JP2019023238A - Tie2 activator, agent for blood vessel maturation, blood vessel stabilizer, and oral composition for tie2 activation - Google Patents

Abstract

To provide a Tie2 activator, a neovascularization inhibitor, an agent for blood vessel maturation, an agent for blood vessel normalization, and a blood vessel stabilizer which have an excellent Tie2 activating action, neovascularization inhibitory action, blood vessel maturation action, blood vessel normalization action, and blood vessel stabilization action, and have high safety, and to provide a pharmaceutical composition.SOLUTION: The invention provides a Tie2 activator, a neovascularization inhibitor, an agent for blood vessel maturation, an agent for blood vessel normalization, and a blood vessel stabilizer which contain as an active ingredient at least one selected from Tilia miqueliana extract, and Aesculus hippocastanum extract, and a pharmaceutical composition containing at least any of them.SELECTED DRAWING: None

Description

  The present invention relates to a Tie2 activator, an angiogenesis inhibitor, a blood vessel maturation agent, a blood vessel normalizing agent, and a blood vessel stabilizer, and a pharmaceutical composition.

  A blood vessel has a structure in which vascular endothelial cells and vascular wall cells (vascular smooth muscle cells and perisite) adhere indirectly or directly via an extracellular matrix, and oxygen and nutrients are absorbed by living bodies. It has a function of supplying waste tissue and removing waste from living tissue.

  In general, the formation of blood vessels consists of vasculogenesis where new blood vessels are formed, and angiogenesis where new blood vessels are formed by the elongation and branching of existing blood vessels. And 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 the subsequent angiogenesis, and the latter is angiopoietin. (Ang) acts and participates 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 vessel endothelial cells and blood vessel wall cells that line the periphery of blood vessels are firmly adhered to each other, and the blood vessel structure is kept stable. Cells can detach from vascular endothelial cells and proliferate disordered blood vessels. Such a phenomenon (angiogenesis) 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 are suppressed by activating a receptor tyrosine kinase Tie2 (Tyrosine kinase with Ig and EGF homology domain 2) expressed in vascular endothelial cells (see Patent Document 1). In an ischemic disease caused by suppression of vascular narrowing or vasodilation, it has been reported that the vascular cavity is enlarged by activation of Tie2 (see Non-Patent Document 1). It has also been reported that the activation of Tie2 suppresses cell death of vascular endothelial cells (see Non-Patent Document 2).

Thus, it is known that the 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 the activation of Tie2 induces adhesion between vascular endothelial cells and vascular wall cells in a blood vessel to mature the blood vessel.
For example, in diseases where vascular wall cells observed in tumors, diabetic retinopathy, etc. do not adhere to vascular endothelial cells, disordered blood vessels grow. By activation of Tie2, vascular wall cells become endothelial cells. It is known to adhere and normalize blood vessels.

In addition, for example, it is known that environmental factors that break down various intracellular and external blood vessel structures suppress the destabilization of blood vessels and stabilize blood vessels by activating Tie2.
As a natural product that suppresses angiogenesis by such activation of Tie2, an extract of cinnamon has been proposed (see Patent Document 1). However, the Tie2 activity of this proposed cinnamon extract is not sufficient. Further, as a substance that suppresses angiogenesis, for example, suramin (polysulfonated naphthyl urea compound) is known (see Patent Document 2). However, this suramin is a synthetic product and has a problem of poor safety.

  Accordingly, there is a strong demand for rapid development of highly safe substances having excellent Tie2 activation action, angiogenesis inhibition action, blood vessel maturation action, blood vessel normalization action, and blood vessel stabilization action. is the current situation.

JP 2009-263358 A JP-T 9-503488

Thurston G. et al. , Science. , 1999 Dec 24; 286 (5449): 2511-4. Cho C.I. H. et al. , Proc. Natl. Acad. Sci. USA, 2004 Apr 13; 101 (15): 5553-8.

