JP6563827B2 - Tie2 activator - Google Patents

Description

  The present invention relates to a Tie2 activator containing an extract (or extract) of ganoderma components selected from ganoderma and / or fermented ganoderma. The present invention also includes an extract (or extract) of ganoderma components selected from ganoderma and / or fermented ganoderma, and an angiogenesis inhibitor, a vascular maturation agent, and a vascular normalization agent. The present invention relates to an agent for use in applications such as a blood vessel stabilizer.

  A blood vessel is composed of vascular endothelial cells and vascular wall cells, and plays a role of carrying oxygen and nutrients into living tissues through blood circulation. Normally, water and low-molecular substances in blood permeate between blood and tissue via blood vessels, but macromolecules such as proteins do not permeate. The permeability of such blood vessels is controlled by vascular endothelial cells, and the stable structure of blood vessels is maintained by adhesion of vascular wall cells to the surroundings in addition to adhesion of vascular endothelial cells.

When oxygen and nutrients in living tissues are insufficient, adhesion between vascular endothelial cells and adhesion between vascular wall cells and vascular endothelial cells are suppressed, and vascular permeability is increased, thereby increasing humoral factors in blood. Molecules reach the tissues.
On the other hand, when the vascular permeability is excessively increased, edema and inflammation of the living tissue are caused, and the living tissue becomes dysfunctional. These phenomena have been observed in pathological conditions such as diabetic retinopathy, tumors, inflammation, and the like, and it is thought that dysregulation of vascular permeability leads to these pathological conditions. Such excessive enhancement of vascular permeability causes further disturbance of the vascular structure, so that maintaining a stable structure of blood vessels is important for improving the above-mentioned pathological condition.

In recent years, for the purpose of stabilizing blood vessels, a search for a factor that activates a receptor tyrosine kinase Tie (tyrosine kinase with Ig and EGF homology domain) 2 has been performed (Patent Document 1). It is known that Tie2 is expressed in vascular endothelial cells and is activated (phosphorylated) to induce adhesion between vascular endothelial cells and vascular wall cells and stabilize blood vessels.
In addition, activation of Tie2 suppresses angiogenesis (for example, formation of a new blood vessel network from existing blood vessels) and maturation of blood vessels (for example, blood vessel endothelial cells form adhesion plaques to form blood vessels. Controls permeability, stabilizes vascular structure by vascular endothelial cells accompanied by lining of vascular wall cells), normalization of blood vessels (for example, blood vessels with ruptured vascular permeability and disordered growth of blood vessels) It is also known that abnormal blood vessels can be made normal by increasing adhesion between vascular endothelial cells and promoting lining of vascular wall cells to inner vascular skin cells (Patent Literature). 1 and 2).
For example, in vascular regenerative medicine and the like, it is known that activation of Tie2 can induce adhesion between vascular endothelial cells and vascular wall cells to mature blood vessels.
In addition, in diseases such as diabetic retinopathy and tumors caused by vascular wall cells not adhering to vascular endothelial cells, Tie2 activation allows vascular wall cells to adhere to vascular endothelial cells and normalize blood vessels It has been known.

  Regarding activation of Tie2, for example, syringaresinol, which is a component of Cinnamum Cassia Blue, has been reported to stabilize the blood vessel structure lowered by ultraviolet B by activating Tie2. (Patent Document 1). Moreover, the Tie2 activation effect | action of the extract of Keika (Osmanthus fragrans var. Aurantiacus), a bodaiju (Tilia cordadata), and a horse chestnut (Aesculus hippocastanum) is reported (patent document 2).

JP 2009-263359 A Japanese Patent Laying-Open No. 2015-168656

An object of the present invention is to provide a new Tie2 activator.
Another object of the present invention is to provide an agent that can achieve both the activation of Tie2 and the inhibitory action of angiotensin converting enzyme (ACE).

  As a result of intensive studies to solve the above problems, the present inventors have found that an extract (or extract) of a ganoderma component selected from ganoderma and / or a fermented product thereof has a Tie2 activation effect. Found to have. In addition to the above, the inventors of the present invention have made further studies and completed the present invention.

