JP2003335690A - Composition having affinity for receptor - Google Patents

Abstract

(57) [Summary] (Modified) [Problem] Composition to improve diseases related to nicotinic and muscarinic acetylcholine receptors, dopamine 1 receptors, benzodiazepine receptors, and GABA receptors with excellent stability, safety, and cost Offer things. A nicotinic and muscarinic acetylcholine receptor, a dopamine 1 receptor, a benzodiazepine receptor, a GABA receptor agonist, schizophrenia, Tourette's syndrome, lack of attention, containing kale, propolis or extracts thereof Composition for treatment or prevention of central nervous system diseases such as disability, anxiety, reading disorders, senile dementia, senile dementia, Parkinsonism including Parkinson's disease, Huntington's chorea, late movement abnormalities, or hyperkinesia,
Brain function improving agent, brain metabolic activator, oral composition, parenteral composition, food and medicine containing these.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to nicotinic and muscarinic acetylcholine receptors, dopamine 1 receptors, benzodiazepine receptors, γ-aminobutyric acid (GAB
A) Compositions useful for diseases caused by disorders involving receptors and oral or parenteral compositions containing them.

[0002]

2. Description of the Related Art Nerve cells in the brain communicate with each other via various chemical mediators.
Regarding signal transduction, when a chain of electric current due to a potential difference at a cell membrane changes an ion channel and further propagates to the tip of an axon, a neurotransmitter is released. Then, it reaches the receptor of the cell on the receiving side through the synapse (contact site between cells).

However, due to inadequate levels of neurotransmitter release, inadequate properties of neurotransmitter receptors, or disorders resulting from interactions between neurotransmitters and neurotransmitter receptors, etc. System (Central Nervous Sy
stem: CNS) Disability is caused. CNS disorders include
Schizophrenia and Tourette's syndrome, attention deficit disorder, anxiety, dyslexia, senile dementia (early onset of Alzheimer's disease), senile dementia (Alzheimer-type dementia), Parkinsonism including Parkinson's disease, Huntington's chorea, late phase Dyskinesia, hyperkinesia and the like.

The acetylcholine system is one of the major neurotransmitters of the central nervous system and is known to play an important role in the regulation of nerve activity in the cerebral cortex and hippocampus.
It has been pointed out that it may be involved in CNS disorders. Therefore, activation of the acetylcholine nervous system may ameliorate these disorders. There are several possible ways to activate the acetylcholine neurotransmission function. The first is to inhibit its degrading enzyme, acetylcholinesterase, in order to increase the concentration of acetylcholine lowered in the brain, and secondly, to promote the release of acetylcholine at synapses. First, it binds to the acetylcholine receptor and activates it. For example, decreased nicotinic receptors in the acetylcholine system have been reported in the cerebral cortex and hippocampus of autopsy brains of Alzheimer's disease patients (Alzheimer's Disease Reviews,
3: 20-34, 1998). Therefore, there is a need to provide therapeutic agents such as directly activating nicotine receptors instead of acetylcholine, or minimizing the loss of these nicotine receptors.

Dopamine (J. Pharm
acol. 47,765,1973), norepinephrine (Biochem. Pha
rmacol. 21,1829,1972), serotonin (Arch. Int. Pharm
acodyn. Ther. It has been reported that neurotransmitters such as 296, 1829, 1972) are accelerated in release. Therefore, it would be desirable to provide a useful method for the prevention and treatment of disorders by administering nicotine compounds to patients susceptible to or already suffering from such disorders. In fact, various nicotine compounds have been used to treat the various CNS disorders mentioned above (Exp. Opin. Invest. Drugs 5
(1), 79-100, 1996).

[0006] However, nicotine generally has problems such as addiction, low bioavailability (bioavailability), and side effects on the circulatory system. Therefore,
It is extremely difficult to provide a pharmaceutical composition which does not have side effects such as an increase in pulse and blood pressure associated with the interaction with a cardiovascular site, and which contains an active ingredient having a pharmacology of nicotine and which interacts with a nicotine receptor. Would be desirable.

Also, attention has been paid to muscarinic acetylcholine receptors, and many studies have been conducted on receptor activating substances. There are two types of muscarinic acetylcholine receptors, and muscarinic 1 receptor and muscarinic 2 receptor are known. Muscarinic 1 receptors are localized in the cerebral cortex and hippocampus, and muscarinic 2 receptors are localized in the thalamus, brain stem, cerebellum, presynaptic region, and heart (Mikoshiba).
Katsuhiko ed .; Term library cranial nerve, pp92, Yodosha, 19
97). As the one that activates the muscarinic 1 receptor,
2- (2-morpholinoethylamino) -9,10-dihydro-oxa-3,4-diazaphenanthrene dihydrochloride (JP-A-1-203387) and morpholinoethylamino-3
-Benzodiclohepta- (5,6-C) -viritadine dihydrochloride (Eur. J. Pharm., 166, 139-147, 1989) and the like have been reported.

In recent years, THA (1,2,3,4-tetrahydro-9) has been used as a therapeutic agent for Alzheimer-type dementia in the United States.
-Aminoacridine) has been developed and has become a hot topic.
However, there are issues with efficacy and side effects. Therefore, there is an urgent need to develop a prophylactic and therapeutic agent for dementia, which exhibits a pharmacological action reliably and has low toxicity.

