Composition for preventing, alleviating or treating stress diseases, containing <i>Platycodon grandiflorum </i>extract as active ingredient

Abstract

The present disclosure relates to a composition for preventing, alleviating or treating stress diseases, containing a Platycodon grandiflorum extract as an active ingredient, and, more specifically, to a food composition for preventing or alleviating stress diseases or a pharmaceutical composition for preventing and treating stress diseases, both of which contain a Platycodon grandiflorum extract and a Citrus aurantium extract as active ingredients. A method of the present disclosure can be used for the preparation of an agent for treating depression and anxiety, which are stress diseases, with few side effects because of the use of a natural material, and a health functional food having effects of alleviating depression, anxiety and fatigue, which are stress diseases.

Claims

1. A method for treating stress diseases wherein an effective amount of a pharmaceutical composition consisting of a Platycodon grandiflorum extract and a Citrus aurantium extract as effective ingredients is administered to an individual in need thereof, wherein a weight ratio of the Platycodon grandiflorum and the Citrus aurantium is (1-10):(1-10), and wherein the stress disease is one or more selected from the group consisting of depression, anxiety, and fatigue.

2. The method of claim 1, comprising administering 100 mg to 500 mg of the pharmaceutical composition per kilogram of body weight of the individual in need thereof daily.

3. The method of claim 2, comprising administering the pharmaceutical composition as a single dose.

4. The method of claim 2, comprising administering the pharmaceutical composition in multiple doses according to a fractionated treatment protocol.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 shows a result of administering 300 mg/kg of SOCG (So-o-chim-tang-gami-bang), linderae radix, licorice, rhizoma cyperi, elecampane, Platycodon grandiflorum and Citrus aurantium extracts to a mouse, inducing depression by applying restraint stress, and then conducting forced swim test (#P<0.05 vs. na, ***P<0.001 vs. Control).

(2) FIG. 2 shows a result of administering 300 mg/kg of an SOCG (So-o-chim-tang-gami-bang) extract and 200 mg/kg of Platycodon grandiflorum and Citrus aurantium extracts from among the drugstuffs constituting SOCG to a mouse with restraint stress-induced depression for 14 days and conducting forced swim test on day 15 (#P<0.05 vs. na, ***P<0.001 vs. Control).

(3) FIG. 3a and FIG. 3b show total global activity (FIG. 3a) and immobility duration (FIG. 3b) as a result of administering 300 mg/kg of an SOCG extract and 150 mg/kg, 300 mg/kg or 600 mg/kg of a Platycodon grandiflorum or Citrus aurantium extract to a mouse with restraint stress-induced depression for 14 days and conducting forced swim test on day 15.

(4) FIG. 4 shows a result of administering 30 mg/kg of amitriptyline, 300 mg/kg of SOCG or 300 mg/kg of a mixture of Platycodon grandiflorum and Citrus aurantium extracts at a weight ratio of 1:1 to a mouse with restraint stress-induced depression for 14 days and conducting forced swim test (#P<0.05 vs. na, *P<0.05, **P<0.01 vs. Control).

(5) FIG. 5 shows a result of administering 10 mg/kg of amitriptyline, 300 mg/kg of SOCG or 300 mg/kg of a mixture of Platycodon grandiflorum and Citrus aurantium to a Wistar-Kyoto rat (WKY) with restraint stress-induced depression for 14 days and conducting forced swim test on day 15 (#P<0.05 vs. na, *P<0.05, **P<0.01 vs. Control).

(6) FIG. 6 shows a result of administering 300 mg/kg of a mixture of Platycodon grandiflorum and Citrus aurantium to a mouse with restraint stress-induced depression for 14 days and measuring the concentration of corticosterone in blood obtained on day 16 by enzyme-linked immunosorbent assay (#P<0.05 vs. na, *P<0.05 vs. control).

(7) FIG. 7 shows a result of administering 10 mg/kg of amitriptyline, 300 mg/kg of SOCG or 300 mg/kg of a mixture of Platycodon grandiflorum and aurantii to WKY with restraint stress-induced depression for 14 days and measuring the concentration of corticosterone in blood obtained on day 16 by enzyme-linked immunosorbent assay (#P<0.05 vs. na, ***P<0.001 vs. control).

