JP2020033374A - Tie2 activator containing olive fruit extract - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a Tie2 activator or the like for inducing adhesion between cells of vascular endothelium and stabilizing vascular endothelial cells to exert an effect of suppressing vascular permeation. By containing an olive fruit extract as an active ingredient, a Tie2 activator or the like can be provided. [Selection diagram] None

Description

  The present invention relates to a Tie2 activator, and more particularly, to a Tie2 activator containing an olive fruit extract as an active ingredient. Also, the present invention provides an olive fruit extract as an active ingredient, a vascular permeability inhibitor, an angiogenesis inhibitor, a vascular maturation agent, a vascular normalizing agent, a vascular stabilizer, and a lymphatic stabilizing agent. It also relates to the agent. Further, the present invention comprises hydroxytyrosol, tyrosol, and one or more components selected from the group consisting of oleuropein as an active ingredient, a vascular permeability inhibitor, a blood vessel maturation agent, a blood vessel normalizing agent, It also relates to vascular stabilizers, lymphatic stabilizers, and Tie2 activators. Furthermore, the present invention also relates to a composition containing said agent.

  Blood vessels not only send oxygen and nutrients to whole body organs through blood, but also carry waste substances from tissues and excrete them outside the body, and are important tissues for the human body to maintain vital activity. Generally, aging or breakdown of blood vessels has a great effect on human health as represented by arteriosclerosis, and also leads to external findings such as wrinkles and swelling. In recent years, attention has been focused on the activation of the receptor tyrosine kinase Tie2 (Tyrosine kinase with Ig and ECF homology domain 2) for the aging of this blood vessel. It is known that when Tie2 is activated by angiopoietin-1 from vascular wall cells, adhesion between vascular endothelial cells is induced and contributes to stabilization of vascular endothelial cells. In recent years, components and foods having this Tie2 activating action have been found, and are used in supplements and beverages, etc., and are used for beauty, swelling, and cooling.

  Specifically, this Tie2 activation is based on Kiraya, Huangkai, Ginkgo, oyster, turmeric, chrysanthemum, jujube, wolfberry, chamomile, butcher bloom, hawthorn, star fruit, ghetto, lotus, rooibos, indians, karin, jujuba guava , Hihatsu, Siberian carrot, Mango ginger, Korai carrot, Akigumi, Okahijiki, Harigiri, Ryobu, Yabukanzo, Jade sweet potato, Mitsuba cypress, Kusagi, Mbe, Sansho-sou, Konara, Kunugi, Akinonogeshi, Suicide galleria , Sawkashi, Ousei, Arctic, Karonin Bald kitten, etc., have been found to have their effects (Patent Documents 1 to 7).

  On the other hand, as components, ursolic acid, corosolic acid, 3-O-galloylprocyanidin B-1, linolenic acid, 13-hydroxy-9Z, 11E, 15E-octadecatrienoic acid, procyanidin B-2, epicatechin- (4β -6) -Epicatechin (4β-8) -epicatechin, procyanidin C-1, astragaloside VIII, soyasaponin I, 3′-O-methylgallocatechin, piperonnarin, syringalesinol, eleuteroside E, eleuteroside E1, Efficacy has been confirmed for sesamin, eudesmin, silvatemin, pinoresinol, yangambin, forcitinol, coumarin and the like (Patent Documents 8 to 10).

JP 2012-236795 A JP-T-2009-154237 JP 2011-201811 A JP 2011-102275 A JP 2011-102274 A JP 2011-102273 A JP 2009-263358A JP 2014-97977 A JP 2013-241356 A JP 2011-102272 A

  An object of the present invention is to provide a novel Tie2 activating effect or the like that enables the vascular endothelial cell to induce adhesion between cells and stabilize the vascular endothelial cell to exert a vascular permeation inhibitory effect. It is intended to provide the ingredients.

  In order to solve the above problems, the present inventors have conducted intensive studies and as a result, newly found that a component obtained from olives, more preferably from olive fruits, has a Tie2 activating action and the like. Hydroxytyrosol, tyrosol, and oleuropein also have vascular permeability inhibitory, vascular maturation, vascular normalization, vascular stabilization, lymphatic stabilization, and Tie2 activation. As a result, the present invention has been completed.

