JP2016006077A - Sleep-inducing and improving composition having gabaa-benzodiazepine receptor activity containing brown algae extract as active ingredient - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a sleep-inducing and improving composition having GABA-benzodiazepine receptor activity by binding to benzodiazepine site of a GABAreceptor to have affinity.SOLUTION: The invention provides a sleep-inducing and improving composition having GABA-benzodiazepine receptor activity containing an extract of brown algae, such as Dictyopteris prolifera, Ecklonia cava, Ecklonia kurome, Ecklonia stolomifera Okamura, Sargassum horneri, Petalonia binghamia, Leathesia disfformis, Myelophycus simplex, Colpomenia sinuosa, Sargassum patens, Dictyota coriacea, Dictyota dichotoma, Fucus vesiculosus, Undaria pinnatifida, and Ishige okamurae, as an active ingredient.

Description

The present invention relates to a composition for GABA _A -benzodiazepine receptor activity comprising brown algae extract as an active ingredient. In particular, the present invention relates to a composition having an affinity for GABA _A -benzodiazepine receptors and having anxiolytic, anticonvulsant, sedative, and sleep-inducing and improving effects.

  In modern society, many people experience sleep disorders due to stress, anxiety and frustration. According to statistics, about 15% of adults are known to need pharmacological treatment for insomnia due to anxiety symptoms, and there are various causes of insomnia such as stress, tension and fear. As such, benzodiazepine drugs and serotonin agonists are used. However, such drugs have severe problems such as tolerance and dependence when used for a long time.

  Therefore, in the case of long-term users of sleeping pills and restrictors, the need for natural supplements is increasing as a means to replace them, and there is a situation in which the demand for health promoting functional foods is increasing.

GABA _A receptor is a pentameric protein that forms a membrane ion channel, and is closely related to regulation of sedation, sleep, anxiety, muscle tone, convulsions, memory loss, etc. Through this, GABA (γ− Aminobutyric acid) acts. Many drugs, including benzodiazepines as anxiety treatments, sedatives or sleep inducers, bind to this receptor for pharmacological action, and if drugs or adjuvants bind to benzodiazepine sites, GABA _A against GABA Increases receptor affinity and increases intracellular entry of chloride ions to show anxiolytic, anticonvulsant, sedative, and sleep-inducing and improving effects. However, it still shows drug dependence and causes serious problems such as muscle relaxation and amnesia.

Therefore, it has a sedative-sleep effect using a substance obtained from a safe natural product that has a risk of side effects while having an affinity for GABA _A -benzodiazepine receptor by binding to the benzodiazepine site of GABA _A receptor. There is a real need for the development of supplements.

By acting on the benzodiazepine site of GABA _A receptors, to activate the chloride channels GABA _A storage body in response to GABA, chlorine is flowed into the cell, neuronal through excessive polarization of the cell membrane potential (neuronal ) There are studies on terrestrial hubs and plant extracts that slow down activity, but reports on phloroglucinol or phlorotannins, which are phenolic compounds specifically present in brown algae extracts or brown algae There was no case worldwide.

The first problem to be solved by the present invention is to provide a composition for inducing and improving sleep having GABA _A -benzodiazepine receptor activity, which has affinity by binding to the benzodiazepine site of GABA _A receptor. .

  The second problem to be solved by the present invention is to provide a composition for anxiolytic, anticonvulsant, sedative, and sleep induction and improvement containing brown algae extract.

In order to achieve the above object, the present invention provides a composition having GABA _A -benzodiazepine receptor activity, which comprises brown algae extract as an active ingredient.

  According to an embodiment of the present invention, the brown algae is selected from the following: Heayahaz, Kajime, Kurome, Tsuruarame, Akamok, Habanori, Nevaimo, Siwahige, Fukuronori, Yatsumatamo, Sanadagusa, Ajigusa, Kombu, Wakame and Ishige It can be any one or more brown algae.

  According to an embodiment of the present invention, the brown algae may be Ecklonia cava or Ecklonia kurome.

  According to one embodiment of the present invention, the brown algae extract is any one selected from a brown algae water extract, a brown algae ethanol extract, a brown algae methanol extract, or water, ethanol and methanol. It can be a brown algae extract with a mixed solvent of more than one species of solvent.

  According to an embodiment of the present invention, the brown algae water extract is produced by extracting brown algae with water at 40 to 100 ° C. for 2 to 48 hours. Extracted with 75% ethanol at 20-60 ° C for 2-36 hours, the brown algae methanol extract is obtained by extracting brown algae with 35-85% methanol at 20-60 ° C for 2-36 hours. Can be manufactured.

  According to one embodiment of the present invention, the brown algae water extract is water added with one or more enzymes selected from the group consisting of cellulase, viscozyme, alcalase, and pectin at 40-55 ° C. at 2-24. It can be produced by extracting time brown algae.

The brown algae extract of the present invention has an affinity for GABA _A -benzodiazepine receptor, and a composition comprising the same has anti-anxiety, anticonvulsant, sedative effects, and sleep induction and improvement, particularly sleep induction and It is effective for improvement, uses a substance obtained from natural products, does not induce side effects, ensures safety, and is very useful as a food composition such as pharmaceutical compositions and health functional foods. .

