KR101336502B1 - Novel use of rice bran extract or rice bran powder for improving, preventing or treating sleep disorders, anxiety, or depression - Google Patents

Abstract

The present invention provides a novel use of rice bran extract or rice bran powder for improving, preventing or treating sleep disorders, anxiety or depression. Rice bran extract or rice bran powder is almost the same level of diazepam, or a drug used as a conventional sleep inducing agent or has an improved effect of reducing the time and sleep time. Rice bran extract or rice bran powder is a natural product, even after long-term use, there are no side effects that form cognitive impairment, resistance or dependence.

Description

New use of rice bran extract or rice bran powder for improving, preventing or treating sleep disorders, anxiety, or depression

The present invention relates to a novel use of rice bran extract or rice bran powder for improving, preventing or treating sleep disorders, anxiety or depression.

Sleep constitutes one-third of human life and is the most fundamental and essential physiological phenomenon and is a very important factor in health maintenance and mental stability. Chronic sleep deprivation and disability negatively impacts overall physical and mental health, including cardiovascular disease and hypertension, memory and learning, metabolic control and weight, immunity and cancer resistance, diabetes, safety concerns, and mood. Currently, about 30% of the world's population is suffering from insomnia, and 10% is suffering from chronic sleep disorders.

According to a survey by the National Sleep Foundation USA, 2008, about 50% of US adults experience insomnia at least once a week, and about 50% of Americans are satisfied with their sleep . In recent years, the prevalence of insomnia and sleep disorders in Korea is rapidly approaching that of developed countries. The number of patients with sleep disorders has increased from 51,000 in 2001 to 228,000 in 2008, 4.5 times more than in the past eight years (National Health Insurance Corporation, 2009 ).

In Korea, there is not much interest in sleeping, and especially, there is no awareness of medical treatment or illness. On the other hand, according to statistics, about 15% of adults are known to need medication due to insomnia due to anxiety symptoms. Insomnia is caused by various factors such as stress, tension, and fear. Drugs include benzodiazepines Serotonin antagonists, etc. However, there is a serious problem in that cognitive impairment, tolerance, and dependence are formed when these drugs are used for a long period of time. Therefore, the necessity of natural supplements as a means to replace the sleeping pills long-term users and limiters, and the demand for sleep-enhancing health functional food is increasing.

Rice is a valuable food resource, one of the world's three major crops, which half of the world's population uses as a staple food, and is a key national resource that cannot be replaced by other crops, especially in Asia. Rice, which is rice husk, which is the surface of rice, is called brown rice, rice rice with rice husk and rice husk is removed and rice is called white rice. Especially, rice bran is a very good material for nutrition though it is a by-product of Jeongbaekmi.

Rice bran (rice bran) contains about 95% of rice nutrients and contains high quality protein, dietary fiber and various vitamins and minerals. Antioxidants, antioxidants, anti-inflammatory, anti-arteriosclerosis, cholesterol-lowering, growth-promoting, digestive and immune-enhancing effects of rice bran have been found. However, the relationship between rice bran and sleep, anxiety or depression has not been identified.

The present invention aims to discover components derived from natural products that can replace existing drugs that have been used for the treatment of sleep disorders, anxiety or depression.

The present inventors have tried to prepare a natural composition having a high effect of preventing or treating sleep disorders, anxiety or depression. As a result, the present invention was completed by elucidating that rice bran is effective in preventing or treating sleep disorders, anxiety or depression.

The present invention is a new use for improving, preventing or treating sleep disorders, anxiety or depression of rice bran extract or rice bran powder, pharmaceutical composition for preventing or treating sleep disorders, anxiety or depression comprising rice bran extract or rice bran powder as an active ingredient Provided is a method of preventing or treating a sleep disorder, anxiety or depression comprising administering to a subject a composition, a therapeutically effective amount of rice bran extract or rice bran powder.

In the present invention, improvement of sleep disorder, prevention or treatment may mean decrease in the time of the face, increase of the sleep duration, or increase of the sleeping surface.

In the following examples, rice bran extract exhibits almost the same level or improved reduction of sleep time and increased sleep duration compared to diazepam, a medicament that is conventionally used as a sleeping, anti-anxiety or antidepressant agent, and increases nonram sleep time. Shows. Therefore, rice bran extract can be used as an active ingredient of the composition for the prevention or treatment of sleep disorders, anxiety or depression. Unlike conventional sleeping pills, rice bran extract is a harmless ingredient approved as a food, has no side effects, and has an excellent effect of inducing sleep and prolonging sleep, and thus can be useful for preventing or treating sleep disorders, anxiety or depression. have. In the examples of the present invention, an experiment for anxiety or depression was not performed separately. However, for example, ingredients used for improvement, prevention, or treatment of sleep disorders, such as diazepam, , Prophylactic or therapeutic, as is well known to those skilled in the art.

In addition, it was found through the following examples that rice bran extract has an antagonistic action on the histamine receptor. Therefore, the present invention also provides an antihistamine comprising rice bran extract or rice bran powder as an active ingredient.

