Manufacturing method of medicinal herb for improving stress depression

Abstract

The present invention is characterized by a method for preparing a herbal medicine extract to improve stress-induced depression, which uses an extract obtained by mixing and extracting herbal medicines Panax ginseng, Astragalus membranaceus, Atractylodes macrocephala, Glycyrrhiza uralensis, Angelica sinensis, Citrus reticulata, Zingiber officinale, and Ziziphus jujuba in a specific weight ratio to improve behavioral patterns and reduce activity levels caused by depression in an animal model of depression induced by stress or corticosterone drugs.

Description

Technology Field

[0001] The present invention relates to a method for preparing a herbal medicine extract for improving stress-induced depression, and more specifically, to ginseng ( Panax ginseng ), Astragalus ( Astragalus membranaceus ), Atractylodes ( Atractylodes macrocephala ), licorice ( Glycyrrhiza uralensis ), Angelica ( Angelica sinensis ), dermis( Citrus reticulata ), ginger( Zingiber officinale ), contrast( Ziziphus jujuba The present invention relates to a method for preparing a herbal medicine extract to improve stress-induced depression, wherein the herbal medicine is mixed in a specific weight ratio and the extract is used to improve behavioral patterns and reduce activity in depression induced by stress or corticosterone drugs. Background Technology

[0002] As society rapidly develops and diversifies, modern people are required to take on various roles, and consequently, the number of people suffering from mental illnesses such as depression, anxiety disorders, or sleep disorders due to various stresses is increasing. According to the 2006 epidemiological survey on the prevalence of mental illness conducted by the Ministry of Health and Welfare targeting adults aged 18 to 64, the one-year prevalence rate of mental illness was found to be 17.1%. Furthermore, the lifetime prevalence rate of mental illness, which is the proportion of the population experiencing one or more mental illnesses during their lifetime, was found to be 30%, or one in three adults (as of 2006). Recently, there has been an increasing trend of adolescent mental illness due to causes such as excessive academic pressure.

[0003] Accordingly, stress refers to a state of physical or psychological pressure; it is triggered by pervasive elements in daily life and contributes to the development of almost all psychiatric and medical illnesses. However, if stress is not managed and is left unchecked, it leads to psychological reactions such as depression, anxiety, fatigue, anger, and mood swings, as well as physiological responses like a decrease in alpha waves in the brain and an increase in blood pressure and heart rate. If these reactions continue to recur, diseases such as depression, anxiety disorders, and sleep disorders appear, causing personal, family, and social losses and lowering an individual's quality of life. Among stress-induced illnesses, depression refers to a psychological state of stagnant mood characterized by feelings of sadness, despair, and frustration. In other words, it signifies a state that has progressed from normal emotions such as "sadness" and dysthymic disorder to major depressive disorder. The major symptoms are characterized by a major depressive episode, which is a period of low mood or loss of interest or pleasure in most activities, combined with some of the following: changes in appetite, weight, or sleep patterns; psychomotor agitation or retardation; reduced ability to think, concentrate, or make decisions; lack of energy and fatigue; feelings of worthlessness; feelings of self-reproach or guilt; frequent thoughts of death or suicide; or plans or attempts to commit suicide.

[0004] Furthermore, depression is a common mental illness affecting over 300 million people worldwide. It not only significantly reduces an individual's quality of life but also increases social and economic burdens. In severe cases, it can lead to suicide, placing it at the forefront of public health issues. Although antidepressants such as Selective Serotonin Reuptake Inhibitors (SSRIs) and Serotonin-Norepinephrine Reuptake Inhibitors (SNRIs) are currently widely used, treatment response rates remain limited to approximately 60–70%, and more than 30% of patients experience insufficient efficacy or serious side effects such as headaches, digestive issues, and insomnia. These limitations stem from the nature of existing drugs, which rely on a single target mechanism rather than addressing the multifaceted pathological mechanisms of depression. Consequently, there is a growing demand for new therapies capable of resolving these complexities. Prior art literature

[0005] Republic of Korea Published Patent No. 10-2019-0073325 Republic of Korea Registered Patent No. 10-1010370 Republic of Korea Registered Patent No. 10-1889479 Republic of Korea Registered Patent No. 10-1282759 The problem to be solved

[0006] The present invention relates to ginseng ( Panax ginseng ), Astragalus ( Astragalus membranaceus ), Atractylodes ( Atractylodes macrocephala ), licorice ( Glycyrrhiza uralensis ), Angelica ( Angelica sinensis ), dermis( Citrus reticulata ), ginger( Zingiber officinale ), contrast( Ziziphus jujuba The purpose is to provide a method for preparing a herbal medicine extract to improve stress-induced depression, which improves behavioral patterns and reduces activity levels caused by depression in an animal model of depression induced by stress or corticosterone drugs, by using an extract obtained by mixing herbal medicines of the above in a certain weight ratio. means of solving the problem

