KR101401612B1 - Composition comprising extract of punica granatum for prevention and treatment of stress diseases - Google Patents

Abstract

The present invention relates to a composition for treating or preventing a stress disorder comprising an extract of pomegranate as an active ingredient, wherein the pomegranate extract of the present invention inhibits the secretion of cortisol to prevent damage to nerve cells by cortisol and inhibits serotonin receptor In addition to inhibiting reabsorption, animal models can improve resistance to stressful diseases such as depression induced by sleep deprivation and the like.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for treating and preventing a stress disorder comprising an extract of pomegranate as an active ingredient,

The present invention relates to a composition for the treatment, prevention or amelioration of stress-related diseases.

Depressive disorder (depressive disorder) is a major depressive disorder that causes a variety of cognitive, mental or physical symptoms, leading to depression of daily function. It is also called depression.

In relation to the term depression, the term melancholia was used in ancient Greece, and the term depression was used from the end of the nineteenth century. In the current psychiatric department, melancholia is classified as a type of depression.

The depression is not a mere feeling of sadness but a long-term depressed mood and various disorders accompanied by the depression. The depression is caused by a decrease in general functions such as thought process, motivation, motivation, This is a difficult situation.

The depressive disorder is known to have a lifetime prevalence of about 15%, in particular about 25% in women, and the World Health Organization (WHO) has predicted that depression will be the world's second disease that afflicts humankind in 2020 . In Korea, about 3.2 million people, or 8% of the total population, have been reported to suffer from depression.

 The depressive disorder or depression is different from a temporary depression and is not an expression of personal weakness or can not be ruled out. Therefore, professional treatment or approach is required for the treatment of depression, but many of them suffer from depression because they do not receive the help of specialists. In particular, the risk of depression is highly correlated with suicide rates.

Specifically, the annual average annual suicide rate in Korea is 5.19% since 1982, and Korea has the highest rate of suicide among OECD countries, and 80% of the suicides are attributed to depression.

The direct cause of depression has not been clearly elucidated, but it is known that biochemical, genetic and environmental factors, such as other mental illnesses, can cause depression.

Regarding these biochemical factors, the imbalance of neurotransmitters or hormones is expected to be closely related to the development of depression. In this regard, it has recently been reported that changes in the brain of patients with depression have been reported through studies using brain imaging devices. Regarding these genetic factors, some studies report that depression is more likely to occur in families with depression, and studies are under way to find genes that produce depression based on these findings. With regard to the above environmental factors, this may be the loss of close acquaintances, economic problems or stress.

Recent studies have shown that excessive mental stress is a major cause of depression. The cause of the mental stress is social factors related to the complicated modern society such as study, work, marriage, child care, and environmental factors such as weather and traffic. Due to various causes closely related to lifetime life, The incidence of depression in modern people is rapidly increasing.

Depression patients suffering from the depression suffer from various stressful diseases or symptoms.

First, almost all depressed patients complain of energy loss to their lives. They complain that they have difficulty completing the task of daily living, have difficulties in school work and normal work at school, I am not motivated to run.

In addition, about 80% of the depressed patients complain of sleeping disorder, which means that the sleeping disorder is not fully sleeping until morning and frequently breaks early during sleep or sleep during the night. Also, the anxiety symptom of the depressed patient is shown, and about 90% of the depressed patient is suffering from an anxiety disorder. In addition, cognitive decline, such as sexual problems such as loss of libido or decreased concentration, can be observed in a significant number of depressed patients. In addition, some depressed patients complain of somatic symptoms. In such cases, it is often the case that the cause of the symptoms is not clear even by medical examination. In this case, when only the medical examination is conducted, depression is suspected because depression diagnosis and treatment are delayed often, and when the unclear physical symptom persists, depression is suspected. The most serious symptom associated with depression is suicide, with about 70% of depressed patients considering suicide, and about 10% to 15% actually suicide.

In relation to symptoms such as sleep disorders, anxiety disorders, and suicidal ideation in patients with depression, studies related to hormones have been carried out in terms of biochemistry.

According to the above study, in the case of excessive mental stress, the corticotropin releasing hormone (CRH) is generated in the hypothalamus of the brain, and the corticotropin releasing hormone stimulates the anterior pituitary gland, The adrenocorticotropic hormone acts on the adrenocortical hormone and promotes the secretion of glucocorticoids such as cortisol, which is a stress hormone among the steroid hormones secreted from the adrenal glands. The hormone secreted by the adrenocorticotropic hormone (ACTH) The glucocorticoid secreted into the body is delivered to each organ of the body.

The secretion of cortisol, the stress hormone, prepares the human body to cope with mental stress, such as tightening muscles and sensitizing the sensory organs, but the sustained release of cortisol hormone due to repetitive mental stress causes the body to feel nervous So that the concentration of the cortisol in the blood is increased, and the sleeping disorder occurs because the user can not take a good sleep. This sleep disorder is again a cause of mental stress, and depression occurs as a vicious cycle occurs.

In addition, when the cortisol secretion control function due to extreme mental stress is lost, excessive cortisol secretion continues to cause brain atrophy and damage throughout the cranial nervous system, resulting in severe depression and the possibility of suicide due to this have.

Cerebral nervous system atrophy (weight and volume reduction) and impairment of depression leading to suicidal suicide, which is the cause of suicide, have been reported to be a direct primary cause of cortisol hormone, which is over secreted by loss of ability to regulate secretion.

However, the mechanism and cause of neuronal damage caused by the cortisol hormone are still unclear, and many researches have been conducted on the subject of interest of many neuroscientists. However, it has been reported that depression is caused by neurodegenerative diseases such as neurodegenerative diseases such as dementia or stroke, which is different from apoptosis, which is known as a mechanism of cell damage caused by DNA damage .

