KR20130115951A - Composition comprising ginsenoside re as an active ingredient for treatment of depression, anxiety or learnig and memory impairments - Google Patents

Abstract

PURPOSE: A pharmaceutical composition and a health food for treating depression, anxiety disorder, or memory loss are provided to treat or alleviate depression, anxiety disorder, and memory loss and to suppress the expression of tyrosine hydroxylase (TH) in neurons. CONSTITUTION: A pharmaceutical composition for treating depression, anxiety disorder, or memory loss contains ginsenoside Re. The composition additionally contains ginsenoside Rg3, Rb2, Rg1, Rd, or Rb1. The content of the additional ginsenoside is less than the content of ginsenoside Re. Depression or anxiety disorder is induced by 5-HT. Ginsenoside Re suppresses the absorption of 5-HT. A health food for treating depression, anxiety disorder, or memory loss contains ginsenoside Re.

Description

Anxiety or memory impairment comprising ginsenoside Re as an active ingredient, and a pharmaceutical composition for treating depression, anxiety disorder or memory impairment comprising ginsenoside Re as an active ingredient,

The present invention relates to a pharmaceutical composition for treating depression, anxiety disorder or memory loss comprising ginsenoside Re as an active ingredient, and a health functional food.

The main active substance of ginseng has been known as ginsenosides extracted from the ginseng saponin compartment. Among them, ginsenoside Re is a compound having the following structure.

[Chemical Formula 1]

Ginseng is classified as an adatogen substance which improves the body's resistance to the harmful effects of physical, chemical and biological factors and helps restore the body's stellar nature irrespective of the direction of abnormal physiological function. There have been many researches on this subject.

It has been reported that PD-type ginsenosides such as ginsenosides Rb1, Rb2, Rb3, Rc, Rd and Rg3 have antioxidative activities (Choi, JH, SY Yoon, EJ Choi, YS Ryu, HS Ko Re, Rg1, Rg2, and Rf and Rf1, Rg1, Rg2, and Rf, respectively, are shown in Table 1. In the case of Ginsenoside Rb1 The same PT-type ginsenosides have been reported to show improvement in learning and memory function (Wang, YZ, J. Chen, SF Chu, YS Wang, XY Wang, 1 NH Chen, and JT Zhang. Rg1 ' s metabolites ginsenoside Rh1 and protopanaxatriol. J. Pharmacol. Sci. 109: 504-510.). As a main active ingredient of PT-type ginsenoside, ginsenoside Re can be extracted mainly from the fruit of ginseng, and it is effective in strengthening immune system, protecting myocardial necrosis, improving digestive function, Have already been reported.

Depression and anxiety disorders are the most common psychiatric dysfunctions. Depression primarily affects chronic depression, most of which is accompanied by sleep disturbances, low self-esteem, shame, and suicidal tendencies. Therefore, depression is considered to be a complex disorder, and the mechanism associated with its onset is unclear. Anxiety disorders are mainly mental illnesses that are manifested by uneasy feelings. The main characteristics of anxiety disorder are continuous (eg, activation or anxiety, catatonia, fear, etc.) associated with syndromes such as autonomic nerves disturbance, muscle rigidity, exercise disturbance, Tense emotions.

Depression and anxiety disorders have been reported to coexist with each other (Mineka, S., and R. Zinbarg, 2006. A contemporary learning theory perspective on the etiology of anxiety disorders: 61: 10-26).

Currently, anti-depression pharmaceuticals have been shown to inhibit the uptake of 5-hydroxytryptamine (5-HT), norepinephrine (NE) and dopamine (DA)  , Paxil  , Zoloft  These agents include selective serotonin reuptake inhibitors (SSRIs), serotonin-norepinephrine reuptake inhibitors (SNRIs), norepinephrine and dopamine reuptake inhibitors (NDRI). The mechanism by which these antidepressant restrictions work is by increasing the amount of neurotransmitters such as 5-HT to relieve the symptoms of depression. However, circulating anti-depressive agents have been associated with increased risk of suicide, headache, dizziness, dizziness, insomnia, excessive sleepiness, tinnitus, thirst, impulse, weight gain, increased blood pressure, stomach upset, regurgitation nausea, They have other types of side effects such as indigestion, diarrhea, constipation, leg pain, skin rash, irritability, seizures, hyperhidrosis, edema, sexual desire, impotence and the like.

The current main anti-anxiety medicine is a mechanism to regulate the activity of gamma-aminobutyric acid (GABA), an inhibitory neurotransmitter, to reduce and stabilize the symptoms. Gt; benzodiazepine < / RTI > However, benzodiazepines have many side effects including insomnia, hypersensitivity, myalgia, weakness, nausea, movement disorders, blurred vision, tiredness, disturbance, and delusions.

The researchers have reported that the decline in memory is due to brain cell death due to brain cell death, and thus correlates between decreased function of cholinergic nervous system and decreased memory.

Currently, the main memory decelerating drugs include acetylcholinesterase inhibitor (ACE), which can inhibit the activity of acetylcholine esterase (ACE), a degrading enzyme of acetylcholine in the brain, or a certain level of acetylcholine Acetylcholine precursors and acetylcholine receptor agonists that can give a large amount of antioxidants. For example, acetylcholine degradation inhibitors include tacrine, aricept and galantamine, which are commercially available under the approval of the FDA, lecithin and acetylcholine receptors for acetylcholine synthesis precursors, Activators include RS-86, nicotine, and the like. However, when the memory decay progresses, not only the acetylcholinergic neurons but also the glutamate-producing neurons, which occupy the majority of the neurons, are degenerated. Therefore, the above method focusing on the activity enhancement of acetylcholine has a problem of being temporary and weak.