[0007] In order to achieve the aforementioned technical objectives, the present invention provides a method for preparing a herbal medicine extract to improve stress-induced depression, wherein ginseng ( Panax ginseng ), Astragalus ( Astragalus membranaceus ), Atractylodes ( Atractylodes macrocephala ), licorice ( Glycyrrhiza uralensis ), Angelica ( Angelica sinensis ), dermis( Citrus reticulata ), ginger( Zingiber officinale ), contrast( Ziziphus jujuba The method comprises the steps of: preparing the herbal ingredients separately; washing the prepared herbal ingredients separately with running water to remove surface dust and foreign substances; drying the washed herbal ingredients separately using a hot air dryer; grinding the dried herbal ingredients separately into fine particles using a grinder and then mixing the herbal ingredients in a certain weight ratio to obtain a mixture; adding 1,000 mL of distilled water, which is 10 times the weight of the herbal ingredient mixture, and performing a first reflux extraction at a certain temperature for a certain period of time to obtain a first extract; adding 1,000 mL of 70% ethanol to the residue remaining after obtaining the first extract and performing a second reflux extraction at a certain temperature for a certain period of time to obtain a second extract; mixing the first extract and the second extract, and then filtering the mixed extract through four layers of gauze to remove impurities; reducing the volume of the impurity-removed mixed extract to 1 / 10 using a vacuum concentrator, and then removing the moisture through a freeze dryer. It is carried out by including the step of removing and preparing an extract powder, which is a specific amount of herbal medicine extract.

[0008] Accordingly, the above ginseng ( Panax ginseng ), Astragalus ( Astragalus membranaceus ), Atractylodes ( Atractylodes macrocephala ), licorice ( Glycyrrhiza uralensis ), Angelica ( Angelica sinensis ), dermis( Citrus reticulata ), ginger( Zingiber officinale ), contrast( Ziziphus jujuba In the step of preparing each of the herbal medicines, the above ginseng ( Panax ginseng ) 12g, Astragalus ( Astragalus membranaceus ) 25g, Atractylodes ( Atractylodes macrocephala ) 15g, licorice ( Glycyrrhiza uralensis ) 5g, Angelica ( Angelica sinensis ) 10g, dried tangerine peel ( Citrus reticulata ) 8g, ginger( Zingiber officinale ) 10g, control ( Ziziphus jujuba It is prepared by preparing herbal medicines at a weight ratio of 15g.

[0009] Here, the temperature and time for the first reflux extraction are 100℃ and 2 hours, and the temperature and time for the second reflux extraction are 80℃ and 1 hour.

[0010] In addition, a herbal medicine extract can be prepared by uniformly adding and mixing 25% by weight of maltodextrin, 10% by weight of citric acid, and 5% by weight of vitamin C based on the weight of the above-mentioned extract powder, thereby formulating it into any one of capsule, tablet, powder, or liquid forms. Effects of the invention

[0011] According to an embodiment of the present invention, ginseng ( Panax ginseng ), Astragalus ( Astragalus membranaceus ), Atractylodes ( Atractylodes macrocephala ), licorice ( Glycyrrhiza uralensis ), Angelica ( Angelica sinensis ), dermis( Citrus reticulata ), ginger( Zingiber officinale ), contrast( Ziziphus jujuba Using an extract obtained by mixing herbal medicines of the following in a specific weight ratio, there is an effect of reducing the corticosterone content increased by stress or drug-induced depression, and not only does the reduced serotonin content increase, but there is also an effect of improving behavioral patterns and reducing activity levels caused by depression in an animal model of depression induction. Brief explanation of the drawing

[0012] Figure 1 is a plan for conducting animal experiments using a herbal medicine extract prepared according to the present invention. Figure 2 is a graph showing behavioral changes following the administration of fluoxetine and a mixture of herbal medicines after inducing depression with corticosterone administration. Figure 3 is a table showing the results of PCA (principal component analysis) and PLS-DA (partial least squares discriminant analysis) analysis following the administration of fluoxetine and a mixture of herbal medicines after inducing depression with corticosterone administration, where A is the PCA result of metabolites analyzed in positive mode, B is the PLS-DA result of metabolites analyzed in positive mode, C is the PCA result of metabolites analyzed in negative mode, and D is the PLS-DA result of metabolites analyzed in negative mode. Figure 4 is a graph showing the changes in individual metabolites (VIP > 1, p < 0.05) following the administration of fluoxetine and a mixture of herbal medicines after inducing depression with corticosterone administration, where A is the metabolite that showed a recovery pattern in the treatment group compared to the normal and control groups, and B is the metabolite that showed an individual pattern without a recovery pattern in the treatment group compared to the normal and control groups. Specific details for implementing the invention

[0013] The advantages and features of the present invention and the methods for achieving them will become clear by referring to the embodiments described below in conjunction with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below but may be implemented in various different forms. These embodiments are provided merely to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the invention, and the present invention is defined only by the scope of the claims.

[0014] The terms used in this specification will be briefly explained, and the invention will be described in detail.

[0015] The terms used in this invention have been selected based on currently widely used general terms, taking into account their functions within the invention; however, these terms may vary depending on the intent of those skilled in the art, case law, the emergence of new technologies, etc. Additionally, in specific cases, terms have been arbitrarily selected by the applicant, and in such cases, their meanings will be described in detail in the relevant description of the invention. Therefore, the terms used in this invention should be defined not merely by their names, but based on their meanings and the overall content of the invention.

[0016] When a part of a specification is described as 'comprising' a certain component, unless specifically stated otherwise, this means that it does not exclude other components but may include additional components, and singular expressions include plural expressions unless the context clearly indicates otherwise.

