Method for Comparing the Efficacy of Niuhuang Qingxin Pills in Treating Depression

Abstract

The present invention discloses a method for comparing the effects of Niuhuang Qingxin Pills in the treatment of depression. Using the method of the present invention can explain the ingredient information and mechanism of action of (Formulary) Niuhuang Qingxin Pills in the treatment of depression, provide a theoretical basis for the clinical application of (Formulary) Niuhuang Qingxin Pills in the treatment of depression, and using this method can compare the therapeutic effects of (Formulary) Niuhuang Qingxin Pills and other existing Niuhuang Qingxin Pills on depression.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically, relates to a method for comparing the therapeutic effect of Niuhuang Qingxin Pills on depression. Background Art

[0002] As a common clinical mental disease, the clinical manifestations of patients with depression include slow thinking, low mood, cognitive function impairment, pessimism and even suicidal behavior. With the development of society, the incidence rate of patients with depression may show an increasing trend in the next few years. According to the statistics of the WHO, about 350 million people worldwide are suffering from depression, and the incidence rate is rising year by year, bringing a serious social and economic burden. The report shows that depression has become the primary and independent factor causing disability in humans. The number of patients in China has exceeded 54 million, ranking second in the disease burden. It is urgent to control depressive symptoms. Depression is a complex disease related to genetic, neurobiological, and psychosocial and cultural factors, and multiple mechanisms are involved in its formation.

[0003] For the treatment of depression, Western medicine still mainly relies on drug treatment. Antidepressant drugs can be roughly divided into the following categories: monoamine oxidase inhibitors (MAOIs), tricyclic and tetracyclic antidepressants (TCAs), selective 5-HT reuptake inhibitors (SSRIs), 5-HT and NE reuptake inhibitors (SNaRIs), etc. However, these drugs have obvious defects, such as drug adverse reactions, drug dependence, and restricted application in special populations such as the elderly, children, pregnant women, and those with abnormal liver and kidney functions. Therefore, it is urgent to find safer, more effective, and cheaper antidepressant drugs. Traditional Chinese medicine has a long history in the treatment of sleep disorders, mental disorders, etc., and the safety and low toxicity of traditional Chinese medicine have shown good advantages in the treatment of depression.

[0004] (Official Prescription) Niuhuang Qingxin Pill is one of the ten classic prescriptions, first recorded in the "Taiping Huimin Heji Bureau Prescription" of the Song Dynasty. The prescription is composed of 29 herbs (yam, ginseng, white atractylodes, poria, licorice, angelica, Chuanxiong, white peony root, donkey-hide gelatin, jujube, dried ginger, ophiopogon, white peony root, bitter almond, platycodon, soybean yellow roll, Liushenqu, cinnamon, saposhnikovia, bupleurum, pollen cattail, bezoar, antelope horn powder, buffalo horn powder, musk, cinnabar, realgar, borneol, scutellaria). Based on the 21-ingredient formula of Shudi Pill recorded in "Jin Kui Yao Lue" by Zhang Zhongjing of the Han Dynasty, which reuses yam to replenish qi and blood and nourish yin, this prescription removes rehmannia and adds pollen cattail, and adds ingredients that clear heat and resolve phlegm, calm the nerves and open the orifices, and extinguish wind and dredge the meridians, forming Niuhuang Qingxin Pill, which has the effects of replenishing qi and nourishing blood, calming the mind, resolving phlegm and extinguishing wind, and strengthening the body and tranquilizing the mind. Modern research shows that (official prescription) Niuhuang Qingxin Pills can relieve symptoms such as insomnia, frequent awakenings, fatigue, irritability, temper tantrums, memory loss, lack of concentration, depression, anxiety, and even a sense of world-weariness, and help people get rid of negative emotions. Various pressures brought about by modern life, work, and interpersonal relationships can easily turn into fire, torment the body and mind, and generate phlegm and heat. Common symptoms include fatigue throughout the body, headaches, depression, muscle and joint pain, memory loss, irritability, and insomnia. The syndrome differentiation is mostly due to excessive psychological pressure leading to excessive five emotions, strong heart fire disturbing the heart, which will dissipate heart qi over time, leading to a mixture of deficiency and excess, with symptoms such as anxiety, depression, irritability, and insomnia. In addition, patients with depression have a long history of illness, with damaged qi and blood, spleen deficiency generating phlegm, blood deficiency and liver hyperactivity, insufficient qi and blood, and phlegm and fire are easy to generate; (official prescription) Niuhuang Qingxin Pills replenish qi and blood, nourish the spleen and liver, and when qi and blood are sufficient, phlegm and fire will not be generated. It can clear the heart and dispel fire, relieve depression and eliminate phlegm, thus curing the disease. Niuhuang Qingxin Pills (Official Prescription) have significant clinical efficacy in treating depression. However, there is currently no research on the chemical component detection method and analysis of Niuhuang Qingxin Pills (Official Prescription) using ultra-performance liquid chromatography-time-of-flight mass spectrometry (UPLC-Q-TOF-MS) to explore the component information and mechanism of action of Niuhuang Qingxin Pills in treating depression.

