JP2014019649A - Gaba-like composition - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a GABA-like composition exhibiting GABA-like activity to GABAreceptor.SOLUTION: A GABA-like composition exhibiting GABA-like activity to GABAreceptor contains a compound having a hydroxy group or a carboxyl group, except for at least 7 substances: GABA, a barbituric acid derivative, a benzodiazepine derivative, muscimol, THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol), isoguvacine and gabapentin. This compound contains at least one of compounds of pyruvic acid, 2-oxoglutaric acid, 2-hydroxy-4-methylvaleric acid, citric acid, malic acid, 2-hydroxyvaleric acid, quinic acid, gluconic acid, glycolic acid, 3-hydroxybutyric acid, mevalonic acid, lactic acid, pantothenic acid and 2-hydroxyvaleric acid ethyl ester.

Description

The present invention relates to a GABA-like composition exhibiting GABA-like activity against GABA (Gamma-aminobutyric acid) _A receptor.

It is well known that neurotransmission in the cranial nervous system is performed by nerve cell excitation. This transmission is caused by activation of a receptor for excitatory neurotransmitters, and the degree of neural excitement is regulated by inhibitory or excitatory neurotransmitters. As excitatory neurotransmitters, acetylcholine and glutamic acid are well known. Examples of inhibitory neurotransmitters include γ-aminobutyric acid (GABA) and glycine. The nerve suppression in the brain is mostly signal transmission by GABA. GABA receptors are receptors that selectively receive GABA, and three types of GABA _A (ion channel type), GABA _B (metabolic type), and GABA _C (ion channel type) receptors have been confirmed ( Non-patent document 1). In addition to the GABA binding site which is a ligand, GABA _A receptor includes a barbiturase binding site, a benzodiazepine binding site, a glucocorticoid binding site, a penicillin binding site, a furosemide binding site, and the like. I am adjusting sex. Since various drugs act on GABA _A receptors, it is known that depending on the degree, peace, mental stability, sleep, anesthesia effect, etc. occur, and it is the main target of substances that affect mood is there. Deficiencies in the expression of GABA _A receptor function cause epilepsy, anxiety disorder, cognitive impairment, schizophrenia, depression, drug dependence, etc. Substances that act on GABA receptor are sedative / hypnotics, anticonvulsants Therefore, research on new substances that act on these receptors is desired.

Bormann J., "The 'ABC' of GABA receptors", Trends Pharmacol Sci. 2000 Jan; 21 (1): 16-9.

Acting on GABA _A receptors, as a substance showing the GABA _A receptor activity, barbituric acid derivatives, benzodiazepine derivatives, muscimol (Muscimol), THIP (4,5,6,7- Tetrahydroisoxazolo [5,4-c] pyridin -3-ol), Isoguvacine, and Gabapentin are known. However, GABA _A receptors include GABA binding site, barbitur binding site, benzodiazepine binding site, glucocorticoid binding site, penicillin binding site, furosemide binding site, and the like. The reactivity is different. Therefore, act on GABA _A receptors, showing the GABA _A receptor activity, or new composition for enhancing is desired.

This invention is made | formed in view of the said situation, and it aims at providing the GABA-like composition which shows GABA-like activity with respect to a GABA _A receptor.

In order to achieve the above object, the GABA-like composition according to the first aspect of the present invention comprises:
At least GABA (Gamma-aminobutyric acid), barbituric acid derivatives, benzodiazepine derivatives, Muscimol, THIP (4,5,6,7-Tetrahydroisoxazolo [5,4-c] pyridin-3-ol), isoguvacine (Isoguvacine) And a GABA-like composition that exhibits a GABA-like activity against a GABA _A receptor, which contains a compound having a hydroxy group or a carboxyl group, excluding the seven substances Gabapentin.