Recent studies on depressed and depressed experimental animal models have shown that cortisol secreted by mental stress causes excessive uptake of calcium ions into nerve cells, and more specifically hippocampal neurons, Calcium ions interfere with intracellular entry of glucose, which is the energy source of the cell, and consequently the amount of adenosine triphosphate (ATP), which is the main energy molecule in the cell, is reduced. As a result, And ultimately induces atrophy of the cranial nervous system and cell death.

Regarding the excessive uptake of calcium ions into the nerve cells by the cortisol, the coritizol induces quantitative increase of nervous system excitatory neurotransmitters such as glutamate, NMDA and kainic acid, and the excitatory neurotransmission It has been reported that the quantitative increase of the substance abnormally activates the excitatory neurotransmitter receptor and excessive influx of calcium ions into the cells through the receptor. In addition, calcium-channel and calcium-pump, which are responsible for electrical signal transmission of neurons, are also overactivated, which causes excessive calcium influx into neurons.

The antidepressant drugs that have been reported and used so far are drugs that induce the therapeutic effects of depression through temporary diversion by increasing serotonin levels in the blood by inhibiting the reabsorption of serotonin, known as the hormone, , And there are about five depressants sold under the FDA approval by the US, including prozac and celexa.

However, in the case of these serotonin reuptake inhibitors, not only do half of the patients improve symptoms, but according to the recommendations of the American Psychiatric Association (APA), a period of at least 4 months is required for symptomatic improvement, There is a problem that it should be continuously taken for 2 to 3 years. In addition, in the case of the above-mentioned serotonin reuptake inhibitor, when the drug is withdrawn, most of the symptoms recur within 6 to 12 months, and the side effects are also known to be severe.

Therefore, there is a continuing demand for development of antidepressants that can treat long-term and effective depression against natural products, as the side effects are unlikely to be a problem.

KR 10-1046126 B KR 10-0008675 B

It is an object of the present invention to provide a composition for treating or preventing stress-related diseases.

It is another object of the present invention to provide a functional food composition for improving or preventing a stressful disease.

In order to achieve the above object, the pharmaceutical composition for prevention or treatment of stress-related disorders in accordance with one embodiment of the present invention, pomegranate (Punica granatum ) as an active ingredient.

In this aspect, the invention pomegranate (Punica granatum ) as an active ingredient. The present invention also relates to a pharmaceutical composition for preventing or treating stress-related diseases.

The stress disorder may be any one selected from the group consisting of depression, sleep disorder, and anxiety disorder.

The pomegranate may be preferably pomegranate.

The pomegranate extract may be one selected from the group consisting of water, an alcohol having 1 to 5 carbon atoms, and a combination thereof, and may be a pomegranate extract.

In addition, the food composition for preventing or improving stress-related disorders in accordance with another embodiment of the present invention to achieve the above object, pomegranate (Punica granatum ) as an active ingredient.

In this aspect, the invention pomegranate (Punica granatum ) extract as an active ingredient.

The pomegranate may be preferably pomegranate.

The food composition may be a health functional food.

In the present invention, a stress disorder refers to a disorder having symptoms caused by stress, for example, depressive disorder and the like.

In the present invention, depressive disorder refers to a disease that causes various cognitive and psychosomatic symptoms due to depression of desire and depression as main symptoms, resulting in deterioration of daily function, and is also referred to as depression.

Hereinafter, the present invention will be described in more detail.

The inventors of the present invention have found that pomegranate extract is effective for cortisol secretion control and inhibition of nerve cell damage while studying natural products effective for stress diseases, especially depression, The present inventors confirmed that the effect of inhibiting the generation of stress and depression by inducing stress mental diseases in rats was excellent, and that the pomegranate hot water extract of the pomegranate extract The inventors confirmed that the therapeutic or preventive effects of stress-induced mental disorders were excellent, and thus completed the present invention.

The present invention relates to a pharmaceutical composition for preventing or treating a stress-related disease comprising an extract of pomegranate as an active ingredient.

The composition according to one embodiment of the present invention is a pharmaceutical composition for the prevention or treatment of stress diseases comprising pomegranate extract as an active ingredient.

Pomegranate ( Punica granatum L.) is a dicotyledonous plant belonging to the Lythraceae family. The pomegranate is native to West Asia and northwest India and is believed to have come from China in Korea early in the Goryeo Dynasty. It is currently grown in the central and southern regions as a garden and fruit plant.

 The pomegranate may be at least one selected from the group consisting of fruit, leaf, flower, bark and root of pomegranate, preferably pomegranate. The pomegranate is called pomegranate and is used for edible or medicinal purposes. The pomegranate can be any one selected from the group consisting of flesh, peel, seed, and combinations thereof, and is not affected by the site.

The pomegranate extract may be prepared according to a conventional plant extract preparation method. For example, the pomegranate may be dried by removing water, pulverized, and then extracted with an extraction solvent, but the present invention is not limited thereto.

The conventional plant extract preparation method may be, for example, a heat extraction method, an ultrasonic extraction method, a reflux extraction method or an ultra high pressure extraction method, but is not limited thereto.

The extract of the present invention may be one prepared by extracting with an extraction solvent or extracting by extracting with an extraction solvent and then fractionating the extract with a fraction solvent. The extraction solvent may be at least one selected from the group consisting of water and an organic solvent. The organic solvent may be a polar solvent such as methanol, ethanol or the like, an alcohol having 1 to 5 carbon atoms, ethyl acetate or acetone, and a non-polar solvent such as hexane or dichloromethane, Ethanol aqueous solution, and combinations thereof. Specifically, the extraction solvent may be 20% ethanol aqueous solution, 80% ethanol aqueous solution or water, preferably water.