In the present invention, the rice bran extract may be extracted using water, an organic solvent or a mixture thereof of rice bran as an extraction solvent. The kind of the organic solvent used at this time 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 alcohols, 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, n-propanol, iso-propanol, n-butanol, 1-pentanol, 2-butoxyethanol or ethylene glycol can be used. Organic solvents include polar solvents such as acetic acid, dimethyl-formamide (DMFO) and dimethyl sulfoxide (DMSO), acetonitrile, ethyl acetate, methyl acetate, fluoroalkane, pentane, 2,2,4-trimethylpentane, and decane. , Cyclohexane, cyclopentane, diisobutylene, 1-pentene, 1-chlorobutane, 1-chloropentane, o-xylene, diisopropyl ether, 2-chloropropane, toluene, 1-chloropropane, chlorobenzene And nonpolar solvents such as benzene, diethyl ether, diethyl sulfide, chloroform, dichloromethane, 1,2-dichloroethane, anneal, diethylamine, ether, carbon tetrachloride and THF (Tetrahydrofuran).

As can be seen in the following examples, when the rice bran is extracted with an organic solvent, the rice bran extract is separated into an upper layer and a lower layer, the upper layer is an oil fraction of the rice bran extract, the lower layer corresponds to a general solvent extract. When the lower layer is separated again by centrifugation or filter paper, it is divided into a liquid phase and a solid residue. The residue is a wax fraction of rice bran extract. Therefore, the rice bran extract of the present invention is interpreted as a concept including both the oil fraction of the rice bran extract, the liquid fraction of the rice bran extract, the wax fraction of the rice bran extract. In one embodiment, the rice bran extract may comprise any one or more selected from the group consisting of oil fraction of rice bran extract, liquid fraction of rice bran extract and wax fraction of rice bran extract.

In one embodiment, the rice bran extract may be a lower alcohol extract of rice bran, preferably an ethanol extract of rice bran.

In one embodiment, the rice bran extract may be an hexane extract of rice bran. Hexane extract of rice bran is generally used as 'rice bran oil'. Thus, in one embodiment of the present invention, the rice bran extract may be uniquely rice bran. As used herein, the term " extract " also includes fractions obtained by further fractionating the extract. That is, the rice bran extract is not only obtained by using the above-described extraction solvent, but also includes a product obtained by applying a purification process to it. In addition, fractions obtained by passing the extract or fraction through an ultrafiltration membrane having a constant molecular weight cut-off value, separation by various chromatography (manufactured for separation according to size, charge, hydrophobicity or affinity), etc. The fraction obtained through the various purification methods described above is also included in the rice bran extract of the present invention.

In one embodiment, the rice bran extract may be a fraction obtained by re-fractionation of the organic solvent extract of the rice bran with a second organic solvent. Here, the organic solvent extract of rice bran broadly includes all of the oil fraction of the rice bran extract, the liquid fraction of the rice bran extract, the wax fraction of the rice bran extract, and narrowly means the liquid fraction of the rice bran extract. do. Therefore, in one embodiment, the fraction re-fractionated from the organic extract of the rice bran with the second organic solvent may mean a fraction from the liquid fraction of the rice bran extract with the second organic solvent. In another embodiment, the rice bran extract may be a fraction obtained by re-fractionation of the lower alcohol extract of rice bran with a second organic solvent. In another embodiment, the rice bran extract may be a fraction of the ethanol extract of the rice bran re-fractionated with hexane. As can be seen in the following examples, the fraction of rice bran extract containing a large amount of fat-soluble components of these rice bran shows a very good surface induction and sleep prolonging effect.

The term 'extract' used in referring to rice bran herein includes not only the crude extract obtained by treating the extraction solvent on the rice bran, but also the processed product of the rice bran extract. For example, rice bran extract may be prepared in powder form by additional processes such as distillation under reduced pressure and freeze drying or spray drying.

In addition, the rice bran extract of the present invention is broadly meant to include a rice bran processed product, such as rice bran powder formulated to administer the rice bran itself to the animal. Although the experiment with the rice bran extract in the present invention, it will be expected to those skilled in the art that the desired effect can be achieved in the same form as the rice bran powder.

On the other hand, the term 'comprising as an active ingredient' as used herein means containing an amount sufficient to achieve the efficacy or activity of the rice bran extract or rice bran powder. In one embodiment of the present invention, the rice bran extract or rice bran powder in the composition of the present invention is for example at least 0.001 mg / kg, preferably at least 0.1 mg / kg, more preferably at least 10 mg / kg, more More preferably at least 100 mg / kg, even more preferably at least 250 mg / kg, most preferably at least 0.1 g / kg. Since rice bran extract or rice bran powder is a natural product, there is no adverse effect on the human body, so the upper limit of the amount of rice bran extract or rice bran powder included in the composition of the present invention can be carried out by those skilled in the art.

The pharmaceutical composition of the present invention can be prepared by using pharmaceutically acceptable and physiologically acceptable adjuvants in addition to the above-mentioned active ingredients. Examples of the adjuvants include excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, A lubricant or a flavoring agent can be used.

The pharmaceutical composition may be formulated into a pharmaceutical composition containing at least one pharmaceutically acceptable carrier in addition to the above-described active ingredients for administration.

The pharmaceutical composition may be in the form of granules, powders, tablets, coated tablets, capsules, suppositories, liquids, syrups, juices, suspensions, emulsions, drops or injectable solutions. For example, for formulation into tablets or capsules, the active ingredient may be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Also, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included as a mixture. Suitable binders include but are not limited to natural and synthetic gums such as starch, gelatin, glucose or beta-lactose, corn sweeteners, acacia, trackercance or sodium oleate, sodium stearate, magnesium stearate, sodium Benzoate, sodium acetate, sodium chloride and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum and the like.

