WO2024057782A1 - Agent for improving brain function and composition for improving brain function - Google Patents
Agent for improving brain function and composition for improving brain function Download PDFInfo
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- WO2024057782A1 WO2024057782A1 PCT/JP2023/028953 JP2023028953W WO2024057782A1 WO 2024057782 A1 WO2024057782 A1 WO 2024057782A1 JP 2023028953 W JP2023028953 W JP 2023028953W WO 2024057782 A1 WO2024057782 A1 WO 2024057782A1
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- brain function
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- 238000003118 sandwich ELISA Methods 0.000 description 1
- 235000019512 sardine Nutrition 0.000 description 1
- 235000020637 scallop Nutrition 0.000 description 1
- HFHDHCJBZVLPGP-UHFFFAOYSA-N schardinger α-dextrin Chemical compound O1C(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(O)C2O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC2C(O)C(O)C1OC2CO HFHDHCJBZVLPGP-UHFFFAOYSA-N 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 235000012046 side dish Nutrition 0.000 description 1
- 235000011888 snacks Nutrition 0.000 description 1
- 235000010234 sodium benzoate Nutrition 0.000 description 1
- 239000004299 sodium benzoate Substances 0.000 description 1
- WXMKPNITSTVMEF-UHFFFAOYSA-M sodium benzoate Chemical compound [Na+].[O-]C(=O)C1=CC=CC=C1 WXMKPNITSTVMEF-UHFFFAOYSA-M 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 235000014347 soups Nutrition 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 235000013547 stew Nutrition 0.000 description 1
- 230000035882 stress Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- FDDDEECHVMSUSB-UHFFFAOYSA-N sulfanilamide Chemical compound NC1=CC=C(S(N)(=O)=O)C=C1 FDDDEECHVMSUSB-UHFFFAOYSA-N 0.000 description 1
- 229940124530 sulfonamide Drugs 0.000 description 1
- 239000000829 suppository Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 239000003765 sweetening agent Substances 0.000 description 1
- 235000013616 tea Nutrition 0.000 description 1
- 210000002435 tendon Anatomy 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 239000000606 toothpaste Substances 0.000 description 1
- 239000006097 ultraviolet radiation absorber Substances 0.000 description 1
- 235000019154 vitamin C Nutrition 0.000 description 1
- 239000011718 vitamin C Substances 0.000 description 1
- 239000008256 whipped cream Substances 0.000 description 1
- 230000002087 whitening effect Effects 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/192—Carboxylic acids, e.g. valproic acid having aromatic groups, e.g. sulindac, 2-aryl-propionic acids, ethacrynic acid
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
- A61P25/16—Anti-Parkinson drugs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/24—Antidepressants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
Definitions
- the present invention relates to a brain function improving agent and a composition for improving brain function.
- Methods to improve brain function that have been studied include improving the supply of nutrients and oxygen to nerve cells in the brain (e.g., raising intracerebral glucose, improving blood flow, etc.); Improved transmission (supply of neurotransmitter precursors, increased release of neurotransmitters, activation of receptors, inhibition of conversion of released neurotransmitters, etc.).
- neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease
- protein waste products such as amyloid- ⁇ and alpha-synuclein are not removed from the brain and accumulate, and the accumulation of such waste products contributes to the neurodegenerative diseases mentioned above. It is believed that there are.
- the lymphatic system contributes to the removal of protein wastes, but since the lymph system does not exist in the brain, it was thought that protein wastes were broken down and removed in the brain.
- protein waste products such as amyloid ⁇ and ⁇ -synuclein can be efficiently removed from the brain, which can be used to treat Alzheimer's disease, Parkinson's disease, Lewy body dementia, and other diseases. It is expected that this will lead to the prevention, treatment, or improvement of neurodegenerative diseases such as system atrophy.
- Astrocytes have also been shown to play an important role in repairing damaged brain tissue. Around the injured area, astrocytes proliferate and increase in number, minimizing the spread of inflammation by surrounding damaged nerve cells, astrocytes themselves, and inflammatory cells that have entered the injured area. It has been reported that On the other hand, it has been pointed out that the proliferative ability of astrocytes after injury decreases with age. It is also becoming clear that astrocyte dysfunction and a decrease in the number of astrocytes are involved in the onset of neurodegenerative diseases such as Alzheimer's disease and depression symptoms.
- glial cell line-derived neurotrophic factor is a type of protein called neurotrophic factor, and plays a role in regulating the growth, functional maintenance, repair, etc. of nerve cells. Therefore, if the expression of GDNF can be promoted, it is thought that brain function can be improved through growth, function maintenance, repair, etc. of nerve cells.
- astrocytes, microglia, etc. are associated with various brain function disorders.
- microglia become activated, leading to increased production of inflammatory cytokines such as interleukin-1 ⁇ (IL-1 ⁇ ) and inflammation-related factors such as nitric oxide (NO). and cause neurological damage.
- IL-1 ⁇ interleukin-1 ⁇
- NO nitric oxide
- the enhancement or chronicity of neurological disorders is associated with neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, as well as cognitive impairment, depression, etc. (see, for example, Non-Patent Document 3). Therefore, if neurological disorders can be suppressed, cognitive functions (memory ability, learning ability, etc.) that have deteriorated due to neurological disorders due to aging etc.
- Non-Patent Document 4 a neurodegenerative diseases such as Parkinson's disease and Parkinson's disease.
- An object of the present invention is to solve the problems in the conventional art and achieve the following objects. That is, an object of the present invention is to provide a brain function improving agent and a brain function improving composition that have an excellent brain function improving effect and are highly safe.
- ⁇ 1> A brain function improving agent characterized by containing at least one of the compounds represented by any of the following structural formulas (1) to (3).
- ⁇ 2> Promoting astrocyte proliferation, promoting glial cell line-derived neurotrophic factor (GDNF) mRNA expression in astrocytes, promoting aquaporin 4 (AQP4) mRNA expression in astrocytes, suppressing nitric oxide (NO) production in microglia
- GDNF glial cell line-derived neurotrophic factor
- AQP4 aquaporin 4
- NO nitric oxide
- TNF- ⁇ tumor necrosis factor- ⁇
- ⁇ 3> A composition for improving brain function, comprising the brain function improving agent according to any one of ⁇ 1
- the brain function improving agent and brain function improving composition of the present invention it is possible to solve the above-mentioned problems in the past, achieve the above-mentioned objectives, have an excellent brain function-improving effect, and be safe.
- the brain function improving agent of the present invention contains at least one of the compounds represented by any of the following structural formulas (1) to (3) as an active ingredient, and further contains other ingredients as necessary.
- improving brain function does not only mean improving the function of a brain whose function has decreased, but also includes preventing a decrease in brain function and enhancing brain function. It will be done.
- the brain function-improving effects of the compounds represented by structural formulas (1) to (3) include, for example, astrocyte proliferation-promoting action and glial cell line-derived neurotrophic factor (GDNF) mRNA expression promoting action in astrocytes. , promoting aquaporin 4 (AQP4) mRNA expression in astrocytes, suppressing nitric oxide (NO) production in microglia, suppressing tumor necrosis factor- ⁇ (TNF- ⁇ ) production in microglia, and inflammation-related gene mRNA expression in microglia. It is preferable that the effect is exerted based on one or more types of effects selected from the group consisting of suppressive effects.
- the brain function-improving effects of the compounds represented by structural formulas (1) to (3) are not limited to the brain function-improving effects exerted based on the above-mentioned effects.
- Inflammation-related genes in microglia include, for example, tumor necrosis factor- ⁇ (TNF- ⁇ ), inducible nitric oxide synthase (iNOS), interleukin-6 (IL-6), and interleukin-1 ⁇ (IL- 1 ⁇ ), interleukin-12 (IL-12), and chemokine (CXC motif) ligand 2 (CXCL2).
- TNF- ⁇ tumor necrosis factor- ⁇
- iNOS inducible nitric oxide synthase
- IL-6 interleukin-6
- IL-1 ⁇ interleukin-1 ⁇
- IL-12 interleukin-12
- CXC motif chemokine ligand 2
- the name of the compound represented by the following structural formula (2) is 3-(4-hydroxyphenyl)propionic acid (English name: 3-(4-Hydroxyphenyl)propionic acid).
- the name of the compound represented by the following structural formula (3) is 3-phenylpropionic acid (English name: 3-Phenylpropionic acid).
- the compounds represented by any of the structural formulas (1) to (3) above are all known compounds, and commercially available products may be used, or those extracted from plants etc. may be used.
- the compounds represented by any of the structural formulas (1) to (3) may be used alone or in combination of two or more.
- the brain function improving agent may consist of at least one compound represented by any one of the structural formulas (1) to (3), or may include only one compound represented by any one of the structural formulas (1) to (3). ) may be a formulation of at least one compound represented by any of the following.
- the compound represented by any of the structural formulas (1) to (3) can be prepared into powder or granules using a conventional method using a pharmaceutically acceptable carrier such as dextrin or cyclodextrin or any other auxiliary agent. It can be formulated into any desired dosage form, such as solid or liquid.
- a pharmaceutically acceptable carrier such as dextrin or cyclodextrin or any other auxiliary agent. It can be formulated into any desired dosage form, such as solid or liquid.
- auxiliary agent for example, an excipient, a binder, a disintegrant, a lubricant, a stabilizer, a flavoring agent, etc. can be used.
- the brain function improving agent can be used in combination with other compositions (for example, compositions for improving brain function described below), and can also be used in tablets, powders, capsules, granules, extracts, It can also be used as oral preparations such as syrup; parenteral preparations such as injections, drops, and suppositories; ointments, eye drops, external solutions, and patches.
- compositions for improving brain function described below can also be used in tablets, powders, capsules, granules, extracts, It can also be used as oral preparations such as syrup; parenteral preparations such as injections, drops, and suppositories; ointments, eye drops, external solutions, and patches.
- the total content of the compounds represented by any of the structural formulas (1) to (3) in the formulated brain function improving agent is not particularly limited and can be appropriately selected depending on the purpose.
- the other ingredients are not particularly limited as long as they do not impair the effects of the present invention, and can be appropriately selected depending on the usage form of the brain function improving agent, such as excipients, moisture proofing agents, and preservatives.
- agent strengthener, thickener, emulsifier, antioxidant, sweetener, acidulant, seasoning, colorant, fragrance, whitening agent, humectant, oily component, ultraviolet absorber, surfactant, thickener
- examples include alcohols, powder components, colorants, aqueous components, water, and skin nutrients. These may be used alone or in combination of two or more.
- the content of the other components in the brain function improving agent is not particularly limited and can be appropriately selected depending on the purpose.
- the usage of the brain function improving agent is not particularly limited and can be appropriately selected depending on the purpose, and examples include oral, parenteral, and external usage.
- the dosage form of the brain function improving agent is not particularly limited, and known dosage forms can be appropriately selected depending on the purpose.
- the method for producing the brain function improving agent in any dosage form is not particularly limited, and any known method can be selected as appropriate.
- the administration method, dosage, administration site, administration period, administration interval, etc. of the brain function improving agent are not particularly limited and can be appropriately selected depending on the purpose.
- the brain function improving agent increases the efficiency of removal and excretion of protein wastes in the brain by the glymphatic system through the brain function improving effect of the compound represented by any of the structural formulas (1) to (3). It also suppresses neurological disorders and further supports, regulates, or protects nerve cells, and through these actions, improves memory and learning abilities; prevents, treats, or improves amnesia and senile cognitive dysfunction; Alzheimer's disease. It can be used for the prevention, treatment, or amelioration of neurodegenerative diseases such as Parkinson's disease, and for the prevention, treatment, or amelioration of mood disorders such as depression.
- the brain function-improving agent of the present invention can be used in all other uses in which it is meaningful to exhibit a brain function-improving effect in addition to these uses.
- the brain function improving agent has an excellent brain function improving effect and is highly safe, so it can be used in a wide range of applications such as medicines, quasi-drugs, food and drinks, etc. It can be suitably used as an active ingredient of a composition for functional improvement.
- at least one compound represented by any one of the above structural formulas (1) to (3) may be blended as is, or a compound represented by any one of the above structural formulas (1) to (3) may be blended as is.
- a formulation of at least one of the compounds listed above may be blended.
- the above-mentioned brain function improving agent can be prepared by blending other ingredients having a brain function-improving effect with the compound represented by any of the structural formulas (1) to (3) above, as necessary. It can also be used as
- the brain function improving agent of the present invention is suitably applied to humans, but as long as its effects are achieved, it can also be applied to animals other than humans (e.g. mice, rats, hamsters, dogs, cats, etc.). It can also be applied to cows, pigs, monkeys, etc.).
- the brain function improving agent of the present invention can also be used as a reagent for research on the mechanism of action of improving brain function.
- composition for improving brain function of the present invention contains the brain function improving agent of the present invention, and further contains other components as necessary.
- the brain function improving agent is the brain function improving agent of the present invention described above.
- the content of the brain function improving agent in the brain function improving composition is not particularly limited and can be adjusted as appropriate depending on the form of the brain function improving composition.
- the amount is preferably 0.0001% by mass to 30% by mass, more preferably 0.0001% by mass to 10% by mass, in terms of the total amount of the compounds represented by any of 1) to (3).
- the brain function improving composition may consist only of the brain function improving agent.
- composition for improving brain function are not particularly limited and can be appropriately selected depending on the usage form of the composition for improving brain function, such as the above-mentioned brain function improving agent.
- Other ingredients similar to those listed in the section above may be mentioned. These may be used alone or in combination of two or more.
- the content of the other components in the composition for improving brain function is not particularly limited and can be appropriately selected depending on the purpose.
- composition for improving brain function is not particularly limited and can be appropriately selected depending on the purpose, and includes, for example, pharmaceuticals, quasi-drugs, food and drink products, and the like.
- the composition for improving brain function of the present invention can be used on a daily basis and improves brain function by the action of the active ingredient, a compound represented by any one of the structural formulas (1) to (3). It can extremely effectively exhibit various physiologically active effects, including the function-improving effect of .
- composition for improving brain function of the present invention is suitably applied to humans, but it can also be applied to animals other than humans (e.g. mice, rats, hamsters, dogs) as long as the respective effects are achieved. , cats, cows, pigs, monkeys, etc.).
- composition for improving brain function of the present invention is not particularly limited and can be appropriately selected depending on the purpose, and examples include oral, parenteral, and external methods, but oral is preferred. .
- Examples of the oral composition include oral preparations and food and drink products.
- food and beverages refer to foods that have little risk of harming human health and are ingested orally or through gastrointestinal administration in normal social life, and are administratively classified foods, drugs, and quasi-drugs. It is not limited to such categories. Therefore, the above-mentioned foods and drinks include general foods that are orally ingested, health foods (functional foods and drinks), foods with health claims (foods for specified health uses, foods with nutritional function claims, foods with functional claims), quasi-drugs, and pharmaceuticals. This term refers to a wide range of food and beverages that make up food and beverages.
- the type of the oral composition is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include beverages such as tea beverages, soft drinks, carbonated beverages, nutritional beverages, fruit beverages, lactic acid beverages, alcoholic beverages, coffee beverages, and coffee-containing soft drinks (including concentrated liquids and powders for adjusting these beverages); cold desserts such as ice cream, ice sorbet, 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, jellies, jams, creams, baked goods, and bread; seafood such as crab, salmon, clams, tuna, sardines, shrimp, bonito, mackerel, whales, oysters, pacific saury, squid, ark shells, scallops, abalone, sea urchins, salmon roe, and tokobushi; kamaboko
- processed seafood and livestock foods such as sausages and 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, mapo tofu, 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, powders, capsules, granules, extracts, syrups, drinks, lozenges, and mouthwash
- oral fresheners and toothpastes used in the oral cavity such as mouth fresheners and breath fresheners.
- the method for producing the composition for improving brain function is not particularly limited, and can be appropriately selected depending on the usage form of the composition for improving brain function.
- the amount, period of use, interval of use, etc. of the composition for improving brain function are not particularly limited and can be appropriately selected depending on the purpose.
- the present invention also relates to a method for improving brain function, which comprises administering to an individual at least one selected from the group consisting of the brain function improving agent and the composition for improving brain function. .