The fraction solvent may be water, butanol, ethyl acetate, chloroform, hexane or a mixture thereof.

The pomegranate extract is prepared by pulverizing and drying the pomegranate tree, preferably pomegranate, and then extracting any one of water, an alcohol having 1 to 5 carbon atoms and a mixed solvent thereof, preferably water or an aqueous ethanol solution And extracting the mixture at a reaction temperature of from 10 캜 to 120 캜 or from 30 캜 to 90 캜 for about 2 hours to 7 days or for 12 hours to 30 hours.

The pomegranate extract may be prepared by extracting the pomegranate extract by the above-mentioned extraction method, preparing an extract, filtering the obtained extract, concentrating under reduced pressure, and / or lyophilizing the concentrated extract.

The stress disorder refers to a disease caused by stress, such as a sleep disorder, a depression, a panic disorder, and a memory loss. , And can be preferably depression.

Such stress-related diseases may be caused by atrophy and damage, in which the capacity for regulating cortisol hormone is lost due to stress and the weight or volume of the cranial nervous system is decreased.

According to the results confirmed by the examples, it was revealed that in the case of the stress disorder, the neurological diseases caused by apoptosis, which is known as a cell damage mechanism due to DNA damage, which is known as a cause of degenerative brain diseases such as dementia or stroke, Respectively.

According to the embodiment of the present invention, in the case of pomegranate extract, cortisol secretion, which is promoted by mental stress, is suppressed and energy metabolism of nerve cells is maintained, thereby preventing neuronal cells from being inactivated or killed, And was not associated with oxidative stress, a major cause of cell damage. In addition, the pomegranate extract was found to have excellent effects such as relieving stress, restoring brain function performance, and restoring reflex behavior.

Depression is a kind of mental illness that is obsessed with depression, loss of motivation, inability, isolation, impatience, guilt, or suicidal impulse, which is caused by the inability of the neurotransmitter to function completely, If the cortisol concentration in the blood is kept high in the body by sustaining cortisol secretion, brain atrophy and damage may be caused throughout the cranial nervous system, resulting in depression.

In the case of pomegranate extract, preferably pomegranate extract, the secretion activity of cortisol, which is one of the hormones in the body, is suppressed and the concentration of cortisol in the blood is normally maintained to inhibit brain atrophy and damage, thereby preventing or treating depression.

The pharmaceutical composition for preventing or treating the stress disorder may contain 0.01 to 99% by weight of the pomegranate extract, preferably pomegranate extract, based on the total weight of the composition. However, the present invention is not limited thereto, and may be different depending on the condition of the patient, the kind of the disease, and the progress of the disease.

The pharmaceutical composition may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions.

The pharmaceutical compositions may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterilized injection solutions according to a conventional method. Examples of carriers, excipients and diluents that may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate and mineral oil.

When the pharmaceutical composition is formulated, it is prepared using a diluent such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, or an excipient usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient, such as starch, calcium carbonate, sucrose, Or lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As a suppository base, witepsol, macrogol, tween 61, cacao paper, laurin, glycerol gelatin and the like can be used.

The preferred dosage of the pharmaceutical composition will vary depending on the condition and the weight of the patient, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the extract is preferably administered at a dose of 0.01 mg / kg to 10 g / kg per day, preferably 1 mg / kg to 1 g / kg per day. The administration may be carried out once a day or divided into several doses. Therefore, the dose is not intended to limit the scope of the present invention in any aspect.

The composition can be administered to mammals such as rats, mice, livestock, humans, and the like in a variety of routes. All the above administration methods can be carried out by a conventional method, for example, oral administration, rectal administration or intravenous administration.

A composition according to another embodiment of the present invention is a food composition for preventing or improving a stress disorder, which comprises pomegranate extract as an active ingredient.

The pomegranate extract may be a pomegranate extract.

The food composition may be a health functional food.

The contents of the pomegranate extract and the stress disorder are the same as those mentioned in the above pharmaceutical composition.

The term " food " means a natural product or a processed product containing one or more nutrients. Preferably, the food means a state in which it can be directly eaten through a certain degree of processing. Food additives and beverage additives.

Examples of the foods include various foods, beverages, gums, tea, vitamin complexes, and health functional foods. In addition, in the present invention, the food may contain special nutritional foods (eg, crude oil, infant formula, etc.), meat products, fish products, tofu, jelly, noodles (eg, (Such as soy sauce, soybean paste, kochujang, mixed potato, etc.), sauces, confectionery (eg, snacks), dairy products (eg fermented milk, cheese, etc.), other processed foods, kimchi, , Fruit, vegetable beverages, edible oils, fermented beverages, etc.), natural seasoning (e.g., ramen soup, etc.).

The food, the health functional food, the beverage, the food additive and the beverage additive can be produced by a usual production method.

In the present invention, the health functional food refers to a food group imparted with added value to function and express the function of the food by physical, biochemical, biotechnological, and the like, Means a food which is processed and designed so that the body control function related to prevention and recovery is sufficiently expressed in the living body.

The health functional food may include food-acceptable food supplementary additives, and may further include suitable carriers, excipients and diluents conventionally used in the production of health functional foods.

In the present invention, beverage is a generic term for drinking or enjoying a taste, and is intended to include a health functional beverage. The beverage is not particularly limited to the ingredients other than the above-mentioned pomegranate extract as an essential ingredient in an indicated ratio as an active ingredient, and may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages .

Examples of such natural carbohydrates include monosaccharides such as disaccharides such as glucose and fructose such as maltose, sucrose and the like and polysaccharides such as dextrins, cyclodextrins and the like, and Xylitol, sorbitol, and erythritol. Natural flavors (tau martin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above The ratio of the natural carbohydrate may be generally from about 1 g to 20 g, preferably from 5 g to 12 g per 100 ml of the food composition of the present invention. In addition, the composition of the present invention may contain natural fruit juice, fruit juice drink, And may further contain pulp for the production of vegetable beverages.

In addition to the above, the food composition of the present invention can be used as a flavoring agent such as a variety of nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and thickening agents (cheese, chocolate etc.), pectic acid and its salts, Salts thereof, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. These components can be used independently or in combination. The proportion of such additives is not so important, but may be selected in the range of 0 to 20 parts by weight per 100 parts by weight of the pomegranate extract of the present invention.

In the present invention, the health functional beverage includes a beverage group to which added value is imparted so that the function of the beverage functions to a specific purpose using physical, biochemical, biotechnological techniques, etc., Means a beverage which is processed by being designed so that the body control function related to recovery and the like is sufficiently expressed to a living body.

The health functional beverage is not particularly limited to the ingredients other than the white bean curd extract of the present invention as an essential ingredient in the indicated ratios and may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages .

Examples of such natural carbohydrates include monosaccharides such as disaccharides such as glucose and fructose such as maltose, sucrose and the like and polysaccharides such as dextrins, cyclodextrins and the like, and xylitol , Sorbitol, and erythritol. Natural flavors (tau martin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably 5 to 12 g per 100 ml of the composition of the present invention.

The composition comprising the pomegranate extract, specifically the pomegranate extract as an active ingredient, has an effect of effectively preventing nerve cell damage caused by mental stress-inducing substances in the neuronal cells of the primary cultured rat, , It was experimentally confirmed that intracellular cAMP is regulated by inhibiting serotonin receptors in 1321N1 cells expressing the 5-HT6 gene and that the suppression effect on stress and depression by inducing mental stress in rats is excellent .

The composition comprising the pomegranate extract of the present invention as an active ingredient has an effect of treating, ameliorating and preventing neuronal cell damage due to excessive secretion of stress hormone and suppressing stress and depression, Prevention or treatment of cancer.

