JP7557765B2 - Tie2 activator and food and drink - Google Patents

Description

The present invention relates to a Tie2 activator, a vascular maturation agent, a vascular stabilizer, and a food and beverage product.

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

In general, blood vessel formation is divided into two stages: vasculogenesis, in which new blood vessels are formed, and angiogenesis, in which existing blood vessels extend and branch to form a new vascular network. The former is mediated by vascular endothelial growth factor (VEGF), which is involved in a very wide range of blood vessel formation, from the initial development of blood vessels known as vasculogenesis to the subsequent angiogenesis. The latter is mediated by angiopoietin (Ang), which is involved in controlling adhesion between vascular endothelial cells and vascular wall cells, and in stabilizing the structure of blood vessels.

Under normal oxygen conditions, vascular endothelial cells are tightly attached to the vascular wall cells that line the surrounding blood vessels, maintaining a stable vascular structure. However, when hypoxia occurs in tissues, vascular wall cells can detach from the vascular endothelial cells, resulting in disordered blood vessel proliferation. This phenomenon (angiogenesis) is often observed in diseases that involve vascular lesions, such as tumors, chronic rheumatoid arthritis, diabetic retinopathy, hyperlipidemia, and hypertension.

It is known that angiogenesis is suppressed by activating Tie2 (tyrosine kinase with Ig and EGF homology domain 2), a receptor tyrosine kinase expressed in vascular endothelial cells (see, for example, Patent Document 1). It has also been reported that in ischemic diseases caused by vascular narrowing or suppression of vascular expansion, activation of Tie2 expands the vascular lumen (see, for example, Non-Patent Document 1). It has also been reported that activation of Tie2 suppresses cell death of vascular endothelial cells (see, for example, Non-Patent Document 2).

Thus, activation of Tie2 is known not only to suppress angiogenesis but also to cause maturation and stabilization of 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 blood vessels, thereby maturing blood vessels. It is also known that the activation of Tie2 suppresses vascular destabilization and stabilizes blood vessels against environmental factors that cause the breakdown of various intracellular and extracellular vascular structures. The stabilization of blood vessels in the present invention also includes the adhesion of vascular wall cells to endothelial cells by the activation of Tie2, which is known to be observed in diseases such as tumors and diabetic retinopathy in which disordered blood vessels proliferate due to the failure of vascular wall cells to adhere to vascular endothelial cells, thereby normalizing blood vessels.

As a naturally occurring substance that inhibits angiogenesis by activating Tie2 as described above, cinnamon extract (see, for example, Patent Document 1) is known. In addition, as a substance that inhibits angiogenesis, suramin (a polysulfonated naphthylurea compound) (see, for example, Patent Document 2) is known.

However, there remains a strong demand for new, safe materials that have superior Tie2 activation, vascular maturation, or vascular stabilization effects, and there is currently a strong demand for their rapid development.

JP 2009-263358 A Special Publication No. 9-503488

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

The present invention aims to solve the above-mentioned conventional problems and to achieve the following objects: That is, the present invention aims to provide a Tie2 activator that has an excellent Tie2 activating effect and is highly safe.
Another object of the present invention is to provide a vascular maturation agent which has an excellent vascular maturation effect and is highly safe.
Another object of the present invention is to provide a vascular stabilizing agent which has an excellent vascular stabilizing effect and is highly safe.
Another object of the present invention is to provide a food or drink that has at least one of an excellent Tie2 activation effect, a blood vessel maturation effect, and a blood vessel stabilization effect, and is highly safe.

As a result of intensive research conducted by the present inventors to solve the above problems, they discovered that astaxanthin has at least one of the following excellent effects: Tie2 activation, vascular maturation, and vascular stabilization, and thus completed the present invention.

The present invention is based on the findings of the present inventors, and the means for solving the above problems are as follows.
<1> A Tie2 activator characterized by containing astaxanthin.
<2> A blood vessel maturation agent characterized by containing astaxanthin.
<3> A blood vessel stabilizer characterized by containing astaxanthin.
<4> A food or drink used for at least one of Tie2 activation, vascular maturation, and vascular stabilization,
This is a food or beverage characterized by containing at least one of the Tie2 activator described in <1>, the vascular maturation agent described in <2>, and the vascular stabilizing agent described in <3>.

