JP6736645B2 - Claudin-5 production promoter and oral composition for promoting claudin-5 production - Google Patents

Description

The present invention provides a Tie2 activator, angiogenesis inhibitor, blood vessel maturation agent, blood vessel normalization agent, blood vessel stabilizer, claudin-5 production promoter, lymphatic vessel stabilizer, eNOS production promotion. Agent, vasodilator, platelet aggregation inhibitor, and pharmaceutical composition.

Blood vessels have a structure in which vascular endothelial cells and vascular wall cells (vascular smooth muscle cells and pericytes) are indirectly or directly adhered via an extracellular matrix, and oxygen and nutrients It has a function of supplying waste to tissues and removing waste products from living tissues.

In general, the formation of blood vessels is angiogenesis, in which new blood vessels are formed (vasculogenesis) and existing blood vessels that have been formed extend and branch to form new blood vessel networks. It is divided into two stages. The former is involved in a very wide range of angiogenesis from the initial development of blood vessels called angiogenesis to subsequent angiogenesis, in which vascular endothelial growth factor (VEGF) acts, and the latter is angiopoietin. (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.

Under normal oxygen conditions, blood vessels have a strong adherence between vascular endothelial cells and the vascular wall cells that line the surrounding cells, maintaining a stable vascular structure. May detach from vascular endothelial cells, resulting in the growth of unregulated 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.

It is known that these angiogenesis is suppressed by activating receptor type 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 the suppression of blood vessel narrowing or blood vessel enlargement, activation of Tie2 enlarges the blood vessel lumen (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).

Thus, activation of Tie2 is known to not only suppress angiogenesis, but also to mature, normalize, and stabilize blood vessels.
For example, in revascularization medicine, it is known that activation of Tie2 induces adhesion between vascular endothelial cells and vascular wall cells in blood vessels to mature the blood vessels.
For example, in a disease such as tumor, diabetic retinopathy, etc. where vascular wall cells do not adhere to vascular endothelial cells and thus disordered blood vessels grow, activation of Tie2 causes vascular wall cells to adhere to 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 with respect to various environmental factors that disrupt internal and external vascular structures.

As a natural product that suppresses angiogenesis by activating Tie2, cinnamon bark extract and the like have been proposed (see, for example, Patent Document 1). However, there is a problem that these activities are insufficient. Further, suramin (a polysulfonated naphthylurea compound) is known as a substance that suppresses angiogenesis (see, for example, Patent Document 2), but it has a problem of poor safety.

One of tight junction constituent proteins called claudin-5 (CLD5) is present in endothelial cells that constitute blood vessels and lymph vessels, and is involved in tight junction between cells. Particularly, in lymphatic endothelial cells, it was reported that the expression of claudin-5 decreased with aging, suggesting a relationship between claudin-5 and stabilization of lymphatic vessels. 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 Ang1/Tie2 signal. , And contributed to the stabilization of lymphatic vessels (see, for example, Non-Patent Document 3).

However, there is no report on a substance that promotes the production of claudin-5, and identification of a substance having such activity is desired.

With respect to nitric oxide, attention has been paid to its physiological action or pharmacological action, and various studies have been conducted. In vivo, nitric oxide uses L-arginine and molecular oxygen as substrates and uses reduced nicotinamide adenine dinucleotide phosphate (NADPH), flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), tetrahydro. It is produced by nitric oxide synthase (NOS) using biopterin as a coenzyme. NOS has three isoforms that differ enzymatically and as protein molecules, and they are inducible nitric oxide synthase (iNOS), vascular endothelial nitric oxide synthase (eNOS), and neurotype one. Nitric oxide synthase (nNOS) is known.

Among these NOSs, eNOS is localized in vascular endothelial cells and bound to cell membranes. When this eNOS is activated, nitric oxide is continuously produced, and the nitric oxide is immediately taken up by vascular smooth muscle to activate guanylyl cyclase. As a result, cyclic guanosine 3′,5′-monophosphate (cGMP) is produced, and vascular smooth muscle is relaxed through activation of cGMP-dependent protein phosphatase. , Blood pressure is kept low. Further, 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 Non-Patent Document 4, for example). ).