Therefore, it is necessary to develop drugs for the treatment of depression, anxiety disorder and memory loss which do not have the above-mentioned side effects and continue to be effective. More recently, there has been a growing interest in the development of natural products and medicinal plants for the treatment of depression, anxiety disorders and memory impairment.

This study investigates whether stressful stimuli in animal models can treat the symptoms such as depression, anxiety disorder and memory decline. In the case of exposed lethal stress, it is necessary to perform forced swimming test (FST), elevated cruciate Experimental (EMP) and active avoidance condition (AAT) tests have been used to test the therapeutic effects of depression, anxiety disorders and memory loss.

It has been reported that the antidepressant effect can be measured using the forced swimming test (FST), and the antifouling effect can be measured using the elevated cruciate test (EMP). In addition, it has been reported that the memory enhancing effect can be measured by an active avoidance condition (AAT) test (Vyas, A., R. Mitra, BS Rao, and S. Chattarji 2002. Chronic stress induces contrasting patterns of dendritic remodeling in hippocampal and amygdaloid neurons, J. Neurosci., 2: 6810-6818).

It has also been reported that the hypothalamic cortical-stimulating hormone-releasing system is associated with the modulation of hypothalamic-pituitary-adrenal (HPA) axis amplification and depression and anxiety-related behavior induced by repeated custodial stress , WM, L. Richter, and DJ Stein, 2004. The effects of repeated intra-amygdala CRF injections on rat behavior and HPA axis function after stress Metab Brain Dis 19: 15-23).

Therefore, the inventors of the present invention conducted experiments on forced swim test (FST), elevated cruciate test (EMP) and active avoidance condition (AAT) test to determine whether ginsenoside Re extracted from the ginseng saponin compartment has depression, And the present inventors have completed the present invention.

The present invention is to provide a pharmaceutical composition and a health functional food for the treatment of depression, anxiety disorder or memory loss comprising ginsenoside Re as an active ingredient.

In order to solve the above problems, the present invention provides a pharmaceutical composition for treating depression, anxiety disorder and memory loss including ginsenoside Re.

The term "ginsenoside Re" used in the present invention is a compound having the following chemical structural formula and is one of the main components of ginseng.

[Chemical Formula 1]

The main active substance of ginseng is known as ginsenosides extracted from the ginseng saponin compartment. The ginsenoside Rx (X = a, b, c, d, e, etc.) ).

The ginsenoside Re used in the present invention can be extracted from ginseng, but not limited thereto, and can be synthesized by a known synthesis method. In the present invention, ginseng used for extracting ginsenoside Re is preferably subjected to heat treatment at a high temperature of 70 to 150 캜 for 2 to 6 hours.

Panax ginseng, Panax quinquefolia, Panax notoginseng, Panax japonica, Panax trifolia, and Panax ginseng can be used to extract ginsenoside Re from ginseng. ), Panax pseudoginseng, Panax vietnamensis, Panax elegatior, Panax wangianus, or Panax bipinratifidus, and the outbreaks, roots, Stem, or leaf.

The term "extract" as used in the present invention is to remove the ginseng using a solvent of the liquid in the solvent is C _1, such as water, methanol, ethanol, butanol, To C ₄ alcohol, ethyl acetate, or a mixed solvent thereof may be used.

The term "depression" as used in the present invention is a type of psychiatric disorder characterized mainly by sadness, tastelessness, loss of emotion, lack of pleasure (lack of pleasure), grief, agitation or delay, guilt and worthlessness, Are accompanied by symptoms such as suicide, hallucinations and delusions.

The term "anxiety disorder" as used herein refers to a variety of psychological and physical anxiety symptoms that manifest themselves as an attack (panic disorder) or a persistent condition (generalized anxiety disorder) .

The term "memory loss" as used in the present invention is a phenomenon caused by brain dysfunction, which occurs in diseases such as dementia.

The pharmaceutical composition for the treatment of depression, anxiety disorder or memory loss according to the present invention can treat symptoms caused by depression, symptoms caused by anxiety disorders, and memory decay symptoms. It is preferred that the depressive or anxiety disorder of the present invention is induced by 5-HT and that ginsenoside Re does not cause side effects while inhibiting absorption of 5-HT.

The pharmaceutical composition for the treatment of depression, anxiety disorder or memory loss according to the present invention contains 0.001 to 99.9% by weight, preferably 0.1 to 99% by weight, more preferably 1 to 99% by weight, of ginsenoside Re, By weight to 50% by weight.

A pharmaceutical composition for the treatment of depression, anxiety disorder or memory loss of the present invention may further comprise ginsenosides Rg3, Rb2, Rg1, Rd or Rb1. The content of the additional ingredient may be preferably added in the range of 0.1 to 20 parts by weight per 100 parts by weight of the therapeutic pharmaceutical composition. Further, the content of the additional component is preferably less than the content of ginsenoside Re.

