KR101789796B1 - Composition for Preventing or Treating Mental Disorder comprising Fomes formentarius extract and Berchemia berche-miaefolia extract - Google Patents
Composition for Preventing or Treating Mental Disorder comprising Fomes formentarius extract and Berchemia berche-miaefolia extract Download PDFInfo
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Abstract
The present invention relates to a composition for preventing or treating mental disorders, which comprises an extract of Horseshoe-mushroom and a mugwort extract as an active ingredient. According to the present invention, the expression of the inflammatory cytokine induced by the hyper-hypothalamic-pituitary-adrenal axis (HPA) is controlled by including the extract of Horseshoe-mushroom and the extract of Aspergillus oryzae as an active ingredient, It is an anti-inflammatory regulatory mechanism by the extract of Mugwort, and has an excellent effect for preventing or treating mental disorder by down-regulating the expression of NFκ-B in the brain. As a natural product, it has few side effects on the human body, There is an effect.
Description
The present invention relates to a composition for preventing or treating mental disorders having an antidepressant activity, comprising a morselia extract of Aspergillus oryzae as an active ingredient.
Depression due to stress is not a temporary feeling of depression, but a pathological level caused by external and internal stress factors. Depression through stress is largely divided into depressive disorder (unipolar disorder) and bipolar disorder by various types of mental disorders (DSM-IV). Depressive disorder is classified into major depressive disorder, acute severe depressive disorder and chronic mild depressive disorder, depending on the degree and duration of depression. Bipolar disorder is manic depressive disorder commonly associated with mania and depression [1]. It is generally reported that bipolar disorder is more difficult to treat than nonpolar, and that treatment with mood stabilizers and antidepressants is difficult, and 30% of depressed patients are not responsive to treatment and go chronic [2]. Therefore, studies on the development of new antidepressant drugs are underway [3,4]. Symptoms usually cause a persistent depressed mood, as well as an anorexia, insensitivity and fatigue. The lifetime prevalence is 5-12% for males and 10-25% for females, and recurrence rate is over 50%, which is very common mental disease [5]. Most treatment drugs are improving symptoms of depression by inhibiting the reabsorption of monoamine hormones based on the monoamine hypothesis. However, single doses of antidepressants have not been shown to have significant effects compared with placebo [6], and the effects of combined treatment with antidepressants and stabilizers are highly individualized and can have many side effects. As a result, the relationship between depression, inflammation, and oxidative stress has been actively researched from the limit of the monoamine hypothesis.
On the other hand, Berchemia berche-miaefolia is a deciduous tree belonging to Rhamnaceae. It is a natural product which is known to be effective for detoxification and diseases such as cirrhosis.
In addition, horseshoe mushrooms are horseshoe-shaped, very hard-barked mushrooms belonging to the Polyporaceae horseshoe (Fomes), and are used for antitumor, antimicrobial, antioxidant, antipyretic and diuretic effects in folk remedies. Recent studies have shown that horseradish mushroom has diabetic improvement, immunomodulation, anti-inflammatory and anti-inflammatory, anticancer and antiviral effects, but the investigation of depression related mental disorders is insufficient.
Thus, the present inventor has made efforts to develop a composition capable of exhibiting an antidepressant effect while containing an effective ingredient derived from a natural material, which has less side effects on the human body than a chemical synthetic medicine and can be easily prepared and ingested. This application of the wood extract was confirmed and the present invention was completed.
Accordingly, it is a technical object of the present invention to provide a pharmaceutical composition for the prevention or treatment of psychiatric disorders comprising an extract of Horsetail mushroom and an extract of Aspergillus as an active ingredient.
Another object of the present invention is to provide a food composition for preventing or ameliorating psychiatric disorders, which comprises an extract of Horseshoe-mushroom and a mugwort extract as an active ingredient.
The present invention relates to a pharmaceutical composition for preventing or treating mental disorders, which comprises an extract of Horseshoe-mushroom and a mugwort extract as an active ingredient.
In the present invention, the Horseshoe-Mushroom Extract and Spruce Extract control the expression of an inflammatory cytokine induced by an activated hypothalamic-pituitary-adrenal axis (HPA) The extract is characterized by down-regulating the expression of NFκ-B in the brain. Wherein the inflammatory cytokine is iNOS or Nrf2.