FIG. 1 is a graph showing the degree of cytotoxicity of pomegranate extract on brain cells according to an embodiment of the present invention. In FIG. 1, the vertical axis represents the degree of cytotoxicity relative to the control group, (Μg / ml) of the extracts of the control group and each solvent. Fig. 1a shows the hippocampal neurons isolated from rat embryo, Fig. 1b shows cytotoxicity on cerebral cortical neurons and Fig. 1c shows cytotoxicity on striatal neurons.
FIG. 2 is a graph showing the protective effect of pomegranate extract against oxidative stress induction by hydrogen peroxide treatment according to an embodiment of the present invention, wherein the vertical axis of the graph represents the cell viability as a relative value to the control group and the horizontal axis of the graph is the control group, treated with hydrogen peroxide, only the negative control (H ₂ O ₂₎ and a pomegranate extract, specifically pomegranate hot-water extract (hot water), pomegranate 20% aqueous ethanol extract (20%) not treated with any sample. (80%) of pomegranate aqueous solution of 80% ethanol and its treatment concentration (mg / ml).
FIG. 3 is a graph showing the induction of intracellular stress through cortisol treatment, which is a mental stress hormone according to an embodiment of the present invention, and the protective effect of nerve cells on the concentration of pomegranate extract, wherein the vertical axis represents cell viability, , And the abscissa of the graph indicates the control group treated with no sample, the negative control group treated with only cortisol, and the pomegranate extract and its treatment concentration (mg / ml). 3b shows the effect of extracting 20% ethanol aqueous solution of pomegranate (20% alcohol extract of pomegranate) and FIG. 3c showing the effect of protecting the nerve cells of 80% ethanol aqueous solution extract of pomegranate (80% alcohol extract of pomegranate) .
FIG. 4 is a photograph showing the cell-stress induction through hydrogen peroxide and cortisol treatment and the protective effect of nerve cell by the hot water extract of pomegranate (0.3 mg / ml) according to ERK phosphorylation and expression of NF-κB according to an embodiment of the present invention FIGS. 4A to 4C show results of confirming the protective effect of hydrogen peroxide on the nerve cells, the degree of phosphorylation of ERK and the expression of NF-κB. FIGS. 4D to 4F show the protective effect of nerve cells against cortisol, The degree of phosphorylation and the expression of NF-κB.
FIG. 5 is a graph showing the cAMP inhibitory effect in order to confirm the effect of the pomegranate extract on the serotonin receptor according to an embodiment of the present invention, wherein the ordinate axis shows the inhibitory effect and the abscissa axis shows the pomegranate extract, (20%), pomegranate 80% ethanol aqueous solution extract (80%) and its treatment concentration (μg / ml).
FIG. 6 is a graph showing the mortality of an experimental animal and the reduction of mortality due to the administration of the pomegranate extract in a depressed animal model state through sleep deprivation stress according to an embodiment of the present invention. The vertical axis of the graph represents the relative value of the survival rate , The abscissa of the graph represents the elapsed time after the experiment, the control group represents the experimental group in which no treatment is performed, the sleep deprivation refers to the negative control group in which sleep deprivation is performed, and the sleep deprivation + pomegranate Refers to an experimental group administered with pomegranate extract to perform sleep deprivation, and the positive control group refers to an experimental group administered sleep deprivation and caffeine.
FIG. 7 is a graph showing changes in body weight of experimental animals and changes in body weight by administration of pomegranate extract in a depressed animal model state through sleep deprivation stress according to an embodiment of the present invention. The control group refers to an experimental group in which no treatment is performed, the sleep deprivation refers to a negative control group in which sleep deprivation is performed, the sleep deprivation + pomegranate performs sleep deprivation, and the pomegranate extract And the pomegranate means a group treated only with pomegranate extract without performing sleep deprivation.
FIG. 8 is a graph showing the degree of sleepiness of a test animal in the depressed animal model state through sleep deprivation stress according to an embodiment of the present invention, wherein the vertical axis of the graph indicates the number of times of repetitive actions The abscissa of the graph represents each group, the sleep deprivation refers to a negative control group in which sleep deprivation is performed, the sleep deprivation + pomegranate refers to an experimental group in which sleep deprivation is performed and pomegranate extract is administered, Deprivation + caffeine refers to a positive control group that performs sleep deprivation and receives caffeine.
FIG. 9 is a graph showing the results of passive avoidance experiments for testing the brain functioning ability and behavioral change of the experimental animals in the depressed animal model state through sleep deprivation stress according to an embodiment of the present invention, The vertical axis of the graph represents time (sec), the abscissa of the graph represents each group, the control group refers to an experimental group in which no treatment is performed, the sleep deprivation refers to a negative control group in which sleep deprivation is performed, The pomegranate refers to a group treated only with pomegranate extract without sleep deprivation, while the sleep deprivation + pomegranate refers to an experimental group administered with pomegranate extract to perform sleep deprivation. The caffeine does not perform sleep deprivation, Treated group, and the sleep deprivation + caffeine refers to a positive control group that performs sleep deprivation and administers caffeine.
FIG. 10 is a graph showing the results of an experiment for confirming the influence of the thermal stimulation of the experimental animals on the sensitivity response behavior in a depressed animal model state through sleep deprivation stress according to an embodiment of the present invention. (° C.) in which the experimental animals exhibit reflex behaviors, and the abscissa of the graph represents each group. The control group represents an experimental group in which no treatment is performed, and the sleep deprivation represents a negative control group for performing sleep deprivation The pomegranate means the group treated only with the pomegranate extract without performing the sleep deprivation, the sleep deprivation + the pomegranate indicates the experimental group in which the sleep deprivation is carried out and the pomegranate extract is administered, and the caffeine does not perform sleep deprivation Caffeine-treated group, the sleep deprivation + caffeine performs sleep deprivation, and the caffeine-administered positive control It means.
FIG. 11 is a graph showing the amount of cortisol secretion in the blood of a laboratory animal in the depressed animal model through sleep deprivation stress according to an embodiment of the present invention. In FIG. 11, the vertical axis represents the cortisol content (mg / ml) , The abscissa of the graph represents each group, the control group represents an experimental group in which no treatment is performed, the sleep deprivation represents a negative control group in which sleep deprivation is performed, the pomegranate does not undergo sleep deprivation, Extract means only the group treated, and sleep deprivation + pomegranate means sleep deprivation and pomegranate extract administration group.

Hereinafter, embodiments of the present invention will be described in detail so that those skilled in the art can easily carry out the present invention. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

[ Manufacturing example : Preparation and extraction of experimental materials]

1. Preparation of experimental material and cell culture

Cell culture media such as RPMI 1640, fetal bovine serum and penicillin-streptomycin were purchased from Gibco BRL (NY, USA). All reagents, including Cortisol, used in the following experimental examples were used for analytical grade or higher.

Primary cells were rapidly separated from the fetal brain (E16-18) and treated with trypsin to separate into single cells such as hippocampal cell (hippocampus), cortical cell (cerebral cortex), and striatal cell (striatal cell) . Cultured for 14 days under the Neurobasal medium (Gibco Co., USA) and B-27 supplement (Gibco, Inc.), according to the purpose of a single cell the USA is separated in the contained culture medium dispensed in a Petri dish and 37 ℃, 5% CO ₂ conditions And then used in the following experiments.

Results were expressed as mean and standard deviation. Significance was tested by using Student's t- test (p <0.05) using GraphPad Prism 5 program. Significance was shown between experimental group and control group.

2. Preparation of pomegranate extract

The pomegranate was cut into 10 equal parts according to size, and then freeze-dried to remove moisture. The water-removed pomegranate was pulverized and homogenized. 5 kg of pomegranate powder was mixed with 50 L of extraction solvent and extracted under the conditions of pressure of 0.7 Kg / ㎠ to 0.75 Kg / ㎠ for 4 hours. The extraction solvent was prepared by extracting 20% ethanol, 80% ethanol and hot water using extracts of 20% ethanol (20% alcohol), 80% ethanol (80% alcohol) and distilled water, respectively .

The prepared hot-water extract was concentrated to 25 Brix at 60 ° C. and 750 mmHg. The ethanol extract of the ethanol was concentrated to 25 Brix at 60 ° C. and 750 mmHg, and then 50 L of purified water was added thereto. Respectively. Each of the concentrated extracts was prepared by freeze-drying at -40 ° C for 48 hours using a freeze dryer.

880 g of pomegranate hot water extract (hereinafter referred to as RR0), 775 g of 20% ethanol extract (hereinafter referred to as RR20E) and 745 g of 80% ethanol extract (hereinafter referred to as RR80E) And used as a test sample.