Binding of GABA _A -benzodiazepine ligand [ ³ H] flumazenil radiolabeled with Kajime water extract (ECK-W), Kajime methanol extract (ECK-M), and Kajime ethanol extract (ECK-E), respectively. It is a graph which shows the suppression effect. Each sample was orally administered to rats, and 45 minutes later, pentobarbital having a concentration of hypnotic (45 mg / kg) was administered by intraperitoneal injection, and is a graph showing the sleep latency. ** has a significant difference at p <0.01 compared to the control group (Dunnet's test). Each sample was orally administered to rats, and 45 minutes later, hypnotic (45 mg / kg) concentration of pentobarbital was administered by intraperitoneal injection, and is a graph showing sleep time. * Is significantly different at p <0.05 compared to the control group, and ** is significantly different at p <0.01 compared to the control group (Dunnet's test). (CON: control group (0.5% CMC-saline 10 mL / kg) after oral administration, pentobarbital administration DZP: diazepam 2 mg / kg, oral administration, pentobarbital administration ECK-E: Kajime ethanol extract 100 , 250, 500, 1000 mg / kg administration, pentobarbital administration ECK-W: Kajime water extract administered 100, 250, 500, 1000 mg / kg administration, pentobarbital administration) It is a schematic diagram which shows the process of manufacturing a solvent fraction from a Kajime ethanol extract. Ecklonia extract fraction of radioactive target positions have been GABA A _- is a graph showing the binding inhibition effect of benzodiazepine ligand ^{[3 H]} flumazenil. Each Kajime extract fraction is orally administered to a rat, and 45 minutes later, pentobarbital having a concentration of hypnotic (45 mg / kg) is administered by intraperitoneal injection, and is a graph showing sleep time. It is a graph showing the correlation between the _{IC 50} for binding of total phenolic contents of Ecklonia extract fraction and (TPC) ^[3 H] flumazenil. It is a graph which shows the correlation with the total phenol content (TPC) of each Kajime extract fraction, and sleep time. It is the structure of phloroglucinol, equol, extronol, trifloretol A, and diacor which are phlorotannins compounds separated from the Kajime extract fraction. It is the structure of fucodifloretol G, 6,6'-biequol, 7-fluoroecol, 6,8'-biequol, 8,8'-biequol. A graph showing the correlation between the total phenol content (TPC) of [Kuromemethanol extract, Kajimemethanol extract, Herayahazumethanol extract and Sanadagusa methanol extract and the binding activity of [ ³ H] flumazenil at 10 mg / mL concentration. It is. It is a graph which shows the correlation with the total phenol content (TPC) and sleep time of each brown algae methanol extract. It is a graph which shows the sleep latency and sleep time by the density | concentration of phloroglucinol (PG), respectively. It is a graph which shows the sleep latency and sleep time by the density | concentration of phloroglucinol (PG), respectively. It is a graph which shows the sleep onset latency by the presence or absence of flumazenil administration of phloroglucinol, and sleep time, respectively. It is a graph which shows the sleep onset latency by the presence or absence of flumazenil administration of phloroglucinol and sleep time, respectively. It is a graph which shows the sleep sleep latency and sleep time by the density | concentration of an extronol (ETN), respectively. It is a graph which shows the sleep sleep latency and sleep time by the density | concentration of an extronol (ETN), respectively. It is a graph which shows the sleep onset latency and sleep time by the presence or absence of flumazenil administration of extronol, respectively. It is a graph which shows the sleep onset latency and sleep time by the presence or absence of flumazenil administration of extronol, respectively. It is a graph which shows the sleep onset latency and sleep time by the density | concentration of trifloretol A (TPE-A), respectively. It is a graph which shows the sleep onset latency and sleep time by the density | concentration of trifloretol A (TPE-A), respectively. It is a graph which shows the sleep latency and sleep time by the presence or absence of the administration of flumazenil of trifloretol A, respectively. It is a graph which shows the sleep latency and sleep time by the presence or absence of the administration of flumazenil of trifloretol A, respectively. It is a graph which shows the sleep onset latency and sleep time by the density | concentration of fucodifloretol G (FDE-G), respectively. It is a graph which shows the sleep onset latency and sleep time by the density | concentration of fucodifloretol G (FDE-G), respectively. It is a graph which shows the sleep latency and sleep time by the presence or absence of the administration of flumazenil of fucodifloretol G, respectively. It is a graph which shows the sleep latency and sleep time by the presence or absence of the administration of flumazenil of fucodifloretol G, respectively. It is a graph which shows the sleep sleep latency and sleep time by the density | concentration of an equol, respectively. It is a graph which shows the sleep sleep latency and sleep time by the density | concentration of an equol, respectively. 2 is a graph showing sleep onset latency and sleep time depending on whether or not equol is administered with flumazenil. 2 is a graph showing sleep onset latency and sleep time depending on whether or not equol is administered with flumazenil. It is a graph which shows the sleep onset latency and sleep time by the density | concentration of 6,6'-biequol, respectively. It is a graph which shows the sleep onset latency and sleep time by the density | concentration of 6,6'-biequol, respectively. It is a graph which shows the sleep onset latency and sleep time by the presence or absence of flumazenil administration of 6,6'-biequol, respectively. It is a graph which shows the sleep onset latency and sleep time by the presence or absence of flumazenil administration of 6,6'-biequol, respectively. It is the graph which confirmed the sleep latency and sleep time by a density | concentration, respectively, in order to confirm the effective density | concentration of diazepam (DZP) and zolpidem (ZPD) used as a positive control group in the confirmation test of the said sleep induction effect. It is the graph which confirmed the sleep latency and sleep time by a density | concentration, respectively, in order to confirm the effective density | concentration of diazepam (DZP) and zolpidem (ZPD) used as a positive control group in the confirmation test of the said sleep induction effect. Chromate methanol extract (EKO-M) of the radioactive mark positions have been GABA _A - is a graph showing the binding inhibition effect of benzodiazepine ligand ^[3 H] flumazenil. It is a graph which shows the sleep latency when the chromate methanol extract (EKO-M) is orally administered to a rat, and pentobarbital with a concentration of hypnotic (45 mg / kg) is administered by intraperitoneal injection after 45 minutes. ** has a significant difference at p <0.01 compared to the control group (Dunnet's test). It is the graph which orally administered the chrome methanol extract (EKO-M) to the rat, and after 45 minutes, hypnotic (45 mg / kg) density | concentration pentobarbital was administered by the intraperitoneal injection, and the sleep time was investigated. * Is significantly different at p <0.05 compared to the control group, and ** is significantly different at p <0.01 compared to the control group (Dunnet's test). (CON: control group (0.5% CMC-saline 10 mL / kg) after oral administration, pentobarbital administration DZP: diazepam 2 mg / kg, oral administration pentobarbital administration ECK-M: Kajime methanol extract 1000 mg each After administration of / kg, administration of pentobarbital EKO-M: After administration of 1000 mg / kg of chrome methanol extract, administration of pentobarbital) In order to investigate the sleep-induced synergy effect of oral administration of Kajime ethanol extract and water extract with diazepam, ECK-E is 100 mg / kg, ECK-W is 100 mg / kg, and diazepam (DZP) is 0. It is a graph which shows the sleep latency and sleep time which were measured by orally administering 0.5 mg / kg 45 minutes before pentobarbital administration (hypnotic dosage 45 mg / kg). In order to investigate the sleep-induced synergy effect of oral administration of Kajime ethanol extract and water extract with diazepam, ECK-E is 100 mg / kg, ECK-W is 100 mg / kg, and diazepam (DZP) is 0. It is a graph which shows the sleep latency and sleep time which were measured by orally administering 0.5 mg / kg 45 minutes before pentobarbital administration (hypnotic dosage 45 mg / kg). To examine the effect of flumazenil (FLU) on sleep-inducing effects of Kajime ethanol extract, water extract and ethyl acetate fraction, ECK-E is 1000 mg / kg, ECK-W is 1000 mg / kg. EtAcO is 200 mg / kg, diazepam (DZP) is 0.5 mg / kg orally administered 45 minutes before administration of pentobarbital (hypotonic dose 45 mg / kg), and flumazenil (FLU) is 8 mg / kg ECK- E, ECK-W, DZP It is the graph which carried out the intraperitoneal injection in advance 10 minutes before oral administration, and measured with respect to the sleep latency and sleep time. To examine the effect of flumazenil (FLU) on sleep-inducing effects of Kajime ethanol extract, water extract and ethyl acetate fraction, ECK-E is 1000 mg / kg, ECK-W is 1000 mg / kg. EtAcO is 200 mg / kg, diazepam (DZP) is 0.5 mg / kg orally administered 45 minutes before administration of pentobarbital (hypotonic dose 45 mg / kg), and flumazenil (FLU) is 8 mg / kg ECK- E, ECK-W, DZP It is the graph which carried out the intraperitoneal injection in advance 10 minutes before oral administration, and measured with respect to the sleep latency and sleep time. In order to examine the effect of Kajime ethanol extract on the sleep structure of rats, the Control group was orally administered with 0.5% CMC-saline solution, and ECK-E was orally administered with 500 mg / kg (po). Then, it is a graph analyzed after measuring the electroencephalogram. In order to examine the effect of Kajime ethanol extract on the sleep structure of rats, the Control group was orally administered with 0.5% CMC-saline solution, and ECK-E was orally administered with 500 mg / kg (po). Then, it is a graph analyzed after measuring the electroencephalogram. * Means that there is a significant difference (Dunnet's test) at p <0.05 compared to the control group.

  Hereinafter, the present invention will be described in detail.

GABA _A receptors are associated with anxiolytic, anticonvulsant, sedative, and sleep-inducing and improving effects. As drugs acting on GABA _A receptors, agonists include benzodiazepine, diazepam ( diazepam (DZP), zolpidem, etc., and antagonists include flumazenil (FLU). As described above, in the case of a drug acting on the GABA _A receptor, there are problems that cause dependency and side effects.

Brown algae extract, unlike green algae and red algae, acts on GABA _A- benzodiazepine (benzodiazepine) receptor to have anxiolytic, anticonvulsant, sedative, and sleep induction and improvement effects It was confirmed. The sleep improvement may be an effect of decreasing sleep latency and increasing sleep duration, but the scope of the present invention is not limited thereto.

An active ingredient that acts on GABA _A -benzodiazepine receptor in the brown algae extract and exhibits anxiolytic, anticonvulsant, sedative, and sleep-inducing and improving effects is a polyphenol compound specifically present only in brown algae. Some phloroglucinol or phlorotannin was confirmed.

