が好ましい。 The extraction site of the plant is not particularly limited and may be appropriately selected depending on the intended purpose. For example, when the plant is a long pepper, the ears, leaves, stems, flowers, roots and the like of the long pepper are used. Can be mentioned. Among these, the fruit spike is preferable.
前記植物の抽出部位の調製方法としては、各部位を乾燥させた後、そのまま又は粗砕機を用い粉砕して溶媒抽出に供することにより得ることができる。前記乾燥は、天日で行ってもよいし、通常使用されている乾燥機を用いて行ってもよい。 The method for preparing the extraction site of the plant can be obtained by drying each site and then crushing the site as it is or using a coarse crusher and subjecting it to solvent extraction. The drying may be performed in the sun or may be performed using a commonly used dryer.
前記植物を抽出する方法としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、室温又は還流加熱下で、任意の抽出装置を用いて抽出する方法などが挙げられ、具体例としては、抽出溶媒を満たした処理槽内に、抽出原料としてのヒハツを投入し、更に必要に応じて時々攪拌しながら、３０分間〜２時間静置して可溶性成分を溶出した後、ろ過して固形物を除去し、得られた抽出液から抽出溶媒を留去し、乾燥することにより抽出する方法などが挙げられる。 The method for extracting the plant is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a method of extracting using an arbitrary extraction device at room temperature or under reflux heating. As an example, Hihatsu as an extraction raw material is put into a treatment tank filled with an extraction solvent, and if necessary, it is allowed to stand for 30 minutes to 2 hours with occasional stirring to elute the soluble components, and then filtered. Then, the solid matter is removed, the extraction solvent is distilled off from the obtained extract, and the extract is dried to extract the extract.
前記植物の抽出に用いる溶媒としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、水、親水性有機溶媒、又はこれらの混合溶媒などが挙げられる The solvent used for extracting the plant is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include water, a hydrophilic organic solvent, and a mixed solvent thereof.
前記植物の抽出溶媒として使用し得る水としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、純水、水道水、井戸水、鉱泉水、鉱水、温泉水、湧水、淡水等の他、これらに各種処理を施したものが含まれる。水に施す処理としては、例えば、精製、加熱、殺菌、ろ過、イオン交換、浸透圧の調整、緩衝化等が含まれる。なお、前記抽出溶媒として使用し得る水には、精製水、熱水、イオン交換水、生理食塩水、リン酸緩衝液、リン酸緩衝生理食塩水等も含まれる。 The water that can be used as the extraction solvent for the plant is not particularly limited and may be appropriately selected depending on the intended purpose. For example, pure water, tap water, well water, mineral spring water, mineral water, hot spring water, spring water, etc. In addition to fresh water, etc., those that have undergone various treatments are included. Treatments applied to water include, for example, purification, heating, sterilization, filtration, ion exchange, osmotic pressure adjustment, buffering and the like. The water that can be used as the extraction solvent includes purified water, hot water, ion-exchanged water, physiological saline, phosphate buffer, phosphate buffered saline and the like.
前記植物の抽出溶媒として使用し得る親水性有機溶媒としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、メタノール、エタノール、プロピルアルコール、イソプロピルアルコール等の炭素数１〜５の低級アルコール；アセトン、メチルエチルケトン等の低級脂肪族ケトン；１，３−ブチレングリコール、プロピレングリコール、グリセリン等の炭素数２〜５の多価アルコールなどが挙げられ、これら親水性有機溶媒と水との混合溶媒なども用いることができる。なお、前記水と前記親水性有機溶媒との混合溶媒を使用する際には、低級アルコールの場合は水１０質量部に対して１質量部〜９０質量部、低級脂肪族ケトンの場合は水１０質量部に対して１質量部〜４０質量部を混合したものを使用することが好ましい。また、多価アルコールの場合は水１０質量部に対して１質量部〜９０質量部を混合したものを使用することが好ましい。 The hydrophilic organic solvent that can be used as the extraction solvent for the plant is not particularly limited and may be appropriately selected depending on the intended purpose. For example, methanol, ethanol, propyl alcohol, isopropyl alcohol and the like have 1 to 5 carbon atoms. Lower alcohols; lower aliphatic ketones such as acetone and methyl ethyl ketone; polyhydric alcohols having 2 to 5 carbon atoms such as 1,3-butylene glycol, propylene glycol and glycerin, and the like, and these hydrophilic organic solvents and water A mixed solvent or the like can also be used. When a mixed solvent of the water and the hydrophilic organic solvent is used, 1 part by mass to 90 parts by mass with respect to 10 parts by mass of water in the case of a lower alcohol, and 10 parts by mass of water in the case of a lower aliphatic ketone. It is preferable to use a mixture of 1 part by mass to 40 parts by mass with respect to parts by mass. In the case of polyhydric alcohol, it is preferable to use a mixture of 1 part by mass to 90 parts by mass with 10 parts by mass of water.
前記植物の抽出条件としては、特に制限はなく、目的に応じて適宜選択することができるが、抽出溶媒量としては、抽出原料の５倍量〜１５倍量（質量比）が好ましく、抽出溶媒として水を用いた場合には、５０℃〜９５℃で１時間〜４時間程度で抽出することが好ましく、抽出溶媒として水とエタノールとの混合溶媒を用いた場合には、４０℃〜９０℃で３０分間〜４時間程度で抽出することが好ましい。 The extraction conditions for the plant are not particularly limited and may be appropriately selected depending on the intended purpose. However, the amount of the extraction solvent is preferably 5 to 15 times the amount (mass ratio) of the extraction raw material, and the extraction solvent is preferable. When water is used, it is preferable to extract at 50 ° C. to 95 ° C. for about 1 hour to 4 hours, and when a mixed solvent of water and ethanol is used as the extraction solvent, 40 ° C. to 90 ° C. is used. It is preferable to extract in about 30 minutes to 4 hours.
