Method for comparing depression treatment effects of calculus bovis heart-clearing pills

Abstract

The invention discloses a method for comparing depression treatment effects of calculus bovis heart-clearing pills. By using the method disclosed by the invention, the component information and the action mechanism of the (local formula) bezoar heart-clearing pill for treating the depression can be explained, a theoretical basis is provided for clinically applying the (local formula) bezoar heart-clearing pill for treating the depression, and the treatment effects of the (local formula) bezoar heart-clearing pill and the existing other bezoar heart-clearing pills on the depression can be compared by using the method.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and in particular, relates to a method for comparing the effects of Niuhuang Qingxin Pills in treating depression. Background Art

[0002] Depression is a common clinical mental illness. The clinical manifestations of patients include slow thinking, low mood, cognitive impairment, pessimism and even suicidal behavior. With the development of society, the incidence of depression patients may show an upward trend in the next few years. According to WHO statistics, about 350 million people in the world are suffering from depression, and the incidence rate is rising year by year, bringing serious socioeconomic burden. The report shows that depression has become the primary and independent factor of human disability. The number of patients in China has exceeded 54 million, ranking second in the burden of disease. It is urgent to control depressive symptoms. Depression is a complex disease related to genetics, neurobiology, and psychosocial and cultural factors, and is caused by multiple mechanisms.

[0003] For the treatment of depression, Western medicine still mainly relies on drug treatment. Antidepressants can be roughly divided into the following categories: monoamine oxidase inhibitors (MAOI), 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 adverse drug reactions, drug dependence, and restrictions on the use of special populations such as the elderly, children, pregnant women, and abnormal liver and kidney function. Therefore, it is urgent to find safer, more effective, more effective, and less expensive antidepressants. 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" in the Song Dynasty. The prescription is composed of 29 herbs (yam, ginseng, atractylodes, poria, licorice, angelica, chuanxiong, white peony, donkey-hide gelatin, jujube, dried ginger, ophiopogon, white peony, bitter almond, platycodon, soybean yellow roll, Liushenqu, cinnamon, saposhnikovia, bupleurum, pollen, bezoar, antelope horn powder, buffalo horn powder, musk, cinnabar, realgar, borneol, scutellaria). Based on the 21-ingredient formula of Shudi Pill recorded in "Golden Chamber Synopsis" by Zhang Zhongjing of the Han Dynasty, this prescription reuses yam to replenish qi and blood and nourish yin, but removes rehmannia and adds pollen, 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 nerves, resolving phlegm and extinguishing wind, and strengthening the body and calming the nerves. Modern research shows that Niuhuang Qingxin Pills can relieve symptoms such as insomnia, dreaminess, easy awakening, fatigue, excitement, temper, memory loss, lack of concentration, depression, anxiety, and even world-weariness, and get rid of negative emotions. Various pressures brought 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, headache, 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, and the dissipation of heart qi over time, resulting in mixed deficiency and excess, and symptoms of anxiety, depression, irritability, and insomnia. In addition, patients with depression have a long history of illness, and their qi and blood have been damaged. Spleen deficiency generates phlegm, blood deficiency and liver hyperactivity, qi and blood deficiency, and phlegm fire are easy to generate; Niuhuang Qingxin Pills replenish qi and blood, nourish the spleen and liver, and phlegm fire will not be generated if qi and blood are sufficient. It can clear the heart and remove fire, and relieve depression and phlegm, thus curing the disease. Niuhuang Qingxin Pills have significant clinical efficacy in treating depression. However, there is currently no research on the chemical composition detection method of Niuhuang Qingxin Pills using ultra-high performance liquid chromatography-time-of-flight mass spectrometry (UPLC-Q-TOF-MS) and analysis of the composition information and mechanism of action of Niuhuang Qingxin Pills in treating depression.