That is, aspects of the present invention include, but are not limited to, the following inventions.
(1) A Tie2 activator containing an olive fruit extract as an active ingredient.
(2) The Tie2 activator according to (1), wherein the olive fruit extract is obtained from olive juice.
(3) The Tie2 activator according to (1) or (2), wherein the olive fruit extract is soluble in an aqueous solvent.
(4) The olive fruit extract contains one or more components selected from the group consisting of polyphenols, compounds containing these in the structure, and secoiridoids derived from the terpene moiety of these compounds. The Tie2 activator according to any of (3).
(5) The Tie2 activator according to any one of (1) to (4), wherein the olive fruit extract contains one or more components selected from the group consisting of hydroxytyrosol, tyrosol, and oleuropein.
(6) A composition comprising the Tie2 activator according to any one of (1) to (5).
(7) The composition according to (6), further comprising an indication of a function exerted by activating Tie2.
(8) A vascular permeability inhibitor containing an olive fruit extract as an active ingredient.
(9) A blood vessel maturation agent containing an olive fruit extract as an active ingredient.
(10) A blood vessel normalizing agent containing an olive fruit extract as an active ingredient.
(11) A blood vessel stabilizer containing an olive fruit extract as an active ingredient.
(12) A lymphatic stabilizer containing an olive fruit extract as an active ingredient.
(13) A composition comprising the agent according to any one of (8) to (12).
(14) Functions exerted by suppressing vascular permeability, functions exerted by maturation of blood vessels, functions exerted by normalizing blood vessels, functions exerted by stabilizing blood vessels, and exerted by stabilizing lymph vessels The composition according to (13), further comprising an indication of one or more functions selected from the group consisting of functions to be performed.
(15) A vascular permeability inhibitor comprising, as an active ingredient, at least one component selected from the group consisting of hydroxytyrosol, tyrosol, and oleuropein.
(16) A blood vessel maturation agent comprising, as an active ingredient, one or more components selected from the group consisting of hydroxytyrosol, tyrosol, and oleuropein.
(17) A blood vessel normalizing agent comprising as an active ingredient one or more components selected from the group consisting of hydroxytyrosol, tyrosol, and oleuropein.
(18) A blood vessel stabilizer comprising, as an active ingredient, one or more components selected from the group consisting of hydroxytyrosol, tyrosol, and oleuropein.
(19) A lymphatic stabilizer comprising, as an active ingredient, one or more components selected from the group consisting of hydroxytyrosol, tyrosol, and oleuropein.
(20) A Tie2 activator comprising, as an active ingredient, one or more components selected from the group consisting of hydroxytyrosol, tyrosol, and oleuropein.
(21) A composition comprising the agent according to any one of (15) to (20).
(22) Functions exerted by suppression of vascular permeability, functions exerted by maturation of blood vessels, functions exerted by normalization of blood vessels, functions exerted by stabilization of blood vessels, exerted by stabilization of lymph vessels The composition according to (21), further comprising an indication of at least one function selected from the group consisting of a function that is activated by activating Tie2 and a function that is exerted by activating Tie2.

  The Tie2 activator in the present invention induces adhesion between vascular endothelial cells by the excellent Tie2 activating action of olive fruit extract, stabilizes vascular endothelial cells, and suppresses vascular permeation, thereby suppressing vascular and lymphatic vessels. It can improve and prevent aging symptoms. For example, Tie2 activation induces the adhesion of vascular endothelial cells to tightly connect vascular cells to provide nutrition to the skin, improve skin firmness and texture, and prevent wrinkles and other cosmetic effects. In addition, the blood flow is improved by closing the gaps between endothelial cells of blood vessels and lymph vessels, water and waste products are collected from tissues, and various symptoms such as cold, stiff shoulders, swelling, and bears are improved. A preventive effect is obtained.