The phlorotannin is an oligomer of phloroglucinol. Fluorotannin may be selected from, for example, equols, fucoles, phloretols, fuhalols and extronol. Examples of the equol include equol, diacol, biequol, fluoroecole, floecole, fucofurocole, florocofloecole, etc., and fucoles are oligomers in which 2 to 10 phloroglucinols are aryl-aryl bonded, Phloretols are aryl-ether-linked oligomers of 2 to 10 phloroglucinols, and fuhalols are ether-bonded at 3 to 10 phloroglucinols in the para or ortho position, with every third ring. Is an oligomer further containing a hydroxyl group. Examples of fluorotannins that act on GABA _A -benzodiazepine receptors and are excellent in anxiolytic, anticonvulsant, sedative, and sleep induction and improvement effects include extronol, trifloretol A, fucodifloretol G, and equol. And 5,6'-Viecol.

  The phloroglucinol or phlorotannin which is an active ingredient of the composition of the present invention is an extract extracted from brown algae separated into individual compounds, or in the state where one or more phlorotannins are mixed. A concentrate obtained by re-fractionating the extract can be used, or an artificially synthesized or semi-synthesized one can be used.

  The brown algae is, for example, any one or more brown algae selected from the following: Preferably, it is caulking or chrome, more preferably caulking.

  The brown algae extract may be extracted using water, an organic solvent, or a mixture thereof as an extraction solvent. At this time, the kind of the organic solvent used and the mixing ratio of water and the organic solvent are not particularly limited.

  For example, the organic solvent may be one or more solvents selected from the group consisting of lower alcohol, hexane, acetone, ethyl acetate, chloroform, and diethyl ether. The lower alcohol may be an alcohol having 1 to 6 carbon atoms. For example, as the lower alcohol, methanol, ethanol, propanol, butanol, normal propanol, isopropanol, normal butanol, 1-pentanol, 2-butoxyethanol or ethylene glycol can be used. Other organic solvents include polar solvents such as acetic acid, DMFO (dimethyl-formamide), DMSO (dimethyl sulfoxide), acetonitrile, ethyl acetate, methyl acetate, fluoroalkane, pentane, 2,2,4-trimethylpentane, Decane, cyclohexane, cyclopentane, diisobutylene, 1-pentene, 1-chlorobutane, 1-chloropentane, o-xylene, diisopropyl ether, 2-chloropropane, toluene, 1-chloropropane, chlorobenzene, benzene, diethyl ether, diethyl sulfate, Use non-polar solvents such as chloroform, dichloromethane, 1,2-dichloroethane, aniline, diethylamine, ether, carbon tetrachloride and THF (Tetrahydrofuran) And it can also be.

  The brown algae extract is a brown algae in a mixed solvent of any two or more solvents selected from a brown algae water extract, a brown algae ethanol extract, a brown algae methanol extract, or water, ethanol and methanol. Although it may be an extract, the scope of the present invention is not limited thereto.

  The water extract is prepared by extracting brown algae with water at 40 to 100 ° C. for 2 to 48 hours, and more desirably, in order to increase the extraction efficiency of water-soluble components by water extraction, cellulase, viscozyme, It can be produced by extracting for 2 to 24 hours at 40 to 55 ° C. with water to which one or more enzymes selected from the group consisting of alcalase and pectin are added.

  The ethanol extract is prepared by extracting brown algae with 35 to 75% ethanol at 20 to 60 ° C. for 2 to 36 hours, preferably at 40 to 50 ° C. for 2.5 to 6 hours, more preferably , And extracted with a 70% aqueous ethanol solution at 45 ° C. for 3 hours.

  The methanol extract is produced by extracting brown algae with 35 to 85% methanol at 20 to 60 ° C. for 2 to 36 hours, preferably extracting at 20 to 30 ° C. for 22 to 26 hours, More preferably, it is produced by extraction with 80% methanol at 25 ° C. for 24 hours.

Among the brown algae extracts of the present invention, the Kajime water extract may have an IC ₅₀ of 1.02 to 1.03 μm as measured by the GABA _A -benzodiazepine receptor binding measurement. Further, the Kajime ethanol extract may have an IC ₅₀ of 0.45 to 0.46 μm as measured by binding of the GABA _A -benzodiazepine receptor. Further, the Kajime methanol extract may have an IC ₅₀ of 1.25 to 1.26 μm as measured by binding of the GABA _A -benzodiazepine receptor. In addition, the chromomeethanol extract may have an IC ₅₀ of 0.62 to 0.63 μm as measured by binding to the GABA _A -benzodiazepine receptor.

  The term 'extract' as used herein also includes a fraction obtained by additionally fractionating the extract. That is, the brown algae extract includes not only those obtained using the extraction solvent described above, but also those obtained by concentrating phlorotannins by additionally applying a tablet process thereto. In addition, fractions obtained by passing the extract or fraction through an ultrafiltration membrane having a certain molecular weight cut-off value, various chromatographies (for separation by size, charge, hydrophobicity or affinity) Fraction obtained through various additional tablet methods such as separation by manufactured) is also included in the brown algae extract of the present invention.

The GABA _A -benzodiazepine receptor active composition containing the phloroglucinol, phlorotannin or brown algae extract of the present invention as an active ingredient can be a pharmaceutical composition for treating anxiolytic, anticonvulsant, sedative and insomnia.

  The pharmaceutical dosage forms of the pharmaceutical composition for the treatment of anti-anxiety, anticonvulsant, sedation and insomnia can be used in the form of their pharmaceutically acceptable salts and can only be combined with other pharmaceutically active compounds alone. Rather, it can be used in the form of a suitable set.

  In addition, the pharmaceutical compositions for the treatment of anxiolytic, anticonvulsant, sedative and insomnia according to the present invention can be applied to powders, granules, inhibitors, capsules, suspensions, emulsions, syrups, aerosols, etc., respectively, by conventional methods. Appropriate carriers and excipients that can be used in the form of oral dosage forms, external preparations, suppositories, and sterile injectable solutions, and are usually used in the manufacture of pharmaceutical compositions for formulation. Or a diluent may be included.

  Examples of the carrier or excipient or diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginic acid, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose And various compounds or mixtures including amorphous cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.

  In the case of formulating, it can be manufactured using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants.

  A solid preparation for oral administration can be prepared by mixing the extract with at least one excipient such as starch, calcium borate, sucrose or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used.

  Liquid preparations for oral use include suspensions, liquids for internal use, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wet Agents, sweeteners, fragrances, preservatives and the like.

  Preparations for parenteral administration include sterile aqueous solutions, water-insoluble agents, suspensions, emulsions, lyophilized formulations, suppositories. As non-aqueous preparations and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate can be used. As a suppository base, hard fat, macrogol, tween 61, cacao butter, lauric fat, glycerol gelatin and the like can be used.

  The desired dosage of the pharmaceutical composition for the treatment of anxiolytic, anticonvulsant, sedative and insomnia according to the present invention varies depending on the patient's condition, body weight, degree of illness, drug form, route of administration, and duration, but may be appropriately determined by those skilled in the art Can be selected. However, for desirable effects, it can be administered at 0.0001 to 2,000 mg / kg per day, preferably 0.001 to 2,000 mg / kg. The administration can be performed once a day or divided into several times. However, the scope of the present invention is not limited by the dose.

  The pharmaceutical composition for treating anxiolytic, anticonvulsant, sedative and insomnia according to the present invention can be administered to mammals such as rats, mice, domestic animals and humans by various routes. Any mode of administration can be administered by, for example, oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dura mater or intracerebral intravascular injection.

  In addition, the present invention is a food composition for anxiolytic, anticonvulsant, sedative, and sleep induction and improvement comprising phloroglucinol, phlorotannin or brown algae extract as an active ingredient.