An object of the present invention is to solve the conventional problems and achieve the following objects. That is, an object of the present invention is to provide a Tie2 activator having an excellent Tie2 activation action and high safety.
An object of the present invention is to provide an angiogenesis inhibitor having an excellent angiogenesis inhibitory action and high safety.
The present invention has an excellent vascular maturation effect, vascular normalization effect, and vascular stabilization effect, and is a highly safe vascular maturation agent, vascular normalization agent, or vascular stabilization. The purpose is to provide an agent.
The present invention has an excellent Tie2 activation action, angiogenesis inhibition action, vascular maturation action, vascular normalization action, and vascular stabilization action, and a highly safe pharmaceutical product An object is to provide a composition.

  As a result of intensive studies by the present inventors in order to solve the above-mentioned problems, at least one selected from an extract of Keika, an acteoside, an extract of Bodaiju and an extract of Maronnier has an excellent Tie2 activation action, blood vessel It was found to have an anti-neoplastic effect, a vascular maturation effect, a vascular normalization effect, and a vascular stabilization effect.

The present invention is based on the above findings by the present inventors, and means for solving the above problems are as follows. That is,
<1> A Tie2 activator characterized by containing as an active ingredient at least one selected from an extract of Keika, an acteoside, an extract of Bodaiju and an extract of Maronnier.
<2> An angiogenesis inhibitor comprising as an active ingredient at least one selected from an extract of Keika, an acteoside, an extract of Bodaiju and an extract of Maronnier.
<3> A vascular maturation agent, a vascular normalization agent characterized by containing as an active ingredient at least one selected from an extract of Keika, Acteoside, an extract of Bodaiju and an extract of Maronnier, or It is a blood vessel stabilizer.
<4> The Tie2 activator according to <1>, the angiogenesis inhibitor according to <2>, and the vascular maturation agent, vascular normalization agent, or vascular A pharmaceutical composition comprising at least one selected from stabilizers.

According to the Tie2 activator of the present invention, the conventional problems can be solved, and an excellent Tie2 activator having an excellent Tie2 activation action and high safety can be provided.
According to the angiogenesis inhibitor of the present invention, the conventional problems can be solved, and an angiogenesis inhibitor having excellent angiogenesis inhibitory action and high safety can be provided.
According to the blood vessel maturation agent, blood vessel normalization agent, or blood vessel stabilization agent of the present invention, the conventional problems can be solved, and an excellent blood vessel maturation action, blood vessel normalization action, In addition, a vascular maturation agent, a vascular normalization agent, or a vascular stabilization agent having a vascular stabilization action and high safety can be provided.
According to the pharmaceutical composition of the present invention, the conventional problems can be solved, and an excellent Tie2 activation action, angiogenesis inhibition action, blood vessel maturation action, blood vessel normalization action, and blood vessel stabilization A pharmaceutical composition having at least one of the actions and having high safety can be provided.

(Tie2 activator, angiogenesis inhibitor, vascular maturation agent, vascular normalization agent, and vascular stabilization agent)
The Tie2 activator, vascular maturation agent, vascular normalization agent, vascular stabilization agent, and angiogenesis inhibitor of the present invention are extracted from Keika extract, Acteoside, Bodaige extract and Maronnier extract. It contains at least one selected as an active ingredient, and further contains other components as necessary.

  The Tie2 activator has a Tie2 activating action of phosphorylating Tie2 to convert it into its active form (phosphorylated Tie2). That is, the Tie2 activator promotes the formation of a Tie2 homodimer, and Tie2 autophosphorylation is induced by the intracellular tyrosine kinase activity of Tie2. Autophosphorylation of Tie2 activates the intracellular signal transduction system through recruitment of adapter protein GRB2 and GRS2-binding guanine nucleotide exchange factor SOS to the vicinity of the cell membrane, and finally vascular endothelial cells And the adhesion between the vascular wall cells are induced. In an ischemic disease caused by suppression of vascular narrowing or vasodilation, the vascular cavity is expanded by activation of Tie2. In addition, activation of Tie2 suppresses 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 the hypoxic state, the activation of Tie2 is temporarily suppressed, the adhesion between the vascular endothelial cell and the vascular wall cell is dissociated, and a new blood vessel network is formed from the vascular endothelial cell from which the adhesion is dissociated. An angiogenesis inhibitor can suppress the disordered growth of blood vessels caused by the adhesion of such vascular wall cells to endothelial cells.