According to the Tie2 activator of the present invention, the above-mentioned conventional problems can be solved, the above-mentioned objects can be achieved, and a Tie2 activator having excellent Tie2 activating activity and high safety can be provided.
According to the vascular maturation agent of the present invention, the above-mentioned problems in the prior art can be solved and the above-mentioned object can be achieved, and a vascular maturation agent having excellent vascular maturation effect and high safety can be provided.
According to the vascular stabilizer of the present invention, the above-mentioned problems in the prior art can be solved and the above-mentioned object can be achieved, and a vascular stabilizer having excellent vascular stabilizing effect and high safety can be provided.
The food and beverage of the present invention can solve the above-mentioned problems in the conventional art and achieve the above-mentioned objective, thereby providing a highly safe food and beverage that has at least one of excellent Tie2 activation effect, vascular maturation effect, and vascular stabilization effect.

(Tie2 activator, vascular maturation agent, and vascular stabilization agent)
The Tie2 activator, vascular maturation agent, and vascular stabilization agent of the present invention contain at least astaxanthin, and further contain other components as necessary.

The Tie2 activator has a Tie2 activation effect of phosphorylating Tie2 and converting it into its active form (phosphorylated Tie2). When the Tie2 is activated, it causes autophosphorylation of the intracellular tyrosine kinase domain, inducing adhesion between vascular endothelial cells and vascular wall cells. In ischemic diseases caused by vascular narrowing or inhibition of vascular dilation, activation of Tie2 causes vascular lumen dilation. Furthermore, activation of Tie2 inhibits cell death of vascular endothelial cells.

The vascular maturation agent induces adhesion between vascular endothelial cells and vascular wall cells, and has a maturation effect of forming focal adhesions between vascular endothelial cells that prevent intravascular environmental factors (cellular and humoral factors) from easily leaking out of the blood vessel. In vascular regenerative medicine, it is possible to induce adhesion between vascular endothelial cells and vascular wall cells in blood vessels by activating Tie2, thereby maturing the blood vessels.

The vascular stabilizer has an action of suppressing damage to existing blood vessels, dissociation between vascular endothelial cells, and dissociation between vascular endothelial cells and vascular wall cells, and a stabilizing action of suppressing cell death of vascular endothelial cells. In addition, it is possible to suppress vascular destabilization and stabilize blood vessels by activating Tie2 against various environmental factors that cause the breakdown of intracellular and extracellular vascular structures.
The effects of the vascular stabilizer also include a normalizing effect of returning abnormal blood vessels, such as those with a breakdown in vascular permeability or those that lead to disordered blood vessel proliferation, to a normal state by increasing adhesion between vascular endothelial cells and promoting the lining of vascular endothelial cells with vascular wall cells. In diseases such as tumors and diabetic retinopathy, in which disordered blood vessel proliferation occurs due to the failure of vascular wall cells to adhere to vascular endothelial cells, activation of Tie2 can cause vascular wall cells to adhere to endothelial cells, thereby normalizing blood vessels.

The fact that astaxanthin has at least one of the excellent effects of Tie2 activation, vascular maturation, and vascular stabilization, and is useful as a Tie2 activator, vascular maturation agent, and vascular stabilizer, was not previously known, and is a new discovery by the present inventors.

<Astaxanthin>
The astaxanthin is not particularly limited, and any known astaxanthin can be appropriately selected. The astaxanthin may be naturally derived astaxanthin extracted and purified from any of fish, crustaceans, plants, algae, bacteria, and fungi, or may be synthetically produced astaxanthin.

Astaxanthin in the narrow sense is a red pigment that has an absorption maximum at 476 nm (ethanol) and 468 nm (hexane), and its chemical structure is 3,3'-dihydroxy-β,β-carotene-4,4'-dione (C ₄₀ H ₅₂ O ₄ , molecular weight 596.82).
Astaxanthin exists in three isomers, 3S,3S'-isomer, 3S,3R'-isomer (meso-isomer), and 3R,3R'-isomer, depending on the configuration of the hydroxyl groups at the 3 (3')-position of the ring structure at both ends of the molecule. In addition, there are also cis- and trans-isomers of the conjugated double bond at the center of the molecule. Any of these isomers can be used in the present invention.