  When the phloroglucinol, phlorotannin or brown algae extract according to the present invention is used as an active ingredient additive for health functional foods or general foods, the extract according to the present invention should be added as it is or with other foods or food ingredients. Can be used properly in the usual way. The mixing amount of the active ingredient can be determined so as to be suitable for each purpose of use such as prevention, health or treatment.

  Generally, the phloroglucinol, phlorotannin or brown algae extract according to the present invention may be added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less based on the raw material during the production of food or beverage. it can. However, in the case of long-term ingestion for the purpose of health and hygiene or for the purpose of health regulation, the amount is below the above range, and the present invention uses an extract from a natural product. Since there is no problem in terms of safety in terms of use, it can be used in an amount exceeding the above range.

  The type of the food is not particularly limited, and examples of foods to which the substance can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, and other noodles , Dairy products including gums, ice creams, various soups, drinking water, tea, drinks, alcoholic beverages and vitamin complexes, etc., and any food in the normal sense can be included.

  Among the health functional foods according to the present invention, the beverage food may contain various flavoring agents or natural carbohydrates as additional components, as in a normal beverage. The above-mentioned natural carbohydrates can be monosaccharides such as sucrose and fructose, disaccharides such as maltose and sucrose, and sugar alcohols such as dextrin and polysaccharides such as cyclodextrin, xylitol, sorbitol, erythritol. As the sweetener, natural sweeteners such as thaumatin and stevia extract, and synthetic sweeteners such as saccharin and aspartame can be used. The ratio of the natural carbohydrate may be about 0.01 to 0.04 g, desirably about 0.02 to 0.03 g, per 100 mL of functional food according to the present invention.

  In addition to the above, the food composition for anxiolytic, anticonvulsant, sedative, and sleep induction and improvement according to the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid And salts thereof, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, and carbonates used in carbonated beverages. In addition, the composition for improving sleep of the present invention can contain a fruit juice for producing a natural fruit juice, a fruit juice drink, and a vegetable drink. Such components can be used independently or in admixture. The ratio of such additives is not limited, but is generally selected in the range of 0.01 to 0.1 parts by weight compared to 100 parts by weight of the composition of the present invention. .

  Hereinafter, the present invention will be described in more detail with reference to preferred examples. However, it is obvious to those skilled in the art that these examples are for explaining the present invention more specifically, and that the scope of the present invention is not limited thereto.

[Experimental Example 1: Search for GABA _A -benzodiazepine receptor binding activity in seaweed extract]
In order to search for seaweeds with anti-anxiety, anticonvulsant, sedative, and sleep-inducing and improving effects, 5 kinds of green algae, 7 kinds of red algae and 6 kinds of brown algae, a total of 18 kinds of seaweed methanol The extract was purchased from Jeju Bioresource Extract Bank, Jeju Technopark, Korea, and GABA _A -benzodiazepine receptor binding capacity was measured. A sample of evidence (Voucher specimen) was deposited with the Jeju Bioresource Extract Bank.

GABA _A -benzodiazepine receptor binding activity was measured by removing the cerebral cortex of SD rats and immediately homogenizing it in 20 mL of 30 mM Tris-HCl buffer (pH 7.4, maintained at 4 ° C.) for 10 seconds. did. Subsequently, after centrifugation at 27,000 × g and 4 ° C. for 15 minutes, the supernatant was discarded, 20 mL of buffer was again taken and centrifuged, and this process was repeated three times. In order to remove GABA in the brain tissue, the mixture was incubated in a 37 ° C. water bath for 30 minutes, centrifuged, and then the pellet was collected and stored frozen at −80 ° C. for use. After thawing the frozen membrane tissue, centrifugation was performed at 27,000 × g and 4 ° C. for 10 minutes, and then the supernatant was discarded, and 50 mM citrate tris buffer (binding buffer, pH 7.1, at 4 ° C.). Maintenance) 20 mL was added and centrifuged. After repeating this process three times, the membrane tissue was suspended in the binding buffer at a concentration of 500 mL buffer / g original tissue and used for the binding strength evaluation test. In 96-well plates, 180 μl of membrane tissue suspension, 10 μl of each extract sample and [ ³ H] flumazenil (1 nM, final concentration) (Ro 15-1788; PerkinElmer Life and Analytical Sciences, Waltham, Mass., USA) 10 μl And incubated for 40 minutes in an ice bath. Subsequently, it was collected using a glass fiber filter (GF / C, Whatman). Sample radiation was measured through a Tri-Carb liquid scintillation analyzer (Perkin-Elmer, Shelton, CT, USA). Nonspecific binding (NSB) was determined using clonazepam (1 uM, final concentration), and the displacement (%) of binding was calculated as in equation (1) below.
Formula (1)
Bond displacement (%) = [1-((Sample DPM-NSB DPM) / (TB DPM-NSB DPM))] × 100
(DPM: disintegrations per minute, TB: total binding, NSB: non-specific binding)
The results of GABA _A -benzodiazepine receptor binding activity of the green algal methanol extract are shown in [Table 1] with different concentrations of the extract to 0.1 mg / mL, 1 mg / mL and 10 mg / mL, respectively. The results of the methanol extract are shown in [Table 2], and the results of the brown algal methanol extract are shown in [Table 3].

GABA _A -benzodiazepine receptor that replaces [ ³ H] -flumazenil binding of 80% or more at a concentration of 10 mg / mL with green algae and button aosa (Ulva conglobata Kjellman) and red algae with Ogonori (Gracilaria verrucosa Papenfus) As shown, most of the remaining green and red algae failed to exhibit effective GABA _A -benzodiazepine receptor binding activity. However, in brown algae, with the exception of Sanadagusa (Dictyota coriacea) and Yatsuma Tamoku (Sargassum patens C. Agardh), most of the binding activity is GABA _A -benzodiazepine receptor binding activity at a concentration of 10 mg / mL and 55% or more. Kajime (Ecklonia cava Kjellman) exhibited greater than 90% GABA _A -benzodiazepine receptor binding activity.

[Experimental Example 2: GABA _A -benzodiazepine receptor binding activity of Kajime extract by extraction solvent]
To confirm whether there is a difference in activity when the extraction solvent is different in Kajime (ECK) having the most excellent GABA _A -benzodiazepine receptor binding activity of 90% or more in Experimental Example 1, different extraction solvents are used. A cajime extract was prepared.

  Kajime ethanol extract was extracted with 70% ethanol at 50 ° C. for 3 hours, Kajime water extract was added with enzymes (viscozyme, alcalase, pectin) and extracted at 45 ° C. for 3 hours. Extraction was performed with 80% methanol at room temperature for 24 hours. All extracts were extracted, filtered and concentrated, and then lyophilized to powder.

FIG. 1 is a graph showing the binding substitution effect of GABA _A -benzodiazepine ligand [ ³ H] flumazenil, which is a radioactively placed extract of Kajime water, ethanol, and methanol. This means that the higher the [ ³ H] flumazenil substitution effect is, the higher the binding activity of the substance is, in order to search for a component that represents the sleep effect.

All of the three extracts of Kajime have an affinity for GABA _A -benzodiazepine receptor that is high in a concentration-dependent manner, and IC ₅₀ values are ECK-W (water extract) = 1.026, ECK-M (methanol extract) ) = 1.256, ECK-E (ethanol extract) = 0.456 mg / mL.

[Experimental Example 3: Sleep-inducing effect of Kajime extract by extraction solvent and concentration]
1) Experimental Materials Pentobarbital was purchased from Yulin Pharmaceutical Company, and GABA _A -benzodiazepine receptor antagonist diazepam (DZP) is one of the well-known sleeping agents, Used as a positive control group for sleep-inducing effect.