  The vascular maturation agent induces adhesion between vascular endothelial cells and vascular wall cells, and between vascular endothelial cells such that intravascular environmental factors (cells and humoral factors) do not easily leak out of the blood vessel. It has a maturation effect to form adhesion spots. In vascular regenerative medicine, activation of Tie2 can induce adhesion between vascular endothelial cells and vascular wall cells in the blood vessel, thereby allowing the blood vessel to mature.

  The blood vessel normalizing agent increases the adhesion between vascular endothelial cells and promotes the lining of the vascular wall cells to the vascular endothelial cells, thereby leading to disordered growth of blood vessels and blood vessels in which vascular permeability is broken. It has a normalizing action to make abnormal blood vessels normal. Also, in diseases where vascular wall cells observed in tumors, diabetic retinopathy, etc. do not adhere to vascular endothelial cells and proliferate disordered blood vessels, Tie2 activation activates vascular wall cells to become endothelial cells. It is possible to adhere and normalize the blood vessels.

  The above-mentioned blood vessel stabilizer has an action to suppress damage to existing blood vessels, dissociation between vascular endothelial cells, and dissociation between vascular endothelial cells and vascular wall cells, and stabilizing effect to suppress cell death of vascular endothelial cells. Have. In addition, with respect to environmental factors that break down various vascular structures inside and outside cells, activation of Tie2 can suppress vascular instability and stabilize blood vessels.

<Keika extract>
The Keika (Keika) is an evergreen small tree belonging to the genus Moxae, and the scientific name is Osmanthus fragrance var. aurantiacus, Japanese name: Kinmokusei, which originates in southern China and can be easily obtained from such areas.
The extraction part of the silica is not particularly limited and can be appropriately selected according to the purpose.For example, the above-ground part such as a leaf part, a branch part, a bark part, a stem part, a stem part, a fruit part, a flower part, The root part or a mixture of these parts can be mentioned, but the flower part is preferred.
Here, the "flower" generally refers to the whole organ involved in sexual reproduction of a seed plant, and is composed of a flower leaf that is a leaf deformation and a flower axis that is a stem deformation. This includes organs such as pupae, petals, stamens, and heart skin. The “floral part” used as an extraction raw material in the present invention includes all of the organs involved in sexual reproduction of seed plants, as well as parts thereof, such as flower leaves, flower coats (buds and corolla), corolla, petals, etc. Is also included.
As a method for preparing the extraction part of the silica, it can be obtained as it is or by pulverizing it using a crusher and subjecting it to solvent extraction.

<Acteoside>
The acteoside is a polyphenol compound having a chemical structure represented by the following structural formula (I).

  The acteoside can be produced by isolation and purification from a plant extract containing acteoside. Such a plant extract containing acteoside can be obtained by a method generally used for plant extraction. Examples of plants containing acteoside include Keika. About the said squid, it is as having mentioned above.

<Bodaige extract>
The body is a deciduous tree belonging to the family Lindenaceae, and its scientific name is Tilia cordata , also called linden. The body is widely distributed in Europe and can be easily obtained from these regions.

The extraction part of the body is not particularly limited and may be appropriately selected according to the purpose. For example, the above-ground part such as a leaf part, a branch part, a stem part, a flower part, a bud part, a fruit part, a fruit skin part, etc. Or a mixture thereof may be mentioned, but among these, the flower portion is preferred.
As a method for preparing the extraction site of the body, it can be obtained as it is or by pulverizing it using a crusher and subjecting it to solvent extraction.

<Marronnier extract>
The said horse chestnut tree is a deciduous broad-leaved tree belonging to the genus Tochinokiaceae , and its scientific name is Aesculus hippocastanum , which is also referred to as “Amanita cypress”. The Maronnier is cultivated in various parts of the world and is easily available from these areas.

The extraction site of the Maronier is not particularly limited and may be appropriately selected depending on the purpose.For example, a leaf part, a branch part, a trunk part, a bark part, a flower part, a seed part, a root part, and the like may be mentioned. Among these, a seed part is preferable.
As a method for preparing the extraction part of the Maronnier, it can be obtained by subjecting it to solvent extraction as it is or after pulverization using a crusher.