According to the forced swimming test according to the embodiment of the present invention, the floating behavior observed in the rat is similar to the state of deceased fragility or helpless depression in humans. According to one embodiment of the present invention, a forced swimming test was used (Getachew, B., SR Hauser, RE Taylor, and Y. Trizabi. 2008. Desipramine blocks alcohol-induced anxiety and depressive-like behaviors in two rat Pharmacol. Biochem, Behav. 91: 97-103), the increase in immobility due to repeated custodial stresses was evident for 2 days in the forced swimming test.

These results suggest that restraint stress increases the duration of immobility on the second day of the forced swimming test compared to the first day (Marks, W., NM Fournier, and LE Kalynchuk LE. 2009. Repeated exposure to corticosterone increases depression-like 98: 67-72). In this study, we compared the results of the two experiments. On the second day, measuring immobility time has been used as a means of measuring depression reactions, and on the second day, a decrease in immobility time indicates depressive activity (Siuciak, JA, DR Lewis, SJ Wiegand, and RM Lindsay, 1997 Antidepressant-like effect of brain-derived neurotrophic factor (BDNF). Pharmacol. Biochem. Biology 55: 131-137).

According to one embodiment of the present invention, administration of GRe (ginsenoside Re) in a forced swimming test reduces immobility and increases avoidance behavior, but has no effect on swimming behavior. It was found that GRe was effective in treating depression without causing any change in motor function.

According to one embodiment of the present invention, in the elevated cruciate ligament test (EMP), the administration of GRe showed an increase in the number of open arm entries and an increase in the time ratio of the open arms to the total time. This study confirmed the therapeutic effect of GRe anxiety. In addition, the anxiety observed in the hyperbolic cruciate lab was reported to be related to the 5-HT overload in the hippocampus (Carvalho, MC, S. Masson, ML Brandao, and MA de Souza Silva. 2008. Anxiolytic like effects of substance P administration into the dorsal, but not ventral, hippocampus and its influence on serotonin. Peptides. 29: 1191-1200). Repeated cramping stress increased the turnover and release of 5-HT, which is associated with behavioral and physiological responses to stimuli in certain areas of the brain, such as the hippocampus. However, administration of GRe was able to inhibit the increase in 5-HT release due to repeated exposure of cadaveric stress in the hippocampus.

In one embodiment of the present invention, tyrosine hydroxylase (TH) is an enzyme involved in stress-induced activity in the central nervous system related to stress-related psychopathological conditions such as depression, anxiety and cognitive disorders. The noradrenergic neurotransmitter system, fundamentally derived from A6 noradrenaline neurons in clinical LC, is a major circuit in the central nervous system associated with stress responses. TH expression in younger children increased with repeated exposure to stress. In one embodiment of the present invention, the TH immunoreactivity in young as a response to repeated cramping stress was further increased in the STR group than in the control group. However, administration of GRe was able to restore increased TH immunoreactivity.

In one embodiment of the present invention, reduced BDNF expression in hippocampus is associated with the onset of cognitive impairment (Naert, G., G. Ixart, T. Maurice, L. Tapia-Arancibia, and L. Givalois 2011. Brain-derived neurotrophic factor and hypothalamic-pituitary-adrenal axis adaptation processes in a depressive-like state induced by chronic restraint stress. Mol.Cell Neurosci. 46: 55-66). According to one embodiment of the present invention, repeated custodial stress caused a decrease in the expression of BDNF mRNA in the hippocampus of the rat as well as learning and memory decline. However, the level of expression of reduced BDNF mRNA was restored in the hippocampus of the rat subjected to cramping stress due to the administration of GRe.

Therefore, according to the embodiment of the present invention, the repetitive cramping stress increased the immobility period in the forced swimming test and decreased the open arm in the high frictional cruciate compared to the unstressed normal control. It is also related to the impairment of cognitive function in the active avoidance condition test. Administration of GRe reduced symptoms such as depression and anxiety and reduced cognitive impairment due to repeated custodial stress. These results indicate that the GRF's central nervous system is regulated by the hypothalamic CRF associated with the noradrenergic system. Thus, GRe may be a useful substance in the development of alternative drugs for the treatment of stress-related disorders such as depression, anxiety disorders and memory loss.

According to an embodiment of the present invention, the dose-dependent activity of GRe (10, 20 or 50 mg / kg) was examined and the most effective in controlling depression, anxiety and memory impairment due to repeated cramping stress induction at 50 mg / kg . Also, according to embodiments of the present invention, repeated cervical stresses indicate that an adrenocorticotropic hormone-releasing (CRF) circuit is activated in the hypothalamus and anxiety and depressive-like behaviors observed by behavioral testing occur.

In addition, the expression and secretion of CRF in the hypothalamic spinal nucleus (PVN) were significantly increased in the STR group compared to the control group in the present study. This is a previous study (Maccari, S., and S. Morley-Fletcher, 2007. Effects of prenatal rrestraint stress on the hypothalamus-pituitary- adrenal axis and related behavioural and neurobiological 24 alterations. Psychoneuroendocrinology 32: S10-15.

Administration of GRe significantly inhibited the increase of CRF immunoreactivity in the hypothalamic spinal nucleus (PVN). The result of regression of CRF in the hypothalamus of GRe due to repeated custodial stress in these rats was found to be related to the therapeutic effect and anxiolytic effect of depression in GRe.

