Use of mind-calming and brain-strengthening liquid in the preparation of drug for treating or preventing depression

Abstract

The present application relates to the field of pharmaceutical uses and particularly relates to the use of a mind-calming and brain-strengthening liquid in the preparation of a drug for treating or preventing depression. For the first time, the present application uses the mind-calming and brain-strengthening liquid in researching of prevention and treatment of depression, and develops a novel use of a traditional compound traditional Chinese medicine from the perspective of "using existing medicines in a new way". By means of a tail suspension test, an open field test and an elevated plus maze test, the present application finds that the mind-calming and brain-strengthening liquid can relieve the anxiety emotion and depression disorders of mice in each group; and further analysis and verification of possible antidepressive targets of the mind-calming and brain-strengthening liquid by means of the network pharmacology indicate that administrating the mind-calming and brain-strengthening liquid can remarkably upregulate the activity of the AKT protein, demonstrating that the mind-calming and brain-strengthening liquid activates a signal pathway downstream of the AKT by means of upregulating the activity of the AKT protein, so as to finally achieve an antidepressive function.

Description

Application of tranquilizing and brain-strengthening liquid in preparing medicine for treating or preventing depression

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to the Chinese patent application filed with the China Patent Office on August 4, 2023, with application number 202310980517.0 and invention name “Application of tranquilizing and brain-strengthening liquid in the preparation of drugs for treating or preventing depression”, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present application relates to the field of medical applications, and specifically to the use of tranquilizing and brain-strengthening liquid in the preparation of drugs for treating or preventing depression. Background Art

[0004] Depression, also known as depressive disorder, is the most common affective mental disorder in society today. Its main clinical features include low mood, slowed thinking, decreased speech and movement, and loss of interest. Severe cases can be accompanied by suicidal tendencies. The pathogenesis of depression is complex, and the underlying process remains unclear. Existing classic theories include the monoaminergic hypothesis, the neurotrophic hypothesis, the neuroplasticity hypothesis, the neuroendocrine hypothesis, and the neuroimmune hypothesis. Currently, the diagnosis of depression is primarily based on clinical manifestations. As of 2020, more than 350 million people worldwide suffer from depression, with 95 million diagnosed in my country by 2022, and this number continues to increase annually. Depression not only causes tremendous suffering to patients and their families, but also increases the medical burden on society and significantly hinders economic development. Currently, research on depression biomarkers both domestically and internationally focuses on inflammatory mediators, neurotrophic factors, and the hypothalamic-pituitary-adrenal (HPA) axis. Existing clinical antidepressant chemical drugs still have problems such as obvious adverse reactions, low efficacy, and poor patient compliance. Therefore, there is an urgent need to expand ideas and try new methods to study and screen effective antidepressants from a multi-target and multi-pathway perspective to intervene in the pathological process of depression as early as possible.

[0005] Compound Chinese medicine has a long history in my country. Long-term use and continuous modification and improvement of the formula have made its formula more reasonable, rich in effective ingredients, and better in pharmacological activity and safety. At the same time, because the formula contains multiple effective ingredients, compound Chinese medicine has the advantage of multi-target and multi-pathway regulation. Beijing Tongrentang Anshen Jiannao Liquid (National Medicine Standard No. Z11020290) is composed of ingredients such as ginseng, Schisandra chinensis, Ophiopogon japonicus, Lycium barbarum, and Salvia miltiorrhiza. It is mainly used to treat insomnia, dizziness, headache, tinnitus, palpitations, fatigue, forgetfulness, dry mouth and tongue, and neurasthenia. However, whether it has a preventive or therapeutic effect on depression has not yet been reported. Therefore, studying the therapeutic effect of Anshen Jiannao Liquid on depression is of great significance, and also provides new ideas for developing compound Chinese medicines for depression from the perspective of "new uses of old drugs".

[0006] Summary of the Invention

[0007] Therefore, the purpose of the present application is to provide a new use of tranquilizing and brain-strengthening liquid, namely, the use of tranquilizing and brain-strengthening liquid in the preparation of a medicine for treating or preventing depression.