Acceptable pharmaceutical carriers for compositions that are formulated into a liquid solution include sterile water and sterile water suitable for the living body such as saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, One or more of these components may be mixed and used. If necessary, other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.

Further, it can be suitably formulated according to each disease or ingredient, using the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA as an appropriate method in the field.

The pharmaceutical composition of the present invention can be administered orally or parenterally. In the case of parenteral administration, the composition can be administered by intravenous injection, subcutaneous injection, muscle injection, intraperitoneal injection, transdermal administration, etc., .

The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate and responsiveness of the patient, Usually, a skilled physician can readily determine and prescribe dosages effective for the desired treatment or prophylaxis. According to a preferred embodiment of the present invention, the daily dosage of the pharmaceutical composition of the present invention is 0.001-10 g / kg.

The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it with a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person skilled in the art to which the present invention belongs Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

The present invention also provides a food composition for preventing or improving sleep disorders, anxiety or depression comprising rice bran extract or rice bran powder as an active ingredient.

The food composition according to the present invention can be formulated in the same manner as the above pharmaceutical composition and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, alcoholic beverages, confectioneries, diet bars, dairy products, meat, chocolates, pizza, ram noodles, other noodles, gums, ice cream, .

The food composition of the present invention may contain, as an active ingredient, a rice bran extract or a rice bran powder, as well as a component that is ordinarily added during the manufacture of a food, for example, a protein, a carbohydrate, a fat, a nutrient, a seasoning agent, . Examples of the above-mentioned carbohydrates are monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, oligosaccharides and the like; And polysaccharides such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavorings such as tau martin and stevia extract (e.g., rebaudioside A and glycyrrhizin) and synthetic flavorings (saccharine, aspartame, etc.) can be used as flavorings. For example, when the food composition of the present invention is prepared with a drink and a beverage, in addition to the rice bran extract or rice bran powder of the present invention, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, and various plant extracts may be further included. Can be.

The present invention provides a health functional food comprising a food composition for preventing or improving sleep disorders, anxiety or depression comprising the rice bran extract or rice bran powder as an active ingredient. Health functional food is a food prepared by adding rice bran extract or rice bran powder to food materials such as beverages, teas, spices, gums, confectionery, or by encapsulating, powdering, and suspension. Means to bring, but unlike the general medicine has the advantage that there is no side effect that can occur when taking a long-term use of the drug as a raw material. The health functional food of the present invention thus obtained is very useful because it can be consumed on a daily basis. The amount of the rice bran extract or the rice bran powder added in such a health functional food can not be uniformly determined depending on the kind of the health functional food to be added, but may be added within a range that does not deteriorate the original taste of the food, Is usually in the range of 0.01 to 50% by weight, preferably 0.1 to 20% by weight. In the case of health functional foods in the form of pills, granules, tablets or capsules, they may be added usually in the range of 0.1 to 100% by weight, preferably 0.5 to 80% by weight. In one embodiment, the health functional food of the present invention may be in the form of a pill, tablet, capsule or beverage.

The invention also provides the use of rice bran extract or rice bran powder for the manufacture of a medicament or food for the prevention, treatment or amelioration of sleep disorders, anxiety or depression. As described above, the rice bran extract or the rice bran powder can be used for prevention, treatment, or improvement of sleep disorder, anxiety or depression.

The invention also provides a method of preventing, treating or ameliorating sleep disorders, anxiety or depression comprising administering to a mammal an effective amount of rice bran extract or rice bran powder.

The term "mammal " as used herein refers to a mammal that is the subject of treatment, observation or experimentation, preferably a human.

As used herein, the term "effective amount" refers to the amount of active ingredient or pharmaceutical composition that elicits a biological or medical response in a tissue system, animal, or human, as contemplated by a researcher, veterinarian, physician or other clinician, ≪ / RTI > inducing a reduction of the symptoms of the disease or disorder. It will be apparent to those skilled in the art that the effective amount and the administration frequency of the active ingredient of the present invention will vary depending on the desired effect. Thus, the optimal dosage to be administered can be readily determined by those skilled in the art and will vary with the nature of the disease, the severity of the disease, the amount of active and other ingredients contained in the composition, the type of formulation, and the age, The age, body weight, sex, diet, time of administration, route of administration and fraction of the composition, duration of treatment, concurrent medication, and the like. In the prophylaxis, treatment or improvement method of the present invention, in the case of an adult, when the rice bran extract or the rice bran powder is administered once to several times a day, it is preferable to administer at a dose of 0.001 mg / kg to 10 g / kg.

The composition comprising rice bran extract or rice bran powder as an active ingredient in the treatment method of the present invention is conventionally oral, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, topical, intraocular or intradermal routes. It may be administered in a phosphorous manner.

Advantages and features of the present invention and methods of achieving them will become apparent with reference to the embodiments described in detail below. However, the present invention is not limited to the embodiments disclosed below, but will be implemented in various forms, and only the embodiments are intended to complete the disclosure of the present invention, and the general knowledge in the technical field to which the present invention pertains. It is provided to fully convey the scope of the invention to those skilled in the art, and the present invention is defined only by the scope of the claims.

Rice bran extract or rice bran powder is almost the same level as diazepam, a medicine used as a conventional sleep inducing agent, provides improved sleep time, increased sleep duration, increased nonram sleep, and is derived from natural rice bran. However, there is no side effect of forming cognitive impairment, tolerance or dependency.