(8) FIG. 8 shows a result of administering 200 mg/kg of a Platycodon grandiflorum extract, 200 mg/kg of a Citrus aurantium extract or 300 mg/kg of a mixture of Platycodon grandiflorum and Citrus aurantium to a mouse with restraint stress-induced depression and measuring the concentration of neurotransmitters in a brain tissue by HPLC.

(9) FIG. 9 shows a result of administering 200 mg/kg of a Platycodon grandiflorum extract, 200 mg/kg of a Citrus aurantium extract or 300 mg/kg of a mixture of Platycodon grandiflorum and Citrus aurantium to a mouse with restraint stress-induced depression and measuring the concentration of neurotransmitters in the brain excluding the hippocampal region by HPLC (#P<0.05 ##P<0.01, ###P<0.001 vs normal group (naive) *P<0.05 **P<0.01, ***P<0.001 vs control group).

(10) FIG. 10 shows a result of administering 200 mg/kg of a Platycodon grandiflorum extract, 200 mg/kg of a Citrus aurantium extract or 300 mg/kg of a mixture of Platycodon grandiflorum and Citrus aurantium to a mouse with restraint stress-induced depression and measuring the concentration of epinephrine in the hippocampal region of the brain by HPLC (###P<0.001 vs normal group (naive), ***P<0.001 vs test group).

BEST MODE

(11) Hereinafter, the present disclosure is described in detail.

(12) However, the following examples merely illustrate the present disclosure and the contents of the present disclosure are not limited by the examples.

(13) <Experimental Methods>

(14) Preparation of Mixture of Platycodon Grandiflorum and Citrus Aurantium

(15) Each of Platycodon grandiflorum (platycodi radix) and Citrus aurantium was added to water of 10 times based on weight and hot-water extracted at 90-100 C. for 3 hours. After filtering the extraction solvent using a filter paper, a Citrus aurantium extract and a Platycodon grandiflorum extract were prepared by conducting concentration under reduced pressure using a rotary evaporator at 60 C. or below. Then, a mixture of Platycodon grandiflorum and Citrus aurantium was prepared by mixing 150 mg/kg of the Platycodon grandiflorum extract and 150 mg/kg of the Citrus aurantium extract at a weight ratio of 1:1.

(16) Administration of Platycodon Grandiflorum Extract, Citrus Aurantium Extract or Mixture of Platycodon Grandiflorum and Citrus Aurantium

(17) The sample was administered orally 2 hours before applying restraint stress. Physiological saline was administered orally to a normal group (na) and a stress control group (control). 300 mg/kg of an SOCG (So-o-chim-tang-gami-bang) extract was administered to an SOCG group. 150, 300 or 600 mg/kg of the Citrus aurantium extract was administered to Citrus aurantium groups. 150, 300 or 600 mg/kg of the Platycodon grandiflorum extract was administered to Platycodon grandiflorum groups. In addition, 150 mg/kg of the Platycodon grandiflorum extract and 150 mg/kg of the Citrus aurantium extract were mixed to prepare a 300 mg/kg mixture. The sample administration volume was 0.1 mL for a mouse and 0.3 mL for WKY.

(18) Stress-Induced Depression Model

(19) C57/BL6 mice (male, 7-week-old) were purchased from Dae Han Bio Link (Korea) and Wistar-Kyoto rats (WKY) were acquired from Kyungpook National University. The mice and WKY were reared in different cages. They were used for experiments after accustoming to the laboratory environment of temperature 222 C., humidity 50-70%, and lighting for 12 hours (08:00-20:00) with illuminance of 150-300 Lux for a week while providing solid feed (Samyang Feed) and water sufficiently.

(20) Restraint stress was applied to induce depression. The C57BL/6 mouse accustomed to the laboratory environment for a week was individually placed in a 50-mL Falcon tube with holes for ventilation. For the WKY, a restraint stress device for rats was used. The animal was restrained for 14 days, 6 hours a day, to induce depression.