[0008] To this end, the present application provides the use of tranquilizing and stimulating brain liquid in the preparation of a medicine for treating, preventing or alleviating depression.

[0009] Furthermore, the raw materials of the tranquilizing and brain-strengthening liquid include ginseng, Schisandra chinensis, Ophiopogon japonicus, Lycium barbarum and Salvia miltiorrhiza.

[0010] Furthermore, in the raw materials of the tranquilizing and brain-strengthening liquid, the mass ratio of ginseng, schisandra chinensis, ophiopogon japonicus, wolfberry and salvia miltiorrhiza is 20-80:100-160:100-160:100-160:60-120.

[0011] The tranquilizing and brain-strengthening liquid can be prepared by conventional methods in the art, or purchased from Beijing Tong Ren Tang Technology Development Co., Ltd. The Schisandra chinensis chinensis is vinegar-fried Schisandra chinensis chinensis.

[0012] Furthermore, the drug can inhibit the increase of anxiety in patients with depression.

[0013] Furthermore, the drug can inhibit the decline of the desire for exploration in patients with depression.

[0014] Furthermore, the depression is depression mediated by the AKT signaling pathway, and the drug can activate the AKT signaling pathway and increase the p-AKT level.

[0015] Furthermore, the drug can reduce the deposition of Aβ protein in the hippocampus of patients with depression.

[0016] Furthermore, the tranquilizing and brain-strengthening liquid is a clinically acceptable oral liquid prepared by conventional process with ginseng, Schisandra chinensis, Radix Ophiopogonis, Lycium barbarum and Salvia miltiorrhiza as active ingredients and conventional auxiliary materials.

[0017] Furthermore, the drug is used to treat, prevent or alleviate neurodegenerative diseases combined with depression.

[0018] Furthermore, the neurodegenerative disease is Alzheimer's disease.

[0019] The present application also provides a method for treating, preventing or alleviating depression, comprising administering a tranquilizing and brain-strengthening liquid to a subject in need thereof.

[0020] Furthermore, the raw materials of the tranquilizing and brain-strengthening liquid include ginseng, Schisandra chinensis, Ophiopogon japonicus, Lycium barbarum and Salvia miltiorrhiza.

[0021] Furthermore, in the raw materials of the tranquilizing and brain-strengthening liquid, the mass ratio of ginseng, schisandra chinensis, ophiopogon japonicus, wolfberry and salvia miltiorrhiza is 20-80:100-160:100-160:100-160:60-120.

[0022] Furthermore, the tranquilizing and brain-strengthening liquid can inhibit the increase of anxiety in patients with depression.

[0023] Furthermore, the tranquilizing and brain-strengthening liquid can inhibit the decline of the desire for exploration in patients with depression.

[0024] Furthermore, the depression is depression mediated by the AKT signaling pathway, and the tranquilizing and brain-strengthening liquid can activate the AKT signaling pathway and increase the p-AKT level.

[0025] Furthermore, the tranquilizing and brain-strengthening liquid can reduce the deposition of Aβ protein in the hippocampus tissue of patients with depression.

[0026] Furthermore, the tranquilizing and brain-strengthening liquid is a clinically acceptable oral liquid prepared by conventional process with ginseng, Schisandra chinensis, Radix Ophiopogonis, Lycium barbarum and Salvia miltiorrhiza as active ingredients and conventional auxiliary materials.

[0027] Furthermore, the dosage of the tranquilizing and brain-strengthening liquid is 3 mL / kg / day to 30 mL / kg / day.