As a natural product that promotes the production of such vascular endothelial nitric oxide synthase, an extract of Hihatu has been proposed (for example, refer to Patent Document 3). However, the active ingredient has not been identified, and a substance having higher activity is desired.

Therefore, excellent Tie2 activating action, angiogenesis suppressing action, blood vessel maturation action, blood vessel normalizing action, blood vessel stabilizing action, claudin-5 production promoting action, lymphatic vessel stabilizing action, eNOS production promoting action At present, there is a strong demand for rapid development of highly safe substances having action, vasodilatory action, and platelet aggregation inhibitory action.

JP, 2009-263358, A Japanese Patent Publication No. 9-503488 JP, 2005-298429, A

Science. 1999 Dec. 24;286(5449):2511-4. P. N. A. S. 2004 Apr. 13; 101(15):5553-8. J. Soc. Cosmet. Chem. Jpn. 46(3) p. 188-196 (2012) Masaki Nakane, "New developments in NO research," Experimental Medicine, 1999, Vol. 17, No. 8, p. 914-917

It is an object of the present invention to solve the above-mentioned conventional problems and achieve the following objects. That is, the object of the present invention is to provide a highly safe Tie2 activator having an excellent Tie2 activating effect.
Another object of the present invention is to provide a highly safe angiogenesis inhibitor having an excellent angiogenesis inhibitory action.
Further, the present invention has an excellent vascular maturation action, vascular normalization action or vascular stabilization action, and is a highly safe vascular maturation agent, vascular normalization agent or vascular stabilization agent. The purpose is to provide.
Another object of the present invention is to provide a highly safe claudin-5 production promoter having an excellent claudin-5 production promoter 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 an eNOS production promoter having an excellent eNOS production promoting action and high safety.
Another object of the present invention is to provide a highly safe vasodilator having an excellent vasodilator effect.
Another object of the present invention is to provide a highly safe platelet aggregation inhibitor having an excellent platelet aggregation inhibitory action.
Further, the present invention provides an excellent Tie2 activating action, angiogenesis suppressing action, vascular maturation action, vascular normalizing action, vascular stabilizing action, claudin-5 production promoting action, lymphatic vessel stabilizing action. , An eNOS production promoting action, a vasodilating action, and a platelet aggregation inhibiting action, and an object thereof is to provide a highly safe pharmaceutical composition.

As a result of extensive studies by the present inventors to solve the above problems, piperine, which is a kind of piperidine alkaloids, has excellent Tie2 activating action, angiogenesis suppressing action, vascular maturation action, vascular normalizing action, The present invention has been completed by discovering that it has a blood vessel stabilizing action, claudin-5 production promoting action, lymphatic vessel stabilizing action, eNOS production promoting action, vasodilating action, and platelet aggregation inhibiting action. ..

The present invention is based on the above findings by the present inventor, and means for solving the above problems are as follows. That is,
<1> A Tie2 activator containing piperine.
<2> An angiogenesis inhibitor containing piperine.
<3> A vascular maturation agent, a vascular normalization agent, or a vascular stabilization agent, characterized by containing piperine.
<4> A claudin-5 production promoter characterized by containing piperine.
<5> A stabilizer for lymphatic vessels, characterized by containing piperine.
<6> A vascular endothelial nitric oxide synthase (eNOS) production promoter characterized by containing piperine.
<7> A vasodilator containing piperine.
<8> A platelet aggregation inhibitor containing piperine.
<9> The Tie2 activator described in <1>, the angiogenesis inhibitor described in <2>, the vascular maturation agent described in <3>, the vascular normalization agent, or the vascular stabilization. Agent, claudin-5 production promoter described in <4>, stabilizer for lymphatic vessel described in <5>, vascular endothelial nitric oxide synthase (eNOS) production described in <6> A pharmaceutical composition comprising at least one of a promoter, the vasodilator described in <7>, and the platelet aggregation inhibitor described in <8>.