- the compounds represented by any of the structural formulas (1) to (3) have an effect of promoting astrocyte proliferation, an effect of promoting GDNF mRNA expression in astrocytes, an effect of promoting AQP4 mRNA expression in astrocytes, and an effect of promoting NO in microglia.
- Astrocyte proliferation promoter, GDNF mRNA expression promoter in astrocytes, and AQP4 mRNA expression promoter in astrocytes by utilizing production suppressing effect, TNF- ⁇ production suppressing effect in microglia, or inflammation-related gene mRNA expression suppressing effect in microglia. It may be used as an active ingredient of an agent, an agent for suppressing NO production in microglia, an agent for suppressing TNF- ⁇ production in microglia, or an agent for suppressing inflammation-related gene mRNA expression in microglia.
- the present invention also provides a method for promoting proliferation of astrocytes, which comprises administering to an individual at least one of the compounds represented by any one of the structural formulas (1) to (3).
- a method of promoting the expression of GDNF mRNA in astrocytes a method of promoting the expression of AQP4 mRNA in astrocytes, a method of suppressing NO production in microglia, a method of suppressing TNF- ⁇ production in microglia, or an inflammation-related method in microglia.
- the present invention also relates to a method of suppressing gene mRNA expression.
- test examples and formulation examples of the present invention will be explained, but the present invention is not limited to these test examples and formulation examples.
- Test sample In each test example described below, the following compounds were used as test samples. - Compound represented by the above structural formula (1) (manufactured by SIGMA) - Compound represented by the above structural formula (2) (manufactured by SIGMA) - Compound represented by the above structural formula (3) (manufactured by Tokyo Chemical Industry Co., Ltd.)
- a mouse-derived astrocyte culture strain (C8-S) was cultured using DMEM containing 10% by mass of FBS, and then the cells were collected by trypsin treatment. The collected cells were diluted with DMEM containing 10 mass% FBS to a concentration of 2.5 x 10 4 cells/mL, then seeded at 100 ⁇ L per well in a 96-well plate, and incubated at 37°C under 5% CO 2 The cells were cultured for 6 hours.
- test sample dissolved in DMEM containing 10% by mass FBS (see Table 1 below for final sample concentration) was added to each well and cultured for 4 days.
- DMEM containing 10% by mass FBS 100 ⁇ L of DMEM containing 10% by mass FBS without addition of the test sample was added and cultured in the same manner.
- a mouse-derived astrocyte culture strain (C8-S) was cultured using DMEM containing 10% by mass of FBS, and then the cells were collected by trypsin treatment. The collected cells were diluted with DMEM containing 10% by mass FBS to a concentration of 5.0 x 10 4 cells/mL, then seeded at 2 mL per well in a 6-well plate, and incubated at 37°C under 5% CO 2 The cells were cultured until confluent.
- RNA After culturing, remove the culture medium, extract total RNA using RNeasy (registered trademark) mini kit (manufactured by Qiagen), calculate the amount of RNA from the absorbance at a wavelength of 260 nm, and adjust the total RNA to 100 ng/ ⁇ L. did.
- RNeasy registered trademark
- mini kit manufactured by Qiagen
- GDNF and GAPDH mRNA which was an internal standard, were measured. Detection of mRNA was performed using a real-time PCR device Thermal Cycler Dice (registered trademark) Real Time System III (manufactured by TaKaRa) using PrimeScript TM RT Master Mix (Perfect Real Time) and TB Gr een (registered trademark) Fast qPCR Mix (manufactured by TaKaRa) ) was carried out using a two-step real-time RT-PCR reaction. The expression level of GDNF mRNA was calculated by correcting the expression level of GAPDH mRNA.
- the promotion rate (%) of GDNF mRNA expression was calculated using the following formula.
- GDNF mRNA expression promotion rate (%) C/D x 100 C to D in the above formula each represent the following.
- a mouse-derived astrocyte culture strain (C8-S) was cultured using DMEM containing 10% by mass of FBS, and then the cells were collected by trypsin treatment. The collected cells were diluted with DMEM containing 10 mass% FBS to a concentration of 5.0 x 10 4 cells/mL, then seeded in 2 mL per well in a 6-well plate, and incubated at 37°C under 5% CO 2 The cells were cultured until confluent.
- test sample (see Table 3 below for final sample concentration) dissolved in DMEM containing 10 mass% FBS containing a final concentration of 0.5% DMSO was added to each well. Cultured for hours. As a control, 2 mL of DMEM containing 10 mass % FBS containing 0.5% DMSO and no test sample was added and cultured in the same manner.
- RNA After culturing, remove the culture medium, extract total RNA using RNeasy (registered trademark) mini kit (manufactured by Qiagen), calculate the amount of RNA from the absorbance at a wavelength of 260 nm, and adjust the total RNA to 100 ng/ ⁇ L. did.
- RNeasy registered trademark
- mini kit manufactured by Qiagen
- RNA was analyzed for the expression levels of AQP4 and GAPDH mRNA, which was an internal standard. Detection of mRNA was performed using a real-time PCR device Thermal Cycler Dice (registered trademark) Real Time System III (manufactured by TaKaRa) using PrimeScript TM RT Master Mix (Perfect Real Time) and TB Gr een (registered trademark) Fast qPCR Mix (manufactured by TaKaRa) ) was carried out using a two-step real-time RT-PCR reaction. The expression level of AQP4 mRNA was calculated by correcting the expression level of GAPDH mRNA.
- Thermal Cycler Dice registered trademark
- Real Time System III manufactured by TaKaRa
- PrimeScript TM RT Master Mix Perfect Real Time
- TB Gr een registered trademark
- Fast qPCR Mix manufactured by TaKaRa
- the AQP4 mRNA expression promotion rate (%) was calculated using the following formula.
- AQP4 mRNA expression promotion rate (%) E/F x 100
- E to F in the above formula each represent the following.
- a mouse-derived microglia culture strain (C8-B4) was cultured using DMEM containing 10% by mass of FBS, and then the cells were collected by trypsin treatment. The collected cells were diluted with DMEM containing 10 mass% FBS to a concentration of 1.0 x 10 5 cells/mL, then seeded at 100 ⁇ L per well in a 96-well plate, and incubated at 37°C under 5% CO 2 The cells were cultured until confluent.
- LPS Lipopolysaccharide
- IFN- ⁇ interferon-gamma
- the amount of nitric oxide (NO) produced was measured using the amount of nitrite ion (NO 2 ⁇ ) as an index. Specifically, after the completion of the culture, the same amount of Griss reagent (5% by mass phosphoric acid solution containing 1% by mass sulfanilamide and 0.1% by mass N-1-naphthyl ethylenediamine dihydrochloride) was added to 100 ⁇ L of the culture solution in each well. was added and reacted for 10 minutes at room temperature. After the reaction, absorbance at a wavelength of 540 nm was measured. A calibration curve was created using NO 2 - as an indicator, and the amount of NO produced in the culture supernatant was determined.
- Griss reagent 5% by mass phosphoric acid solution containing 1% by mass sulfanilamide and 0.1% by mass N-1-naphthyl ethylenediamine dihydrochloride
- TNF- ⁇ Tumor necrosis factor- ⁇ (TNF- ⁇ ) production inhibition test in microglia
- a mouse-derived microglia culture strain (C8-B4) was cultured using DMEM containing 10% by mass of FBS, and then the cells were collected by trypsin treatment. The collected cells were diluted with DMEM containing 10 mass% FBS to a concentration of 1.0 x 10 5 cells/mL, then seeded at 100 ⁇ L per well in a 96-well plate, and incubated at 37°C under 5% CO 2 The cells were cultured until confluent.
- LPS Lipopolysaccharide
- TNF- ⁇ production inhibition rate (%) ⁇ (J-I)/J ⁇ 100 I to J in the above formula each represent the following. I: TNF- ⁇ amount when test sample is added J: TNF- ⁇ amount when test sample is not added
- a mouse-derived microglia culture strain (C8-B4) was cultured using DMEM containing 10% by mass of FBS, and then the cells were collected by trypsin treatment. The collected cells were diluted with DMEM containing 10 mass% FBS to a concentration of 1.0 x 10 5 cells/mL, then seeded in 2 mL per well in a 6-well plate, and incubated at 37°C under 5% CO 2 The cells were cultured until confluent.
- LPS Lipopolysaccharide
- FBS interferon-gamma
- TNF- ⁇ tumor necrosis factor- ⁇
- iNOS inducible nitric oxide synthase
- IL-6 interleukin-6
- IL-1 ⁇ interleukin-1 ⁇
- IL-12 interleukin-12
- CXC motif chemokine ligand 2
- Detection of mRNA was performed using a real-time PCR device Thermal Cycler Dice (registered trademark) Real Time System III (manufactured by TaKaRa) using PrimeScript TM RT Master Mix (Perfect Real Time) and TB Gr een (registered trademark) Fast qPCR Mix (manufactured by TaKaRa) ) was carried out using a two-step real-time RT-PCR reaction.
- TNF- ⁇ , iNOS, IL-6, IL-1 ⁇ , IL12, and CXCL2 are known to be inflammation-inducing factors, and when stimulated with LPS and IFN- ⁇ , they are The expression of these mRNAs was increased compared to .
- mRNA expression rate (%) of each gene K/L x 100 K to L in the above formula each represent the following. K: Correction value when test sample is added, LPS and IFN- ⁇ stimulation L: Correction value when test sample is not added, LPS and IFN- ⁇ stimulation
- Combination example 1 Tablets having the following composition were manufactured by a conventional method.
- - Compound represented by the above structural formula (1) 5.0 mg ⁇ Dolomite 83.4mg (Contains 20% calcium and 10% magnesium) ⁇ Casein phosphopeptide 16.7mg ⁇ Vitamin C 33.4mg ⁇ Maltitol 136.8mg ⁇ Collagen 12.7mg ⁇ Sucrose fatty acid ester 12.0mg
- Combination example 2 An oral liquid preparation having the following composition was produced by a conventional method. ⁇ Composition in 1 ampoule (100 mL per bottle)> - Compound represented by the above structural formula (2) 0.3% by mass ⁇ Sorvit 12.0% by mass ⁇ Sodium benzoate 0.1% by mass ⁇ Fragrance 1.0% by mass ⁇ Calcium sulfate 0.5% by mass ⁇ Remaining purified water
- Combination example 4 Capsules having the following composition were manufactured by a conventional method. Note that a No. 1 hard gelatin capsule was used as the capsule. ⁇ Composition in 1 capsule (1 tablet 200mg)> - Compound represented by the above structural formula (1) 30.0 mg ⁇ Corn starch 70.0mg ⁇ Lactose 80.0mg ⁇ Calcium lactate 10.0mg ⁇ Hydroxypropylcellulose (HPC-L) 10.0mg
Abstract
This agent for improving brain function comprises at least any of compounds represented by structural formulae (1)-(3).
Description
本発明は、脳の機能改善剤及び脳の機能改善用組成物に関する。
The present invention relates to a brain function improving agent and a composition for improving brain function.
高齢化社会となりつつある近年においては、心身ともに健康でありたいという願望がますます高まっている。ヒトは、高齢になるにつれ記憶力や学習能力が徐々に衰えていく。また、アルツハイマー病やパーキンソン病等においては、神経の変性による認知機能の障害が大きな問題となっている。一方、仕事環境、家庭的事情、人間関係などの様々なストレスによるうつ病等の気分障害をはじめ、高齢でなくとも脳の機能に問題を抱えている患者数は年々増加している。
In recent years, as society has become an aging society, the desire to be healthy both physically and mentally is increasing. As humans grow older, their memory and learning abilities gradually decline. Furthermore, in Alzheimer's disease, Parkinson's disease, and the like, impairment of cognitive function due to neurodegeneration is a major problem. On the other hand, the number of patients who are not elderly and are suffering from brain function problems, including mood disorders such as depression due to various stresses such as work environment, family circumstances, and human relationships, is increasing year by year.
従来から研究されている脳の機能の改善方法としては、脳における神経細胞への栄養や酸素の供給の改善（例えば、脳内グルコースの上昇、血流の改善等）；シナプス間隙で行われる神経伝達の改善（神経伝達物質の前駆体の供給、神経伝達物質の放出の増加、受容体の活性化、放出された神経伝達物質の変換阻害等）などが挙げられる。
Methods to improve brain function that have been studied include improving the supply of nutrients and oxygen to nerve cells in the brain (e.g., raising intracerebral glucose, improving blood flow, etc.); Improved transmission (supply of neurotransmitter precursors, increased release of neurotransmitters, activation of receptors, inhibition of conversion of released neurotransmitters, etc.).
アルツハイマー病やパーキンソン病等の神経変性疾患においては、アミロイドβやαシヌクレイン等のタンパク質老廃物が脳から除去されずに蓄積し、かかる老廃物の蓄積が前述した神経変性疾患の一因になっていると考えられている。身体の他の部位ではリンパ系がタンパク質老廃物の除去に寄与しているが、リンパ系は脳には存在しないため、脳においてはタンパク質老廃物は分解除去されているものと考えられていた。しかし、近年、脳において、血管周囲腔やアストロサイトを流れる脳脊髄液等がタンパク質老廃物の除去に寄与していること、アストロサイトに高発現した水チャネルであるアクアポリン４（ＡＱＰ４）が脳脊髄液等の流量に大きく寄与していることが発見された（例えば、非特許文献１参照）。さらに、睡眠や麻酔時には脳脊髄液等の流量が増加しアミロイドβの除去速度も高まることが報告され（例えば、非特許文献２参照）、これら一連の脳内経路は「グリンパティック系」と名付けられ注目を集めている。そのため、かかるグリンパティック系の流量を増加させることができれば、アミロイドβやαシヌクレイン等のタンパク質老廃物を脳から効率的に除去することができ、アルツハイマー病、パーキンソン病、レビー小体型認知症、多系統萎縮症といった神経変性疾患の予防、治療又は改善につながるものと期待されている。
In neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, protein waste products such as amyloid-β and alpha-synuclein are not removed from the brain and accumulate, and the accumulation of such waste products contributes to the neurodegenerative diseases mentioned above. It is believed that there are. In other parts of the body, the lymphatic system contributes to the removal of protein wastes, but since the lymph system does not exist in the brain, it was thought that protein wastes were broken down and removed in the brain. However, in recent years, it has been discovered that in the brain, cerebrospinal fluid flowing through the perivascular space and astrocytes contributes to the removal of protein wastes, and that aquaporin 4 (AQP4), a water channel highly expressed in astrocytes, It was discovered that it greatly contributes to the flow rate of liquid etc. (for example, see Non-Patent Document 1). Furthermore, it has been reported that during sleep and anesthesia, the flow rate of cerebrospinal fluid increases and the removal rate of amyloid β also increases (see, for example, Non-Patent Document 2), and this series of intracerebral pathways has been named the "glymphatic system." It is attracting attention. Therefore, if the flow rate of the glymphatic system can be increased, protein waste products such as amyloid β and α-synuclein can be efficiently removed from the brain, which can be used to treat Alzheimer's disease, Parkinson's disease, Lewy body dementia, and other diseases. It is expected that this will lead to the prevention, treatment, or improvement of neurodegenerative diseases such as system atrophy.
アストロサイトは、損傷を受けた脳組織の修復においても重要な働きをもつことが明らかになっている。損傷部周囲では、アストロサイトが増殖して数を増やし、損傷部でダメージを受けた神経細胞、アストロサイト自身や損傷部に進入した炎症細胞などを取り囲むことで、炎症の拡大を最小限にとどめていることが報告されている。一方、損傷後のアストロサイトの増殖能は加齢とともに低下することが指摘されている。
また、アストロサイトの機能異常やアストロサイト数の減少がアルツハイマー病やうつ症状などの神経変性疾患の発症に関与することも明らかになりつつある。
そのため、アストロサイトの増殖を促進することができれば、頭部外傷や脳梗塞などによる脳損傷時に神経組織が受けるダメージを最小限に食い止め、再生を促したり、アルツハイマー病やうつ症状などの神経変性疾患の予防、治療又は改善につながると考えられる。 Astrocytes have also been shown to play an important role in repairing damaged brain tissue. Around the injured area, astrocytes proliferate and increase in number, minimizing the spread of inflammation by surrounding damaged nerve cells, astrocytes themselves, and inflammatory cells that have entered the injured area. It has been reported that On the other hand, it has been pointed out that the proliferative ability of astrocytes after injury decreases with age.