As used herein, the term "treatment" means any action that improves or alleviates a symptom of a disease upon administration of a composition comprising ginsenoside Re.

For the administration of the composition of the present invention, the composition of the present invention may contain a pharmaceutically acceptable carrier, excipient or diluent in addition to the above-mentioned effective ingredient. Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The composition of the present invention may be formulated in the form of oral, granule, tablet, capsule, suspension, emulsion, syrup, aerosol or the like oral preparation, external preparation, suppository or sterilized injection solution according to a conventional method. In detail, when formulating, it can be prepared by using diluents or excipients such as fillers, weighing agents, binders, humectants, disintegrants, surfactants and the like which are generally used. Solid formulations for oral administration include, but are not limited to, tablets, pills, powders, granules, capsules, and the like. Such a solid preparation can be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose, lactose, gelatin and the like, in the ginsenoside Re. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration, liquid paraffin, and various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like. Examples of the suppository base include withexol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The composition of the present invention may be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) depending on the desired method, and the dose may be determined depending on the condition and weight of the patient, The mode of administration, the route of administration, and the time, but may be suitably selected by those skilled in the art. The daily dose of ginsenoside Re is preferably 1 mg / kg to 500 mg / kg, and may be administered once or several times a day, if necessary.

The present invention also provides a health functional food for improving depression, anxiety disorder or memory loss including ginsenoside Re.

The ginsenoside Re that can be used in the health functional food of the present invention is the same as that described above in the pharmaceutical composition for treating depression, anxiety disorder or memory loss.

The symptoms of depression, anxiety disorder or memory loss which can be improved through the health functional food of the present invention are the same as those described above in the pharmaceutical composition for treating depression, anxiety disorder and memory loss.

The term "improvement" as used in the present invention means any action that improves the symptom of the disease by the administration of the composition containing the ginsenoside Re.

The health functional food may contain flavoring agents such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and thickening agents (cheese, chocolate etc.), pectic acid and its salts, alginic acid and its salts, Organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. In addition, it may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks. These components may be used independently or in combination. In addition, the health functional food may be in the form of any one of meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, gum, ice cream, soup, beverage, tea, functional water, Lt; / RTI >

In addition, the health functional food may further include food additives, and the suitability of the food functional food as a "food additive" Standards and standards.

Examples of the products listed in the above-mentioned "food additives" include natural products such as ketones, chemical products such as glycine, potassium citrate, nicotinic acid and cinnamic acid, coloring matter, licorice extract, crystalline cellulosic, high- , A sodium L-glutamate preparation, a noodle-added alkaline agent, a preservative preparation, a tar coloring agent, and the like.

The present invention is not only pharmaceutically acceptable as a composition for the treatment or amelioration of depression, anxiety disorder and memory loss including ginsenoside Re derived from a natural substance, but also useful as a health functional food.

Figure 1 shows an experimental schedule for the treatment or amelioration effect of depression, anxiety disorder or memory loss in the case of administration of GRe in the case of inducing repeated cramping stress on the rat.
Fig. 2 shows the result of examining the immobility time in the forced swimming test for the rat. * P < Day 1 group (B); * p < CON group (C); ** p < CON group and # p < STR group (D).
FIG. 3 shows the result of examining the time of the climbing behavior in the forced swimming test for the lattice. * P < Day 1 group (B); * p < CON group (C); ** p < CON group and # p < STR group (D).
Figure 4 shows the time spent in the open arm and the number of times it enters the open arm in the elevated cruciform experiment with the lattice. * P < 0.05, ** p < CON group; # p < STR group.
Fig. 5 shows waiting time A and escape failure (B) required to enter the room in the opposite direction as a conditional avoidance reaction in the active avoidance condition test for the rat. * P < 0.05, ** p < CON group; # p < STR group.
Figure 6 shows CRF expression in hypothalamic PVN versus lattice and TH expression in LC. (A), STR-CRF (B), STR + GRe50-CRF (C), CON-TH (D), STR-TH (E) and STR + GRe50-TH (F). The unit bar represents 50 mu m.
FIG. 7 shows the effect of GRe on the expression of CRF in the hypothalamic PVN and the effect of GR on the TH expression in LC for a 10-day repeated lethal stress. ** p <0.001 vs. CON group, #p < STR group.
Figure 8 shows RT-PCR bands (A) and their relative intensities (B) of the two brain innervation factor (BNDF) mRNAs in the hippocampus of a rat subjected to repeated cramping stress for 10 days. ** p < 0.05 and ** p < CON group, # p < STR group.

Hereinafter, the present invention will be described in more detail with reference to the following examples. The following examples are for illustrative purposes only and are not intended to limit the scope of the invention.

Example  1: Materials and Methods

Animal preparation

A 240-280 g male Sprague-Dawley rats were purchased from Samtaco Animal Co. (Seoul, Korea). Rets were kept in a limited access facility for up to five animals per polycarbonate cage. The cage temperature was maintained at 22 ° C ± 2 ° C and the relative humidity was maintained at 55% ± 15%. The cage was ignited by artificial light for 12 hours daily. Sterile drinking water and a standard feeding diet were randomly fed into each cage during the experiment. Animal testing was conducted in accordance with the National Institutes Health Guide to Laboratory Animals Management and Use (NIH Publication No. 80-23), revised in 1996. All animals were used at least 7 days after the animal arrived.