That is, the Tie2 activator (agent for activating Tie2) of the present invention contains an extract (or extract) of a ganoderma component selected from ganoderma and / or fermented ganoderma.
Such an agent has various functions (for example, angiogenesis suppression function, blood vessel maturation function, blood vessel stabilization function, etc.) due to the Tie2 activation effect.
Therefore, the present invention includes agents for use in various applications based on such functions. Such an agent contains, for example, an extract of a ganoderma component selected from ganoderma and / or a ganoderma fermented product, an angiogenesis inhibitor, a vascular maturation agent, a vascular normalization agent, and a vasculature It may be an agent for use in at least one or more applications selected from the group consisting of:
In the agent of the present invention, the ganoderma component may particularly contain a fermented ganoderma.
In the agent of the present invention, the extract may be a water extract.
As described above, since the agent of the present invention uses an extract of ganoderma component as an active ingredient, it has not only a Tie2 activation function but also other functions that are not present in components having a conventional Tie2 activation function. doing. Therefore, the agent of the present invention may further be an agent for imparting such other functions [for example, an agent for inhibiting angiotensin converting enzyme (ACE)]. Specifically, the agent of the present invention further includes an agent for lowering blood pressure and / or an agent for improving blood flow (for at least one use selected from a blood pressure lowering agent and a blood flow improving agent). Agent for use). Further, the agent of the present invention may be an agent for improving the body temperature regulation function (an agent for use in a body temperature regulation function improving agent).
The present invention includes the above-mentioned agent (Tie2 activator or the like) or a composition (such as a food or drink, a medicine or a pharmaceutical composition) containing the ganoderma component. Such a composition contains a ganoderma component and has, for example, a Tie2 activation function (and further an ACE inhibition function) as described above. Therefore, the composition of the present invention may be, for example, a composition for activating Tie2 (further inhibiting ACE) (composition for activating Tie2). More specifically, the composition of the present invention is a disease caused by detachment of vascular wall cells from vascular endothelial cells (eg, tumor, rheumatoid arthritis, diabetic retinopathy, hyperlipidemia, hypertension, etc.). It may be a composition (pharmaceutical composition) for the prevention, amelioration and / or treatment.

  Furthermore, the present invention also includes a method of activating Tie2 by administering the agent (or ganoderma component) or the composition to an animal (human, non-human animal). Such a method may be at least one method selected from a method of suppressing angiogenesis, a method of maturating a blood vessel, and a method of stabilizing a blood vessel. The method of the present invention may further inhibit ACE. Specifically, in the method of the present invention, blood pressure may be lowered or blood flow may be improved. Moreover, the agent of the present invention may further improve the body temperature regulation function.

According to the agent or composition of the present invention, Tie2 can be effectively activated. Therefore, in the present invention, due to the activation of Tie2, agents for improving or preventing various symptoms and diseases (for example, angiogenesis inhibitors, blood vessel maturation agents, blood vessel normalizing agents, blood vessel normalizing agents, Stabilizers, etc.). In a more specific aspect, since the agent or composition of the present invention can induce adhesion between vascular endothelial cells and vascular wall cells, diseases caused by detachment of vascular wall cells from vascular endothelial cells (for example, Tumor, rheumatoid arthritis, diabetic retinopathy, hyperlipidemia, hypertension and the like).
Moreover, in this invention, in addition to a Tie2 activation function, the function which is not in the conventional Tie2 activation component, such as the inhibitory action of angiotensin converting enzyme (ACE), is realizable. For example, the agent of the present invention is extremely useful because it also has a blood pressure lowering effect, a blood flow improving effect, a body temperature regulating function improving effect, and the like.

The Tie2 activation evaluation result of the reishi of Reishi and Reishi is shown. The Tie2 activation evaluation result of the reishi of Reishi and Reishi is shown.

Hereinafter, the present invention will be described in detail.
The agent of the present invention (Tie2 activator, angiogenesis inhibitor, blood vessel maturation agent, blood vessel normalizing agent, blood vessel stabilizer, etc.) is selected from ganoderma and / or fermented products thereof. It contains a component extract (extract or extract).
Ganoderuma lingzhi is a mushroom belonging to the genus Mannentake belonging to the genus Amanitaceae.
The ganoderma is not particularly limited, and examples thereof include red ganoderma, blue ganoderma, yellow ganoderma, black ganoderma, white ganoderma, purple ganoderma, deer ganoderma and the like. These can use 1 type (s) or 2 or more types. The reishi may be a natural product or a cultivated product, and those obtained by a conventionally known acquisition method can be used.

  The part of the ganoderma is not particularly limited, and any part can be used. Examples of the ganoderma part include an umbrella, a fold, a brim, a handle, and an acupoint. These can use 1 type (s) or 2 or more types.

The fermented product of Ganoderma is not particularly limited, but is typically Ganoderma self-digested product.
The self-digested product of Ganoderma can be obtained, for example, by fermenting or maturing Ganoderma at a predetermined temperature (for example, about 35 to 40 ° C.). Fermentation time is not specifically limited, For example, it is 1-48 hours, Preferably it is 1-30 hours. The fermentation may be performed in the presence of bacteria that contribute to the reishi fermentation.