[0005] Currently, there are several Niuhuang Qingxin Pills available on the market, each with different ingredients. It is speculated that different Niuhuang Qingxin Pills have different mechanisms of action and efficacy in treating depression. For example, some Niuhuang Qingxin Pills contain bezoar, cinnabar, astragalus, coptis root, gardenia, and turmeric; others contain artificial bezoar, artificial musk, buffalo horn concentrate powder, antelope horn, astragalus, angelica, Chinese yam, licorice, jujube, and Poria. (Official Prescription) Niuhuang Qingxin Pills differ from these other pills in that they contain bezoar, musk, buffalo horn concentrate powder, antelope horn, astragalus, angelica, Chinese yam, licorice, jujube, Poria, realgar, and cinnabar. Because (Official Prescription) Niuhuang Qingxin Pills contain bezoar, musk, realgar, and cinnabar, patients with depression often prefer the pills without realgar or cinnabar. However, there is currently no unified evaluation method to determine which Niuhuang Qingxin Pill is more effective in treating depression.

[0006] Therefore, there is an urgent need in the art for a method to compare the efficacy of Niuhuang Qingxin Pills in treating depression. Summary of the Invention

[0007] The object of the present invention is to compare the efficacy of Niuhuang Qingxin Pills in treating depression by using network pharmacology, UPLC-Q-TOF-MS and molecular docking techniques, and thus, the present invention is completed.

[0008] In a first aspect, the present invention provides a method for comparing the efficacy of Niuhuang Qingxin Pills in treating depression, and the method comprises the following steps:

[0009] S1. Prepare multiple test sample solutions:

[0010] Take various Niuhuang Qingxin Pills, chop them respectively, add diatomaceous earth and grind evenly until dispersed uniformly, add methanol solution, and perform ultrasonic treatment to obtain various mixtures of Niuhuang Qingxin Pills;

[0011] S2. Detect the component information of various Niuhuang Qingxin Pills:

[0012] Use ultra-high performance liquid chromatography coupled with high-resolution time-of-flight mass spectrometry to determine the component information of the various mixtures of Niuhuang Qingxin Pills in step S1 respectively;

[0013] S3. Screen the active components of various Niuhuang Qingxin Pills:

[0014] Perform duplicate removal and sorting on the component information obtained in step S2, and establish active component data sets for various Niuhuang Qingxin Pills respectively;

[0015] S4. Predict active component targets and collect disease targets:

[0016] Retrieve the action targets corresponding to the active components of various Niuhuang Qingxin Pills in step S3 through the pubchem and Swiss Target Prediction databases, and establish an active component-action target data set through duplicate removal and sorting; use the Genecards and OMIM databases to collect the action targets of depression to obtain a disease-action target data set;

[0017] S5. Construct an "active component-action target-disease" network:

[0018] Use the active component-action target data set and the disease-action target data set in step S4 to construct an "active component-action target-disease" network;

[0019] S6. Construct a protein-protein interaction network:

[0020] Obtain the intersection genes of the active component-action target data set and the disease-action target data set in step S4, and use the intersection genes to obtain a protein-protein interaction network;

[0021] S7. GO and KEGG enrichment analysis:

[0022] Perform GO and KEGG enrichment analysis on the intersection genes in step S6;

[0023] S8. Molecular docking:

[0024] Take the 5, 10, 15 or 20 proteins with the highest degree in the protein - protein interaction network in step S6 as receptor proteins, take the active ingredients corresponding to the receptor proteins in the "active ingredient - action target - disease" network in step S5 as ligand molecules, obtain the structures of the receptor proteins and the ligand molecules, and perform molecular docking;

[0025] S9. Calculate the network efficiency of the protein - protein interaction network in step S6, compare the network efficiencies of various Niuhuang Qingxin Pills, and the greater the network efficiency value, the better the therapeutic effect on depression.

[0026] In one embodiment, post - process the mixtures in step S1 respectively, and the steps are as follows: Centrifuge the mixtures at high speed respectively, and then filter through a 0.22 μm microporous filter membrane to obtain the test samples of Niuhuang Qingxin Pills methanol extracts, and then dilute them 1 - fold with the initial mobile phase.

[0027] In one embodiment, the chromatographic conditions in step S2 are as follows: Thermo Hypersil GOLD C18 chromatographic column (2.1 * 100 mm, 1.9 μm); Column temperature: 35 °C; Sample manager temperature: 15 °C; Injection volume: 1 μL; Flow rate: 0.3 mL / min; Mobile phase: Phase A: acetonitrile, Phase B: 0.1% formic acid aqueous solution.

[0028] In one embodiment, the gradient elution conditions in step S2 are as follows: 0 min, 1.0% mobile phase A, 99.0% mobile phase B; 1 min, 1.0% mobile phase A, 99.0% mobile phase B; 4 min, 10.0% mobile phase A, 90.0% mobile phase B; 9 min, 22.0% mobile phase A, 78.0% mobile phase B; 15 min, 33.0% mobile phase A, 67.0% mobile phase B; 20 min, 50.0% mobile phase A, 50.0% mobile phase B; 27 min, 55.0% mobile phase A, 45.0% mobile phase B; 32 min, 90.0% mobile phase A, 10.0% mobile phase B; 34 min, 90.0% mobile phase A, 10.0% mobile phase B; 35 min, 1.0% mobile phase A, 99.0% mobile phase B; 38 min, 1.0% mobile phase A, 99.0% mobile phase B.

[0029] In one embodiment, the mass spectrometry detection conditions in step S2 are as follows: electrospray ionization source, positive and negative ion scanning modes, scanning range 100 - 1200 amu, ion source temperature 120 °C, scanning time 0.2 s, capillary voltage 2500 V, desolvation temperature 400 °C, desolvation gas flow rate 1000 L / h.

[0030] In one embodiment, step S3 further includes: searching for relevant literature to supplement the components that were not selected but have relevant activity research.