[0017] Embodiments of the present invention are described below in detail so that those skilled in the art can easily implement them. Furthermore, in order to clearly explain the present invention in the drawings, parts unrelated to the description are omitted. Additionally, when describing with reference to the attached drawings, identical components are given the same reference numerals regardless of drawing symbols, and redundant descriptions thereof are omitted. Moreover, in describing the present invention, if it is determined that a detailed description of related known technology may unnecessarily obscure the essence of the present invention, such detailed description is omitted.

[0018] First, the method for preparing a herbal medicine extract for improving stress-induced depression according to the present invention is ginseng ( Panax ginseng ) 12g, Astragalus ( Astragalus membranaceus ) 25g, Atractylodes ( Atractylodes macrocephala ) 15g, licorice ( Glycyrrhiza uralensis ) 5g, Angelica ( Angelica sinensis ) 10g, dried tangerine peel ( Citrus reticulata) 8g, ginger( Zingiber officinale ) 10g, control ( Ziziphus jujuba Each herbal medicine is prepared in a weight ratio of 15g, and each prepared herbal medicine is washed with running water to remove dust and foreign substances from the surface, and each washed herbal medicine is dried using a hot air dryer, and each dried herbal medicine is ground into fine particles using a grinder, and then the herbal medicines are mixed in a constant weight ratio as shown in Table 1 below to obtain a mixture.

[0019] Korean medicine name scientific name Indicator components weight(%) ginseng Panax ginseng Ginsenoside Rg1, Rb1, Re 12 Astragalus Astragalus membranaceus Astragaloside IV 25 Atractylodes Atractylodes macrocephala Atractyion 15 licorice Glycyrrhiza uralensis Glycyrrhizin 5 Angelica Angelica sinensis Ferulic Acid 10 dermis Citrus reticulata Hesperidin, Naringin 8 ginger Zingiber officinale Gingerol, Shogaol 10 contrast Ziziphus jujuba Jujuboside A 15

[0020] 1000 mL of distilled water, which is 10 times the weight of the above herbal medicine mixture, is added and a first reflux extraction is performed at a temperature of 100°C for 2 hours to obtain a first extract, and 1000 mL of 70% ethanol is added to the residue remaining after obtaining the first extract and a second reflux extraction is performed at a temperature of 80°C for 1 hour to obtain a second extract.

[0021] Here, the first extract and the second extract are mixed, and then the mixed extract is filtered through four layers of gauze to remove impurities, and then the volume is reduced to 1 / 10 using a vacuum concentrator, and then moisture is removed through a freeze dryer to produce a certain amount of extract powder, which is a herbal medicine extract.

[0022] Accordingly, a herbal medicine extract can be prepared by uniformly adding and mixing 25% by weight of maltodextrin, 10% by weight of citric acid, and 5% by weight of vitamin C based on the weight of the above-mentioned extract powder, thereby formulating it into any one of capsule, tablet, powder, or liquid forms.

[0023] That is, when the above-prepared extract powder is 20g, 5g of maltodextrin (25% by weight), 2g of citric acid (10% by weight), and 1g of vitamin C (5% by weight) are added and uniformly mixed based on the weight of 20g of the extract powder. At this time, the product is completed by molding and packaging suitable for each formulation, and below, the herbal medicine extract was used in the experiment.

[0024] The values ​​established through the above explanation are the result of research, and if they fall outside the set range, there is a problem in that the herbal medicine extract cannot be properly manufactured.

[0025] Animal experiments and the like were conducted to confirm that the herbal medicine extract prepared according to the present invention improves stress-induced depression.

[0026] 1. Animal testing

[0027] The animal experiments were supervised and approved by the Dongshin University Animal Ethics Committee (DSU2024-06-01) and were conducted according to the animal experiment plan shown in the attached Figure 1. Seven-week-old male C57BL / 6 rats were purchased from Samtaco Co., Ltd. (Osan, South Korea). Prior to the experiment, the animals were housed in a facility equipped with a constant temperature and humidity control system. Four rats were placed per cage, and they were allowed to eat and drink freely, with light and dark controlled on a 12-hour cycle. An adaptation period of at least seven days was observed prior to the experiment. Depression was induced by dissolving corticosterone (Sigma-Aldrich, MO, USA) in 0.9% (w / v) saline containing 1% Tween-80 and administering it via subcutaneous injection on days 7, 14, 21, and 28 after the start of the experiment. The groups were divided into a normal group (Normal), a depression-induced group (Control), a group administered fluoxetine after depression induction (Fluoxetine), a group administered a low concentration (100 mg / kg) of a herbal medicine mixture after depression induction (Low), and a group administered a high concentration (200 mg / kg) of a herbal medicine mixture after depression induction (High). The positive control group, Fluoxetine, was administered after dissolving it in a 0.9% saline solution at a concentration of 1.5 mg / mL. The experiment was conducted for a total of 4 weeks, and behavioral tests were performed at 3-day intervals to monitor behavioral changes following the induction of depression.