According to the Tie2 activator of the present invention, it is possible to provide the Tie2 activator which solves the above-mentioned problems in the related art, has an excellent Tie2 activating effect, and is highly safe.
According to the angiogenesis inhibitor of the present invention, it is possible to provide a highly safe angiogenesis inhibitor that solves the above-mentioned problems in the related art, has an excellent angiogenesis inhibitory action.
The vascular maturating agent, vascular normalizing agent or vascular stabilizing agent of the present invention solves the above-mentioned problems in the related art, and has an excellent vascular maturation effect, vascular normalizing effect or vascular stabilization effect It is possible to provide a vascular maturating agent, a vascular normalizing agent, or a vascular stabilizing agent that has an action and is highly safe.
According to the claudin-5 production promoter of the present invention, it is possible to provide a claudin-5 production promoter which solves the above-mentioned problems in the related art, has an excellent claudin-5 production promoting action, and is highly safe. You can
According to the lymphatic vessel stabilizer of the present invention, it is possible to provide a lymphatic vessel stabilizer that solves the above-mentioned problems in the related art, has an excellent lymphatic vessel stabilizing action, and is highly safe.
According to the eNOS production promoter of the present invention, it is possible to provide an eNOS production promoter that solves the above-mentioned problems in the related art, has an excellent eNOS production promoting action, and is highly safe.
According to the vasodilator of the present invention, it is possible to solve the above-mentioned problems in the related art, provide a vasodilator having an excellent vasodilator action and high safety.
According to the platelet aggregation inhibitor of the present invention, it is possible to solve the above-mentioned problems in the related art, to provide a platelet aggregation inhibitor having an excellent platelet aggregation inhibitory action and high safety.
The pharmaceutical composition of the present invention solves the above-mentioned problems in the related art, and has an excellent Tie2 activating action, angiogenesis suppressing action, vascular maturation action, vascular normalizing action, vascular stabilizing action, claudin -5 It has at least one of a production promoting action, a lymphatic vessel stabilizing action, an eNOS production promoting action, a vasodilatory action, and a platelet aggregation inhibiting action, and can provide a highly safe pharmaceutical composition. ..

(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 Expanding agent 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 The vasodilator, and the platelet aggregation inhibitor contain piperine and, if necessary, other components.

The Tie2 activator has a Tie2 activating effect of converting Tie2 into its active form (phosphorylated Tie2) by phosphorylating Tie2. The activation of Tie2 induces autophosphorylation of the intracellular tyrosine kinase domain and induces adhesion between vascular endothelial cells and vascular mural cells. In ischemic diseases caused by the suppression of blood vessel narrowing or blood vessel enlargement, activation of Tie2 enlarges the blood vessel lumen. In addition, activation of Tie2 can suppress cell death of vascular endothelial cells.

The angiogenesis inhibitor has an angiogenesis inhibitory action of inhibiting a network of new blood vessels formed from existing blood vessels. In hypoxia, the activation of Tie2 is temporarily suppressed, the adhesion between vascular endothelial cells and vascular wall cells is dissociated, and a new blood vessel network is formed from the vascular endothelial cells with dissociated adhesion. The angiogenesis inhibitor can suppress unregulated vascular hyperplasia due to such vascular wall cells not adhering to endothelial cells.

The vascular maturation agent induces adhesion between vascular endothelial cells and vascular mural cells, and adhesion between vascular endothelial cells prevents intravascular environmental factors (cells and humoral factors) from easily leaking out of the blood vessel. It has a maturation effect of forming plaques. Further, in revascularization medicine, activation of Tie2 can induce the adhesion between vascular endothelial cells and vascular wall cells in the blood vessel to mature the blood vessel.

The blood vessel normalizing agent enhances adhesion between vascular endothelial cells and promotes lining of vascular wall cells to vascular endothelial cells, thereby causing abnormal growth of broken blood vessels or unregulated blood vessels. It has a normalizing effect of making blood vessels normal. In addition, in a disease such as that observed in tumors and diabetic retinopathy that causes unregulated vascular hyperplasia due to non-adhesion of vascular wall cells to vascular endothelial cells, activation of Tie2 causes vascular wall cells to become endothelial cells. Can be adhered and normalize blood vessels.