  The compound includes pyruvic acid, 2-oxoglutaric acid, 2-hydroxy-4-methylvaleric acid (DL-leucic acid), citric acid ( Citric acid, malic acid, 2-hydroxyvaleric acid, quinic acid, gluconic acid, glycolic acid, 3-hydroxybutyric acid (3 -Hydroxybutyric acid, Mevalonic acid, Lactic acid, Pantothenic acid, 2-Hydroxyvaleric acid ethyl ester; 2-ethylhydroxyvaleric acid (Ethyl 2- hydroxyvalerate)), p-Hydroxyphenylacetic acid, Shikimic acid, Citramalic acid, Citraconic acid, 2-Isopropylmalic acid, Huma Nucleic acid (Fumaric acid), N-Acetylmuramic acid (Glyceric acid), Benzoic acid, Homovanillic acid, Glutaric acid, 6- Hydroxyhexanoic acid (6-Hydroxyhexanoic acid), Glucuronic acid, Galacturonic acid, 2-Hydroxybutyric acid, 5-oxo-2-tetrahydrofurancarboxylic acid (5-Oxo-2) -tetrahydrofurancarboxylic acid, Phthalic acid, Terephthalic acid, Succinic acid, Isocitric acid, Threonic acid, Crotonic acid, Malonic acid ( Malonic acid), Isethionic acid, cis-Aconitic acid, N-Acetylaspartic acid, Maleic acid , Propionic acid, Vanillic acid, 3-Hydroxy-3-methylglutaric acid, Digalacturonic acid, 6-phosphogluconic acid (6 -Phosphogluconic acid), Isobutyric acid, Cysteic acid, Methanesulfonic acid, Glyoxylic acid, N-acetylmethionine, biotin, p -Sulfobenzoic acid, N-acetylglucosamine-1-phosphate, 5-Oxoproline, N-acetyl-β-alanine (N-Acetyl) -β-alanine), Glycerol 3-phosphate, N-Acetylglutamic acid, Glucose 6-phosphate, O-Succinylhomoserine N-acetylleucine, fructose 6-phosphate, succinic semialdehyde, trehalose 6-phosphate, and glucose 1-phosphate You may contain at least any one among the compounds of (Glucose 1-phosphate).

  The concentration of the compound may be 10 μM to 10 mM.

  The compound may be separated and purified from sake.

  The compound may be separated and purified from the organic acid fraction of the sake.

According to the present invention, the GABA _A receptor can have the same activity as GABA.

It is a figure which shows the structure of a GABA _A receptor. It is a figure for demonstrating the fractionation method of sake. It is a figure which shows the GABA _A receptor activity of each A fraction. It is a figure which shows the GABA _A receptor activity of A fraction and A fraction of 10 time dilution. It is a figure which shows the GABA _A receptor activity of a Sephadex G-10 fraction. It is a figure which shows the standard measurement state of GABA _A receptor. It is a figure which shows the GABA density | concentration response curve with respect to (alpha) 1 / (beta) 1 subtype GABA _A receptor of GABA. It is a figure which shows GABA _A receptor activity when the density | concentration of a compound is 200 micromol. It is a figure which shows GABA _A receptor operation activity when the density | concentration of a compound is 500 micromol. It is a figure which shows an elevated plus maze test apparatus. It is a figure which shows the stay time of an open arm. It is a figure which shows the frequency | count of approach to an open arm.

Before describing embodiments of the present invention, the GABA _A receptor will be described in detail, and the effects of acting on the activity of this receptor will be described.

GABA receptors are receptors that selectively receive GABA, and three types of GABA _A (ion channel type), GABA _B (metabolic type), and GABA _C (ion channel type) receptors have been confirmed. Among these receptors, GABA _A receptor has seven subfamilies of α, β, γ, δ, ε, θ, and π, and among them, five types of subunits form a pentamer. FIG. 1 is a diagram showing the structure of GABA _A receptor. The GABA _A receptor expressed in the brain is considered to be composed of two α subunits, two β subunits, and one γ subunit, as shown in FIG. When GABA binds between the α and β subunits, the channel is opened and Cl ⁻ ions are transmitted. When this Cl ⁻ ion flows into the cell from the outside of the cell, the nerve cell is hyperpolarized and the excitation is suppressed.

The GABA _A receptor has a GABA binding site that is a ligand formed by combining a part of five types of subunits. In addition to this GABA binding site, a barbituric binding site, a benzodiazepine binding site, a glucocorticoid binding site, a penicillin binding site, a furosemide binding site, and the like are known, and the reactivity with GABA is regulated. For example, picrotoxin binds to the pore side of the β subunit and inhibits the activity non-competitively. Benzodiazepines known for pharmacological actions such as sleep sedation and anxiolytic actions bind to the α subunit and enhance the action of GABA. In addition, ethanol binding to the γ subunit enhances the action of GABA. Thus, for acting various drugs on the GABA _A receptor, depending on the extent, comfort, tranquilizers, sleeping, it is known that such anesthetic effect occurs, GABA _A receptors mood It is the main target of the affected substance. Deficiencies in the expression of GABA _A receptor function cause epilepsy, anxiety disorder, cognitive impairment, schizophrenia, depression, drug dependence, etc. Substances that act on GABA receptor are sedative / hypnotics, anticonvulsants It becomes a target for the development of sedative neurological drugs such as anti-anxiety drugs. From the above, the substance (composition) that acts on the GABA _A receptor is important.

  Based on the above knowledge, embodiments of the present invention will be described in detail with reference to the drawings. In addition, embodiment described below is an illustration of this invention and this invention is not limited to this example.

First, a method for measuring the activity of GABA _A receptor will be described.