The mental disorder may also be anxiety, depression, mood disorder, insomnia, delusional disorder, obsessive compulsive disorder, migraine, stress, memory disorder, cognitive disorder, senile dementia related disorder, Alzheimer's disease related disorder, Parkinson ' s disease, Related disorders, attention disorders, insomnia disorders and ischemic or brain trauma related disorders.
In addition, the present invention relates to a food composition for preventing or ameliorating mental disorders, which comprises an extract of Horseshoe-mushroom and a mugwort extract as an active ingredient.
According to the present invention described above, the expression of inflammatory cytokines induced by hyperactivity hypothalamic-pituitary-adrenal axis (HPA) is suppressed by the addition of mushroom extract of Horsetail mushroom and extract of mugwort , And the extract of the mugwort has an excellent effect in preventing or treating mental disorders by down-regulating the expression of NFκ-B in the brain, and is a natural product having few side effects on the human body and being easily produced and ingested .
1 is a schematic representation of a test method and plan according to an embodiment of the present invention.
FIG. 2 shows the result of the measurement of the immobility time of the forced swimming test in the oral administration of the extract of Horsetail mushroom in an embodiment of the present invention (Vei: FST induced Group, 1X: FST + FEE 100mg / kg treated Group, 4X: FST + FEE 25 mg / kg treated Group, Po: FST +
FIG. 3 shows the results of mobility evaluation according to total entries and total distance in Y-maze when Oral administration of horseradish mushroom extract in an embodiment of the present invention (Vei: FST induced Group, 1: FST +
FIG. 4 shows the results of analysis of signal transduction protein expression in soft tissue and adrenal gland when oral horseradish extract was orally administered in an embodiment of the present invention (I: Control Group, II: FST induced Group, III, IV: FST +
FIG. 5 is a graph showing the result of the measurement of the immobility time of the forced swimming test in the case of oral administration of the extract of the mulberry tree in one embodiment of the present invention. (Group II: only FST, Group III: FST +
FIG. 6 is a graph showing the results of cytokine (A) IL-1β (B) IL-6 (C) TNF-α concentration analysis in the case of oral administration of extract of Angelica keiskei koidz. ), Group Ⅱ: only FST, Group Ⅲ: FST +
FIG. 7 shows the results of the expression of phosphorylated -NFkBp65 protein in (A) adrenal (B) soft tissue (C) brain during orally administration of the extract of Fusarium oxysporum according to an embodiment of the present invention (Note that Group II: only FST, Group III : FST +
FIG. 8 shows the result of the measurement of the immobility time of the forced swimming test (FF: Fomes Fomentarius, BB: Berchemia berchemiaefolia) in oral administration of horseradish or mushroom extract mixture in an embodiment of the present invention. , High: 100 mg / kg, Low: 25 mg / kg, Positive:
FIG. 9 shows the results of analysis of signal transduction protein expression in soft water when oral administration of horseradish or mushroom extract mixture (FF: Fomes Fomentarius, BB: Berchemia berchemiaefolia High: 100mg / kg, Low: 25mg / kg, Positive: Sodium tianeptine 10mg / kg).
The present invention relates to a composition for preventing or treating psychiatric disorders having an antidepressant activity including an extract of Horseshoe mushroom and an extract of Aspergillus oryzae as an active ingredient, and the present invention will be described in detail below.
First, the term 'extract' as used herein means that it is used as a crude extract in the art, but it also includes fractions in which the extract is further fractionated. That is, the horsetail-mushroom extract and the broad-leaved tree extract used in the present invention include not only those obtained by using an extraction solvent but also those obtained by additionally applying a purification process thereto. For example, a fraction obtained by passing the above extract through an ultrafiltration membrane having a constant molecular weight cut-off value, a separation by various chromatotactypes (made for separation according to size, charge, hydrophobicity or affinity) Fractions obtained through various purification methods are also included in the extract of the present invention. In addition, the extract of Horseshoe Mushroom and Capsicum extract used in the present invention can be prepared in powder form by an additional process such as vacuum distillation, freeze-drying or spray-drying.
Also, the term " comprising as an active ingredient " is used herein to include an amount sufficient to achieve the efficacy or activity of the following extract of Horseshoe-mushroom and Spruce tree. Since the present invention is a composition extracted from natural horticultural mushrooms and moss trees, there is no adverse effect on the human body even when administered in an excessive amount. Therefore, the quantitative upper limit of the amount of the horsetail mushroom extract and chestnut tree extract contained in the composition of the present invention is .