前記植物抽出物の精製方法としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、液−液分配抽出、各種クロマトグラフィー、膜分離などの精製方法が挙げられる。 The method for purifying the plant extract is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include liquid-liquid partition extraction, various chromatographies, and membrane separation.
−植物抽出物溶出工程−
前記植物抽出物溶出工程は、前記植物抽出物調製工程により得られた前記植物抽出物からピペリンを含有する成分を溶出して、ピペリンを含有する溶出物を得る工程である。
-Plant extract elution process-
The plant extract elution step is a step of eluting a piperine-containing component from the plant extract obtained by the plant extract preparation step to obtain a piperine-containing eluate.
前記植物抽出物からピペリンを含有する成分を溶出する方法としては、特に制限はなく、目的に応じて適宜選択することができるが、前記ヒハツ抽出物を極性溶媒により溶出する方法が好ましく、具体的には、前記ヒハツ抽出物をＯＤＳカラムに付して、５０質量％エタノール、７０質量％エタノール、メタノール、及びアセトンにより順次溶出する方法がより好ましい。 The method for eluting the component containing piperin from the plant extract is not particularly limited and may be appropriately selected depending on the intended purpose. However, a method for eluting the Hihatsu extract with a polar solvent is preferable and specific. More preferably, the Hihatsu extract is subjected to an ODS column and eluted with 50% by mass ethanol, 70% by mass ethanol, methanol, and acetone in order.
−溶出物精製工程−
前記溶出物精製工程は、前記植物抽出物溶出工程により得られた溶出物からピペリンを精製する工程である。
-Eluent purification process-
The eluate purification step is a step of purifying piperine from the eluate obtained by the plant extract elution step.
前記溶出物からピペリンを精製する方法としては、特に制限はなく、目的に応じて適宜選択することができるが、前記極性溶媒により得られた溶出物を粗精製して分取ＨＰＬＣを用いてピペリンを分離精製する方法が好ましく、具体的には、得られた５０質量％エタノール溶出物、７０質量％メタノール溶出物、メタノール溶出物、及びアセトン溶出物のうち、メタノール溶出物をＳｉＯ２カラムにより粗精製し、前記粗精製することにより得られた粗精製物から分取ＨＰＬＣを用いてピペリンを単離して精製する方法が好ましい。 The method for purifying piperin from the eluate is not particularly limited and may be appropriately selected depending on the intended purpose. However, the eluate obtained from the polar solvent is roughly purified and piperin is purified by preparative HPLC. Is preferable, and specifically, among the obtained 50% by mass ethanol eluate, 70% by mass methanol eluent, methanol eluate, and acetone eluate, the methanol eluate is crudely prepared by a SiO 2 column. A method of isolating and purifying piperin by preparative HPLC from the crude product obtained by purification and the crude purification is preferable.
＜＜ピペリンが製造されたことを確認する方法＞＞
前記ピペリンが製造されたことを確認する方法としては、特に制限はなく、目的に応じて適宜選択することができるが、単離された目的物の１３ＣＮＭＲスペクトルを、超伝導多核種核磁気共鳴装置（ＪＥＯＬ ＪＮＭ ＡＬ−４００、日本電子株式会社製）を用いて解析することにより確認する方法が好ましい。
なお、１３ＣＮＭＲスペクトルは、以下に示す通りである。
１３Ｃ−ＮＭＲスペクトル（１００ＭＨｚ，ＣＤＣｌ３）δＣ：１６５．３（Ｃ−１），１４８．１（Ｃ−４’），１４８．０（Ｃ−３’），１４２．４（Ｃ−３），１３８．１（Ｃ−５），１３１．０（Ｃ−１’），１２５．３（Ｃ−４），１２２．４（Ｃ−６’），１２０．０（Ｃ−２），１０８．４（Ｃ−５’），１０５．７（Ｃ−２’），１０１．２（ＯＣＨ２Ｏ），４６．９（Ｃ−５’’），４３．１（Ｃ−１’’），２６．５（Ｃ−４’’），２５．３（Ｃ−２’’），２４．７（Ｃ−３’’）．
<< How to confirm that piperine was manufactured >>
The method for confirming that the piperin has been produced is not particularly limited and may be appropriately selected depending on the intended purpose. However, the 13 CNMR spectrum of the isolated object is obtained from the superconducting polynuclear species nuclear magnetic resonance. A method of confirmation by analysis using an apparatus (JEOL JNM AL-400, manufactured by JEOL Ltd.) is preferable.
The 13 CNMR spectrum is as shown below.
13 C-NMR spectrum (100 MHz, CDCl 3 ) δC: 165.3 (C-1), 148.1 (C-4'), 148.0 (C-3'), 142.4 (C-3) , 138.1 (C-5), 131.0 (C-1'), 125.3 (C-4), 122.4 (C-6'), 120.0 (C-2), 108. 4 (C-5'), 105.7 (C-2'), 101.2 (OCH2O), 46.9 (C-5''), 43.1 (C-1''), 26.5 (C-4''), 25.3 (C-2''), 24.7 (C-3'').