As experimental animals, male ICR mice weighing 18 to 22 g (Koate Animal) were freely ingested under conditions of a temperature of 24 ° C., a humidity of 55%, and a fluorescent light / dark 12 hour cycle (lighting at 8 am). In order to adapt to a new environment, they were raised for one week before the experiment. In addition, SD rats having a weight of 200 to 250 g were used for preparation of a membrane for evaluation of GABA _A -benzodiazepine receptor binding. All procedures related to animal experiments were conducted based on the guidelines for the use of experimental animals by the Korea Food Researchers Animal Control Committee (KFRI-IACUC, Korea Food Research Institute Animal Care and Use Committee).

2) Experimental method All experiments proceeded between 1 pm and 5 pm, and mice were phagocytosed 24 hours before the experiment. For oral administration, Kajime ethanol extract was diluted in 0.5% carboxymethylcellulose (0.5%, CMC-saline 10 mL / kg) and the positive control group diazepam (DZP) was also Diluted in 0.5% CMC-saline. Kajime ethanol extract and water extract (100, 250, 500, 1000 mg / kg, respectively) and diazepam (DZP, 2 mg / kg) were orally administered 45 minutes before pentobarbital administration. In addition, as a negative control group (Control group, CON), a separate drug was not administered, and 0.5% CMC-saline 10 mL / kg was administered.

Each mouse was intraperitoneally injected with pentobarbital (sub-hypnotic dose 30 mg / kg, hypnotic dose 45 mg / kg), and the sleep latency and sleep time were measured. After pentobarbital was injected into the abdominal cavity, leaning to the bottom on the back, the elapsed time until the head position reflex was lost, that is, the time until sleep onset after pentobarbital injection It was regarded as a latency, and if there was no movement for more than 1 minute after administration of pentobarbital from mice in the box, it was regarded as sleeping. Sleep time was taken as the time from when the mouse was moved to the box until there was no movement until it recovered autonomous behavior. In the experiment of pentobarbital sub-hypnotic dose, the sleep onset calculation method is as shown in the following equation (2).
Formula (2)
Sleep start (%) = (number of sleep states / total number) × 100

3) Sleep-inducing effect of Kajime ethanol and water extract in rats induced by hypnotic concentrations of pentobarbital Kajime ethanol extract (ECK-E) and water extract (ECK-W) were 100, 250, 500, 1 The effect of pentobarbital (45 mg / kg, ip) on the sleep induction effect after oral administration (po) at a concentration of 1,000,000 mg / kg is shown in FIGS. 2a and 2b below. Each graph represents an average value (means ± SEM, n = 10).

  * Means p <0.05 compared to the control group, ** means that there is a significant difference (Dunnet's test) compared to the control group, p <0.001, and # Means p <0.05 and ## means there is a significant difference between the co-administered group and the single-administered group at p <0.01.

  Kajime ethanol extract (ECK-E) decreased sleep-onset latency in a dose-dependent manner and increased sleep time in a dose-dependent manner at concentrations other than 100 mg / kg, and exhibited a sleep-enhancing effect. In the case of Kajime water extract (ECK-W), the sleep effect was not exhibited at low concentrations, but a significant increase (p <0. 0) in a dose-dependent manner at concentrations of 500 and 1,000 mg / kg. 01) and a significant decrease in sleep latency (p <0.01). In particular, at the concentration of Kajime water extract (ECK-W) of 1,000 mg / kg, it is significantly decreased (p <0. 0) compared with Kajime ethanol extract (ECK-E) and DZP which is a positive control. 01) The sleep latency was confirmed.

4) Sleep-inducing effect of Kajimeethanol and water extract in rats induced by Sub-hypnotic concentrations of pentobarbital Kajimeethanol and water extract were at sub-hypnotic (pentobarbital 30 mg / kg, intraperitoneal injection) concentration The sleep latency and sleep time were measured, and the influence on the sleep induction effect was shown in [Table 4] and [Table 5] below. As a result of the experiment, Kajime ethanol extract was pentobarbital at a sub-hypnotic concentration, and the control group was not able to induce sleep, whereas diazepam (2 mg / kg, oral administration) was 100% onset of sleep. The highest measured, all went to sleep. Kajime ethanol and water extract showed an increase in sleep onset latency and an increase in sleep time depending on the concentration. In particular, at a concentration of 1000 mg / kg, sleep onset was 92% and 90%, respectively. Appeared high. Sleep time also showed a significant increase (p <0.05) at 38.7 ± 6.3 minutes and 39.93 ± 4.7 minutes, respectively.

[Table 4] and [Table 5] are: CON (0.5%, CMC-saline 10 mL / kg), Kajime ethanol extract (ECK-E, 100, 250, 500, 1000 mg / kg, respectively), Kajime A water extract (ECK-W, 100, 250, 500, 1000 mg / kg, respectively) and DZP (2 mg / kg) were orally administered 45 minutes before pentobarbital administration,
Sleep start (%) = (number of sleep states / total number) × 100
The sleep time is an average value (mean ± SEM, n = 12)
* Is significantly different at p <0.05 compared to the control group, and ** is significantly different at p <0.01 compared to the control group (Dunnet's test).
CON: Control group (0.5% CMC-saline 10 mL / kg) after oral administration, pentobarbital administration DZP: Diazepam 2 mg / kg, oral administration, pentobarbital administration ECK-E: Kajime ethanol extract 100, 250 , 500, 1000 mg / kg orally, then pentobarbital administered ECK-W: Kajime water extract 100, 250, 500, 1000 mg / kg orally administered, then pentobarbital administered

  As can be seen from the above [Table 4] and [Table 5], it can be confirmed that Kajime ethanol and the water extract have an increase in sleep latency and an increase in sleep time depending on the concentration.

[Experimental Example 4: GABA _A -benzodiazepine receptor binding activity and sleep-inducing effect of Kajime extract fraction]
1) Preparation of Kajime Extract Fraction In order to confirm active ingredients that bind to GABA _A -benzodiazepine receptors and exhibit activities such as sedation and sleep induction, as shown in FIG. 3, n-hexane, ethyl acetate N-butanol and water fractions were prepared.

2) GABA A _- benzodiazepine receptor binding activity and the experimental results of sleep-inducing effects GABA A _- binding displacement effects of benzodiazepines ligand ^{[3 H]} flumazenil is confirmed by the method in Experiment 1, each Ecklonia extract fraction The binding displacement effect of [ ³ H] flumazenil is shown in FIG. 4 a, and the sleep-inducing effect of the Kajime extract fraction was confirmed by the method of Experimental Example 3 in the rat induced by the hypotonic concentration of pentobarbital. This is shown in 4b.

The ethyl acetate (EtOAc) fraction represents the lowest IC ₅₀ value (0.0185 mg / mL), and the n-butanol, n-hexane and moisture fractions have IC ₅₀ values of 0.1033, 0.1405 and 0. 9607 was represented (FIG. 4a).

In relation to the sleep-inducing effect, the same tendency as the binding replacement activity of [ ³ H] flumazenil was exhibited. The ethyl acetate fraction significantly increased sleep time at both 100 and 200 mg / mL, and the water fraction failed to exhibit meaningful sleep-inducing activity even at 200 mg / mL (FIG. 4b).

From the above results, it was determined that the ethyl acetate fraction of Kajime extract has an affinity for GABA _A -benzodiazepine receptors and has a high content of active ingredients representing anxiolytic, anticonvulsant, sedative, and sleep induction and improvement effects. It was.

[Experimental Example 5: Correlation between the total phenol content of the Kajime extract fraction, GABAA-benzodiazepine receptor binding activity and sleep-inducing effect]
Since the ethyl acetate fraction of Kajime extract contains a large amount of fluorotannin, a polyphenol peculiar to brown algae, the GABA _A -benzodiazepine ligand [ ³ H] flumazenil binding substitution effect and sleep of the ethyl acetate fraction In order to confirm the possibility that the induction effect is due to phlorotannin, the total phenol content of each of the Kajime extract fractions was analyzed, and the total phenol content, GABA _A -benzodiazepine receptor binding activity and sleep induction effect were analyzed. And the correlation was analyzed.