(21) Forced Swim Test (FST)

(22) After filling a transparent acrylic cylindrical water bath (Vision Scientific Co., Korea) with water at room temperature, the depression-induced mouse or WKY was forced to swim for 15 minutes. 24 hours later, the duration of immobility was measured while forcing the animal to swim for 6 minutes under the same condition. The measurement was made for the last 4 minutes and recording was made conducted with a video camera. The total global activity of the animal in water was analyzed with a video-tracking software (SMART 3.0, Panlab, Spain).

(23) Enzyme-Linked Immunosorbent Assay

(24) Serum was obtained by centrifuging (Labogene, Gyrozen, Daejeon, Korea) blood extracted from the depression-induced mouse or rat. A corticosterone ELISA kit (Enzo Life Science, Cat. ADI-900-097) was used for enzyme-linked immunosorbent assay. After adding 100 L of the mouse or rat serum and standard to a pre-coated 96-well plate included in the kit, 50 L of the blue conjugate reagent was added to each well containing the serum and standard. After adding 50 L of a capture antibody to the well containing the serum and standard, incubation was performed on a shaker at room temperature and 500 rpm for 2 hours. After draining the well and washing 3 times with a wash solution, 200 L of the pNpp substrate solution was added to all wells and incubation was performed at room temperature for 1 hour. Then, after treating with 50 L of a stop solution, absorbance was measured at 405 nm using a microspectrophotometer (Molecular Device, Sunnyvale, Calif., USA).

(25) Preparation of Sample for Specific Brain Regions

(26) According to a method specified for the hippocampal and cortical region of the brain (Adams, 2013), the brain tissue was homogenized by adding a phosphate buffer of about 2 times of weight. 100 L of the homogenate was immediately added to 300 L of cold perchloric acid (0.1 M in PBS), and the mixture was completely homogenized by sonicating for 1 minute. Then, after keeping the homogenate on ice for 10 minutes, centrifugation was performed at 4 C. and 12000 rpm for 15 minutes. Subsequently, the supernatant was filtered through a 0.2-m syringe filter and then subjected to HPLC (high-performance liquid chromatography) analysis.

(27) Chromatographic Analysis

(28) Neurotransmitters were analyzed using a liquid chromatographic water system (Waters Corp., Milford, Mass., USA) coupled with a photodiode array detector and a separation module (e2695) according to a specified method (Church 2005). For the analysis of neurotransmitters, a mixture of 1% citric acid, 2% K.sub.2HPO.sub.4, 1 mM EDTA, 7 mg/mL sodium octyl sulfate and 1.2% methanol and a C18 column (250 mm, 4.6 mm, 5 m) were used. 20 L of an isocratic mobile phase was injected at a flow rate of 0.5 mL/min at a wavelength of 254 nm. Epinephrine, norepinephrine, dopamine and serotonin were used as standards at a concentration of 1 mg/mL.

(29) Statistical Analysis

(30) All data were represented as meanstandard error (SEM). The result for each test group was statistically analyzed using a statistical program (one-way ANOVA; post-hoc Tukey test), and significance was tested at a level of P<0.05 (*P<0.05, **P<0.01, ***P<0.001).

EXAMPLE 1

(31) Effect of Extracts of Drugstuffs Constituting SOCG on Depression

(32) Experiment was conducted as follows in order to investigate the effect of each of the drugstuffs constituting SOCG (So-o-chim-tang-gami-bang), linderae radix, licorice, rhizoma cyperi, elecampane, Platycodon grandiflorum and Citrus aurantium, on depression.

(33) After orally administering physiological saline to C57/BL6 mice (male, 7-week-old) of a normal group and a stress control group, 300 mg/kg of each of an SOCG extract, a linderae radix extract, a licorice extract, a rhizoma cyperi extract, an elecampane extract, a Platycodon grandiflorum extract and a Citrus aurantium extract was administered. Restraint stress was applied 2 hours later. After applying the restraint stress for 14 days, 6 hours a day, forced swim test was conducted on day 15.

(34) As seen from FIG. 1, it was confirmed that immobility duration was decreased in the group treated with each extract as compared to the stress control group, and the groups treated with the Platycodon grandiflorum extract, the Citrus aurantium extract and the rhizoma cyperi extract showed shorter immobility duration than the group treated with the SOCG extract.