[0005] At present, there are many kinds of Niuhuang Qingxin Pills on the market, but their ingredients are different. It is speculated that the mechanism and effect of Niuhuang Qingxin Pills with different ingredients in treating depression are different. For example, some Niuhuang Qingxin Pills contain bezoar, cinnabar, astragalus, coptis, gardenia and turmeric; some Niuhuang Qingxin Pills contain artificial bezoar, artificial musk, buffalo horn concentrated powder, antelope horn, astragalus, angelica, yam, licorice, jujube, Poria, etc.; (Ju Fang) Niuhuang Qingxin Pills are different from the above drugs in that their ingredients include bezoar, musk, buffalo horn concentrated powder, antelope horn, astragalus, angelica, yam, licorice, jujube, Poria, realgar and cinnabar. Because (Ju Fang) Niuhuang Qingxin Pills contain bezoar, musk, realgar and cinnabar, in real life, patients with depression prefer to use Niuhuang Qingxin Pills without realgar and cinnabar. However, there is no unified evaluation method for which Niuhuang Qingxin Pills are more effective in treating depression.

[0006] Therefore, there is an urgent need in the art for a method for comparing the effects of Niuhuang Qingxin Pills in treating depression. Summary of the invention

[0007] The purpose of the present invention is to compare the effects of Niuhuang Qingxin Pills in treating depression by using network pharmacology, UPLC-Q-TOF-MS and molecular docking technology, thereby completing the present invention.

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

[0009] S1. Prepare multiple test sample solutions:

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

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

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

[0013] S3. Screening of active ingredients of various Niuhuang Qingxin Pills:

[0014] The component information obtained in step S2 is deduplicated and sorted, and multiple Niuhuang Qingxin Pills active component data sets are established respectively;

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

[0016] The targets corresponding to the active ingredients of various Niuhuang Qingxin Pills in step S3 were retrieved through pubchem and Swiss Target Prediction databases, and the active ingredient-target data set was established after deduplication and sorting; the targets of depression were collected using Genecards and OMIM databases to obtain the disease-target data set;

[0017] S5. Constructing the “active ingredient-target-disease” network:

[0018] Using the active ingredient-target data set and the disease-target data set in step S4, constructing an “active ingredient-target-disease” network;

[0019] S6. Construction of protein interaction network:

[0020] 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;

[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] Taking the 5, 10, 15 or 20 proteins with the largest degrees in the protein interaction network in step S6 as receptor proteins, taking 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;

[0025] 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.

[0026] In one embodiment, the mixture in step S1 is post-treated separately, the steps are as follows: the mixture is centrifuged at high speed, filtered through a 0.22 μm microporous filter membrane, used as the methanol extract of Niuhuang Qingxin Pills as a test sample, and then diluted 1 time 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 in, 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.

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

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

[0031] In one embodiment, in step S8, the structure of the receptor protein is obtained using the PDB database.

[0032] In a second aspect, the present invention proposes a method for comparing the effects of Niuhuang Qingxin Pills in treating depression, the method comprising using the method described in the first aspect to compare the network efficiency of the local Niuhuang Qingxin Pills and other Niuhuang Qingxin Pills in treating depression.

[0033] In one embodiment, the fingerprint of the official prescription Niuhuang Qingxin Pills has 7 common peaks, and the retention time of each common peak is:

[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: using network pharmacology, UPLC-Q-TOF-MS and molecular docking technology to analyze the component information of Niuhuang Qingxin Pills and the mechanism of action for treating depression, providing a theoretical basis for the clinical application of (local prescription) Niuhuang Qingxin Pills to treat depression, and using this method can compare the therapeutic effects of (local prescription) Niuhuang Qingxin Pills and other existing Niuhuang Qingxin Pills on depression. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] In order to more clearly illustrate the technical solutions in the specific implementation manner of the present invention, the drawings in the specific implementation manner are briefly introduced below.

[0043] Figure 1AD show the UPLC-Q-TOF-MS results of A Niuhuang Qingxin Pills, B Niuhuang Qingxin Pills, C Niuhuang Qingxin Pills and (official prescription) Niuhuang Qingxin Pills, respectively.

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

[0045] Figure 3 The protein interaction network of Niuhuang Qingxin Pill in treating depression is shown.

[0046] Figure 4 A GO enrichment analysis bar chart of the intersection genes of the target of Niuhuang Qingxin Pill (official prescription) and the target of depression is shown, where A, B, and C represent biological process, molecular function, and cellular localization, respectively.