＜その他の成分＞
前記その他の成分としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、賦形剤、防湿剤、防腐剤、強化剤、増粘剤、乳化剤、酸化防止剤、甘味料、酸味料、調味料、着色料、香料等、美白剤、保湿剤、油性成分、紫外線吸収剤、界面活性剤、増粘剤、アルコール類、粉末成分、色剤、水性成分、水、皮膚栄養剤などが挙げられる。
また、前記その他の成分の具体例としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、ヒアルロン酸、ヒアルロン酸加水分解物、コラーゲン、コラーゲン加水分解物、アスコルビン酸、アスコルビン酸誘導体、アスコルビン酸配糖体、コエンザイムＱ１０、プロポリス、ローヤルゼリー、ローヤルゼリー蛋白分解物、フコイダン、アロエ粉末、アロエ抽出物、ブルーベリー粉末、ブルーベリー抽出物、イソフラボン、ノニ粉末、ノニ抽出物、ニンニク粉末、ニンニク抽出物、ウコン粉末、ウコン抽出物、キトサン、グルコサミン、クロレラ粉末、クロレラ抽出物、カルニチン、マカ粉末、マカ抽出物、カシス粉末、カシス抽出物、ハナビラタケ粉末、ハナビラタケ抽出物、その他の植物の粉末及び／又は抽出物などが挙げられる。
<Other ingredients>
The other components are not particularly limited and may be appropriately selected depending on the intended purpose. For example, excipients, moisture-proofing agents, preservatives, strengthening agents, thickeners, emulsifiers, antioxidants and sweeteners. , Acidifiers, Seasonings, Colorants, Fragrances, Whitening Agents, Moisturizers, Oily Ingredients, UV Absorbents, Surfactants, Thickeners, Alcohols, Powder Ingredients, Coloring Agents, Aqueous Ingredients, Water, Skin Nutrition Examples include agents.
The specific examples of the other components are not particularly limited and may be appropriately selected depending on the intended purpose. For example, hyaluronic acid, hyaluronic acid hydrolyzate, collagen, collagen hydrolyzate, ascorbic acid, ascorbin. Acid derivative, ascorbic acid glycoside, coenzyme Q10, propolis, royal jelly, royal jelly proteolytic product, fucoidan, aloe powder, aloe extract, blueberry powder, blueberry extract, isoflavone, noni powder, noni extract, garlic powder, garlic Extracts, Ukon powder, Ukon extract, Chitosan, Glucosamine, Chlorella powder, Chlorella extract, Carnitine, Maca powder, Maca extract, Cassis powder, Cassis extract, Hanabiratake powder, Hanabiratake extract, and other plant powders / Or an extract or the like.
＜用途＞
本発明のＴｉｅ２活性化剤、血管新生抑制剤、血管の成熟化剤、血管の正常化剤、血管の安定化剤、クローディン−５産生促進剤、リンパ管の安定化剤、ｅＮＯＳ産生促進剤、血管拡張剤、及び血小板凝集抑制剤は、優れたＴｉｅ２活性化作用、血管新生抑制作用、血管の成熟化作用、血管の正常化作用、血管の安定化作用、クローディン−５産生促進作用、リンパ管の安定化作用、ｅＮＯＳ産生促進作用、血管拡張作用、及び血小板凝集抑制作用を有するため、腫瘍、慢性関節リウマチ、糖尿病網膜症、高脂血症、高血圧等の血管病変を主体とした疾患、アトピー性皮膚炎、及び花粉症などのアレルギー性疾患に関する医薬品、並びにこれらの疾患に関する安全な予防薬として好適に用いることができる。
また、本発明のＴｉｅ２活性化剤、血管新生抑制剤、血管の成熟化剤、血管の正常化剤、血管の安定化剤、クローディン−５産生促進剤、リンパ管の安定化剤、ｅＮＯＳ産生促進剤、血管拡張剤、及び血小板凝集抑制剤は、消化管で消化されるようなものでないことが確認されているため、美容用飲食品、健康用飲食品等の飲食品として、幅広く用いることができる。
<Use>
Tie2 activator, angiogenesis inhibitor, blood vessel maturation agent, blood vessel normalizing agent, blood vessel stabilizing agent, claudin-5 production promoting agent, lymphatic vessel stabilizing agent, eNOS production promoting agent of the present invention. , Blood vessel dilator, and platelet aggregation inhibitor have excellent Tie2 activation action, angiogenesis inhibitory action, blood vessel maturation action, blood vessel normalization action, blood vessel stabilizing action, claudin-5 production promoting action, Diseases mainly caused by vascular lesions such as tumors, rheumatoid arthritis, diabetic retinopathy, hyperlipidemia, and hypertension because they have lymphatic vessel stabilizing action, eNOS production promoting action, vasodilatory action, and platelet aggregation inhibitory action. , Atopic dermatitis, and pharmaceuticals for allergic diseases such as pollinosis, and safe preventive agents for these diseases.
In addition, the Tie2 activator, angiogenesis inhibitor, blood vessel maturation agent, blood vessel normalizing agent, blood vessel stabilizer, claudin-5 production promoter, lymphatic vessel stabilizer, eNOS production of the present invention. Since it has been confirmed that accelerators, vasodilators, and platelet aggregation inhibitors are not digestible in the gastrointestinal tract, they should be widely used as foods and drinks such as beauty foods and drinks and health foods and drinks. Can be done.