[0028] 2. Behavioral Experiment

[0029] Open field test (OFT)

[0030] The OFT measuring device consisted of a square box measuring 50 × 50 × 35 cm and was divided into 16 equally sized zones. The central area of ​​the device was defined as a 25 cm × 25 cm square, and the remaining parts were designated as the surrounding areas. Each experimental mouse was placed in one corner of the box, and the total distance traveled during the 5-minute test was recorded. The OFT was performed in a quiet environment, and lighting conditions were maintained without significant changes. After each experiment, the device was cleaned with 75% ethanol to remove odors.

[0031] Tail suspension test (TST)

[0032] Rats were individually placed in clear glass cylinders 30 cm high and 18 cm in diameter, filled with 20 cm of water (24 ± 2 °C). During the total 6-minute test, the first 2 minutes were used for acclimatization, followed by 4 minutes of immobility. Inactivity was defined as when the rats floated motionless or moved only minimally to keep their heads above the water. The FST was conducted in a quiet environment, and lighting conditions were maintained without significant changes. After each experiment, the rats were removed from the water and returned to their cages, and the water in the cylinders was replaced with fresh water.

[0033] Forced swim test (FST)

[0034] After attaching adhesive tape to the posterior one-third of the tail of each experimental rat, they were suspended approximately 40 cm from the floor for 6 minutes. Inactivity was measured during the last 4 minutes. Inactivity was defined as maintaining a vertical posture while suspended without any active escape movements.

[0035] 3. UPLC-QTOF-MS Analysis

[0036] Serum metabolite analysis was performed using a UPLC-QTOF-MS system (Xevo G3 QTOF, Waters Corp., CT, USA) and an ACQUITY UPLC HSS T3 column (150 mm × 2.1 mm, 1.8 μm) at a temperature maintained at 25°C. Elution was performed using a gradient program consisting of mobile phases A (water containing 0.1% formic acid) and B (acetonitrile containing 0.1% formic acid): 0-2 min, 10% B; 2-40 min, 10-60% B; 40-45 min, 60-85% B; 45-52 min, 85-95% B; 52-53 min, 95-100% B. The flow rate was set to 0.3 ml / min, and the injection volume was 2 μl. Mass spectrometry (MS) analysis was performed in cation and anion modes, respectively, with the mass range set to m / z 50–1200 Da. The optimized ionization conditions were as follows: capillary voltage 3.0 kV, source temperature 120°C, desolvate gas flow rate 900 L / h, desolvate temperature 450°C, cone voltage 40 V, cone gas flow 10 L / h, collision energy 4.0 V, transfer collision energy 2.0 V, and ramp collision energy 20 eV–40 eV. During the detection process, a scan time of 0.3 seconds and an interval scan time of 0.1 seconds were used. Additionally, to ensure the accuracy of the MS analysis, a solution of Leucine-enkephalin (m / z 556.2771, [M + H]+) at a concentration of 200 ng / μl was used as an external reference at a flow rate of 10 μl / min. The data were analyzed using TOF MS E Mass spectroscopic data of the mother ion and decomposition ion of each detected analyte were collected simultaneously in MS at elevated fragmentation energy mode.

[0037] 4. Data Processing

[0038] All MS E The results were processed using the UNIFI program (Waters Corp., Milford, USA). MS E The results were processed using vertex-peak detection and alignment algorithms and analyzed using Progenesis QI software (Waters Corp., Milford, USA). The intensity of each ion was calculated as a peak area by normalizing the total number of ions to retention time and m / z values, thereby generating a data matrix. Metabolites corresponding to the generated peaks were identified through spectral comparison using the Human Metabolome Database (HMDB) and the MassBank of North America (MoNA) databases. The processed data was exported to Metaboanalyst online software for multivariate analysis. After performing mean scaling on the data, Principal Component Analysis (PCA) and Partial Least Squares Discriminant Analysis (PLS-DA) were conducted. To identify metabolites contributing to differences between groups, metabolites with VIP values ​​greater than 1.0 were selected as candidate biomarkers, and their significance was verified using t-tests.

[0039] 5. Statistical Analysis

[0040] All data statistics were analyzed using Graphpad Prism 9.0, and all data were displayed as mean and standard deviation. Differences between groups were tested for significance using the Mann-Whitney-Wilcoxon (MWW) test.

[0041] Figure 2 of the attached drawing is a graph showing behavioral changes following the administration of fluoxetine and a mixture of herbal medicines after depression was induced by the administration of corticosterone, which will be explained in detail below.

[0042] Open Field Test

[0043] The Open Field Test is an experiment designed to evaluate the voluntary activity and anxiety levels of animals. Total Distance (TD) indicates behavioral activity by measuring the distance a rat travels within the box. An increase in travel distance signifies higher activity or decreased anxiety.

[0044] The experimental results showed no difference in travel distance between the normal and control groups, but the group treated with fluoxetine after inducing depression showed a significant difference from the control group. This suggests that while CORT-induced depression did not directly affect the results of the open field test, treatment with fluoxetine did influence activity levels.

[0045] When a mixture of herbal medicines was administered after inducing depression with CORT, higher activity was observed compared to the control group, but no significant difference was found.

[0046] The results of the open field test suggest that the induction of depression (CORT treatment) did not have a direct effect on activity (Total Distance, TD). The absence of a difference in travel distance between the normal and control groups indicates that the depressive state did not exhibit a clear effect in reducing activity indicators such as travel distance. This implies that the open field test may be more sensitive to evaluating changes in anxiety levels than changes in activity levels in the CORT-induced depression model.