As described above, studies on acetylcholine disorders have been advanced, but their efficacy, duration, and side effects are not always sufficient, and excellent therapeutic agents and therapeutic methods have not been found until now. The current situation is that it has not been done.

Dopamine is a kind of in-vivo amine such as norepinephrine and epinephrine contained in sympathetic postganglionic fibers and adrenal medulla, and is an important neurotransmitter of the central nervous system. Its actions are involved in mental and motor functions, including schizophrenia, Parkinson's disease, depression and eating disorders,
It is thought to be associated with the development of disorders such as sleep, learning, memory, sexual behavior, and blood pressure.

In patients with Parkinson's disease, striatal dopamine in the basal ganglia, which is a region involved in motor control of the brain, is decreased, so that L- is converted into dopamine in the brain.
Dopamine replacement therapy with dopa (3,4-dihydroxyphenylalanine) is used. In addition to L-dopa, dopamine agonist bromocriptine and the like are also used.

On the other hand, excess dopamine in the brain causes schizophrenia with impaired thought processes, hallucinations and realistic loss of touch. Anti-schizophrenia drugs block excessive receptor stimulation by blocking dopamine receptors (De
velopments in the drug treatment of shizophrenia, T
IPS, 13, 116-121, 1992).

Dopamine receptors are currently D ₁ -like receptors (D ₁ , D ₅ ) based on biochemical and pharmacological differences.
And D ₂ -like receptors (D ₂ , D ₃ , D ₄ ) (Molecu
larBiology of Dopamine receputors, TIPS, 13,61-69,19
92). Heretofore, an acid addition salt of a 2,3,4,5-tetrahydro-1H-3-benzazepine compound has been used as a composition for treating an indication associated with dysfunction of the D ₁ receptor system (see Table 1
1-512403), piperazine, piperidine, and 1,2,5,6-tetrahydropyridine compound (Japanese Patent Laid-Open No. 08-325257) have been reported for D ₄ receptors.

Thus, for dopamine receptors,
Those having an affinity have been developed and used for various diseases, but often have a problem of side effects. Side effects include tardive dyskinesia, malignant neurological syndrome, hyperprolactinemia, amenorrhea, involuntary movements, extrapyramidal action, and hypotension. Benzodiazepine (BZD) compounds have anxiolytic action, anticonvulsant action, sedative / hypnotic action, and the like, and are widely clinically applied as anxiolytic drug, hypnotic drug, muscle relaxant drug, antiepileptic drug, and the like. It has been clarified that there are three subtypes of the BZD receptor, which are named ω ₁ , ω ₂ and ω ₃ receptors, respectively. The ω ₁ receptor is deeply involved in the expression of sedative / sleeping actions and anxiolytic actions, the ω ₂ receptor is involved in muscle relaxation action, and the ω ₃ receptor is deeply involved in pharmacological actions such as antianginal action and tolerance formation. Has been suggested. Conventional BZD compounds are considered to bind to ω ₁ and ω ₂ receptors and exert their pharmacological actions. The pharmacological actions of BZD compounds are not expressed by independent mechanisms of action, but are interpreted to be caused by closely related neuropharmacological mechanisms. Its mechanism of action is the phenomenon of enhancement of the γ-aminobutyric acid (GABA) ergic neuronal signaling mechanism in the central nervous system by BZD drugs. GABA, an inhibitory neurotransmitter
GABA-A receptor, GABA-B receptor, etc. exist in the receptor. GABA-A receptors are coupled to Cl ^- channels and are associated with anxiolytic and sleep-inducing actions, while GABA-B receptors are coupled to G proteins and also via G proteins. It is linked to Ca ²⁺ and K ⁺ channels and is thought to be associated with depression (CLINICALNEUROSCIENCE, 14 (4) 404-40
7, 1996). BZD drugs are GABA-A receptor BZD
It binds to the binding site and enhances the action of GABA.

A compound having an affinity for the BZD receptor is a 5-heteroarylindole derivative (Table 9-50
1949) and 1,2,4, -oxadiazolylquinolone derivatives (JP-A-11-279176). Further, β-carboline derivative (European Patent Publication No. 499527) is described as a drug useful in the treatment of degenerative central nervous system disorders (for example, Alzheimer's disease).

However, BZD-based compounds have many side effects such as staggering, drowsiness, muscle relaxation, or deterioration of cognitive ability and reflex motor capacity, and problems such as tolerance and dependence formation that should be improved. Therefore, for the purpose of solving various problems, there is a need for a BZD receptor agonist which is stable, safe, and inexpensive in terms of price.