In one aspect, the present invention relates to a pharmaceutical composition for preventing or treating psychiatric disorders, comprising an extract of Horsetail mushroom and an extract of Aspergillus oryzae as an active ingredient.
Wherein the mental disorder is selected from the group consisting of anxiety, depression, mood disorders, delusional disorders, obsessive compulsive disorders, migraine, stress, memory disorders, cognitive disorders, senile dementia, dementia related disorders, Parkinsonism related disorders, attention disorders, Or a brain trauma-related disorder.
In association with the mental stress response, the hypothalamic-pituitary-adrenal axis (HPA) is activated and a large amount of glucocorticoids are secreted. The HPA axis regulates the body's response to the regulation of the immune system, homeostasis, emotions and stress in the body. Abnormal overexpression of the HPA axis due to stress is known to be a direct cause of depression, and chronic stress has a significant impact on the neuroplasticity of the limbic system , Which is known to contribute to the pathophysiology of depression.
In addition, inflammation is an immune response caused by external and internal stimuli, which also causes an inflammatory response in psychiatric disorders. That is, when the HPA axis is activated and secretion of glucocorticoid is continued, the susceptibility to glucocorticoid decreases, adrenal exhaustion and receptor sensitivity decrease, and inflammation is exacerbated without controlling inflammation. Allostasis occurs in response to this physiological change. Repeated stress requires the cost of adaptation, called allostatic load, to maintain homeostasis, and causes damage to the body. In addition, the default mode is adjusted in the state of the damage, and even if the stress is reduced, the restoration is not performed well and the inflammation continues.
Thus, when the hyperpolarization of the HPA axis is induced by the stress stimulation, the secretion of the glucocorticoid increases, and when the activation of the HPA axis is continuously induced by the stress, the receptor sensitivity decreases and the inflammatory change eventually occurs , MAPK pathways stimulated by inflammation and oxidative stress (ROS) secrete IL-1, IL-2, IL-6, IFN-γ and TNF-α which are associated with inflammation-promoting activation of Nrf2 and NFκB Nrf2 and NFκB, which enter into the nucleus, induce protein transcription such as HO-1, which is antioxidant, and COX2, iNOS, and C reactive protein (CRP), which induce inflammation [7]. Specifically, increased secretion of inflammatory mediators such as proinflammatory cytokines, chemokines, acute inflammatory factors, and the like ultimately activates inflammatory signaling pathways such as NFκ-B in the brain, and thus NFκ-B signaling activity IL-1, TNF-α, and reactive oxygen intermediates (ROIs) directly affect the signaling pathway through the TLR and TNF receptors and phosphorylate p65, which is ultimately bound to the inhibitory κB (inhibitor κB) To enter the nucleus.
In accordance with one embodiment of the present invention, the extracts of P. pallens showed a decrease in the concentration of cytokines IL-1 and TNF-a in the blood, a decrease in the cortisol content in the adrenal gland and an effect of inhibiting p-p65 expression, It was concluded that the wood extract inhibited NFκ-B phosphorylation by the anti-inflammatory regulatory mechanism and down-regulated NFκ-B expression. In addition, according to one embodiment of the present invention, the expression of iNOS and Nrf2 was significantly reduced in the soft water, compared with the depression-induced group, and the extracts of horseshoe-mushroom extract, In the case of oral administration of the mixture of extracts, the expression of Nrf2 was significantly decreased compared to the case of oral administration of each extract.
Therefore, the composition of the present invention controls the inflammatory action by controlling the expression of the inflammatory cytokine induced by the hyperactive HPA axis due to stress, down-regulates NFκ-B expression in the brain, Can be effectively used for prevention and treatment of disorders.
In addition, in one embodiment of the present invention, the forced swimming test (FST), which measures the time taken for the rats to fall into the frustrated state and take the immobile state at the same time as inducing the depression as a fear state, The inventors of the present invention have found that both the administration of the compound and the administration of the combination of the compounds of the present invention show resistance to stress-induced depression, .
In the case of the Horseshoe mushroom extract, in order to investigate how fast the rats recovered in the desperate state recovered their motility, the total number of entries and the total movement distance were measured and compared with each other through the Y-maze experiment. As a result, As a result, the composition of the present invention can be more effectively used for prevention and treatment of psychiatric disorders by including the extract of Horsetail mushroom.