  The agent containing the olive fruit extract as an active ingredient in the present invention exerts a vascular permeability inhibitory action, a vascular maturation action, a blood vessel normalization action, a blood vessel stabilization action, and a lymph vessel stabilization action. be able to. Furthermore, an agent containing one or more components selected from the group consisting of hydroxytyrosol, tyrosol, and oleuropein according to the present invention is also effective in suppressing vascular permeability, maturating blood vessels, normalizing blood vessels, It can exert a stabilizing effect, a lymphatic stabilizing effect, and a Tie2 activating effect.

FIG. 1 shows the results of Western blotting of activated Tie2 in each sample, and a graph of the Tie2 activation rate as compared to a control. FIG. 2 shows the results of the Miles assay of each sample and a graph of the vascular permeability inhibition rate.

  Hereinafter, embodiments of the present invention will be described in detail.

Tie2 activator Activation of a receptor called Tie2 present in vascular endothelial cells plays an important role in the structural stabilization of capillaries. This Tie2 is shown to be present not only in vascular endothelial cells but also in lymphatic endothelial cells, and is activated by angiopoietin-1 released from parietal cells, strengthening adhesion between endothelial cells and adhesion with parietal cells, and Involved in stabilization. In the present invention, “Tie2 activator” refers to an agent having the ability to convert Tie2 into its active form (phosphorylated Tie2) by phosphorylation, and the Tie2 activator in the present invention activates Tie2. An olive fruit extract, in addition to polyphenols such as hydroxytyrosol and tyrosol as active ingredients, oleuropein, oleacein, acteoside, 3,4-DHPEA-EA, 3,4-DHPEA-EDA, and hydroxyurethane containing these in the structure. Examples include tyrosol glycoside and 4-Ep-coumaroyl secologanoside having p-coumaroyl acid in the structure. Further, many of these polyphenol derivatives have a compound derived from terpene in the molecule, and the terpene portion is secoiridoids. Other components include elenolic acid and terpene glycosides such as lengiosides and oleosides. The active ingredient in the Tie2 activator of the present invention is not particularly limited, but is selected from the group consisting of hydroxytyrosol, tyrosol, oleuropein, oleacein, acteoside, 3,4-DHPEA-EA, and 3,4-DHPEA-EDA. It is preferably one or more components, and more preferably one or more components selected from the group consisting of hydroxytyrosol, tyrosol, and oleuropein.

  It has also been reported that the activity of Tie2 decreases with age, for example, and it has been clarified that blood vessels and lymph vessels also age with this. Therefore, the Tie2 activator of the present invention can prevent and improve aging of blood vessels and lymphatic vessels by the excellent Tie2 activating action of olive fruit extract.

  In addition, when the blood vessel structure becomes unstable due to aging of blood vessels, nutrient components such as plasma components leak from the blood vessels, and the necessary components cannot be carried to necessary places. As a result, various troubles such as a decrease in skin condition may occur. Therefore, the Tie2 activator in the present invention can stabilize vascular endothelial cells by the excellent Tie2 activating action of the olive fruit extract, and can exert a vascular permeation inhibitory action. The tight connection of blood vessel cells provides nutrition to the skin, improves skin firmness and texture, and prevents wrinkles and other cosmetic effects. Furthermore, the blood flow is improved by closing the gaps between the endothelial cells of blood vessels and lymphatic vessels, water and waste products are collected from the tissues, and various symptoms such as cold, stiff shoulders, swelling, and bears are improved. A preventive effect is obtained.

<Active ingredient (olive fruit extract)>
In the present invention, “olive fruit extract” refers to an extract obtained by extracting from olive fruit, and has a Tie2 activity, a vascular permeability inhibitory action, a vascular maturation action, a vascular normalization action, and a vascular stabilization action. A component having an action and a lymphatic stabilization action. More preferably, it is a component group obtained from olive juice. Here, in the present invention, “olive juice” refers to an extract obtained from an aqueous layer obtained by separating a liquid phase obtained from olive fruits into an oil layer and an aqueous layer. The olive fruit extract is, for example, a water-soluble component and can be extracted from olives using an aqueous solvent. Examples of the aqueous solvent include water and alcohols (eg, ethanol). As the olive fruit extract, olive juice or an aqueous solvent extract may be used as it is, or an extract obtained by filtering these, concentrating the polyphenol in an adsorption column, and spray-drying may be used.