  Table 6 shows the total phenol content of the Kajime extract fraction, and it was confirmed that the total phenol content (TPC) was highest in the ethyl acetate fraction and lowest in the water fraction. The total phenol content of the Kajime ethanol extract (ECK-E) was 138.5 mg PEG / g, and the ethyl acetate fraction and the n-butanol fraction had a higher total phenol content than the Kajime ethanol extract.

The correlation between the total phenol content (TPC) of each Kajime extract fraction and the IC ₅₀ for binding of [ ³ H] flumazenil is represented in FIG. 5a, and the total phenol content (TPC) of each Kajime extract fraction and The correlation with sleep time is shown in FIG.

According to FIGS. 5a and 5b, the binding activity and sleep-inducing effect of each extract fraction were found to be proportional to the total phenol content. The coefficient (R ² ) by non-linear regression analysis of binding activity is 0.9544, and R ² is 0.8396 at 100 mg / kg concentration and R at ²⁰⁰ mg / kg concentration between sleep time and total phenol content. ² was 0.9051, indicating that the correlation was high.

  Moreover, the kind and content (mg / g) of the fluoro tannin compound contained in the said Kajime extract fraction and Kajime ethanol extract were analyzed, and it represented in [Table 7].

  The fluorotannin compounds detected from the respective Kajime extract fractions and Kajime ethanol extract were phloroglucinol, equol, extronol, trifluoroletol A, and diacor. The structure of each fluorotannin compound is shown in FIG.

  The ethyl acetate fraction and n-butanol fraction, which had high binding activity and sleep-inducing effect, had a total fluorotannin compound content of 367.42 mg / g and 177.11 mg / g, n-hexane fraction and water fraction It was significantly higher than

Moreover, the correlation between the content of the sleep time of total phlorotannin compounds each fraction, the 0.9682,200mg / kg concentration ^{R 2} in 100 mg / kg concentrations a ^{R 2} is 0.9434, very I was able to confirm that it was very high.

[Experimental Example 6: GABA _A -benzodiazepine receptor binding activity of fluorotannin compound]
IC ₅₀ values and binding affinities (K _i ) representing the binding displacement effect of GABA _A -benzodiazepine ligand [ ³ H] flumazenil of the fluorotannin compound present in each Kajime extract fraction were calculated, [Table 8] Expressed in The binding affinity (K _i ) was calculated as in the following formula (3).
Formula (3)
K _i = (IC ₅₀ ) / (1 + ([L] / K _d ))
([L]: concentration of radioactive ligand used ([ ³ H] flumazenil), K _d : competator-ligand dissociation equilibrium constant of [ ³ H] flumazenil, K _d value is 1.6 nM. )

  Diazepam (DZP) was used as a positive control to confirm the relative strength of the binding affinity of the fluorotannin compound. Ecole and Extronol have higher binding affinity than Trifloretol A and Daiechol, and in the case of phloroglucinol, which is the basic skeleton of phlorotannin, it shows 42.25% inhibition at 1000 μM It was not high compared to fluorotannin with different affinity.

[Experimental example 7: Correlation between total phenol content of brown algae extract, GABAA-benzodiazepine receptor binding activity and sleep-inducing effect]
Among the brown algae of [Table 3], Sanadagusa, Herayahaz (Dictyopteris prolifera Okamura), and Kajime, which have different GABA _A -benzodiazepine receptor binding activities, were screened, and the same genus as Kajime, which had high GABA _A -benzodiazepine receptor binding activity. The total phenol content of each methanol extract was measured, and the correlation between the total phenol content, GABAA-benzodiazepine receptor binding activity and sleep-inducing effect was confirmed.

  [Table 9] shows the total phenol content of four brown algae methanol extracts, and represents the total phenol content of the Kajime extract fraction. The content was similar, and the content decreased in the order of Herayahazu and Sanadagusa.

  The correlation between the total phenol content (TPC) of the Chromome methanol extract, Kajime methanol extract, Herayahazu methanol extract and Sanadagusa methanol extract and [3H] flumazenil binding activity at 10 mg / mL concentration is shown in FIG. The correlation between the total phenol content (TPC) of each brown algae methanol extract and sleep time is shown in FIG. 7b.

  In FIGS. 5a and 5b, the binding activity and sleep-inducing effect are proportional to the total phenol content depending on the total phenol content of the Kajime extract fraction. In FIGS. 7a and 7b, the total phenol content of each brown algae is also shown. It was confirmed that the binding activity and sleep-inducing effect are proportional to each other.

[Experimental Example 8: Sleep-inducing effect of phloroglucinol or phlorotannin and the effect of flumazenil on sleep induction]
Among phloroglucinol (PG) and phlorotannin, echtronol (ETN), trifloretol A (TPE-A), fucodifluorethol G (FDE-G), The sleep-inducing effect due to the concentrations of equol and 6,6′-biequol and the effect of flumazenil on sleep induction were confirmed.

  In order to confirm the sleep induction effect by the concentration of phloroglucinol or phlorotannin, the method of Experimental Example 3 is used. Oral administration of phloroglucinol or phlorotannin at concentrations of 5, 10, 25, and 50 mg / kg ( p.o.), the sleep induction effect by pentobarbital (45 mg / kg, ip) was confirmed. The negative control group (CON) was 0.5% and CMC-saline was 10 mL / kg. As a positive control group, 2 mg / kg of diazepam (DZP) and 10 mg / kg of zolpidem (ZPD) were orally administered 45 minutes before pentobarbital administration. After administration, sleep latency and sleep time were measured.

  In order to examine the influence of flumazenil on sleep-inducing effects, phloroglucinol or phlorotannin was administered with 50 mg / kg, DZP with 2 mg / kg, and ZPD with 10 mg / kg of pentobarbital 45 mg / kg. kg) was orally administered 45 minutes before, and FLU was injected intraperitoneally at 8 mg / kg 10 minutes before oral administration of the sample, and the sleep latency and sleep time were measured.

  8a and 8b are graphs showing sleep onset latency and sleep time depending on the concentration of phloroglucinol (PG), respectively. FIGS. 8c and 8d show sleep onset latency and sleep with and without administration of phloroglucinol flumazenil. It is a graph which shows time, respectively.

  9a and 9b are graphs showing sleep onset latency and sleep time depending on the concentration of extronol (ETN), respectively. FIGS. 9c and 9d show sleep onset latency and sleep with and without extronol flumazenil administration. It is a graph which shows time, respectively.

  FIGS. 10a and 10b are graphs showing sleep onset latency and sleep time depending on the concentration of trifloretol A (TPE-A), respectively, and FIGS. 10c and 10d show sleep onset with and without administration of trifluretol A flumazenil. It is a graph which shows a latency and sleep time, respectively.

  11a and 11b are graphs showing sleep onset latency and sleep time depending on the concentration of fucodifloretol G (FDE-G), respectively, and FIGS. 11c and 11d show the presence or absence of flumazenil administration of fucodifloretol G. It is a graph which shows the sleep sleep latency and sleep time by respectively.

  12a and 12b are graphs showing sleep onset latency and sleep time according to the concentration of equol, respectively. FIGS. 12c and 12d are graphs showing sleep onset latency and sleep time with and without equol administered by flumazenil, respectively. .

  FIGS. 13a and 13b are graphs showing sleep onset latency and sleep time according to the concentration of 6,6′-biequol, respectively. FIGS. 13c and 13d show sleep onset latency with and without the administration of 6,6′-biequol flumazenil. It is a graph which shows time and sleep time, respectively.

  FIG. 14a and FIG. 14b show the sleep latency and sleep time according to the concentration in order to confirm the effective concentration of diazepam (DZP) and zolpidem (ZPD) used as positive control groups in the confirmation test of the sleep induction effect. Each is a confirmed graph.

  According to the above results, the phloroglucinol and phlorotannin of the present invention exhibited a significant decrease in sleep latency and increase in sleep time at very low concentrations of 5 to 25 mg / kg.