EXAMPLE 2

(35) Effect of Platycodon Grandiflorum Extract and Citrus Aurantium Extract on Depression

(36) <2-1> Effect of Platycodon Grandiflorum Extract and Citrus Aurantium Extract on Depression

(37) Experiment was conducted as follows in order to investigate the effect of the Platycodon grandiflorum extract and the Citrus aurantium extract alone on depression.

(38) Physiological saline was orally administered to C57/BL6 mice (male, 7-week-old) of a normal group and a stress control group, 300 mg/kg of the SOCG extract was administered to an SOCG group, and 200 mg/kg of each of the Platycodon grandiflorum extract and the Citrus aurantium extract was administered. Restraint stress was applied 2 hours later. After applying the restraint stress for 14 days, 6 hours a day, forced swim test was conducted on day 15.

(39) As seen from FIG. 2, total global activity was increased and immobility duration was decreased as compared to the stress control group. In addition, it was confirmed that the group treated with the Platycodon grandiflorum extract or the Citrus aurantium extract alone showed increased total global activity and decreased immobility duration as compared to the group treated with the SOCG extract.

(40) Through this, it was confirmed that the Platycodon grandiflorum extract and the Citrus aurantium extract are effective in alleviating depression.

(41) <2-2> Effect of Platycodon Grandiflorum Extract and Citrus Aurantium Extract on Depression Depending on Concentration

(42) Experiment was conducted as follows in order to investigate the effect of the Platycodon grandiflorum extract and the Citrus aurantium extract depending on concentration.

(43) Physiological saline was orally administered to C57/BL6 mice (male, 7-week-old) of a normal group and a stress control group, 300 mg/kg of the SOCG extract was administered to an SOCG group, and 150, 300 or 600 mg/kg of each of the Platycodon grandiflorum extract and the Citrus aurantium extract was administered. Restraint stress was applied 2 hours later. After applying the restraint stress for 14 days, 6 hours a day, forced swim test was conducted on day 15.

(44) As seen from FIG. 3a and FIG. 3b, the group treated with 300 mg/kg of the Citrus aurantium extract and the group treated with 300 mg/kg of the Platycodon grandiflorum extract showed the most active movement (FIG. 3a). In addition, the group treated with 300 or 600 mg/kg of the Citrus aurantium extract showed the shortest immobility duration, and the group treated with 300 mg/kg of the Platycodon grandiflorum showed the shortest immobility duration.

(45) Through this, it was confirmed that administration of 300 mg/kg of the Platycodon grandiflorum extract or the Citrus aurantium extract has the largest effect of alleviating depression.

EXAMPLE 3

(46) Effect of Mixture of Platycodon Grandiflorum and Citrus Aurantium on Depression

(47) Experiment was conducted as follows in order to investigate the effect of a mixture of Platycodon grandiflorum and Citrus aurantium on depression.

(48) <3-1> Effect of Mixture of Platycodon Grandiflorum and Citrus Aurantium on Depression-Induced Mouse

(49) 300 mg/kg of a mixture of Platycodon grandiflorum and Citrus aurantium was prepared by mixing 150 mg/kg of a Platycodon grandiflorum extract and 150 mg/kg of a Citrus aurantium extract. Physiological saline was orally administered to C57/BL6 mice (male, 7-week-old) of a normal group and a stress control group, 30 mg/kg of amitriptyline (A8404-10G, Sigma-Aldrich, USA) was administered to a positive control group, 300 mg/kg of the SOCG extract was administered to an SOCG group, and 300 mg/kg of the mixture of Platycodon grandiflorum and Citrus aurantium was administered. Restraint stress was applied 2 hours later. After applying the restraint stress for 14 days, 6 hours a day, forced swim test was conducted on day 15.

(50) As seen from FIG. 4, it was confirmed that immobility duration was decreased in the group to which SOCG was administered and the group to which the mixture of Platycodon grandiflorum and Citrus aurantium was administered, and the group to which the mixture of Platycodon grandiflorum and Citrus aurantium was administered showed shorter immobility duration than the group to which SOCG was administered.