  The content of the olive fruit extract contained in the Tie2 activator is 0.01% by weight to 50% by weight, preferably 0.1% by weight to 40% by weight, and more preferably the total amount of the Tie2 activator. It is 1% to 30% by weight. The dose of the Tie2 activator in the present invention can be appropriately determined according to the content of the olive fruit extract contained in the Tie2 activator, and can be appropriately determined based on the age, weight, and health status of the subject. However, for example, the daily intake of a human adult is 0.1 mg to 400 mg, preferably 1 mg to 200 mg, more preferably 10 mg to 100 mg, and can be ingested and administered in single or divided doses.

  The components of the olive fruit extract according to the present invention include, in addition to polyphenols, one or more components selected from compounds containing these in the structure and sequoiridoids derived from the terpene portion of these compounds. Examples of the polyphenols include polyphenols such as hydroxytyrosol and tyrosol, and oleuropein, oleacein, acteoside, 3,4-DHPEA-EA, 3,4-DHPEA-EDA, and hydroxytyrosol glycoside containing these in the structure. And 4-Ep-coumaroyl secologanoside having p-coumaroyl acid in the structure. Further, many of these polyphenol derivatives have a compound derived from terpene in the molecule, and the terpene portion is secoiridoids. Other components include elenolic acid and terpene glycosides such as lengiosides and oleosides. Although the active ingredient in the present invention is not particularly limited, one or more ingredients selected from the group consisting of hydroxytyrosol, tyrosol, oleuropein, oleacein, acteoside, 3,4-DHPEA-EA, and 3,4-DHPEA-EDA And more preferably one or more components selected from the group consisting of hydroxytyrosol, tyrosol, and oleuropein.

<Raw material of olive fruit extract>
Olive, which is a raw material of olive fruit extract, is a plant of the Oleaceae family, and olive oil collected from olive fruit is widely used as edible oil and the like. On the other hand, olive fruit extract is one of the industrial wastes produced when olives are crushed, and in recent years, health foods and supplement materials for processed olives have been developed and sold. Olive fruit can be used as a main raw material of the olive fruit extract, but leaves, seeds, leaves, stems and the like may be mixed. When using olive fruit as a raw material, it may be used as it is, or may be dried by freeze-drying or the like. In addition, the residue after squeezing oil from olive fruits can be used as it is or in a dried state. It is preferable to use only olive juice, which is an aqueous layer obtained by separating a liquid phase obtained from olive fruits into an oil layer and an aqueous layer, as a raw material.

  The varieties of olives are not particularly limited, but varieties such as Manzanillo, Lucca, Nevadillo Blanco, Mission, Picual, Albequina, Ohiblanca, Cornicabra, Gordal, Moroiolo, Frantoio, Cortina, Retino and the like can be suitably used.

<Method for preparing olive fruit extract>
The olive fruit extract can be obtained through a separation step and a purification step.
(Separation process)
The raw olive fruit is squeezed and separated into oil and juice by centrifugation to obtain olive juice. Squeezing and centrifugation can be performed using methods known to those skilled in the art.
(Purification process)
An extract obtained by filtering and concentrating the olive juice obtained in the separation step may be used as the olive fruit extract in the present invention. In addition, the filtrate obtained by filtration of olive juice is loaded on a column filled with an adsorption resin to remove components that are not adsorbed on the adsorption resin such as saccharides, minerals, and organic acids, and then an aqueous solvent (eg, water, ethanol, Or a mixture thereof) to obtain a fraction abundantly containing a certain component such as olive-derived Tie2 activating action. The fraction rich in a certain component having Tie2 activating action or the like may be used as the olive fruit extract in the present invention. Those skilled in the art can appropriately set the conditions of the type of the adsorption resin and the elution solvent of the column according to the type and form of the olive to be used.