[0047] Figure 5 A bar chart of KEGG pathway enrichment analysis of the intersection genes of the targets of Niuhuang Qingxin Pills (official prescription) and the targets of depression is shown.

[0048] Figure 6 A protein interaction network of Niuhuang Qingxin Pill in treating depression is shown.

[0049] Figure 7 The figure shows the comparison of the reduction of MMN latency in the four groups of patients before and after treatment. DETAILED DESCRIPTION

[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 to the embodiments set forth herein.

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

[0052] Example 1

[0053] This embodiment shows an example of comparing the effects of (bureau prescription) Niuhuang Qingxin Pills (purchased from Shanxi Guangyuyuan Chinese Medicine Co., Ltd.), a certain brand of Niuhuang Qingxin Pills sold on the market that does not contain realgar and cinnabar (referred to as A Niuhuang Qingxin Pills in this article), and B Niuhuang Qingxin Pills and C Niuhuang Qingxin Pills prepared by the inventor on treating depression, wherein the difference between the ingredients of B Niuhuang Qingxin Pills and (bureau prescription) Niuhuang Qingxin Pills is that it does not contain realgar, and the difference between the ingredients of C Niuhuang Qingxin Pills and (bureau prescription) Niuhuang Qingxin Pills is that it does not contain cinnabar. This embodiment includes the following steps:

[0054] S1. Prepare the test solution:

[0055] Take Niuhuang Qingxin Pills (prescription), A Niuhuang Qingxin Pills, B Niuhuang Qingxin Pills, and C Niuhuang Qingxin Pills, chop them separately, add diatomaceous earth and grind until evenly dispersed, add methanol solution, and perform ultrasonic treatment to obtain four mixtures, i.e., test solution;

[0056] The four test sample solutions were respectively centrifuged at high speed and then filtered through a 0.22 μm microporous filter membrane to serve as the methanol extract of Niuhuang Qingxin Pills test sample, and then diluted 1 time with the initial mobile phase.

[0057] S2. Test (bureau prescription) Niuhuang Qingxin Pills, A Niuhuang Qingxin Pills, B Niuhuang Qingxin Pills, C Niuhuang Qingxin Pills ingredient information:

[0058] The composition information of the mixture in step S1 was determined by ultra-high performance liquid chromatography coupled with a high-resolution time-of-flight mass spectrometer; the chromatographic conditions were 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; gradient elution conditions 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 Mobile phase A, 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; mass spectrometry detection conditions are as follows: electrospray ion source, positive and negative ion scanning mode, 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 follows: Figure 1 As shown, the test results show that the fingerprint of Niuhuang Qingxin Pills (prescription) has 7 common peaks, and the retention time of each common peak is:

[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, wogonin, taurocholic acid, ginsenoside Rg1, ginsenoside Rb1, tauroursodeoxycholic acid, and cholic acid, respectively.

[0067] The test information of some ingredients of Niuhuang Qingxin Pills (official prescription) is shown in Table 1:

[0068] Table 1: Test information of some ingredients of Niuhuang Qingxin Pills (official prescription)

[0069]

[0070] Carry out the following steps for Niuhuang Qingxin Pills:

[0071] S3. Screening of the active ingredients of Niuhuang Qingxin Pills (official prescription):

[0072] The ingredient information obtained in step S2 was deduplicated and sorted to establish a dataset of active ingredients of Niuhuang Qingxin Pills (official prescription); in addition, relevant literature was searched to supplement the ingredients that were not selected but had relevant activity studies; after screening, a total of 67 active ingredients were obtained (Table 2).

[0073] Table 2: Active ingredients of Niuhuang Qingxin Pills (official prescription)

[0074]

[0075]

[0076]

[0077] In addition, since UPLC-Q-TOF-MS failed to detect realgar and cinnabar, it was necessary to manually supplement the component information corresponding to these substances, among which realgar corresponds to tetraarsenic tetrasulfide and cinnabar corresponds to mercuric sulfide.