本発明のＴｉｅ２活性化剤、血管新生抑制剤、血管の成熟化剤、血管の正常化剤、血管の安定化剤、クローディン−５産生促進剤、リンパ管の安定化剤、ｅＮＯＳ産生促進剤、血管拡張剤、及び血小板凝集抑制剤における前記ピペリンの含有量としては、特に制限はなく、目的に応じて適宜選択することができる。前記ピペリンそのものを本発明のＴｉｅ２活性化剤、血管新生抑制剤、血管の成熟化剤、血管の正常化剤、血管の安定化剤、クローディン−５産生促進剤、リンパ管の安定化剤、ｅＮＯＳ産生促進剤、血管拡張剤、及び血小板凝集抑制剤として用いてもよい。Ｔｉｅ２活性化剤、血管新生抑制剤、血管の成熟化剤、血管の正常化剤、血管の安定化剤、クローディン−５産生促進剤、リンパ管の安定化剤、ｅＮＯＳ産生促進剤、血管拡張剤、及び血小板凝集抑制剤１ｍＬあたりの前記ピペリンの含有量としては、０．０１μｇ以上が好ましく、０．１μｇ以上１００μｇ以下がより好ましく、１μｇ以上３０μｇ以下が更に好ましく、５μｇ以上１５μｇ以下が特に好ましい。 Tie2 activator, angiogenesis inhibitor, blood vessel maturation agent, blood vessel normalizing agent, blood vessel stabilizer, claudin-5 production promoter, lymphatic vessel stabilizer, eNOS production promoter of the present invention. The content of the piperin in the vasodilator and the platelet aggregation inhibitor is not particularly limited and may be appropriately selected depending on the intended purpose. The piperin itself is used as a Tie2 activator, an angiogenesis inhibitor, a blood vessel maturation agent, a blood vessel normalizing agent, a blood vessel stabilizer, a claudin-5 production promoter, a lymphatic vessel stabilizer of the present invention. It may be used as an eNOS production promoter, a vasodilator, and a platelet aggregation inhibitor. Tie2 activator, angiogenesis inhibitor, blood vessel maturation agent, blood vessel normalizing agent, blood vessel stabilizer, claudin-5 production promoter, lymphatic vessel stabilizer, eNOS production promoter, vasodilator The content of the piperin per 1 mL of the agent and the platelet aggregation inhibitor is preferably 0.01 μg or more, more preferably 0.1 μg or more and 100 μg or less, further preferably 1 μg or more and 30 μg or less, and particularly preferably 5 μg or more and 15 μg or less. ..
本発明のＴｉｅ２活性化剤、血管新生抑制剤、血管の成熟化剤、血管の正常化剤、血管の安定化剤、クローディン−５産生促進剤、リンパ管の安定化剤、ｅＮＯＳ産生促進剤、血管拡張剤、及び血小板凝集抑制剤の投与形態としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、経口、非経口、外用などが挙げられる。 Tie2 activator, angiogenesis inhibitor, blood vessel maturation agent, blood vessel normalizing agent, blood vessel stabilizer, claudin-5 production promoter, lymphatic vessel stabilizer, eNOS production promoter of the present invention. The administration form of the vasodilator and the platelet aggregation inhibitor is not particularly limited and may be appropriately selected depending on the intended purpose, and examples thereof include oral, parenteral and external use.
本発明のＴｉｅ２活性化剤、血管新生抑制剤、血管の成熟化剤、血管の正常化剤、血管の安定化剤、クローディン−５産生促進剤、リンパ管の安定化剤、ｅＮＯＳ産生促進剤、血管拡張剤、及び血小板凝集抑制剤の剤型としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、錠剤、粉剤、カプセル剤、顆粒剤、エキス剤、シロップ剤等の経口投与剤；注射剤、点滴剤、坐剤等の非経口投与剤；軟膏、クリーム、乳液、ローション、パック、浴用剤、頭髪化粧料等の外用剤などが挙げられる。 Tie2 activator, angiogenesis inhibitor, blood vessel maturation agent, blood vessel normalizing agent, blood vessel stabilizer, claudin-5 production promoter, lymphatic vessel stabilizer, eNOS production promoter of the present invention. The dosage form of the blood vessel dilator and the platelet aggregation inhibitor is not particularly limited and may be appropriately selected depending on the intended purpose. For example, tablets, powders, capsules, granules, extracts, syrups and the like. Oral administration agents; parenteral administration agents such as injections, instillations, suppositories; ointments, creams, emulsions, lotions, packs, bath preparations, external preparations such as hair cosmetics, and the like.
（医薬品組成物）
本発明の医薬品組成物は、上述した本発明のＴｉｅ２活性化剤、血管新生抑制剤、血管の成熟化剤、血管の正常化剤、血管の安定化剤、クローディン−５産生促進剤、リンパ管の安定化剤、ｅＮＯＳ産生促進剤、血管拡張剤、及び血小板凝集抑制剤の少なくともいずれかを含有し、更に必要に応じて医薬品に通常使用される添加剤を含有してもよい。
(Pharmaceutical composition)
The pharmaceutical composition of the present invention comprises the above-mentioned Tie2 activator, angiogenesis inhibitor, blood vessel maturation agent, blood vessel normalizing agent, blood vessel stabilizer, claudin-5 production promoter, and lymph of the present invention. It may contain at least one of a tube stabilizer, an eNOS production promoter, a vasodilator, and a platelet aggregation inhibitor, and if necessary, an additive usually used in pharmaceutical products.
本発明の医薬品組成物は、優れたＴｉｅ２活性化作用、血管新生抑制作用、血管の成熟化作用、血管の正常化作用、血管の安定化作用、クローディン−５産生促進作用、リンパ管の安定化作用、ｅＮＯＳ産生促進作用、血管拡張作用、及び血小板凝集抑制作用の少なくともいずれかの作用を有するため、腫瘍、慢性関節リウマチ、糖尿病網膜症、高脂血症、高血圧等の血管病変を主体とした疾患、アトピー性皮膚炎、及び花粉症などのアレルギー性疾患に関する医薬品、並びにこれらの疾患に関する安全な予防薬として好適に用いることができる。
また、本発明の医薬品組成物は、消化管で消化されるようなものでないことが確認されているため、美容用飲食品、健康用飲食品等の飲食品として、幅広く用いることができる。
The pharmaceutical composition of the present invention has excellent Tie2 activating action, angiogenesis inhibitory action, blood vessel maturation action, blood vessel normalizing action, blood vessel stabilizing action, claudin-5 production promoting action, and lymphatic vessel stabilizing action. It mainly has vascular lesions such as tumors, rheumatoid arthritis, diabetic retinopathy, hyperlipidemia, and hypertension because it has at least one of the effects of bacteriostatic, eNOS production promoting, vasodilatory, and platelet aggregation inhibitory effects. It can be suitably used as a drug for allergic diseases such as diabetic retinopathy, atopic dermatitis, and pollinosis, and as a safe preventive drug for these diseases.