Other Agents Another embodiment of the present invention includes a vascular permeability inhibitor, an angiogenesis inhibitor, a vascular maturation agent, a vascular normalizing agent, a vascular stabilizer, a lymphatic stabilizer, and the like. These agents contain an olive fruit extract, which is also an active ingredient of the Tie2 activator, as an active ingredient. In addition, these agents include, as an active ingredient, at least one component selected from the group consisting of hydroxytyrosol, tyrosol, oleuropein, oleacein, acteoside, 3,4-DHPEA-EA, and 3,4-DHPEA-EDA. The active ingredient may be contained, and more preferably contains, as an active ingredient, one or more components selected from the group consisting of hydroxytyrosol, tyrosol, and oleuropein.

  In the present invention, the “vascular permeability inhibitor” refers to an agent having an inhibitory effect on the enhancement of vascular permeability caused by VEGF (vascular endothelial cell growth factor). The “angiogenesis inhibitor” refers to an agent having an action of suppressing a network of new blood vessels formed from existing blood vessels. "Vascular maturation agent" induces adhesion between vascular endothelial cells and vascular wall cells to form plaques between vascular endothelial cells such that intravascular environmental factors do not easily leak out of blood vessels An agent having a maturation effect. "Vascular normalizing agents" cause the disordered growth of blood vessels with broken vascular permeability and blood vessels by promoting adhesion between vascular endothelial cells and promoting lining of vascular wall cells to vascular endothelial cells. It refers to an agent having a normalizing action to bring such abnormal blood vessels into a normal state. "Vascular stabilizer" refers to the action of suppressing damage to existing blood vessels, dissociation between vascular endothelial cells, and vascular endothelial cells from vascular wall cells, and stabilizing vascular endothelial cell death An agent having an action. The “lymphatic vessel stabilizing agent” refers to an agent having an effect of stabilizing lymphatic vessels so that lymphatic endothelial cells are appropriately arranged and lined, and maintaining a function of rapidly collecting interstitial fluid.

  The type, dosage, production method, etc. of the components used for vascular permeability inhibitors, angiogenesis inhibitors, blood vessel maturation agents, blood vessel normalizing agents, blood vessel stabilizers, and lymphatic vessel stabilizers are Tie2 activation The content described for the agent can be applied as it is.

Composition As one embodiment of the present invention, the Tie2 activator, vascular permeability inhibitor, angiogenesis inhibitor, vascular maturation agent, vascular normalizing agent, vascular stabilizer, lymphatic vessel stabilizer Compositions containing one or more agents selected from the group consisting of:

  In addition to the Tie2 activator and the like, known additives used in cosmetics, pharmaceuticals and the like can be added to the composition. The additives are not particularly limited, and for example, additives such as excipients, binders, disintegrants, lubricants, antioxidants, preservatives, flavoring agents, flavoring agents, coloring agents, fragrances and the like can be used. For example, L-ascorbic acid, catechin, dextrin, cyclodextrin, calcium carbonate, trehalose and the like can be used. In addition, a known component having a Tie2 activating action or the like may be used in combination with the composition, or a component known to be effective in preventing aging of blood vessels or cosmetics may be used in combination. For example, Kiraya, Huangkai, Ginkgo, oyster, turmeric, chrysanthemum, jujube, wolfberry, chamomile, butcher bloom, hawthorn, star fruit, ghetto, lotus, rooibos, indian dates, karin, siuju, which have been reported to activate Tie2 Guava, Hihats, Siberian carrot, Mango ginger, Goryeo carrot, Akigumi, Okahijiki, Harigiri, Ryobu, Yabukanzo, Jade, Mitsubatsugi, Kusagi, Mbe, Sanshoso, Konara, Kunugi, Akinonogashi, Suicidae Extracts such as grape seeds, pine bark, peanut seed coat, and litchi seed coat that contain extracts of Nikkei, Saw beetle, Ousei, Arctic, and Karonin Baldock and procyanidins that are effective in improving vascular endothelium, L-citrulline, L-arginine It is possible to use the amino acid.