The blood vessel stabilizer suppresses damage to existing blood vessels, dissociation of vascular endothelial cells from each other, and dissociation of vascular endothelial cells from vascular mural cells, and suppresses cell death of vascular endothelial cells. Have an effect. Further, with respect to various environmental factors that disrupt internal and external vascular structures, activation of Tie2 can suppress destabilization of blood vessels and stabilize the blood vessels.

The claudin-5 production promoter has an action of promoting claudin-5 production in endothelial cells that form blood vessels and lymph 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.

The lymphatic vessel stabilizer has an action of increasing adhesion between lymphatic endothelial cells to maintain and stabilize the structure of the lymphatic vessel. By promoting the production of claudin-5, adhesion between endothelial cells can be stabilized and lymph vessels can be stabilized. Also, activation of Tie2 expressed in lymphatic endothelial cells can promote the production of claudin-5, suppress the instability of lymphatic vessels, and stabilize the lymphatic vessels.

The eNOS production promoter has an action of promoting the production of vascular endothelial nitric oxide synthase (eNOS) localized in vascular endothelial cells. By promoting the production of eNOS, production of carbon monoxide in vascular endothelium is promoted, and vascular smooth muscle is relaxed through activation of cGMP-dependent protein kinase, thereby expanding blood vessels and lowering blood pressure. To be kept. Further, nitric oxide produced by eNOS acts on platelets in blood to suppress the aggregation of platelets and also inhibit the adhesion of coagulated blood to the blood vessel wall.

The vasodilator has the effect of dilating blood vessels and keeping blood pressure low by relaxing vascular smooth muscles with nitric oxide produced by the eNOS.

The platelet aggregation inhibitor has an action of inhibiting platelet aggregation by increasing the cGMP level in platelets by nitric oxide produced by the eNOS.

<Piperine>
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 a vendor code No. 162-17241 (Wako Pure Chemical Industries, Ltd.) and the like.

<<Production method of piperine>>
The method for producing piperine is not particularly limited and may be appropriately selected depending on the intended purpose, and includes, for example, a plant extract preparation step, a plant extract elution step, and an eluate purification step, and further if necessary. And other steps.

-Plant extract preparation step-
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 from which the piperine can be extracted, and can be appropriately selected depending on the purpose. Pepper (Piper guienense) and the like. Among these, hihatsu is preferable.

Hihatu, which is an extraction raw material of the plant extract, is a vine of evergreen wood of the genus Pepper of the family Pepperidaceae, and its scientific name is Piper longum or Piper retrofructum. The hihatsu is easily available from the regions of India and Southeast Asia. The fruit ears of the hihatsu are succulent cylinders, and dried products are widely used as spices.

The plant extract can be easily obtained by using a method generally used for plant extraction. The plant extract includes an extract of the plant, a diluent or concentrate of the extract, a dried product of the extract, or a crudely purified product or a purified product thereof.

The extraction site of the plant is not particularly limited and can be appropriately selected depending on the purpose.For example, when the plant is hihat, the ear part of the hihat, leaf part, stem part, flower part, root part, etc. Can be mentioned. Of these, the fruit ears are preferable.

As a method of preparing the extracted part of the plant, it can be obtained by drying each part and then subjecting it to solvent extraction either as it is or after crushing with a crusher. The drying may be performed in the sun or using a commonly used dryer.

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 any extraction device at room temperature or under reflux heating, and the like. As an example, the hihat as an extraction raw material is put into a treatment tank filled with an extraction solvent, and the mixture is allowed to stand for 30 minutes to 2 hours while eluting the soluble component while occasionally stirring, if necessary, 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 solvent used for extracting the plant is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include water, hydrophilic organic solvents, and mixed solvents thereof.