1 is a graph showing the effect of BWE on the elevation time of mice administered the pentobarbital sleeping dose (45 mg / kg, ip), Figure 2 is a pentobarbital sleeping dose (45 mg / kg, ip) It is a graph showing the effect of BWE on the sleep time of the mice administered. After 45 minutes of oral administration (po) with DZP (2 mg / kg) and BWE (50, 100, 250, and 500 mg / kg), pentobarbital was administered in the control (0.5% CMC-saline 10 mL / kg) Each graph represents the mean value (means ± SEM, n = 10). * Indicates p <0.05 compared to the control, ** indicates that there is a significant difference (Dunnet's test) at p <0.01 compared to the control. CON is the control; DZP stands for diazepam.
Figure 3 is a graph showing the effect of BEE on the elevation of the mice administered the pentobarbital sleeping dose (45 mg / kg, ip), Figure 4 is a pentobarbital sleeping dose (45 mg / kg, ip) It is a graph showing the effect of BEE on the sleep time of the mice administered. After 45 minutes of oral administration (po) with DZP (2 mg / kg) and BEE (50, 100, 250, 500 mg / kg) in the control group (0.5% CMC-saline 10 mL / kg) Each graph represents the mean value (means ± SEM, n = 10). * Indicates p <0.05 compared to the control, ** indicates that there is a significant difference (Dunnet's test) at p <0.01 compared to the control. CON is the control; DZP represents diazepam.
5 is a graph showing the effect of BEE-Wax on the elevation time of the mice administered the pentobarbital sleeping dose (45 mg / kg, ip), Figure 6 is a pentobarbital sleeping dose (45 mg / kg, ip ) Is a graph showing the effect of BEE-Wax on the sleep time of mice administered. After 45 minutes of oral administration (po), DZP (2 mg / kg) and BEE-Wax (50, 100, 250, 500 mg / kg) were administered to the control (0.5% CMC-saline 10 mL / kg) . Each graph represents the mean value (means ± SEM, n = 10). * Indicates p <0.05 compared to the control, ** indicates that there is a significant difference (Dunnet's test) at p <0.01 compared to the control. CON is the control; DZP represents diazepam.
Figure 7 is a graph showing the effect of BEE-Oil on the elevation time of mice administered pentobarbital sleep dose (45 mg / kg, ip), Figure 8 is a pentobarbital sleep dose (45 mg / kg, ip ) Is a graph showing the effect of BEE-Oil on the sleep time of mice administered. After 45 minutes of oral administration (po), control (0.5% CMC-saline 10 mL / kg), DZP (2 mg / kg) and BEE-Oil (50, 100, 250, 500 mg / kg) Each graph represents the mean value (± SEM, n = 10). * Indicates p <0.05 compared to the control, ** indicates that there is a significant difference (Dunnet's test) at p <0.01 compared to the control. CON is the control; DZP represents diazepam.
9 is a graph showing the effect of BEE (500 mg / kg) on elevation time and total sleep time in SD rats. Each graph represents the mean value (± SEM, n = 8). * Indicates p <0.05 compared to the control, ** indicates that there is a significant difference (Dunnet's test) at p <0.01 compared to the control. CON represents a control group.
10 shows the effect of BEE (500 mg / kg) on sleep structure in SD rats. CON represents a control group.
FIG. 11 is a graph showing changes in wakefulness, nonremem sleep, and REM sleep over time. CON represents a control group.
12 is a diagram showing the fractionation process of BEE (EtOH extract). EtOAc is ethyl acetate; n-BuOH is n-butanol; H2O represents distilled water.
Figure 13 is a graph showing the effect of the BEE fraction on the elevation of the mice administered the pentobarbital sleeping dose (45 mg / kg, ip), Figure 14 is a pentobarbital sleeping dose (45 mg / kg, ip) It is a graph showing the effect of the BEE fraction on the sleep time of the mice administered. (Control) (0.5% CMC-saline 10 mL / kg), DZP (2 mg / kg) BEE-H, BEE-B, BEE-W and BEE- Forty-five minutes later, pentobarbital was administered. Each graph represents the mean value (± SEM, n = 10). * Indicates p <0.05 compared to the control, ** indicates that there is a significant difference (Dunnet's test) at p <0.01 compared to the control. CON is the control; DZP is diazepam; H is hexane extract; B is butanol extract; W is water extract; E represents ethyl acetate extract.
FIG. 15 is a diagram illustrating a process of separating a subfraction of n-hexane extract from BEE. EtOAc is ethyl acetate; H2O represents distilled water.
FIG. 16 is a graph showing the effect of subfractions obtained from BEE-H on the elevation time of mice administered pentobarbital sleep dose (45 mg / kg, ip), and FIG. 17 is a pentobarbital sleep dose (45 mg). / kg, ip) is a graph showing the effect of the subfraction obtained from BEE-H on the sleep time of mice administered. Pentavaval was administered 45 minutes after oral administration (po) of control (0.5% CMC-saline 10 mL / kg), DZP (2 mg / kg) and BEE-H subfraction (50 mg / kg). Each graph represents the mean value (± SEM, n = 10). * Means p <0.05 compared with the control, ** means that there is a significant difference (Dunnet's test) at p <0.01 compared with the control group. C is the control; D represents diazepam.
FIG. 18 is a graph showing the effect of subfractions obtained from BEE-H on the elevation time of mice administered pentobarbital sleep dose (45 mg / kg, ip), and FIG. 19 is a pentobarbital sleep dose (45 mg). / kg, ip) is a graph showing the effect of the subfraction obtained from BEE-H on the sleep time of mice administered. Pentavaval was administered 45 minutes after oral administration (po) of control (0.5% CMC-saline 10 mL / kg), DZP (2 mg / kg) and BEE-H subfraction (250 mg / kg). Each graph represents the mean value (± SEM, n = 10). * Indicates p <0.05 compared to the control, ** indicates that there is a significant difference (Dunnet's test) at p <0.01 compared to the control. C is the control; D represents diazepam.
20 is a diagram illustrating a process of separating a small fraction of n-hexane extract from BEE. MeOH is methanol; EtOAc represents ethyl acetate.
21 is a diagram illustrating a process of separating a small fraction of n-hexane extract. EtOAc is ethyl acetate; CHCl3 is chloroform; MeOH is methanol; H2O represents distilled water.
22 is a diagram illustrating a process of separating a small fraction of n-hexane extract. EtOAc is ethyl acetate; CHCl3 is chloroform; MeOH represents methanol.
23 is a graph showing the effect of the BEE-H-2 fraction on the activity of G-Protein Coupled receptor (GPCR).