Further, since it has been confirmed that the pharmaceutical composition of the present invention is not digestible in the digestive tract, it can be widely used as foods and drinks such as cosmetic foods and drinks and health foods and drinks.
本発明の医薬品組成物における前記ピペリンの含有量としては、特に制限はなく、目的に応じて適宜選択することができ、前記ピペリンそのものを本発明の医薬品組成物として用いてもよいが、医薬品組成物１ｍＬあたり、０．０１μｇ以上が好ましく、０．１μｇ以上１００μｇ以下がより好ましく、１μｇ以上３０μｇ以下が更に好ましく、５μｇ以上１５μｇ以下が特に好ましい。 The content of the piperine in the pharmaceutical composition of the present invention is not particularly limited and may be appropriately selected depending on the intended purpose. The piperine itself may be used as the pharmaceutical composition of the present invention, but the pharmaceutical composition. Per 1 mL of the product, 0.01 μg or more is preferable, 0.1 μg or more and 100 μg or less is more preferable, 1 μg or more and 30 μg or less is further preferable, and 5 μg or more and 15 μg or less is particularly preferable.
本発明の医薬品組成物の投与形態としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、経口、非経口、外用などが挙げられる。 The administration form of the pharmaceutical composition of the present invention is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include oral, parenteral and external use.
本発明の医薬品組成物の剤型としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、錠剤、粉剤、カプセル剤、顆粒剤、エキス剤、シロップ剤等の経口投与剤；注射剤、点滴剤、坐剤等の非経口投与剤；軟膏等の外用剤などが挙げられる。 The dosage form of the pharmaceutical composition of the present invention is not particularly limited and may be appropriately selected depending on the intended purpose. For example, oral administration agents such as tablets, powders, capsules, granules, extracts and syrups. Parenteral administrations such as injections, instillations and suppositories; external preparations such as ointments.
以下、本発明の実施例を説明するが、本発明は、これらの実施例に何ら限定されるものではない。 Hereinafter, examples of the present invention will be described, but the present invention is not limited to these examples.
［試験例１：Ｔｉｅ２活性化作用（イムノアッセイ）試験］
（実施例１：ピペリン）
コンフルエントまで培養した正常ヒト臍帯静脈内皮細胞（ＨＵＶＥＣ）を、９６ウェルプレートへ２．０×１０４細胞／０．１ｍＬ／ウェルとなるように播種し、低血清血管内皮細胞増殖用培地（倉敷紡績株式会社製、Ｈｕｍｅｄｉａ−ＥＧ２）を用いて一晩培養した。次に、一晩培養後の前記ＨＵＶＥＣを、細胞刺激（被験試料添加）の３時間前に０．１ｍＬの血管内皮細胞基礎培地（倉敷紡績株式会社製、Ｈｕｍｅｄｉａ−ＥＢ２）に置換し、再度培養を行った。その後、前記ウェル内に、被験試料として前記Ｈｕｍｅｄｉａ−ＥＢ２で表１に記載の濃度に調製したピペリン（和光純薬工業株式会社製）を０．１ｍＬ添加し、１０分間のインキュベーションを行った。インキュベーション後、イムノアッセイキット（Ｒ＆Ｄ Ｓｙｓｔｅｍｓ社製、Ｈｕｍａｎ Ｐｈｏｓｐｈｏ−Ｔｉｅ２（Ｙ９９２）Ｉｍｍｕｎｏａｓｓａｙ）を用いてプロトコールに従い、細胞内のリン酸化型Ｔｉｅ２量及び総Ｔｉｅ２量を測定し、総Ｔｉｅ２に対するリン酸化型Ｔｉｅ２の比率を計算した。
また、陰性コントロールとして用いたジメチルスルホキシド（ＤＭＳＯ）についても、同様に総Ｔｉｅ２に対するリン酸化型Ｔｉｅ２の比率を計算した。
Ｔｉｅ２活性化率は、下記式（１）を用いて計算した。そして、得られた活性化率をもとに、リン酸化作用を評価した。結果を表１に示す。
(Example 1: Piperine)
Normal human umbilical vein endothelial cells were cultured to confluency (HUVEC), were seeded such that 2.0 × 10 4 cells /0.1mL/ well into 96-well plates, low serum vascular endothelial cell growth medium (Kurabo Industries It was cultured overnight using Human-EG2) manufactured by Humedia-EG2). Next, the HUVEC after overnight culturing was replaced with 0.1 mL of vascular endothelial cell basal medium (Humedia-EB2 manufactured by Kurashiki Spinning Co., Ltd.) 3 hours before cell stimulation (addition of test sample), and cultured again. Was done. Then, in the well, 0.1 mL of piperine (manufactured by Wako Pure Chemical Industries, Ltd.) prepared in Table 1 with the Humania-EB2 as a test sample was added, and incubation was carried out for 10 minutes. After incubation, the intracellular phosphorylated Tie2 amount and total Tie2 amount were measured using an immunoassay kit (Human Phospho-Tie2 (Y992) Immunoassay manufactured by R & D Systems) according to the protocol, and the phosphorylated Tie2 amount was measured with respect to the total Tie2. The ratio was calculated.
Also, for dimethyl sulfoxide (DMSO) used as a negative control, the ratio of phosphorylated Tie2 to total Tie2 was calculated in the same manner.
The Tie2 activation rate was calculated using the following formula (1). Then, the phosphorylation action was evaluated based on the obtained activation rate. The results are shown in Table 1.