Accordingly, the composition of the present invention can reduce the inflammation and the antioxidant function by regulating the resistance to mental disorders induced by stress, regeneration of mobility and expression of inflammatory cytokines and controlling inflammation-related signal transduction through oral administration, Stress, and psychiatric disorders.
In addition, when the extract used in the composition of the present invention is obtained by treating an extraction solvent, various extraction solvents may be used. Preferably, a polar solvent or a non-polar solvent can be used. Suitable polar solvents are (a) water, (b) alcohols (preferably methanol, ethanol, propanol, butanol, n-propanol, iso-propanol, n-butanol, Or ethylene glycol), (c) acetic acid, (d) dimethylformamide (DMFO), and (e) dimethyl sulfoxide (DMSO). Suitable nonpolar solvents are acetone, cyclohexane, cyclopentane, diisobutylene, 1-pentene, 1-chlorobutane, 1- chloropentane, xylene, diisopropyl ether, 2- chloropropane, Chlorobenzene, benzene, diethyl ether, diethyl sulfide, chloroform, dichloromethane, 1,2-dichloroethane, aniline, diethylamine, ether, carbon tetrachloride and THF. More preferably, the extraction solvent used in the present invention is (i) water, (ii) a dihydric or hydric alcohol having 1 to 4 carbon atoms (methanol, ethanol, propanol, butanol, etc.) (V) ethyl acetate, (vi) chloroform, (ⅶ) butyl acetate, (ⅷ) 1,3-butylene glycol, (ⅸ) hexane and (x) diethyl ether. do. Most preferably, the horseradish mushroom extract of the present invention is obtained by treating water, ethanol or a combination thereof with horseradish mushroom.
In addition, the extracts of Horseshoe-mushroom and Spruce tree contained in the composition of the present invention can be used in the form of powders or extracts, and their amounts and ratios can be varied depending on the amount of horseshoe mushrooms and mosses used for extraction, . For example, the composition of the present invention can be used in the form of a mixture of Horseshoe Mushroom Extract and Spruce Extract at a ratio of 10: 1 to 1:10, preferably 4: 1 to 1: 4 And most preferably, the horseradish extract may be used in a mixed ratio of 4: 1.
In preparing the pharmaceutical composition of the present invention, (a) a pharmaceutically effective amount of the above-described extract of Horsetail mushroom of the present invention and the extract of Spruce tree; And (b) a pharmaceutically acceptable carrier. The term "pharmaceutically effective amount" means an amount sufficient to achieve the efficacy or activity of the above-described mushroom extract and moss extract.
Herein, the pharmaceutically acceptable carriers are those conventionally used in pharmaceutical preparations such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose But are not limited to, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
In addition, the pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc., in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).
In addition, the pharmaceutical composition of the present invention can be administered orally or parenterally. At this time, the appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate and responsiveness of the patient . ≪ / RTI > Typical dosages of the pharmaceutical compositions of the invention are in the range of 0.001 to 100 mg / kg on an adult basis.
In addition, the pharmaceutical composition of the present invention may be formulated into a unit dosage form by using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or may be manufactured by penetrating into a multi-dose container. The formulations may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or may be in the form of excipients, powders, powders, granules, tablets or capsules, and may additionally contain dispersing agents or safeners.
In another aspect of the present invention, the present invention relates to a food composition for preventing or ameliorating mental disorders, which comprises a mushroom extract of horseradish and a mushroom extract.
The food composition means a natural product or a processed product containing one or more nutrients, preferably a state of being able to be directly eaten through a certain degree of processing, and as a general meaning, , Beverages, food additives, and beverage additives.
At this time, the food composition may contain, as an active ingredient, horseradish and mushroom extract and a component that is usually added in food production. For example, proteins, carbohydrates, fats, nutrients, flavoring agents and flavoring agents. Examples of the above-mentioned carbohydrates are monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, oligosaccharides and the like; And polysaccharides such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavorings such as tau martin and stevia extract (e.g., rebaudioside A and glycyrrhizin) and synthetic flavorings (saccharine, aspartame, etc.) can be used as flavorings.
In addition, when the food composition of the present invention is prepared with a drink, it may further contain citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, juice, mulberry extract, jujube extract, licorice extract, have.