Moreover, the sleep effect of the phloroglucinol and the fluorotannin of the present invention was suppressed by flumazenil, which is a GABA _A -benzodiazepine antagonist, like diazepam.

[Experimental Example 9: GABA _A -benzodiazepine receptor binding activity and sleep-inducing effect of chrome extract]
Similar to the Kajime extract, it was confirmed whether the chrome belonging to the same genus exhibits GABA _A -benzodiazepine receptor binding activity and sleep-inducing effect.

Figure 15a, GABA _A radioactively target positions of chromate methanol extract - a graph showing the binding inhibition effect of benzodiazepine ligand ^[3 H] flumazenil, when chromate methanol extract of _{(EKO-M), GABA A} - The affinity for the benzodiazepine receptor increased in a concentration-dependent manner, and the IC ₅₀ value was 0.626 mg / mL.

  15b and 15c show that the sleep latency and sleep of Kajimemethanol extract (ECK-M) and Chromomeethanol extract (EKO-M) with pentobarbital (45 mg / kg, intraperitoneal injection) at a hypnotic concentration are shown. It can be confirmed that the chrome methanol extract has a significant effect on sleep time.

[Experimental Example 10: Pentobarbital sleep-induced synergy effect for oral administration of brown algae extract and diazepam]
In order to investigate the sleep-induced synergy effect of oral administration of Kazime ethanol and water extract and diazepam, ECK-E is 100 mg / kg, ECK at a low concentration that does not affect the sleep-inducing effect of pentobarbital. -W is 100 mg / kg, DZP is 0.5 mg / kg orally administered 45 minutes before administration of pentobarbital (hypnotic dosage 45 mg / kg), and the results of measuring the sleep latency and sleep time, This is shown in FIGS. 16a and 16b. Each graph represents an average value (means ± SEM, n = 10).

  * Means p <0.05 compared to the control group, *** means that there is a significant difference at p <0.001 compared to the control group (Dunnet's test), # Means p <0.05 and ## means that there is a significant difference between the co-administered group and the single-administered group at p <0.01 (unpaired Student t Test). NS means that there is no significant difference.

  As can be seen from FIGS. 16a and 16b, it can be confirmed that when Kajime ethanol and water extract according to the present invention are administered in combination with DZP, there is a significant effect on sleep time.

[Experimental example 11: Effect of flumazenil on sleep induction effect of brown algae extract]
Flumazenil (FLU), which is a GABA _A -benzodiazepine antagonist, was produced by Kajime ethanol extract (ECK-E), Kajime water extract (ECK-W) produced by the method of Experimental Example 2, and by the method of Experimental Example 4. In order to investigate the effect of the ethyl acetate fraction (EtOAc) on the sleep-inducing effect, ECK-E was 1000 mg / kg, ECK-W was 1000 mg / kg, EtOAc was 200 mg / kg, and DZP was , 0.5 mg / kg is orally administered 45 minutes before pentobarbital administration (hypnotic dose 45 mg / kg), and FLU is 8 mg / kg in advance 10 minutes before oral administration of ECK-E, ECK-W, EtOAc, DZP. An intraperitoneal injection was performed to measure sleep latency and sleep time. The results are shown in FIGS. 17a and 17b below. Each graph represents an average value (means ± SEM, n = 10).

  * Indicates p <0.05, ** indicates p <0.01, and *** indicates p <0.001 compared to the control group (Dunnet's test) Where # is p <0.05, ## is p <0.01, and ## is p <0.001 between the group treated with flumazenil and the group not treated Means there is a significant difference (unpaired Student's t-test). NS means that there is no significant difference.

As can be seen from FIGS. 17a and 17b, diazepam, one of the representative sleeping agents, is a well-known GABA _A -benzodiazepine active agent, and diazepam's sleeping effect is GABA _A -benzodiazepine antagonist. It was suppressed by the agent flumazenil.

In addition, Kajime water extract (ECK-W), ethanol extract (ECK-E) and ethyl acetate fraction (EtOAc) according to the present invention were also produced by flumazenil, a GABA _A -benzodiazepine antagonist, as well as the sleeping agent diazepam. The effect was suppressed.

From the results, it can be confirmed that the brown algae extract has an affinity for the GABA _A -benzodiazepine receptor and plays the role of an efficacy agent, like diazepam, which is an existing sleeping agent. And even in animal experiments using mice, it shows a sleep-inducing effect and, like diazepam, it can be seen that GABA _A -benzodiazepine antagonist reduces the effect.

[Experimental example 12: Effect of brown algae extract on sleep structure of rats]
After SD rats (200-250 g) were adapted for 1 week, electrode insertion surgery was performed for electroencephalogram (EEG) and electromyogram (Electromyogram, EMG) measurements. Rat was intoxicated with pentobarbital (50 mg / kg, ip), and the head was fixed to a stereotaxic instrument. After incising the subcutaneous head connective tissue, stainless steel screws and silver electrode lines were inserted for EEG and EMG measurements. Subsequently, it was fixed with dental cement and sutured. Surgical site disinfection and antibiotics were administered for 3 days to prevent surgery-induced inflammation and a 7-day recovery period. In order to adapt to the measurement environment, 0.5% CMC-saline solution used for the control experiment group was orally administered (po) from 4 days before the measurement, and then connected to the memory device to adapt to the experimental process. Induced.

  EEG and EMG were measured for 6 hours from 10:00 to 16:00 using PAL-8200 series (Pincle Technology Inc, Oregon, USA). The sampling rate of EEG and EMG was set to 200 Hz (epoch time: 10 seconds), EEG was set to a filter area of 0.1-25 Hz, and EMG was set to a filter area of 10-100 Hz, and data was recorded.

  Sleep structure analysis was performed by a Fast Fourier Transform (FFT) algorithm and utilized the SleepSign program (Ver. 3.0, Kissei Comtec, Nagano, Japan).

  The analysis results were divided into wake, REM (REM sleep, theta band: 6-10 Hz), and NREM (non-REM sleep, delta band: 0.65-4 Hz). The sleep onset latency was set as the time taken for NREM sleep in 10-second epoch units to appear continuously 12 times or more.

  As shown in FIG. 18a, the sleep structure was analyzed by oral administration of 500 mg / kg of Kajime ethanol extract according to the present invention. As a result, the sleep latency was significantly reduced by about 9 minutes from 40.7 minutes to 31.8 minutes. It was done.

  Further, with the sleep structure of FIG. 18b, the NREM sleep time was increased by about 12% from 42.5% to 54.6%, and the sleep enhancement was effective.

[Experimental Example 13: Anticonvulsant effect of flourotannin concentrated fraction]
In Experimental Example 1, an enzyme extract using an enzyme was obtained in Kajime (ECK) having the most excellent GABA _A -benzodiazepine receptor binding activity of 90% or more, and a fluorotannin-enriched fraction was obtained from the enzyme extract. The total phenol content and anticonvulsant effect of the enzyme extract (ECEE) and the fluorotannin concentrated fraction (PTRF) were confirmed.

1) Production of Enzyme Extract (ECEE) and Fluorotannin Concentrated Fraction (PTRF) After mixing 1 kg of freeze-dried caulking powder and 10 L of distilled water, 10 mL of cellulase (Celluclast, Novo Nordisk, Bagsvaard, Denmark) is added. , Reacted at 50 ° C. for 24 hours, and inactivated the enzyme at 100 ° C. for 10 minutes. Centrifugation was performed at 3000 × g for 20 minutes to obtain a filtrate obtained by removing a residue that was not hydrolyzed. The filtrate was concentrated and then freeze-dried to obtain an enzyme extract (ECEE).

  The concentrate before lyophilization of the enzyme extract was mixed with 70% ethanol and then stirred for 24 hours. The stirred mixture was centrifuged at 3500 × g for 20 minutes, and then Whatman filter paper No. 1 was used. The residue was removed by 1 to obtain a filtrate, which was concentrated and then lyophilized to obtain a fluorotannin-concentrated fraction (PTRF).