GABA is considered to be one of the most important inhibitory neurotransmitters in mammals because it produces an inhibitory postsynaptic potential and is distributed in high concentrations in the brain and spinal cord of mammals. There is. When the brain is abnormally excited by stress, GABA in the brain functions to calm the brain's excitement and calm the mind. This GAB
It has been revealed that A exerts its action for the first time by binding to the GABA receptor in the brain. Furthermore, G
ABA activates cerebral blood flow, increases oxygen supply to enhance cerebral metabolic function, acts on vasomotor center of spinal cord to lower blood pressure, and suppresses secretion of vasopressin, an antidiuretic hormone Have been reported to be involved in promoting blood glucose expansion by expanding blood vessels, lowering blood pressure, and increasing the hexokinase activity required at the introduction site of the TCA cycle [Eiichi Otomo: New Edition: Brain Metabolism. Activator,
Pharmaceutical Journal (1987)].

[0018] So far, GABA transaminase (GABA transamination) has been used for the purpose of enhancing the concentration of GABA in the brain to improve brain metabolism, antidepressant, sedation, anxiety, sleep disorders, and lower blood pressure. A drug that inhibits an enzyme or has a GABA receptor activating effect (JP-A-60-3
6448, JP-A-2-32029, JP8-509238, JP11-509194, JP2001-515033) and G
Foods containing a high amount of ABA (JP 2000-14356, JP 2000-3
21100, JP 2000-60536, JP 2001-352940, JP 2001-1
20179 etc.) has been developed, but no drug has been developed that is safe and can be sufficiently satisfied to take a reliable treatment even if taken for a long period of time. Although many have been developed, as far as the inventor knows, no food derived from a natural product having a GABA receptor activating effect has been reported.

[0019]

DISCLOSURE OF THE INVENTION The present invention provides a composition for improving diseases relating to nicotinic and muscarinic acetylcholine receptors, dopamine 1 receptors, benzodiazepine receptors and GABA receptors which are excellent in stability, safety and cost. It is to provide things.

[0020]

Means for Solving the Problems The present inventors have conducted extensive studies to solve the above problems, and as a result, kale and propolis are nicotinic and muscarinic acetylcholine receptors, dopamine 1 receptors, benzodiazepine receptors,
The inventors have found that they have an affinity for GABA receptors and completed the present invention.

That is, the present invention comprises a nicotinic and muscarinic acetylcholine receptor, a dopamine 1 receptor, a benzodiazepine receptor, a GABA receptor agonist, which contains kale, propolis or an extract thereof. , Central nervous system such as schizophrenia, Tourette's syndrome, attention deficit disorder, anxiety, dyslexia, senile dementia, senile dementia, Parkinsonism including Parkinson's disease, Huntington's chorea, late dyskinesia, or hyperkinesia The present invention relates to a composition for treating or preventing a systemic disease, a brain function improving agent, a brain metabolism activating agent, an oral composition containing the same, a parenteral composition, and further, these compositions in the form of food or medicine.

[0022]

BEST MODE FOR CARRYING OUT THE INVENTION The kale (Bras used in the present invention
sicca Oleracea L.var.acephala DC.) includes kitchen kale, maro kale, bush kale, tree kale, collard and green leaf citrus. The cruciferous plant is originally a vegetable originally from Southern Europe and is said to be the original cabbage species. The leaves may be those usually used for food, and the cultivation method and cultivation place are not particularly limited.

As the kale and the extract thereof, a dried product obtained by drying the kale itself, a crushed product thereof, a squeezed juice, a crude extract with water or an organic solvent such as alcohol, ether or acetone, and a crude extract are distributed, It includes all of the extract fractions obtained by stepwise purification by various chromatography such as column chromatography. These may be used alone or in combination of two or more.

For example, 3 L of 99.5% ethanol extract was added to 1 kg of dried matter such as leaves, stems, flowers and roots of kale, and the extract obtained by soaking overnight at room temperature was used as it was to give nicotinic and muscarinic acetylcholine. Receptor, dopamine
1 Receptor, benzodiazepine receptor, GABA receptor may be used as an activator, or the extract may be purified by combining various chromatographies.

Propolis is a resin-like black mass collected by bees. Propolis from Brazil, Propolis from China, Propolis from Australia, Propolis from Uruguay,
It may be produced in any place such as Japanese propolis, and is not particularly limited, but from the viewpoint of versatility, Brazilian propolis and Chinese propolis are preferable.

The propolis in the present invention is a dried product obtained by drying propolis itself, a pulverized product thereof, a supercritical extract,
Includes all, such as crude extract with water or organic solvent such as alcohol, ether, acetone, etc., and extract fraction obtained by partitioning the crude extract and stepwise purification by various chromatography such as column chromatography . These may be used alone or in combination of two or more.

For example, the extract obtained by adding 3 L of a 99.5% ethanol extract to 1 kg of a dried raw product of propolis from Brazil and immersing it at room temperature overnight may be used as it is, or may be subjected to various chromatographies. You may use it, combining and refine | purifying.

The concentration of the extract in the solution of the extracted kale and propolis extract is not particularly limited, but is preferably 15 to 70% by mass, preferably about 20 to 60% by mass. If this concentration is less than 15% by mass, it is necessary to evaporate a large amount of solution such as ethanol or water at the time of drying.
When it exceeds, the viscosity of the solution becomes too high, which may deteriorate the processability.

The kale, propolis or an extract thereof has an activating effect on nicotinic acetylcholine receptor, muscarinic acetylcholine receptor, dopamine 1 receptor, benzodiazepine receptor and GABA receptor.