It is also becoming clear that astrocyte dysfunction and a decrease in the number of astrocytes are involved in the onset of neurodegenerative diseases such as Alzheimer's disease and depression symptoms.
Therefore, if we can promote the proliferation of astrocytes, it will be possible to minimize damage to neural tissue during brain damage caused by head trauma or cerebral infarction, and promote regeneration, as well as neurodegenerative diseases such as Alzheimer's disease and depression symptoms. It is thought that this will lead to the prevention, treatment, or improvement of.
また、アストロサイトの機能異常やアストロサイト数の減少がアルツハイマー病やうつ症状などの神経変性疾患の発症に関与することも明らかになりつつある。
そのため、アストロサイトの増殖を促進することができれば、頭部外傷や脳梗塞などによる脳損傷時に神経組織が受けるダメージを最小限に食い止め、再生を促したり、アルツハイマー病やうつ症状などの神経変性疾患の予防、治療又は改善につながると考えられる。 Astrocytes have also been shown to play an important role in repairing damaged brain tissue. Around the injured area, astrocytes proliferate and increase in number, minimizing the spread of inflammation by surrounding damaged nerve cells, astrocytes themselves, and inflammatory cells that have entered the injured area. It has been reported that On the other hand, it has been pointed out that the proliferative ability of astrocytes after injury decreases with age.
It is also becoming clear that astrocyte dysfunction and a decrease in the number of astrocytes are involved in the onset of neurodegenerative diseases such as Alzheimer's disease and depression symptoms.
Therefore, if we can promote the proliferation of astrocytes, it will be possible to minimize damage to neural tissue during brain damage caused by head trauma or cerebral infarction, and promote regeneration, as well as neurodegenerative diseases such as Alzheimer's disease and depression symptoms. It is thought that this will lead to the prevention, treatment, or improvement of.
また、グリア細胞株由来神経栄養因子（ＧＤＮＦ）は、神経栄養因子と呼ばれるタンパク質の一種であり、神経細胞の成長、機能維持、修復などを調節する役割を担っている。
そのため、ＧＤＮＦの発現を促進することができれば、神経細胞の成長、機能維持、修復などを通じて、脳の機能を改善することができると考えられる。 Furthermore, glial cell line-derived neurotrophic factor (GDNF) is a type of protein called neurotrophic factor, and plays a role in regulating the growth, functional maintenance, repair, etc. of nerve cells.
Therefore, if the expression of GDNF can be promoted, it is thought that brain function can be improved through growth, function maintenance, repair, etc. of nerve cells.
そのため、ＧＤＮＦの発現を促進することができれば、神経細胞の成長、機能維持、修復などを通じて、脳の機能を改善することができると考えられる。 Furthermore, glial cell line-derived neurotrophic factor (GDNF) is a type of protein called neurotrophic factor, and plays a role in regulating the growth, functional maintenance, repair, etc. of nerve cells.
Therefore, if the expression of GDNF can be promoted, it is thought that brain function can be improved through growth, function maintenance, repair, etc. of nerve cells.
また、アストロサイトやミクログリア等の活性化が、様々な脳の機能の障害に関連することが明らかになりつつある。例えば、老化した脳においては、ミクログリアが活性化した状態となって、インターロイキン－１β（ＩＬ－１β）等の炎症性サイトカインや一酸化窒素（ＮＯ）などの炎症関連因子の産生亢進が起こっており、神経障害の原因となる。ここで、神経障害の亢進又は慢性化は、認知障害、抑うつ等のほか、アルツハイマー病やパーキンソン病等の神経変性疾患と関連があることが分かっている（例えば、非特許文献３参照）。そのため、神経障害を抑制することができれば、老化等による神経障害に起因して低下した認知機能（記憶能力や学習能力等）を改善させることができ、また認知機能障害、気分障害、さらにはアルツハイマー病やパーキンソン病等の神経変性疾患の予防、治療又は改善につながるものと考えられている（例えば、非特許文献４参照）。
Additionally, it is becoming clear that activation of astrocytes, microglia, etc. is associated with various brain function disorders. For example, in the aging brain, microglia become activated, leading to increased production of inflammatory cytokines such as interleukin-1β (IL-1β) and inflammation-related factors such as nitric oxide (NO). and cause neurological damage. Here, it is known that the enhancement or chronicity of neurological disorders is associated with neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, as well as cognitive impairment, depression, etc. (see, for example, Non-Patent Document 3). Therefore, if neurological disorders can be suppressed, cognitive functions (memory ability, learning ability, etc.) that have deteriorated due to neurological disorders due to aging etc. can be improved, and cognitive dysfunction, mood disorders, and even Alzheimer's disease can be improved. It is believed that this will lead to the prevention, treatment, or improvement of neurodegenerative diseases such as Parkinson's disease and Parkinson's disease (for example, see Non-Patent Document 4).
一方、食品成分が脳の機能に影響を及ぼすことが近年の研究で明らかになりつつあり、脳の機能改善、抗うつ、抗認知症などに関する効果を有する食品成分が注目されている。例えば、イチョウ葉エキスについては、脳の細胞の活性化作用等が知られている（例えば、特許文献１参照）。
On the other hand, recent research has revealed that food ingredients affect brain function, and food ingredients that have effects on improving brain function, anti-depression, anti-dementia, etc. are attracting attention. For example, ginkgo biloba extract is known to have an activating effect on brain cells (see, for example, Patent Document 1).
しかしながら、脳の機能改善作用を有し、かつ安全性が高く、そのため、飲食品、医薬品、研究用試薬などの成分として広く利用が可能な新たな素材に対する要望は依然として強く、その速やかな開発が求められているのが現状である。
However, there is still a strong demand for new materials that improve brain function, are highly safe, and can be widely used as ingredients in foods and drinks, pharmaceuticals, research reagents, etc., and rapid development is essential. This is what is currently required.
本発明は、前記従来における諸問題を解決し、以下の目的を達成することを課題とする。即ち、本発明は、優れた脳の機能改善作用を有し、かつ安全性が高い脳の機能改善剤及び脳の機能改善用組成物を提供することを目的とする。
An object of the present invention is to solve the problems in the conventional art and achieve the following objects. That is, an object of the present invention is to provide a brain function improving agent and a brain function improving composition that have an excellent brain function improving effect and are highly safe.
前記課題を解決するために本発明者らが鋭意検討を重ねた結果、下記構造式（１）～（３）のいずれかで表される化合物が、優れた脳の機能改善作用を有し、かつ安全性が高く、脳の機能改善に有用であることを知見した。
As a result of extensive studies by the present inventors to solve the above problems, it was found that a compound represented by any of the following structural formulas (1) to (3) has an excellent effect on improving brain function, They also found that it is highly safe and useful for improving brain function.
本発明は、本発明者らの前記知見に基づくものであり、前記課題を解決するための手段としては、以下の通りである。即ち、
＜１＞ 下記構造式（１）～（３）のいずれかで表される化合物の少なくともいずれかを含むことを特徴とする脳の機能改善剤である。
＜２＞ アストロサイト増殖促進作用、アストロサイトにおけるグリア細胞株由来神経栄養因子（ＧＤＮＦ）ｍＲＮＡ発現促進作用、アストロサイトにおけるアクアポリン４（ＡＱＰ４）ｍＲＮＡ発現促進作用、ミクログリアにおける一酸化窒素（ＮＯ）産生抑制作用、ミクログリアにおける腫瘍壊死因子－α（ＴＮＦ－α）産生抑制作用、及びミクログリアにおける炎症関連遺伝子ｍＲＮＡ発現抑制作用からなる群から選択される１種以上の作用に基づく脳の機能改善用途に用いられる前記＜１＞に記載の脳の機能改善剤である。
＜３＞ 前記＜１＞から＜２＞のいずれかに記載の脳の機能改善剤を含むことを特徴とする脳の機能改善用組成物である。 The present invention is based on the above findings of the present inventors, and means for solving the above problems are as follows. That is,
<1> A brain function improving agent characterized by containing at least one of the compounds represented by any of the following structural formulas (1) to (3).
<2> Promoting astrocyte proliferation, promoting glial cell line-derived neurotrophic factor (GDNF) mRNA expression in astrocytes, promoting aquaporin 4 (AQP4) mRNA expression in astrocytes, suppressing nitric oxide (NO) production in microglia It is used for purposes of improving brain function based on one or more effects selected from the group consisting of the following: action, suppressing effect on tumor necrosis factor-α (TNF-α) production in microglia, and suppressing effect on inflammation-related gene mRNA expression in microglia. The brain function improving agent according to <1> above.
<3> A composition for improving brain function, comprising the brain function improving agent according to any one of <1> to <2>.
＜１＞ 下記構造式（１）～（３）のいずれかで表される化合物の少なくともいずれかを含むことを特徴とする脳の機能改善剤である。
＜３＞ 前記＜１＞から＜２＞のいずれかに記載の脳の機能改善剤を含むことを特徴とする脳の機能改善用組成物である。 The present invention is based on the above findings of the present inventors, and means for solving the above problems are as follows. That is,
<1> A brain function improving agent characterized by containing at least one of the compounds represented by any of the following structural formulas (1) to (3).
<3> A composition for improving brain function, comprising the brain function improving agent according to any one of <1> to <2>.
本発明の脳の機能改善剤及び脳の機能改善用組成物によると、従来における前記諸問題を解決し、前記目的を達成することができ、優れた脳の機能改善作用を有し、かつ安全性が高い脳の機能改善剤及び脳の機能改善用組成物を提供することができる。
According to the brain function improving agent and brain function improving composition of the present invention, it is possible to solve the above-mentioned problems in the past, achieve the above-mentioned objectives, have an excellent brain function-improving effect, and be safe. Thus, it is possible to provide a brain function improving agent and a composition for improving brain function with high efficacy.
（脳の機能改善剤）
本発明の脳の機能改善剤は、下記構造式（１）～（３）のいずれかで表される化合物の少なくともいずれかを有効成分として含み、更に必要に応じてその他の成分を含む。
(Brain function improving agent)
The brain function improving agent of the present invention contains at least one of the compounds represented by any of the following structural formulas (1) to (3) as an active ingredient, and further contains other ingredients as necessary.
本発明の脳の機能改善剤は、下記構造式（１）～（３）のいずれかで表される化合物の少なくともいずれかを有効成分として含み、更に必要に応じてその他の成分を含む。
The brain function improving agent of the present invention contains at least one of the compounds represented by any of the following structural formulas (1) to (3) as an active ingredient, and further contains other ingredients as necessary.
本明細書において、脳の機能を改善するとは、機能が低下した脳においてその機能を向上させることだけではなく、脳の機能が低下することを防いだり、脳の機能を高めたりすることも含まれる。
In this specification, improving brain function does not only mean improving the function of a brain whose function has decreased, but also includes preventing a decrease in brain function and enhancing brain function. It will be done.
前記構造式（１）～（３）で表される化合物が、優れた脳の機能改善作用を有し、脳の機能改善剤として有用であることは、従来は全く知られておらず、本発明者らによる新たな知見である。
It was not previously known that the compounds represented by the above structural formulas (1) to (3) have excellent brain function-improving effects and are useful as brain function-improving agents. This is a new finding by the inventors.
前記構造式（１）～（３）で表される化合物が有する脳の機能改善作用としては、例えば、アストロサイト増殖促進作用、アストロサイトにおけるグリア細胞株由来神経栄養因子（ＧＤＮＦ）ｍＲＮＡ発現促進作用、アストロサイトにおけるアクアポリン４（ＡＱＰ４）ｍＲＮＡ発現促進作用、ミクログリアにおける一酸化窒素（ＮＯ）産生抑制作用、ミクログリアにおける腫瘍壊死因子－α（ＴＮＦ－α）産生抑制作用、及びミクログリアにおける炎症関連遺伝子ｍＲＮＡ発現抑制作用からなる群から選択される１種以上の作用に基づいて発揮されるものであることが好ましい。ただし、前記構造式（１）～（３）で表される化合物が有する脳の機能改善作用は、上記作用に基づいて発揮される脳の機能改善作用に限定されるものではない。
The brain function-improving effects of the compounds represented by structural formulas (1) to (3) include, for example, astrocyte proliferation-promoting action and glial cell line-derived neurotrophic factor (GDNF) mRNA expression promoting action in astrocytes. , promoting aquaporin 4 (AQP4) mRNA expression in astrocytes, suppressing nitric oxide (NO) production in microglia, suppressing tumor necrosis factor-α (TNF-α) production in microglia, and inflammation-related gene mRNA expression in microglia. It is preferable that the effect is exerted based on one or more types of effects selected from the group consisting of suppressive effects. However, the brain function-improving effects of the compounds represented by structural formulas (1) to (3) are not limited to the brain function-improving effects exerted based on the above-mentioned effects.
前記ミクログリアにおける炎症関連遺伝子としては、例えば、腫瘍壊死因子－α（ＴＮＦ－α）、誘導型一酸化窒素合成酵素（ｉＮＯＳ）、インターロイキン－６（ＩＬ－６）、インターロイキン－１β（ＩＬ－１β）、インターロイキン－１２（ＩＬ－１２）、ケモカイン（Ｃ－Ｘ－Ｃモチーフ）リガンド２（ＣＸＣＬ２）などが挙げられる。
Inflammation-related genes in microglia include, for example, tumor necrosis factor-α (TNF-α), inducible nitric oxide synthase (iNOS), interleukin-6 (IL-6), and interleukin-1β (IL- 1β), interleukin-12 (IL-12), and chemokine (CXC motif) ligand 2 (CXCL2).
＜構造式（１）～（３）のいずれかで表される化合物＞
下記構造式（１）で表される化合物の名称は、３，４－ジヒドロキシヒドロ桂皮酸（英名：３，４－Ｄｉｈｙｄｒｏｘｙｈｙｄｒｏｃｉｎｎａｍｉｃ ａｃｉｄ）である。
<Compound represented by any of structural formulas (1) to (3)>
The name of the compound represented by the following structural formula (1) is 3,4-dihydroxyhydrocinnamic acid (English name: 3,4-Dihydroxyhydrocinnamic acid).
下記構造式（１）で表される化合物の名称は、３，４－ジヒドロキシヒドロ桂皮酸（英名：３，４－Ｄｉｈｙｄｒｏｘｙｈｙｄｒｏｃｉｎｎａｍｉｃ ａｃｉｄ）である。
The name of the compound represented by the following structural formula (1) is 3,4-dihydroxyhydrocinnamic acid (English name: 3,4-Dihydroxyhydrocinnamic acid).
下記構造式（２）で表される化合物の名称は、３－（４－ヒドロキシフェニル）プロピオン酸（英名：３－（４－Ｈｙｄｒｏｘｙｐｈｅｎｙｌ）ｐｒｏｐｉｏｎｉｃ ａｃｉｄ）である。
The name of the compound represented by the following structural formula (2) is 3-(4-hydroxyphenyl)propionic acid (English name: 3-(4-Hydroxyphenyl)propionic acid).
下記構造式（３）で表される化合物の名称は、３－フェニルプロピオン酸（英名：３－Ｐｈｅｎｙｌｐｒｏｐｉｏｎｉｃ ａｃｉｄ）である。
The name of the compound represented by the following structural formula (3) is 3-phenylpropionic acid (English name: 3-Phenylpropionic acid).
前記構造式（１）～（３）のいずれかで表される化合物はいずれも公知の化合物であり、市販品を用いてもよいし、植物等から抽出したものを用いることもできる。
The compounds represented by any of the structural formulas (1) to (3) above are all known compounds, and commercially available products may be used, or those extracted from plants etc. may be used.
前記構造式（１）～（３）のいずれかで表される化合物は、いずれか１種を用いてもよいし、２種以上を併用してもよい。
The compounds represented by any of the structural formulas (1) to (3) may be used alone or in combination of two or more.