[0047] The significant increase in travel distance in the fluoxetine group compared to the control group indicates that fluoxetine had a positive effect on restoring behavioral activity.

[0048] This suggests that fluoxetine is effective in inducing behavioral recovery in depression models and implies the possibility that the drug improved activity through mechanisms related to neurotransmitter regulation. Therefore, it can be concluded that fluoxetine acted as a therapeutic agent demonstrating a significant therapeutic effect in the open field test in depression models.

[0049] In the groups administered the herbal mixture (Low and High), travel distance increased compared to the control group, but no significant difference was observed. This suggests that the herbal mixture may have been less potent than fluoxetine in fully restoring reduced activity levels caused by depression. However, the observation of increased activity in the groups administered the herbal mixture suggests that the mixture may possess a certain level of behavioral improvement. In particular, the higher activity levels observed in the high-concentration group (High) support the possibility of a concentration-dependent effect of the herbal mixture.

[0050] These results show that although the herbal medicine mixture exhibited relatively limited effects compared to fluoxetine in open field tests, it still possesses a certain level of therapeutic potential.

[0051] Forced Swim Test

[0052] The forced swim test is an experiment designed to assess lethargy by measuring the time rats remain in an inactive state in water. An increase in activity time leads to a decrease in lethargy and indicates an antidepressant effect.

[0053] The experimental results showed that, similar to the open field test, there was no significant difference between the normal and control groups in the forced swim test; however, the control group tended to have lower values ​​than the normal group, while the fluoxetine and herbal medicine mixture treatment group showed an increasing pattern compared to the control group. In particular, the low-concentration (Low) administration group of the herbal medicine mixture showed a significantly increased result.

[0054] Although no significant difference was observed between the normal and control groups, the fact that the control group showed a lower tendency than the normal group suggests the possibility that CORT treatment induced slight lethargy.

[0055] The pattern of increased activity time in the fluoxetine treatment group and the herbal medicine mixture treatment group is interpreted as a result reflecting the therapeutic effect as an antidepressant. In particular, fluoxetine appears to have contributed to alleviating lethargy caused by depression through its primary mechanism of action, which involves inhibiting the reabsorption of neurotransmitters (especially serotonin). The increase in activity time in the fluoxetine group compared to the control group reflects this mechanism and supports the behavioral recovery effect of antidepressants.

[0056] The significant increase in activity time in the low-concentration group of the herbal mixture indicates that the mixture may be effective in alleviating lethargy associated with depression. This suggests that the herbal mixture may have contributed to behavioral recovery through multiple mechanisms (anti-inflammatory, neuroprotective, immunomodulatory, etc.) and demonstrates that it can exert a meaningful therapeutic effect, particularly at low concentrations. This suggests the possibility that the herbal mixture improves depression-related behavioral indicators through a mechanism of action different from fluoxetine, highlighting its therapeutic potential as a traditional herbal medicine.

[0057] However, the fact that the increase in activity in the high-concentration herbal mixture administration group (High) was not distinct compared to the low-concentration group (Low) suggests that the concentration-dependent effects of the herbal mixture may act non-linearly on specific behavioral indicators.

[0058] Tail Suspension Test

[0059] The tail suspension test is an experiment that measures the time a rat remains motionless while suspended, and this period of inactivity is used as an indicator to assess lethargy. A shorter period of inactivity indicates an antidepressant effect.

[0060] The experimental results showed that the control group exhibited a decreasing trend compared to the normal group, but no significant difference was observed. However, the fluoxetine and herbal medicine mixture showed an increasing trend, and notably, the group administered a low concentration of the fluoxetine and herbal medicine mixture showed a significant difference compared to the control group.

[0061] The fact that the control group showed a slight decreasing trend compared to the normal group suggests the possibility that lethargy may have increased somewhat due to CORT treatment.

[0062] The significant increase in inactivity time observed in the fluoxetine treatment group and the low-concentration herbal mixture group is interpreted as an important indicator reflecting antidepressant effects. Fluoxetine is known to reduce lethargy associated with depression by inhibiting serotonin reuptake, and this study also demonstrated a significant improvement compared to the control group. This implies that fluoxetine demonstrated a clear behavioral recovery effect in the TST.

[0063] The significant increase in inactivity time in the low-concentration herbal mixture administration group suggests that the herbal mixture may be effective in alleviating lethargy associated with CORT-induced depression. This implies that the herbal mixture has the potential to improve lethargy in depressive states through multiple mechanisms (anti-inflammatory, neuroprotective, etc.) distinct from fluoxetine. In particular, the fact that meaningful results were obtained even at low concentrations demonstrates that the herbal mixture is a practical treatment option capable of exerting efficacy even at relatively low levels.

[0064] On the other hand, the increase in inactivity time was not pronounced in the high-concentration (High) group of the herbal medicine mixture compared to the low-concentration (Low) group. This suggests that the concentration-dependent effects of the herbal medicine mixture may reach a saturation state above a certain level for specific behavioral indicators, or that high concentrations may exhibit limited effects on behavioral indicators. These results emphasize the need for further research to identify the optimal concentration of the herbal medicine mixture and to more clearly understand the metabolic and physiological changes at high concentrations.