The water that can be used as the plant extraction solvent is not particularly limited and can be appropriately selected depending on the purpose, for example, pure water, tap water, well water, mineral water, mineral water, hot spring water, spring water, In addition to fresh water and the like, those subjected to various treatments are included. Examples of the treatment applied to water include purification, heating, sterilization, filtration, ion exchange, adjustment of osmotic pressure, buffering and the like. The water that can be used as the extraction solvent also includes purified water, hot water, ion-exchanged water, physiological saline, phosphate buffer, phosphate buffered saline and the like.

The hydrophilic organic solvent that can be used as the plant extraction solvent is appropriately selected depending on the intended purpose without any limitation, and examples thereof include methanol, ethanol, propyl alcohol, and isopropyl alcohol having 1 to 5 carbon atoms. Lower alcohols such as acetone and methyl ethyl ketone; and polyhydric alcohols having 2 to 5 carbon atoms such as 1,3-butylene glycol, propylene glycol and glycerin, and the like. 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 lower alcohol, and 10 parts by mass of water in the case of lower aliphatic ketone. It is preferable to use a mixture of 1 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.

The plant extraction conditions are not particularly limited and may be appropriately selected depending on the intended purpose, but the amount of extraction solvent is preferably 5 to 15 times the amount of the extraction raw material (mass ratio), and the extraction solvent When water is used as the solvent, it is preferable to perform extraction at 50°C to 95°C for about 1 to 4 hours, and when a mixed solvent of water and ethanol is used as the extraction solvent, 40°C to 90°C. It is preferable to perform the extraction in 30 minutes to 4 hours.

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 chromatography methods, and membrane separation purification methods.

-Plant extract elution step-
The plant extract elution step is a step of eluting a component containing piperine from the plant extract obtained in the plant extract preparation step to obtain an eluate containing piperine.

The method of eluting the component containing piperine from the plant extract is not particularly limited and may be appropriately selected depending on the purpose, but a method of eluting the hihatsu extract with a polar solvent is preferable, and More preferable is a method in which the hihatsu extract is applied to an ODS column and eluted sequentially with 50% by mass ethanol, 70% by mass ethanol, methanol, and acetone.

-Eluate purification step-
The eluate purification step is a step of purifying piperine from the eluate obtained in the plant extract elution step.

The method for purifying piperine from the eluate is not particularly limited and may be appropriately selected depending on the intended purpose, but the eluate obtained with the polar solvent is roughly purified and then piperine using preparative HPLC. Of the obtained 50% by mass ethanol eluate, 70% by mass methanol eluate, methanol eluate, and acetone eluate, the methanol eluate is roughly purified by a SiO ₂ column. A method in which piperine is purified and the piperine is isolated and purified from the crude product obtained by the crude purification is preferred.

<< How to confirm that piperine was manufactured >>
The method for confirming that the piperine has been produced is not particularly limited and may be appropriately selected depending on the intended purpose. However, the ¹³ C NMR spectrum of the isolated target product is determined by superconducting multinuclide nuclear magnetic resonance. A method of confirming by analysis using a device (JEOL JNM AL-400, manufactured by JEOL Ltd.) is preferable.
The ¹³ C NMR 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″).

<Other ingredients>
The other components are not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include excipients, moisture-proofing agents, preservatives, toughening agents, thickeners, emulsifiers, antioxidants and sweeteners. , Acidulants, seasonings, colorings, fragrances, whitening agents, moisturizers, oily ingredients, UV absorbers, surfactants, thickeners, alcohols, powder ingredients, coloring agents, aqueous ingredients, water, skin nutrition Agents and the like.
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, ascorbine. Acid derivative, ascorbic acid glycoside, coenzyme Q10, propolis, royal jelly, royal jelly proteolysis product, fucoidan, aloe powder, aloe extract, blueberry powder, blueberry extract, isoflavone, noni powder, noni extract, garlic powder, garlic Extract, turmeric powder, turmeric extract, chitosan, glucosamine, chlorella powder, chlorella extract, carnitine, maca powder, maca extract, cassis powder, cassis extract, Hanabitake mushroom powder, Hanabitake mushroom extract, other plant powder and And/or an extract and the like.