The hydroxyl groups at the 3,3'-positions can form esters with fatty acids. Astaxanthin obtained from krill is a diester with two fatty acids bonded, while astaxanthin obtained from H. pluvialis is a 3S,3S'-form, and contains a large amount of monoester with one fatty acid bonded. Astaxanthin obtained from Phaffia Rhodozyma is a 3R,3R'-form, which has the opposite structure to the 3S,3S'-form normally found in nature.

In the present invention, unless otherwise specified, the astaxanthin includes astaxanthin and derivatives such as astaxanthin esters. Furthermore, in addition to the above-mentioned naturally occurring astaxanthin esters, the derivatives include appropriately synthesized astaxanthin salts, oxidized forms, reduced forms, geometric isomers, optical isomers, glycosides, esters, etc., and all of these are referred to as "astaxanthin," and any of them can be used.

The astaxanthin is not particularly limited and can be appropriately selected depending on the purpose. For example, astaxanthin extracted from natural products such as krill, salmon, trout, Adonis ramosa, red yeast, and Haematococcus algae, or synthetic products can be used. These may be used alone or in combination of two or more types.

The extraction solvent used when obtaining astaxanthin from the natural product may be an aqueous solvent or an organic solvent.
The organic solvent is not particularly limited and may be appropriately selected depending on the purpose, and examples thereof include methanol, ethanol, isopropanol, acetone, 1,3-butylene glycol, ethylene glycol, propylene glycol, glycerin, ethyl acetate, ether, and hexane. Carbon dioxide in a supercritical state may also be used. These solvents may be used alone or in combination of two or more.

There are no particular limitations on the method for producing the synthetic astaxanthin product, and any known method can be appropriately selected.

The content of astaxanthin in the Tie2 activator, vascular maturation agent, and vascular stabilizer of the present invention is not particularly limited and can be appropriately selected depending on the purpose. The Tie2 activator, vascular maturation agent, and vascular stabilizer may consist only of astaxanthin.

<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 use form of the Tie2 activator, vascular maturation agent, and vascular stabilizer. For example, excipients, moisture-proofing agents, preservatives, strengthening agents, thickeners, emulsifiers, antioxidants, sweeteners, acidulants, seasonings, colorants, fragrances, whitening agents, moisturizers, oily components, UV absorbers, surfactants, thickeners, alcohols, powder components, colorants, aqueous components, water, skin nutrients, etc. These may be used alone or in combination of two or more.
The contents of the other components in the Tie2 activator, vascular maturation agent, and vascular stabilizing agent of the present invention are not particularly limited and can be appropriately selected depending on the purpose.

<Applications>
The uses of the Tie2 activator, vascular maturation agent, and vascular stabilizer of the present invention are not particularly limited and can be appropriately selected depending on the purpose. For example, since it has been confirmed that the Tie2 activator, vascular maturation agent, and vascular stabilizer are not digested in the digestive tract, they can be suitably used as foods and beverages for at least one of Tie2 activation, vascular maturation, and vascular stabilization, and the amount, method of use, and dosage form thereof can be appropriately selected depending on the purpose of use.
The amount of at least any one of the Tie2 activator, vascular maturation agent, and vascular stabilizer in the food or beverage is not particularly limited and can be appropriately selected depending on the purpose. When converted into the amount of astaxanthin, the amount is preferably 0.0001% by mass to 20% by mass, and more preferably 0.0001% by mass to 10% by mass.

The method of use is not particularly limited and can be selected appropriately depending on the purpose, and examples of administration include oral, parenteral, and topical.

The dosage form is not particularly limited and can be appropriately selected depending on the purpose. Examples of the dosage form include oral administration agents such as tablets, powders, capsules, granules, extracts, and syrups; parenteral administration agents such as injections, drops, and suppositories; and external applications such as skin lotions, milky lotions, creams, ointments, beauty essences, lotions, packs, jellies, lip balm, lipstick, foundations, bath additives, soaps, body soaps, astringents, hair tonics, hair lotions, hair creams, hair liquids, pomades, shampoos, rinses, and conditioners.
The method for producing each of the above-mentioned dosage forms of the Tie2 activator, vascular maturation agent, and vascular stabilizing agent is not particularly limited, and any known method can be appropriately selected.

There are no particular limitations on the amount, duration, and other usage methods of the Tie2 activator, vascular maturation agent, and vascular stabilizer, and these can be appropriately selected depending on the purpose.