[0065] In conclusion, the Tail Suspension Test results demonstrate that a mixture of fluoxetine and herbal medicines (particularly at low concentrations) may be effective in alleviating lethargy in a CORT-induced depression model. Unlike fluoxetine, the herbal mixture has the potential to exert antidepressant effects through multiple mechanisms, which supports the therapeutic potential of traditional Korean medicine. Future research needs to more closely analyze the dose-response relationships of the herbal mixtures and elucidate their biochemical mechanisms to expand their applicability to the treatment of depression.

[0066] Figure 3 of the attached drawing is a table showing the results of PCA (principal component analysis) and PLS-DA (partial least squares discriminant analysis) analysis following the administration of fluoxetine and a mixture of herbal medicines after inducing depression with corticosterone administration, where A represents the PCA results of metabolites analyzed in positive mode, B represents the PLS-DA results of metabolites analyzed in positive mode, C represents the PCA results of metabolites analyzed in negative mode, and D represents the PLS-DA results of metabolites analyzed in negative mode, which will be explained in detail below.

[0067] Principal Component Analysis (PCA) is an unsupervised learning technique used to explain the major variability of high-dimensional data by reducing it to a lower dimension. It generates new axes (principal components, PCs) that best represent the variability of the original data, visualizing the data to make it easier to understand and interpret.

[0068] The purpose of the analysis is to extract key features from high-dimensional data and identify similarities and differences between data. Additionally, it aims to reduce complexity by decreasing data dimensionality, enable data visualization, and explore key patterns by analyzing correlations between variables.

[0069] Partial Least Squares Discriminant Analysis (PLS-DA) is a supervised learning technique, unlike PCA. It generates new axes (PLS components) by linearly combining data variables to maximize discrimination between groups based on given class information. This analysis is used to effectively distinguish specific groups (e.g., control group and treatment group).

[0070] Upon reviewing the PCA results in positive mode (Figure 3A), the two axes of PC1 (29.9%) and PC2 (23.8%) explained 53.7% of the total data variability. The control group was found to show some differences compared to the normal group and the group treated with fluoxetine. The group administered the herbal mixture showed a greater difference than fluoxetine, exhibiting the same pattern across both concentration ranges.

[0071] Figure 3B shows the results of metabolite data analysis via PLS-DA, where component 1 (29.5%) and component 2 (5.6%) explain the overall data variability. The control group is located near the center of the graph and formed an independent cluster distinct from other groups (normal and treatment groups). This indicates that depression induction (CORT treatment) caused changes in the metabolite profile. The normal group is located in the upper right corner and formed a cluster distinct from other groups. This suggests that the normal group possesses metabolite characteristics clearly distinguishable from the control and treatment groups. The group treated for depression with fluoxetine shifted toward the normal group compared to the control group, suggesting a metabolite recovery effect. The group treated with a mixture of herbal medicines showed a distinct pattern compared to the group treated for depression with fluoxetine and the normal group. This can be interpreted as exhibiting greater metabolic differences than the group treated with fluoxetine.

[0072] Figure 3C shows the results of PCA in negative mode, where PC1 (42%) and PC2 (29.4%) explain 71.4% of the total data variability. The control group is located near the center of the graph and shows a distribution that partially overlaps with the normal and treatment groups. This suggests that while CORT-induced depression in negative mode caused some changes at the metabolite level, clear differentiation may be difficult. The normal group forms a cluster relatively distinct from the other groups. This indicates that the normal group is differentiated from other groups in terms of metabolomic characteristics. The pattern of the treatment group (fluoxetine and herbal mixture) did not show a clear difference from the control group. The results of PLS-DA in Figure 3D also showed similar findings to PCA.

[0073] Synthesizing the analysis results of the Positive and Negative modes, it can be seen that the Positive mode acted more sensitively in reflecting the induction and therapeutic effects of depression. This suggests that metabolites analyzed in the Positive mode may serve as more important biomarkers in depression models, and further research should be conducted focusing on these metabolites.

[0074] The herbal medicine mixture induced metabolite changes through a mechanism different from that of fluoxetine and showed a certain level of effect in positive mode. However, the fact that the difference from the normal group compared to fluoxetine was greater suggests that the mechanism of action of the herbal medicine mixture may be focused on inducing metabolite changes rather than restoring the normal state. To confirm this, it is necessary to elucidate the mechanism of action of the herbal medicine mixture by conducting an in-depth analysis of metabolite data and identifying key metabolites and pathways.

[0075] In conclusion, the herbal medicine mixture induces metabolic changes associated with the induction and treatment of depression in positive mode metabolite analysis, and has the potential to exhibit effects similar to fluoxetine.

[0076] Figure 4 of the attached drawings is a graph showing the changes in individual metabolites (VIP > 1, p < 0.05) following the administration of fluoxetine and a mixture of herbal medicines after inducing depression with corticosterone administration, where A represents metabolites that showed a recovery pattern in the treatment group compared to the normal group and control group, and B represents metabolites that did not show a recovery pattern and showed an individual pattern in the treatment group compared to the normal group and control group, which will be explained in detail below.