By binding to the acetylcholine receptor and activating it, the concentration of acetylcholine lowered in the brain can be increased, and such an increase in acetylcholine concentration activates the acetylcholine neurotransmission function. be able to. Therefore, the CNS disorder can be improved. For example, it can be applied to Alzheimer's disease patients in which a decrease in nicotine receptors among acetylcholine receptors occurs.

By activating the muscarinic acetylcholine receptor, it can be used for symptoms due to cholinergic deficiency in the cerebral cortex and Alzheimer-type dementia.

By the dopamine 1 receptor activating effect,
It is used for central nervous system diseases related to dopamine, such as depression and memory disorders, such as schizophrenia due to hyperactivity of dopaminergic system, Parkinson's disease due to its hypoactivity.

By activating benzodiazepine receptors, senile dementia is used as an anti-anxiety drug and a therapeutic agent for sleep disorders in which benzodiazepine agonists have been conventionally used, and in addition, inverse agonists are used as brain stimulants. , Can be used as a remedy for memory disorders such as Alzheimer-type dementia.

By activating the GABA receptor,
It can be used for amelioration of cerebral dysfunction induced by reduction of GABA concentration.

Furthermore, kale, propolis or an extract thereof, by activating the above-mentioned various receptors, has been pointed out to be related to Tourette's syndrome, attention deficit disorder, reading disorder, and senile dementia ( Early onset of Alzheimer-type dementia), treatment of Parkinson's syndrome, Huntington's chorea, late movement disorders, hyperkinesia, etc.,
Useful for prevention.

Compositions having an affinity for nicotinic and muscarinic acetylcholine receptors, dopamine 1 receptors, benzodiazepine receptors and GABA receptors containing the kale or propolis of the present invention and extracts thereof are for oral administration. Can be manufactured as a food, oral or parenteral drug.

As an application method as a medicine, either oral administration or parenteral administration can be adopted. Upon administration, the active ingredient can be mixed with a solid or liquid non-toxic pharmaceutical carrier suitable for administration methods such as oral administration, rectal administration and injection, and then administered in the form of a conventional pharmaceutical preparation.
Examples of such preparations include solid preparations such as tablets, granules, powders and capsules, liquid preparations such as solutions, suspensions and emulsions, and freeze-dried preparations. It can be prepared by conventional means. Examples of the non-toxic carrier for pharmaceutical use include glucose, lactose, sucrose, starch, mannitol, dextrin, fatty acid glyceride, polyethylene glycol, hydroxyethyl starch, ethylene glycol, polyoxyethylene sorbitan fatty acid ester, amino acid, gelatin, Albumin, water, physiological saline and the like can be mentioned. Further, if necessary, conventional additives such as a stabilizer, a wetting agent, an emulsifier, a binder and an isotonicity agent can be appropriately added.

As foods, dried products or extracts of kale or propolis, as they are, or with various nutritional components added or contained in foods and drinks, nicotinic and muscarinic acetylcholine, dopamine 1, benzodiazepines. , GABA can be used as a food or food material for health useful for treating and preventing diseases associated with GABA. For example, starch, lactose, maltose, vegetable oil and fat powder, cocoa butter powder, after adding a suitable auxiliary agent such as stearic acid, using a conventional means, an edible form, for example, granular,
Granules, tablets, capsules, may be formed into paste or the like and provided for edible use, and various foods, for example, processed meat products such as ham and sausage, fish paste products such as kamaboko and chikuwa, bread, confectionery, butter, It may be used by adding it to milk powder or fermented dairy products, or by adding it to beverages such as water, fruit juice, milk and soft drinks. The blending amount of the present invention is appropriately set depending on the type and condition of the edible composition.

The effective dose of the kale or propolis or the extract thereof of the present invention can be appropriately determined according to the age, body weight, symptom of the patient, degree of the patient, administration route, administration schedule, formulation form, etc. For example, in the case of oral administration of kale extract, the dry weight is usually calculated as an adult.
0.1g / kg body weight or more. It may be administered in several divided doses a day. Oral administration of propolis extract generally 1
The daily dose is about 10 to 500 mg / kg body weight, preferably about 150 to 350 mg / kg body weight per day, which may be administered in several divided doses per day.

The kale or propolis or the extract thereof of the present invention is a natural product and therefore has low toxicity and no adverse side effects have been reported. Also, since kale is a vegetable that can be obtained by cultivation, kale, the composition of the present invention containing a kale extract as an active ingredient is safe and
It is an excellent product with no problems in terms of price. Also,
Since propolis is generally used as a dietary supplement in Japan and around the world, the composition of the present invention containing propolis and a propolis extract as an active ingredient is safe and excellent in terms of price. It is a thing.

[0041]

EXAMPLES The present invention will be specifically described below with reference to examples.
It is not limited to this.

[Production Example 1] Kale extract Kale leaves were dried at 90 ° C to deactivate the enzyme which is a bitter and astringent ingredient. Regarding the kale leaf drying method,
It is not particularly limited. The crushed product (4 kg) was subjected to extraction with 20 L of 99.5% ethanol (Wako Pure Chemical Industries, Ltd.) while heating under reflux with an electrically heated water bath to obtain 80 g of kale extract.