<Use>
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 have excellent Tie2 activating action, angiogenesis inhibiting action, vascular maturation action, vascular normalizing action, vascular stabilizing action, claudin-5 production promoting action, Since it has a stabilizing action on lymphatic vessels, an eNOS production promoting action, a vasodilatory action, and a platelet aggregation inhibiting action, diseases mainly caused by vascular lesions such as tumors, rheumatoid arthritis, diabetic retinopathy, hyperlipidemia, and hypertension. , Atopic dermatitis, hay fever and other allergic diseases, and safe preventive agents for these diseases can be preferably used.
In addition, the Tie2 activator, angiogenesis inhibitor, blood vessel maturation agent, blood vessel normalization agent, blood vessel stabilizer, claudin-5 production promoter, lymphatic vessel stabilizer, eNOS production of the present invention Promoters, vasodilators, and platelet aggregation inhibitors have been confirmed not to be digested in the digestive tract, so they should be widely used as food and drink such as beauty food and drink and health food and drink. You can

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 piperine in the vasodilator, and the platelet aggregation inhibitor is not particularly limited and may be appropriately selected depending on the purpose. The piperine itself is used as a Tie2 activator, an angiogenesis inhibitor, a blood vessel maturation agent, a blood vessel normalization agent, a blood vessel stabilizer, a claudin-5 production promoter, a lymph vessel stabilizer of the present invention, You may use as an eNOS production promoter, a vasodilator, and a platelet aggregation inhibitor. Tie2 activator, angiogenesis inhibitor, blood vessel maturation agent, blood vessel normalization agent, blood vessel stabilizer, claudin-5 production promoter, lymphatic vessel stabilizer, eNOS production promoter, vasodilation The content of the piperine 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 ..

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 purpose, and examples thereof include oral, parenteral, and external use.

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 vasodilator, and the platelet aggregation inhibitor is not particularly limited and may be appropriately selected according to the purpose. Examples thereof include tablets, powders, capsules, granules, extracts and syrups. Oral administration agents; parenteral administration agents such as injections, infusions, suppositories; ointments, creams, emulsions, lotions, packs, bath agents, external preparations such as hair cosmetics.

(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, lymph of the present invention described above. It contains at least one of a vascular stabilizer, an eNOS production promoter, a vasodilator, and a platelet aggregation inhibitor, and may further contain, if necessary, additives usually used in pharmaceuticals.

The pharmaceutical composition of the present invention has an excellent Tie2 activating action, angiogenesis suppressing action, vascular maturation action, vascular normalizing action, vascular stabilizing action, claudin-5 production promoting action, and lymphatic vessel stability. Since it has at least one of the following effects: oxidative action, eNOS production promoting action, vasodilating action, and platelet aggregation inhibiting action, it is mainly used for vascular lesions such as tumors, rheumatoid arthritis, diabetic retinopathy, hyperlipidemia, and hypertension It can be suitably used as a drug for allergic diseases such as the above-mentioned diseases, atopic dermatitis, and hay fever, and a safe preventive agent for these diseases.
Moreover, since it has been confirmed that the pharmaceutical composition of the present invention is not digested in the digestive tract, it can be widely used as food and drink such as beauty food and drink and health food and drink.

The content of the piperine in the pharmaceutical composition of the present invention is not particularly limited and can be appropriately selected depending on the purpose, and the piperine itself may be used as the pharmaceutical composition of the present invention. The amount is preferably 0.01 μg or more, more preferably 0.1 μg or more and 100 μg or less, still more preferably 1 μg or more and 30 μg or less, and particularly preferably 5 μg or more and 15 μg or less, per 1 mL of the product.

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. Examples thereof include oral administration, parenteral administration and external use.

The dosage form of the pharmaceutical composition of the present invention is not particularly limited and may be appropriately selected depending on the purpose. For example, oral administration agents such as tablets, powders, capsules, granules, extracts and syrups Parenteral administration agents such as injections, infusions and suppositories; external preparations such as ointments.

Examples of the present invention will be described below, but the present invention is not limited to these examples.