<Extraction method>
The Keika extract, the Bodaige extract, and the Maronier extract can be easily obtained by using a method generally used for plant extraction. Moreover, you may use a commercial item as said Keika extract, said Bodaige extract, and said Maronier extract. In addition, the extract of the Keika, the extract of the Bodaiju, and the extract of the Maronier include the extract of the Keika, the Bodaiju, and the Maronier, a diluted solution or a concentrated solution of the extract, A dry product, or any of these crudely purified product or purified product is included.

There are no particular limitations on the extraction solvent for the extract of the cayca, the extract of the bodaige, and the extract of the maronier, and can be appropriately selected according to the purpose. For example, water, a hydrophilic solvent, or these The mixed solvent is mentioned.
The water is not particularly limited and may be appropriately selected according to the purpose.For example, pure water, tap water, well water, mineral spring water, mineral water, hot spring water, spring water, fresh water, purified water, hot water, Examples include ion-exchanged water, physiological saline, phosphate buffer, and phosphate buffered saline.

There is no restriction | limiting in particular as said hydrophilic solvent, According to the objective, it can select suitably, For example, C1-C5 lower alcohols, such as methanol, ethanol, propyl alcohol, isopropyl alcohol; Acetone, methyl ethyl ketone, etc. Lower aliphatic ketones; C2-C5 polyhydric alcohols such as 1,3-butylene glycol, propylene glycol, and glycerin.
There is no restriction | limiting in particular as a mixed solvent of the said water and the said hydrophilic solvent, Although it can select suitably according to the objective, When using a lower alcohol, it is 1 mass part-with respect to 10 mass parts of water. When 90 parts by weight, lower aliphatic ketone is used, 1 part by weight to 40 parts by weight with respect to 10 parts by weight of water, and when polyhydric alcohol is used, 1 part by weight to 90 parts by weight with respect to 10 parts by weight of water. It is preferable to add part by mass. The extraction solvent is preferably used at a temperature between room temperature and the boiling point of the solvent.
Among these, water and water-containing ethanol are preferable as the above.

As an extraction method of the extract of the silica, the extract of the bodaiju, and the extract of the maronier, the fat-soluble component contained in the raw material for the extraction of the silica, the bodaiju, and the maroonier can be eluted in the solvent. If possible, it is not particularly limited and can be carried out according to a conventional method. In the extraction process, it is not necessary to employ a special extraction method, and any apparatus can be used at room temperature or under reflux heating.
Specifically, as an extraction method of the extract of the silica, the extract of the bodaiju, and the extract of the maronier, for example, in the treatment tank filled with the solvent such as an ethanol aqueous solution, the silica, the bodaige, Then, the raw material for extraction of the above-mentioned Maronier is added, heated and extracted with a reflux extractor at 80 ° C. for 2 hours while being appropriately stirred, and filtered while hot to elute fat-soluble components, and then using an evaporator. The method of concentrating under reduced pressure and performing the same filtration process further is mentioned.
At this time, the extraction conditions can be appropriately adjusted according to the extraction raw material and the like, but the amount of the extraction solvent is preferably 5 to 20 times (mass ratio) with respect to the extraction raw material, and the extraction time is 1 Time to 3 hours are preferable, and the extraction temperature is preferably 20 ° C to 95 ° C.

The obtained extract of the squid, the extract of the bodaiju, and the extract of the maronier are diluted with the extract of the squid, the extract of the bodaige, and the extract of the melonier, a concentrate, In order to obtain a dried product, a roughly purified product, a purified product, etc., treatments such as dilution, concentration, drying, and purification may be performed according to a conventional method.
Further, the obtained extract of Keika, the extract of Bodaiju, and the extract of Maronnier are the Tie2 activator, the angiogenesis inhibitor, the vascular maturation agent, and the normal blood vessel as they are. The concentrated solution and the dried product are preferable because they can be used as any of the agent and the blood vessel stabilizer. In obtaining the dried product, a carrier such as dextrin or cyclodextrin may be added to improve hygroscopicity.

<Other ingredients>
The other components are not particularly limited as long as they do not impair the effects of the present invention, and can be appropriately selected according to the purpose. For example, excipients, binders, disintegrants, lubricants Agents, stabilizers, flavoring agents, flavoring agents, and the like.