[ Experimental Example  One: In vivo  Stress Relaxation Activity of Pomegranate Extracts on Rats]

1-1. Cytotoxicity to pomegranate extract

MTT assay was used to determine the cytotoxicity of the extracts of 20% and 80% ethanol (alcohol) in primary cultured rat brain cells.

Brain cells were isolated from the rat embryo of Preparation Example 1, treated with the pomegranate extract of the above Preparation Example, and the cell viability was measured. The cytotoxicity results of each of the pomegranate extracts are shown in FIG.

More specifically, the brain tissue of rat embryo (E16-18) was rapidly separated and treated with trypsin to separate into single cells such as hippocampal cell (hippocampus), cortical cell (cerebral cortex), striatal cell do. Single cells were cultured in a culture medium containing Neurobasal medium (Gibco) and B-27 supplement (Gibco) for 14 days at 37 ° C and 5% CO ₂ according to purpose. Each sample was treated and cell death was observed after 24 hours. The pomegranate extract was tested by adjusting the concentration of each of the pomegranate hot water extract and ethanol aqueous solution extract of Preparation Example 2 to 0.3 μg / ml, 1 μg / ml, 3 μg / ml, 10 μg / ml and 30 μg / The measurement results are shown in FIG.

As shown in Fig. 1, no toxicity was observed in cells isolated from hippocampus, cerebral cortex, and striatum. Specifically, the cytotoxicity was confirmed using MTT after 24 hours. As a result, the pomegranate extract, specifically the hot water extract, the 20% alcohol extract and the 80% alcohol extract did not show a significant decrease, and the highest concentration of 30 .mu.g / ml did not show cytotoxicity.

1-2. Oxidative  Confirming the protective effect of pomegranate extract on stress

FIG. 2 shows the protective effect of nerve cells due to oxidative stress by the pomegranate extract treatment according to the extraction solvent of the above Preparation Example.

The cells used in the experiment were hippocampal neurons of the primary cultured rat. Pomegranate extracts were pretreated with concentrations of 0.3 mg / ml and 10 mg / ml for 24 hours, treated with H ₂ O ₂ at a concentration of 100 μM for 24 hours and treated with pomegranate extract for a total of 48 hours The survival rate was compared by the MTT method to observe the protective effect of the sample on the nerve cell. The results are shown in FIG.

As shown in FIG. 2, the cell viability was reduced to about 90% when treated with only hydrogen peroxide (H ₂ O ₂ ) compared with the control without any treatment, and the cytoprotective effect of pomegranate extract by pre- It was confirmed that no protective effect of nerve cell due to oxidative stress was observed under all conditions regardless of concentration.

1-3. Identification of cell viability by pomegranate extract concentration

In order to confirm the resistance activity of the pomegranate extract against the mental stress at the cellular level, the stimulation of the stimulation by the treatment of the neuronal cell damage inducer of the stress hormone cortisol was performed.

FIG. 3 shows the protective effect of nerve cells by treating pomegranate extract according to the extraction solvent of the above Preparation Example.

The primary cultured rat hippocampal neurons of the above-mentioned preparations were prepared and divided into a control group in which nothing was treated, a negative control group in which cortisol was treated for the cells, and an experimental group in which the cortisol was treated and the pomegranate extract of the above-mentioned example was treated Respectively.

The crude hippocampal neurons cultured in the above-mentioned preparations were divided into extractive solvents for 24 hours, and then cortisol was added for 24 hours, followed by pretreatment at 1, 3, 10, 30, 100 and 300 mg / , And MTT (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolane bromide) were treated for 4 hours and then the medium was removed. The survival rate was confirmed by dissolving purple crystals produced by mitochondrial dehydrogenases in living cells and measuring the absorbance at 540 nm, and is shown in FIG.

As shown in FIG. 3, the survival rate was similar to that of the control group at all other concentrations except for the cell survival rate of 93.30% at the concentration of 0.1 mg / ml in the case of the pomegranate hot water extract. The 20% But the cell viability was increased. In the case of 80% ethanol extracts, the concentrations of the extracts were 86.42% (0.1 mg / ml), 89.25% (0.3 mg / ml), 83.70% (1 mg / ml), 77.98% mg / ml), indicating that the protective effect was insignificant.

Therefore, the pomegranate hydrothermal extract and 20% ethanol extract have excellent protective effect against nerve cell damage due to mental stress, and especially, the effect of hydrothermal extract is most excellent. Hereinafter, the following experiment was carried out using pomegranate hot water extract.

1-4. Oxidative  stress( H2O2 ) And mental stress (cortisol) Intracellular  Identification of expression factors ( ERK -P, NF ΚB)

When physiological and chemical stresses such as active oxygen species or UV are brought into the cell, the protein kinase of various signaling pathways is activated and activates the next step anoxic enzyme. Activation of a series of intracellular protein kinases activates the activated protein to migrate into the nucleus and increase gene expression of the target protein, resulting in regulation of cellular physiology such as cell proliferation, survival, differentiation, and death .

Expression of NF-κB, phosphorylation of EKR, and changes in pomegranate extract were observed. The expression of NF-κB and ERK was examined by HTS when cortisol or H ₂ O ₂ (hydrogen peroxide) was pretreated (0.3 mg / ml) in rat hippocampal neurons cultured in primary culture of pomegranate hydrothermal extract 4a to 4f. Blue was stained with hoechst dye and green was stained with HTS by fluorescent staining of NF-κB and ERK using an antibody. The intesity of each cell was measured, and the expression and changes of various factors were analyzed by extract treatment. Statistical analysis was performed by Student's t-test.