In the present invention, the composition of the present embodiment in Xenopus oocytes expressing the GABA _A receptor (compounds) to act on refluxing GABA _A receptor, this receptor is activated Cl ^- ions receptor Since current flows through the channel, receptor activity is measured using a current measurement method based on the two-electrode membrane potential fixation method. In the Xenopus oocyte expression system, it is known that when exogenous mRNA is injected, the protein can be expressed efficiently. GABA _A receptors are all expressed in neurons, and include GABA _B (metabolic type) and GABA _C (ion channel type) receptors activated by GABA. Therefore, it is very difficult to see the activity of GABA _A receptor alone by direct administration of GABA to nerve cells. Therefore, by using the Xenopus oocyte expression system, only the target GABA _A receptor protein can be expressed in the oocyte, and the activity can be measured efficiently.

With respect to the GABA-like composition (GABA _A receptor agonist) according to this embodiment, GABA _A receptor activity can be measured by the activity measurement method described above. GABA-like compositions that are shown to activate (enhance) GABA _A receptors include pyruvic acid, 2-Oxoglutaric acid, 2-hydroxy-4-methylvaleric acid (2 -Hydroxy-4-methylvaleric acid (DL-leucic acid), citric acid, malic acid, 2-hydroxyvaleric acid, quinic acid, gluconic acid ( Gluconic acid, Glycolic acid, 3-Hydroxybutyric acid, Mevalonic acid, Lactic acid, Pantothenic acid, 2-hydroxyvaleric acid ethyl ester ( 2-Hydroxyvaleric acid ethyl ester; 2-ethylhydroxyvalerate, p-hydroxyphenylacetic acid, shikimic acid, citramalic acid, citraconic acid C itraconic acid), 2-Isopropylmalic acid, Fumaric acid, N-Acetylmuramic acid, Glyceric acid, Benzoic acid, Homovanillic acid, Glutaric acid, 6-Hydroxyhexanoic acid, Glucuronic acid, Galacturonic acid, 2-Hydroxybutyric acid , 5-Oxo-2-tetrahydrofurancarboxylic acid, Phthalic acid, Terephthalic acid, Succinic acid, Isocitric acid, Threon Threonic acid, Crotonic acid, Malonic acid, Isethionic acid, cis-Aconitic acid, N-acetic acid Ruaspartic acid (N-Acetylaspartic acid), maleic acid (Maleic acid), propionic acid (Propionic acid), vanillic acid (Vanillic acid), 3-hydroxy-3-methylglutaric acid (3-Hydroxy-3-methylglutaric acid) , Digalacturonic acid, 6-phosphogluconic acid, isobutyric acid, cysteic acid, methanesulfonic acid, glyoxylic acid, N-Acetylmethionine, Biotin, p-Sulfobenzoic acid, N-Acetylglucosamine 1-phosphate, 5-oxoproline ( 5-Oxoproline), N-Acetyl-β-alanine, Glycerol 3-phosphate, N-Acetylglutamic acid, Glucose 6-phosphate (Glucose 6-phosphate), O-Succinylhomoserine, N-Acetylleucine, Fructose 6-phosphate, Succinic semialdehyde, Trehalose 6 -A composition containing at least one of phosphoric acid (Trehalose 6-phosphate) and glucose 1-phosphate (Glucose 1-phosphate) compounds.
More preferably as an agonist of GABA _A receptor, pyruvic acid, 2-oxoglutaric acid, 2-hydroxy-4-methylvaleric acid, citric acid, malic acid, 2-hydroxyvaleric acid, quinic acid, gluconic acid, glycolic acid, 3-hydroxybutyric acid, mevalonic acid, lactic acid, pantothenic acid, 2-hydroxyvaleric acid ethyl ester, p-hydroxyphenylacetic acid, shikimic acid, citramalic acid, citraconic acid, 2-isopropylmalic acid, fumaric acid, N-acetylmuramine Acid, glyceric acid, benzoic acid, homovanillic acid, glutaric acid, 6-hydroxyhexanoic acid, glucuronic acid, galacturonic acid, 2-hydroxybutyric acid, 5-oxo-2-tetrahydrofurancarboxylic acid, phthalic acid, terephthalic acid, succinic acid, Isocitric acid, threonic acid, crotonic acid, malonic acid, isethionic acid, cis-aconitic acid, N-acetic acid Ruaspartic acid, maleic acid, propionic acid, vanillic acid, 3-hydroxy-3-methylglutaric acid, digalacturonic acid, 6-phosphogluconic acid, isobutyric acid, cysteic acid, methanesulfonic acid, and glyoxylic acid It is a composition containing at least one of them.
More preferably as an agonist of GABA _A receptor, pyruvic acid, 2-oxoglutaric acid, 2-hydroxy-4-methylvaleric acid, citric acid, malic acid, 2-hydroxyvaleric acid, quinic acid, gluconic acid, glycolic acid, It is a composition containing at least one of compounds of 3-hydroxybutyric acid, mevalonic acid, lactic acid, pantothenic acid, and 2-hydroxyvaleric acid ethyl ester.