[0031] In one embodiment, in step S8, the PDB database is used to obtain the structure of the receptor protein.

[0032] In a second aspect, the present invention proposes a method for comparing the therapeutic effects of Niuhuang Qingxin Pills in treating depression. The method includes comparing the network efficiency of Jufang Niuhuang Qingxin Pills and other Niuhuang Qingxin Pills in treating depression by using the method described in the first aspect.

[0033] In one embodiment, the fingerprint of Jufang Niuhuang Qingxin Pills has 7 common peaks, and the retention times of each common peak are as follows:

[0034] Peak 1: relative retention time 12.59;

[0035] Peak 2: relative retention time 14.86;

[0036] Peak 3: relative retention time 16.33;

[0037] Peak 4: relative retention time 16.67;

[0038] Peak 5: relative retention time 17.60;

[0039] Peak 6: relative retention time 18.95;

[0040] Peak 7: relative retention time 19.60;

[0041] The beneficial effects of the present invention are as follows: analyzing the component information of Niuhuang Qingxin Pills and the mechanism of action in treating depression by using network pharmacology, UPLC-Q-TOF-MS and molecular docking technologies, providing a theoretical basis for the clinical application of (Jufang) Niuhuang Qingxin Pills in treating depression, and using this method to compare the therapeutic effects of (Jufang) Niuhuang Qingxin Pills and existing other Niuhuang Qingxin Pills on depression. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] In order to more clearly illustrate the technical solutions in the specific embodiments of the present invention, the drawings in the specific embodiments will be briefly introduced below.

[0043] Figure 1UPLC-Q-TOF-MS results of Niuhuang Qingxin Pills A, B, C, and (Jufang) Niuhuang Qingxin Pills are shown separately as A, B, C, and D.

[0044] Figure 2 The active ingredient-action target network of (Jufang) Niuhuang Qingxin Pills and depression is shown, where blue nodes represent action targets and pink nodes represent active ingredients.

[0045] Figure 3 The protein interaction network of (Jufang) Niuhuang Qingxin Pills in the treatment of depression is shown.

[0046] Figure 4 The column chart of GO enrichment analysis of the intersection genes of the action targets of (Jufang) Niuhuang Qingxin Pills and the action targets of depression is shown. A, B, and C are biological process, molecular function, and cellular component respectively.

[0047] Figure 5 The column chart of KEGG pathway enrichment analysis of the intersection genes of the action targets of (Jufang) Niuhuang Qingxin Pills and the action targets of depression is shown.

[0048] Figure 6 The protein interaction network of Niuhuang Qingxin Pills A in the treatment of depression is shown.

[0049] Figure 7 The comparison of the reduction amplitude of MMN latency before and after treatment in four groups of patients is shown. Detailed implementation mode

[0050] The preferred embodiments of the present invention will be described in more detail below. Although the preferred embodiments of the present invention are described below, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein.

[0051] The present inventors proposed a method for comparing the effects of Niuhuang Qingxin Pills in the treatment of depression. This method uses new methods of modern research such as network pharmacology, UPLC-Q-TOF-MS, and molecular docking technology, laying a solid theoretical foundation for studying the disease targets and action mechanisms of the active ingredients of traditional Chinese medicine compounds.

[0052] Example 1

[0053] This embodiment shows an example of comparing the therapeutic effects of (official formula) Niuhuang Qingxin Pills (purchased from Shanxi Guangyuyuan Traditional Chinese Medicine Co., Ltd.), a certain brand of Niuhuang Qingxin Pills without realgar and cinnabar sold on the market (referred to as A Niuhuang Qingxin Pills in this article), and B Niuhuang Qingxin Pills and C Niuhuang Qingxin Pills prepared by the inventor. Among them, the difference between the ingredients of B Niuhuang Qingxin Pills and (official formula) Niuhuang Qingxin Pills is that they do not contain realgar, and the difference between the ingredients of C Niuhuang Qingxin Pills and (official formula) Niuhuang Qingxin Pills is that they do not contain cinnabar. This embodiment includes the following steps:

[0054] S1. Prepare the test solution:

[0055] Take (official formula) Niuhuang Qingxin Pills, A Niuhuang Qingxin Pills, B Niuhuang Qingxin Pills, and C Niuhuang Qingxin Pills, chop them separately, add diatomaceous earth and grind evenly until dispersed uniformly, add methanol solution, and perform ultrasonic treatment to obtain four mixtures, namely the test solutions;

[0056] Centrifuge the four test solutions at high speed respectively, and then filter them through a 0.22 μm microporous membrane to obtain the Niuhuang Qingxin Pills methanol extract test samples, and dilute them 1-fold with the initial mobile phase.