  Examples of the excipient include lactose, sucrose, sodium chloride, glucose, starch, calcium carbonate, kaolin, microcrystalline cellulose, and silicic acid. Examples of the binder include water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropyl starch, methylcellulose, ethylcellulose, shellac, calcium phosphate, polyvinylpyrrolidone, and the like. Can be mentioned. Examples of the disintegrant include dry starch, sodium alginate, agar powder, sodium bicarbonate, calcium carbonate, sodium lauryl sulfate, stearic acid monoglyceride, and lactose. Examples of the lubricant include purified talc, stearate, borax, and polyethylene glycol. Examples of the stabilizer include sodium pyrosulfite, EDTA, thioglycolic acid, thiolactic acid, and the like. Examples of the flavoring agent or flavoring agent include sucrose, orange peel, citric acid, and tartaric acid.

<Method for producing acteoside>
The acteoside can be produced by isolation and purification from a plant extract containing acteoside (for example, an extract of Keika). The plant extract containing such acteoside is as described above.
The method for isolating and purifying acteoside from the obtained extract, the concentrate of the extract or the dried product of the extract is not particularly limited and can be appropriately selected according to the purpose. For example, plant extraction The product is subjected to column chromatography using a porous material such as silica gel or alumina, a porous resin such as styrene-divinylbenzene copolymer or polymethacrylate, and eluted in the order of water and alcohol. Acteoside can be obtained as a fraction eluted with alcohol.
The alcohol used as the eluent in the column chromatography is not particularly limited, and examples thereof include lower aliphatic alcohols having 1 to 5 carbon atoms such as methanol, ethanol, propyl alcohol, isopropyl alcohol, and their aqueous solutions. Can be mentioned. Further, the alcohol fraction obtained by column chromatography can be purified by any organic compound such as reverse phase silica gel chromatography using ODS, recrystallization, liquid-liquid countercurrent extraction, column chromatography using ion exchange resin, etc. It may be purified using means.

  The Keika extract, Acteoside, Bodaiju extract, and Maronnier extract obtained as described above have Tie2 activation action, angiogenesis inhibition action, blood vessel maturation action, and blood vessel normalization. And / or a blood vessel stabilizing action, and based on these actions, the Tie2 activator, angiogenesis inhibitor, blood vessel maturation agent, blood vessel normalizing agent, and blood vessel It can be suitably used as an active ingredient of at least one of the stabilizers.

The Tie2 activator, angiogenesis inhibitor, blood vessel maturation agent, blood vessel normalizing agent, and blood vessel stabilizer of the present invention have excellent Tie2 activation action, angiogenesis inhibition action, and blood vessel maturation action. Since it has a blood vessel normalizing action and a blood vessel stabilizing action, it is a disease mainly consisting of vascular lesions such as tumor, rheumatoid arthritis, diabetic retinopathy, hyperlipidemia, hypertension, atopic dermatitis, and pollen. Can be suitably used as pharmaceuticals for allergic diseases such as infectious diseases and safe preventive drugs for these diseases, and the compounding amount, usage, and dosage form can be appropriately selected according to the purpose of use. .
In addition, it was confirmed that the Tie2 activator, vascular maturation agent, vascular normalization agent, vascular stabilization agent, and angiogenesis inhibitor of the present invention are not digested in the digestive tract. Therefore, it can be suitably used as a food / beverage product such as a beauty food / beverage product or a health food / beverage product, and the blending amount, usage, and dosage form can be appropriately selected according to the purpose of use.

  The blending amount can be adjusted as appropriate according to the physiological activity of the extract of the squid, the acteoside, the extract of the bodaige, and the extract of the maronier, etc., but the extract of the squid, the extraction of the bodaige Product, purified product of the extract of Maronier, and acteoside, 0.0001% by mass to 20% by mass is preferable, and 0.0001% by mass to 10% by mass is more preferable.

  There is no restriction | limiting in particular as said usage, According to the objective, it can select suitably, For example, usage, such as oral, parenteral, and external use, is mentioned.

  The dosage form is not particularly limited and may be appropriately selected depending on the intended purpose. For example, oral dosage forms such as tablets, powders, capsules, granules, extracts, and syrups, injections, and infusions And oral preparations such as suppositories, ointments, creams, emulsions, lotions, packs, bath preparations, hair cosmetics, and the like.