EXAMPLES Hereinafter, the present invention will be described in detail with reference to Examples. However, the scope of the present invention is not limited to the following Examples.
Example 1 Oral Administration and Analysis of Horseshoe Mushroom Extract in Induced Depression Rat Model
Test subject
The animals were housed in a Sprague-Dawley (SD) female rats purchased from Oriental Biotech Co., Ltd. for 15 weeks at 10 weeks (220-280g). The temperature of the kennel was 25 ℃, 50% Dark circulation was maintained. Purchased rats were allowed to adapt for one week after the transfer, and feed and drinking water were provided freely without limitation.
Production of mushroom extract (FFE)
The horseradish mushroom was ground with a grinder and mixed with 70% ethanol and extracted with a rounded mixed machine for 18 hours. After the extraction, the residue was filtered with filter paper, centrifuged and the supernatant was collected and concentrated by evaporation in a bath at 40 ° C. The remaining material was repeated twice in the same manner and the yield was 7.8%.
Preparation of depression-induced rat model
An animal model to induce depression is an animal model that attempts to escape in the early stages when the animal is trapped in a water filled cylinder, but forced swimming tests to measure the immobility time, , FST) were performed. Specifically, the depression-inducing model used in the test is Detke et al. [Detke MJ, Rickels M, Lucki I (1995). Psychopharm 121: 66-72]. For this purpose, a glass cylinder having a height of 50 cm and a diameter of 25 cm was filled with water at 23 ° C., and water was added to the water for 15 minutes before the experiment for 24 hours Brought to you by forced swimming. Behavioral tests were performed on the 1st and 5th days of oral administration of the drug to evaluate the acute and chronic effects of the drug.
(N = 3), Group II: Depression-induced group (n = 3), Group III: Depression + Horseradish Mushroom extract 100 mg / kg orally (N = 3), Group Ⅳ: Depression + Horsetail mushroom extract 25 mg / kg orally (n = 3), Group V: Depression +
Analysis method
(1) Forced swimming experiment
We measured the immobility, climbing, and swimming position of the rat for 5 minutes, and measured the immobility time except the climbing posture and swimming posture. Floating posture measurements were performed 30 minutes after oral administration of horseradish mushroom extract and thiacenetin sodium. The results were analyzed on the 1st and 5th day of drug administration to confirm the acute effect and chronic effect.
(2) Total number of Y-maze entry, total travel distance measurement
Immediately after the floating time measurement, the Y-maze test was performed for 3 minutes. The total number of times the rats in each group entered the three arms were compared between the groups, and the total distance traveled by the rats in the maze was statistically analyzed to determine the difference between the groups. To evaluate the acute and chronic effects as well as the immobility time measurement, the study was performed on the 1st and 5th day of the drug administration.
(3) Quantitative analysis of protein
The drug was orally administered for 5 days and the rats on which the behavioral tests were performed were immediately sacrificed and the soft water and the adrenal gland were excised. The lysis buffer (PRO-Prep ™ , protein Extration solution) was added to appropriate amount of tissue and homogenized homogenously with a homogenizer (Intron Biotechnology, Gyeonggi-do, Korea) And centrifuged at 13,000 rpm for 10 minutes to obtain a cell extract. The total protein content of the cell extract was measured by Bradfod method using a Bio-Rad Protein assay kit (Bio-Rad Hercules, CA, USA) at 595 nm. Each sample was quantitated with equivalent amount of protein, heated at 95 ° C for 10 minutes, cooled at -20 ° C for 3 minutes, loaded with 18.5 μl of sample in 10% -12% sodium dodecyl sulfate-polyacrylamide gel, Electrophoresis was performed for 30 minutes at 100 V for 1 hour and 30 minutes and at 110 V for 1 hour and 40 minutes. The electrophoresed proteins were transferred to a nitrocellulose membrane under refrigeration at 100 V for 1 hour and 30 minutes. Protein was stained with Ponceau solution and blocked with 5% skim milk for 1 hour. The primary antibodies iNOS, Nrf2, and β-actin were diluted to 1: 500, 1: 500, and 1: 4000, respectively, and reacted at 4 ° C for 24 hours. The cells were washed five times with 1 × PBST (10 × PBST, DW, 0.1% Tween 20) once every 7 min. The secondary antibody (rabbit, mouse) was reacted for 2 hours in accordance with the primary antibody species, . ECL prime (Amersham Pharmacia Biotech, Buckinghamshire, UK) was used to measure and analyze the amount of protein expressed in darkness.