Fermentation may be performed in the presence of water. The mode in which water is present is not particularly limited, and includes spraying and dipping.
In addition, after fermentation, you may perform a sterilization process (for example, heat sterilization, high-pressure sterilization, filtration sterilization, etc.) as needed.

Examples of the extract of the ganoderma component include a solvent extract of the ganoderma component.
Although it does not specifically limit as an extraction solvent, For example, water (for example, distilled water, ion-exchange water, physiological saline etc.), an organic solvent [For example, lower alcohol (for example, methanol, ethanol, n-propanol, isopropanol, t) -Butanol, etc.), polyhydric alcohols (eg, glycerin, propylene glycol, etc.), ketones (eg, acetone, etc.), esters (eg, ethyl acetate, etc.), ethers (eg, diethyl ether, tetrahydrofuran, etc.), etc. Etc., preferably water, lower alcohol and the like, more preferably water, ethanol and the like. These can be used alone or in combination of two or more.

The method for producing the solvent extract of the ganoderma component is not particularly limited, and may be a conventionally known method used in the production of plant solvent extracts.
The solvent extract of the ganoderma component can be obtained, for example, by stirring a mixture of the ganoderma component and the extraction solvent as necessary.
In addition, as a ganoderma component used for solvent extraction, ganoderma and / or its fermented product may be used as it is, or ganoderma and / or its fermented product may be used as a pulverized product, powder, paste or the like. Good.

  In the production of the solvent extract of the ganoderma component, the mixing ratio of the ganoderma component and the extraction solvent is not particularly limited. For example, the weight ratio of the ganoderma component / extraction solvent is preferably 1/2 to 1/100, More preferably, it is 1/2 to 1/50, and more preferably 1/5 to 1/20.

The solvent extraction temperature can be appropriately selected depending on the weight of the mixture of the ganoderma component and the extraction solvent, and is, for example, 15 to 150 ° C.
The solvent extraction time can be appropriately selected depending on the weight of the mixture, the processing temperature, and the like, and is, for example, 0.1 to 48 hours.

  Among the solvent extracts of the ganoderma component, a water extract of the ganoderma component is preferable, and a hot water extract (for example, a product obtained by heating a mixture of a ganoderma component and water at 90 to 130 ° C.) is preferable.

  The extract of the ganoderma component may be further purified or purified (eg, filtration, centrifugation, etc.), sterilized (eg, heat sterilization, high-pressure sterilization, filter sterilization, etc.), diluted, concentrated, and dried as necessary. Freeze-drying etc.), crystallization, recrystallization, etc. may be performed. These treatment methods are not particularly limited, and may be according to conventional methods.

The present invention also includes a composition comprising a ganoderma component (or the agent of the present invention).
Such a composition may be, for example, a food or drink (food composition), a pharmaceutical composition, or the like.

  The food and drink includes health foods and supplements. Examples of health foods include foods intended for health, health maintenance and promotion (for example, approved specific functional foods, so-called health foods without specific functional foods approved), foods for specific health, etc. is there. Examples of supplements include nutritional supplements for supplementing nutrients and the like, nutritional functional foods, health supplements having functions useful for health maintenance, recovery, enhancement, and the like, health functional foods, and the like.

  The composition of the present invention may contain an additive. Additives (other additives) are not particularly limited, and additives used in ordinary foods and pharmaceuticals (for example, excipients, lubricants, disintegrants, binders, solubilizers, sweeteners) , Coloring agents, preservatives, thickeners, antioxidants, fragrances, acidulants, seasonings, emulsifiers, pH adjusters, vitamins, minerals, amino acids, pigments, and the like). The additives may be used alone or in combination of two or more.

  The dosage form of the agent or composition of the present invention is not particularly limited and is, for example, oral administration, parenteral administration, external use, etc., preferably oral administration.

  The dosage form of the agent or composition of the present invention is not particularly limited. In the case of oral administration, for example, it is a solid agent (eg, tablet, coated tablet, granule, powder, pill, troche, capsule, etc.) And liquid agents (for example, elixir, syrup, suspension, etc.). The dosage form for parenteral administration is not particularly limited, and examples thereof include injections, transdermal agents, enteral agents, instillations, external preparations, suppositories, and the like.

The dose (or intake) of the agent or composition of the present invention is not particularly limited, but for example, in terms of the dry weight of the extract of ganoderma components contained in the agent or composition per day for an adult, Preferably, it is about 1-1000 mg / kg (body weight), more preferably about 10-100 mg / kg (body weight). Administration in such an amount is preferable in that the Tie2 activation effect, the angiogenesis suppression effect, the blood vessel maturation effect, the blood vessel normalization effect, the blood vessel stabilization effect, and the like can be obtained effectively.
In addition, the number of administrations (or the number of intakes) of the Tie2 activator is appropriately selected according to the dosage form, the administration subject, etc., and may be a single administration or a continuous administration at a certain interval. . In the case of continuous administration, the administration interval may be once a day to once every several months.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited to these Examples, A various change and correction are possible in the range which does not deviate from the technical scope of this invention.