[0078] S4. Predict active ingredient targets and collect disease targets:

[0079] The targets corresponding to the active ingredients of Niuhuang Qingxin Wan (official prescription) in step S3 were retrieved through pubchem and Swiss Target Prediction databases, and the active ingredient-target data set was established after deduplication and sorting; the targets of depression were collected using Genecards and OMIM databases to obtain the disease-target data set;

[0080] Specifically, the active ingredients of Niuhuang Qingxin Pills were input into pubchem and Swiss TargetPrediction databases respectively, and targets were acquired with "human" as the screening condition. Targets with Probability of 0 were deleted, and 595 targets were obtained. After de-duplication, the targets were input into Uniprot database, and standardized protein names were obtained with "verified, human" as the screening condition, and 487 targets corresponding to the active ingredients of Niuhuang Qingxin Pills were obtained; "depression" was used as the keyword to search in Genecards and OMIM databases to obtain gene targets related to depression, the obtained genes were unioned, de-duplicationd, and screened, and then 978 standardized protein names were obtained through Uniprot database with "verified, human" as the condition, which can be used for subsequent network analysis.

[0081] In addition, for tetraarsenic tetrasulfide, it can be entered into the CTD (Comparative Toxicogenomics Database) 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. Constructing the “active ingredient-target” network:

[0083] The “active ingredient-target” network was constructed using the active ingredient-target dataset and the disease-target dataset in step S4, and visualized using Cytoscape software ( Figure 2 ).

[0084] S6. Construction of protein interaction network:

[0085] Obtain the intersection genes of the active ingredient-target dataset and the disease-target dataset in step S4, import the 125 intersection genes into the String database, set the search target to "Multiple proteins", select the species to "Homo sapiens" to obtain the protein interaction network ( Figure 3), the network has 122 nodes and 1256 edges; the network was analyzed using Cytoscape software, and the "CytoNCA" plug-in was used to calculate the score of each node according to the betweenness centrality to obtain the core node, and the nodes were sorted according to the betweenness centrality. The larger the node, the more critical the role of the gene in the network, and the core targets were obtained, such as GAPDH, TNF, CNR1, PPARG, etc.

[0086] S7.GO and KEGG enrichment analysis:

[0087] The intersection genes in step S6 were subjected to GO and KEGG enrichment analysis, and the results were as follows: Figure 4 and Figure 5 As shown;

[0088] Specifically, the intersection genes were input into Metascape, and "human" was selected as the species for personalized analysis. As a result, 20 KEGG pathways were obtained, including cancer pathways, neuroactive ligand-receptor interactions, calcium signaling pathways, serotonin-containing synapses, proteoglycans in cancer, hepatitis B, diabetic cardiomyopathy, dopaminergic synapses, etc.; 20 GO biological processes, GO molecular functions, and GO cell localizations were obtained. GO biological processes mainly included hormone response, behavior, positive regulation of phosphorus metabolism, circulatory system processes, response to external stimuli, secretion regulation, regulation of membrane potential, response to extracellular stimuli, etc.; GO molecular functions mainly included neurotransmitter receptor activity, hormone binding, G protein-coupled amine receptor activity, alcohol-based receptor phosphotransferase activity, steroid binding, transcription factor binding, protein kinase binding, dopamine neurotransmitter receptor activity, etc.; GO cell localizations mainly included receptor complexes, dendrites, membrane rafts, postsynaptic membranes, etc. The darker the color of the bar graph, the more significant the P value is, the higher the credibility of gene enrichment in the pathway or biological process, molecular function, and cell localization is, and the more important the signaling pathway, biological process, molecular function, or cell localization is.

[0089] S8. Molecular docking:

[0090] Using Chem 3D, PyMOL, AutoDock Tools, and vina, we conducted docking simulations on the core target GAPDH and the chemical components peony glucoside or isomers, TNF and glycocholic acid, CNR1 and glycocholic acid, and PPARG and flavonols of glycyrrhizae to predict the binding mode and strength, determine the docking binding sites and names, and plot the molecular docking results (using the PDB database to obtain the protein structure of the core target). The minimum binding energies of the ligand receptors are GAPDH-peony glucoside or isomers-5.3kcal / mol, TNF-glycocholic acid-7.4kcal / mol, CNR1-glycocholic acid-4.0kcal / mol, and PPARG-flavonols of glycyrrhizae-8.7kcal / mol.