(4) Statistical processing
The forced swimming test float time and Y - maze total travel distance were analyzed by ANOVA using SPSS Version 18. Protein quantitation results were quantified using Vision Works Image Software and analyzed with ANOVA.
Analysis
(1) Forced swimming test
Fig. 2 shows the reduction in the immobility time according to the oral administration of the extract of horseradish peroxidase. In the acute effect, the oral administration group of 100 and 25 mg / kg of horseradish extract showed a significant decrease in immobility time (p <0.01) compared with the FST induction group and the chronic effect of 25 mg / kg of horseradish extract Showed a significant decrease (p <0.05). It showed better immune time reduction effect from acute than chronic.
(2) Y-maze total number of entries, total movement distance measurement result
FIG. 3 shows mobility evaluation results according to the total number of entries and total travel distance in the Y-maze. As a result, the total number of admittances was increased 3 times in the FST-induced group, 100 and 25 mg / kg in the horseradish peroxidase group, and 5 times and 4 times in the acute effect, respectively. . Chronic effects also showed an increase in the number of entries. In the acute effects, total migration distance was significantly increased in the 25 mg / kg group of horseradish extract (p <0.05) and in the chronic effect, 100 and 25 mg / kg of horseradish extract were significantly increased compared to the FST induction group (p < 0.01). It is considered that the increase of the total number of entry and movement distance of acute effect and chronic effect increased due to adaptation by repeated experiment.
(3) Protein quantitative analysis results
FIG. 4 shows the results of analysis of signal transduction protein expression in soft tissue and adrenal glands. The expression of iNOS and Nrf2, an antioxidant signal transduction protein in the horseshoe mushroom extract, was significantly higher in Group Ⅱ than in Group Ⅰ, (P <0.01), and Nrf2 showed the same pattern as iNOS. In addition, the expression of iNOS and Nrf2 in the horseshoe mushroom extract in the adrenal gland was significantly reduced in Group Ⅳ compared to Group Ⅱ (p <0.05), and the expression of Nrf2 was significantly decreased in Group Ⅲ (p < 0.01).
<Example 2> Oral Administration and Analysis of Extract of Extracts from Depressed Rat Model
Test subject
Twenty four-week-old (150-170 g) female rats were used for the Sprague-Dawley (SD) female rats purchased from Korean Central Animal. The temperature of the kennels was 20 ㅁ 2 ℃, humidity 55-60% Main / night circulation was maintained. Purchased rats were allowed to adapt for one week after the transfer, and feed and drinking water were provided freely without limitation.
Manufacture of Extracts
Spruce root was purchased from Palmtec Bio in Busan. The completely dried roots of chestnut were chopped finely using a grinder and lyophilized. 2 g of the lyophilized sample and 10 mL of 70% ethanol were placed in a 15 mL tube and dissolved in a mixer for 18 hours. The supernatant was collected by centrifugation at 3000 rpm. The collected supernatant was evaporated at high temperature and the remaining sample was concentrated and lyophilized. The chestnut samples used in this experiment were extracted with 6.3% yield from the raw material of chestnut tree.
Preparation of depression-induced rat model
The first forced swim test (FST) was carried out for 15 minutes after having the incubator environmental adaptation for one week.
Twenty - four hours later, the second FST experiment was carried out for 5 minutes. Animals were sacrificed immediately after 5 minutes of the last FST experiment for 5 days. Fasted the day before sacrifice and water was supplied in sufficient quantity. Immediately after the last FST experiment, blood was collected over 6 ml in the abdominal vein after ether anesthesia. Blood collected from the abdominal vein was stored in a 5 ml SST tube. SST tube blood specimens were centrifuged at 3000 rpm for 15 min and the serum was stored in an E-tube at -70 ° C. After perfusion with saline through the ascending aorta, tissues of brain, soft tissue and adrenal glands were excised and specimens were transferred to E-tubes and stored at -70 ° C until homogenization.
(N = 5), Group III: Forced swimming test + Sprague-Dawley extract (86 mg / kg) orally (n = 5) ), And ④ Group IV: The test was conducted with the forced swimming test + the extract of Spruce (256 mg / kg) orally (n = 5).