  The form of the composition can be appropriately determined according to the purpose of use, but it can be used in the form of a cosmetic composition, a pharmaceutical composition, or the like. Pharmaceutical compositions include pharmaceuticals and quasi-drugs, which do not belong to the Pharmaceutical Affairs Law, but with the expectation of the preventive and ameliorating effects of the Tie2 activator of the present invention, like pharmaceuticals and quasi-drugs. Also included are compositions to be purchased and compositions that explicitly or implicitly claim the preventive / improving effects of the Tie2 activator of the present invention. Although the dosage form of the composition is not particularly limited, it is typically an oral preparation such as a granule, tablet, capsule, or liquid, and a capsule is preferred.

  The present invention, in another aspect, vascular permeability suppression, maturation of blood vessels, normalization of blood vessels, stabilization of blood vessels, lymphatic stabilization, or with the indication of the function exerted by Tie2 activation, The present invention relates to a composition containing the agent of the present invention. Such a display or a functional display is not particularly limited. For example, for example, “suppress vascular hyperpermeability”, “suppress the leakage of intravascular factors out of blood vessels”, “improve blood vessels to a normal state” , "Maintain blood vessels in a normal state", "Suppress vascular endothelial cell dissociation", "Suppress cell death of vascular endothelial cells", or "Keep a rapid recovery function of interstitial fluid" And the like. In the present invention, the display such as the display and the function display may be provided on the composition itself, or may be provided on a container or a package of the composition.

  In a composition according to an embodiment of the present invention, the Tie2 activator is added to the composition in an amount of 0.01% to 99% by weight, preferably 0.1% to 50% by weight, more preferably 0.1% to 50% by weight. 1 to 30% by weight, even more preferably 10 to 30% by weight, and the olive fruit extract is present in an amount of 0.05 to 50% by weight, preferably 0 to 50% by weight, based on the total amount of the composition. 0.5 to 25% by weight, more preferably 5 to 20% by weight. The dose of the composition of one embodiment of the present invention varies depending on the content of the olive fruit extract contained in the Tie2 activator, and can be appropriately determined based on the age, weight, and health status of the subject. The daily intake of a human adult is 0.1 mg to 400 mg, preferably 1 mg to 300 mg, more preferably 10 mg to 200 mg, and can be ingested and administered in single or divided doses.

  Further, as one embodiment of the present invention, a composition containing the above vascular permeability inhibitor, angiogenesis inhibitor, vascular maturation agent, vascular normalizing agent, vascular stabilizer, or lymphatic vessel stabilizer Is mentioned. Regarding the type, form, dosage form, dose, and the like of the components of these compositions, the contents described for the composition containing the Tie2 activator can be applied as they are.

  Hereinafter, the present invention will be described more specifically based on examples. Note that the present invention is not limited to these examples.

Example 1: Evaluation of Tie2 activating action of olive fruit extract Tie2 activating action was evaluated using olive fruit extract.
<Preparation of sample (olive fruit extract)>
The olive fruit is pressed, centrifuged and separated into oil and juice. After filtering the juice obtained here, the components that are not adsorbed to the adsorption resin, such as sugars, minerals, and organic acids, are removed by an adsorption column, and the olive components that act on Tie2 activation are concentrated. The spray-dried one was used as an olive fruit extract. The obtained olive fruit extract was dissolved in dimethyl sulfoxide (DMSO) to a concentration of 50 μg / mL and 100 μg / mL to prepare a test sample. A test sample in which angiopoietin-1 was dissolved in DMSO to a concentration of 500 ng / mL was prepared and used as a positive control.
<Test method>
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 growth (manufactured by Kurashiki Boseki Co., Ltd. The cells were cultured overnight using Humedia-EG2). Next, the HUVEC after the overnight culture was replaced with 0.1 mL of vascular endothelial cell basal medium (Kurashiki Spinning Co., Ltd., Humedia-EB2) 3 hours before the cell stimulation (addition of the test sample), and the culture was performed again. Was. Thereafter, the wells were dissolved in Humedia-EB2, 0.1 mL of a test sample was added, and incubation was performed for 20 minutes. After the incubation, the amount of phosphorylated (active) Tie2 in the cells was measured using an immunoassay kit (Human Phospho-Tie2 (Y992) Immunoassay, manufactured by R & D Systems) according to the protocol.