[0057] S2. Detect the ingredient information of (official formula) Niuhuang Qingxin Pills, A Niuhuang Qingxin Pills, B Niuhuang Qingxin Pills, and C Niuhuang Qingxin Pills:

[0058] Use a ultra-high performance liquid chromatography coupled with high-resolution time-of-flight mass spectrometer to determine the composition information of the mixture in step S1 respectively; the chromatographic conditions are as follows: Thermo Hypersil GOLD C18 chromatographic column (2.1*100mm, 1.9μm); column temperature: 35°C; sample manager temperature: 15°C; injection volume: 1μL; flow rate: 0.3mL / min; mobile phase: phase A: acetonitrile, phase B: 0.1% formic acid aqueous solution; gradient elution conditions are as follows: 0min, 1.0% mobile phase A, 99.0% mobile phase B; 1min, 1.0% mobile phase A, 99.0% mobile phase B; 4min, 10.0% mobile phase A, 90.0% mobile phase B; 9min, 22.0% mobile phase A, 78.0% mobile phase B; 15min, 33.0% mobile phase A, 67.0% mobile phase B; 20min, 50.0% mobile phase A, 50.0% mobile phase B; 27min, 55.0% mobile phase A, 45.0% mobile phase B; 32min, 90.0% mobile phase A, 10.0% mobile phase B; 34min, 90.0% mobile phase A, 10.0% mobile phase B; 35min, 1.0% mobile phase A, 99.0% mobile phase B; 38min, 1.0% mobile phase A, 99.0% mobile phase B; the mass spectrometry detection conditions are as follows: electrospray ionization source, positive and negative ion scanning modes, scanning range 100-1200amu, ion source temperature 120°C, scanning time 0.2s, capillary voltage 2500V, desolvation temperature: 400°C, desolvation gas flow rate 1000L / h; the detection results of the above four Niuhuang Qingxin Pills are as Figure 1 shown, the detection results show that the fingerprint of Niuhuang Qingxin Pills (Pharmacopoeia) has 7 common peaks, and the retention times of each common peak are respectively:

[0059] Peak 1: relative retention time 12.59;

[0060] Peak 2: relative retention time 14.86;

[0061] Peak 3: relative retention time 16.33;

[0062] Peak 4: relative retention time 16.67;

[0063] Peak 5: relative retention time 17.60;

[0064] Peak 6: relative retention time 18.95;

[0065] Peak 7: relative retention time 19.60;

[0066] Peaks 1-7 correspond to baicalin, wogonoside, taurocholic acid, ginsenoside Rg1, ginsenoside Rb1, tauroursodeoxycholic acid, and cholic acid respectively.

[0067] The detection information of some components of Niuhuang Qingxin Pills (Pharmacopoeia) is shown in Table 1:

[0068] Table 1: Detection information of some components of Niuhuang Qingxin Pills (Pharmacopoeia)

[0069]

[0070] The following steps are carried out for Niuhuang Qingxin Pills (Pharmacopoeia):

[0071] S3. Screen the active components of Niuhuang Qingxin Pills (Pharmacopoeia):

[0072] Deduplicate and sort out the component information obtained in step S2 to establish an active component dataset of Niuhuang Qingxin Pills (Pharmacopoeia); in addition, search relevant literature to supplement the components that are not selected but have relevant activity research; after screening, a total of 67 active components are obtained (Table 2).

[0073] Table 2: Active components of Niuhuang Qingxin Pills (Pharmacopoeia)

[0074]

[0075]

[0076]

[0077] In addition, since realgar and cinnabar cannot be detected by UPLC-Q-TOF-MS, the component information corresponding to these substances needs to be manually supplemented, where realgar corresponds to tetraarsenic tetrasulfide and cinnabar corresponds to mercury sulfide.

[0078] S4. Predict the targets of active components and collect disease targets:

[0079] Retrieve the action targets corresponding to the active components of Niuhuang Qingxin Pills (Pharmacopoeia) in step S3 through the pubchem and Swiss Target Prediction databases, and establish an active component-action target dataset after deduplication and sorting; use the Genecards and OMIM databases to collect the action targets of depression to obtain a disease-action target dataset;

[0080] Specifically, the active ingredients of (official formula) Niuhuang Qingxin Pills were separately input into the pubchem and Swiss TargetPrediction databases, and targets were obtained with the screening condition of "human". Targets with a probability of 0 were deleted, and 595 acting targets were obtained. After de-duplicating and sorting out the acting targets, they were input into the Uniprot database, and standardized protein names were obtained with the screening conditions of "verified, human", and 487 acting targets corresponding to the active ingredients of (official formula) Niuhuang Qingxin Pills were obtained; Search was conducted in the Genecards and OMIM databases with the keyword "depression" to obtain gene targets related to depression. After taking the union of the obtained genes, de-duplicating, sorting out and screening, 978 standardized protein names were obtained through the Uniprot database with the condition of "verified, human", which can be used for subsequent network analysis.

[0081] In addition, for tetraarsenic tetrasulfide, it can be input into the CTD (The Comparative Toxicogenomics Database) for retrieval, and its targets include AQP9, MYC, CASP3, CDKN1B, RARB, etc. (screening threshold interactions≥3, a total of 21); Similarly, the targets of mercuric sulfide include SKR-1, SLC15A1 (screening threshold interactions≥3, a total of 2).

[0082] S5. Construct the "active ingredient - acting target" network:

[0083] Use the active ingredient - acting target data set and disease - acting target data set in step S4 to construct the "active ingredient - acting target" network, and visualize it using Cytoscape software ( Figure 2 )

[0084] S6. Construct the protein - protein interaction network:

[0085] Obtain the intersection genes of the active ingredient - acting target data set and disease - acting target data set in step S4, import the 125 intersection genes into the String database, set the search target to "Multiple proteins", and select the species as "Homo sapiens" to obtain the protein - protein interaction network ( Figure 3) The network has 122 nodes and 1256 edges. Then, use Cytoscape software to analyze the network. Use the "CytoNCA" plugin to calculate the scores of each node according to betweenness centrality to obtain the core nodes, and sort the nodes according to betweenness centrality. The larger the node, the more crucial the role of the gene in the network. Thus, the core targets can be obtained, such as GAPDH, TNF, CNR1, PPARG, etc.