前記脳の機能改善剤は、前記構造式（１）～（３）のいずれかで表される化合物の少なくとも１種のみからなるものであってもよいし、前記構造式（１）～（３）のいずれかで表される化合物の少なくとも１種を製剤化したものであってもよい。
The brain function improving agent may consist of at least one compound represented by any one of the structural formulas (1) to (3), or may include only one compound represented by any one of the structural formulas (1) to (3). ) may be a formulation of at least one compound represented by any of the following.
前記構造式（１）～（３）のいずれかで表される化合物は、デキストリン、シクロデキストリン等の薬学的に許容し得るキャリアーその他任意の助剤を用いて、常法に従い、粉末状、顆粒状、液状等の任意の剤形に製剤化することができる。この際、助剤としては、例えば、賦形剤、結合剤、崩壊剤、滑沢剤、安定剤、矯味・矯臭剤等を用いることができる。
前記脳の機能改善剤は、他の組成物（例えば、後述する脳の機能改善用組成物等）に配合して使用することができるほか、錠剤、粉剤、カプセル剤、顆粒剤、エキス剤、シロップ剤等の経口投与剤；注射剤、点滴剤、坐剤等の非経口投与剤；軟膏剤、点眼剤、外用液剤、貼付剤などとして使用することもできる。 The compound represented by any of the structural formulas (1) to (3) can be prepared into powder or granules using a conventional method using a pharmaceutically acceptable carrier such as dextrin or cyclodextrin or any other auxiliary agent. It can be formulated into any desired dosage form, such as solid or liquid. In this case, as the auxiliary agent, for example, an excipient, a binder, a disintegrant, a lubricant, a stabilizer, a flavoring agent, etc. can be used.
The brain function improving agent can be used in combination with other compositions (for example, compositions for improving brain function described below), and can also be used in tablets, powders, capsules, granules, extracts, It can also be used as oral preparations such as syrup; parenteral preparations such as injections, drops, and suppositories; ointments, eye drops, external solutions, and patches.
前記脳の機能改善剤は、他の組成物（例えば、後述する脳の機能改善用組成物等）に配合して使用することができるほか、錠剤、粉剤、カプセル剤、顆粒剤、エキス剤、シロップ剤等の経口投与剤；注射剤、点滴剤、坐剤等の非経口投与剤；軟膏剤、点眼剤、外用液剤、貼付剤などとして使用することもできる。 The compound represented by any of the structural formulas (1) to (3) can be prepared into powder or granules using a conventional method using a pharmaceutically acceptable carrier such as dextrin or cyclodextrin or any other auxiliary agent. It can be formulated into any desired dosage form, such as solid or liquid. In this case, as the auxiliary agent, for example, an excipient, a binder, a disintegrant, a lubricant, a stabilizer, a flavoring agent, etc. can be used.
The brain function improving agent can be used in combination with other compositions (for example, compositions for improving brain function described below), and can also be used in tablets, powders, capsules, granules, extracts, It can also be used as oral preparations such as syrup; parenteral preparations such as injections, drops, and suppositories; ointments, eye drops, external solutions, and patches.
製剤化した脳の機能改善剤における前記構造式（１）～（３）のいずれかで表される化合物の合計含有量としては、特に制限はなく、目的に応じて適宜選択することができる。
The total content of the compounds represented by any of the structural formulas (1) to (3) in the formulated brain function improving agent is not particularly limited and can be appropriately selected depending on the purpose.
＜その他の成分＞
前記その他の成分としては、本発明の効果を損なわない限り、特に制限はなく、前記脳の機能改善剤の利用形態に応じて適宜選択することができ、例えば、賦形剤、防湿剤、防腐剤、強化剤、増粘剤、乳化剤、酸化防止剤、甘味料、酸味料、調味料、着色料、香料、美白剤、保湿剤、油性成分、紫外線吸収剤、界面活性剤、増粘剤、アルコール類、粉末成分、色剤、水性成分、水、皮膚栄養剤などが挙げられる。これらは、１種単独で使用してもよいし、２種以上を併用してもよい。 <Other ingredients>
The other ingredients are not particularly limited as long as they do not impair the effects of the present invention, and can be appropriately selected depending on the usage form of the brain function improving agent, such as excipients, moisture proofing agents, and preservatives. agent, strengthener, thickener, emulsifier, antioxidant, sweetener, acidulant, seasoning, colorant, fragrance, whitening agent, humectant, oily component, ultraviolet absorber, surfactant, thickener, Examples include alcohols, powder components, colorants, aqueous components, water, and skin nutrients. These may be used alone or in combination of two or more.
前記その他の成分としては、本発明の効果を損なわない限り、特に制限はなく、前記脳の機能改善剤の利用形態に応じて適宜選択することができ、例えば、賦形剤、防湿剤、防腐剤、強化剤、増粘剤、乳化剤、酸化防止剤、甘味料、酸味料、調味料、着色料、香料、美白剤、保湿剤、油性成分、紫外線吸収剤、界面活性剤、増粘剤、アルコール類、粉末成分、色剤、水性成分、水、皮膚栄養剤などが挙げられる。これらは、１種単独で使用してもよいし、２種以上を併用してもよい。 <Other ingredients>
The other ingredients are not particularly limited as long as they do not impair the effects of the present invention, and can be appropriately selected depending on the usage form of the brain function improving agent, such as excipients, moisture proofing agents, and preservatives. agent, strengthener, thickener, emulsifier, antioxidant, sweetener, acidulant, seasoning, colorant, fragrance, whitening agent, humectant, oily component, ultraviolet absorber, surfactant, thickener, Examples include alcohols, powder components, colorants, aqueous components, water, and skin nutrients. These may be used alone or in combination of two or more.
前記その他の成分の前記脳の機能改善剤における含有量としては、特に制限はなく、目的に応じて適宜選択することができる。
The content of the other components in the brain function improving agent is not particularly limited and can be appropriately selected depending on the purpose.
前記脳の機能改善剤の用法としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、経口、非経口、外用などの用法が挙げられる。
The usage of the brain function improving agent is not particularly limited and can be appropriately selected depending on the purpose, and examples include oral, parenteral, and external usage.
前記脳の機能改善剤の剤形としては、特に制限はなく、公知の剤形を目的に応じて適宜選択することができる。
任意の剤形の前記脳の機能改善剤の製造方法としては、特に制限はなく、公知の方法を適宜選択することができる。 The dosage form of the brain function improving agent is not particularly limited, and known dosage forms can be appropriately selected depending on the purpose.
The method for producing the brain function improving agent in any dosage form is not particularly limited, and any known method can be selected as appropriate.
任意の剤形の前記脳の機能改善剤の製造方法としては、特に制限はなく、公知の方法を適宜選択することができる。 The dosage form of the brain function improving agent is not particularly limited, and known dosage forms can be appropriately selected depending on the purpose.
The method for producing the brain function improving agent in any dosage form is not particularly limited, and any known method can be selected as appropriate.
前記脳の機能改善剤の投与方法、投与量、投与部位、投与期間、投与間隔などとしては、特に制限はなく、目的に応じて適宜選択することができる。
The administration method, dosage, administration site, administration period, administration interval, etc. of the brain function improving agent are not particularly limited and can be appropriately selected depending on the purpose.
前記脳の機能改善剤は、前記構造式（１）～（３）のいずれかで表される化合物が有する脳の機能改善作用を通じて、脳におけるタンパク質老廃物のグリンパティック系による除去排出効率を高めると共に、神経障害を抑制し、さらに神経細胞をサポート、調節又は保護し、これらの作用により、記憶能力や学習能力の向上；健忘症や老年性認知機能障害の予防、治療、又は改善；アルツハイマー病やパーキンソン病等の神経変性疾患の予防、治療、又は改善；うつ病等の気分障害の予防、治療、又は改善などに用いることができる。ただし、本発明の脳の機能改善剤は、これらの用途以外にも脳の機能改善作用を発揮することに意義のあるすべての用途に用いることができる。
The brain function improving agent increases the efficiency of removal and excretion of protein wastes in the brain by the glymphatic system through the brain function improving effect of the compound represented by any of the structural formulas (1) to (3). It also suppresses neurological disorders and further supports, regulates, or protects nerve cells, and through these actions, improves memory and learning abilities; prevents, treats, or improves amnesia and senile cognitive dysfunction; Alzheimer's disease. It can be used for the prevention, treatment, or amelioration of neurodegenerative diseases such as Parkinson's disease, and for the prevention, treatment, or amelioration of mood disorders such as depression. However, the brain function-improving agent of the present invention can be used in all other uses in which it is meaningful to exhibit a brain function-improving effect in addition to these uses.
前記脳の機能改善剤は、優れた脳の機能改善作用を有し、安全性が高いので、医薬品、医薬部外品、飲食品などの幅広い用途に用いることができ、例えば、後述する脳の機能改善用組成物の有効成分として好適に用いることができる。この場合、前記構造式（１）～（３）のいずれかで表される化合物の少なくとも１種をそのまま配合してもよいし、前記構造式（１）～（３）のいずれかで表される化合物の少なくとも１種を製剤化したものを配合してもよい。
The brain function improving agent has an excellent brain function improving effect and is highly safe, so it can be used in a wide range of applications such as medicines, quasi-drugs, food and drinks, etc. It can be suitably used as an active ingredient of a composition for functional improvement. In this case, at least one compound represented by any one of the above structural formulas (1) to (3) may be blended as is, or a compound represented by any one of the above structural formulas (1) to (3) may be blended as is. A formulation of at least one of the compounds listed above may be blended.
なお、前記脳の機能改善剤は、必要に応じて、脳の機能改善作用を有する他の成分を前記構造式（１）～（３）のいずれかで表される化合物と共に配合して有効成分として用いることもできる。
The above-mentioned brain function improving agent can be prepared by blending other ingredients having a brain function-improving effect with the compound represented by any of the structural formulas (1) to (3) above, as necessary. It can also be used as
本発明の脳の機能改善剤は、ヒトに対して好適に適用されるものであるが、その作用効果が奏される限り、ヒト以外の動物（例えば、マウス、ラット、ハムスター、イヌ、ネコ、ウシ、ブタ、サルなど）に対して適用することもできる。
The brain function improving agent of the present invention is suitably applied to humans, but as long as its effects are achieved, it can also be applied to animals other than humans (e.g. mice, rats, hamsters, dogs, cats, etc.). It can also be applied to cows, pigs, monkeys, etc.).
また、本発明の脳の機能改善剤は、脳の機能改善作用の作用機構に関する研究のための試薬としても用いることができる。
Furthermore, the brain function improving agent of the present invention can also be used as a reagent for research on the mechanism of action of improving brain function.
（脳の機能改善用組成物）
本発明の脳の機能改善用組成物は、本発明の脳の機能改善剤を含み、更に必要に応じてその他の成分を含む。 (Composition for improving brain function)
The composition for improving brain function of the present invention contains the brain function improving agent of the present invention, and further contains other components as necessary.
本発明の脳の機能改善用組成物は、本発明の脳の機能改善剤を含み、更に必要に応じてその他の成分を含む。 (Composition for improving brain function)
The composition for improving brain function of the present invention contains the brain function improving agent of the present invention, and further contains other components as necessary.
＜脳の機能改善剤＞
前記脳の機能改善剤は、上述した本発明の脳の機能改善剤である。 <Brain function improving agent>
The brain function improving agent is the brain function improving agent of the present invention described above.
前記脳の機能改善剤は、上述した本発明の脳の機能改善剤である。 <Brain function improving agent>
The brain function improving agent is the brain function improving agent of the present invention described above.
前記脳の機能改善用組成物における前記脳の機能改善剤の含有量としては、特に制限はなく、前記脳の機能改善用組成物の形態などによって適宜調整することができるが、前記構造式（１）～（３）のいずれかで表される化合物の合計量に換算して、０．０００１質量％～３０質量％が好ましく、０．０００１質量％～１０質量％がより好ましい。前記脳の機能改善用組成物は、前記脳の機能改善剤のみからなるものであってもよい。
The content of the brain function improving agent in the brain function improving composition is not particularly limited and can be adjusted as appropriate depending on the form of the brain function improving composition. The amount is preferably 0.0001% by mass to 30% by mass, more preferably 0.0001% by mass to 10% by mass, in terms of the total amount of the compounds represented by any of 1) to (3). The brain function improving composition may consist only of the brain function improving agent.
＜その他の成分＞
前記脳の機能改善用組成物におけるその他の成分としては、特に制限はなく、前記脳の機能改善用組成物の利用形態に応じて適宜選択することができ、例えば、上記した脳の機能改善剤の項目に記載したその他の成分と同様のものなどが挙げられる。これらは、１種単独で使用してもよいし、２種以上を併用してもよい。 <Other ingredients>
Other components in the composition for improving brain function are not particularly limited and can be appropriately selected depending on the usage form of the composition for improving brain function, such as the above-mentioned brain function improving agent. Other ingredients similar to those listed in the section above may be mentioned. These may be used alone or in combination of two or more.
前記脳の機能改善用組成物におけるその他の成分としては、特に制限はなく、前記脳の機能改善用組成物の利用形態に応じて適宜選択することができ、例えば、上記した脳の機能改善剤の項目に記載したその他の成分と同様のものなどが挙げられる。これらは、１種単独で使用してもよいし、２種以上を併用してもよい。 <Other ingredients>
Other components in the composition for improving brain function are not particularly limited and can be appropriately selected depending on the usage form of the composition for improving brain function, such as the above-mentioned brain function improving agent. Other ingredients similar to those listed in the section above may be mentioned. These may be used alone or in combination of two or more.
前記その他の成分の前記脳の機能改善用組成物における含有量としては、特に制限はなく、目的に応じて適宜選択することができる。
The content of the other components in the composition for improving brain function is not particularly limited and can be appropriately selected depending on the purpose.
＜態様＞
前記脳の機能改善用組成物の態様としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、医薬品、医薬部外品、飲食品などが挙げられる。
本発明の脳の機能改善用組成物は、日常的に使用することが可能であり、有効成分である前記構造式（１）～（３）のいずれかで表される化合物の働きによって、脳の機能改善作用をはじめとする様々な生理活性作用を極めて効果的に発揮させることができる。 <Aspects>
The form of the composition for improving brain function is not particularly limited and can be appropriately selected depending on the purpose, and includes, for example, pharmaceuticals, quasi-drugs, food and drink products, and the like.
The composition for improving brain function of the present invention can be used on a daily basis and improves brain function by the action of the active ingredient, a compound represented by any one of the structural formulas (1) to (3). It can extremely effectively exhibit various physiologically active effects, including the function-improving effect of .
前記脳の機能改善用組成物の態様としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、医薬品、医薬部外品、飲食品などが挙げられる。
本発明の脳の機能改善用組成物は、日常的に使用することが可能であり、有効成分である前記構造式（１）～（３）のいずれかで表される化合物の働きによって、脳の機能改善作用をはじめとする様々な生理活性作用を極めて効果的に発揮させることができる。 <Aspects>
The form of the composition for improving brain function is not particularly limited and can be appropriately selected depending on the purpose, and includes, for example, pharmaceuticals, quasi-drugs, food and drink products, and the like.
The composition for improving brain function of the present invention can be used on a daily basis and improves brain function by the action of the active ingredient, a compound represented by any one of the structural formulas (1) to (3). It can extremely effectively exhibit various physiologically active effects, including the function-improving effect of .
本発明の脳の機能改善用組成物は、ヒトに対して好適に適用されるものであるが、それぞれの作用効果が奏される限り、ヒト以外の動物（例えば、マウス、ラット、ハムスター、イヌ、ネコ、ウシ、ブタ、サルなど）に対して適用することもできる。
The composition for improving brain function of the present invention is suitably applied to humans, but it can also be applied to animals other than humans (e.g. mice, rats, hamsters, dogs) as long as the respective effects are achieved. , cats, cows, pigs, monkeys, etc.).
本発明の脳の機能改善用組成物の用法としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、経口、非経口、外用などの用法が挙げられるが、経口が好ましい。
The method of use of the composition for improving brain function of the present invention is not particularly limited and can be appropriately selected depending on the purpose, and examples include oral, parenteral, and external methods, but oral is preferred. .