実施例１におけるＴｉｅ２活性化作用（リン酸化作用）の結果について説明する。
前記イムノアッセイキットによりＴｉｅ２活性化作用の評価を実施したところ、ピペリンにより、Ｔｉｅ２がリン酸化して活性化されることが認められた。なお、陰性コントロールであるＤＭＳＯを添加した系では、Ｔｉｅ２の顕著なリン酸化は認められなかった。なお、Ｔｉｅ２のリン酸化により、血管成熟化、血管正常化、及び血管安定化がもたらされ、血管新生が抑制されることが知られている。
以上より、ピペリンが、Ｔｉｅ２リン酸化効果を有することにより、血管成熟化、血管正常化、及び血管安定化がもたらされ、血管新生が抑制されることが示唆された。
The result of the Tie2 activation action (phosphorylation action) in Example 1 will be described.
When the evaluation of the Tie2 activating effect was carried out using the immunoassay kit, it was confirmed that Tie2 was phosphorylated and activated by piperine. No significant phosphorylation of Tie2 was observed in the system to which DMSO, which is a negative control, was added. It is known that phosphorylation of Tie2 brings about vascular maturation, vascular normalization, and vascular stabilization, and suppresses angiogenesis.
From the above, it was suggested that piperine has a Tie2 phosphorylation effect, which leads to vascular maturation, vascular normalization, and vascular stabilization, and suppresses angiogenesis.
［試験例２：クローディン−５産生促進作用試験］
（実施例２：ピペリン）
以下の手順により、リンパ管内皮細胞におけるクローディン−５産生促進作用を評価した。
正常ヒト皮膚微小リンパ管内皮細胞（ＨＭＶＥＣ−ｄＬｙ）を、微小血管内皮細胞増殖培地（ＥＧＭ）を用いて培養した後、トリプシン処理により細胞を回収した。回収した細胞をＥＧＭを用いて１．０×１０５細胞／ｍＬの濃度に希釈した後、６ウェルコラーゲンコートプレートに２．０ｍＬ播種し、一晩培養した。培養終了後培地を捨て、ＥＧＭで表２に記載の濃度に調製したピペリン（和光純薬工業株式会社製）を１．０ｍＬ添加し、１０分間培養した。培養終了後、１．０ｍＬのＰＢＳ（−）（１３７ｍｍｏｌ／Ｌ ＮａＣｌ，８．１ｍｍｏｌ／Ｌ Ｎａ２ＨＰＯ４，２．６８ｍｍｏｌ／Ｌ ＫＣｌ，１．４７ｍｍｏｌ／Ｌ ＫＨ２ＰＯ４，ｐＨ７．４）で洗浄後、２００μＬのＭ−ＰＥＲ（登録商標）Ｍａｍｍａｌｉａｎ Ｐｒｏｔｅｉｎ Ｅｘｔｒａｃｔｉｏｎ Ｒｅａｇｅｎｔ（サーモフィッシャーサインティフィック株式会社製）を加えタンパク抽出液を得、常法により電気泳動用の試料を調製した。
調製した試料のタンパク量をそろえて（４μｇ／レーン）、Ｌａｅｍｍｌｉ法に従いＳＤＳポリアクリル電気泳動を行い、抗クローディン−５抗体（Ａｓｓａｙ Ｂｉｏｔｅｃｈ社製）及びペルオキシダーゼ標識２次抗体（Ｊａｃｋｓｏｎ社製）を用いてウエスタンブロッティングを行い、ＥＣＬ Ｐｒｉｍｅ Ｗｅｓｔｅｒｎ Ｂｌｏｔｔｉｎｇ Ｄｅｔｅｃｔｉｏｎ Ｒｅａｇｅｎｔｓ（ＧＥ Ｈｅａｌｔｈｃａｒｅ社製）の発光により画像撮影装置ＣｈｅｍｉＤｏｃ ＸＲＳ Ｐｌｕｓ（Ｂｉｏ−Ｒａｄ Ｌａｂｏｒａｔｏｒｉｅｓ社製）を用いてクローディン−５の検出を行った。クローディン−５産生促進効果の評価は、検出したバンドをＩｍａｇｅ Ｌａｂ Ｓｏｆｔｗａｒｅ ｖｅｒｓｉｏｎ２．０（Ｂｉｏ−Ｒａｄ Ｌａｂｏｒａｔｏｒｉｅｓ社製）にて定量的に測定してクローディン−５のバンド強度を数値化し、クローディン−５産生促進率を算出した。
[Test Example 2: Claudin-5 production promoting action test]
(Example 2: Piperine)
The claudin-5 production-promoting effect on lymphatic endothelial cells was evaluated by the following procedure.
Normal human skin microlymphatic vessel endothelial cells (HMVEC-dLy) were cultured in microvascular endothelial cell growth medium (EGM), and then the cells were recovered by trypsin treatment. The collected cells were diluted with EGM to a concentration of 1.0 × 10 5 cells / mL, 2.0 mL was seeded on a 6-well collagen coated plate, and the cells were cultured overnight. After completion of the culture, the medium was discarded, 1.0 mL of piperine (manufactured by Wako Pure Chemical Industries, Ltd.) prepared by EGM to the concentration shown in Table 2 was added, and the cells were cultured for 10 minutes. After completion of the culture, use 1.0 mL of PBS (-) (137 mmol / L NaCl, 8.1 mmol / L Na 2 HPO 4 , 2.68 mmol / L KCl, 1.47 mmol / L KH 2 PO 4 , pH 7.4). After washing, 200 μL of M-PER (registered trademark) Mammalian Protein Execution Reagent (manufactured by Thermofisher Intific Co., Ltd.) was added to obtain a protein extract, and a sample for electrophoresis was prepared by a conventional method.
Align the amount of protein in the prepared sample (4 μg / lane), perform SDS polyacrylic electrophoresis according to the Laemmli method, and obtain an anti-claudin-5 antibody (manufactured by Assay Biotech) and a peroxidase-labeled secondary antibody (manufactured by Jackson). Western blotting was performed using ECL Prime Western Blotting Detection Reagents (manufactured by GE Healthcare), and an imaging apparatus ChemiDoc XRS Plus (manufactured by Bio-Rad Laboratories) was used to detect claudin-5. To evaluate the claudin-5 production promoting effect, the detected band was quantitatively measured with Image Lab Software version 2.0 (manufactured by Bio-Rad Laboratories) to quantify the band intensity of claudin-5, and the claudin-5 was evaluated. -5 Production promotion rate was calculated.