[0086] S7. GO and KEGG enrichment analysis:

[0087] Perform GO and KEGG enrichment analysis on the intersection genes in step S6, and the results are as Figure 4 and Figure 5 shown;

[0088] Specifically, input the intersection genes into Metascape, select "Human" for species for personalized analysis. The results show 20 KEGG pathways, and the main pathways are cancer pathway, neuroactive ligand-receptor interaction, calcium signaling pathway, serotonin-containing synapse, proteoglycans in cancer, hepatitis B, diabetic cardiomyopathy, dopaminergic synapse, etc.; 20 GO biological processes, 20 GO molecular functions, and 20 GO cellular localizations are obtained. The GO biological processes mainly include hormone response, behavior, positive regulation of phosphorus metabolic process, circulatory system process, response to external stimuli, secretion regulation, regulation of membrane potential, response to extracellular stimuli, etc.; The GO molecular functions mainly include neurotransmitter receptor activity, hormone binding, G protein-coupled amine receptor activity, phosphotransferase activity with alcohol group as receptor, steroid binding, transcription factor binding, protein kinase binding, dopamine neurotransmitter receptor activity, etc.; The GO cellular localizations mainly include receptor complex, dendrite, membrane raft, postsynaptic membrane, etc. The darker the color of the bar graph, the more significant the P value, and the higher the credibility that the gene is enriched in this pathway or biological process, molecular function, cellular localization, and the more important this signaling pathway, biological process, molecular function or cellular localization is.

[0089] S8. Molecular docking:

[0090] Use Chem 3D, PyMOL, AutoDock Tools, vina, etc. to perform docking simulations on the core targets GAPDH with paeonol glucoside or its isomers, TNF with glycocholic acid, CNR1 with glycocholic acid, and PPARG with liquiritigenin, predict the binding mode and strength, determine the docking binding sites and names, and draw the molecular docking results into figures (where the protein structures of the core targets are obtained using the PDB database). The lowest binding energies of their ligand-receptor pairs are GAPDH - paeonol glucoside or its isomers -5.3 kcal / mol, TNF - glycocholic acid -7.4 kcal / mol, CNR1 - glycocholic acid -4.0 kcal / mol, and PPARG - liquiritigenin -8.7 kcal / mol.

[0091] S9. Calculate the network efficiency of the protein interaction network in step S6. The calculation formula is as follows:

[0092]

[0093] where E represents the network efficiency, G represents the protein interaction network, N represents the number of nodes in network G, and d ij represents the length of the shortest path between nodes i and j. The network efficiency index measures the average ease of communication between node pairs in the network through the shortest path. In this invention, the network efficiency of the protein interaction network for treating depression with drugs is used to compare the therapeutic effect of Niuhuang Qingxin Pills on depression. The higher the network efficiency, the more robust the protein interaction network for treating depression with drugs, the higher the communication efficiency, and the better the therapeutic effect of the drug. After calculation, the network efficiency corresponding to (Jufang) Niuhuang Qingxin Pills is 0.259.

[0094] According to Figure 1 it can be seen that there are differences between (Jufang) Niuhuang Qingxin Pills and A Niuhuang Qingxin Pills. There are more peaks in A Niuhuang Qingxin Pills in the positive ion mode, and more peaks in (Jufang) Niuhuang Qingxin Pills in the negative ion mode. Comparing the component information of the two, it is found that: compared with A Niuhuang Qingxin Pills, (Jufang) Niuhuang Qingxin Pills contain more chemical substances such as taurodeoxycholic acid, deoxycholic acid, dichloronaphthoquinone, methyl 3,5-di-tert-butyl-4-hydroxybenzoate, ibuprofen, lupenone, and kushenol in their components. At the same time, the content of curcumin is higher than that of A Niuhuang Qingxin Pills, and other components are similar. Use the component information of A Niuhuang Qingxin Pills for steps S3 - S9. The protein interaction network of A Niuhuang Qingxin Pills is as Figure 6As shown, its network efficiency is calculated to be 0.105, and the results show that the network efficiency of (the official formula) Niuhuang Qingxin Pills is higher than that of A Niuhuang Qingxin Pills. Further, using the ingredient information of B Niuhuang Qingxin Pills and C Niuhuang Qingxin Pills for steps S3 - S9, the network efficiency of the two is calculated to be between (the official formula) Niuhuang Qingxin Pills and A Niuhuang Qingxin Pills. The above results indicate that the method of the present invention shows that (the official formula) Niuhuang Qingxin Pills have the best effect in treating depression.

[0095] Example 2

[0096] To confirm the reliability of the method of the present invention, the inventor conducted the following experiment:

[0097] 50 adult male ICR mice were purchased and raised for one week under the conditions of 12h light / 12h cycle, relative humidity of 55% - 65%, and constant temperature of 23 ± 2°C. All mice were subjected to 7 different chronic stressors for 4 weeks to complete the model establishment. Among them, the 7 different chronic stressors included: ice bath (0°C ice - water mixture, 5 min); tail suspension (15 min); tail clamping (5 min); wet bedding (24 h); voltage stimulation (32 V, 10 min); forced swimming (24°C, 15 min); restraint (30 min).