[0091] S9. Calculate the network efficiency of the protein interaction network in step S6, and 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 the 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 pairs of nodes in the network through the shortest path. In the present invention, the network efficiency of the protein interaction network of drug treatment for depression 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 of drug treatment for depression, the higher the communication efficiency, and the better the therapeutic effect of the drug. After calculation, the network efficiency corresponding to (bureau prescription) Niuhuang Qingxin Pills is 0.259.

[0094] according to Figure 1 It can be seen that there are differences between (Ju Fang) Niuhuang Qingxin Pills and A Niuhuang Qingxin Pills. In the positive ion mode, A Niuhuang Qingxin Pills have more peaks, while in the negative ion mode, (Ju Fang) Niuhuang Qingxin Pills have more peaks. Comparing the ingredient information of the two, it was found that compared with A Niuhuang Qingxin Pills, (Ju Fang) Niuhuang Qingxin Pills have more chemical substances such as taurodeoxycholic acid, deoxycholic acid, dichloronaphthoquinone, 3,5-di-tert-butyl-4-hydroxybenzoic acid methyl ester, isobutylpropionic acid, lupenone, and berberine alcohol. At the same time, the curcumin content is higher than that of A Niuhuang Qingxin Pills, and other ingredients are similar. Steps S3-S9 are performed using the ingredient information of A Niuhuang Qingxin Pills. The protein interaction network of A Niuhuang Qingxin Pills is shown in the figure. Figure 6As shown, the network efficiency is calculated to be 0.105, and the results show that the network efficiency of (Ju Fang) 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 to perform steps S3-S9, the network efficiency of the two is calculated to be between (Ju Fang) Niuhuang Qingxin Pills and A Niuhuang Qingxin Pills. The above results show that the method of the present invention is used to calculate that (Ju Fang) Niuhuang Qingxin Pills has the best effect in treating depression.

[0095] Example 2

[0096] In order to verify the reliability of the method of the present invention, the inventors conducted the following experiments:

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

[0098] 50 mice were randomly divided into 5 groups, including a control group, a local prescription group, a group A, a group B, and a group C. The 5 groups of mice were treated with drugs by gavage, and the drugs were normal saline, (local prescription) Niuhuang Qingxin Pills, A Niuhuang Qingxin Pills, B Niuhuang Qingxin Pills, and C Niuhuang Qingxin Pills, respectively. Each drug was ground into granules, dissolved in water, and then gavage was performed. The daily dose was 0.5g / kg, and the gavage volume was 0.2ml. The 5 groups of mice were free to eat and drink 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 taking the drugs (local prescription group, group A, group B, and group C) were improved to varying degrees, mainly manifested in a significant reduction in the immobility time of tail suspension and forced swimming, an increase in the time of entering the middle area in the 10-minute open field test, and a stronger exploratory nature, that is, the depression and anxiety-like behaviors of the 4 groups of mice were significantly improved, and the mice in the local prescription group showed the best therapeutic effect (Table 3).

[0099] Table 3: Time taken by the five groups of mice to enter the middle area in the open field test (mean ± standard error)

[0100] Group Time (minutes) Control group 1.26±0.34 Bureau 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 speculate that this may be because (official prescription) Niuhuang Qingxin Pills contain realgar and cinnabar, which have a calming and tranquilizing effect. These two substances together lead to more efficient signal transduction to depression targets in the protein interaction network. Therefore, the therapeutic effect is better than A Niuhuang Qingxin Pills without realgar and cinnabar and Niuhuang Qingxin Pills containing realgar or cinnabar alone.

[0102] Example 3

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

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

[0105] The Western medical diagnostic criteria for depressive cognitive impairment in this embodiment are as follows: refer to the fifth edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-V). There is a depressed 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) hyperactivity or hypoactivity; (4) fatigue or lack of energy; (5) feeling worthless or guilty; (6) decreased concentration and thinking ability, indecisiveness in making decisions; (7) suicidal thoughts or suicidal plans, suicidal behavior. Symptoms last for at least 2 weeks, accompanied by severe psychological distress and obvious social function impairment.