(51) <3-2> Effect of Mixture of Platycodon Grandiflorum and Citrus Aurantium on Depression-Induced Rat

(52) Physiological saline was orally administered to depression-induced Wistar-Kyoto rats (WKY) of a normal group and a stress control group, 10 mg/kg of amitriptyline (A8404-10G, Sigma-Aldrich) was administered to a positive control group, 300 mg/kg of the SOCG extract was administered to an SOCG group, and 300 mg/kg of the mixture of Platycodon grandiflorum and Citrus aurantium was administered. Restraint stress was applied 2 hours later. After applying the restraint stress for 14 days, 6 hours a day, forced swim test was conducted on day 15.

(53) As seen from FIG. 5, it was confirmed that the group to which the SOCG mixture was administered and the group to which the mixture of Platycodon grandiflorum and Citrus aurantium was administered showed shorter immobility duration as compared to the control group, and the group to which the mixture of Platycodon grandiflorum and Citrus aurantium was administered showed the shortest immobility duration.

(54) Through this, it was confirmed that the mixture of Platycodon grandiflorum and Citrus aurantium is more effective in alleviating depression than the SOCG mixture.

EXAMPLE 4

(55) Effect of Mixture of Platycodon Grandiflorum and Citrus Aurantium on Hormone

(56) Experiment was conducted as follows in order to investigate the effect of a mixture of Platycodon grandiflorum and Citrus aurantium on the corticosterone level in the blood of depression-induced mouse or rat.

(57) <4-1> Effect of Mixture of Platycodon Grandiflorum and Citrus Aurantium on Corticosterone Hormone of Depression-Induced Mouse

(58) Physiological saline was orally administered to C57/BL6 mice (male, 7-week-old) of a normal group and a stress control group, and 300 mg/kg of the mixture of Platycodon grandiflorum and Citrus aurantium was administered. Restraint stress was applied 2 hours later. After centrifuging blood taken from the depression-induced mouse, the corticosterone level in serum was measured by enzyme-linked immunosorbent assay.

(59) As seen from FIG. 6, it was confirmed that the stress control group showed increased blood corticosterone level, and the group to which the mixture of Platycodon grandiflorum and Citrus aurantium was administered showed decreased corticosterone level.

(60) <4-2> Effect of Mixture of Platycodon Grandiflorum and Citrus Aurantium on Corticosterone Hormone of Depression-Induced Rat

(61) Physiological saline was orally administered to Wistar-Kyoto rats (WKY) of a normal group and a stress control group. After administering 10 mg/kg of amitriptyline, 300 mg/kg of SOCG, or 300 mg/kg of the mixture of Platycodon grandiflorum and Citrus aurantium, restraint stress was applied 2 hours later. Blood taken from the depression-induced rat was centrifuged and the corticosterone level in serum was measured by enzyme-linked immunosorbent assay.

(62) As seen from FIG. 7, it was confirmed that the stress control group showed increased corticosterone level in blood, whereas the group to which the SOCG mixture was administered and the group to which the mixture of Platycodon grandiflorum and Citrus aurantium was administered showed decreased corticosterone level. In addition, it was confirmed that the group to which the mixture of Platycodon grandiflorum and Citrus aurantium was administered showed a lower corticosterone level than the group to which the SOCG mixture was administered.

(63) Through this, it was confirmed that the mixture of Platycodon grandiflorum and Citrus aurantium decreases the blood level of corticosterone, which is a stress hormone.

EXAMPLE 5

(64) Effect of Mixture of Platycodon Grandiflorum and Citrus Aurantium on Neurotransmitters

(65) Experiment was conducted as follows in order to investigate the effect of a mixture of Platycodon grandiflorum and Citrus aurantium on the contents of neurotransmitters in a specific brain region of a mouse with stress-induced depression.

(66) Physiological saline was orally administered to C57/BL6 mice (male, 7-week-old) of a normal group and a stress control group. After administering 200 mg/kg of a Platycodon grandiflorum extract, 200 mg/kg of a Citrus aurantium extract, 300 mg/kg of a mixture of Platycodon grandiflorum and Citrus aurantium or the tricyclic antidepressant AMT (amitriptyline) as a positive control to different groups, restraint stress was applied 2 hours later.

(67) After harvesting the hippocampal region and the cortical region from the brain of the mouse with stress-induced depression, the tissue was homogenized and then centrifuged after mixing with perchloric acid. The concentration of the neurotransmitters, norepinephrine, epinephrine, dopamine and serotonin, in the obtained supernatant was measured by HPLC.