[0028] The technical solution of this application has the following advantages:

[0029] This application is the first to use Anshen Jiannao Liquid for the research on the prevention and treatment of depression, and to develop new uses for traditional compound Chinese medicine from the perspective of "new uses for old medicines". This application uses the tail suspension test, open field test and cross elevated maze test to find that Anshen Jiannao Liquid can improve the anxiety and depressive disorders of each group of mice. Further analysis and verification of the possible anti-depressant targets of Anshen Jiannao Liquid through network pharmacology found that the administration of Anshen Jiannao Liquid can significantly upregulate the activity of AKT protein, indicating that Anshen Jiannao Liquid is by upregulating the activity of AKT, thereby activating the signaling pathway downstream of AKT, and ultimately achieving the anti-depressant effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the following is a brief introduction to the drawings required for use in the specific implementation methods or the description of the prior art. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0031] Figure 1 shows the behavioral assessment of depression and anxiety in mice in Experimental Example 1. A: Time spent in tail suspension; B: Number of grids crossed by mice; C: Number of times mice stood up; D: Number of times mice defecated; E: Time spent in an open arm; F: Number of open arm entries; G: Number of open arm entries / total number of arm entries (n=8, comparison between wild type (WT) group and model (AD) group, ## P<0.01, ### P<0.001; *P<0.05, **P<0.01, ***P<0.001 compared with the model group).

[0032] Figure 2 shows the detection of Aβ pathology-related proteins in mouse hippocampus tissue in Experimental Example 1. A: Western Blot detection of Aβ in mouse hippocampus 1-42 Protein expression level; B: Aβ in mouse hippocampus 1-42 Gray value analysis of protein (n=4); C: Immunofluorescence detection of Aβ expression levels in CA1 and CA3 regions of hippocampus of mice in each group (bar: 100μm). ## P<0.01, ### P<0.001; *P<0.05, **P<0.01 compared with the model group.

[0033] Figure 3 shows the screening of depression and tranquilizing and brain-strengthening liquid targets and the common target PPI network in Experimental Example 1, A: Venn diagram of depression and tranquilizing and brain-strengthening liquid component targets; B: common target PPI network.

[0034] Figure 4 is a bar chart of GO analysis of common targets in Experimental Example 1 of this application.

[0035] Figure 5 is the detection of Aβ pathological protein in mouse hippocampus tissue in Experimental Example 1 of the present application; A: Western Blot detection of the expression levels of p-AKT and AKT protein in mouse hippocampus; B: Gray value analysis of p-AKT / AKT in mouse hippocampus (n=4); Comparison between wild-type group and model group, # P<0.05; *P<0.05, **P<0.01 compared with the model group. DETAILED DESCRIPTION

[0036] The following examples are provided to further better understand the present application, but are not limited to the best implementation mode described herein, and do not limit the content and protection scope of the present application. Any product identical or similar to the present application obtained by anyone under the inspiration of the present application or by combining the features of the present application with other prior arts shall fall within the scope of protection of the present application.

[0037] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in this field can be used. If the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional reagents.

[0038] Experimental Example 1

[0039] Using triple transgenic AD (3×Tg-AD) mice as the research subjects, different doses of Anshen Jiannao Liquid were administered to compare and analyze the antidepressant effects and mechanisms of Anshen Jiannao Liquid. The Anshen Jiannao Liquid used in this study (National Medicine Approval No. Z11020290) was provided by Beijing Tong Ren Tang Science and Technology Development Co., Ltd., batch number: 21260815.

[0040] 3×Tg-AD model mice and wild-type (WT) mice of the same age were fed to 3 months of age. Model mice were then randomly divided into four groups (15 per group): low-dose, medium-dose, high-dose, and model groups. 0.15 mL of Anshen Jiannao solution was added to 1 mL of ddH2O and mixed thoroughly to obtain the test drug solution. The low-dose (AD+Low), medium-dose (AD+Middle), and high-dose (AD+High) groups were all gavaged with the test drug solution at doses of 3-6 mL / kg / day, 8-12 mL / kg / day, and 20-30 mL / kg / day, respectively. Drug administration began at 3 months of age and continued for 4 consecutive months until the mice reached 7 months of age. No drug administration was administered to the WT and model groups. All mice were housed under standard conditions, with the animal room temperature maintained at 22 ± 2°C. They were maintained under standard experimental conditions with a 12-hour day-night cycle and fed a standard, certified diet purchased from the Guangdong Medical Laboratory Animal Center (Guangzhou, China). Then conduct subsequent indicator testing.