前記経口用の組成物としては、例えば、経口投与剤や飲食品が挙げられる。ここで、飲食品とは、人の健康に危害を加えるおそれが少なく、通常の社会生活において、経口又は消化管投与により摂取されるものをいい、行政区分上の食品、医薬品、医薬部外品などの区分に制限されるものではない。したがって、前記飲食品は、経口的に摂取される一般食品、健康食品（機能性飲食品）、保健機能食品（特定保健用食品、栄養機能食品、機能性表示食品）、医薬部外品、医薬品等を構成する飲食品を幅広く含むものを意味する。
Examples of the oral composition include oral preparations and food and drink products. Here, food and beverages refer to foods that have little risk of harming human health and are ingested orally or through gastrointestinal administration in normal social life, and are administratively classified foods, drugs, and quasi-drugs. It is not limited to such categories. Therefore, the above-mentioned foods and drinks include general foods that are orally ingested, health foods (functional foods and drinks), foods with health claims (foods for specified health uses, foods with nutritional function claims, foods with functional claims), quasi-drugs, and pharmaceuticals. This term refers to a wide range of food and beverages that make up food and beverages.
前記経口用の組成物の種類としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、茶飲料、清涼飲料、炭酸飲料、栄養飲料、果実飲料、乳酸飲料、アルコール飲料、コーヒー飲料、コーヒー入り清涼飲料等の飲料（これらの飲料の濃縮原液及び調整用粉末を含む）；アイスクリーム、アイスシャーベット、かき氷等の冷菓；そば、うどん、はるさめ、ぎょうざの皮、しゅうまいの皮、中華麺、即席麺等の麺類；飴、キャンディー、ガム、チョコレート、錠菓、スナック菓子、ビスケット、ゼリー、ジャム、クリーム、焼き菓子、パン等の菓子類；カニ、サケ、アサリ、マグロ、イワシ、エビ、カツオ、サバ、クジラ、カキ、サンマ、イカ、アカガイ、ホタテ、アワビ、ウニ、イクラ、トコブシ等の水産物；かまぼこ、ハム、ソーセージ等の水産・畜産加工食品；加工乳、発酵乳等の乳製品；サラダ油、てんぷら油、マーガリン、マヨネーズ、ショートニング、ホイップクリーム、ドレッシング等の油脂及び油脂加工食品；ソース、たれ等の調味料；カレー、シチュー、親子丼、お粥、雑炊、中華丼、かつ丼、天丼、うな丼、ハヤシライス、おでん、マーボードーフ、牛丼、ミートソース、玉子スープ、オムライス、餃子、シューマイ、ハンバーグ、ミートボール等のレトルトパウチ食品；サラダ、漬物等の惣菜；種々の形態の健康・美容・栄養補助食品；錠剤、粉剤、カプセル剤、顆粒剤、エキス剤、シロップ剤、ドリンク剤、トローチ、うがい薬等の医薬品、医薬部外品；口中清涼剤、口臭防止剤等の口腔内で使用する口腔清涼剤、歯磨剤などが挙げられる。
The type of the oral composition is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include beverages such as tea beverages, soft drinks, carbonated beverages, nutritional beverages, fruit beverages, lactic acid beverages, alcoholic beverages, coffee beverages, and coffee-containing soft drinks (including concentrated liquids and powders for adjusting these beverages); cold desserts such as ice cream, ice sorbet, 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, jellies, jams, creams, baked goods, and bread; seafood such as crab, salmon, clams, tuna, sardines, shrimp, bonito, mackerel, whales, oysters, pacific saury, squid, ark shells, scallops, abalone, sea urchins, salmon roe, and tokobushi; kamaboko, halibut, and other seafood. processed seafood and livestock foods such as sausages and 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, mapo tofu, 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, powders, capsules, granules, extracts, syrups, drinks, lozenges, and mouthwash; oral fresheners and toothpastes used in the oral cavity, such as mouth fresheners and breath fresheners.
前記脳の機能改善用組成物の製造方法としては、特に制限はなく、前記脳の機能改善用組成物の利用形態などに応じて適宜選択することができる。
The method for producing the composition for improving brain function is not particularly limited, and can be appropriately selected depending on the usage form of the composition for improving brain function.
前記脳の機能改善用組成物の使用量、使用期間、使用間隔等としては、特に制限はなく、目的に応じて適宜選択することができる。
The amount, period of use, interval of use, etc. of the composition for improving brain function are not particularly limited and can be appropriately selected depending on the purpose.
上述したように、本発明の脳の機能改善剤及び脳の機能改善用組成物は、優れた脳の機能改善作用を有する。
したがって、本発明は、個体に前記脳の機能改善剤及び脳の機能改善用組成物からなる群から選択される少なくとも１種を投与することを特徴とする脳の機能を改善する方法にも関する。 As described above, the brain function improving agent and brain function improving composition of the present invention have an excellent brain function improving effect.
Therefore, the present invention also relates to a method for improving brain function, which comprises administering to an individual at least one selected from the group consisting of the brain function improving agent and the composition for improving brain function. .
したがって、本発明は、個体に前記脳の機能改善剤及び脳の機能改善用組成物からなる群から選択される少なくとも１種を投与することを特徴とする脳の機能を改善する方法にも関する。 As described above, the brain function improving agent and brain function improving composition of the present invention have an excellent brain function improving effect.
Therefore, the present invention also relates to a method for improving brain function, which comprises administering to an individual at least one selected from the group consisting of the brain function improving agent and the composition for improving brain function. .
また、前記構造式（１）～（３）のいずれかで表される化合物は、そのアストロサイト増殖促進作用、アストロサイトにおけるＧＤＮＦ ｍＲＮＡ発現促進作用、アストロサイトにおけるＡＱＰ４ ｍＲＮＡ発現促進作用、ミクログリアにおけるＮＯ産生抑制作用、ミクログリアにおけるＴＮＦ－α産生抑制作用、又はミクログリアにおける炎症関連遺伝子ｍＲＮＡ発現抑制作用を利用して、アストロサイト増殖促進剤、アストロサイトにおけるＧＤＮＦ ｍＲＮＡ発現促進剤、アストロサイトにおけるＡＱＰ４ ｍＲＮＡ発現促進剤、ミクログリアにおけるＮＯ産生抑制剤、ミクログリアにおけるＴＮＦ－α産生抑制剤、又はミクログリアにおける炎症関連遺伝子ｍＲＮＡ発現抑制剤の有効成分として使用してもよい。
In addition, the compounds represented by any of the structural formulas (1) to (3) have an effect of promoting astrocyte proliferation, an effect of promoting GDNF mRNA expression in astrocytes, an effect of promoting AQP4 mRNA expression in astrocytes, and an effect of promoting NO in microglia. Astrocyte proliferation promoter, GDNF mRNA expression promoter in astrocytes, and AQP4 mRNA expression promoter in astrocytes by utilizing production suppressing effect, TNF-α production suppressing effect in microglia, or inflammation-related gene mRNA expression suppressing effect in microglia. It may be used as an active ingredient of an agent, an agent for suppressing NO production in microglia, an agent for suppressing TNF-α production in microglia, or an agent for suppressing inflammation-related gene mRNA expression in microglia.
また、本発明は、個体に、前記構造式（１）～（３）のいずれかで表される化合物の少なくともいずれかを投与することを特徴とする、アストロサイトの増殖を促進する方法、アストロサイトにおけるＧＤＮＦ ｍＲＮＡの発現を促進する方法、アストロサイトにおけるＡＱＰ４ ｍＲＮＡの発現を促進する方法、ミクログリアにおけるＮＯの産生を抑制する方法、ミクログリアにおけるＴＮＦ－αの産生を抑制する方法、又はミクログリアにおける炎症関連遺伝子ｍＲＮＡの発現を抑制する方法にも関する。
The present invention also provides a method for promoting proliferation of astrocytes, which comprises administering to an individual at least one of the compounds represented by any one of the structural formulas (1) to (3). A method of promoting the expression of GDNF mRNA in astrocytes, a method of promoting the expression of AQP4 mRNA in astrocytes, a method of suppressing NO production in microglia, a method of suppressing TNF-α production in microglia, or an inflammation-related method in microglia. The present invention also relates to a method of suppressing gene mRNA expression.
以下、本発明の試験例、配合例を説明するが、本発明は、これらの試験例、配合例に何ら限定されるものではない。
Hereinafter, test examples and formulation examples of the present invention will be explained, but the present invention is not limited to these test examples and formulation examples.
（被験試料）
後述の各試験例では、以下の化合物を被験試料として用いた。
・ 前記構造式（１）で表される化合物（ＳＩＧＭＡ製）
・ 前記構造式（２）で表される化合物（ＳＩＧＭＡ製）
・ 前記構造式（３）で表される化合物（東京化成工業製） (Test sample)
In each test example described below, the following compounds were used as test samples.
- Compound represented by the above structural formula (1) (manufactured by SIGMA)
- Compound represented by the above structural formula (2) (manufactured by SIGMA)
- Compound represented by the above structural formula (3) (manufactured by Tokyo Chemical Industry Co., Ltd.)
後述の各試験例では、以下の化合物を被験試料として用いた。
・ 前記構造式（１）で表される化合物（ＳＩＧＭＡ製）
・ 前記構造式（２）で表される化合物（ＳＩＧＭＡ製）
・ 前記構造式（３）で表される化合物（東京化成工業製） (Test sample)
In each test example described below, the following compounds were used as test samples.
- Compound represented by the above structural formula (1) (manufactured by SIGMA)
- Compound represented by the above structural formula (2) (manufactured by SIGMA)
- Compound represented by the above structural formula (3) (manufactured by Tokyo Chemical Industry Co., Ltd.)
（試験例１：アストロサイト増殖促進作用試験）
アストロサイト増殖促進作用を以下のように試験した。 (Test Example 1: Astrocyte proliferation promotion effect test)
The astrocyte proliferation promoting effect was tested as follows.
アストロサイト増殖促進作用を以下のように試験した。 (Test Example 1: Astrocyte proliferation promotion effect test)
The astrocyte proliferation promoting effect was tested as follows.
マウス由来アストロサイト培養株（Ｃ８－Ｓ）を、１０質量％ＦＢＳ含有ＤＭＥＭを用いて培養した後、トリプシン処理により細胞を回収した。回収した細胞を２．５×１０４ｃｅｌｌｓ／ｍＬの濃度になるように１０質量％ＦＢＳ含有ＤＭＥＭで希釈した後、９６ウェルプレートに１ウェル当たり１００μＬずつ播種し、３７℃、５％ＣＯ２下で６時間培養した。
A mouse-derived astrocyte culture strain (C8-S) was cultured using DMEM containing 10% by mass of FBS, and then the cells were collected by trypsin treatment. The collected cells were diluted with DMEM containing 10 mass% FBS to a concentration of 2.5 x 10 4 cells/mL, then seeded at 100 μL per well in a 96-well plate, and incubated at 37°C under 5% CO 2 The cells were cultured for 6 hours.
培養終了後、１０質量％ＦＢＳ含有ＤＭＥＭに溶解した被験試料（最終試料濃度は下記表１を参照）を各ウェルに１００μＬ添加し、４日間培養した。なお、対照として、被験試料無添加の１０質量％ＦＢＳ含有ＤＭＥＭ １００μＬを加え、同様に培養した。
After completion of the culture, 100 μL of the test sample dissolved in DMEM containing 10% by mass FBS (see Table 1 below for final sample concentration) was added to each well and cultured for 4 days. As a control, 100 μL of DMEM containing 10% by mass FBS without addition of the test sample was added and cultured in the same manner.
細胞増殖促進作用は、ＭＴＴアッセイ法を用いて測定した。
具体的には、培養終了後、培地を除去し、終濃度０．４ｍｇ／ｍＬでＰＢＳ（－）に溶解したＭＴＴを各ウェルに１００μＬずつ添加した。２時間培養した後に、細胞内に生成したブルーホルマザンを２－プロパノール１００μＬで抽出した。抽出後、波長５７０ｎｍにおける吸光度を測定した。同時に濁度として波長６５０ｎｍにおける吸光度を測定し、両者の差をもってブルーホルマザン生成量とした。
アストロサイト増殖促進率（％）は、下記式により算出した。結果を表１に示す。
アストロサイト増殖促進率（％）＝Ａ／Ｂ×１００
上記式中のＡ～Ｂは、それぞれ以下を表す。
Ａ ： 被験試料添加時の細胞でのブルーホルマザン生成量
Ｂ ： 被験試料無添加時の細胞でのブルーホルマザン生成量 The cell proliferation promoting effect was measured using the MTT assay method.
Specifically, after the culture was completed, the medium was removed, and 100 μL of MTT dissolved in PBS(-) at a final concentration of 0.4 mg/mL was added to each well. After culturing for 2 hours, blue formazan produced within the cells was extracted with 100 μL of 2-propanol. After extraction, absorbance at a wavelength of 570 nm was measured. At the same time, absorbance at a wavelength of 650 nm was measured as turbidity, and the difference between the two was determined as the amount of blue formazan produced.
Astrocyte proliferation promotion rate (%) was calculated using the following formula. The results are shown in Table 1.
Astrocyte proliferation promotion rate (%) = A/B x 100
A to B in the above formula each represent the following.
A: Amount of blue formazan produced in cells when the test sample is added B: Amount of blue formazan produced in cells when the test sample is not added
具体的には、培養終了後、培地を除去し、終濃度０．４ｍｇ／ｍＬでＰＢＳ（－）に溶解したＭＴＴを各ウェルに１００μＬずつ添加した。２時間培養した後に、細胞内に生成したブルーホルマザンを２－プロパノール１００μＬで抽出した。抽出後、波長５７０ｎｍにおける吸光度を測定した。同時に濁度として波長６５０ｎｍにおける吸光度を測定し、両者の差をもってブルーホルマザン生成量とした。
アストロサイト増殖促進率（％）は、下記式により算出した。結果を表１に示す。
アストロサイト増殖促進率（％）＝Ａ／Ｂ×１００
上記式中のＡ～Ｂは、それぞれ以下を表す。
Ａ ： 被験試料添加時の細胞でのブルーホルマザン生成量
Ｂ ： 被験試料無添加時の細胞でのブルーホルマザン生成量 The cell proliferation promoting effect was measured using the MTT assay method.
Specifically, after the culture was completed, the medium was removed, and 100 μL of MTT dissolved in PBS(-) at a final concentration of 0.4 mg/mL was added to each well. After culturing for 2 hours, blue formazan produced within the cells was extracted with 100 μL of 2-propanol. After extraction, absorbance at a wavelength of 570 nm was measured. At the same time, absorbance at a wavelength of 650 nm was measured as turbidity, and the difference between the two was determined as the amount of blue formazan produced.
Astrocyte proliferation promotion rate (%) was calculated using the following formula. The results are shown in Table 1.
Astrocyte proliferation promotion rate (%) = A/B x 100
A to B in the above formula each represent the following.
A: Amount of blue formazan produced in cells when the test sample is added B: Amount of blue formazan produced in cells when the test sample is not added
（試験例２：アストロサイトにおけるグリア細胞株由来神経栄養因子（ＧＤＮＦ）ｍＲＮＡ発現促進作用試験）
アストロサイトにおけるＧＤＮＦ ｍＲＮＡ発現促進作用を以下のように試験した。 (Test Example 2: Glial cell line-derived neurotrophic factor (GDNF) mRNA expression promotion test in astrocytes)
The effect of promoting GDNF mRNA expression in astrocytes was tested as follows.
アストロサイトにおけるＧＤＮＦ ｍＲＮＡ発現促進作用を以下のように試験した。 (Test Example 2: Glial cell line-derived neurotrophic factor (GDNF) mRNA expression promotion test in astrocytes)
The effect of promoting GDNF mRNA expression in astrocytes was tested as follows.