<Examples 1-3>
[Reishi's solvent extract]
500 g of water was added to 25 g of ganoderma powder obtained by pulverizing ganoderma (red ganoderma, fruit body) and heated at 121 ° C. for 2 hours to extract ganoderma into water. The liquid after extraction was filtered and then frozen to obtain a powder extract of Ganoderma lucidum.

[Cell culture]
Normal human umbilical vein endothelial cells (HUVECs) were cultured in tissue culture low serum medium Humedia-EG2 (manufactured by Kurashiki Boseki Co., Ltd.), seeded in a 6-well plate at 0.5 × 10 ⁶ cells / 2 mL / well, The cells were cultured overnight at 37 ° C. in a 5% CO ₂ environment.
To the cultured HUVEC, the solvent extract of Ganoderma was added to the medium so that the final concentrations would be 100, 300, and 1000 μg / mL, respectively, and further cultured for 30 minutes.

[Tie2 activation evaluation method]
Using the RIPA buffer (Millipore), the HUVEC obtained above was recovered, and the SDS sample buffer [0.25M Tris-HCl (pH 6.8), 10% SDS, 30% glycerol, 0.5M DDT, 0.03% BPB] was added and SDS-PAGE was performed under the following conditions.
Gel: 10% acrylamide gel Electrophoretic conditions: 200 mA, 60 minutes Tie2 antibody: Cell signaling Phospho-Tie2 antibody: Cell signaling Co. Next, Western blotting was performed using iBlot (Invitrogen) PVDF (polyvinylidene fluoride) ) Transferred to the membrane. Blocking with 5% skim milk / TBST for 1 hour, followed by washing 3 times with TBST, followed by addition of Tie2 antibody (a-Tie2) or Phospho-Tie2 antibody (a-pTie2) and antibody reaction for 1 hour, HRP A labeled secondary antibody was added and reacted for 1 hour, and a protein band was detected by chemiluminescence detection (ECL).

Further, as a positive target, Angiopoietin (Ang) 1 (manufactured by R & D systems) was added to the cultured HUVEC so that the final concentration was 100 ng / mL, and then cultured at 37 ° C. in a 5% CO ₂ environment for 30 minutes. Using the cells obtained in this manner, Tie2 activation evaluation was performed in the same manner as described above. Angiopoietin 1 is known to activate (phosphorylate) Tie2.

<Examples 4 to 6>
Instead of Ganoderma solvent extract, add 12 L of water to 800 g of Ganoderma powder, stir for 24 hours at 37 degrees, heat sterilize at 121 ° C. for 15 minutes, filter and extract Was lyophilized to a powder to obtain fermented ganoderma. Water (500 mL) was added to 25 g of this Reishi fermented product and heated at 121 ° C. for 2 hours to extract the Reishi fermented product into water. The liquid after extraction was filtered and then freeze-dried to obtain a powder, and a solvent extract of the fermented ganoderma was obtained.
Tie2 activation evaluation was performed in the same manner as in Examples 1 to 3, except that this was added so that the final concentrations were 100, 300, and 1000 μg / mL.

The Tie2 activation evaluation results of Examples 1 to 6 are shown in FIG. 1 and FIG. In FIG. 2 and Table 1, “pTie2 / Tie2” indicates the detected concentration ratio of Phospho-Tie2 antibody / Tie2 antibody.
Moreover, in FIG.1 and FIG.2, Ang1 shows the result of a positive object. 1 and 2, Ganoderma Ex represents a solvent extract of Ganoderma lucidum, and Fermented ganoderma Ex represents a solvent extract of Ganoderma lucidum fermented product.

As shown in FIG. 1, FIG. 2, and Table 1, it was confirmed that the extract of a ganoderma component phosphorylates Tie2, and has a Tie2 activation effect.
In particular, as shown in Example 5, it is an extremely excellent effect that the Tie2 activation effect is 1.9 times that of the control.

  The agent of the present invention can effectively activate Tie2. Further, the agent of the present invention can be used as an angiogenesis inhibitor, a vascular maturation agent, a vascular normalization agent, a vascular stabilization agent, or the like.

Claims (3)

  A Tie2 activator containing an extract of a reishi fermented product.   An extract of fermented ganoderma extract, for use in at least one application selected from the group consisting of an angiogenesis inhibitor, a vascular maturation agent, a vascular normalization agent, and a vascular stabilization agent Agent.   The agent of Claim 1 or 2 except the case where it is an angiogenesis inhibitor.