[0098] The 50 mice were randomly divided into 5 groups, including a control group, an official formula group, group A, group B, and group C. The 5 groups of mice were respectively given drug gavage treatment. The drugs were normal saline, (the official formula) Niuhuang Qingxin Pills, A Niuhuang Qingxin Pills, B Niuhuang Qingxin Pills, and C Niuhuang Qingxin Pills. Each drug was ground into granules and dissolved in water for gavage treatment. The daily dose was 0.5 g / kg, and the gavage volume was 0.2 ml. The 5 groups of mice had free access to food and water. After 4 weeks of treatment, the inventor tested the behavioral phenotypes of the 5 groups of mice. Compared with the control group, the depressive - like behaviors of the mice administered drugs (the official formula group, group A, group B, and group C) were improved in varying degrees, mainly manifested as a significant reduction in the immobile time of tail suspension and forced swimming, and an increase in the time entering the middle area in a 10 - minute open - field experiment, showing stronger exploratory ability. That is, the depressive and anxiety - like behaviors of the 4 groups of mice were significantly improved, and the mice in the official formula group showed the best treatment effect (Table 3).

[0099] Table 3: Time (mean ± standard error) of the 5 groups of mice entering the middle area in the open - field experiment

[0100] Group Time (minutes) Control group 1.26±0.34 Official formula group 3.55±0.20 Group A 2.24±0.61 Group B 2.50±0.14 Group C 2.48±0.12

[0101] Based on the above results, the inventors speculated that this might be because the Niuhuang Qingxin Pills (authorized by the authorities) contain realgar and cinnabar which have the effect of calming the mind, and the combination of these two substances makes the signal transduction of the depression targets in the protein interaction network more efficient. Therefore, it has a better therapeutic effect than Niuhuang Qingxin Pills A which do not contain realgar and cinnabar, and Niuhuang Qingxin Pills that contain only realgar or cinnabar alone.

[0102] Example 3

[0103] The inventors further compared the therapeutic effects of the above four kinds of Niuhuang Qingxin Pills on depression in clinical practice.

[0104] A total of 131 patients who visited the outpatient department of the Second Department of Encephalopathy in Shaanxi Provincial Hospital of Traditional Chinese Medicine and the outpatient department of the Department of Psychiatry in Xianyang Hospital of Yan'an University from April 2023 to December 2023, and were diagnosed with depressive cognitive disorder by Western medicine and depressive syndrome of phlegm-heat harassing the mind by traditional Chinese medicine, were selected. This study has obtained the approval of the hospital ethics committee, and all participants have signed the informed consent form.

[0105] The Western medicine diagnostic criteria for depressive cognitive disorder in this example are as follows: Refer to the fifth edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-V). There is a depressive mood or loss of interest and pleasure in daily activities, accompanied by at least 4 of the following: (1) significant weight loss or gain; (2) insomnia or excessive sleep; (3) psychomotor agitation or retardation; (4) fatigue or lack of energy; (5) feeling worthless or self-deprecating; (6) decreased attention and thinking ability, indecision in making decisions; (7) suicidal thoughts or suicide plans, suicide attempts. The symptoms persist for at least 2 weeks, accompanied by severe psychological distress and obvious impairment of social function.

[0106] The traditional Chinese medicine diagnostic criteria for depressive syndrome of phlegm-heat harassing the mind are as follows:

[0107] (1) Disease name diagnosis: Refer to the "Diagnostic Criteria and Treatment Plan for Traditional Chinese Medicine Syndromes of Depression" formulated by the Encephalopathy Professional Committee of the Chinese Association of Traditional Chinese Medicine:

[0108] Main symptoms: Mainly mental depression, accompanied by lack of interest, restlessness, slow thinking, fatigue, insomnia, forgetfulness, decreased libido, and decreased appetite.

[0109] Patients must first have mental depression, and need to have more than 4 other symptoms in the main symptoms at the same time, and the symptoms persist for at least 2 weeks, then they can be diagnosed with depressive syndrome.

[0110] (2) Syndrome type diagnosis: Refer to the "Expert Consensus on the Integrated Traditional Chinese and Western Medicine Diagnosis and Treatment of Depression":

[0111] Diagnostic criteria for phlegm-heat harassing the mind syndrome:

[0112] Main symptoms: Restlessness, chest distress and abdominal distension, sticky mouth and bad breath.

[0113] Secondary symptoms: nightmare, drowsiness and lethargy, heaviness and soreness of limbs, nausea, constipation, flushed face and greasy complexion. Tongue and pulse: red tongue proper, yellow greasy tongue coating, string-taut and slippery or slippery and rapid pulse.

[0114] Diagnostic criteria: having all the main symptoms and more than 1 secondary symptom.

[0115] The inclusion criteria for patients in this embodiment are as follows:

[0116] ① Meeting the diagnostic criteria for depressive disorder episodes in DSM-V;

[0117] ② Meeting the diagnostic criteria for traditional Chinese medicine syndrome differentiation, and being diagnosed as depressive disorder with the syndrome of "phlegm-heat disturbing the mind";

[0118] ③ Screening that the score of the 17-item Hamilton Depression Scale (HAMD) is 7 ≤ score ≤ 24, and in HAMD, the score of depressive mood is ≥ 2 points and the score of suicide is < 2 points; the self-rating scale of cognitive function deficiency (PDQ-D) ≥ 21 points;

[0119] ④ Patients with depressive disorder who are first-episode and have not received any treatment, or have not received any treatment in the past 2 months;

[0120] ⑤ Aged 18 - 75 years, both males and females are included;

[0121] ⑥ Those who are informed and consent to participate in this research.