Here, the compounds according to the following structural formulas (1) to (14) are pyruvic acid, 2-oxoglutaric acid, 2-hydroxy-4-methylvaleric acid, citric acid, malic acid, 2-hydroxyvaleric acid, quina Acid, gluconic acid, glycolic acid, 3-hydroxybutyric acid, mevalonic acid, lactic acid, pantothenic acid, 2-hydroxyvaleric acid ethyl ester (2-ethylhydroxyvaleric acid). All the 65 compounds including the compounds according to the structural formulas (1) to (14) are compounds having a hydroxy group or a carboxyl group. Accordingly, GABA _A composition receptors is effective to enhance the, GABA _A GABA receptor activity are already known, barbituric acid derivatives, benzodiazepine derivatives, muscimol, THIP, isoguvacine, and gabapentin at least 7 The composition containing the compound which has a hydroxyl group or a carboxyl group except a substance may be sufficient.

Table 1 below is a table showing the molecular weights of the compounds according to GABA and structural formulas (1) to (14). As shown in the table, the molecular weight of the compounds according to structural formulas (1) to (14) is not far from the molecular weight (103.12) of GABA. Therefore, the molecular weight of the composition exhibiting GABA _A receptor activity is preferably not far from the molecular weight of GABA, more preferably 75 to 220.

The concentration of the composition exhibiting GABA _A receptor activity is preferably 10 μM to 10 mM, more preferably 13 μM to 6.7 mM.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, an Example does not limit this invention.

(Purification extraction of compounds)
First, a method for purifying and extracting a composition (compound) exhibiting GABA _A receptor activity from sake will be described. Animal behavior experiments have shown that alcoholic beverages such as sake, whiskey and beer have a relaxing effect. For example, sake contains 1000 or more kinds of components including amino acids and organic acids, and it is considered that components that exhibit GABA _A receptor activity are also included. Therefore, the corresponding compound was purified and extracted from sake. In this example, the compound was purified and extracted from three pure rice sakes: A1, A2, A3, one pure rice ginjo sake: A4, one mountain waste pure rice sake: A5, and one ordinary sake: A6.

FIG. 2 is a diagram for explaining a method for fractionating sake. In order to purify and extract compounds, sake was fractionated using 3 types of ion exchange resins (manufactured by Organo Co., Ltd.) (Sato Nobu) “Study on changes in sake by light and storage. Reference), lyophilized, dissolved in H ₂ O, A fraction (organic acid etc.), R fraction (medieval / acidic amino acid, peptide etc.), C fraction (basic amino acid, peptide etc.), N fraction A 50-fold concentrated solution (sugar etc.) was obtained. Then, the 50-fold concentrated solution of the A fraction was returned to the original concentration of sake, and the GABA _A receptor activity of each fraction was measured. At that time, the pH was adjusted to the same pH 7.5 as the buffer solution used for the measurement, and the measurement was performed.

FIG. 3 is a diagram showing GABA _A receptor activity of each A fraction. In this figure, GABA _A receptor activity of GABA 200 μM is assumed to be 100%, and each pure rice sake (A1, A2, A3), pure rice ginjo sake (A4), mountain waste pure rice sake (A5), ordinary sake (A6) A The GABA _A receptor activity of the fractions was compared. As a result, it was shown that the A fraction containing an organic acid or the like contains a compound showing GABA _A receptor activity.

When the A fraction was analyzed, it was found that the A fraction did not contain GABA. For this reason, it was confirmed that the A fraction contains compounds having GABA _A receptor activity other than GABA.

Next, the activity of the A fraction was compared with that obtained by diluting the A fraction 10 times in order to proceed with purification of the activated component and clarify the difference in the presence of the activated component. FIG. 4 is a graph showing GABA _A receptor activity of the A fraction and the 10-fold diluted A fraction. As in FIG. 3, the activity was compared with GABA _A receptor activity of 200 μM GABA as 100%. In the figure, “A fraction” represents the activity of the A fraction alone. As a result, even when the A fraction was diluted 10 times (“A1 (× 10), A2 (× 10), A3 (× 10), A4 (× 10)” in the figure), 3 brands ( The A fractions of A1, A2, A3) showed activity. Therefore, it became clear that the A fraction of sake contains a compound having GABA activity even at a low concentration.