Gin Senocide Re

Ginsenoside Re was isolated from red ginseng (RG, purchased from Korea Tobacco Ginseng Corporation). Red ginseng was extracted 4 times with 80% ethanol under reflux conditions at 80 ° C for 2 hours. Next, saponin components were extracted by Diaion HP-20 (Mitsubishi Jasei, Kasei, Japan) adsorption chromatography. The saponin extract was freeze-dried and made into fine powder. The powder of the saponin extract was dissolved in water and adsorbed to benzene ethylene resin (Mitsubishi Kasei, Kasei, Japan 2.8 x 20 cm) with 200 times (v / w) water of saponin weight. The total saponin adsorbed on the resin phase was washed with 10 times the weight of the resin, and the non-saponin component was extracted with 20% ethanol of 8 times the weight of the resin. Thereafter, ginsenoside Re was extracted with 30% ethanol which was 8 times the resin weight. All analyzes were performed on an HPLC system equipped with an automated gradient controller, a Waters ™ 510 pump and a Water 484 UV detector (MA, USA). Separation was carried out using a Waters Nova-Pak C ₁₈ (3.9 × 150 mm, 5 μm) column (MA, USA) and a mobile phase with a mixture of acetonitrile and water in the eluent.

Experimental group  Classification

Six groups of 7 rats were randomly divided as follows.

(CON, n = 7) in which saline (0.9% NaCl) was administered daily instead of GRe (ginsenoside Re)

- Stretch stress group (STR, n = 7) treated daily with saline instead of GRe

- cervical stress and 10 mg / kg GRe-treated group (STR + GRe 10, n = 7)

- cervical stress and 20 mg / kg GRe-treated group (STR + GRe 20, n = 7)

- cervical stress and 50 mg / kg GRe-treated group (STR + GRe 50, n = 7)

- custodial stress and 10 mg / kg fluoxetine-administered group (STR + FLX, n = 7 as positive group).

Fluoxetine (FLX) was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Rette administered peritoneal with either GRe or fluoxetine for 30 minutes before giving them daily cramping stress for 10 days. GRe and fluoxetine were dissolved in 0.9% physiological saline prior to use.

Custodial stress was applied from 10:00 AM to 12:00 PM for two hours, once a day, for 10 consecutive days in lethal cigarette bags. The rats were placed in a transparent plastic tube (20 x 7 cm) with one end conical and a 3 mm bore for breathing and the other end open. The animals had sufficient air, but it was difficult to move within the tubes.

Statistical analysis

All measurements were performed by independent observers. The results of the numerical values were expressed as means ± standard error of means. Within the behavior values in the FST test, Levine's test was used to compare the first and second days using Student's t-test to make an appropriate assumption of the variance averages.

Differences between and within mean variance data were analyzed by ANOVA using SPSS (Version 13.0; SPSS, Inc., Chicago, IL, USA) and then Tukey's post-hoc test was performed. Statistical significance was set at p <0.05.

Example  2-1: Forced swimming test ( FST )

The forced swimming test is used to measure the activity of potential depressive remedies in the rats. Forced immersion of mice in water for long periods of time causes floating motion. GRe reduces float motion with escape reactions and increased escape reactions such as swimming.

At 25 占 폚, a transparent plexiglass cylinder (20 cm diameter x 50 cm high) was filled with water at a height of 30 cm. At this depth, the litt does not touch the bottom or bottom of the cylinder's tail or two legs. On the first day, the rats in all groups were placed in a water-filled cylinder and tested for 15 minutes. After the test, the latt was dried and returned to the home cage of the latt.

Twenty-four hours later, the rats were forced to swim for 5 min / day for 2 consecutive days and observed for escape behavior (avoidance reaction and swimming). The dwell time was measured during the test time of 5 minutes.

The avoidance behavior was defined as the upward-directional motion of the paw on the side of the swimming pool, and the swimming was regarded as a behavior across the swimming pool, including crossing other quadrants.

Floating behavior was measured as the length of time the animal did not exhibit escape behavior (for example, the behavior of the test total time immobility was measured as the length of time the animal did not see the escape behavior (eg, Response and time to swim action). Animal behavior was recorded continuously by a video camera on the head.

After the test, the latt was removed from the tank, dried with a towel, and sent back to the home cage.

Example  2-2: Forced swimming test ( FST ) result

We measured whether exposure to custodial stress accelerated depressive symptoms associated with forced swimming testing. An increase in immobility during the first two days of a forced swimming test (eg, a floating rate in two days compared to a floating rate in one day) was greater in the stressed lattice compared to the non- Respectively. Normal mice in the control (CON) did not measure an increase in immobility during the first two days of the forced swimming test (Fig. 2A; Student's t-test, p = 0.036). This was normalized to the value of the immobility at day 1 (FIG. 2C) and quantified as the value of the immobility at day 2.

It was confirmed that the normal rats in the control group did not show an increase in immobility. The level of immobility in the lattice in the STR group was analyzed by injecting repetitive cramping stress for 10 days. Twenty-four hours after repeated custody, the stressed rats in the STR group showed a significant increase in immobility during the first two days in the forced swimming test (Fig. 2B; Student's t-test, p &lt; 0.05).

The normalized immobility period of the rats in the STR group showed a significant increase in the immobility period during the forced swimming test as compared to the control (Figure 2C; Student's t-test, p &lt; 0.05).