マウス由来アストロサイト培養株（Ｃ８－Ｓ）を、１０質量％ＦＢＳ含有ＤＭＥＭを用いて培養した後、トリプシン処理により細胞を回収した。回収した細胞を５．０×１０４ｃｅｌｌｓ／ｍＬの濃度になるように１０質量％ＦＢＳ含有ＤＭＥＭで希釈した後、６ウェルプレートに１ウェル当たり２ｍＬずつ播種し、３７℃、５％ＣＯ２下でコンフルエントになるまで培養した。
A mouse-derived astrocyte culture strain (C8-S) was cultured using DMEM containing 10% by mass of FBS, and then the cells were collected by trypsin treatment. The collected cells were diluted with DMEM containing 10% by mass FBS to a concentration of 5.0 x 10 4 cells/mL, then seeded at 2 mL per well in a 6-well plate, and incubated at 37°C under 5% CO 2 The cells were cultured until confluent.
培養終了後、培地を除去し、終濃度０．５％ＤＭＳＯを含む１０質量％ＦＢＳ含有ＤＭＥＭに溶解した被験試料（最終試料濃度は下記表２を参照）を各ウェルに２ｍＬずつ添加し、２４時間培養した。なお、対照として、被験試料無添加の０．５％ＤＭＳＯを含む１０質量％ＦＢＳ含有ＤＭＥＭ ２ｍＬを加え、同様に培養した。
After the culture was completed, the medium was removed, and 2 mL of the test sample (see Table 2 below for the final sample concentration) dissolved in DMEM containing 10 mass% FBS containing a final concentration of 0.5% DMSO was added to each well. Cultured for hours. As a control, 2 mL of DMEM containing 10 mass % FBS containing 0.5% DMSO and no test sample was added and cultured in the same manner.
培養後、培養液を除去し、ＲＮｅａｓｙ（登録商標） ｍｉｎｉ ｋｉｔ（Ｑｉａｇｅｎ製）にて総ＲＮＡを抽出し、波長２６０ｎｍにおける吸光度からＲＮＡ量を計算し、１００ｎｇ／μＬになるように総ＲＮＡを調製した。
After culturing, remove the culture medium, extract total RNA using RNeasy (registered trademark) mini kit (manufactured by Qiagen), calculate the amount of RNA from the absorbance at a wavelength of 260 nm, and adjust the total RNA to 100 ng/μL. did.
この総ＲＮＡを鋳型とし、ＧＤＮＦ及び内部標準であるＧＡＰＤＨのｍＲＮＡの発現量を測定した。
ｍＲＮＡの検出は、リアルタイムＰＣＲ装置Ｔｈｅｒｍａｌ Ｃｙｃｌｅｒ Ｄｉｃｅ（登録商標） Ｒｅａｌ Ｔｉｍｅ Ｓｙｓｔｅｍ ＩＩＩ（ＴａＫａＲａ製）を用いて、ＰｒｉｍｅＳｃｒｉｐｔＴＭ ＲＴ Ｍａｓｔｅｒ Ｍｉｘ（Ｐｅｒｆｅｃｔ Ｒｅａｌ Ｔｉｍｅ）及びＴＢ Ｇｒｅｅｎ（登録商標） Ｆａｓｔ ｑＰＣＲ Ｍｉｘ（ＴａＫａＲａ製）による２ステップリアルタイムＲＴ－ＰＣＲ反応により行った。
ＧＤＮＦ ｍＲＮＡの発現量は、ＧＡＰＤＨ ｍＲＮＡの発現量で補正し算出した。得られた値から、下記式によりＧＤＮＦのｍＲＮＡ発現促進率（％）を算出した。
ＧＤＮＦ ｍＲＮＡ発現促進率（％）＝Ｃ／Ｄ×１００
上記式中のＣ～Ｄは、それぞれ以下を表す。
Ｃ ： 被験試料添加時の補正値
Ｄ ： 被験試料無添加時の補正値 Using this total RNA as a template, the expression levels of GDNF and GAPDH mRNA, which was an internal standard, were measured.
Detection of mRNA was performed using a real-time PCR device Thermal Cycler Dice (registered trademark) Real Time System III (manufactured by TaKaRa) using PrimeScript TM RT Master Mix (Perfect Real Time) and TB Gr een (registered trademark) Fast qPCR Mix (manufactured by TaKaRa) ) was carried out using a two-step real-time RT-PCR reaction.
The expression level of GDNF mRNA was calculated by correcting the expression level of GAPDH mRNA. From the obtained values, the promotion rate (%) of GDNF mRNA expression was calculated using the following formula.
GDNF mRNA expression promotion rate (%) = C/D x 100
C to D in the above formula each represent the following.
C: Correction value when test sample is added D: Correction value when test sample is not added
ｍＲＮＡの検出は、リアルタイムＰＣＲ装置Ｔｈｅｒｍａｌ Ｃｙｃｌｅｒ Ｄｉｃｅ（登録商標） Ｒｅａｌ Ｔｉｍｅ Ｓｙｓｔｅｍ ＩＩＩ（ＴａＫａＲａ製）を用いて、ＰｒｉｍｅＳｃｒｉｐｔＴＭ ＲＴ Ｍａｓｔｅｒ Ｍｉｘ（Ｐｅｒｆｅｃｔ Ｒｅａｌ Ｔｉｍｅ）及びＴＢ Ｇｒｅｅｎ（登録商標） Ｆａｓｔ ｑＰＣＲ Ｍｉｘ（ＴａＫａＲａ製）による２ステップリアルタイムＲＴ－ＰＣＲ反応により行った。
ＧＤＮＦ ｍＲＮＡの発現量は、ＧＡＰＤＨ ｍＲＮＡの発現量で補正し算出した。得られた値から、下記式によりＧＤＮＦのｍＲＮＡ発現促進率（％）を算出した。
ＧＤＮＦ ｍＲＮＡ発現促進率（％）＝Ｃ／Ｄ×１００
上記式中のＣ～Ｄは、それぞれ以下を表す。
Ｃ ： 被験試料添加時の補正値
Ｄ ： 被験試料無添加時の補正値 Using this total RNA as a template, the expression levels of GDNF and GAPDH mRNA, which was an internal standard, were measured.
Detection of mRNA was performed using a real-time PCR device Thermal Cycler Dice (registered trademark) Real Time System III (manufactured by TaKaRa) using PrimeScript TM RT Master Mix (Perfect Real Time) and TB Gr een (registered trademark) Fast qPCR Mix (manufactured by TaKaRa) ) was carried out using a two-step real-time RT-PCR reaction.
The expression level of GDNF mRNA was calculated by correcting the expression level of GAPDH mRNA. From the obtained values, the promotion rate (%) of GDNF mRNA expression was calculated using the following formula.
GDNF mRNA expression promotion rate (%) = C/D x 100
C to D in the above formula each represent the following.
C: Correction value when test sample is added D: Correction value when test sample is not added
（試験例３：アストロサイトにおけるアクアポリン４（ＡＱＰ４）ｍＲＮＡ発現促進作用試験）
アストロサイトにおけるＡＱＰ４ ｍＲＮＡ発現促進作用を以下のように試験した。 (Test Example 3: Aquaporin 4 (AQP4) mRNA expression promotion effect test in astrocytes)
The effect of promoting AQP4 mRNA expression in astrocytes was tested as follows.
アストロサイトにおけるＡＱＰ４ ｍＲＮＡ発現促進作用を以下のように試験した。 (Test Example 3: Aquaporin 4 (AQP4) mRNA expression promotion effect test in astrocytes)
The effect of promoting AQP4 mRNA expression in astrocytes was tested as follows.
マウス由来アストロサイト培養株（Ｃ８－Ｓ）を、１０質量％ＦＢＳ含有ＤＭＥＭを用いて培養した後、トリプシン処理により細胞を回収した。回収した細胞を５．０×１０４ｃｅｌｌｓ／ｍＬの濃度になるように１０質量％ＦＢＳ含有ＤＭＥＭで希釈した後、６ウェルプレートに１ウェル当たり２ｍＬずつ播種し、３７℃、５％ＣＯ２下でコンフルエントになるまで培養した。
A mouse-derived astrocyte culture strain (C8-S) was cultured using DMEM containing 10% by mass of FBS, and then the cells were collected by trypsin treatment. The collected cells were diluted with DMEM containing 10 mass% FBS to a concentration of 5.0 x 10 4 cells/mL, then seeded in 2 mL per well in a 6-well plate, and incubated at 37°C under 5% CO 2 The cells were cultured until confluent.
培養終了後、培地を除去し、終濃度０．５％ＤＭＳＯを含む１０質量％ＦＢＳ含有ＤＭＥＭに溶解した被験試料（最終試料濃度は下記表３を参照）を各ウェルに２ｍＬずつ添加し、２４時間培養した。なお、対照として、被験試料無添加の０．５％ＤＭＳＯを含む１０質量％ＦＢＳ含有ＤＭＥＭ ２ｍＬを加え、同様に培養した。
After the culture was completed, the medium was removed, and 2 mL of the test sample (see Table 3 below for final sample concentration) dissolved in DMEM containing 10 mass% FBS containing a final concentration of 0.5% DMSO was added to each well. Cultured for hours. As a control, 2 mL of DMEM containing 10 mass % FBS containing 0.5% DMSO and no test sample was added and cultured in the same manner.
培養後、培養液を除去し、ＲＮｅａｓｙ（登録商標） ｍｉｎｉ ｋｉｔ（Ｑｉａｇｅｎ製）にて総ＲＮＡを抽出し、波長２６０ｎｍにおける吸光度からＲＮＡ量を計算し、１００ｎｇ／μＬになるように総ＲＮＡを調製した。
After culturing, remove the culture medium, extract total RNA using RNeasy (registered trademark) mini kit (manufactured by Qiagen), calculate the amount of RNA from the absorbance at a wavelength of 260 nm, and adjust the total RNA to 100 ng/μL. did.
この総ＲＮＡを鋳型とし、ＡＱＰ４及び内部標準であるＧＡＰＤＨのｍＲＮＡの発現量を測定した。
ｍＲＮＡの検出は、リアルタイムＰＣＲ装置Ｔｈｅｒｍａｌ Ｃｙｃｌｅｒ Ｄｉｃｅ（登録商標） Ｒｅａｌ Ｔｉｍｅ Ｓｙｓｔｅｍ ＩＩＩ（ＴａＫａＲａ製）を用いて、ＰｒｉｍｅＳｃｒｉｐｔＴＭ ＲＴ Ｍａｓｔｅｒ Ｍｉｘ（Ｐｅｒｆｅｃｔ Ｒｅａｌ Ｔｉｍｅ）及びＴＢ Ｇｒｅｅｎ（登録商標） Ｆａｓｔ ｑＰＣＲ Ｍｉｘ（ＴａＫａＲａ製）による２ステップリアルタイムＲＴ－ＰＣＲ反応により行った。
ＡＱＰ４ ｍＲＮＡの発現量は、ＧＡＰＤＨ ｍＲＮＡの発現量で補正し算出した。得られた値から、下記式によりＡＱＰ４のｍＲＮＡ発現促進率（％）を算出した。
ＡＱＰ４ ｍＲＮＡ発現促進率（％）＝Ｅ／Ｆ×１００
上記式中のＥ～Ｆは、それぞれ以下を表す。
Ｅ ： 被験試料添加時の補正値
Ｆ ： 被験試料無添加時の補正値 Using this total RNA as a template, the expression levels of AQP4 and GAPDH mRNA, which was an internal standard, were measured.
Detection of mRNA was performed using a real-time PCR device Thermal Cycler Dice (registered trademark) Real Time System III (manufactured by TaKaRa) using PrimeScript TM RT Master Mix (Perfect Real Time) and TB Gr een (registered trademark) Fast qPCR Mix (manufactured by TaKaRa) ) was carried out using a two-step real-time RT-PCR reaction.
The expression level of AQP4 mRNA was calculated by correcting the expression level of GAPDH mRNA. From the obtained values, the AQP4 mRNA expression promotion rate (%) was calculated using the following formula.
AQP4 mRNA expression promotion rate (%) = E/F x 100
E to F in the above formula each represent the following.
E: Correction value when test sample is added F: Correction value when test sample is not added
ｍＲＮＡの検出は、リアルタイムＰＣＲ装置Ｔｈｅｒｍａｌ Ｃｙｃｌｅｒ Ｄｉｃｅ（登録商標） Ｒｅａｌ Ｔｉｍｅ Ｓｙｓｔｅｍ ＩＩＩ（ＴａＫａＲａ製）を用いて、ＰｒｉｍｅＳｃｒｉｐｔＴＭ ＲＴ Ｍａｓｔｅｒ Ｍｉｘ（Ｐｅｒｆｅｃｔ Ｒｅａｌ Ｔｉｍｅ）及びＴＢ Ｇｒｅｅｎ（登録商標） Ｆａｓｔ ｑＰＣＲ Ｍｉｘ（ＴａＫａＲａ製）による２ステップリアルタイムＲＴ－ＰＣＲ反応により行った。
ＡＱＰ４ ｍＲＮＡの発現量は、ＧＡＰＤＨ ｍＲＮＡの発現量で補正し算出した。得られた値から、下記式によりＡＱＰ４のｍＲＮＡ発現促進率（％）を算出した。
ＡＱＰ４ ｍＲＮＡ発現促進率（％）＝Ｅ／Ｆ×１００
上記式中のＥ～Ｆは、それぞれ以下を表す。
Ｅ ： 被験試料添加時の補正値
Ｆ ： 被験試料無添加時の補正値 Using this total RNA as a template, the expression levels of AQP4 and GAPDH mRNA, which was an internal standard, were measured.
Detection of mRNA was performed using a real-time PCR device Thermal Cycler Dice (registered trademark) Real Time System III (manufactured by TaKaRa) using PrimeScript TM RT Master Mix (Perfect Real Time) and TB Gr een (registered trademark) Fast qPCR Mix (manufactured by TaKaRa) ) was carried out using a two-step real-time RT-PCR reaction.
The expression level of AQP4 mRNA was calculated by correcting the expression level of GAPDH mRNA. From the obtained values, the AQP4 mRNA expression promotion rate (%) was calculated using the following formula.
AQP4 mRNA expression promotion rate (%) = E/F x 100
E to F in the above formula each represent the following.
E: Correction value when test sample is added F: Correction value when test sample is not added
（試験例４：ミクログリアにおける一酸化窒素（ＮＯ）産生抑制作用試験）
ミクログリアにおけるＮＯ産生抑制作用を以下のように試験した。 (Test Example 4: Nitric oxide (NO) production inhibition test in microglia)
The inhibitory effect on NO production in microglia was tested as follows.
ミクログリアにおけるＮＯ産生抑制作用を以下のように試験した。 (Test Example 4: Nitric oxide (NO) production inhibition test in microglia)
The inhibitory effect on NO production in microglia was tested as follows.
マウス由来ミクログリア培養株（Ｃ８－Ｂ４）を、１０質量％ＦＢＳ含有ＤＭＥＭを用いて培養した後、トリプシン処理により細胞を回収した。回収した細胞を１．０×１０５ｃｅｌｌｓ／ｍＬの濃度になるように１０質量％ＦＢＳ含有ＤＭＥＭで希釈した後、９６ウェルプレートに１ウェル当たり１００μＬずつ播種し、３７℃、５％ＣＯ２下でコンフルエントになるまで培養した。
A mouse-derived microglia culture strain (C8-B4) was cultured using DMEM containing 10% by mass of FBS, and then the cells were collected by trypsin treatment. The collected cells were diluted with DMEM containing 10 mass% FBS to a concentration of 1.0 x 10 5 cells/mL, then seeded at 100 μL per well in a 96-well plate, and incubated at 37°C under 5% CO 2 The cells were cultured until confluent.