[ Example ]

Experimental Method

Experimental animal

ICR mice (18-22 g, male) and SD rats (200-250 g, male) were distributed from Koatech and Orient Bio Co., respectively, and were adapted for one week in an experimental animal breeding box. Respectively. Animals were fed at a temperature of 23 ± 1 ℃, a humidity of 55 ± 5%, a light - dark cycle (9:00 am - 9:00 pm) and an illuminance of 3000 Lux. All animals were maintained in accordance with the Guidelines for the Use of Laboratory Animals by the Korea Food Research Institute (KFRI-IACUC, Korea Food Research Institutional Animal Care and Use Committee).

Pentobaby  Sleep induction experiment Pentobarbital - induced sleep test )

The pentobarbital sleep induction experiment was conducted within a certain time from 1 pm to 5 pm, and 10 mice (n = 10) per group were used after fasting for 24 hours before the experiment. All samples were prepared using 0.5% CMC-saline solution and administered orally (po) to mice 45 minutes before pentobarbital administration. In the general control group, 0.5% CMC-saline solution was treated at a concentration of 10 mg / kg, and diazepam was used as a positive control to compare the effect of the extract on sleep. Pentovavital (Hanlim Pharmaceutical Co., Ltd.) was administered via intraperitoneal injection (ip) at a concentration of 45 mg / kg (hypnotic dosage) according to the experimental design. After the pentobarbital treatment, each individual was moved to a separate space to measure sleep latency and sleep duration. The time of the elevation was set as the elapsed time until the righting reflex was lost for more than 1 minute after the peritoneal injection of pentobarbital, and the sleeping time was set as the time until the regular reflection was restored again. Mice that did not show sleep behavior after 10 minutes of pentobarbital administration were excluded from the experiment.

Sleep structure analysis

Electroencephalogram (EEG) and Electromyogram (EMG) measurements were performed after an adaptation of SD rats (Sprague-Dawley Rat, 200-250 g) for 1 week. The rats were anesthetized with pentobarbital (50 mg / kg, ip) and the head was fixed in a stereotaxic instrument. After cutting the subcutaneous connective tissue, a stainless-steel screw and a silver electrode line were inserted to measure EEG and EMG. After that, it was fixed with dental cement and sutured. Surgical site disinfection and antibiotic treatment were performed for 3 days to prevent the inflammation caused by the operation, and the recovery period was 7 days. In order to adapt to the measurement environment, the recording medium was connected with the 0.5% CMC-saline solution (po) used for the control group for 4 days prior to the measurement and was induced to conform to the experimental procedure. Electroencephalography (EEG) and electromyogram (EMG) were performed for 6 hours from 10:00 to 16:00 using PAL-8200 series (Pinnacle Technology Inc, Oregon, USA) after stabilization for 5 minutes after oral administration. ] Were measured. The sampling rate of the EEG and EMG was set to 200 Hz (epoch time: 10 s), the EEG was set to 0.1 to 25 Hz, and the EMG was set to a filter area of 10 to 100 Hz. Sleep structure analysis was performed by FFT (fast Fourier transform) algorithm and SleepSign program (Ver. 3.0, Kissei Comtec, Nagono, Japan) was used. The results of the analysis were divided into wake, REM sleep, rapid eye movement, theta band (6-10 Hz), NREM sleep, non-rapid eye movement, and delta band (0.65-4 Hz) . The elevation time was set as the time taken for a continuous 10 seconds epoch unit to appear more than 12 times.

< Manufacturing example  1> Preparation of Rice Bran Extract

Water and ethanol extracts were prepared from selected raw materials to explore the effects of sleep enhancement. Rice bran extract (BWE) was prepared by adding 1 L of distilled water to 100 g of the ground sample and heating to 100 ° C. for 1 hour. Rice ethanol extract (BEE) was added 10 times (w / v) of 70% ethanol to the ground sample. Prepared by extraction for 1 day in a 50 ℃ incubator. All the extracts were filtered and concentrated under reduced pressure, lyophilized and powdered for use in the experiment. BEE had a lot of oils in the extraction process, and in addition to BEE, two separate fractions could be identified. These were named BEE-Wax and BEE-Oil.