[Production Example 2] Propolis extract 99.
After adding 3 L of 5% ethanol and immersing at 50 ° C. overnight, ethanol was removed by a rotary evaporator to obtain 324 mg and 216 mg of propolis extract, respectively.

[Example 1] Receiving nicotinic acetylcholine
Incorporation inhibition test The nicotinic acetylcholine receptor binding inhibition test was performed by Laur
ie A. Pabreza et al. (Molecular Pharmacology, 39, 9
-12, 1991). 50 mM rat cerebral cortex
Squirrel (hydroxymethyl) aminomethane, 120 mM sodium chloride
Thorium, 5 mM potassium chloride, 1 mM magnesium chloride
Buffer containing 2.5 mM calcium chloride (pH 7.0)
Homogenized and suspended. Then 1 at 40,000 × g
After centrifuging for 0 min (4 ° C), pellet again
It was homogenized with an impact liquid. Centrifuge again under the same conditions
After centrifugation, centrifuge 2 times of supernatant at 40,000 xg for 10 minutes (4 ℃).
separated. Then, the precipitate is suspended in the above buffer solution and
It was used as a membrane preparation of the brain cortex. This membrane preparation (400-600 μg protein)
(Including white matter), [³H] Cytisine (final concentration 1.5 nM), made
The kale extract obtained in Production Example 1 or each obtained in Production Example 2
Propolis extract (final concentration: 500 μg / ml) each
Add (final volume 250 μl) and incubate at 4 ° C for 75 minutes.
I broke up. The reaction is quick, Brandel Selha
-Using the Bester, Whatman GF / B filter (0.
0.5% polyethyleneimine containing 1% bovine serum albumin
Pretreat to aqueous solution for a minimum of 3 hours), suction filter over and cool
Washed 3 times with the above buffer. Filter in vial
Put in, add liquid scintillator and then bind to the filter
Liquid radioactivity (tritium) liquid scintillation cow
Measurement. In addition, non-specific binding is 10 μM
(-)-Measured in the presence of nicotine. The results are shown in Table 1.
However, the inhibition rate (%) shown here is calculated by the following formula.
It was Inhibition rate (%) = 100-[(C₁-B₀) / (C₀ -B₀)] × 1
00 (In the formula, C₁ With a known amount of sample and [³H] Cytisine
In the state of coexistence [³[H] for the membrane fraction of cytisine
Represents the amount of binding, C₀ Is [when the sample is removed ³H]
The amount of cytisine bound to the membrane fraction is expressed as B₀Is over
In the presence of excess nicotine (10 μM) [³H] Cytisine membrane
Represents the amount bound to the fraction. ) In addition, in this reaction system
Nicotinic acetyl as a positive control
IC of nicotine, an agonist of cholinergic receptors₅₀Value (Nico
(Concentration that inhibits binding of tin acetylcholine receptor by 50%)
Was 8.0 nM. From Table 1, the kale extract of the present invention and
And propolis extract showed nicotinic acetylcholine receptor
Nicotinic by competitively inhibiting cytisine binding to the body
Is it known to have an acetylcholine receptor activating effect?
It

[0045]

[Table 1]

Example 2 Muscarinic Acetylcholine Receptor Binding Assay The muscarinic receptor binding assay was evaluated by FRANK DORJ.
E. et al. (J. Pharmaco. Exp. Ther., 256 (2), 727
-733, 1990) with some modifications.

[Cell culture] Chinese hamster ovary cells (hereinafter abbreviated as CHO cells) were transformed into ATCC (American Ty).
pe Culture Collection). Until the introduction operation, the cells under 5% CO _2, 10% fetal calf serum, 100 u
Monolayer culture was performed in DMEM medium (manufactured by Gibco) containing nits / ml penicillin G, 100 units / ml streptomycin, and 4 mM glutamine (manufactured by MA Bioproducts).

[Introduction Operation] Chen and Okayama's method (Chen,
C. AND Okayama, H .: Mol. Cell. Biol. 7, 2745-27.
52, 1987), the improved calcium phosphate method using pcD neo as a selection marker was used. CHO cells were transformed with the plasmid into which the pcD expression vector containing the human muscarinic M1 receptor gene sequence was inserted. G-418, an analog of neomycin, an antibiotic
The selection operation using 600 μg / ml (manufactured by Gibco) is 7
It started from the 2nd hour and continued for 2 to 3 weeks. The medium was changed every 3 days. The clonal cell line was obtained by performing limiting dilution cloning. That is, 1 per well as theoretical density
The cells were transferred to a 96-well plate, and after about 2 weeks, a single colony, ie, pure cells having the same properties was subcloned, and [ ³ H] pirenzepine binding ability was measured.