(Pharmaceutical composition)
The pharmaceutical composition of the present invention contains at least one of the above-described Tie2 activator, angiogenesis inhibitor, vascular maturation agent, vascular normalization agent, and vascular stabilization agent of the present invention, You may contain the additive normally used for a pharmaceutical as needed.

Since the pharmaceutical composition of the present invention has at least one of the excellent Tie2 activation action, angiogenesis inhibition action, blood vessel maturation action, blood vessel normalization action, and blood vessel stabilization action, As a drug for allergic diseases such as rheumatoid arthritis, diabetic retinopathy, hyperlipidemia, hypertension and other vascular lesions, atopic dermatitis, and hay fever, and as a safe preventive drug for these diseases It can be used suitably.
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 a food or drink such as a beauty food or a health food.

  The content of at least one of the Tie2 activator, angiogenesis inhibitor, blood vessel maturation agent, blood vessel normalizing agent, and blood vessel stabilizer in the pharmaceutical composition of the present invention is not particularly limited. The Tie2 activator, the angiogenesis inhibitor, the vascular maturation agent, the vascular normalization agent, and the vascular stabilization agent itself can be selected according to the purpose. Although it may be used as a pharmaceutical composition, it is preferably 0.01 μg or more, more preferably 0.1 μg or more and 1,000 μg or less, further preferably 1 μg or more and 500 μg or less, and particularly preferably 3 μg or more and 400 μg or less per mL of the pharmaceutical composition. .

  There is no restriction | limiting in particular as an administration form of the pharmaceutical composition of this invention, According to the objective, it can select suitably, For example, oral, parenteral, external use, etc. are mentioned.

  There is no restriction | limiting in particular as a dosage form of the pharmaceutical composition of this invention, According to the objective, it can select suitably, For example, oral administration agents, such as a tablet, a powder agent, a capsule, a granule, an extract, a syrup agent, etc. ; Parenteral administration agents such as injections, instillations, suppositories; and external preparations such as ointments.

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

(Production Example 1)
-Production of Acteoside-
8.9 g of Katsura Extract Powder (Maruzen Pharmaceutical Co., Ltd.) dissolved in a mixed solution of chloroform / methanol / water = 10: 5: 1 and filled with silica gel (trade name: Silica Gel 60, manufactured by Merck) It flowed in from the top of the column and was adsorbed on silica gel. Chloroform / methanol / water = 10: 5: 1 was passed as a moving bed through a glass column, the eluate was collected, and the solvent was distilled off to obtain a purified product (2.5 g).

The purified product obtained by purification as described above was subjected to mass spectral analysis, ¹ H-NMR analysis and ¹³ C-NMR analysis. The analysis results are shown below.

<Mass spectrum (ESI-MS)>
[M−H] ⁻ m / z 623 (theoretical value: C ₂₉ H ₃₆ O ₁₅ —H = 623)
[M + Na] ⁺ m / z 647 (theoretical value: C ₂₉ H ₃₆ O ₁₅ + Na = 647)

< ¹ H-NMR chemical shift δ (assigned hydrogen):>
6.69 (1H, d, J = 2.0 Hz, H-2), 6.67 (1H, d, J = 8.1 Hz, H-5), 6.55 (1H, dd, J = 2. 0, 8.1 Hz, H-6), 2.78 (2H, t-like, H-7), 4.01 (1H, overlapped, H-8a), 3.70 (1H, overlapped, H-8b) ), 7.05 (1H, d, J = 2.2 Hz, H-2 ′), 6.77 (1H, d, J = 8.3 Hz, H-5 ′), 6.94 (1H, dd, J = 2.2, 8.3 Hz, H-6 ′), 7.58 (1H, d, J = 15.9 Hz, H-7 ′), 6.27 (1H, d, J = 15.9 Hz, H-8 ′), 4.36 (1H, d, J = 7.8 Hz, GlcH−1), 3.39 (1H, dd, J = 7.8, 8.1 Hz, Glc H−2), 3 .80 (1H, rt, J = 9.0 Hz, Glc H-3), 4.88 (1H, br.t, J = 9.2 Hz, Glc H-4), 3.58 (1H, overlapped, Glc H-5) ), 3.48 (2H, overlapped, Glc H-6), 5.18 (1H, br.s, Rha H-1), 3.9 (1H, m, Rha H-2), 3.58 ( 2H, overlapped, Rha-H-3 and Rha H-5), 3.30 (1H, overlapped, Rha-H-4), 1.08. (3H, d, J = 6.1 Hz, Rha H-6)