Next, identification of a compound exhibiting GABA _A receptor activity contained in the A fraction of sake was attempted. First, sake once fractionated was further fractionated using a low molecular sieve Sephadex G-10 (manufactured by GE Healthcare). For the fractionation, a 10 ml Terumo syringe (manufactured by Terumo Corporation) was used. A column was created by connecting the tip of an infusion tube to the tip of a syringe. Cotton was packed to a 1 ml scale, resin (Sephadex G-10) was added to a 7 ml scale, and fractionation was performed using a buffer solution ND96 at a flow rate of 10 ml / min. At that time, fractionation was performed using p-nitrophenol (molecular weight: 139.11) as an index of molecular weight. 500 μl of 50-fold concentrated sake was used, and 1 ml each was collected from 14 Eppenes. Each was diluted 20 times, pH was adjusted to 7.5, and it used for the measurement. As a result, as shown in FIG. 5, A2, A3, and A4 showed the maximum activity in the same fraction 6 as p-nitrophenol.

For buffer solution ND96, 950 ml of MQ water was added with purity of 99% NaCl 5.61 g (final concentration: 96 mM), 1M KCl 2.0 ml (final concentration: 2 mM), 1M CaCl ₂ 1.8 ml (final concentration: 1.8 mM), 1M MgCl ₂ 1.0 mL (final concentration: 1 mM), 1M HEPES 5.0 ml (final concentration: 5 mM) are dissolved, and the pH is adjusted to 7.5 using 2M NaOH (special grade), then 1000 ml Created by measuring up.

Table 2 below shows the metabolome of the A fraction of Japanese sake (3 pure rice sake: A1, A2, A3, 1 pure rice ginjo sake: A4, 1 mountain waste pure rice sake: A5, 1 ordinary sake: A6). It is a table | surface which shows the relative area value of the peak in each compound normalized by the area value of the internal standard substance by the result of analysis (a request to Human Metabolome Technologies, Inc.). In the table, ND means Not Detected. As shown in the table, 64 compounds were identified from the A fraction of sake. These 64 compounds had a hydroxy group or a carboxyl group, and had a characteristic that the molecular weight was not far from the molecular weight 103.12 of GABA. Muscimol, which has been shown to enhance the GABA _A receptor, has a hydroxy group and a molecular weight of 114.1, and thus has a characteristic that it is not far from the molecular weight of GABA. GABA has a carboxyl group. For this reason, 64 compounds had the same characteristics as muscimol and GABA which show GABA _A receptor activity. As shown in FIGS. 3 and 4, it is known that the A fraction of sake contains a compound having the same activity as GABA with respect to the GABA _A receptor. From these facts, 64 compounds identified by metabolomic analysis are considered to be compounds having GABA action.

Table 3 below is a table showing the concentration of the compound contained in the A fraction obtained from the results of the metabolome analysis. As shown in the table, the concentration of the compound was in the range of 10 μM to 10 mM. For this reason, the concentration of the compound exhibiting GABA _A receptor activity is preferably 10 μM to 10 mM, more preferably 13 μM to 6.7 mM. The concentration range of 13 μM to 6.7 mM is the range indicated by the concentrations of gluconic acid and lactic acid that showed high GABA _A receptor activity in FIGS.

Next, a method for expressing the GABA _A receptor in Xenopus oocytes and a method for measuring the activity of the GABA _A receptor will be described. Using this method for measuring the activity of GABA _A receptor, the activity of each compound was measured.

(Ingestion of Xenopus oocytes)
Xenopus laevis is left to sleep in ice for about 30 minutes, then a 3-5 mm hole is made in the abdomen using an injection needle and surgical scissors. Mother cells were removed. The extracted oocyte was placed in a collagenase solution and allowed to stand for about 2 hours, and then the follicular membrane was removed using forceps under a microscope. Peel the required amount of eggs from the weekly measurement, pick up the color, shape, and scratches and place them in a petri dish. In a thermo plate (FTP-28190 made by ASONE) set at 20 ° C in Barth solution. saved.

Here, the Barth solution and the collagenase solution were prepared as follows.
About 950 ml of ultrapure water (MQ water) is placed in a 1-beaker, and each reagent (reagent concentration required amount / 1 ?; NaCl 88 mM 51.4 g, KCl 1.0 mM 0.75 g, MgSO ₄ 0.82 mM 2.0 g, Tris 5.0 mM) 6.0 g and NaHCO ₃ 2.4 mM (2.0 g) were weighed, adjusted to pH 7.5, and made up to 1000 ml to produce a Barth 10A solution. The Barth 10A solution was put in 50 ml in a 500 ml graduated cylinder and diluted 10 times with MQ water. The 10-fold diluted solution was placed in a culture bottle, covered with aluminum foil, and autoclaved to produce Barth A solution. About 95 ml of MQ water is put in a 100 ml beaker, and each reagent (reagent concentration required amount / 1 ?; CaCl 0.41 mM 0.6 g, Ca (NO ₃ ) ₂ 0.31 mM 0.78 g) is weighed and added, and the volume is adjusted to 100 ml. After up, it was stored in a culture bottle and autoclaved to produce Barth 100B solution. An autoclaved Barth A solution was weighed with a spatula in which Pnicillin G 25 mg and Streptomycin 12.5 mg were disinfected with alcohol, and 5 ml of Barth 100B solution was added with a sterilized measuring pipette to prepare a Barth solution.
35 mg of collagenase (manufactured by SIGMA) was placed in a beaker and 10 ml of Barth A solution was added to produce (adjust) a collagenase solution.