培養終了後、培地を除去し、終濃度０．５％ＤＭＳＯを含む１０質量％ＦＢＳ含有ＤＭＥＭに溶解した被験試料（最終試料濃度は下記表４を参照）を各ウェルに１００μＬ添加し、続けて１０質量％ＦＢＳ含有ＤＭＥＭに溶解した終濃度０．５μｇ／ｍＬのリポポリサッカライド（ＬＰＳ）（Ｅ．ｃｏｌｉ Ｏ１１１；Ｂ４、ＳＩＧＭＡ製）及び５ｎｇ／ｍＬのインターフェロン－ガンマ（ＩＦＮ－γ）（マウス由来、Ｒ＆Ｄ ｓｙｓｔｅｍｓ製）を１００μＬ加え、２４時間培養した。なお、対照として、被験試料無添加の０．５％ＤＭＳＯを含む１０質量％ＦＢＳ含有ＤＭＥＭ １００μＬ及び１０質量％ＦＢＳ含有・ＬＰＳ・ＩＦＮ－γ含有ＤＭＥＭ １００μＬを加え、同様に培養した。
After culturing, remove the medium and add 100 μL of the test sample (see Table 4 below for final sample concentration) dissolved in DMEM containing 10 mass% FBS containing 0.5% DMSO to each well. Lipopolysaccharide (LPS) (E. coli O111; B4, manufactured by SIGMA) with a final concentration of 0.5 μg/mL dissolved in DMEM containing 10% by mass FBS and 5 ng/mL interferon-gamma (IFN-γ) (derived from mouse) , manufactured by R&D systems) was added and cultured for 24 hours. As a control, 100 μL of 10 mass % FBS-containing DMEM containing 0.5% DMSO without the addition of the test sample and 100 μL of 10 mass % FBS-containing, LPS, and IFN-γ-containing DMEM were added and cultured in the same manner.
一酸化窒素（ＮＯ）産生量は、亜硝酸イオン（ＮＯ２
－）量を指標に測定した。
具体的には、培養終了後、各ウェルの培養液１００μＬに、同量のグリス試薬（１質量％スルファニルアミド、０．１質量％ Ｎ－１－ｎａｐｈｔｈｙｌ ｅｔｈｙｌｅｎｅｄｉａｍｉｎｅ ｄｉｈｙｄｒｏｃｈｌｏｒｉｄｅを含む５質量％リン酸溶液）を添加し、１０分間室温にて反応させた。反応後、波長５４０ｎｍにおける吸光度を測定した。ＮＯ２ －を指標として検量線を作成し、培養上清中のＮＯ産生量を求めた。
ＮＯ産生抑制率（％）は、被験試料無添加時（対照）のＮＯ産生量をもとに、下記式により算出した。結果を表４に示す。
ＮＯ産生抑制率（％）＝｛（Ｈ－Ｇ）／Ｈ｝×１００
上記式中のＧ～Ｈは、それぞれ以下を表す。
Ｇ ： 被験試料添加時のＮＯ量
Ｈ ： 被験試料無添加時のＮＯ量 The amount of nitric oxide (NO) produced was measured using the amount of nitrite ion (NO 2 − ) as an index.
Specifically, after the completion of the culture, the same amount of Griss reagent (5% by mass phosphoric acid solution containing 1% by mass sulfanilamide and 0.1% by mass N-1-naphthyl ethylenediamine dihydrochloride) was added to 100 μL of the culture solution in each well. was added and reacted for 10 minutes at room temperature. After the reaction, absorbance at a wavelength of 540 nm was measured. A calibration curve was created using NO 2 - as an indicator, and the amount of NO produced in the culture supernatant was determined.
The NO production suppression rate (%) was calculated by the following formula based on the NO production amount when the test sample was not added (control). The results are shown in Table 4.
NO production suppression rate (%) = {(HG)/H}×100
G to H in the above formula each represent the following.
G: NO amount when test sample is added H: NO amount when test sample is not added
具体的には、培養終了後、各ウェルの培養液１００μＬに、同量のグリス試薬（１質量％スルファニルアミド、０．１質量％ Ｎ－１－ｎａｐｈｔｈｙｌ ｅｔｈｙｌｅｎｅｄｉａｍｉｎｅ ｄｉｈｙｄｒｏｃｈｌｏｒｉｄｅを含む５質量％リン酸溶液）を添加し、１０分間室温にて反応させた。反応後、波長５４０ｎｍにおける吸光度を測定した。ＮＯ２ －を指標として検量線を作成し、培養上清中のＮＯ産生量を求めた。
ＮＯ産生抑制率（％）は、被験試料無添加時（対照）のＮＯ産生量をもとに、下記式により算出した。結果を表４に示す。
ＮＯ産生抑制率（％）＝｛（Ｈ－Ｇ）／Ｈ｝×１００
上記式中のＧ～Ｈは、それぞれ以下を表す。
Ｇ ： 被験試料添加時のＮＯ量
Ｈ ： 被験試料無添加時のＮＯ量 The amount of nitric oxide (NO) produced was measured using the amount of nitrite ion (NO 2 − ) as an index.
Specifically, after the completion of the culture, the same amount of Griss reagent (5% by mass phosphoric acid solution containing 1% by mass sulfanilamide and 0.1% by mass N-1-naphthyl ethylenediamine dihydrochloride) was added to 100 μL of the culture solution in each well. was added and reacted for 10 minutes at room temperature. After the reaction, absorbance at a wavelength of 540 nm was measured. A calibration curve was created using NO 2 - as an indicator, and the amount of NO produced in the culture supernatant was determined.
The NO production suppression rate (%) was calculated by the following formula based on the NO production amount when the test sample was not added (control). The results are shown in Table 4.
NO production suppression rate (%) = {(HG)/H}×100
G to H in the above formula each represent the following.
G: NO amount when test sample is added H: NO amount when test sample is not added
（試験例５：ミクログリアにおける腫瘍壊死因子－α（ＴＮＦ－α）産生抑制作用試験）
ミクログリアにおけるＴＮＦ－α産生抑制作用を以下のように試験した。 (Test Example 5: Tumor necrosis factor-α (TNF-α) production inhibition test in microglia)
The inhibitory effect on TNF-α production in microglia was tested as follows.
ミクログリアにおけるＴＮＦ－α産生抑制作用を以下のように試験した。 (Test Example 5: Tumor necrosis factor-α (TNF-α) production inhibition test in microglia)
The inhibitory effect on TNF-α production in microglia was tested as follows.
マウス由来ミクログリア培養株（Ｃ８－Ｂ４）を、１０質量％ＦＢＳ含有ＤＭＥＭを用いて培養した後、トリプシン処理により細胞を回収した。回収した細胞を１．０×１０５ｃｅｌｌｓ／ｍＬの濃度になるように１０質量％ＦＢＳ含有ＤＭＥＭで希釈した後、９６ウェルプレートに１ウェル当たり１００μＬずつ播種し、３７℃、５％ＣＯ２下でコンフルエントになるまで培養した。
A mouse-derived microglia culture strain (C8-B4) was cultured using DMEM containing 10% by mass of FBS, and then the cells were collected by trypsin treatment. The collected cells were diluted with DMEM containing 10 mass% FBS to a concentration of 1.0 x 10 5 cells/mL, then seeded at 100 μL per well in a 96-well plate, and incubated at 37°C under 5% CO 2 The cells were cultured until confluent.
培養終了後、培地を除去し、終濃度０．５％ＤＭＳＯを含む１０質量％ＦＢＳ含有ＤＭＥＭに溶解した被験試料（最終試料濃度は下記表５を参照）を各ウェルに１００μＬ添加し、続けて１０質量％ＦＢＳ含有ＤＭＥＭに溶解した終濃度０．５μｇ／ｍＬのリポポリサッカライド（ＬＰＳ）（Ｅ．ｃｏｌｉ Ｏ１１１；Ｂ４、ＳＩＧＭＡ製）及び５ｎｇ／ｍＬのインターフェロン－ガンマ（ＩＦＮ－γ）（マウス由来、Ｒ＆Ｄ ｓｙｓｔｅｍｓ製）を１００μＬ加え、２４時間培養した。なお、対照として、被験試料無添加の０．５％ＤＭＳＯを含む１０質量％ＦＢＳ含有ＤＭＥＭ １００μＬ及び１０質量％ＦＢＳ含有・ＬＰＳ・ＩＦＮ－γ含有ＤＭＥＭ １００μＬを加え、同様に培養した。
After the culture was completed, the medium was removed, and 100 μL of the test sample (see Table 5 below for final sample concentration) dissolved in DMEM containing 10 mass% FBS containing a final concentration of 0.5% DMSO was added to each well. Lipopolysaccharide (LPS) (E. coli O111; B4, manufactured by SIGMA) with a final concentration of 0.5 μg/mL dissolved in DMEM containing 10% by mass FBS and 5 ng/mL interferon-gamma (IFN-γ) (derived from mouse) , manufactured by R&D systems) was added and cultured for 24 hours. As a control, 100 μL of 10 mass % FBS-containing DMEM containing 0.5% DMSO without the addition of the test sample and 100 μL of 10 mass % FBS-containing, LPS, and IFN-γ-containing DMEM were added and cultured in the same manner.
培養終了後、各ウェルの培養上清中のＴＮＦ－α量を、サンドイッチＥＬＩＳＡ法を用いて測定した。
ＴＮＦ－α産生抑制率（％）は、被験試料無添加時（対照）のＴＮＦ－α産生量をもとに、下記式により算出した。結果を表５に示す。
ＴＮＦ－α産生抑制率（％）＝｛（Ｊ－Ｉ）／Ｊ｝×１００
上記式中のＩ～Ｊは、それぞれ以下を表す。
Ｉ ： 被験試料添加時のＴＮＦ－α量
Ｊ ： 被験試料無添加時のＴＮＦ－α量 After completion of the culture, the amount of TNF-α in the culture supernatant of each well was measured using a sandwich ELISA method.
The TNF-α production inhibition rate (%) was calculated by the following formula based on the amount of TNF-α produced when the test sample was not added (control). The results are shown in Table 5.
TNF-α production inhibition rate (%) = {(J-I)/J}×100
I to J in the above formula each represent the following.
I: TNF-α amount when test sample is added J: TNF-α amount when test sample is not added
ＴＮＦ－α産生抑制率（％）は、被験試料無添加時（対照）のＴＮＦ－α産生量をもとに、下記式により算出した。結果を表５に示す。
ＴＮＦ－α産生抑制率（％）＝｛（Ｊ－Ｉ）／Ｊ｝×１００
上記式中のＩ～Ｊは、それぞれ以下を表す。
Ｉ ： 被験試料添加時のＴＮＦ－α量
Ｊ ： 被験試料無添加時のＴＮＦ－α量 After completion of the culture, the amount of TNF-α in the culture supernatant of each well was measured using a sandwich ELISA method.
The TNF-α production inhibition rate (%) was calculated by the following formula based on the amount of TNF-α produced when the test sample was not added (control). The results are shown in Table 5.
TNF-α production inhibition rate (%) = {(J-I)/J}×100
I to J in the above formula each represent the following.
I: TNF-α amount when test sample is added J: TNF-α amount when test sample is not added
（試験例６：ミクログリアにおける炎症関連遺伝子ｍＲＮＡ発現抑制作用試験）
ミクログリアにおける炎症関連遺伝子ｍＲＮＡ発現抑制作用を以下のように試験した。 (Test Example 6: Inflammation-related gene mRNA expression suppression test in microglia)
The inhibitory effect on inflammation-related gene mRNA expression in microglia was tested as follows.
ミクログリアにおける炎症関連遺伝子ｍＲＮＡ発現抑制作用を以下のように試験した。 (Test Example 6: Inflammation-related gene mRNA expression suppression test in microglia)
The inhibitory effect on inflammation-related gene mRNA expression in microglia was tested as follows.
マウス由来ミクログリア培養株（Ｃ８－Ｂ４）を、１０質量％ＦＢＳ含有ＤＭＥＭを用いて培養した後、トリプシン処理により細胞を回収した。回収した細胞を１．０×１０５ｃｅｌｌｓ／ｍＬの濃度になるように１０質量％ＦＢＳ含有ＤＭＥＭで希釈した後、６ウェルプレートに１ウェル当たり２ｍＬずつ播種し、３７℃、５％ＣＯ２下でコンフルエントになるまで培養した。
A mouse-derived microglia culture strain (C8-B4) was cultured using DMEM containing 10% by mass of FBS, and then the cells were collected by trypsin treatment. The collected cells were diluted with DMEM containing 10 mass% FBS to a concentration of 1.0 x 10 5 cells/mL, then seeded in 2 mL per well in a 6-well plate, and incubated at 37°C under 5% CO 2 The cells were cultured until confluent.
培養終了後、培地を除去し、終濃度０．５％ＤＭＳＯを含む１０質量％ＦＢＳ含有ＤＭＥＭに溶解した被験試料（最終試料濃度は下記表６を参照）を各ウェルに１ｍＬずつ添加し、続けて１０質量％ＦＢＳ含有ＤＭＥＭに溶解した終濃度０．５μｇ／ｍＬのリポポリサッカライド（ＬＰＳ）（Ｅ．ｃｏｌｉ Ｏ１１１；Ｂ４、ＳＩＧＭＡ製）及び５ｎｇ／ｍＬのインターフェロン－ガンマ（ＩＦＮ－γ）（マウス由来、Ｒ＆Ｄ ｓｙｓｔｅｍｓ製）を１ｍＬ加え、２４時間培養した。なお、対照として、被験試料無添加の０．５％ＤＭＳＯを含む１０質量％ＦＢＳ含有ＤＭＥＭ １ｍＬ及び１０質量％ＦＢＳ含有・ＬＰＳ・ＩＦＮ－γ含有ＤＭＥＭ １ｍＬを加え、同様に培養した。
After culturing, remove the medium, add 1 mL of the test sample (see Table 6 below for final sample concentration) dissolved in DMEM containing 10 mass% FBS containing a final concentration of 0.5% DMSO to each well, and continue. Lipopolysaccharide (LPS) (E. coli O111; B4, manufactured by SIGMA) at a final concentration of 0.5 μg/mL and 5 ng/mL interferon-gamma (IFN-γ) (mouse) were dissolved in DMEM containing 10% by mass of FBS. 1 mL of R&D Systems) was added and cultured for 24 hours. As a control, 1 mL of 10 mass % FBS-containing DMEM containing 0.5% DMSO and 1 mL of 10 mass % FBS-containing/LPS/IFN-γ-containing DMEM containing no test sample were added and cultured in the same manner.
培養後、培養液を除去し、ＩＳＯＧＥＮＥ ＩＩ（ＮＩＰＰＯＮ ＧＥＮＥ製）にて総ＲＮＡを抽出し、波長２６０ｎｍにおける吸光度からＲＮＡ量を計算し、１００ｎｇ／μＬになるように総ＲＮＡを調製した。
After culturing, the culture solution was removed, total RNA was extracted using ISOGENE II (manufactured by NIPPON GENE), the amount of RNA was calculated from the absorbance at a wavelength of 260 nm, and the total RNA was adjusted to 100 ng/μL.
この総ＲＮＡを鋳型とし、各種炎症関連遺伝子（腫瘍壊死因子－α（ＴＮＦ－α）、誘導型一酸化窒素合成酵素（ｉＮＯＳ）、インターロイキン－６（ＩＬ－６）、インターロイキン－１β（ＩＬ－１β）、インターロイキン－１２（ＩＬ－１２）、ケモカイン（Ｃ－Ｘ－Ｃモチーフ）リガンド２（ＣＸＣＬ２））及び内部標準であるＧＡＰＤＨのｍＲＮＡの発現量を測定した。
ｍＲＮＡの検出は、リアルタイムＰＣＲ装置Ｔｈｅｒｍａｌ Ｃｙｃｌｅｒ Ｄｉｃｅ（登録商標） Ｒｅａｌ Ｔｉｍｅ Ｓｙｓｔｅｍ ＩＩＩ（ＴａＫａＲａ製）を用いて、ＰｒｉｍｅＳｃｒｉｐｔＴＭ ＲＴ Ｍａｓｔｅｒ Ｍｉｘ（Ｐｅｒｆｅｃｔ Ｒｅａｌ Ｔｉｍｅ）及びＴＢ Ｇｒｅｅｎ（登録商標） Ｆａｓｔ ｑＰＣＲ Ｍｉｘ（ＴａＫａＲａ製）による２ステップリアルタイムＲＴ－ＰＣＲ反応により行った。
各遺伝子ｍＲＮＡの発現量は、ＧＡＰＤＨ ｍＲＮＡの発現量で補正し算出した。得られた値から、下記式により各遺伝子のｍＲＮＡ発現率（％）を算出した（対照＝１００％）。結果を表６に示す。なお、ＴＮＦ－α、ｉＮＯＳ、ＩＬ－６、ＩＬ－１β、ＩＬ１２、及びＣＸＣＬ２は炎症誘導因子であることが知られており、ＬＰＳ及びＩＦＮ－γ刺激により、ＬＰＳ及びＩＦＮ－γ未刺激の場合と比べて、これらのｍＲＮＡの発現は上昇した。
各遺伝子のｍＲＮＡ発現率（％）＝Ｋ／Ｌ×１００
上記式中のＫ～Ｌは、それぞれ以下を表す。
Ｋ ： 被験試料添加、ＬＰＳ及びＩＦＮ－γ刺激時の補正値
Ｌ ： 被験試料無添加、ＬＰＳ及びＩＦＮ－γ刺激時の補正値 Using this total RNA as a template, various inflammation-related genes (tumor necrosis factor-α (TNF-α), inducible nitric oxide synthase (iNOS), interleukin-6 (IL-6), interleukin-1β (IL -1β), interleukin-12 (IL-12), chemokine (CXC motif) ligand 2 (CXCL2)), and the internal standard GAPDH mRNA expression levels were measured.