Specifically, the rice bran ethanol extract (BEE) was divided into three types during the manufacturing process. The first rice bran extract is divided into the supernatant and the lower layer. The supernatant is the oil fraction (BEE-Oil) of the rice bran extract, and the lower layer corresponds to the normal ethanol extract. When the lower layer is separated again through centrifugation or filter paper, it is divided into liquid phase (liquid fraction of rice bran extract) and solid residue, which corresponds to the wax fraction (BEE-Wax) of rice bran extract.

< Experimental Example  1> Using Rice Bran Extract Pentobaby  Sleep induction experiment

Water extract (BWE) and ethanol extract (BEE) of rice bran (BE) were orally administered (po) at concentrations of 50, 100, 250 and 500 mg / kg, followed by pentobarbital (45 mg / kg, ip). Sleep induction effects were analyzed. As a result, all the elevation time in BWE was significantly reduced (p <0.01) (Fig. 1), it was confirmed that the elevation time was reduced than the positive control DZP. In addition, dose-dependent increase in sleep time was observed at concentrations of 250 mg / kg and 500 mg / kg except for 50 mg / kg and 100 mg / kg in BWE (FIG. 2). In BEE, as in BWE, there was a decreased elevation time than DZP (FIG. 3) and a dose-dependent increase in sleep time at concentrations of 250 mg / kg and 500 mg / kg (FIG. 4). Sleep inducer BE showed significantly reduced elevation times and significantly increased sleep times at 250 mg / kg and 500 mg / kg without difference in water and ethanol extracts.

BEE-Wax was administered at a concentration of 50, 100, 250 and 500 mg / kg, BEE-Oil at a concentration of 500 and 1,000 mg / kg, and oral administration of BEE- After the administration (po), pentobarbital (45 mg / kg, ip) was intraperitoneally injected to measure the changes of the time of surface and sleeping time. Experimental results show that BEE-Wax has remarkable results. A dose-dependent decrease in elevation time (p <0.01) (FIG. 5) and an increase in sleep time ( p <0.01) (FIG. 6) were shown, with a decreased elevation time and increased sleep time than BEE. In addition, the BEE-Oil concentration of 1,000 mg / kg significantly showed a significant decrease in elevation time ( p <0.01) (Fig. 7) and an increase in sleep time ( p <0.01) (Fig. 8). Although BEE and BEE-Wax did not induce much sleep inducing effect, BEE-Oil was able to confirm sleep enhancement effect, and BEE-Wax is expected to have a very large ripple effect in the future.

< Experimental Example  2> Sleep structure analysis using rice bran extract

Elevation time and total sleep time after oral administration of BEE 500 mg / kg are shown in FIG. 9. The mean elevation time of the control group was 31.9 minutes and the BEE elevation time was 26.2 minutes, which was shortened by about 5.7 minutes. In the total sleep time, the control group was 165.4 minutes, and when BEE 500 mg / kg was orally administered, it increased by about 83 minutes to 248.6 minutes.

The arousal, rem and non-remme time of the SD rats were analyzed and shown in FIG. 10. In the BEE-treated group, the increase in the number of nonremes decreased relative awakening, the control group had a normalization of 153.8 minutes, BEE 500 mg / kg of nonem increased 234.8 minutes by 81 minutes, and most of the increase in total sleep time .

After measuring oral administration of 500 mg / kg of BEE, the ratios of arousal, nonremem and rem each hour are shown in FIG. After 3 hours of oral administration, the nonremem increased significantly, then decreased slightly and then increased again. This led to sleeping in the first 3 hours of digestion of BEE, and overall the rate of nonem at all times was greater than in the control group, indicating that the sleep effect persisted for 6 hours. BEE did not significantly reduce sleep induction time, but it was judged to increase sleep persistence with longer total sleep time.

< Manufacturing example  2> Preparation of Solvent Fraction of Rice Bran Ethanol Extract

BEE (liquid fraction of rice bran ethanol extract of Preparation Example 1) was separated and purified using four solvents of hexane, butanol, water and ethyl acetate in order to obtain a more accurate single sleep inducing substance using BEE having a large amount of oils. 12)

200 kg of rice bran ethanol extract (BEE) was extracted at 60 ° C in a 60% aqueous EtOH solution for 43 hours. The extract was filtered and the residue was extracted three more times in the same manner. The resulting filtrate was combined and concentrated under reduced pressure to obtain 20 L of an aqueous EtOH extract. To the obtained EtOH extract, 7 L of H ₂ O was added and the mixture was extracted with n-hexane (27 L × 2) / H ₂ O (27 L) The H ₂ O fraction was extracted again with EtOAc (27 L × 2), and the H ₂ O fraction was again extracted with n-BuOH (25 L × 2). Each layer was concentrated under reduced pressure, n-hexane fraction (BEE-H, 2176 g), EtOAc fraction (BEE-E, 1458 g), n-BuOH fraction (BEE-B, 1223 g) and H ₂ O fraction (BEE- W, 6.74 kg) (FIG. 12)

< Experimental Example  3> Solvent Fraction of Rice Bran Ethanol Extract Pentobaby  Sleep induction experiment

Oral doses of 50 mg / kg and 250 mg / kg to confirm sleep-inducing effects of BEE-H (hexane), BEE-B (butanol), BEE-W (water) and BEE-E (ethyl acetate) After administration (po), pentobarbital (45 mg / kg, ip) was injected intraperitoneally to determine the changes in elevation time and sleep time (FIGS. 13 and 14).