(Synthesis and regulation of mRNA)
GABA _A receptor mRNA was synthesized in vitro using SP6 RNA polymerase (manufactured by RiboMAX Promega) using pSP6 plasmid containing α1 and β1 of GABA _A receptor cloned from bovine brain as a template. The synthesized GABA _A receptor α1, β1 mRNA was diluted to preset concentrations (α1: 0.8 μg / μl, β1: 0.8 μg / μl). MQ water, Eppendorf tubes, tips, etc. were all sterilized.

(MRNA injection)
A glass tube for injection was made with NARISHIGE PP-83 (manufactured by Narishige Co., Ltd.) and sterilized by dry heat at 200 ° C. for about 1 hour. The sterilized glass tube was filled with mineral oil and set in an injection manipulator (NARISHIGE MMN-8: manufactured by Narishige Co., Ltd.), and the adjusted mRNA was sucked into the glass bottle. A petri dish with a net was filled with Barth solution, and the eggs from which the follicular membrane had been removed were placed and injected one by one (α1: 30.0 ng / oocyte, β1: 30.0 ng / oocyte). α1 and β1 were mixed by 1 μl each, and mRNA was injected at about 50 nl per egg. The injected eggs were incubated for about 18-24 hours in a thermoplate set at 20 ° C.

(Two-electrode membrane potential fixation method)
The membrane potential was fixed at -70 mV by the two-electrode membrane potential fixing method, and ions passing through the cell membrane were measured with an amperometric electrode. The amplifier used an ausite clamp device (TEV-200A: made by DAGAN), and the current value was AD (Analog to Digital) converted by Power Lab / 200 and captured by software chart. FIG. 6 is a diagram showing a standard measurement state of GABA _A receptor. The horizontal axis represents time (s) and the vertical axis represents current (A). To the eye, the ligand GABA was refluxed. Here, the activity of GABA _A receptor was measured by refluxing the A fraction (compound contained therein) instead of GABA.

(Measurement of GABA _A receptor activity)
Figure 7 is a representation of the concentration response curves of GABA for [alpha] 1 / .beta.1 subtype GABA _A receptors of GABA agonists of GABA _A receptors. The 50% effective concentration (EC ₅₀ ) was 65.1 μM. From the results shown in the figure, it was found that the composition that enhances the GABA _A receptor exhibits activity at a GABA concentration of 65 μM to 500 μM, more preferably 100 μM to 500 μM. Further, from this figure, it was found that the concentration showing the maximum activity of GABA was 200 μM. Based on this result, GABA _A receptor activity of compounds contained in the A fraction was examined using GABA 200 μM as a control. Further, instead of 2-hydroxyvaleric acid contained in the A fraction, it is a compound having a similar structural formula and molecular weight (a compound having a hydroxy group or a carboxyl group and not far from the molecular weight of GABA). GABA _A receptor activity was also investigated for 2-hydroxyvaleric acid ethyl ester (Ethyl 2-hydroxyvalerate).

FIG. 8 is a graph showing GABA _A receptor activity when the compound concentration is 200 μM. In the figure, 100% of GABA _A receptor activity GABA200myuM, were compared GABA _A receptor activity of each compound. As a result, as shown in the figure, lactic acid, quinic acid, glycolic acid, pyruvic acid and 3-hydroxybutyric acid showed high GABA _A receptor activity. In addition, 2-hydroxyvaleric acid ethyl ester, gluconic acid, malic acid, 2-hydroxy-4-methylvaleric acid, citric acid, 2-oxoglutaric acid, pantothenic acid, and mevalonic acid also have GABA _A receptor activity. It has been shown.

Next, FIG. 9 is a graph showing GABA _A receptor activity when the compound concentration is 500 μM. As shown in the figure, quinic acid, gluconic acid, malic acid, pyruvic acid and 3-hydroxybutyric acid showed high GABA _A receptor activity. In addition, 2-hydroxyvaleric acid ethyl ester, 2-hydroxy-4-methylvaleric acid, citric acid, glycolic acid, and 2-oxoglutaric acid were also shown to have GABA _A receptor activity.