Analysis method
(1) forced swimming assay, (4) quantitative analysis of proteins, and analysis of liver function markers, inflammatory markers, cytokines and hormones were carried out in the same manner as in Example 1 Respectively.
(2) Analysis of liver function markers and inflammatory markers
(AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) were measured by ECLIA (Chemiluminescence) using a Cobas 6000 analyzer (Roche Diagnostics, Switzerland) immunoassay. The anti-inflammatory C-reactive protein, an inflammatory marker, was measured by the ECLIA principle using HITACHI 7600-210 (Hitachi, Japan).
(3) cytokine and hormone analysis
IL-6, TNF-α and IL-1β were measured using an ELISA kit (R & D systems, America). Serum cortisol and adrenocorticotropic hormone (ACTH) concentrations were measured by the ECLIA principle using a Beckman Coulter AU5800 Chemistry system (Bechman Coulter, America).
Analysis
(1) Forced swimming test
FIG. 5 shows the decrease in the immobility time according to the oral administration of the extract of the Japanese apricot. (P <0.05). Compared to the second forced swimming experiment (A) and the forced swimming experiment (B) just before the animal sacrifice, group III and group IV showed a significant decrease The control group was 144 seconds and 138 seconds, respectively, but the high concentration (256 mg / kg) was significantly different between 71 and 11.4 seconds and 68 and 8.7 seconds, respectively.
(2) Analysis of liver function markers and inflammatory markers
Table 1 below shows the results of liver damage marker and CRP concentration evaluation. There was no significant difference in liver function markers (ALT, AST, ALP) from the control group (p> 0.05). The concentration of the highly sensitive C protein (hS-CRP) was not significantly different from the test group (p> 0.05).
(3) cytokine and hormone analysis results
The results of the cytokine (A) IL-1 beta (B) IL-6 (C) TNF-alpha concentration analysis between the groups are shown in FIG.
There was a significant difference in IL-1β between the experimental group (Group III, IV: 105.7 ㅁ 10.5, 72.8 ㅁ 9.8) and the group II (vehicle: Group III, IV: 162.8 15 15.9) (P <0.05), and the lowest level of IL-1β was found in group I (34.7 ㅁ 21.9). IL-6 showed the same tendency as IL-1β, and the values of groups I, II, III and IV were 76.0, 39.8, 551.6, 28.1, 124.8, 17.9 and 116.9, 24.1, respectively, There was a statistically significant difference (p <0.05). TNF-α also showed the highest level of group II (199.7 ㅁ 38.2) and there was a significant difference (p <0.05) with other groups.
Table 2 shows the results of the assay of cortisol and ACTH as a result of hormone analysis. As a result, the concentration of cortisol was the highest in group II, and the result of adreno corticotrophic hormone (ACTH) also showed the highest concentration in group II.
(4) Quantitative analysis of protein
Adrenal, soft tissue, and brain tissues were quantitatively analyzed by Western blotting. The results are shown in FIG. 7 in terms of the ratio of p-p65 / total p65.
7 (A), the adrenal gland extract group (group II, 1.73) showed about twice the activity as the control group (group I 0.86), and the group IV 0.375), which was significantly reduced compared to the control group.
As shown in FIGS. 7 (B) and 7 (C), the adrenal glands showed similar tendency in the soft tissues (B) and brain (C) (1.15 / 0.30) showed a 6.42-fold and 3.83-fold increase, respectively, compared to the control group. Similarly, the rapid decrease of p-p65 expression was observed in the high concentration group of extracts from the mugwort extract.
Example 3 Oral Administration and Analysis of Horseshoe Mushroom Extract Mixture in Depressed Rat Model
Analysis method
The same experiment was carried out as in Example 1, except that a mixture of mushroom extract of Morschach and extract of Mugwort was used. The ratio of the mixture of the mushroom extract of the horseradish to the extract of the mushroom was 4: 1.
(1) Forced swimming test Floating time measurement analysis result
FIG. 8 shows the result of the measurement of the immobility time of the forced swimming test in the oral administration of the horseradish / mushroom extract mixture. The results showed that the immobility time was significantly lowered in both acute and chronic effects compared with the control group, and showed a similar reduction effect as compared with the positive control group, and showed similar floating time reduction effect in acute and chronic.