< ¹³ C-NMR chemical shift δ (assigned carbon):>
131.1 (s, C-1), 116.1 (d, C-2), 146.5 (s, C-3), 144.3 (s, C-4), 116.8 (d, C-5), 121.0 (d, C-6), 36.4 (t, C-7), 72.0 (t, C-8), 127.3 (s, C-1 ′), 114.9 (d, C-2 ′), 145.8 (s, C-3 ′), 149.4 (s, C-4 ′), 116.2 (d, C-5 ′), 122. 9 (d, C-6 ′), 147.7 (d, C-7 ′), 114.4 (d, C-8 ′), 167.9 (s, C-9 ′), 103.9 ( d, Glc C-1), 75.9 (d, Glc C-2), 81.4 (d, Glc C-3), 70.7 (d, Glc C-4), 75.8 (d, Glc C-5), 62.1 (t, Glc C-6), 102.7 (d, Rha C-1), 7 .1 (d, Rha C-2), 71.8 (d, Rha C-3), 73.5 (d, Rha C-4), 70.2 (d, Rha C-5), 18.3 ( q, Rha C-6)

From the analysis results described above, it was confirmed that the compound obtained from the extract of the flower part of cinnamon moth (Keika extract powder) was Acteoside represented by the following structural formula (I).

Example 1
-Tie2 activation promoting test by ELISA method (Keika extract)-
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 medium for low serum vascular endothelial cell proliferation (Kurashiki Spinning) The culture was carried out overnight using Humedia-EG2). Next, the HUVEC after overnight culture was replaced with 0.1 mL of vascular endothelial cell basal medium (Humdia-EB2 manufactured by Kurashiki Boseki Co., Ltd.) 3 hours before cell stimulation (addition of test sample) and cultured again. Went. Thereafter, 0.1 mL of Keihana extract powder (manufactured by Maruzen Pharmaceutical Co., Ltd.) prepared as a test sample to the concentration shown in Table 1 as a test sample was added and incubated for 20 minutes. After the incubation, the amount of phosphorylated Tie2 in the cells was measured using an immunoassay kit (manufactured by R & D Systems, Human Phospho-Tie2 (Y992) Immunoassay) according to the protocol.
Moreover, the amount of phosphorylated Tie2 was similarly measured for dimethyl sulfoxide (DMSO) used for dissolving the test sample as a negative control.
And according to following formula (1), Tie2 activation promotion rate was calculated and the phosphorylation effect was evaluated. The results are shown in Table 1.

(Example 2)
-Tie2 activation promoting action test by ELISA method (acteoside)-
In Example 1, the Tei2 phosphorylating action was evaluated in the same manner as in Example 1 except that the Katsura extract powder was changed to Acteoside of Production Example 1 and the concentrations shown in Table 1 were used. The results are shown in Table 1.

(Example 3)
-Test of Tie2 activation promoting action by ELISA (Bodaige extract)-
In Example 1, the Tei2 phosphorylating action was evaluated in the same manner as in Example 1 except that the Katsura extract powder was changed to Bodaiju extract (manufactured by Maruzen Pharmaceutical Co., Ltd.) and the concentrations shown in Table 1 were used. did. The results are shown in Table 1.

Example 4
-Tie2 activation promoting action test by ELISA method (Maronier extract)-
In Example 1, the katsura flower extract powder was changed to a freeze-dried product of Maronnier extract (manufactured by Maruzen Pharmaceutical Co., Ltd.) and the concentrations shown in Table 1 were used. The phosphorylation effect was evaluated. The results are shown in Table 1.