[0122] The exclusion criteria for patients are as follows:

[0123] (1) Severe aphasia, agnosia, unable to communicate;

[0124] (2) Clinically diagnosed with other mental illnesses (such as obsessive-compulsive disorder, schizophrenia, etc.);

[0125] (3) There is evidence that anxiety or depression has an etiological relationship with a certain somatic disease or drug use;

[0126] (4) Patients who belong to those with recurrent anxiety or depression;

[0127] (5) Known alcohol or drug dependents;

[0128] (6) Those with unstable vital signs and suffering from severe or unstable internal medical diseases such as heart, liver, kidney, endocrine, blood, and respiration;

[0129] (7) Pregnant and lactating women.

[0130] The criteria for excluding and withdrawing patients are as follows:

[0131] (1) Those with poor compliance during the treatment process; those who have severe adverse reactions during the treatment process or whose condition worsens and requires active intervention;

[0132] (2) Those who took other anti - anxiety and antidepressant drugs on their own or increased the dosage of drugs by themselves during the treatment process;

[0133] (3) Patients who were uncooperative and disobeyed treatment, and whose condition remained ineffective after repeated explanations by clinicians;

[0134] (4) Those who actively requested to stop treatment during the treatment process.

[0135] The 131 patients included in the study were randomly divided into group A (n = 33), group B (n = 33), group C (n = 32), and the official - formula group (n = 33). Among them, 5 patients in group A, 2 patients in group B, 2 patients in group C, and 3 patients in the official - formula group dropped out during the study because they did not take medicine according to the research regulations. A total of 119 patients completed the trial, with 28 in group A, 31 in group B, 30 in group C, and 30 in the official - formula group. There were no statistically significant differences in gender, age, disease course, HAMD score, and PDQ - D score among the four groups of patients (P > 0.05).

[0136] The treatment methods are as follows: Patients in group A, group B, group C, and the official - formula group took Bolus of Calculus Bovis and Borneol (A), Bolus of Calculus Bovis and Borneol (B), Bolus of Calculus Bovis and Borneol (C), and Bolus of Calculus Bovis and Borneol (official - formula) orally respectively (the four drugs were made into pills of the same weight), 1 pill each time, once a day, continuously for 3 weeks, stopped for 1 week, then continued for 3 weeks, and stopped for 1 week again. The total treatment course was 8 weeks. During the experiment, acupuncture and other Chinese and Western drugs for treating depressive disorders were strictly prohibited.

[0137] Observation indicators included:

[0138] (1) Main efficacy index: Self - rating Scale of Cognitive Function Deficiency (PDQ - D);

[0139] (2) Secondary efficacy indexes: Beck Depression Inventory (BDI), Beck Anxiety Inventory (BAI);

[0140] (3) Biological index: Event - related potential (ERP);

[0141] (4) Safety indexes: Vital signs, electrocardiogram, blood routine, urine analysis, coagulation function, liver function, kidney function, adverse reactions;

[0142] (5) Observation time points: The four groups of patients were each evaluated once at baseline and 8 weeks after treatment.

[0143] The efficacy evaluation is as follows:

[0144] The efficacy was evaluated according to the reduction rate of the score of the Self - rating Scale of Cognitive Function Deficiency after treatment. Reduction rate = (total score before treatment - total score after treatment) / total score before treatment × 100%.

[0145] Evaluation criteria:

[0146] Recovery: Mental symptoms disappear, and the reduction rate is ≥ 75%;

[0147] Marked improvement: Mental symptoms basically disappear, and the reduction rate is ≥ 50%;

[0148] Effective: Mental symptoms are alleviated, and the reduction rate is ≥ 25%;

[0149] Ineffective: The reduction rate < 25%.

[0150] Event-related potential (ERP) detection:

[0151] MMN mainly reflects the reaction speed of the subject to deviant stimuli and depressive mood. The passive auditory oddball stimulation mode is adopted in the test. The standard stimulus is a pure tone of 500 Hz and 80 dB, with an appearance probability of 0.8; the deviant stimulus is a pure tone of 2000 Hz and 85 dB, with an appearance probability of 0.2. The patient is not required to respond to the target stimulus and non-target stimulus, and it is superimposed 100 times. Measure the latency of MMN.

[0152] After 8 weeks of treatment, the clinical efficacy of the four groups of patients is shown in Table 4, and the total effective rate of the formula group is the highest.

[0153] Table 4: Comparison of the clinical efficacy of PDQ-D in four groups of patients

[0154]

[0155] Analysis of PDQ-D, BDI, and BAI scores before and after treatment:

[0156] Before treatment, there were no significant differences in the PDQ-D, BDI, and BAI scores among the four groups of patients (all P > 0.05). After treatment, the PDQ-D, BDI, and BAI scores of the four groups of patients all decreased significantly (all P < 0.05), and the reduction amplitudes of the PDQ-D, BDI, and BAI scores in the formula group were significantly greater than those in Group A, Group B, and Group C, with statistically significant differences (all P < 0.05).

[0157] Comparison of MMN before and after treatment:

[0158] Before treatment, there were no significant differences in the MMN latency among the four groups of patients (all P > 0.05). After treatment, the MMN latency of the four groups of patients all decreased significantly (all P < 0.05), and the reduction amplitude of the MMN latency in the formula group was significantly greater than that in the other three groups, with statistically significant differences (P < 0.05), while there were no significant differences in the reduction amplitude of the MMN latency among Group A, Group B, and Group C (Table 5 and Figure 7 )

[0159] Table 5: Comparison of MMN latency before and after treatment in four groups of patients

[0160]

[0161] The results of the above clinical trials are the same as the calculation results of the method provided by the present invention and the experimental results of the mice in Example 2, further confirming the reliability of the method provided by the present invention.