(2) Protein quantitative analysis results
FIG. 9 shows the results of analysis of signal transduction protein expression in soft water when orally administered with horseradish or mushroom extract.
The expression of iNOS and Nrf2 in inflammation-regulated and antioxidant signaling proteins was significantly increased in the Vehicle group than in the Control group. The expression of iNOS and Nrf2 in the Vehicle group was significantly higher than that in the Control group. The expression of iNOS and Nrf2 was decreased in all groups treated with extracts. Especially, the expression of Nrf2 was significantly decreased in the group treated with extracts of horseradish and mugwort. It is considered that the effect of controlling the expression of iNOS and Nrf2, an inflammation - regulating and antioxidant signal transduction protein, is excellent in the horseradish / mushroom extract mixture.
As described above, through the examples of the present invention, it was confirmed that the mushroom extracts of the horseshoe crab were subjected to the forced swimming test (FST) , INOS and Nrf2, which are inflammatory and antioxidant signal transduction proteins, and adrenal gland expression. As a result, the immersion time was significantly decreased in the horseradish mushroom extract group, and the total number of entries and total travel distance were also significantly increased compared to the forced swimming test induction group. Expression of iNOS and Nrf2 was also significantly decreased. These results suggest that the extract of horseshoe mushroom has an anti - depressive effect on the regulation of inflammatory response and anti - stress through inhibition of the signal transduction proteins iNOS and Nrf2.
In addition, the liver function test and hS-CRP were performed in order to confirm the presence of cytotoxicity of the natural product itself and the fact that there was no specific inflammation in rats before the experiment. It is confirmed that there is no difference, and it is confirmed that the immersion time experiment shows a decrease in immobility time. In addition, a rapid decrease in NFκ-B signaling-related cytokines IL-1β, IL-6 and TNF-α was observed in the extract-treated group, . In addition, the hormone analysis showed that cortisol secretion was significantly decreased in the treated group compared to the non - treated group, and the ACTH concentration was maintained in a proper manner. As a result, the extract of Corynebacterium reticulatum decreased the concentration of excess cortisol Not only reduced stress hormones, but also had positive effects that helped maintain HPA axis feedback. In addition, the expression of NFκ-B was evaluated by analysis of phosphorylated p65 (p-p65). As a result, rapid p-p65 expression inhibition effect was exhibited in the group of the callus, especially in the high concentration group, (B) and (B), respectively. These results suggest that the extracts of Phellinus linteus may have the effect of anti-psychotic disorder by acting as a NFκ-B phosphorylation inhibitor by the anti-inflammatory regulatory mechanism.
In addition, floating time measurement and the expression of iNOS and Nrf2 in inflammatory and antioxidative signal transduction proteins in the extracts of the extracts of Horseshoe mushroom extract and Quercus variabilis extract showed a decrease in immobility time, In particular, the oral administration of horseradish mushroom extract showed a marked decrease in the expression of Nrf2 as compared with the iNOS and Nrf2 expression as well as the single extract.
Therefore, it can be concluded from the results of these examples that the extract of Horseradish Mushroom Extract and Mugwort extract of the present invention can exhibit anti-stress and anti-psychotic effects by controlling anti-inflammatory and antioxidant functions in mental disorders including depression.
Claims (5)
Wherein the mental disorder is a psychiatric disorder selected from the group consisting of anxiety, depression, mood disorders, insomnia, obsessive compulsive disorder and stress.
The Horsetail mushroom extract and Quercus mongolica extract control the expression of an inflammatory cytokine induced by an activated hypothalamic-pituitary-adrenal axis (HPA) Lt; RTI ID = 0.0 > of-B < / RTI >
Wherein the inflammatory cytokine is iNOS or Nrf2.
Wherein the mental disorder is a psychiatric disorder selected from the group consisting of anxiety, depression, mood disorders, insomnia, obsessive compulsive disorder and stress.
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| US15/761,969 US11338004B2 (en) | 2015-09-22 | 2016-09-22 | Composition for preventing or treating cranial nerve disease comprising fomes fomentarius extract, fraction thereof, or compound isolated therefrom as active ingredient |
| PCT/KR2016/010583 WO2017052227A1 (en) | 2015-09-22 | 2016-09-22 | Composition for preventing or treating cranial nerve disease comprising fomes fomentarius extract, fraction thereof or compound isolated therefrom as active ingredient |
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