From the results of Examples 1 to 4, it was confirmed that the Keika extract, Acteoside, Bodaiju extract and Maronier extract all induced phosphorylation of Tie2, that is, activation of Tie2. Although no specific data is shown, no significant phosphorylation of Tie2 was observed in the system to which DMSO, which is a negative control, was added. It was confirmed that Tie2 was phosphorylated in a system to which Angiopoietin-1 as a positive control was added. Therefore, Tie2 is phosphorylated and activated by Keika extract, Acteoside, Bodaiju extract and Maronnier extract, resulting in vascular maturation, vascular normalization, and vascular stabilization, and inhibition of angiogenesis It was suggested that

(Example 5)
-Tie2 activation promoting action test by Western blotting method (Maronier extract)-
Cells obtained by overexpressing human Tie2 (Ba / F3-human Tie2) in mouse pro-B cells (Ba / F3) were used for Tie2 phosphorylation analysis. The stimulation of Ba / F3-human Tie2 by the test sample was performed by lyophilizing a Maronier extract (manufactured by Maruzen Pharmaceutical Co., Ltd.) as a test sample dissolved in RPMI-1640 medium (manufactured by SIGMA-ALDRICH) without FBS. After 10 minutes from the addition, the cells were washed with PBS, and the cell extract was collected with PhosphoSafe ^™ Extraction Reagent (manufactured by Novagen). This was electrophoresed on a 7.5% SDS gel and transferred to a PVDF membrane. After blocking non-specific protein with blocking one-P (manufactured by Nacalai Tesque) for 60 minutes, a band of phosphorylated Tie2 was obtained using an anti-phosphorylated Tie2 antibody (manufactured by Cell Signaling Technology) and an HRP-labeled secondary antibody. Detected. In addition, a phosphorylated Tie2 band was similarly detected for dimethyl sulfoxide (DMSO) used to dissolve the test sample as a negative control. Band detection and analysis were performed with an imaging device ChemiDoc XRS Plus (manufactured by Bio-Rad Laboratories) and Image Lab Software version 2.0 (manufactured by Bio-Rad Laboratories), and Tie2 activity according to the following formula (2). The rate of activation was determined. The results are shown in Table 2.
Tie2 activity promotion rate (%) =
[(Band intensity of phosphorylated Tie2 when test sample is added) / (Band intensity of phosphorylated Tie2 in negative control)] × 100 Formula (2)

From the results of Table 2, it was confirmed that the extract of Maronnier had an excellent Tie activation action depending on the concentration.

  From the above, the extract of Keika, Acteoside, the extract of Bodaiju and the extract of Maronnier have Tie2 phosphorylation effects, thereby suppressing angiogenesis, vascular maturation, vascular normalization, and vascular It was suggested that stabilization can be induced.

The Tie2 activator, angiogenesis inhibitor, blood vessel maturation agent, blood vessel normalizing agent, blood vessel stabilizer, and pharmaceutical composition of the present invention have excellent Tie2 activation action, angiogenesis inhibition action, Drugs related to diseases mainly composed of vascular lesions such as tumors, rheumatoid arthritis, diabetic retinopathy, hyperlipidemia, hypertension and the like because they have vascular maturation action, vascular normalization action, and vascular stabilization action It can be widely used as a safe preventive drug for these diseases.
In addition, it was confirmed that the Tie2 activator, vascular maturation agent, vascular normalization agent, vascular stabilization agent, and angiogenesis inhibitor of the present invention are not digested in the digestive tract. Therefore, it can be widely used as food and drink such as beauty food and drink and health food and drink.

Claims (4)

  A Tie2 activator comprising, as an active ingredient, a water extract of a seed part of Maronnier, a hydrophilic solvent extract, or a mixed solvent extract of water and a hydrophilic solvent.   The Tie2 activator according to claim 1, which has a Tie2 activating action of phosphorylating Tie2 and converting it into phosphorylated Tie2.   A maturation action or a blood vessel stabilization based on Tie2 activation action, containing, as an active ingredient, an aqueous extract of a seed part of Maronnier, a hydrophilic solvent extract, or a mixed solvent extract of water and a hydrophilic solvent A blood vessel maturation agent or a blood vessel stabilizer characterized by having an action.   An oral composition for activating Tie2, comprising the Tie2 activator according to any one of claims 1 to 2.