クローディン−５産生促進率の計算方法は、以下のとおりである。
クローディン−５産生促進率（％）＝（Ａ／Ｂ）×１００
Ａ：被験試料添加時のバンド強度
Ｂ：被験試料無添加時（対照）のバンド強度
The calculation method of the claudin-5 production promotion rate is as follows.
Claudin-5 production promotion rate (%) = (A / B) x 100
A: Band strength when test sample is added B: Band strength when no test sample is added (control)
（参考例１：ヒハツ抽出物）
実施例２において、ピペリンを、ヒハツ抽出物（Ｔｉｅ２ヒハツエキスパウダーＭＦ、丸善製薬株式会社製）に変更し、表２に記載の濃度を用いたこと以外は、実施例２と同様にして、クローディン−５産生促進作用を評価した。結果を表２に示す。
(Reference example 1: Long pepper extract)
In Example 2, the piperine was changed to a long pepper extract (Tie2 long pepper extract powder MF, manufactured by Maruzen Pharmaceuticals Co., Ltd.), and claudins were used in the same manner as in Example 2 except that the concentrations shown in Table 2 were used. -5 The production promoting effect was evaluated. The results are shown in Table 2.
以上より、ピペリンが、クローディン−５産生促進効果を有することが示唆された。また、ヒハツ抽出物にクローディン−５産生促進作用が認められ、ピペリンにも低濃度でクローディン−５産生促進作用が認められたことから、ピペリンが有効成分の一つであることが示唆された。 From the above, it was suggested that piperine has a claudin-5 production promoting effect. In addition, the long pepper extract was found to have a claudin-5 production promoting effect, and piperine was also found to have a claudin-5 production promoting effect at a low concentration, suggesting that piperine is one of the active ingredients. rice field.
［試験例３：ｅＮＯＳ産生促進作用試験］
（実施例３：ピペリン）
以下の手順により、ヒト皮膚微小血管内皮細胞における血管内皮型一酸化窒素合成酵素（ｅＮＯＳ）産生促進作用を評価した。
ヒト微小血管由来内皮細胞（ＨＭＶＥＣ、倉敷紡績株式会社製）は、専用培地ＨｕＭｅｄｉａ−ＭｖＧ（倉敷紡績株式会社製）を用いて、３７℃、５％ＣＯ２条件下で培養を行った。細胞はあらかじめコラーゲンコートした８０ｃｍ２フラスコで８０％コンフルエントになるまで培養した後、トリプシン処理により細胞を回収し、６ウェルプレートに３．０×１０５細胞／ウェルの密度で細胞を播種した。翌日、表３に記載の各濃度に調製した被験試料（ピペリン、和光純薬工業株式会社製）を含む培地に交換し、さらに培養を続けた。同時にサンプルを含まない培地で同様の培養を行い、これをコントロールとした。なお、被験試料は５０ｖ／ｖ％ＤＭＳＯに溶解後ろ過滅菌し、培地中におけるＤＭＳＯの最終濃度が０．５ｖ／ｖ％になるように調製した。
培養２４時間後に細胞を回収し、この細胞のタンパク量をそろえて（１０μｇ／レーン）、Ｌａｅｍｍｌｉ法に従いＳＤＳポリアクリル電気泳動を行い、抗ｅＮＯＳ抗血清（ＣＡＹＭＡＮ社製）及びアルカリホスファターゼ標識２次抗体（ＣＨＥＭＩＣＯＮ社製）を用いてウエスタンブロッティングを行い、ｅＮＯＳの検出を行った。ｅＮＯＳ産生促進効果の評価は、画像解析ソフト（Ｋｏｄａｋ（登録商標）１Ｄ Ｉｍａｇｅ Ａｎａｌｙｓｉｓ Ｓｏｆｔｗａｒｅ、Ｋｏｄａｋ社製）を用いてｅＮＯＳのバンド強度を数値化し、ｅＮＯＳ産生促進率を算出した。
[Test Example 3: eNOS production promoting action test]
(Example 3: Piperine)
The vascular endothelial nitric oxide synthase (eNOS) production promoting action in human skin microvascular endothelial cells was evaluated by the following procedure.
Human microvessel-derived endothelial cells (HMVEC, manufactured by Kurabo Industries, Ltd.) were cultured using a special medium HuMedia-MvG (manufactured by Kurabo Industries, Ltd.) under 37 ° C. and 5% CO 2 conditions. After the cells were cultured to 80% confluence in 80 cm 2 flasks previously coated with collagen, the cells were harvested by trypsinization, cells were seeded at a density of 3.0 × 10 5 cells / well in 6-well plates. The next day, the medium was replaced with a medium containing a test sample (piperine, manufactured by Wako Pure Chemical Industries, Ltd.) prepared at each concentration shown in Table 3, and further culturing was continued. At the same time, the same culture was performed in a medium containing no sample, and this was used as a control. The test sample was dissolved in 50 v / v% DMSO and then sterilized by filtration so that the final concentration of DMSO in the medium was 0.5 v / v%.
After 24 hours of culturing, the cells were collected, the protein content of the cells was adjusted (10 μg / lane), SDS polyacrylic electrophoresis was performed according to the Laemmli method, and anti-eNOS antiserum (manufactured by CAYMAN) and alkaline phosphatase-labeled secondary antibody were performed. Western blotting was performed using (manufactured by CHEMICON) to detect eNOS. For the evaluation of the eNOS production promoting effect, the band intensity of eNOS was quantified using image analysis software (Kodak (registered trademark) 1D Image Analysis Software, manufactured by Kodak), and the eNOS production promoting rate was calculated.