  Phosphorylated Tie2 was similarly measured for DMSO used for dissolution of the test sample as a negative control.

  Then, the Tie2 activation rate was calculated according to the following formula, and the phosphorylation action was evaluated.

Tie2 activation rate (%) =
[(Measured value of phosphorylated Tie2 when test sample is added) / (Measured value of phosphorylated Tie2 in negative control)] × 100
<Result>
Table 1 shows the results. As shown in Table 1, the Tie2 activating effect was confirmed in the olive fruit extract.

Example 2: Tie2 activating action of olive fruit extract As a test sample, a sample containing the olive fruit extract prepared in Example 1 at a concentration of 3.125 μg / mL, 6.25 μg / mL, and 12.5 μg / mL in DMSO was used. Tie2 activation was examined using Western blotting.

<Test method>
Cells in which human Tie2 was overexpressed in mouse pro-B cells (Ba / F3) (Ba / F3-human Tie2) were used for Tie2 phosphorylation analysis. Stimulation of Ba / F3-human Tie2 by the test sample was carried out by adding RPMI-1640 medium without FBS and the test sample, washing the cells 10 minutes after the addition of PBS, and then adding the cells to PhosphoSafe ™ Extraction Reagent (Novagen). To collect the cell extract. This was electrophoresed on a 7.5% SDS gel and transferred to a PVDF membrane. After blocking the nonspecific protein with blocking one-P (Nacalai Tesque) for 60 minutes, the band of phosphorylated Tie2 was detected using an anti-phosphorylated Tie2 antibody (Cell Signaling Technology) and an HRP-labeled secondary antibody. In addition, phosphorylated Tie2 was similarly evaluated for DMSO used for dissolution of the test sample as a negative control, and as a positive control, a sample containing angiopoietin-1 at a concentration of 0.4 μg / mL in DMSO was used. Similarly, phosphorylated Tie2 was evaluated. Band detection and analysis were performed using an image capturing device ChemiDoc XRS Plus (Bio-Rad Laboratories) and Image Lab Software version 2.0 (Bio-Rad Laboratories), and the Tie2 activating effect was evaluated according to the following formula.

Tie2 activation rate (%) =
[(Band intensity of phosphorylated Tie2 when test sample is added) / (band intensity of phosphorylated Tie2 in negative control)] × 100
<Result>
FIG. 1 shows the results of Western blotting. The band intensity of the negative control was set to 100%, and the relative value was shown in a graph. From the results in FIG. 1, the Tie2 activating effect was confirmed in the olive fruit extract.

Example 3 Inhibition of Vascular Permeability of Olive Fruit Extract As a test sample, a sample containing the olive fruit extract prepared in Example 1 at a concentration of 25 μg / mL and 100 μg / mL in DMSO was used, and VEGF (vascular endothelial) was used. growth factor) was evaluated for its inhibitory effect on increased vascular permeability.

<Test method>
Six-week-old ICR mice were purchased from Shimizu Experimental Materials Co., Ltd., and were reared for at least one week. Three days before the test, the back of the mouse was shaved, and a mouse with no visible back damage was used for the experiment. In addition, eight administration compartments were set for each mouse (four at each of the left and right sides of the midline of the back), and the evaluation was made at n = 4.

A mouse under pentobarbital anesthesia was intravenously injected with 100 μL of 1% Evans blue dye prepared in saline, and 15 minutes later, vascular endothelial growth factor (VEGF 300 ng / mL final) and each test sample were Equal volumes were mixed, and 20 μL thereof was administered intradermally using a 30G injection needle. After 40 minutes, the skin on the back was peeled off, and the dye leaked into the skin was observed and photographed. After that, the skin tissue was cut out, the dye was extracted using formamide, and OD ₆₂₀ was measured with an absorbance meter. Each mouse was provided with a compartment to which neither the test sample nor VEGF was administered (control only with PBS (-)) and a compartment to which only VEGF was administered, and the vascular permeability inhibitory effect of the test sample was evaluated. The measurement results were subjected to a significant difference test by Tukey's multiple comparison test.