As a result of the experiment, BEE-E (ethyl acetate) showed a significant decrease in the time of the face, but no increase in the sleeping time was found, and it was confirmed that there was no improvement in the sleep induction. BEE-H (hexane) and BEE-B (butanol) showed a significant decrease in the surface time ( p <0.01) and an increase in the sleeping time ( p <0.01) ). ( P <0.01) and concentration-dependent sleeping time ( p <0.01) in BEE-H (hexane) and BEE-B (butanol) BEE-H (hexane) was found to be effective in promoting sleep induction. Finally, BEE-H (hexane) was used to proceed with more detailed separation and purification.

< Manufacturing example  3> Rice bran ethanol extract Hexane  Preparation of sub-fraction of fraction

In the solvent fraction of rice bran extract ethanol (BEE) In vivo experiments, a sub-fraction of the n-hexane fraction was prepared because the activity of the n-hexane fraction was significantly better than the other fractions. 365 g of n-hexane fraction (BEE-H, 2176 g) was subjected to SiO ₂ column chromatography (hereinafter referred to as cc), in which the column was packed with a silica gel resin having a diameter of 13 cm and a height of 15 cm. The eluting solvent was gradually increased in polarity from the non-polar solvent of n-hexane: EtOAc = 10: 1 and used in the ratio of n-hexane: EtOAc = 7: 1 2: 1. As a result, a total of 12 subfractions (BEE-H-1 BEE-H-12) (see FIG. 15).

< Experimental Example  4> BEE For the sub-fraction of -H fraction Pentobaby  Sleep induction experiment

To confirm the effect of BEE-H-1 to BEE-H-11 sub-fractions of the BEE-H fraction on the induction of sleep induction, oral administration (po) was carried out at a concentration of 50 mg / kg and 250 mg / kg, (45 mg / kg, ip) was injected intraperitoneally.

As shown in FIG. 16 and FIG. 17, at a concentration of 50 mg / kg, only 2 fractions of BEE-hexane extract showed a significant increase in sleep time ( p <0.01), and the elevation time was BEE-hexane. Only 2, 6, 10 and 11 fractions of extract showed a significant decrease ( p <0.05). At concentrations of 250 mg / kg, as seen in FIGS. 18 and 19, significant increase in sleep time ( p <0.01) was observed in BEE-hexane extracts 1, 2, 4, 7, 10 and 11 fractions. And BEE-hexane extract. ( P <0.05, p <0.01) in all fractions except 9 and 10 fractions. In the 11 fractions, it was found that the extracts of BEE-hexane extracts 2, 10 and 11 had a prolonged sleep inducing effect.

< Manufacturing example  4> BEE From the sub-fraction of -H Small fraction  Produce

(One) BEE -H-2 fraction

A total of 12 fractions (BEE-H-1 to BEE-H-12) were obtained by fractionation of BEE-H. It showed a high activity resulting in a result BEE-H-2, BEE- H-10 and BEE-H-11 fractions were tested for in vivo activity against these, 2 Preparation of tea small fraction of that of BEE-H-2 Proceeded. ODS cc (φ 4 × 7 cm, acetone: H ₂ O = 1: 1) was performed on BEE-H-2 (8.2 g), and the same fractions were collected again and concentrated to 5 fractions (BEE-H-2- 1 ~ BEE-H-2-5), and confirmed the material by SiO ₂ and ODS TLC (Fig. 20).

(2) BEE -H-10 fraction

A total of 12 fractions (BEE-H-1 to BEE-H-12) were obtained by fractionation of BEE-H. As a result of the in vivo activity test for these, BEE-H-2, BEE-H-10 and BEE-H-11 fractions showed high activity results, among which the preparation of the second small fraction of BEE-H-10 Proceeded. SiO ₂ cc (φ 6 × 17 cm, n-hexane: EtOAc = 10: 1 6: 1 4: 1 2: 1 1: 1 CHCl ₃ : MeOH = 8: 1 for BEE-H-10 (9.5 g) 1: 1) was carried out and divided into 23 fractions (BEE-H-10-1 ~ BEE-H-10-23), the material was confirmed by SiO ₂ and ODS TLC (see Fig. 21).

(3) BEE -H-11 fraction

A total of 12 fractions (BEE-H-1 to BEE-H-12) were obtained by fractionation of BEE-H. The activity of BEE-H-2, BEE-H-10 and BEE-H-11 fraction was higher than that of BEE-H-11. Respectively. SiO ₂ cc (φ 5 × 10 cm, n-hexane: EtOAc = 10: 1 5: 1 3: 1 1: 1 CHCl ₃ -MeOH = 8: 1 5: 1 for BEE-H-11 (810 mg) 1: 1) was carried out and divided into 13 fractions (BEE-H-11-1 ~ BEE-H-11-13), the material was confirmed by SiO2 and ODS TLC (see Fig. 22).

< Experimental Example  5> BEE Of the -H-2 fraction GPCR (G- Protein Coupled receptor ) Active inhibition

Whether the BEE-H-2 fraction inhibited the activity of GPCR was commissioned by Millipore Corporation (USA) and analyzed according to the GPCRProfiler ™ method. Experimental results showed that at least 50% inhibitory effect on seven GPCR receptors (Serotonin 1A receptor, Adenosine 1 receptor, Histamine 1 receptor, Histamine 2 receptor, Acetylcholine 2 receptor, Vasopressin 1A receptor and Neuropeptide Y2 receptor). ).

