JP6969041B2 - Vascular Endothelial Nitric Oxide Synthetic Enzyme Production Promoter and Oral Composition - Google Patents

Description

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.

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.

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.

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.

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).

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.

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.

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).

However, there have been no reports on substances that promote the production of claudin-5, and identification of substances having such activity is desired.

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.

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). ).

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.

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.

Japanese Unexamined Patent Publication No. 2009-263358 Special Table No. 9-503488 Gazette Japanese Unexamined Patent Publication No. 2005-298429

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

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.

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. ..

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>.

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 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

<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 the distributor code No. 162-17241 (Wako Pure Chemical Industries, Ltd.) and the like can be mentioned.

<< 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.

-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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

-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.

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.

-Eluent purification process-
The eluate purification step is a step of purifying piperine from the eluate obtained by the plant extract elution step.

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.

<< 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'').

<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.

<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.

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. ..

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.

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.

(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.

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.

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.

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.

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.

Hereinafter, examples of the present invention will be described, 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 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.

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.

[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.

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)

(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.

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.

[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.

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)

(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.

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.

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)

Vascular endothelial type nitric oxide synthase production promoter characterized by containing piperin (however, those used for individuals with decreased eNOS activity, those used for vasodilation, those used for lowering blood pressure, And those used to suppress platelet aggregation). 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).