The Tie2 activator, vascular maturation agent, and vascular stabilizer can also be used as reagents for research into the mechanism of action of Tie2 activation, vascular maturation, or vascular stabilization.

(Food and beverages)
The food and beverage is used for at least one of Tie2 activation, vascular maturation, and vascular stabilization, and contains at least one of the Tie2 activator, vascular maturation agent, and vascular stabilizer of the present invention, and further contains other ingredients as necessary.

The content of at least one of the Tie2 activator, vascular maturation agent, and vascular stabilizer in the food and beverage of the present invention is not particularly limited and can be appropriately selected depending on the purpose. The food and beverage may consist of at least one of the Tie2 activator, vascular maturation agent, and vascular stabilizer.

The other components in the food and drink of the present invention are not particularly limited, and can be appropriately selected from components used in food and drink. The other components may be used alone or in combination of two or more.
The contents of other ingredients in the food and drink are not particularly limited and can be appropriately selected depending on the purpose.

The food and drink are not particularly limited and can be appropriately selected according to the purpose, and examples of such food and drink include beverages such as tea drinks, soft drinks, carbonated drinks, nutritional drinks, fruit drinks, and lactic acid drinks; cold desserts such as ice cream, ice sherbet, and shaved ice; noodles such as soba, udon, harusame, gyoza wrappers, shumai wrappers, Chinese noodles, and instant noodles; sweets such as candy, candy, gum, chocolate, tablet candy, snacks, biscuits, jelly, jam, cream, baked goods, and bread; seafood such as crab, salmon, clams, tuna, sardines, shrimp, bonito, mackerel, whale, oysters, pacific saury, squid, ark shells, scallops, abalone, sea urchin, salmon roe, and tokobushi; kamaboko, ham, Examples of such foods include processed seafood and livestock foods such as sausages; dairy products such as processed milk and fermented milk; oils and fats and oil-based processed foods such as salad oil, tempura oil, margarine, mayonnaise, shortening, whipped cream, and dressings; seasonings such as sauces and sauces; retort pouch foods such as curry, stew, oyakodon, porridge, porridge, Chinese rice bowl, katsudon, tendon, unadon, hayashi rice, oden, mabo dolph, beef bowl, meat sauce, egg soup, omelet rice, gyoza, shumai, hamburger steak, and meatballs; side dishes such as salads and pickles; health, beauty, and nutritional supplements in various forms; medicines and quasi-drugs such as tablets, granules, capsules, drinks, and lozenges. The foods and beverages are not limited to the above examples.

The use of the food or drink is not particularly limited, so long as it is used for at least one of Tie2 activation, vascular maturation, and vascular stabilization, and can be appropriately selected depending on the purpose.
The food and beverage products of the present invention can be ingested on a daily basis, and due to the action of the active ingredient astaxanthin, can extremely effectively exert various physiological activities, including at least one of the actions of activating Tie2, maturing blood vessels, and stabilizing blood vessels.

The Tie2 activator, vascular maturation agent, vascular stabilizer, and food and beverage of the present invention are preferably applied to humans, but can also be applied to animals other than humans (e.g., mice, rats, hamsters, dogs, cats, cows, pigs, monkeys, etc.) as long as their respective action effects are achieved.

Below, test examples and formulation examples of the present invention are explained, but the present invention is not limited to these test examples and formulation examples.

(Test Example 1: Tie2 Activation Test-1)
Astaxanthin (manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.) was used as a test sample, and the Tie2 activation effect of astaxanthin was tested by the following test method.

Normal human umbilical vein endothelial cells (HUVEC) cultured to confluence were seeded on a 96-well plate at 2.0 x ¹⁰⁴ cells/0.1 mL/well, and cultured overnight using a low-serum vascular endothelial cell growth medium (Kurashiki Boseki Co., Ltd., Humedia-EG2). After culture, 3 hours before cell stimulation (addition of test sample), the medium was replaced with 0.1 mL of vascular endothelial cell basal medium (Kurashiki Boseki Co., Ltd., Humedia-EB2), and cultured again. Then, 0.1 mL of the test sample solution prepared with the Humedia-EB2 at twice the final concentration was added to the well, and incubation was performed for 10 minutes. After incubation, the amounts of phosphorylated Tie2 and total Tie2 in the cells were measured using an immunoassay kit (R&D Systems, Human Phospho-Tie2 (Y992) Immunoassay) according to the protocol, and the ratio of phosphorylated Tie2 to total Tie2 was calculated.
Dimethyl sulfoxide (DMSO) was used as a negative control, and the ratio of phosphorylated Tie2 to total Tie2 was calculated in the same manner.
The Tie2 activation rate was then calculated according to the following formula 1 to evaluate the phosphorylation effect. The results are shown in Table 1.
<Formula 1>
Tie2 activation rate (%) = [{(A)/(B)}/{(C)/(D)}]×100
In the above formula 1, A to D represent the following.
A: Measured value of phosphorylated Tie2 when test sample was added. B: Measured value of total Tie2 when test sample was added. C: Measured value of phosphorylated Tie2 in negative control. D: Measured value of total Tie2 in negative control.