(68) As seen from FIG. 9, it was confirmed that norepinephrine, epinephrine, dopamine and serotonin were decreased in the cerebral region except the hippocampal region, for the control group as compared to the normal group. When the Platycodon grandiflorum extract, the Citrus aurantium extract or the mixture of Platycodon grandiflorum and Citrus aurantium was administered, the concentration of norepinephrine, dopamine and serotonin was increased as compared to the control group.

(69) In addition, as seen from FIG. 10, in the hippocampal region which is known as a region where cell death is induced by stress, epinephrine was significantly decreased in the control group as compared to the normal group. The concentration of epinephrine was increased when the Platycodon grandiflorum extract, the Citrus aurantium extract or the mixture of Platycodon grandiflorum and Citrus aurantium was administered as compared to the control group.

(70) Through this, it was confirmed that the Platycodon grandiflorum extract, the Citrus aurantium extract and the mixture of Platycodon grandiflorum and Citrus aurantium is effective in inhibiting the decrease of neurotransmitters in the brain of a mouse with restraint stress-induced depression.

(71) Accordingly, considering that the mixture of Platycodon grandiflorum and Citrus aurantium increases the level of norepinephrine, epinephrine, dopamine and serotonin, the effect of overcoming stress and inhibiting stress-induced cell death in the hippocampal region may be expected through their interaction.

EXAMPLE 6

(72) Effect of Mixture of Platycodon Grandiflorum and Citrus Aurantium on Fatigue

(73) Experiment was conducted as follows in order to investigate the effect of a mixture of Platycodon grandiflorum and Citrus aurantium on the improvement of fatigue in a mouse with stress-induced fatigue.

(74) <6-1> Expression of Genes Affecting Improvement of Fatigue

(75) First, the known ingredients of Platycodon grandiflorum and Citrus aurantium, their effects and genes targeted by the ingredients were investigated, and experiment was conducted based thereon. While inducing depression of a mouse by applying restraint stress, a mixture of Platycodon grandiflorum and Citrus aurantium was administered for 14 days. On the last day of experiment, the mouse was sacrificed. After removing blood through cardiac puncture, the brain tissue was extracted. The extracted brain tissue was homogenized using TRIzol. After mixing with chloroform of the volume of 1/5 of the TRIzol, the homogenate was kept at room temperature for 3 minutes. Then, after centrifuging at 12,000 rpm and 4 C. for 15 minutes, the supernatant was taken and 2-propanol of the same volume was added. RNA was precipitated by conducting incubation at room temperature for 10 minutes. Subsequently, after centrifuging at 12,000 rpm and 4 C. for 10 minutes, the supernatant was removed and RNA pellet was washed with 80% ethanol. Then, after removing the supernatant by centrifuging at 7500 g for 5 minutes and spontaneously drying in the air, 50 L of DEPC water was added and the mixture was stored at 80 C. The prepared RNA was subjected to microarray assay by BioCore.

(76) As a result, a total of 33,793 genes were identified. Differentially expressed genes (DEGs) were screened from among them. Specifically, gene expression level was compared between the normal group and the control group, and gene expression level was compared between the control group and the group to which the mixture of Platycodon grandiflorum and Citrus aurantium was administered. The genes with the absolute value of log.sub.2 (ratio of expression levels) being greater than 0.6 were screened as DEGs.

(77) As a result, a total of 202 DEGs were screened, and signaling pathways that can affect the improvement of fatigue were identified based thereon. The result is shown in Table 1.

(78) TABLE-US-00001 TABLE 1 Substance Exemplary compounds All possible pathways Citrus aurantium (S)-Linalool 65,662 Naringin 16,770 Synephrine 4,806 Platycodon grandiflorum Playtogenic acid A 8 Platycodigenin 890

(79) <6-2> Pathways Affecting Improvement of Fatigue

(80) The signaling pathways affecting the improvement of fatigue were analyzed based on the result of Example <6-1>.