[Test Example 1: Tie2 activating action (immunoassay) test]
(Example 1: Piperine)
Normal human umbilical vein endothelial cells (HUVEC) cultured to confluence were seeded in a 96-well plate at 2.0×10 ⁴ cells/0.1 mL/well, and a low serum vascular endothelial cell growth medium (Kurashiki Spinning Co., Ltd. It was cultured overnight using Humedia-EG2) manufactured by Co., Ltd. Next, the HUVECs after overnight culture were replaced with 0.1 mL of a vascular endothelial cell basal medium (Kurashiki Spinning Co., Ltd., Humedia-EB2) 3 hours before cell stimulation (addition of test sample), and the cells were cultured again. I went. Then, 0.1 mL of piperine (manufactured by Wako Pure Chemical Industries, Ltd.) prepared as the test sample with Humedia-EB2 at a concentration shown in Table 1 was added to the well and incubated for 10 minutes. After the incubation, the amount of intracellular phosphorylated Tie2 and the amount of total Tie2 were measured according to the protocol using an immunoassay kit (manufactured by R&D Systems, Human Phospho-Tie2 (Y992) Immunoassay), and the amount of phosphorylated Tie2 with respect to the total Tie2 was measured. 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 effect was evaluated based on the obtained activation rate. The results are shown in Table 1.

The result of the Tie2 activating action (phosphorylation action) in Example 1 will be described.
When the Tie2 activating effect was evaluated by the immunoassay kit, it was found that Tie2 was phosphorylated and activated by piperine. In addition, in the system in which DMSO, which is a negative control, was added, no remarkable phosphorylation of Tie2 was observed. It is known that phosphorylation of Tie2 causes 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.

[Test Example 2: Claudin-5 production promoting action test]
(Example 2: Piperine)
The action of promoting claudin-5 production in lymphatic endothelial cells was evaluated by the following procedure.
Normal human skin microlymphatic endothelial cells (HMVEC-dLy) were cultured in microvascular endothelial cell growth medium (EGM), and then cells were collected by trypsin treatment. The collected cells were diluted with EGM to a concentration of 1.0×10 ⁵ cells/mL, and 2.0 mL was seeded on a 6-well collagen-coated plate and cultured overnight. After the culture was completed, the medium was discarded, 1.0 mL of piperine (manufactured by Wako Pure Chemical Industries, Ltd.) adjusted to the concentration shown in Table 2 with EGM was added, and the mixture was cultured for 10 minutes. After the completion of the culture, 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) was used. After washing, 200 μL of M-PER (registered trademark) Mammalin Protein Extraction Reagent (manufactured by Thermo Fisher Scientific) was added to obtain a protein extract, and a sample for electrophoresis was prepared by a conventional method.
The amount of protein in the prepared sample was adjusted (4 μg/lane), and SDS polyacryl electrophoresis was performed according to the Laemmli method to obtain anti-claudin-5 antibody (manufactured by Assay Biotech) and peroxidase-labeled secondary antibody (manufactured by Jackson). Western blotting was carried out using ECL Prime Western Blotting Detection Reagents (manufactured by GE Healthcare) and light emitted from an image capturing device ChemiDoc XRS Plus (manufactured by Bio-Rad Laboratories) was used to detect claudin-5. The evaluation of the claudin-5 production promoting effect was performed by quantitatively measuring the detected band with Image Lab Software version 2.0 (manufactured by Bio-Rad Laboratories) to quantify the band intensity of claudin-5, and using claudin. -5 The production promotion rate was calculated.

The method for calculating the claudin-5 production promoting rate is as follows.
Claudin-5 production promotion rate (%)=(A/B)×100
A: Band intensity when test sample was added B: Band intensity when test sample was not added (control)

(Reference example 1: Hihatu extract)
Claudin was prepared in the same manner as in Example 2 except that piperine was changed to a Hihatu extract (Tie2 Hihatu extract powder MF, manufactured by Maruzen Pharmaceutical Co., Ltd.) and the concentrations shown in Table 2 were used. -5 The production promoting action was evaluated. The results are shown in Table 2.