This study also measures the effect of GRe on immobility in depression - like behavior induced by custodial stress. Rette in the STR + GRe50 group showed a significant reduction in immobility time in the forced swimming test for 5 minutes compared to the STR group (p <0.05), indicating that administration of GRe is effective in reducing depressant-like behavior. The results also showed that the decrease in immobility to depressive-like behavior in the STR + GRe50 group was nearly similar to that in the STR + FLX group (Fig. 2D).

Likewise, we investigated another key behavior that appeared as "avoidance behavior". During the first two days of the forced swimming test, we found that avoidance behavior increased in the control group (Fig. 3A; Student's t-test, p = 0.012). Furthermore, the Rets at repeated custodial stresses showed a significant decrease in climbing behavior (Fig. 3B; Student's t-test, p &lt; 0.05).

In order to measure the normalized value of the avoidance behavior, the ratio of the time spent on the avoidance action on the 1st day to the time spent on the avoidance action on the 2nd day was measured. The cervical stressed rats (STR group) showed significant avoidance behavior reduction during the forced swimming test as compared to the control (Figure 3C; Student's t-test, p &lt; 0.05).

Compared to the STR group (p <0.05) in the forced swimming test, the lett in the STR + GRe50 group showed a significant recovery of avoidance behavior for 5 minutes, indicating that administration of GRe reduces depressant-like behavior. This also showed that restoration of avoidance behavior in the STR + GRe50 group was almost similar to recovery of the STR + FLX group (FIG. 3D). However, repeated restraint stress did not induce significant differences in swimming behavior in any group during the forced swimming test.

Example  3-1. High-priced meal A cross maze  ( EPM ) Test

The high-cost cruciate test is a widely performed behavioral test to measure the effects of anxiety or anxiety relief in drug preparations (Walf, AA, and CA Frye. 2007. The use of the assay for anxiety -related behavior in rodents, Nat. Protoc. 2: 322-328).

The elevated cruciform test was performed for 12 days. The device consists of two open arms (50 × 10 cm each), two closed arms (50 × 10 cm each) and a central platform (10 × 10 cm) with two arms of each type located in opposite directions do. The labyrinth was made of black plexiglass and was made 50 cm high from the floor. Exploration on the open arm side was performed with indirect faint light (2 × 60 W).

At the beginning of each attempt, the animal was placed in the middle of the maze, facing a closed arm. During the 5 minute test time, the following parameters were recorded:

b) the number of times it enters the closed arm, c) the time spent by the open arm, and d) the time spent by the closed arm.

The entry of an animal into an arm is defined as the state where the animal's four feet are located on the arm. After each of the rats had finished the experiment, the maze was washed with alcohol. Behavior in the labyrinth was recorded using a video camera mounted on the ceiling above the center of the labyrinth and delivered to the S-MART program (PanLab, Barcelona, Spain). In an elevated crucifix, the reduction in anxiety displayed by an open arm exploration is defined as an increase in the number of times an open arm enters the open arm or the total number of times it enters a closed arm. It is also defined as an increase in the rate of time spent by the open arm over the total time spent by the open arm or closed arm. Also, the number of times to enter the whole arm is used as an index for the change of the movement ability of the rat.

Example  3-2. High-priced meal A cross maze  ( EPM ) Test results

The effect of GRe administration on anxiety, expressed by a decrease in open arm exploration in a high-cost crossmaze test, was measured (Figure 4). A post-analysis comparison confirmed a significant reduction in the percentage of time spent in the open arms of the labyrinth after repeated cramping stresses for 10 days compared to the control (Figure 4). However, compared to the STR group (p <0.05; Fig. 4A) in the STR + GRe50 group, for the open arm of the labyrinth, Rette showed a significant recovery in the ratio of time spent in the open arm of the labyrinth by repeated custody .

In addition, post-analysis comparisons show a significant reduction in the number of maze open-arm accesses after exposure to repeated cortical stress for 10 days compared to the control (p <0.05).

In the STR + GRe50 group, Rette also showed significant recovery in total number of accesses to and from the open arm of the labyrinth compared to the STR group (p &lt;0.05; Figure 4B). As a significant difference in the number of times of entering and leaving a closed arm in a high-cost crossmaze test was not observed among the groups, we found that the anxiety-like behavior of the Lets due to repeated custodial stress was due to differences in their athletic capacities I could.

Example  4-1. Active avoidance condition test ( AAT )

An active avoidance condition test measures the ability of an animal to avoid an abomination event. It also provides an approach related to learning and memory (Grauer, SM, VL Pulito, RL Navarra, MP Kelly, C. Kelley, R. Graf, B. Langen, S. Logue, J. Brennan, L. Jiang, E. Charych, U. Egerland, F. Liu, KL Marquis, M. Malamas, T. Hage, TAComery, and NJ Brandon. 2009. Phosphodiesterase 10A inhibitor activity in preclinical models of the positive, cognitive, and negative symptoms of schizophrenia. Pharmacol. Exp. Ther., 331: 574-590). In this experiment, a Gemini Avoidance System (SD Instruments) was used. Each RET was placed in a two-way shuttle box (23 cm x 50 cm x 23 cm) consisting of two stainless steel bars (3 mm diameter, 1 cm apart) and two 28 V DC lights. The electric shock was transmitted to the floor by an isolated impact generator (Behbood Pardaz Co. Iran).