From the above results, pyruvic acid, 2-oxoglutaric acid, 2-hydroxy-4-methylvaleric acid, citric acid, malic acid, 2-hydroxyvaleric acid ethyl ester, quinic acid, gluconic acid, glycolic acid, 3-hydroxybutyric acid Mevalonic acid, lactic acid, and pantothenic acid have been shown to have agonist activity at the GABA _A receptor. In addition, quinic acid, pyruvic acid, and 3-hydroxybutyric acid were shown to have high GABA _A receptor activity at any concentration of 200 μM and 500 μM. In addition, quinic acid, gluconic acid, malic acid, pyruvic acid, and 3-hydroxybutyric acid were shown to increase GABA _A receptor activity as the concentration increased. Moreover, even if it was a compound which is not contained in A fraction, if it was a compound with a similar structural formula and molecular weight, it was shown that it has agonist activity with respect to a GABA _A receptor.

(Elevated cross maze test)
Next, the action of quinic acid and gluconic acid on GABA _A receptor activity was examined by an elevated plus maze test.

FIG. 10 is a diagram showing the elevated plus maze test apparatus used in the experiment. The elevated plus maze testing apparatus is 40 cm above the floor, and consists of four arms (6 x 30 cm) that run straight and a platform (9 x 9 cm) where they intersect. The two open arms (the road without walls) have a 2 cm high edge but no side walls, and the two closed arms (the road with walls) have a gray opaque side wall with a height of 10 cm The other floors are also gray opaque. In this test, it was observed whether the mouse searches without fear of an open arm by utilizing the property that the mouse likes the wall. When an anti-anxiety agent is administered to mice, the search for an open arm increases, so the action on GABA _A receptor activity was examined using the time spent in the open arm and the number of times of entry as an index.

  Nine-week-old C57BL / 6NCrlCrlj male mice (purchased from Charles River Japan) were acclimated in an animal breeding room, and then an elevated plus maze test was conducted. Five mice were placed in a polycarbonate cage (30 × 20 × 15 cm) with paper chip bedding, and were allowed to freely feed and tap water. As the feed, animal solid feed MF (manufactured by Oriental Yeast Co., Ltd.) was used. The animal laboratory was adjusted to light and dark every 12 hours (light period: 8 o'clock to 20 o'clock, dark period: 20 o'clock to 8 o'clock), room temperature 23 ° C and humidity 60%.

  In performing the elevated plus maze test, the mice were allowed to freely take water, but the mice were fasted for 3 to 5 hours from before administration of the compound until the completion of the elevated plus maze test. When physiological saline, diazepam, muscimol, quinic acid, and gluconic acid were administered to mice, eppendorf tubes, tips, injection needles, syringes, etc. were all sterilized. The intraperitoneal dose (mg / kg body weight) of each compound to mice was diazepam = 1, muscimol = 0.3, quinic acid = 1, and gluconic acid = 10. Place the mouse on the platform part of the elevated cross maze test device, start the test with the head facing the open arm, and observe the number of intrusions and staying time for each of the open and closed arms for 10 minutes. And recorded. The case where all the limbs came out of the platform into the arm part was recorded as the transition to the arm.

FIGS. 11 and 12 are diagrams comparing the ratio of the stay time of the open arm and the number of times of entry when physiological saline, diazepam, muscimol, quinic acid, and gluconic acid were each administered to mice. When quinic acid and gluconic acid were administered to mice, as shown in the figure, the stay time and the number of times of entry of the open arm were higher than those when physiological saline was administered. As a result, it was found that quinic acid and gluconic acid have an action on GABA _A receptor activity and have a function as an anxiolytic agent. In addition, as shown in the figure, in the case of administering diazepam and muscimol, which are known to act on GABA _A receptor activity, compared to the case of administration of physiological saline, The number of times of entry has exceeded. From this, it was found that this elevated plus maze test was conducted properly.

From the above results, it was shown that quinic acid and gluconic acid have agonist activity with respect to the GABA _A receptor.

  In addition, this invention is not limited to said embodiment and Example, A various deformation | transformation and application are possible.

The GABA-like composition (compound) exhibiting GABA-like activity with respect to the GABA _A receptor according to the above embodiment is not limited to those purified and extracted from sake, but purified and extracted from other liquors such as whiskey and beer. It may also be produced by chemical synthesis. In addition, sake can be any type of sake, such as ginjo sake, pure rice sake, mountain abandonment, or regular sake.

Examples of the dosage form of the composition exhibiting GABA _A receptor activity include internal medicines, injections, suppositories, and inhalants. The composition is a known method that can be normally used in the pharmaceutical formulation technical field such as excipients, binders, disintegrants, lubricants, flavoring agents, solubilizers, suspension agents, coating agents, etc., according to conventional methods. The drug is formulated into a dosage form suitable for oral administration, intra-tissue administration (subcutaneous administration, intramuscular administration, intravenous administration, etc.), local administration (transdermal administration, etc.), transrectal administration, etc. be able to. In addition to the above-mentioned auxiliary agents, coloring agents, preservatives, fragrances, flavoring agents, sweetening agents, and other auxiliary agents as well as other pharmaceuticals may be included as necessary. Of course, the composition is administered in dosage forms suitable for these methods of administration. The dose of the drug is arbitrary, and can be determined in consideration of the patient's condition such as age and weight, the administration route and the like.