(Test Example 2: Tie2 Activation Test-2)
Astaxanthin (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was used as a test sample, and the Tie2 activation test (Western blotting method) using Ba/F3-human Tie2 described below was carried out to test the Tie2 activation effect of astaxanthin.

Mouse pro-B cells (Ba/F3) in which human Tie2 (Ba/F3-human Tie2) was forcibly expressed were used for the evaluation.
The cells were seeded in a 10 cm petri dish and cultured for 3 days using RPMI-1640 (Sigma-Aldrich) medium containing 10% FBS and 100 pg/mL mouse IL-3. After the culture, 3 hours before cell stimulation (addition of the test sample), the medium was replaced with 10 mL of RPMI-1640 medium containing no FBS or IL-3, and the cells were cultured again. Thereafter, the cells were adjusted to 4 x ¹⁰⁶ cells/mL using RPMI-1640 medium containing no FBS or IL-3, and dispensed into microtubes in 0.5 mL portions. 0.5 mL of the test sample solution, which had been adjusted to twice the final concentration with the RPMI-1640 medium containing no FBS or IL-3, was added to the microtube, and incubation was performed for 15 minutes. After incubation, the cells were washed with PBS, and then a cell extract was collected using PhosphoSafe ^™ Extraction Reagent (Novagen).
The collected cell extract was electrophoresed on a 7.5% SDS gel and transferred to a PVDF membrane. After blocking, Western blotting was performed using an anti-phosphorylated Tie2 antibody (R&D Systems), an anti-Tie2 antibody (Santa Cruz Biotechnology), and an HRP-labeled secondary antibody (Jackson ImmunoResearch).
As a negative control, dimethyl sulfoxide (DMSO) was used and the evaluation was carried out in the same manner.
The detection and analysis of the bands were performed using an imaging device ChemiDoc XRS Plus and Image Lab Software version 2.0 (manufactured by Bio-Rad Laboratories), and the Tie2 activation rate was calculated according to the following formula 2 to evaluate the Tie2 activation effect. The results are shown in Table 2.
<Formula 2>
Tie2 activation rate (%) = [{(E)/(F)}]/{(G)/(H)}]×100
In the above formula 2, E to H represent the following.
E: Band intensity of phosphorylated Tie2 when test sample was added. F: Band intensity of total Tie2 when test sample was added. G: Band intensity of phosphorylated Tie2 in negative control. H: Band intensity of total Tie2 in negative control.

The results of Test Examples 1 and 2 confirmed that astaxanthin phosphorylates Tie2, i.e., activates Tie2. This suggests that astaxanthin phosphorylates and activates Tie2, leading to vascular maturation and stabilization.

(Formulation Example 1)
Tablets having the following composition were prepared by a conventional method.
・ Astaxanthin: 5.0 mg
Dolomite: 83.4 mg
(Contains 20% calcium and 10% magnesium)
・ Casein phosphopeptide: 16.7 mg
Vitamin C: 33.4 mg
Maltitol: 136.8 mg
・Collagen: 12.7mg
・ Sucrose fatty acid esters: 12.0 mg

Claims (2)

A Tie2 activator containing astaxanthin,
The astaxanthin is 3,3'-dihydroxy-β,β-carotene-4,4'-dione,
A Tie2 activator, wherein the activation of Tie2 is achieved by phosphorylating Tie2 and converting it into phosphorylated Tie2 . A food or drink used for activating Tie2,
A composition comprising the Tie2 activator according to claim 1,
A food or drink, characterized in that the activation of Tie2 is achieved by phosphorylating Tie2 and converting it into phosphorylated Tie2.