Detection of mRNA was performed using a real-time PCR device Thermal Cycler Dice (registered trademark) Real Time System III (manufactured by TaKaRa) using PrimeScript TM RT Master Mix (Perfect Real Time) and TB Gr een (registered trademark) Fast qPCR Mix (manufactured by TaKaRa) ) was carried out using a two-step real-time RT-PCR reaction.
The expression level of each gene mRNA was calculated by correcting the expression level of GAPDH mRNA. From the obtained values, the mRNA expression rate (%) of each gene was calculated using the following formula (control = 100%). The results are shown in Table 6. Furthermore, TNF-α, iNOS, IL-6, IL-1β, IL12, and CXCL2 are known to be inflammation-inducing factors, and when stimulated with LPS and IFN-γ, they are The expression of these mRNAs was increased compared to .
mRNA expression rate (%) of each gene = K/L x 100
K to L in the above formula each represent the following.
K: Correction value when test sample is added, LPS and IFN-γ stimulation L: Correction value when test sample is not added, LPS and IFN-γ stimulation
ｍＲＮＡの検出は、リアルタイムＰＣＲ装置Ｔｈｅｒｍａｌ Ｃｙｃｌｅｒ Ｄｉｃｅ（登録商標） Ｒｅａｌ Ｔｉｍｅ Ｓｙｓｔｅｍ ＩＩＩ（ＴａＫａＲａ製）を用いて、ＰｒｉｍｅＳｃｒｉｐｔＴＭ ＲＴ Ｍａｓｔｅｒ Ｍｉｘ（Ｐｅｒｆｅｃｔ Ｒｅａｌ Ｔｉｍｅ）及びＴＢ Ｇｒｅｅｎ（登録商標） Ｆａｓｔ ｑＰＣＲ Ｍｉｘ（ＴａＫａＲａ製）による２ステップリアルタイムＲＴ－ＰＣＲ反応により行った。
各遺伝子ｍＲＮＡの発現量は、ＧＡＰＤＨ ｍＲＮＡの発現量で補正し算出した。得られた値から、下記式により各遺伝子のｍＲＮＡ発現率（％）を算出した（対照＝１００％）。結果を表６に示す。なお、ＴＮＦ－α、ｉＮＯＳ、ＩＬ－６、ＩＬ－１β、ＩＬ１２、及びＣＸＣＬ２は炎症誘導因子であることが知られており、ＬＰＳ及びＩＦＮ－γ刺激により、ＬＰＳ及びＩＦＮ－γ未刺激の場合と比べて、これらのｍＲＮＡの発現は上昇した。
各遺伝子のｍＲＮＡ発現率（％）＝Ｋ／Ｌ×１００
上記式中のＫ～Ｌは、それぞれ以下を表す。
Ｋ ： 被験試料添加、ＬＰＳ及びＩＦＮ－γ刺激時の補正値
Ｌ ： 被験試料無添加、ＬＰＳ及びＩＦＮ－γ刺激時の補正値 Using this total RNA as a template, various inflammation-related genes (tumor necrosis factor-α (TNF-α), inducible nitric oxide synthase (iNOS), interleukin-6 (IL-6), interleukin-1β (IL -1β), interleukin-12 (IL-12), chemokine (CXC motif) ligand 2 (CXCL2)), and the internal standard GAPDH mRNA expression levels were measured.
Detection of mRNA was performed using a real-time PCR device Thermal Cycler Dice (registered trademark) Real Time System III (manufactured by TaKaRa) using PrimeScript TM RT Master Mix (Perfect Real Time) and TB Gr een (registered trademark) Fast qPCR Mix (manufactured by TaKaRa) ) was carried out using a two-step real-time RT-PCR reaction.
The expression level of each gene mRNA was calculated by correcting the expression level of GAPDH mRNA. From the obtained values, the mRNA expression rate (%) of each gene was calculated using the following formula (control = 100%). The results are shown in Table 6. Furthermore, TNF-α, iNOS, IL-6, IL-1β, IL12, and CXCL2 are known to be inflammation-inducing factors, and when stimulated with LPS and IFN-γ, they are The expression of these mRNAs was increased compared to .
mRNA expression rate (%) of each gene = K/L x 100
K to L in the above formula each represent the following.
K: Correction value when test sample is added, LPS and IFN-γ stimulation L: Correction value when test sample is not added, LPS and IFN-γ stimulation
試験例１～６で示したように、構造式（１）～（３）のいずれかで表される化合物は、優れた各種作用を示し、脳の機能改善剤として有用であることが確認された。
As shown in Test Examples 1 to 6, it has been confirmed that compounds represented by any of structural formulas (1) to (3) exhibit various excellent effects and are useful as brain function improving agents. Ta.
（配合例１）
常法により、以下の組成を有する錠剤を製造した。
・ 前記構造式（１）で表される化合物 ５．０ｍｇ
・ ドロマイト ８３．４ｍｇ
（カルシウム２０％、マグネシウム１０％含有）
・ カゼインホスホペプチド １６．７ｍｇ
・ ビタミンＣ ３３．４ｍｇ
・ マルチトール １３６．８ｍｇ
・ コラーゲン １２．７ｍｇ
・ ショ糖脂肪酸エステル １２．０ｍｇ (Combination example 1)
Tablets having the following composition were manufactured by a conventional method.
- Compound represented by the above structural formula (1) 5.0 mg
・Dolomite 83.4mg
(Contains 20% calcium and 10% magnesium)
・Casein phosphopeptide 16.7mg
・Vitamin C 33.4mg
・Maltitol 136.8mg
・Collagen 12.7mg
・Sucrose fatty acid ester 12.0mg
常法により、以下の組成を有する錠剤を製造した。
・ 前記構造式（１）で表される化合物 ５．０ｍｇ
・ ドロマイト ８３．４ｍｇ
（カルシウム２０％、マグネシウム１０％含有）
・ カゼインホスホペプチド １６．７ｍｇ
・ ビタミンＣ ３３．４ｍｇ
・ マルチトール １３６．８ｍｇ
・ コラーゲン １２．７ｍｇ
・ ショ糖脂肪酸エステル １２．０ｍｇ (Combination example 1)
Tablets having the following composition were manufactured by a conventional method.
- Compound represented by the above structural formula (1) 5.0 mg
・Dolomite 83.4mg
(Contains 20% calcium and 10% magnesium)
・Casein phosphopeptide 16.7mg
・Vitamin C 33.4mg
・Maltitol 136.8mg
・Collagen 12.7mg
・Sucrose fatty acid ester 12.0mg
（配合例２）
常法により、以下の組成を有する経口液状製剤を製造した。
＜１アンプル（１本１００ｍＬ）中の組成＞
・ 前記構造式（２）で表される化合物 ０．３質量％
・ ソルビット １２．０質量％
・ 安息香酸ナトリウム ０．１質量％
・ 香料 １．０質量％
・ 硫酸カルシウム ０．５質量％
・ 精製水 残部 (Combination example 2)
An oral liquid preparation having the following composition was produced by a conventional method.
<Composition in 1 ampoule (100 mL per bottle)>
- Compound represented by the above structural formula (2) 0.3% by mass
・Sorvit 12.0% by mass
・Sodium benzoate 0.1% by mass
・Fragrance 1.0% by mass
・Calcium sulfate 0.5% by mass
・Remaining purified water
常法により、以下の組成を有する経口液状製剤を製造した。
＜１アンプル（１本１００ｍＬ）中の組成＞
・ 前記構造式（２）で表される化合物 ０．３質量％
・ ソルビット １２．０質量％
・ 安息香酸ナトリウム ０．１質量％
・ 香料 １．０質量％
・ 硫酸カルシウム ０．５質量％
・ 精製水 残部 (Combination example 2)
An oral liquid preparation having the following composition was produced by a conventional method.
<Composition in 1 ampoule (100 mL per bottle)>
- Compound represented by the above structural formula (2) 0.3% by mass
・Sorvit 12.0% by mass
・Sodium benzoate 0.1% by mass
・Fragrance 1.0% by mass
・Calcium sulfate 0.5% by mass
・Remaining purified water
（配合例３）
常法により、以下の組成を有するコーヒー飲料を製造した。
・ 前記構造式（３）で表される化合物 ０．１質量％
・ コーヒー抽出液 ４０質量％
（Ｌ（明度）＝２０、Ｂｒｉｘ＝３）
・ マルチトール ２質量％
・ 香料 適量
・ 水 残部 (Combination example 3)
A coffee beverage having the following composition was produced by a conventional method.
- Compound represented by the above structural formula (3) 0.1% by mass
・Coffee extract 40% by mass
(L (lightness) = 20, Brix = 3)
・Maltitol 2% by mass
- Appropriate amount of fragrance - Remaining water
常法により、以下の組成を有するコーヒー飲料を製造した。
・ 前記構造式（３）で表される化合物 ０．１質量％
・ コーヒー抽出液 ４０質量％
（Ｌ（明度）＝２０、Ｂｒｉｘ＝３）
・ マルチトール ２質量％
・ 香料 適量
・ 水 残部 (Combination example 3)
A coffee beverage having the following composition was produced by a conventional method.
- Compound represented by the above structural formula (3) 0.1% by mass
・Coffee extract 40% by mass
(L (lightness) = 20, Brix = 3)
・Maltitol 2% by mass
- Appropriate amount of fragrance - Remaining water
（配合例４）
常法により、以下の組成を有するカプセル剤を製造した。なお、カプセルとしては、１号ハードゼラチンカプセルを使用した。
＜１カプセル（１錠２００ｍｇ）中の組成＞
・ 前記構造式（１）で表される化合物 ３０．０ｍｇ
・ コーンスターチ ７０．０ｍｇ
・ 乳糖 ８０．０ｍｇ
・ 乳酸カルシウム １０．０ｍｇ
・ ヒドロキシプロピルセルロース（ＨＰＣ－Ｌ） １０．０ｍｇ (Combination example 4)
Capsules having the following composition were manufactured by a conventional method. Note that a No. 1 hard gelatin capsule was used as the capsule.
<Composition in 1 capsule (1 tablet 200mg)>
- Compound represented by the above structural formula (1) 30.0 mg
・Corn starch 70.0mg
・Lactose 80.0mg
・Calcium lactate 10.0mg
・Hydroxypropylcellulose (HPC-L) 10.0mg
常法により、以下の組成を有するカプセル剤を製造した。なお、カプセルとしては、１号ハードゼラチンカプセルを使用した。
＜１カプセル（１錠２００ｍｇ）中の組成＞
・ 前記構造式（１）で表される化合物 ３０．０ｍｇ
・ コーンスターチ ７０．０ｍｇ
・ 乳糖 ８０．０ｍｇ
・ 乳酸カルシウム １０．０ｍｇ
・ ヒドロキシプロピルセルロース（ＨＰＣ－Ｌ） １０．０ｍｇ (Combination example 4)
Capsules having the following composition were manufactured by a conventional method. Note that a No. 1 hard gelatin capsule was used as the capsule.
<Composition in 1 capsule (1 tablet 200mg)>
- Compound represented by the above structural formula (1) 30.0 mg
・Corn starch 70.0mg
・Lactose 80.0mg
・Calcium lactate 10.0mg
・Hydroxypropylcellulose (HPC-L) 10.0mg
本出願は、２０２２年９月１６日に出願した日本国特許出願２０２２－１４７６４４号に基づく優先権を主張するものであり、日本国特許出願２０２２－１４７６４４号の全内容を本出願に援用する。
This application claims priority based on Japanese Patent Application No. 2022-147644 filed on September 16, 2022, and the entire content of Japanese Patent Application No. 2022-147644 is incorporated into this application.
Claims (3)
- 下記構造式（１）～（３）のいずれかで表される化合物の少なくともいずれかを含むことを特徴とする脳の機能改善剤。
- アストロサイト増殖促進作用、アストロサイトにおけるグリア細胞株由来神経栄養因子（ＧＤＮＦ）ｍＲＮＡ発現促進作用、アストロサイトにおけるアクアポリン４（ＡＱＰ４）ｍＲＮＡ発現促進作用、ミクログリアにおける一酸化窒素（ＮＯ）産生抑制作用、ミクログリアにおける腫瘍壊死因子－α（ＴＮＦ－α）産生抑制作用、及びミクログリアにおける炎症関連遺伝子ｍＲＮＡ発現抑制作用からなる群から選択される１種以上の作用に基づく脳の機能改善用途に用いられる請求項１に記載の脳の機能改善剤。 Promoting astrocyte proliferation, promoting glial cell line-derived neurotrophic factor (GDNF) mRNA expression in astrocytes, promoting aquaporin 4 (AQP4) mRNA expression in astrocytes, suppressing nitric oxide (NO) production in microglia, microglia Claim 1: Used for brain function improvement based on one or more effects selected from the group consisting of tumor necrosis factor-α (TNF-α) production suppressing effect in microglia and inflammation-related gene mRNA expression suppressing effect in microglia. A brain function improving agent described in .
- 請求項１から２のいずれかに記載の脳の機能改善剤を含むことを特徴とする脳の機能改善用組成物。 A composition for improving brain function, comprising the brain function improving agent according to any one of claims 1 to 2.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2022-147644 | 2022-09-16 | ||
| JP2022147644 | 2022-09-16 |
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| WO2024057782A1 true WO2024057782A1 (en) | 2024-03-21 |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020175690A1 (en) * | 2019-02-28 | 2020-09-03 | 森永乳業株式会社 | Composition for inducing pili formation in bacterium of genus bifidobacterium |
| CN116059194A (en) * | 2023-03-21 | 2023-05-05 | 河北医科大学 | Application of parahydroxybenzoic acid in preparation of protein amyloid fibrosis inhibitor |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020175690A1 (en) * | 2019-02-28 | 2020-09-03 | 森永乳業株式会社 | Composition for inducing pili formation in bacterium of genus bifidobacterium |
| CN116059194A (en) * | 2023-03-21 | 2023-05-05 | 河北医科大学 | Application of parahydroxybenzoic acid in preparation of protein amyloid fibrosis inhibitor |
Non-Patent Citations (2)
| Title |
|---|
| GONZALEZ-SARRIAS ET AL.: "Neuroprotective Effects of Bioavailable Polyphenol-Derived Metabolites against Oxidative Stress-Induced Cytotoxicity in Human Neuroblastoma SH-SY5Y Cells", JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY., vol. 65, no. 4, 14 November 2016 (2016-11-14), pages 752 - 758, XP002768227, DOI: 10.1021/acs.jafc.6b04538 * |
| SEISUKE MIMORI, YASUNOBU OKUMA, MASAYUKI KANEKO, KOICHI KAWADA, TORU HOSOI, KOICHIRO OZAWA, YASUYUKI NOMURA, HIROSHI HAMANA: "Protective Effects of 4-Phenylbutyrate Derivatives on the Neuronal Cell Death and Endoplasmic Reticulum Stress", BIOLOGICAL & PHARMACEUTICAL BULLETIN, PHARMACEUTICAL SOCIETY OF JAPAN, TOKYO., JP, vol. 35, no. 1, 1 January 2012 (2012-01-01), JP , pages 84 - 90, XP055340337, ISSN: 0918-6158, DOI: 10.1248/bpb.35.84 * |
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