[Preparation of Membrane Fraction] The cells were cultured to a confluence of about 80%, washed, and then ice-cooled in a binding buffer (pH 7.4) consisting of 25 mM sodium phosphate containing 5 mM magnesium chloride (hereinafter referred to as "pH 7.4"). , Binding buffer) and stripped using a Brinkmann Homogenizer (adjustment position 5).
Homogenized for seconds. The membrane was pelleted by centrifugation at 16,000 xg for 15 minutes and the pellet was homogenized again under the same conditions. Protein concentration is determined by the method of Bradford (Br
Adford, MM: Anal. Biochem. 72, 248-254, 1976), using a Bio-Rad protein assay kit (manufactured by Bio-Rad). The membrane fraction was stored frozen at −80 ° C. until use. The assay was carried out in a total volume of 1 ml using the binding buffer and the membrane fraction was adjusted to a protein concentration of 6 μg / ml. The saturation binding test of [ ³ H] pirenzepine was carried out by dividing the concentration of the radioligand (2-1400 pM) into 8 to 10 concentrations. In the binding inhibition test, [ ³
H] pirenzepine (final concentration 2 nM) and the kale extract obtained in Preparation Example 1 or each propolis extract obtained in Preparation Example 2 (final concentration: 500 μg / ml) were simultaneously added. Non-specific binding was defined as the difference in [ ³ H] pirenzepine binding in the presence of 1 μM atropine. Incubation was performed at 22 ° C. for 60 minutes, and the reaction was stopped by rapid filtration using a glass filter (Model 7019, manufactured by Skatron Inc.). The membrane fraction bound on the filter was washed 3 times with 5 ml of ice-cold binding buffer, dried and washed with scintillation cocktail (N
ew England Nuclear Aquasol) Transfer to 10 ml, LKB β-
Radioactivity was measured using a counter. Show the result
Shown in 2. The inhibition rate (%) was calculated by the following formula. Inhibition rate (%) = 100 - _{[(C 3 -B 1) / (} C 2 -B 1) ] × 10
0 (wherein, C ₃ represents the amount of binding ^[3 H] pirenzepine membrane fraction in a state in which a known amount of the sample and ^[3 H] pirenzepine coexist, C ₂ is to remove the sample of time[
³ H] represents the amount of pirenzepine bound to the membrane fraction, B ₁
Represents the amount of [ ³ H] pirenzepine bound to the membrane fraction in the presence of excess atropine (1 μM). )

The IC ₅₀ value of pirenzepine, a muscarinic 1 receptor antagonist as a positive control in this reaction system, was 13 nM (4.57 × 10 ⁻³ μg / ml). It can be seen from Table 2 that the kale extract and propolis extract of the present invention competitively inhibit pirenzepine binding to the muscarinic 1 receptor and have a strong muscarinic receptor activating effect.

[0051]

[Table 2]

[Example 3] Inhibition of dopamine 1 receptor binding
test For the dopamine 1 receptor binding inhibition test, see the method of Zhou et al.
I went to the study (Nature, 347,76-80,1990). 3.0 kb EcoRl
-The Sacl fragment was cloned into the Hind lll BamH l sequence of the pBC12Bl vector.
COS- by the calcium phosphate method
7 cells were introduced. Then, prepare the membrane preparation by using the existing method.
Method, and finally TEM buffer [25 mM Tris buffer
Liquid pH 7.4, 6 mM magnesium chloride, 1 mM ethylenedia
It was suspended in mintetraacetic acid (EDTA) to prepare a receptor membrane preparation.
In each test tube, the kale extract obtained in Production Example 1 or production
Each propolis extract obtained in Example 2 (final concentration: 500 μg
/ Ml) respectively, and is a dopamine 1 receptor antagonist
[³H] SCH 23390 (Amersham, final concentration 0.3 n
M), a receptor membrane preparation (containing 20 to 30 μg of protein) and
Add the above buffer solution to make a reaction solution (total volume 500 μl),
Was initiated by the addition of a membrane preparation. 30 ° C, 60 minutes
Labeled ligand bound to the receptor after incubation
Using a cell harvester (Brandell)
Suction filtered on the Man GF / B glass fiber filter
Reaction to stop the reaction and immediately cool with ice-cold 50 mM Tris-HCl.
It was washed 3 times with 5 ml of the buffer solution (pH 7.7). Then the filter
The radioactivity on the screen to a liquid scintillation counter
Then, the total amount of binding was determined. Also measured at the same time
Non-specific binding in the presence of 1 μM SCH 23390
Specific amount by subtracting this from the total binding amount.
The amount of binding was determined. The results are shown in Table 3. Inhibition rate shown here
(%) Was calculated by the following formula. Inhibition rate (%) = 100-[(C_Five-B₂) / (C_Four -B₂)) × 10
0 (In the formula, C_FiveWith a known amount of sample and [³H] SCH 23390
In the state of coexistence [³H] For the membrane fraction of SCH 23390
Represents the amount of binding_FourIs [when the sample is removed ³H]
Represents the amount of SCH 23390 bound to the membrane fraction,₂Is over
In the presence of excess SCH 23390 (1 μM) [³H] SCH 23390 membrane
Represents the amount bound to the fraction. ) In addition, in this reaction system
Dopamine 1 receptor as a positive control
IC of SCH 23390, an antagonist of₅₀Value (dopamine 1 receptor
The concentration that inhibits binding by 50%) was 1.2 nM. Table 3
As can be seen from the above, the kale extract and propolysaccharide of the present invention
The extract has a dopamine 1 receptor activating effect.
I understand.