After a 5 minute adaptation period in the shuttle box, Rette underwent 50 avoidance trails by variable spacing (range = 7.5-22.5 s). Each walkway consisted of a warning tone and a stimulus light (control stimulus) for 10 seconds. If the animal crossed the archway for the first 10 seconds of each attempt, the warning tone and light were terminated. Conditional avoidance responses were defined by crossing the opposite room within the first 10 seconds of each attempt.

After exactly 24 hours, the latt was put back into the shuttle box. Each trial consisted of a 10 second warning tone and an electric shock (0.5 mA; unregulated stimulus) through the side and floor where the latt was located. After the start of the electric shock, if the animal crosses the archway, the warning tone and electric shock are terminated. An escape reaction is defined as a crossing into the opposite room within the first 10 seconds of each attempt. The memory retention test (escape reaction) was carried out with 20 avoidance trails. If there was no escape reaction within 10 seconds after the electric shock was applied, the electric shock and tone-controlled stimulus was interrupted and the escape was recorded as failure.

Example  4-2. Active avoidance condition test ( AAT ) result

To prove the correlation between repeated custodial stress-induction and cognitive memory impairment and also to confirm the cognitive decline healing effect of Ginenoside Re, we conducted an active avoidance condition test on the caretak stress and the lette treated with ginsenoside Re Respectively. Lit of the STR group showed a decrease in latency to enter the opposite room compared to the control group (p <0.05; Fig. 5A). On the other hand, the RET of STR + GRe20 and STR + GRe50 groups showed an increase in waiting time to enter the room in the opposite direction as compared with the STR group.

After 24 hours, the effect of GRe administration on the waiting time to enter the room in the opposite direction was measured by applying an electric shock to the bottom of the shuttle box in the active avoidance condition test. Regarding memory, Rets in the STR group showed a significant increase in waiting time to enter the room in the opposite direction to the escape failure compared to the control group's waiting time (p <0.01; Fig. 5B). However, in the STR + GRe50 group, compared to the waiting time of the STR group (p &lt; 0.05), it showed a significant decrease in waiting time to enter the room in the opposite direction.

According to this experiment, repeated exposures to custodial stress impaired short-term memory in the lattice. And depressed stress - induced memory loss due to administration of GRe. In the STR group, conditional avoidance response decreased and escape failure increased. In addition, the percent of conditional active avoidance response or escape failure observed in the STR + GRe50 group was equal to the percent of control. Recovery of cognitive function of stress-related memory impairment in STR + GRe50 group was substantially similar to STR + FLX group.

Example  5-1. Corticotropin-releasing factor ( CRF ) And Tyrosine  Immunohistochemistry of hydroxylase

For immunohistochemical studies, animals were sacrificed with sodium pentobarbital (80 mg / kg, intraperitoneal injection), perfused through the aorta with normal saline (0.9%) and resuspended in 0.1 M phosphate- buffered saline Was treated with 300 ml of 4% paraformaldehyde (per liter).

After the brain of the rat was removed, it was stored frozen at 4 ° C in 0.1 M PBS with 20% sucrose through post fixation at night. The coronal section of the brain at 30 μm thickness was cut across the hypothalamus and LC (locus coeruleus) using a microtome (Leica CM1850; Leica Microsystems Ltd., Nussloch, Germany).

The cleaved portion was stained with antibodies for CRF and TH expression using the avidin-biotin-peroxidase complex (ABC) method. The cleaved portion was washed 3 times with PBS for 5 minutes. (1: 2000 dilution, 1: 2000 dilution, Santa Cruz Biotechnology Inc., California, CA, USA) in PBST (PBS plus 0.3% Triton X-100) (sheep) anti-TH antibody (1: 2000 dilution, Chemicon International Inc., Temecular, CA, USA).

The cleaved portion was washed in PBS for 5 minutes and incubated with a biotin-conjugated rabbit anti-goto IgG secondary antibody (for anti-CRF antibody) and a biotin-conjugated goat anti-sheep IgG secondary antibody (anti- TH antibody). Secondary antibodies were obtained from Vector Laboratories Co. (Burlingame, CA, USA) and diluted 1: 200 in PBST containing 2% normal serum.

To identify areas of immune response, the cuts were incubated in ABC reagent (Vectastain Elite ABC kit; Vector Labs Co., Burlingame, Calif., USA) for 90 minutes, washed three times in PBS for 5 minutes, (DAB; Sigma-Aldrich Chemical Co., St. Louis, Mo., USA) and 0.01% H ₂ O ₂ for 1 minute.

Finally, after briefly washing in distilled water, tissues were washed with PBS and mounted on slides, respectively. The slides were dried and covered. Images were acquired using an AxioVision 3.0 imaging system (Carl Zeiss, Inc., Oberkochen, Germany) and processed using Adobe Photoshop (Adobe Systems, Inc., San Jose, CA, USA). The cut portion was enlarged (× 200), and the number of cells in ² × 100 × 100 μm was measured.