[0106] The TCM diagnostic criteria for depression syndrome of phlegm-heat disturbing the mind are as follows:

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

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

[0109] The patient must first have mental depression and at least 4 other symptoms among the main symptoms, and the symptoms last for at least 2 weeks, then he can be diagnosed with depression.

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

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

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

[0113] Secondary symptoms: nightmare, drowsiness, heaviness and soreness of limbs, nausea, constipation, red and greasy face. Tongue and pulse: red tongue, yellow and greasy tongue coating, stringy or slippery pulse.

[0114] Diagnostic criteria: all main symptoms and one or more secondary symptoms.

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

[0116] ①Meet the diagnostic criteria of DSM-V for depressive disorder;

[0117] ② Those who meet the diagnostic criteria of TCM syndrome differentiation and are diagnosed with depression disorder due to “phlegm-heat disturbing the mind” syndrome;

[0118] ③ Screening score 7 ≤ Hamilton Depression Rating Scale (HAMD) 17-item score ≤ 24 points, with depression item ≥ 2 points and suicide item < 2 points in HAMD; PDQ-D score ≥ 21 points;

[0119] ④ Patients with depressive disorder who have just started the disease and have not received any treatment, or have not received any treatment in the past two months;

[0120] ⑤Age 18-75, regardless of gender;

[0121] ⑥ Those who are informed and agree to participate in the study.

[0122] The patient exclusion criteria were as follows:

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

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

[0125] (3) There is evidence that anxiety or depression is causally related to a physical illness or drug use;

[0126] (4) Patients with recurrent anxiety or depression;

[0127] (5) known alcohol or drug addicts;

[0128] (6) Patients with unstable vital signs and severe or unstable heart, liver, kidney, endocrine, blood, respiratory and other medical diseases;

[0129] (7) Pregnant women and breastfeeding women.

[0130] The criteria for patient exclusion and dropout are as follows:

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

[0132] (2) Those who take other anti-anxiety and depression drugs or increase the dosage of drugs on their own during treatment;

[0133] (3) The patient is uncooperative and refuses to comply with treatment, and repeated explanations by the clinician are ineffective;

[0134] (4) Those who actively request 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 local prescription group (n=33). Among them, 5 cases in group A, 2 cases in group B, 2 cases in group C, and 3 cases in the local prescription group did not take the medicine according to the study regulations and dropped out during the study. A total of 119 cases completed the trial, including 28 cases in group A, 31 cases in group B, 30 cases in group C, and 30 cases in the local prescription group. There was no statistically significant difference in gender, age, disease course, HAMD score, and PDQ-D score among the four groups (P>0.05).

[0136] The treatment method is as follows: patients in group A, group B, group C and the local prescription group were orally treated with A Niuhuang Qingxin Pills, B Niuhuang Qingxin Pills, C Niuhuang Qingxin Pills, and (local prescription) Niuhuang Qingxin Pills (the four drugs were made into pills of the same weight), 1 pill / time, 1 time / day, for 3 consecutive weeks, and then stopped for 1 week, and then continued to take for 3 weeks, and stopped for 1 week, for a total course of 8 weeks. Acupuncture and other Chinese and Western medicines for the treatment of depressive disorders were strictly prohibited during the experiment.

[0137] Observation indicators include:

[0138] (1) Main efficacy indicators: PDQ-D;

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

[0140] (3) Biological indicators: event-related potential (ERP);

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

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

[0143] The efficacy was evaluated as follows:

[0144] The efficacy was evaluated based on the reduction rate of the self-rating scale for cognitive deficits after treatment: reduction rate = (total score before treatment - total score after treatment) / total score before treatment × 100%.