< Experimental Example  6> Sleep Mechanism of Rice Bran Ethanol Extract: Histamine Receptor Efficacy agent , 2-p Ridylethylene Min Dihydrochloride  Effects of Rice Bran Ethanol Extracts on Sleep Induction Effects

In the United States, antihistamine and natural herb products are the most representative of nonprescription sleep aids. In particular, antihistamines are antagonists of histamine receptors and are representative of cold medicines, and are the only hypnotics that can be easily purchased without a doctor's prescription. This is possible because the pharmacological side effects and dependence is low.

The effect of the histamine receptor antagonist on the sleep-inducing effect is inhibited by histamine receptor agonists. In this experiment, the mechanism of action of rice bran ethanol extract was demonstrated using pyrilamine maleate salt (PMS) and 2-pyridylethylamine dihydrochloride (PD) acting on histamine receptor.

To investigate the effect of agonist PD on the sleep induction effect of rice bran ethanol extract, 500 mg / kg of rice ethanol extract (BEE) and 70 mg / kg of antagonist PMS were administered pentobarbital (hypnotic dosage 45 mg / kg). 45 minutes before oral administration, agonist PD was injected 20 mg / kg BEE, PMS orally 10 minutes prior to oral administration to measure sleep latency and sleep duration.

Experimental results, as can be seen in Figure 24, the sleeping effect of PMS, an antagonist of histamine receptors, as is well known, was completely inhibited by the agonist PD. In addition, the effect of rice bran ethanol extract (BEE) was inhibited by PD, a histamine receptor agonist, similar to PMS.

From the above results, rice bran ethanol extract (BEE) has a antagonistic action on histamine receptors, like PMS, an antagonist of histamine, and the effect was reduced by agonists. The development of natural antihistamines having a sleep inducing effect in rice bran is very important in terms of science and technology and the industrial economy.

Claims (29)

Pharmaceutical composition for the prevention or treatment of sleep disorders, anxiety or depression comprising rice bran extract or rice bran powder as an active ingredient.
The method of claim 1,
Wherein said prevention or treatment of a sleep disorder is for a reduction in sleep time, an increase in sleep duration, or an increase in non-sleep sleep.
The method of claim 1,
The rice bran extract is a pharmaceutical composition which is an extract of water, organic solvent or a mixture thereof of rice bran.
The method of claim 3,
The organic solvent is at least one solvent selected from the group consisting of lower alcohols having 1 to 6 carbon atoms, hexane, acetone, ethyl acetate, chloroform, and diethyl ether.
The method of claim 1,
The rice bran extract is a pharmaceutical composition comprising at least one selected from the group consisting of oil fraction of rice bran extract, liquid fraction of rice bran extract and wax fraction of rice bran extract.
The method of claim 1,
The rice bran extract is a pharmaceutical composition of the lower alcohol extract having 1 to 6 carbon atoms of rice bran.
The method of claim 1,
The rice bran extract is a pharmaceutical composition of the ethanol extract of rice bran.
The method of claim 1,
The rice bran extract is a pharmaceutical composition of hexane extract of rice bran.
The method of claim 1,
The extract is rice bran oil pharmaceutical composition.
The method of claim 1,
The rice bran extract is a pharmaceutical composition which is a fraction obtained by re-fractionation of the organic solvent extract of rice bran with a second organic solvent.
The method of claim 1,
The rice bran extract is a pharmaceutical composition of the fraction re-fractionated from the lower alcohol extract of 1 to 6 carbon atoms of rice bran with a second organic solvent.
The method of claim 1,
The rice bran extract is a pharmaceutical composition which is a fraction of the ethanol extract of rice bran re-fractionated with hexane.

Food composition for the prevention or improvement of sleep disorders, anxiety or depression comprising rice bran extract as an active ingredient.
The method of claim 13,
The prevention or improvement of the sleep disorder is for reducing the time of sleep, increase in sleep duration or increase in non-sleep sleep.
The method of claim 13,
The rice bran extract is a water composition of rice bran, an organic solvent or an extract of a mixture thereof.
16. The method of claim 15,
The organic solvent is one or more solvents selected from the group consisting of lower alcohols having 1 to 6 carbon atoms, hexane, acetone, ethyl acetate, chloroform, and diethyl ether.
The method of claim 13,
The rice bran extract is a food composition comprising one or more selected from the group consisting of oil fraction of rice bran extract, liquid fraction of rice bran extract and wax fraction of rice bran extract.
The method of claim 13,
The rice bran extract is a food composition of a lower alcohol extract having 1 to 6 carbon atoms of rice bran.
The method of claim 13,
The rice bran extract is a food composition of ethanol extract of rice bran.
The method of claim 13,
The rice bran extract is a food composition of hexane extract of rice bran.
The method of claim 13,
The extract is a food composition of rice bran oil.
The method of claim 13,
The rice bran extract is a food composition of the fraction re-fractionated from the organic solvent extract of the rice bran with a second organic solvent.
The method of claim 13,
The rice bran extract is a food composition of the fraction re-fractionated from the lower alcohol extract having 1 to 6 carbon atoms of rice bran with a second organic solvent.
The method of claim 13,
The rice bran extract is a food composition that is a fraction of the ethanol extract of rice bran re-fractionated with hexane.


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