[0077] This represents the metabolites that showed a VIP of 1 or higher and a p-value of 0.05 or lower by comparing PLS-DA based on metabolites identified in positive mode and negative mode to confirm individual metabolite changes.

[0078] In other words, the results were confirmed to be classified into a total of two patterns. 2-O-Acetly-20-hydroxyecdysone, phenylalanine, 3-formylindole, voacamine, LPC 18:2, and dauricine were metabolites that showed a pattern of recovery to the normal group in the treatment group (fluoxetine and herbal medicine mixture) compared to the normal and control groups (see attached Figure 4A).

[0079] Figure 4B of the attached drawing shows the metabolites that showed individual patterns in the treatment group compared to the normal group and the control group, and the metabolites included therein were (E,2S,3R,4R,5S)-4-acetyloxy-2-amino-3,5,14-trihydroxyicos-6-enoic acid, LPC 16:0, LPC 18:3, 1-stearoyl-sn-glycero-3-phosphocholine, PC(16:0 / 0:0), LPE 18:1, histidine conjugated cholic acid, LysoPC(0:0 / 18:0).

[0080] 2-O-Acetyl-20-hydroxyecdysone is a steroid derivative that may contribute to anti-inflammatory and immunomodulatory effects. This may be related to the recovery effects observed in the treatment group.

[0081] Phenylalanine is an essential amino acid that acts as a precursor for the synthesis of monoamine neurotransmitters (particularly dopamine and norepinephrine). The recovery patterns following treatment suggest that the imbalance of neurotransmitters associated with depression has been restored.

[0082] LPC 18:2 is a phospholipid metabolite that plays an important role in regulating inflammation and cell signaling. The fact that the treatment group recovered to levels similar to the normal group suggests that the treatment may have regulated the inflammatory response.

[0083] Metabolites in this pattern reflect a metabolite profile restored to a normal state following treatment and have the potential to serve as biomarkers indicating the efficacy of depression treatment. In particular, the recovery of metabolites related to inflammation regulation, neurotransmitter metabolism, and cell signaling is highly likely to be associated with the behavioral improvements observed in the treatment group.

[0084] LPC 16:0, LPC 18:3, and LysoPC(0:0 / 18:0)** belong to the lysophosphatidylcholine (LysoPC) family and play important roles in cell membrane metabolism and inflammatory responses. Individual patterns in the treatment group suggest that the herbal mixture and Fluoxetine may regulate cell membrane lipid metabolism differently.

[0085] Histidine conjugated cholic acid is a bile acid metabolite that may be involved in the regulation of the gut-brain axis. This suggests that herbal medicine mixtures may influence the treatment of depression through the regulation of metabolites.

[0086] (E,2S,3R,4R,5S)-4-acetyloxy-2-amino-3,5,14-trihydroxyicos-6-enoic acid is a fatty acid metabolite with a complex structure that has the potential to play an important role in anti-inflammation and cell signaling regulation.

[0087] These metabolites exhibit metabolic changes unique to the treatment group that are distinct from the control group, demonstrating that the fluoxetine and herbal mixture can exert therapeutic effects on depression through different mechanisms. In particular, the unique metabolite profile of the herbal mixture is worthy of exploring mechanisms of action related to inflammatory responses, cell membrane metabolism, and the gut-brain axis.

[0088] Metabolites identified in positive and negative modes exhibit two major patterns in the treatment group. The first pattern consists of metabolites that have recovered to the normal group level, suggesting their potential to serve as key biomarkers directly indicating therapeutic effects. The second pattern represents metabolite changes unique to the treatment group, reflecting the distinct mechanisms of action of the herbal mixture and Fluoxetine.

[0089] As described above, a study using a mixture of herbal extracts according to the present invention evaluated the antidepressant effects of the herbal mixture and Fluoxetine in a corticosterone (CORT)-induced depression model using a metabolomic approach. Metabolite analysis results in positive and negative modes classified the treatment group into two main patterns: a pattern of recovery to the normal group and metabolite changes unique to the treatment group. Metabolites that recovered to the normal group (e.g., 2-O-Acetyl-20-hydroxyecdysone, phenylalanine, LPC 18:2, etc.) played important roles in inflammation regulation, neurotransmitter metabolism, and cell signaling, reflecting the antidepressant effect. On the other hand, metabolites unique to the treatment group (e.g., LPC 16:0, histidine conjugated cholic acid, etc.) suggested that the herbal mixture and Fluoxetine contribute to the treatment of depression through different mechanisms of action. Through this, it was confirmed that the herbal medicine mixture possesses unique therapeutic effects through multiple mechanisms and can be evaluated as a promising candidate for the treatment of depression.

[0090] Finally, when using the herbal medicine mixture prepared according to the present invention, it is applicable to TCM, corticosterone (CORT), and metabolomics.

[0091] In other words, TCM is a promising complementary and alternative therapy capable of providing a multi-targeted approach to treating depression, and TCM herbal prescriptions have demonstrated efficacy in regulating the complex pathophysiology of depression through multiple mechanisms. For example, Xiaoyaosan has demonstrated efficacy in alleviating depression-like behaviors in preclinical models by regulating the hypothalamic-pituitary-adrenal (HPA) axis and relieving oxidative stress. Additionally, Kaixinsan has been reported to stabilize mood and relieve stress by regulating monoamine neurotransmitters such as serotonin and dopamine.