[0053]

[Table 3]

Example 4 Benzodiazepine Receptor Binding Inhibition Test The benzodiazepine receptor binding inhibition test was conducted by Robert C. Sp.
The method of eth et al. was used as reference (Life Sciences, 24, 351-35).
8,1979). A crude synaptosome membrane fraction prepared from the brains of Wistar rats aged 7 to 8 weeks was treated with 15 mM Tris-HCl buffer (pH 7 containing 118 mM sodium chloride, 4.8 mM potassium chloride, 1.28 mM calcium chloride and 1.2 mM magnesium sulfate). .4) suspension (1g brain wet weight / 20ml)
And used as a receptor membrane preparation. [ ³ H] flunitrazepam was used as the labeled ligand. Manufacturing example 1 for each test tube
The kale extract obtained in Example 1 or each propolis extract obtained in Production Example 2 (final concentration: 500 μg / ml) was added, and [ ³ H] flunitrazepam (final concentration 0.4 nM), receptor membrane preparation and Add the above buffer to the reaction mixture (total volume 1 m
l) and the reaction was started by adding a membrane preparation. 0
After incubation at ℃ for 20 minutes, labeled ligand bound to the receptor was used as cell harvester (Brandell).
With Whatman GF / B glass fiber filter by suction filtration to stop the reaction, and immediately cool to 50 m on ice.
It was washed 3 times with 5 ml of M Tris-hydrochloric acid buffer (pH 7.7).
Then, the radioactivity on the filter was measured by a liquid scintillation counter to determine the total amount bound. Further, the binding amount in the presence of 1 μM diazepam measured at the same time was defined as the non-specific binding amount, and the specific binding amount was determined by subtracting this from the total binding amount. The results are shown in Table 4. The inhibition rate (%) shown here was calculated by the following formula. Inhibition rate (%) = 100 - _{[(C 7 -B 3) / (} C 6 -B 3) ] × 10
0 (In the formula, C ₇ represents the binding amount to the membrane fraction of [ ³ H] fluni in the state where a known amount of sample and [ ³ H] flunitrazepam coexist, and C ₆ excludes the sample. Represents the amount of [ ³ H] flunitrazepam bound to the membrane fraction, and B ₃ was [ ³ H] in the presence of excess diazepam (1 μM).
It represents the amount of flunitrazepam bound to the membrane fraction. )
The IC ₅₀ value of diazepam (concentration that inhibits benzodiazepine receptor binding by 50%), which is a benzodiazepine receptor agonist as a positive control in this reaction system, was 10 nM. From Table 4, it can be seen that the kale extract and propolis extract of the present invention competitively inhibit the flunitrazepam binding to the benzodiazepine receptor and have a benzodiazepine receptor activating effect.

[0055]

[Table 4]

[Example 5] (GABA receptor binding inhibition test) [ ³ H] GABA of kale extract or propolis extract
The binding inhibition test for rat-derived cerebral cortex GABA receptor was carried out by the method of AKIRA TSUJI et al. (ANTIMICROB. AGENTS CHEMOT
HER., 32 (2) 190-194, 1988) with some modifications.

That is, the cerebral cortex of an SD rat (CLEA Japan, Inc.) having a body weight of 230-270 kg was excised, and a polytron homogenizer (manufactured by Brinkman Instrument Co. Inc.) was used at a speed of 5 for 30 seconds. Homogenization was performed in 50 mM Tris buffer (pH 7.4) (hereinafter, abbreviated as buffer A) containing a double volume of 1 mM ascorbic acid and 1 mM disodium tetraacetate ethylenediamine. Tissues were maintained at 0 ° C for all manipulations. Then, the homogenate was centrifuged at 40,000 xg for 15 minutes, the obtained precipitate was resuspended in 50 times volume (original weight) of buffer A, and homogenized with Polytron under the same conditions as above. . Centrifugation and resuspension was repeated 3 times. Then 37
Incubation was performed at 10 ° C for 10 minutes. This homogenate was centrifuged at 40,000 xg for 15 minutes, resuspended in 10 volumes (original weight) of buffer A (hereinafter abbreviated as membrane fraction), and stored at -20 ° C or lower until measurement. did.

In conducting a binding test on rat-derived cerebral cortex GABA receptor, the above-mentioned membrane fraction was thawed to 37 ° C., and then 136 mM sodium chloride, 5 mM potassium chloride, 2 mM magnesium sulfate, 2 mM was added. 20 m consisting of 20 mM Tris buffer (pH 7.4) containing potassium dihydrogen phosphate, 2 mM calcium chloride and 1 mM ascorbic acid, 1 mM disodium tetraacetate ethylenediamine
M Tris-Krebs buffer (hereinafter abbreviated as buffer B)
The volume has been increased to 20 volumes (original weight). The membrane fraction was washed with a polytron homogenizer at a speed of 5
Homogenize for 2 seconds, incubate at 37 ° C. for 15 minutes, and centrifuge at 20,000 × g for 10 minutes. The precipitate thus obtained is washed and resuspended twice or more,
The finally obtained precipitate was resuspended in 100 times volume (original weight) of buffer solution B and used in the following test.