As shown in FIGS. 4A to 4C, when the oxidative stress was induced by treatment with hydrogen peroxide, the expression of intracellular NF-κB and phosphorylated ERK was increased, but the reduction effect by the pretreatment of pomegranate hydrothermal extract It did not appear.

As shown in FIG. 4D to FIG. 4F, it was also confirmed that the experimental group in which mental stress was induced by treatment with cortisol also increased the expression of intracellular NF-κB and phosphorylated ERK. Expression by the pretreatment of pomegranate hydrothermal extract Was observed to be suppressed.

The pomegranate extract showed that the mechanism of neuronal protective effect was different from that of cell damage caused by oxidative stress such as hydrogen peroxide, and the protective effect of cell death on mental stress hormone, cortisol, was specifically exerted Respectively.

1-5. Pomegranate hot water extract  Confirm the effects on serotonin receptors

To confirm the association between pomegranate hydrothermal extract and depressive disorder, pomegranate extracts were treated with a cell line overexpressing type 6 serotonin receptor (5-HT6R), a selective receptor for the treatment of depression, and a specific signal by serotonin receptor The increase / inhibition effect of cyclic AMP, which is a delivery route, was confirmed.

Human astrocytoma 1321N1 cells expressing the 5-HT6 receptor gene were used. Cells were cultured for 24 hours, and the supernatant medium was removed. Cells were treated with phosphodiesterase inhibitors IBMX (0.5 mM, SIGMA) and Ro 20-1724 (Pectin) extract, and 20% ethanol (pectin) extract of pomegranate and 80% ethanol (pectin) extract of pomegranate were treated for 30 minutes. After 15 minutes treatment with pomegranate extract, the cells treated with serotonin were dissolved and assayed according to the cAMP Assay kit (R & D Systems). The results are shown in FIG.

As shown in FIG. 5, it was confirmed that the extract of Pomegranate hydrothermal extract had an excellent inhibitory effect of 23% at 100 μg / ml and 50% at 30 μg / ml. %, Respectively. However, it was confirmed that the effect of 80% pomegranate extract was insignificant.

[ Experimental Example  2: In vivo  Stress Relaxation Activity of Pomegranate Extract on Animal Model of Depression]

2-1. Preparation and method of experiment

To investigate the effect of pomegranate extract on the stress - relieving activity, an experiment using an animal model of depression which is induced by extreme stress induction through long - term sleep deprivation was conducted.

The sleep deprivation is made by separately preparing a cage capable of depriving the user of the water for a long period of time (21 × 25 × 27 cm), placing a small cylinder in the middle of the cage where the animal can stand only and filling the water, And it was not possible to sleep deeply. Diets were fed arbitrarily for general laboratory animals and no water was provided separately, but instead water was introduced into the cage on a daily basis.

Depression Animal model rats were male Sprague-Dawley rats (SD rats) of about 150 ± 10 g (samtaco). A temperature of 22 to 24 캜 and a humidity of 60 to 80% were maintained, and day and night cycles were set at intervals of 12 hours one week before the experiment.

The control group was free of sleep, dieting, and negative water on a wide pedestal. The negative control group was orally administered saline to the sleep deprived animal model rats and the positive control group was orally administered caffeine at a concentration of 10 mg / kg to the sleep deprived animal model rats.

In the experimental group, the pomegranate hot water extract was dissolved in physiological saline at a concentration of 0.1 mg / ml and orally administered twice a day to the deprived animal model mice.

2-2. in vivo  Identification of the stress-relieving effects of pomegranate extracts in depressed animal models

6 to 8 show the survival rate, body weight and various behavioral changes of the experimental animals in order to ascertain the accumulation of stress due to sleep deprivation and the occurrence of depression and various changes and symptoms thereof during the experiment.

As shown in FIG. 6, as the sleep deprivation continued, the survival rates of the control group and the experimental group as well as the survival rate of the positive and negative control groups were significantly different. The control group, which was provided free of sleep, did not die during the experiment, but the negative control group, which received saline deprivation, showed a survival rate of 40% on the 6th day of the experiment and died on the 10th day. The survival rate of the pomegranate extract was 80% on the 5th day and 60% on the 10th day until the end of the experiment. The caffeine-treated control group had a 20% survival rate at 5 days and a 100% mortality at 15 days.

Based on the above results, it was confirmed that when the pomegranate extract is administered to sleep deprived rats, death by accumulation of stress is prevented.

As shown in FIG. 7, the body weight of each experimental animal was measured daily and the increase rate relative to the weight measured before the start of the experiment was measured. As a result, the body weight of the control group tended to increase with the lapse of time, Respectively. However, no increase in body weight was observed in the pomegranate extract-treated group in the sleep deprived state, but it was not significantly decreased compared to the SDPD group.

The sleepiness of the rodents was observed during the day. The number of repetition for 10 minutes was measured only for the sleep deprived experimental group and is shown in FIG.

As shown in FIG. 8, it was confirmed that the puddling of the experimental group administered with the pomegranate extract was significantly reduced (* p <0.05 vs SD group) as compared with the control group. There was no significant difference between the caffeine - treated group and the control group.

As a result of observing the survival rate, body weight and various behavioral changes of the animal model depression animal, it was confirmed that pomegranate extract has a remarkable effect for relieving depression by increasing the resistance to stress.

2-3. in vivo  Assessment of brain function performance in depressed animal models

Depression through sleep deprivation stress A passive avoidance test, which is used to evaluate brain function performance and behavioral changes in experimental groups by pomegranate extracts in animal model conditions, is shown in FIG.