[0041] (1) The method of using the classic antidepressant drug evaluation model - the tail suspension test to test the depression level of each group of mice. The specific method is as follows: 1-2 hours in advance, the mice are transferred from the animal room to the behavioral laboratory. The light in the laboratory is dim (based on the minimum brightness that can distinguish the subtle activities of mice at a distance of 1.5m) and kept constant. The room temperature is about 24℃. Keep quiet and let the mice adapt to the laboratory environment and return to a calm state. Before the experiment begins, the end of the mouse's tail is fixed on the tail suspension test equipment so that the mouse's head is kept hanging down, about 15 cm from the ground. The behavior of the mouse is recorded using behavioral software and video equipment. Each mouse is tested for 6 minutes, and the total duration of the mouse's immobility in the last 2 minutes of the experiment is recorded. The final experiment uses the duration of the mouse's immobility in the tail suspension state to show the mouse's depressive-like despair behavior. The results are shown in Figure 1. The tail suspension immobility times of the wild-type group, model group, low-dose group, medium-dose group, and high-dose group were 30.58, 119.89, 94.77, 79.82, and 79.38 seconds, respectively.

[0042] This shows that the immobility time of mice in the model group was significantly longer than that of mice in the wild group, and the mice in the model group showed certain depressive-like behaviors; while the immobility time of mice in each group treated with tranquilizing and brain-enhancing liquid shortened successively with increasing doses, indicating that tranquilizing and brain-enhancing liquid can alleviate depressive disorders in mice.

[0043] (2) The open field test was used to assess the anxiety of mice in a new environment. The specific method was as follows: 1-2 hours in advance, the mice were transferred from the animal room to the behavioral laboratory. The light in the laboratory was dim (based on the minimum brightness that could distinguish the subtle movements of mice at a distance of 1.5m) and kept constant. The room temperature was around 24℃. The mice were kept quiet and allowed to adapt to the laboratory environment and return to a calm state. At the beginning of the experiment, the mice were placed in the central grid of the open field, with their heads kept in the same direction as much as possible. At the same time, the timer was pressed to start the countdown, and the mice were allowed to move freely for 5 minutes. The number of grids crossed, the number of times the mice stood up, and the number of times they defecated were recorded and counted. The results are shown in Figure 1. The number of grids crossed by the wild group, model group, low dose group, medium dose group, and high dose group were 74.25, 9.75, 26.25, 30.12, and 34.75, respectively. The number of times the wild group, model group, low dose group, medium dose group, and high dose group stood up were 6.12, 2.12, 2.87, 2.12, and 5.75, respectively. The number of bowel movements in the wild group, model group, low-dose group, medium-dose group, and high-dose group were 2.62, 3.12, 2.50, 1.87, and 2.37 times, respectively.

[0044] Compared with wild-type mice, the model group mice crossed significantly fewer grids and stood up fewer times, indicating a weakened ability for spontaneous activity and a decreased desire to explore the outside world. Furthermore, the model group mice had an increased defecation frequency compared to the wild-type mice, suggesting increased anxiety and tension in the new environment. Mice treated with tranquilizing and brain-strengthening fluid showed a significant increase in the number of grids crossed and the number of times they stood up, and a significant decrease in the number of defecations. This suggests that tranquilizing and brain-strengthening fluid significantly improved the exploratory ability and anxiety of the model group mice.

[0045] (3) The elevated plus maze uses the animal's exploratory characteristics of a novel environment and its fear of high-hanging open arms to form an exploration-avoidance conflict behavior to examine the animal's anxiety state. The elevated plus maze experimental test can be used to examine the effect of the drug of the present application on anxiety. The number and time that animals enter the open arms in the elevated plus maze are negatively correlated with the animal's anxiety. The specific method is as follows: transfer the mice from the animal room to the behavioral laboratory 1-2 hours in advance. The light in the laboratory is dim (based on the minimum brightness that can distinguish the subtle activities of mice at a distance of 1.5m) and kept constant. The room temperature is about 24℃. Keep it quiet and let the mice adapt to the laboratory environment and return to a calm state. Before the experiment begins, put the mice in an empty cage and let them explore freely for 5 minutes. At the beginning of the experiment, quickly transfer the mice to the central platform of the plus maze so that their heads are facing one of the open arms. After being released, start recording various behavioral indicators immediately. Each mouse is tested for 5 minutes. If the mouse accidentally falls from the elevated platform, it will not be counted in the final experimental data.