Example  5-2. Corticotropin-releasing factor ( CRF ) And Tyrosine  Immunohistochemical results of hydroxylase

Corticotropin-releasing factor (CRF) -like immunoreactivity was analyzed in the hypothalamus part of the cell body containing the hypothalamic paraventricular nucleus (PVN) (FIG. 6). In the STR group, the number of adrenocorticotropic hormone-releasing factor immunoreactive fibers in the hypothalamic brainstem (PVN) increased to 145.60%. Due to repeated cramping stress, there was a significant increase in CRF expression when compared to the control group. Compared with the STR group (FIG. 7), the number of CRF-immunoreactive neurons was significantly reduced in the hypothalamic chamber in the STR + GRe50 group (p <0.05). The increase in CRF-immunoreactivity due to repeated cramping stress was significantly restored by GRe administration, and the number of CRF-immunoreactive neurons in the STR + GRe50 group was significantly similar to that of the control group.

Tyrosine hydroxylase (TH) -like immunoreactivity was analyzed in the adrenalin region of the cell organs containing the locus coeruleus (Fig. 6). In the STR group, the brain of LETT increased the number of tyrosine hydroxylase immunoreactive fibers to 138.76% in young. RET repeatedly exposed to custodial stress showed a significant increase in tyrosine hydroxylase expression compared to the control (p < 0.01). In STR + GRe50 group (p < 0.05), a significant number of TH-I immunoreactive neurons decreased in the central adrenaline region compared to the number of neurons in the STR group (Figure 7).

These results indicate that the increase in the number of TH-immunoreactive neurons in the rat with repeated cramping stress is restored by GRe administration. And that the number of TH-immunoreactive neurons in the STR + GRe50 group is similar to the number of TH-immunoreactive neurons in the STR + FLX group.

Example  6-1. all RNA  Manufacturing and RT - PCR  Analysis method

The hippocampus was collected from four rats in each group. Total RNA was isolated from TRIzol  Were obtained from samples of the brain using reagents (Invitrogen Co., Carlsbad, Calif., USA) and used to extract RNA according to the supplier's instructions. The complementary DNA was synthesized from total RNA with reverse transcriptase.

BDNF mRNA expression levels were measured by reverse transcription-polymerase chain reaction (RT-PCR). RT-PCR was performed using a PTC-100 programmed thermal modulator (MJ Research, Inc., Watertown, Mass., USA).

The operating conditions were as follows:

For glyceraldehyde-3-phosphate dehydrogenase (GAPDH), denaturation at 30 cycles at 95 ° C. for 30 seconds, annealing at 58 ° C. for 30 seconds, and extension at 72 ° C. for 30 seconds,

For BDNF, denaturation to 27 cycles at 95 DEG C for 30 seconds, annealing at 57 DEG C for 30 seconds, and extension at 72 DEG C for 30 seconds.

All primers were designed using known mRNA sequences and primer design software (Primer 3, available from www.white.mit.edu, Cambridge, MA, USA), which is provided through the website. The following sequence was used:

CCT CCT TCT TCC AG-3 'and (reverse) 5'-CCT GCT TCA CCA CCT TCT TG-3' (forward direction)

; For BDNF (153 bp), 5'-CAG GGG CAT AGA CAA AAG-3 'and 5'-CTT CCC CTT TTA ATG GTC-3' (forward).

The product by PCR was separated on 1.2% agarose gel, stained with ethidium bromide, and the density of each band was analyzed using an image-analysis system (i-MaxTM7, CoreBio System Co., Seoul, Korea) . The complementary DNA expression level was calculated as the relative density of each BDNF band for GAPDH.

Example  6-2. all RNA  Manufacturing and RT - PCR  Analysis method result

The effect of GRe administration on the expression level of BDFN mRNA was investigated in the rat with repeated hatching stress induced hippocampal changes using RT-PCR analysis (Figure 8). BDNF mRNA expression levels were averaged by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA and housekeeping genes were used as internal modulators. In the hypothalamus of the rats, BDNF mRNA expression in the STR group was significantly reduced compared to the control group (p <0.01). Reduced expression of BDNF mRNA in the STR group was restored in the STR + GRe50 group (p <0.05). The restored levels were almost similar to normal rats in the control group. In the STR + GRe50 group, the recovery of the expression level of the marker of the neuron was almost similar to that of the STR + FLX group.

Claims (8)

A pharmaceutical composition for the treatment of depression, anxiety disorder or memory loss comprising ginsenoside Re.
The pharmaceutical composition according to claim 1, further comprising ginsenoside Rg3, Rb2, Rg1, Rd or Rb1, wherein the added ginsenoside content is less than the content of ginsenoside Re.
2. The pharmaceutical composition according to claim 1, wherein the depressive or anxiety disorder is induced by 5-HT and the ginsenoside Re inhibits the absorption of 5-HT.
2. The pharmaceutical composition according to claim 1, wherein the ginsenoside Re is extracted from ginseng.
5. The pharmaceutical composition according to claim 4, wherein the ginseng is heat-treated at a high temperature of 70 to 150 DEG C for 2 to 6 hours.
The method according to claim 4, wherein the ginseng is selected from the group consisting of Panax ginseng, Panax quinquefolia, Panax notoginseng, Panax japonica, Panax trifolia, Panax pseudoginseng Root, stem, or leaf of Panax vietnamensis, Panax elegatior, Panax wangianus, or Panax bipinratifidus. &Lt; / RTI &gt;
A health functional food for improving depression, anxiety disorder or memory loss including ginsenoside Re.
The health functional food according to claim 7, wherein the ginsenoside Re is extracted from ginseng.

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