Evaluation of brain function performance by sleep deprivation and administration of pomegranate extract was performed by measuring the time taken for electrical stimulation (75 V, 0.2 mA), which was disgusted by the experiment animals, to reach the box in which electrical stimulation was given after 24 hours Respectively.

As shown in FIG. 9, it was confirmed that the brain function was decreased in the negative control group as compared with the control group, and the brain function was restored to the control level again by the experimental group. As a result, the pomegranate extract was effective for the brain function treatment .

2-4. in vivo  Evaluation of the effects of pomegranate extract on reflex behavior in depressed animal models

Depression through Sleep Deprivation Stress Hot plate experiments were conducted to confirm the effects of pomegranate extract on sensitive reflex behaviors of pain, which is the main symptom of depression in the experimental group, and the results are shown in FIG. 10 .

Experiments were carried out with the equipment equipped with a temperature controllable metal plate so that the temperature was increased from 30 ° C to 50 ° C by 2.5 ° C per minute, and the experimental animals were placed on a metal plate. While the temperature was rising, the temperature at which the action such as licking or footing was observed was observed by observing the experimental animal.

As shown in FIG. 10, in the case of the control group, the group treated with only pomegranate extract, and the group treated with caffeine alone, the temperature was detected at an order of 46 ° C in the case of the non-sleep deprivation group, , And the sensory temperature was lowered in the experimental group to which the pomegranate extract was administered together with the deprivation of sleep than in the negative control group.

Therefore, it is shown that the ability of reflex behaviors is decreased due to decrease of physical ability by sleep deprivation, but the reflex behavior ability is restored when pomegranate extract is administered.

2-5. in vivo  Depression in animal models Animals in experimental animals Cortisol  Content check

The blood of the test animal of the depressed animal model was sampled to isolate plasma, and the content of cortisol known as stress hormone was measured, and the result is shown in FIG.

As shown in Fig. 11, when the pomegranate extract alone was administered alone, the cortisol content of the pomegranate extract did not change, and the cortisol content of the negative control (sleep deprivation) in the depression animal model through sleep deprivation stress (** p <0.01), and this tendency was significantly decreased (p <0.05 compared to the negative control) by the pomegranate extract administration.

Therefore, it has been experimentally confirmed that the effect of suppressing the secretion of cortisol in the pomegranate extract and the effect of preventing or treating depression through the suppression of secretion.

[ Formulation example ]

Formulation example  One. Sanje  Produce

RR0 200 mg

Lactose 100 mg

Talc 10 mg

The above ingredients can be mixed and filled into an airtight can to produce powders.

Formulation example  2. Preparation of tablets

RR20E 200 mg

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.

Formulation example  3. Preparation of capsules

RR80E 200 mg

Crystalline cellulose 3 mg

Lactose 14.8 mg

Magnesium stearate 0.2 mg

The above components are mixed in accordance with a conventional method for producing a capsule, and filled in a gelatin capsule to prepare a capsule.

Formulation example  4. Preparation of injections

RRO 200 mg

180 mg mannitol

Sterile sterilized water for injection 2974 mg

Na ₂ HPO _{4 ,} 12H ₂ O 26 mg

(2 ml) per ampoule in accordance with the usual injection method.

Formulation example  5. Liquid  Produce

RRO 200 mg

10 g per isomer

5 g mannitol

Purified water quantity

Each component was added and dissolved in purified water according to the usual liquid preparation method, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto. The whole was added with purified water to adjust the total volume to 100 ml, And sterilized to prepare a liquid preparation.

Formulation example  6. Manufacture of health food

RR80E 1000 mg

Vitamin mixture quantity

70 [mu] g of vitamin A acetate

Vitamin E 1.0 mg

0.13 mg vitamin B1

0.15 mg of vitamin B2

0.5 mg vitamin B6

0.2 [mu] g vitamin B12

10 mg vitamin C

Biotin 10 μg

Nicotinic acid amide 1.7 mg

50 ㎍ of folic acid

Calcium pantothenate 0.5 mg

Mineral mixture quantity

1.75 mg of ferrous sulfate

0.82 mg of zinc oxide

Magnesium carbonate 25.3 mg

15 mg of potassium phosphate monobasic

Secondary calcium phosphate 55 mg

Potassium citrate 90 mg

100 mg of calcium carbonate

24.8 mg of magnesium chloride

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a suitable ingredient for the health food, it is possible to arbitrarily modify the blending ratio of the vitamin and mineral mixture, and the above ingredients are mixed according to a general health food manufacturing method, Granules can be prepared and used in the manufacture of a health food composition according to conventional methods.

Formulation example  7. Manufacture of health drinks

RRO 1000 mg

Citric acid 1000 mg

100 g of oligosaccharide

Taurine 1 g

Purified water was added to a total of 900 ml

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was stirred and heated at 85 캜 for about 1 hour. The resulting solution was filtered to obtain a sterilized 2-liter container, sealed sterilized, Used in the manufacture of health beverage compositions.

Although the composition ratio is relatively mixed with a component suitable for a favorite beverage as a preferred embodiment, the blending ratio can be arbitrarily varied according to the regional and national preference such as demand level, demand country, use purpose, and the like.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, Of the right.

Claims (8)

A pharmaceutical composition for the prevention or treatment of a stress-related disease, comprising a pomegranate hydrothermal extract as an active ingredient. The method according to claim 1,
Wherein the stress disorder is any one selected from the group consisting of depression, sleep disorder, and anxiety disorder. delete delete delete A food composition for preventing or ameliorating a stress disorder, comprising a pomegranate hydrothermal extract as an active ingredient. delete The method according to claim 6,
Wherein said food composition is a health functional food.

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