Next, 50 μl of the kale extract obtained in Production Example 1 or each propolis extract obtained in Production Example 2
(Final concentration: 500 μg / ml) and 50 μl of [ ³ H] GABA (final concentration 10 nM) were added, and 900 μl of the above membrane fraction was added to this.
The reaction was started by adding. After incubating at 22 ° C for 20 minutes, use a Brandell Cell Harvester under reduced pressure for 2 ×.
Whatman GF with 7.5 ml of filter wash
The reaction was stopped by filtration through a / B filter. After filtration, the radioactivity retained on the filter was analyzed by a liquid scintillation counter (Packard Model 2425, 30-
40% efficiency).

The binding amount in the presence of 100 μM non-radiolabeled GABA measured at the same time was defined as the non-specific binding amount,
The specific binding amount was determined by subtracting this from the total binding amount. The results are shown in Table 5. Inhibition rate (%) shown here
Was calculated by the following formula. Inhibition rate (%) = 100 - _{[(C 9 -B 4) / (} C 8 -B 4) ] × 10
0 (In the formula, C ₉ represents the binding amount of [ ³ H] GABA to the membrane fraction in the presence of a known amount of sample and [ ³ H] GABA, and C ₈ excludes the sample. [ ³ H] GA at the time
It represents the amount of BA bound to the membrane fraction, and B ₄ is an excess of G
The amount of [ ³ H] GABA bound to the membrane fraction in the presence of ABA (100 μM) is shown. )

[0061] Incidentally, IC ₅₀ values of GABA as a positive control in this assay system (GABA receptor binding the concentration which inhibits ^{50%), 11 nM (1.13 × 10 -3} μg / m
l) was.

As can be seen from Table 5, the kale extract and propolis extract of the present invention have a strong GABA receptor activating effect.

[0063]

[Table 5]

Prescription examples are shown below. [Production of feed] Using the ethanolic extract of kale obtained in Production Example 1, a feed having the following composition was produced according to a conventional method. (Composition) (Composition: mass%) Kale extract 10 Soy protein 10 Mineral mixture 4 Vitamin mixture 1 Corn oil 5 Cellulose 5 Starch 65

[Production of Tablet] Using the ethanolic extract of Brazilian propolis obtained in Production Example 2, a tablet having the following composition was produced according to a conventional method. (Composition) (Blending:% by mass) Brazilian propolis extract 24 Lactose 63 Corn starch 12 Guar gum 1

[Manufacture of Juice] Using the extract of Chinese propolis obtained in Manufacturing Example 2, a juice having the following composition was manufactured by a conventional method. (Composition) (Composition:% by mass) Frozen concentrated Unshu mandarin orange juice 5.0 5.0 Fructose glucose liquid sugar 11.0 Citric acid 0.2 L-ascorbic acid 0.02 Perfume 0.2 Pigment 0.1 Chinese propolis extract 0.2 Water 83.28

[0067]

EFFECTS OF THE INVENTION The kale and propolis of the present invention and their extracts are nicotinic and muscarinic acetylcholine receptors, dopamine 1 receptors, benzodiazepine receptors,
Since they have an affinity for GABA receptors, diseases related to these receptors, such as schizophrenia and Tourette's syndrome, attention deficit disorder, anxiety, dyslexia, senile dementia (early onset of Alzheimer's disease), senile dementia It can be used for (Alzheimer's dementia), Parkinsonism including Parkinson's disease, Huntington's chorea, late dyskinesia, hyperkinesia and the like.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 25/14 A61P 25/14 25/16 25/16 25/18 25/18 25/22 25/22 25 / 28 25/28 F term (reference) 4B018 LB08 MD07 MD53 MD78 ME02 ME10 MF01 4C087 AA01 AA02 BB22 MA52 NA52 ZA02 ZA05 ZA16 ZA18 ZA22 4C088 AB15 BA07 BA08 MA52 NA14 ZA02 ZA05 ZA16 ZA18 ZA22

Claims (11)

[Claims] 1. A nicotinic acetylcholine receptor activator comprising kale, propolis or an extract thereof. 2. A muscarinic acetylcholine receptor agonist which contains kale, propolis or an extract thereof. 3. A dopamine 1 receptor activator comprising kale, propolis or an extract thereof. 4. A benzodiazepine receptor activator containing kale, propolis or an extract thereof. 5. A γ-aminobutyric acid receptor activator containing kale, propolis or an extract thereof. 6. A schizophrenia, Tourette's syndrome, attention deficit disorder, anxiety, dyslexia, senile dementia, senile dementia, Parkinson's disease, characterized by containing kale, propolis or an extract thereof. A composition for treating or preventing central nervous system diseases such as Parkinsonism, Huntington's disease, late movement disorders, or hyperkinesia. 7. A brain function improving agent comprising kale, propolis or an extract thereof. 8. A brain metabolism activating agent comprising kale, propolis or an extract thereof. 9. An oral or parenteral composition containing the agent according to claim 1. 10. The oral composition according to claim 1, which is in the form of a food. 11. The composition for oral and parenteral administration according to claim 1, which is in the form of a medicine.