[0046] The results are shown in Figure 1. The open arm entry times for animals in the wild-type, model, low-dose, medium-dose, and high-dose groups were 19.96, 4.37, 6.71, 9.38, and 11.45 seconds, respectively. The open arm entries for animals in the wild-type, model, low-dose, medium-dose, and high-dose groups were 9.12, 1.5, 4.37, 7.00, and 3.50, respectively. The percentages of open arm entries and total arm entries for the wild-type, model, low-dose, medium-dose, and high-dose groups were 39.95%, 15.93%, 41.46%, 54.14%, and 51.52%, respectively.

[0047] The results showed that the number of times and time that the model group mice entered the open arms of the elevated rack were significantly lower than those of the wild group mice, indicating that the model group mice showed obvious anxiety; while the number of times and time that the mice in the tranquilizer and brain-enhancing fluid-treated group entered the open arms were significantly increased, indicating that tranquilizer and brain-enhancing fluid treatment could effectively relieve the anxiety of the model group mice.

[0048] (4) Detection of Aβ pathological indicators in mouse brain

[0049] A large amount of Aβ aggregates and deposits outside neuronal cells to form senile plaques, which has a toxic effect on nerve cells and is one of the typical pathological characteristics of AD. Western-Blot (immunoblotting) was used to detect the hippocampal tissues of mice in each group.

[0050] The results are shown in Figure 2. Treatment with Anshen Jiannao Fluid can reduce the deposition of Aβ in the brain. Further immunofluorescence staining of mouse brain sections also revealed a significant decrease in the number of Aβ plaques in the CA1 and CA3 regions of the mouse hippocampus.

[0051] (5) Using R software, a Venn diagram was created between 3751 depression disease targets and the targets of 480 active ingredients of Anshen Jiannao Liquid, resulting in 253 common disease-active ingredient targets (see Figure 3A). The PPI interaction network was obtained after analysis using the STRING data platform, with a total of 53 interaction nodes and 431 interaction relationships (see Figure 3B).

[0052] (6) Using R software, GO biological function annotation was performed on 253 common target proteins of the components of Anshen Jiannaoye and diseases, and visual analysis was performed to obtain a GO analysis bar chart (Figure 4). It was found that the common targets were mainly enriched in biological processes such as modulation of chemical synaptic transmission, regulation of trans-synaptic signaling, protein autophosphorylation, and positive regulation of protein serine / threonine kinase activity. This suggests that Anshen Jiannaoye may improve depressive symptoms through biological processes such as modulation of chemical synaptic transmission, regulation of trans-synaptic signaling, protein autophosphorylation, and positive regulation of protein serine / threonine kinase activity.

[0053] (7) By analyzing the common target PPI network and GO analysis histogram, it was predicted that AKT1 may be the key target gene for the antidepressant effect of Anshen Jiannaoye. AKT1 is an isomer of protein kinase B (Akt). Using AKT pathway protein as a detection indicator, the mechanism by which Anshen Jiannaoye exerts its antidepressant effect by intervening in the AKT signaling pathway was explored. Western Blot was used to test the levels of AKT and p-AKT in the hippocampal tissue of each group of mice. The results are shown in Figure 5. The AKT activity of the model group mice was significantly lower than that of the wild group mice. The AKT activity of the mice treated with Anshen Jiannaoye was significantly increased. This indicates that Anshen Jiannaoye may achieve its antidepressant effect by activating the AKT signaling pathway.