[0092] Furthermore, corticosterone (CORT) is a glucocorticoid hormone commonly used to study stress-induced depression in rodent models and corresponds to human cortisol. Corticosterone induces hyperactivation of the hypothalamic-pituitary-adrenal axis, which is similar to the pathological mechanism by which depression develops due to chronic stress. Hyperactivation of the hypothalamic-pituitary-adrenal axis leads to neurotransmitter imbalance, an increase in inflammatory cytokines (such as IL-6 and TNF-α), suppression of brain-derived neurotrophic factor expression, and hippocampal damage. These changes induce neuroinflammation and reduced neuroplasticity, exacerbating symptoms of depression by inhibiting the synthesis of neurotransmitters such as serotonin, dopamine, and norepinephrine. Experimentally, corticosterone is administered for a certain period to mimic chronic stress and is used to evaluate symptoms of depression in behavioral tests such as the Forced Swim Test (FST), Tail Suspension Test (TST), and Open Field Test (OFT). Furthermore, this allows for the comparison of the therapeutic effects and mechanisms of action of antidepressants and herbal medicines.

[0093] Furthermore, metabolomics serves as a powerful tool for exploring biochemical pathways and metabolic disturbances in complex conditions such as depression, and for evaluating therapeutic effects. Depression is associated with metabolic changes closely related to neurotransmitter synthesis, lipid metabolism, and energy homeostasis. Advanced technologies, such as ultra-high performance liquid chromatography and quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS), can comprehensively analyze metabolic changes and pathways regulated by therapeutic interventions. This approach is suitable for multi-target therapies, such as herbal mixtures, and can contribute to understanding the diverse effects of herbal mixtures on depression.

[0094] Accordingly, the present invention allows for the confirmation of an antidepressant effect by applying a herbal medicine extract composition in a corticosterone-induced depression model, and for the identification of the mechanism of action through metabolite analysis.

[0095] The above description is merely an illustrative explanation of the technical concept of the present invention, and those skilled in the art to which the present invention pertains will be able to make various modifications and variations within the scope of the essential quality of the present invention. Accordingly, the embodiments disclosed in the present invention are intended to explain, not limit, the technical concept of the present invention, and the scope of the technical concept of the present invention is not limited by such embodiments. The scope of protection of the present invention shall be interpreted by the claims below, and all technical concepts within the equivalent scope shall be interpreted as being included within the scope of rights of the present invention.

Claims

Claim 1 ginseng( Panax ginseng ), Astragalus ( Astragalus membranaceus ), Atractylodes ( Atractylodes macrocephala ), licorice ( Glycyrrhiza uralensis ), Angelica ( Angelica sinensis ), dermis( Citrus reticulata ), ginger( Zingiber officinale ), contrast( Ziziphus jujuba A step of preparing the herbal ingredients separately; a step of washing the prepared herbal ingredients separately with running water to remove surface dust and foreign substances; a step of drying the washed herbal ingredients separately using a hot air dryer; a step of grinding the dried herbal ingredients separately into fine particles using a grinder and then mixing the herbal ingredients in a certain weight ratio to obtain a mixture; a step of adding 1,000 mL of distilled water, which is 10 times the weight of the herbal ingredient mixture, and performing a first reflux extraction at a certain temperature for a certain time to obtain a first extract; a step of adding 1,000 mL of 70% ethanol to the residue remaining after obtaining the first extract and performing a second reflux extraction at a certain temperature for a certain time to obtain a second extract; a step of mixing the first extract and the second extract, and then filtering the mixed extract through four layers of gauze to remove impurities; a step of reducing the volume of the impurity-removed mixed extract to 1 / 10 using a vacuum concentrator and then removing moisture through a freeze dryer to obtain a certain amount of herbal ingredients A method for preparing a herbal medicine extract for improving stress-induced depression, comprising the step of preparing an extract powder. Claim 2 In paragraph 1, the above ginseng ( Panax ginseng ), Astragalus ( Astragalus membranaceus ), Atractylodes ( Atractylodes macrocephala ), licorice ( Glycyrrhiza uralensis ), Angelica ( Angelica sinensis ), dermis( Citrus reticulata ), ginger( Zingiber officinale ), contrast( Ziziphus jujuba In the step of preparing each of the herbal medicines, the above ginseng ( Panax ginseng ) 12g, Astragalus ( Astragalus membranaceus ) 25g, Atractylodes ( Atractylodes macrocephala ) 15g, licorice ( Glycyrrhiza uralensis ) 5g, Angelica ( Angelica sinensis ) 10g, dried tangerine peel ( Citrus reticulata ) 8g, ginger( Zingiber officinale ) 10g, control ( Ziziphus jujuba A method for preparing a herbal medicine extract to improve stress-induced depression, characterized by preparing the herbal medicine at a weight ratio of 15g. Claim 3 A method for preparing a herbal medicine extract for improving stress-induced depression, characterized in that, in claim 1, the temperature and time for the first reflux extraction are 100℃ and 2 hours, and the temperature and time for the second reflux extraction are 80℃ and 1 hour.

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