The composition exhibiting GABA _A receptor activity may be in the form of liquid, solid, slurry, powder, etc., and is preferably mixed with various known foods so as to be easy to eat. The ingredients include sweeteners, umami seasonings, inorganic salts, acidulants, amino acids, nucleic acids, sugars, excipients, spices, seasonings other than umami, antioxidants, colorants, preservatives as necessary. Various additives usable for foods such as fortifiers, emulsifiers, herbs, spices and ethanol can be used.

Examples of patients and patients who can administer and ingest compositions that exhibit GABA _A receptor activity include humans, mammals other than humans (mouse, rat, guinea pig, hamster, rabbit, cat, dog, sheep, pig, goat, Cattle, monkeys, etc.), but birds, reptiles, amphibians, fish, invertebrates, etc. are optional.

The combination and ratio of 65 compounds exhibiting GABA _A receptor activity (64 compounds contained in fraction A + 2-hydroxyvaleric acid ethyl ester) are arbitrary. For example, pyruvic acid and 2-oxoglutaric acid can be combined with 1 : 1 or a combination of pyruvic acid, quinic acid and gluconic acid at a ratio of 1: 2: 3.

As described above, the GABA-like composition according to the present invention produces a tranquilizing / hypnotic drug that exerts a neuroprotective effect and the like because a GABA _A receptor acts to cause mental stability, sleep, an anesthetic effect, etc. It is useful as an anticonvulsant, anxiolytic, or sedative neuroactive composition.

Claims (5)

At least GABA (Gamma-aminobutyric acid), barbituric acid derivatives, benzodiazepine derivatives, Muscimol, THIP (4,5,6,7-tetrahydroisoxazolo [5,4-c] pyridin-3-ol), isoguvacine (Isoguvacine) And a GABA-like composition that exhibits a GABA-like activity with respect to a GABA _A receptor, comprising a compound having a hydroxy group or a carboxyl group, excluding seven substances of Gabapentin.   The compound includes pyruvic acid, 2-oxoglutaric acid, 2-hydroxy-4-methylvaleric acid (DL-leucic acid), citric acid ( Citric acid, malic acid, 2-hydroxyvaleric acid, quinic acid, gluconic acid, glycolic acid, 3-hydroxybutyric acid (3 -Hydroxybutyric acid, Mevalonic acid, Lactic acid, Pantothenic acid, 2-Hydroxyvaleric acid ethyl ester; 2-ethylhydroxyvaleric acid (Ethyl 2- hydroxyvalerate)), p-Hydroxyphenylacetic acid, Shikimic acid, Citramalic acid, Citraconic acid, 2-Isopropylmalic acid, Huma Nucleic acid (Fumaric acid), N-Acetylmuramic acid (Glyceric acid), Benzoic acid, Homovanillic acid, Glutaric acid, 6- Hydroxyhexanoic acid (6-Hydroxyhexanoic acid), Glucuronic acid, Galacturonic acid, 2-Hydroxybutyric acid, 5-oxo-2-tetrahydrofurancarboxylic acid (5-Oxo-2) -tetrahydrofurancarboxylic acid, Phthalic acid, Terephthalic acid, Succinic acid, Isocitric acid, Threonic acid, Crotonic acid, Malonic acid ( Malonic acid), Isethionic acid, cis-Aconitic acid, N-Acetylaspartic acid, Maleic acid , Propionic acid, Vanillic acid, 3-Hydroxy-3-methylglutaric acid, Digalacturonic acid, 6-phosphogluconic acid (6 -Phosphogluconic acid), Isobutyric acid, Cysteic acid, Methanesulfonic acid, Glyoxylic acid, N-acetylmethionine, biotin, p -Sulfobenzoic acid, N-acetylglucosamine-1-phosphate, 5-Oxoproline, N-acetyl-β-alanine (N-Acetyl) -β-alanine), Glycerol 3-phosphate, N-Acetylglutamic acid, Glucose 6-phosphate, O-Succinylhomoserine N-acetylleucine, fructose 6-phosphate, succinic semialdehyde, trehalose 6-phosphate, and glucose 1-phosphate The GABA-like composition according to claim 1, comprising at least one of compounds of (Glucose 1-phosphate).   The GABA-like composition according to claim 1, wherein the concentration of the compound is 10 μM to 10 mM.   The GABA-like composition according to any one of claims 1 to 3, wherein the compound is separated and purified from sake.   The GABA-like composition according to claim 4, wherein the compound is separated and purified from the organic acid fraction of the sake.