2) Picrotoxin-induced seizure test Experimental animals were prepared as in Experimental Example 3, and rats were given a control group (CON, 0.5%, CMC-saline 10 mL / kg), ECEE and DZP. Orally administered, 45 minutes after oral administration, seizures were induced by intravenous injection of 7 mg / kg picrotoxin. Rats receiving picrotoxin were immediately placed in individual cages and observed for 90 minutes to record the number of convulsions and the seizure latency from onset of picrotoxin until the onset of seizures. If there was no seizure for 90 minutes, the seizure latency was recorded as 0 minutes.

3) Experimental results Table 10 shows the total phenol content of enzyme extract (ECEE) and fluorotannin-enriched fraction (PTRF), and the residue means the ECEE part excluding RTRF. . Fluorotannin-enriched fractions represented a total phenol content that was more than 6 times higher than the enzyme extract.

  The anticonvulsant effect of the enzyme extract (ECEE) is shown in [Table 11], and the anticonvulsant effect of the fluorotannin concentrated fraction (PTRF) is shown in [Table 12].

** has a significant difference at p <0.01 compared to the control group (Dunnet's test).

  In [Table 11], the final mortality of the control group (CON) was 80%, and diazepam (DZP) used as a positive control group had a significant delay in seizure latency. Enzyme extract (ECEE) also showed the effect of lowering the final mortality rate and delaying the seizure latency in a dose-dependent manner, especially the significant increase in seizure latency at 1000 mg / kg capacity. .

  In [Table 12], the fluorotannin-enriched fraction (PTRF), like the enzyme extract (ECEE), has the effect of lowering the final mortality rate and delaying the seizure latency. Even a volume of about / 4 to 1/10 exhibited an anticonvulsant effect at the same level as ECEE.

[Experimental example 14: Statistical analysis]
The 50% inhibitory concentration (IC ₅₀ ) value was calculated by applying the one-site compression binding model using the Prism 5.0 program. Results of all experiments were expressed as mean and standard error (means ± SEM), and statistical comparisons between each group and control were assessed through one-way analysis of variance (ANOVA) with Dunnet's test. For the display of the significance level, the results showing the significance at the p <0.05 (*), p <0.01 (**), and p <0.001 (***) levels are shown in a separate table. Data comparison between the two groups was analyzed by unpaired Student's t-test, p <0.05 (#), p <0.01 (##), p <0.001 (####) levels It was shown that there was a statistically significant difference between the two groups. The statistical analysis program was performed using Prism 5.0 software.

  Hereinafter, formulation examples of the composition containing the phloroglucinol, phlorotannin or brown algae extract of the present invention will be described. However, the present invention is not intended to limit the present invention, but will be specifically described. It is to do.

<Formulation Example 1. Production of powder>
Kajime ethanol extract powder 20 mg obtained in Experimental Example 1
Lactose 100mg
Talc 10mg
The above ingredients are mixed and filled into an airtight cloth to produce a powder.

<Formulation Example 2. Manufacture of tablets>
Ecole of Experimental Example 8 10mg
Corn starch 100mg
Lactose 100mg
Magnesium stearate 2mg
After mixing the above-mentioned components, tablets are produced by tableting according to a conventional tablet production method.

<Formulation Example 3. Manufacture of capsules>
Phloroglucinol 10 mg from Experimental Example 8
Crystalline cellulose 3mg
Lactose 14.8mg
Magnesium stearate 0.2mg
According to a normal capsule manufacturing method, the above-mentioned components are mixed and charged into a gelatin capsule to prepare a capsule.

<Formulation Example 4. Manufacture of injections>
Extronol 10 mg of Experimental Example 8
Mannitol 180mg
Sterile distilled water for injection 2974mg
Na ₂ HPO ₄ , 12H ₂ O 26 mg
It is manufactured at the above-mentioned component content per ampoule by an ordinary injection manufacturing method.

<Formulation Example 5. Manufacture of liquid agent>
Kajime ethanol extract powder 20 mg obtained in Experimental Example 1
Isomerized sugar 10g
Mannitol 5g
Purified water Appropriate amount Each component is added and dissolved in purified water by the usual method for producing liquid medicine, and after adding a proper amount of lemon flavor, the above components are mixed, and then the whole is added with purified water to make a total of 100. After adjustment, fill the brown bottle and sterilize to produce the solution.

<Formulation Example 6. Production of health functional food>
Kajime ethanol extract powder 1,000 mg obtained in Experimental Example 1
Vitamin mixture appropriate amount vitamin A acetate 70μg
Vitamin E 1.0mg
Vitamin B1 0.13mg
Vitamin B2 0.15mg
Vitamin B6 0.5mg
Vitamin B12 0.2μg
Vitamin C 10mg
Biotin 10 μg
Nicotinamide 1.7mg
50 μg of folic acid
Calcium pantothenate 0.5mg
Inorganic mixture Appropriate amount of ferrous sulfate 1.75mg
Zinc oxide 0.82mg
Magnesium carbonate 25.3mg
Monobasic potassium phosphate 15mg
Dicalcium phosphate 55mg
Potassium citrate 90mg
Calcium carbonate 100mg
Magnesium chloride 24.8mg
The composition ratio of the above-mentioned vitamin and mineral mixture is a composition suitable for relatively healthy function foods in the preferred embodiment. However, the composition ratio may be arbitrarily changed to produce a normal health function food. After mixing the above-mentioned components by a method, a granule is manufactured and can be used for manufacturing a health functional food composition by a conventional method.

<Formulation example 7. Production of functional beverages>
Kajime ethanol extract powder 1,000 mg obtained in Experimental Example 1
Citric acid 1,000mg
Oligosaccharide 100g
Plum concentrate 2g
Taurine 1g
900 mL of purified water
After mixing the above ingredients and stirring and heating at 85 ° C. for about 1 hour by a normal health drink manufacturing method, the resulting solution is filtered, obtained in a sterilized 2 L container, and sealed and sterilized After refrigerated storage, it is used for the production of the functional beverage composition of the present invention.

  The composition ratio is a composition that is a composition suitable for preference taste ingredients in a desirable embodiment, but the composition ratio is arbitrarily changed depending on the regional and ethnic preference levels such as demand hierarchy, demand country, and intended use. May be.

Claims (6)

_A composition for inducing and improving sleep having GABA _A -benzodiazepine receptor activity, comprising a brown algae extract as an active ingredient.   The brown algae is any one or more brown algae selected from Herayahaz, Kajime, Kurome, Tsuruarame, Akamok, Habanori, Nevaimo, Siwahige, Fukuronori, Yatsumatamo, Sanadagusa, Ajigususa, Kombu, Wakame and Ishige The composition for inducing and improving sleep according to claim 1.   The composition for inducing and improving sleep according to claim 2, wherein the brown algae is Ecklonia cava or Ecklonia kurome.   The brown algae extract is a brown algae in a mixed solvent of any two or more solvents selected from a brown algae water extract, a brown algae ethanol extract, a brown algae methanol extract, or water, ethanol and methanol. The composition for sleep induction and improvement according to claim 1, wherein the composition is an extract.   The brown algae water extract is produced by extracting brown algae with water at 40 to 100 ° C. for 2 to 48 hours, and the brown algae ethanol extract is obtained at 20 to 60 ° C. with 35 to 75% ethanol. The brown algae methanol extract is produced by extracting for 2 to 36 hours, and the brown algae is produced by extracting brown algae with 35 to 85% methanol at 20 to 60 ° C for 2 to 36 hours. 5. The composition for inducing and improving sleep according to 4.   The brown algae water extract is produced by extracting brown algae at 40 to 55 ° C. for 2 to 24 hours with water to which one or more enzymes selected from the group consisting of cellulase, viscozyme, alcalase and pectin are added. The composition for inducing and improving sleep according to claim 5.