ｅＮＯＳ産生促進率の計算方法は、以下のとおりである。
ｅＮＯＳ産生促進率（％）＝（Ａ／Ｂ）×１００
Ａ：被験試料添加時のバンド強度
Ｂ：被験試料無添加時（対照）のバンド強度
The calculation method of the eNOS production promotion rate is as follows.
eNOS production promotion rate (%) = (A / B) x 100
A: Band strength when test sample is added B: Band strength when no test sample is added (control)
（参考例２：ヒハツ抽出物）
実施例３において、ピペリンを、ヒハツ抽出物（Ｔｉｅ２ヒハツエキスパウダーＭＦ、丸善製薬株式会社製）に変更し、表３に記載の濃度を用いたこと以外は、実施例３と同様にして、ｅＮＯＳ産生促進作用を評価した。結果を表３に示す。
(Reference example 2: Long pepper extract)
In Example 3, piperine was changed to a long pepper extract (Tie2 long pepper extract powder MF, manufactured by Maruzen Pharmaceuticals Co., Ltd.), and eNOS was produced in the same manner as in Example 3 except that the concentrations shown in Table 3 were used. The promoting effect was evaluated. The results are shown in Table 3.
以上より、ピペリンが、ｅＮＯＳ産生促進効果を有することが示唆された。また、ヒハツ抽出物にｅＮＯＳ産生促進作用が認められ、ピペリンにも低濃度でｅＮＯＳ産生促進作用が認められたことから、ピペリンが有効成分の一つであることが示唆された。ここで、ｅＮＯＳ産生促進により、血管内の血流が促進されることが知られている。
以上より、ヒハツ抽出物から単離されたピペリンが、ｅＮＯＳ産生促進効果を有することにより、血流促進効果がもたらされることが示唆された。
From the above, it was suggested that piperine has an eNOS production promoting effect. In addition, the long pepper extract was found to have an eNOS production-promoting effect, and piperine was also found to have an eNOS production-promoting effect at a low concentration, suggesting that piperine is one of the active ingredients. Here, it is known that the promotion of eNOS production promotes blood flow in blood vessels.
From the above, it was suggested that piperine isolated from the long pepper extract has a blood flow promoting effect by having an eNOS production promoting effect.
本発明のＴｉｅ２活性化剤、血管新生抑制剤、血管の成熟化剤、血管の正常化剤、血管の安定化剤、クローディン−５産生促進剤、リンパ管の安定化剤、ｅＮＯＳ産生促進剤、血管拡張剤、及び血小板凝集抑制剤、並びに医薬品組成物は、優れたＴｉｅ２活性化作用、血管新生抑制作用、血管の成熟化作用、血管の正常化作用、血管の安定化作用、クローディン−５産生促進作用、リンパ管の安定化作用、ｅＮＯＳ産生促進作用、血管拡張作用、及び血小板凝集抑制作用の少なくともいずれかの作用を有するため、腫瘍、慢性関節リウマチ、糖尿病網膜症、高脂血症、高血圧などの血管病変を主体とした疾患に関する医薬品及びこれらの疾患に関する安全な予防薬として、幅広く用いることができる。
また、本発明のＴｉｅ２活性化剤、血管新生抑制剤、血管の成熟化剤、血管の正常化剤、血管の安定化剤、クローディン−５産生促進剤、リンパ管の安定化剤、ｅＮＯＳ産生促進剤、血管拡張剤、及び血小板凝集抑制剤、並びに医薬品組成物は、消化管で消化されるようなものでないことが確認されているため、美容用飲食品、健康用飲食品等の飲食品として、幅広く用いることができる。
Tie2 activator, angiogenesis inhibitor, blood vessel maturation agent, blood vessel normalizing agent, blood vessel stabilizing agent, claudin-5 production promoting agent, lymphatic vessel stabilizing agent, eNOS production promoting agent of the present invention. , Vascular dilators, and platelet aggregation inhibitors, and pharmaceutical compositions have excellent Tie2 activating, angiogenesis-suppressing, vascular maturation, vascular normalization, vascular stabilizing, claudin- 5 Tumor, chronic rheumatoid arthritis, diabetic retinopathy, hyperlipidemia because it has at least one of production promoting action, lymphatic vessel stabilizing action, eNOS production promoting action, vascular dilation action, and platelet aggregation inhibitory action. It can be widely used as a drug for diseases mainly related to vascular lesions such as high blood pressure and as a safe preventive drug for these diseases.
In addition, the Tie2 activator, angiogenesis inhibitor, blood vessel maturation agent, blood vessel normalizing agent, blood vessel stabilizer, claudin-5 production promoter, lymphatic vessel stabilizer, eNOS production of the present invention. Since it has been confirmed that accelerators, vasodilators, platelet aggregation inhibitors, and pharmaceutical compositions are not digestible in the gastrointestinal tract, foods and drinks such as beauty foods and drinks and health foods and drinks Can be widely used.
Claims (2)
請求項１に記載の血管内皮型一酸化窒素合成酵素産生促進剤（ただし、ｅＮＯＳ活性が低下している個体に用いられるもの、血管拡張に用いられるもの、血圧低下に用いられるもの、及び血小板凝集抑制に用いられるものを除く）を含有することを特徴とする経口用組成物。 An oral composition used to promote the production of vascular endothelial nitric oxide synthase (excluding those used for vasodilation, blood pressure lowering, and platelet aggregation suppression). hand,
The vascular endothelial type nitric oxide synthase production promoter according to claim 1 (however, those used for individuals with decreased eNOS activity, those used for vasodilation, those used for lowering blood pressure, and platelet aggregation. An oral composition comprising (excluding those used for inhibition).
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