From the above, it was suggested that piperine has a claudin-5 production promoting effect. In addition, the clover-5 production-promoting action was observed in the hihatu extract, and the claudin-5 production-promoting action was also observed at low concentrations in piperine, suggesting that piperine is one of the active ingredients. It was

[Test Example 3: eNOS production promoting action test]
(Example 3: Piperine)
The action of promoting vascular endothelial nitric oxide synthase (eNOS) production in human skin microvascular endothelial cells was evaluated by the following procedure.
Human microvessel-derived endothelial cells (HMVEC, manufactured by Kurashiki Spinning Co., Ltd.) were cultured at 37° C. under 5% CO ₂ conditions using a dedicated medium HuMedia-MvG (manufactured by Kurashiki Spinning Co., Ltd.). The cells were cultured in an 80 cm ² flask previously coated with collagen until they became 80% confluent, then the cells were collected by trypsin treatment, and the cells were seeded on a 6-well plate at a density of 3.0×10 ⁵ cells/well. The next day, the medium was replaced with a medium containing a test sample (piperine, manufactured by Wako Pure Chemical Industries, Ltd.) prepared to each concentration shown in Table 3, and the culture was further continued. At the same time, the same culture was performed in a medium containing no sample, which was used as a control. The test sample was dissolved in 50 v/v% DMSO and sterilized by filtration to prepare a final concentration of DMSO in the medium of 0.5 v/v %.
After 24 hours of culturing, the cells were collected, the protein amount of the cells was adjusted (10 μg/lane), and SDS polyacryl electrophoresis was performed according to the Laemmli method to obtain anti-eNOS antiserum (manufactured by CAYMAN) and alkaline phosphatase-labeled secondary antibody. Western blotting was performed using (manufactured by CHEMICON) to detect eNOS. For 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.

The calculation method of the eNOS production promotion rate is as follows.
eNOS production promotion rate (%)=(A/B)×100
A: Band intensity when test sample was added B: Band intensity when test sample was not added (control)

(Reference Example 2: Hihatu extract)
Production of eNOS was performed in the same manner as in Example 3 except that piperine was changed to a Hihatu extract (Tie2 Hihatu extract powder MF, manufactured by Maruzen Pharmaceutical Co., Ltd.), and the concentrations shown in Table 3 were used. The promoting action was evaluated. The results are shown in Table 3.

From the above, it was suggested that piperine has an eNOS production promoting effect. Moreover, the eNOS production promoting action was observed in the hihatu extract, and the eNOS production promoting action was also observed in the piperine at a low concentration, suggesting that piperine is one of the active ingredients. Here, it is known that blood flow in blood vessels is promoted by promoting production of eNOS.
From the above, it was suggested that the piperine isolated from the Hihatu extract has a blood flow promoting effect by having an eNOS production promoting effect.

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 , A vasodilator, a platelet aggregation inhibitor, and a pharmaceutical composition have excellent Tie2 activating action, angiogenesis inhibiting action, vascular maturation action, vascular normalizing action, vascular stabilizing action, claudin- 5. Tumor, rheumatoid arthritis, diabetic retinopathy, hyperlipidemia, because it has at least one of a production promoting action, a lymphatic vessel stabilizing action, an eNOS production promoting action, a vasodilatory action, and a platelet aggregation inhibiting action. It can be widely used as a drug for diseases mainly caused by vascular lesions such as hypertension and a safe preventive drug for these diseases.
In addition, the Tie2 activator, angiogenesis inhibitor, blood vessel maturation agent, blood vessel normalization agent, blood vessel stabilizer, claudin-5 production promoter, lymphatic vessel stabilizer, eNOS production of the present invention Since the promoter, vasodilator, platelet aggregation inhibitor, and pharmaceutical composition have been confirmed not to be digested in the digestive tract, food and drink such as beauty food and drink and health food and drink. Can be widely used as

Claims (2)

Claudin-5 production promoter characterized by containing piperine as an active ingredient (provided that Tie2 is activated, a product containing hihatsu extract as an active ingredient, and for treating or preventing tumor or allergy) Excluding those used ). An oral composition for promoting claudin-5 production, comprising the claudin-5 production promoter according to claim 1.