(81) Microarray assay was contacted using DNA obtained from a mouse to which a mixture of Platycodon grandiflorum and Citrus aurantium was administered. Based on the result, the gene expression level change ratio of the normal group and the control group, and the gene expression level change ratio of the control group and the group to which a mixture of Platycodon grandiflorum and Citrus aurantium was administered were derived. Recovery rate was designated for each pathway using the gene expression level change ratio. The recovery rate was determined to be closer to 0 as the gene expression level of the group to which a mixture of Platycodon grandiflorum and Citrus aurantium was administered is closer to the gene expression level of the normal group.

(82) The total change ratio was calculated by summing all change ratios of gene nodes belonging to each pathway. For example, if a specific pathway is compound.fwdarw.gene 1.fwdarw.gene 2.fwdarw.gene 3.fwdarw.depression and the expression ratio of each gene is 0.3, 0.5, and 0.2 in order, the expression ratio of the pathway is 1.0. In this manner, the pathway change ratio of the normal group and the control group, and the pathway change ratio of the control group and the group to which a mixture of Platycodon grandiflorum and Citrus aurantium was administered were calculated. The recovery rate for each pathway was defined as follows.
Recovery rate=|(pathway change ratio.sub.(normal group vs. control group)+pathway change ratio.sub.(control group vs. group to which mixture of Platycodon grandiflorum and Citrus aurantium was administered))/(pathway change ratio.sub.(normal group vs. control group))|<Equation 1>

(83) For example, for a specific pathway, if the change ratio between the normal group and the control group is 4 and the change ratio between the control group and the group to which a mixture of Platycodon grandiflorum and Citrus aurantium was administered is 2, the recovery rate is 0.5.

(84) As a result of measuring the recovery rate of the group to which a mixture of Platycodon grandiflorum and Citrus aurantium was administered, it was confirmed that, as the absolute value of the recovery rate is closer to 0, the DNA expression profile of the mouse to which a mixture of Platycodon grandiflorum and Citrus aurantium was administered is similar to that of the normal group. Based on this, from among the pathways expected to affect the recovery of the fatigue-improving genes, 15 pathways that recover the DEG expression level of the depression-induced mouse similarly to that of the control group were screened

(85) As show in Table 2, it was confirmed that the mixture of Platycodon grandiflorum and Citrus aurantium is associated with Casp3, Fos and BDNF in many pathways, from among the genes suspected with the disease. In addition, it was confirmed that the GOs associated with dopamine, epinephrine and norepinephrine are involved in the recovery from fatigue, as the node 4 of priorities 4, 5, 13, 14 and 15.

(86) Through this, it was confirmed that the mixture of Platycodon grandiflorum and Citrus aurantium can improve stress-induced fatigue by promoting the expression of the genes affecting the improvement of fatigue.

(87) TABLE-US-00002 TABLE 2 Prior- ity Node 1 Node 2 Node 3 Node 4 Disease 1 MDM2 MRPL43 UBC CASP3 Depression 2 CDKN1A HMGXB3 UBC CASP3 Depression 3 KAT2B VHL UBC FOS Depression 4 MARCKS ELAVL1 GNB1 GO:0007191.sup.1 Depression 5 FN1 TUFM SNCG GO:0014059.sup.2 Depression 6 KAT2B CTBP1 CREBBP FOS Depression 7 HSPB1 ZBTB1 UBC FOS Depression 8 AKT1 UBC CASP8 CASP3 Depression 9 AKT1 UBC CASP8 CASP3 Depression 10 EGFR ARF6 UBC FOS Depression 11 EGFR ETF1 UBC BDNF Depression 12 FOS UBC LY6E GO:0042415.sup.3 Depression 13 FOS UBC LY6E GO:0048242.sup.4 Depression 14 FOS UBC LY6E GO:0042415.sup.3 Depression 15 OGDH PLSCR1 EP300 FOS Depression .sup.1GO:0007191 = Adenylate cyclase-activating dopamine receptor signaling pathway .sup.2GO:0014059 = Regulation of dopamine secretion .sup.3GO:0042415 = Norepinephrine metabolic process .sup.4GO:0048242 = Epinephrine secret ion

INDUSTRIAL APPLICABILITY

(88) As described above, the present disclosure can be usefully utilized in preparation of a therapeutic agent for depression and anxiety, which are stress diseases, with few side effects using a natural product, and a health functional food effective in alleviation of depression and anxiety, which are stress diseases, and fatigue.