[0145] Evaluation criteria:

[0146] Recovery: Psychiatric symptoms disappear, with a score reduction rate of ≥75%;

[0147] Markedly effective: Psychiatric symptoms basically disappeared, with a score reduction rate of ≥50%;

[0148] Effective: Mental symptoms are alleviated, with a reduction rate of ≥25%;

[0149] Ineffective: reduction rate <25%.

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

[0151] MMN mainly reflects the subject's reaction speed to deviant stimuli and depression. The test uses a passive auditory oddball stimulation mode. The standard stimulus is a pure tone of 500Hz and 80dB with a probability of 0.8; the deviant stimulus is a pure tone of 2000Hz and 85dB with a probability of 0.2. The patient is not required to respond to the target stimulus and non-target stimulus, which are superimposed 100 times. The latency of MMN is measured.

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

[0153] Table 4: Comparison of 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] There were no significant differences in PDQ-D, BDI and BAI scores among the four groups before treatment (all P>0.05). After treatment, PDQ-D, BDI and BAI scores of the four groups decreased significantly (all P<0.05), and the reduction of PDQ-D, BDI and BAI scores in the Jufang group was significantly greater than that in groups A, B and C, with statistically significant differences (all P<0.05).

[0157] Comparison of MMN before and after treatment:

[0158] Before treatment, there was no significant difference in the MMN latency between the four groups (all P>0.05). After treatment, the MMN latency of the four groups was significantly shortened (all P<0.05), and the shortening of the MMN latency in the Jufang group was significantly greater than that in the other three groups, with a statistically significant difference (P<0.05), while there was no significant difference in the shortening of the MMN latency between groups A, B, and C (Table 5 and Figure 7 ).

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

[0160]

[0161] The results of the above clinical trials are consistent with the calculation results of the method provided by the present invention and the mouse experiment results in Example 2, further confirming the reliability of the method provided by the present invention.

[0162] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

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 respectively, add diatomaceous earth and grind them evenly until they are evenly dispersed, add methanol solution, and perform ultrasonic treatment to obtain a mixture of various Niuhuang Qingxin Pills; S2. Detect the ingredient information of various Niuhuang Qingxin Pills: Using ultra-high performance liquid chromatography coupled with a high-resolution time-of-flight mass spectrometer to respectively determine the composition information of the multiple Niuhuang Qingxin Pills mixtures in step S1; S3. Screening of active ingredients of various Niuhuang Qingxin Pills: The component information obtained in step S2 is deduplicated and sorted, and multiple Niuhuang Qingxin Pills active component data sets are established respectively; S4. Predict active ingredient targets and collect disease targets: The targets corresponding to the active ingredients of various Niuhuang Qingxin Pills in step S3 were retrieved through pubchem and Swiss Target Prediction databases, and the active ingredient-target data set was established after deduplication and sorting; the targets of depression were collected using Genecards and OMIM databases to obtain the disease-target data set; S5. Constructing the "active ingredient-target-disease" network: Using the active ingredient-target data set and the disease-target data set in step S4, constructing an "active ingredient-target-disease" network; 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: Taking the 5, 10, 15 or 20 proteins with the largest degrees in the protein interaction network in step S6 as receptor proteins, taking 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 mixture in step S1 is post-treated respectively, the steps are as follows: the mixture is subjected to high-speed centrifugation, filtered through a 0.22 μm microporous filter membrane, used as the methanol extract of Niuhuang Qingxin Pills as a test sample, and then diluted 1 time with the initial mobile phase.

3. The method according to claim 1 or 2, characterized in that: The chromatographic conditions in step S2 are as follows: ThermoHypersil 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.

4. The method according to any one of claims 1 to 3, characterized in that 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.

5. The method according to any one of claims 1 to 4, characterized in that The mass spectrometry detection conditions in step S2 are as follows: electrospray ion 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.

6. The method according to any one of claims 1 to 5, characterized in that Step S3 also includes: searching for relevant literature to supplement the ingredients that were not selected but have relevant activity studies.

7. The method according to any one of claims 1 to 6, characterized in that In step S8, the structure of the receptor protein is obtained using the PDB database.

8. 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 Pills and other Niuhuang Qingxin Pills in treating depression using the method described in any one of claims 1-7.

9. The method according to claim 8, 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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