[0162] This specific embodiment is only an interpretation of the present application, and it does not limit the present application. After reading this specification, those skilled in the art can make modifications to this embodiment without creative contributions as needed, but as long as it is within the scope of the claims of the present application, it is protected by the patent law.

Claims

1. A method for comparing the effects of Niuhuang Qingxin Pills in treating depression, characterized in that: The method comprises the following steps: S1. Prepare multiple test sample solutions: Take various Niuhuang Qingxin Pills, chop them separately, add diatomaceous earth and grind until they are evenly dispersed, add methanol solution, and perform ultrasonic treatment to obtain a mixture of various Niuhuang Qingxin Pills; The various Niuhuang Qingxin Pills mixtures were post-processed separately, and the steps were as follows: the mixtures were respectively subjected to high-speed centrifugation, and then filtered through a 0.22 μm microporous filter membrane to serve as Niuhuang Qingxin Pills methanol extract test samples, and then diluted 1-fold with the initial mobile phase; S2. Detect the ingredient information of various Niuhuang Qingxin Pills: Use ultra-high performance liquid chromatography coupled with a high-resolution time-of-flight mass spectrometer to respectively determine the ingredient information of the multiple Niuhuang Qingxin Pills mixtures in step S1; S3. Screening of various active ingredients of Niuhuang Qingxin Pills: The component information obtained in step S2 is deduplicated and reorganized to establish multiple Niuhuang Qingxin Pills active ingredient data sets; S4. Predict active ingredient targets and collect disease targets: The targets corresponding to the active ingredients of the various Niuhuang Qingxin Wan pills in step S3 were retrieved using the Pubchem and Swiss Target Prediction databases, and an active ingredient-target dataset was established after deduplication and sorting. The targets of depression were collected using the Genecards and OMIM databases to obtain a disease-target dataset. S5. Constructing the "active ingredient-target-disease" network: Constructing an "active ingredient-target-disease" network using the active ingredient-target dataset and the disease-target dataset in step S4; S6. Construction of protein interaction network: Obtaining the intersection genes of the active ingredient-target dataset and the disease-target dataset in step S4, and obtaining a protein interaction network using the intersection genes; S7.GO and KEGG enrichment analysis: Perform GO and KEGG enrichment analysis on the intersection genes in step S6; S8. Molecular Docking: Selecting the 5, 10, 15, or 20 proteins with the largest degrees in the protein interaction network in step S6 as receptor proteins, and selecting the active ingredient corresponding to the receptor protein in the "active ingredient-target-disease" network in step S5 as the ligand molecule, obtaining the structures of the receptor protein and the ligand molecule, and performing molecular docking; S9. Calculate the network efficiency of the protein interaction network in step S6, and compare the network efficiencies of various Niuhuang Qingxin Pills. The larger the network efficiency value, the better the effect of treating depression.

2. The method according to claim 1, characterized in that The chromatographic conditions in step S2 were as follows: ThermoHypersil GOLD C18 column (2.1*100 mm, 1.9 μm); column temperature: 35°C; sample manager temperature: 15°C; injection volume: 1 μL; flow rate: 0.3 mL / min; Mobile phase: Phase A: acetonitrile, Phase B: 0.1% formic acid aqueous solution.

3. The method according to claim 1 or 2, characterized in that The gradient elution conditions in step S2 were as follows: 0 min, 1.0% mobile phase A, 99.0% mobile phase B; 1 min, 1.0% mobile phase A, 99.0% mobile phase B; 4 min, 10.0% mobile phase A, 90.0% mobile phase B; 9 min, 22.0% mobile phase A, 78.0% mobile phase B; 15 min, 33.0% mobile phase A, 67.0% mobile phase B; 20 min, 50.0% mobile phase A, 50.0% mobile phase B; 27 min, 55.0% mobile phase A, 45.0% mobile phase B; 32 min, 90.0% mobile phase A, 10.0% mobile phase B; 34 min, 90.0% mobile phase A, 10.0% mobile phase B; 35 min, 1.0% mobile phase A, 99.0% mobile phase B; 38 min, 1.0% mobile phase A, 99.0% mobile phase B.

4. The method according to claim 1 or 2, characterized in that The mass spectrometry detection conditions in step S2 are as follows: electrospray ion source, positive and negative ion scan modes, scan range 100-1200 amu, ion source temperature 120°C, scan time 0.2 s, capillary voltage 2500 V, desolvation temperature 400°C, and desolvation gas flow rate 1000 L / h.

5. The method according to claim 1 or 2, characterized in that Step S3 also includes searching for relevant literature to supplement the ingredients that were not selected but have relevant activity studies.

6. The method according to claim 1 or 2, characterized in that In step S8, the structure of the receptor protein is obtained using the PDB database.

7. A method for comparing the effects of Niuhuang Qingxin Pills in treating depression, characterized in that: The method comprises comparing the network efficiency of the local Niuhuang Qingxin Pill and other Niuhuang Qingxin Pills in treating depression using the method described in any one of claims 1 to 6.

8. The method according to claim 7, characterized in that The fingerprint of the official prescription Niuhuang Qingxin Pill has 7 common peaks, and the retention time of each common peak is: Peak 1: relative retention time 12.59; Peak 2: relative retention time 14.86; Peak 3: relative retention time 16.33; Peak 4: relative retention time 16.67; Peak 5: relative retention time 17.60; Peak 6: relative retention time 18.95; Peak 7: relative retention time 19.60.

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