[0054] In summary, this application is the first to use Anshen Jiannao Liquid for the research on the prevention and treatment of depression, and to develop new uses for traditional compound Chinese medicine from the perspective of "new uses for old medicines". This application found through behavioral testing that treatment with Anshen Jiannao Liquid can significantly improve the depression and anxiety of mice in the model group. Further analysis and verification of the possible anti-depressant targets of Anshen Jiannao Liquid through network pharmacology found that the administration of Anshen Jiannao Liquid can significantly upregulate the activity of AKT protein, indicating that Anshen Jiannao Liquid achieves its anti-depressant effect by upregulating the activity of AKT and then activating the signaling pathway downstream of AKT.

[0055] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of this application.

Claims

1. Use of tranquilizing and brain-strengthening liquid in the preparation of medicines for treating, preventing or alleviating depression.

2. The use according to claim 1, characterized in that: The raw materials of the tranquilizing and brain-strengthening liquid include ginseng, schisandra chinensis, ophiopogon japonicus, wolfberry and salvia miltiorrhiza.

3. The use according to claim 2, characterized in that: In the raw materials of the tranquilizing and brain-strengthening liquid, the mass ratio of ginseng, schisandra chinensis, ophiopogon japonicus, wolfberry and salvia miltiorrhiza is 20-80:100-160:100-160:100-160:60-120.

4. The use according to any one of claims 1 to 3, characterized in that: The drug can inhibit the increase of anxiety in patients with depression.

5. The use according to any one of claims 1 to 4, characterized in that: The drug can inhibit the decline in the desire to explore in patients with depression.

6. The use according to any one of claims 1 to 5, characterized in that: The depression is depression mediated by the AKT signaling pathway, and the drug can activate the AKT signaling pathway and increase the p-AKT level.

7. The use according to any one of claims 1 to 6, characterized in that: The drug can reduce the deposition of Aβ protein in the hippocampus tissue of patients with depression.

8. The use according to any one of claims 1 to 7, characterized in that: The tranquilizing and brain-strengthening liquid is a clinically acceptable oral liquid prepared by conventional processes with ginseng, schisandra chinensis, ophiopogon japonicus, wolfberry and salvia miltiorrhiza as active ingredients and conventional auxiliary materials.

9. A method for treating, preventing or alleviating depression, characterized in that: The method comprises administering a tranquilizing and brain-strengthening solution to a subject in need thereof.

10. The method according to claim 9, characterized in that The raw materials of the tranquilizing and brain-strengthening liquid include ginseng, schisandra chinensis, ophiopogon japonicus, wolfberry and salvia miltiorrhiza.

11. The method according to claim 10, characterized in that In the raw materials of the tranquilizing and brain-strengthening liquid, the mass ratio of ginseng, schisandra chinensis, ophiopogon japonicus, wolfberry and salvia miltiorrhiza is 20-80:100-160:100-160:100-160:60-120.

12. The method according to any one of claims 9 to 11, characterized in that: The tranquilizing and brain-strengthening liquid can inhibit the increase of anxiety in patients with depression.

13. The method according to any one of claims 9 to 12, characterized in that: The tranquilizing and brain-strengthening liquid can inhibit the decline of the desire for exploration in patients with depression.

14. The method according to any one of claims 9 to 13, characterized in that: The depression is depression mediated by the AKT signaling pathway, and the tranquilizing and brain-strengthening solution can activate the AKT signaling pathway and increase the p-AKT level.

15. The method according to any one of claims 9 to 14, characterized in that: The tranquilizing and brain-strengthening liquid can reduce the deposition of Aβ protein in the hippocampal tissue of patients with depression.

16. The method according to any one of claims 9 to 15, characterized in that: The tranquilizing and brain-strengthening liquid is a clinically acceptable oral liquid prepared by conventional processes with ginseng, schisandra chinensis, ophiopogon japonicus, wolfberry and salvia miltiorrhiza as active ingredients and conventional auxiliary materials.

17. The method according to any one of claims 9 to 16, characterized in that: The dosage of the tranquilizing and brain-strengthening liquid is 3 mL / kg / day to 30 mL / kg / day.

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