<Result>
FIG. 2 shows the results of the Miles assay for each test sample. The horizontal axis of the graph represents the test sample and the concentration, and the vertical axis represents the amount of Evans blue dye leaked from the blood vessel. The photograph also shows the state of leakage of the pigment from the back skin at that time. Further, based on the amount of leaked Evans blue, the vascular permeability inhibition rate of the test sample was determined and is shown in Table 2. As shown in Table 2, the olive fruit extract was confirmed to have a concentration-dependent permeability-inhibiting action from blood vessels.

Example 4: Evaluation of Tie2 activating action of hydroxytyrosol, tyrosol and oleuropein derived from olive fruit extract Hydroxytyrosol, tyrosol and oleuropein contained in olive fruit extract were separated and purified, and the Tie2 activating action of those components was determined. evaluated.

<Separation and purification of hydroxytyrosol, tyrosol and oleuropein>
After subjecting the olive fruit extract prepared in Example 1 to adsorption column chromatography (Diaion HP-20, manufactured by Mitsubishi Chemical Corporation), the fraction in which Tie2 activity was aggregated was subjected to separation and purification by preparative HPLC (ODS). Hydroxythisolole, tyrosol and oleuropein were isolated. The three compounds isolated were identified by matching the retention times with the standards in HPLC.

<Test method>
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 growth (manufactured by Kurashiki Boseki Co., Ltd. The cells were cultured overnight using Humedia-EG2).

  Next, the HUVEC after the overnight culture was replaced with 0.1 mL of vascular endothelial cell basal medium (Kurashiki Spinning Co., Ltd., Humedia-EB2) 3 hours before the cell stimulation (addition of the test sample), and the culture was performed again. Was. Thereafter, the wells were dissolved in Humedia-EB2, 0.1 mL of a test sample was added, and incubation was performed for 20 minutes. After the incubation, the amount of phosphorylated (active) Tie2 in the cells was measured using an immunoassay kit (Human Phospho-Tie2 (Y992) Immunoassay, manufactured by R & D Systems) according to the protocol.

  Phosphorylated Tie2 was similarly measured for DMSO used for dissolution of the test sample as a negative control.

  Then, the Tie2 activation rate was calculated according to the following formula, and the phosphorylation action was evaluated.

Tie2 activation rate (%) =
[(Measured value of phosphorylated Tie2 when test sample is added) / (Measured value of phosphorylated Tie2 in negative control)] × 100
<Result>
Table 3 shows the results. As shown in Table 1, Tie2 activating action was confirmed in hydroxytyrosol, tyrosol, and oleuropein.

Production Example 1: Capsules Olive fruit extract and additives having the composition shown in Table 4 below are weighed and mixed so as to be uniform to prepare a mixture. The obtained mixture is a soft capsule prepared according to the composition shown in Table 5. The capsule was filled in a usual manner to obtain a soft capsule.

Industrial applicability

  The Tie2 activator or the like in the present invention induces adhesion between vascular endothelial cells to stabilize vascular endothelial cells, suppresses vascular permeation, etc. It can improve and prevent symptoms caused by lymphatic aging.

Claims (4)

A vascular permeability inhibitor containing an olive fruit extract as an active ingredient,
The raw material of the olive fruit extract is olive juice,
Containing one or more components selected from the group consisting of hydroxytyrosol, tyrosol, and oleuropein,
The vascular permeability inhibitor.   The vascular permeability inhibitor according to claim 1, wherein the olive fruit extract is soluble in an aqueous solvent.   The olive fruit extract according to claim 1 or 2, wherein the olive fruit extract contains one or more components selected from the group consisting of polyphenols, compounds containing them in the structure, and secoiridoids derived from the terpene moiety of these compounds. Vascular permeability inhibitor.   A vascular permeability inhibitor comprising, as an active ingredient, one or more components selected from the group consisting of hydroxytyrosol, tyrosol, and oleuropein.