Application of grass aconite in preparation of anti-epilepsy drugs and analysis method of mechanism of action

Abstract

The application provides application of grass aconite in preparation of anti-epilepsy drugs and a mechanism analysis method, relates to the technical field of anti-epilepsy drugs, and discloses a "drug active ingredient-key target point-disease-pathway" network diagram of the grass aconite anti-epilepsy by starting from the chemical structure of the effective component of the grass aconite, applying network pharmacology and molecular docking technology, revealing how the active component of the grass aconite plays the drug efficacy through "multi-target point, multi-pathway and multi-pathway" combined regulation, predicting the anti-epilepsy target point and mechanism of the grass aconite, and providing certain theoretical reference for research and development of the grass aconite as an anti-epilepsy drug and a functional food.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of anti-epileptic drugs, and particularly relates to application of aconite in preparation of an anti-epileptic drug and a mechanism analysis method. BACKGROUND

[0002] Epilepsy is a chronic central nervous system disease characterized by repeated seizures, which not only seriously affects the daily life of patients, but also the risk of premature death of epilepsy patients is three times that of normal people. Sudden recurrence and short-term sensory or behavioral disorders caused by highly synchronized cortical neuronal networks cause individuals to experience long periods of abnormal discharges in the brain. Due to high disability rate, long course of disease, easy recurrence, and long-term drug treatment, patients and their families have suffered huge economic burden. Therefore, it is urgent for the clinic to find new anti-epileptic drugs to solve the current situation of shortage of treatment drugs and high cost pressure, which is of great significance to patients and their families and the whole society. The main treatment method for epilepsy is oral drug, and the development of the drug is upgrading, because as many as one-third of epilepsy patients are resistant to drugs, and the current focus of treatment development is to find new mechanisms of action. Many traditional Chinese medicines have anti-epileptic effects, and currently found main effective traditional Chinese medicines for treating epilepsy include turmeric, scorpion, rhizoma acori graminei, Uncaria, Ganoderma lucidum, etc. The main compounds having therapeutic effects on epilepsy include curcumin, gastrodin, ligustrazine, baicalin and uncarine, etc. These traditional Chinese medicines play an important role in the clinical treatment of epilepsy in traditional Chinese medicine. Now a new traditional Chinese medicine is explored, and a new mechanism of action is explored to treat epilepsy. SUMMARY

[0003] Therefore, the application provides application of aconite in preparation of an anti-epileptic drug.

[0004] It is also necessary to provide an anti-epileptic mechanism analysis method of aconite.

[0005] The technical solution adopted by the application to solve the technical problem is:

[0006] Application of aconite in preparation of an anti-epileptic drug.

[0007] Preferably, the active ingredients of aconite are N-dimethyl-1-phenylmethylamine (BDMA), 2-(3,4-dimethoxyphenyl)-N-methyl ethylamine (N-Methylhomoveratrylamine), Izoteolin, palmitic acid.

[0008] Preferably, aconite plays an anti-epileptic role through CHRM1, ADRA2A, CHRM2, OPRD1 and OPRM1 key related disease targets, and regulates G-protein coupled receptor molecular signal pathways of a nerve active ligand-receptor pathway.

[0009] The method for analyzing the anti-epilepsy mechanism of the grass wu as described above comprises the following steps:

[0010] S1 screening of active ingredient targets of the grass wu: screening is performed through a traditional Chinese medicine systematic pharmacology database and analysis platform to predict the action targets of the active ingredients of the grass wu;

[0011] S2 screening of key targets of the grass wu against epilepsy: screening is performed through a disease target database Disgenet to obtain gene targets of epilepsy diseases, and then the action targets of the active ingredients of the grass wu obtained in S1 are intersected with the gene targets of epilepsy diseases to obtain intersection targets of the grass wu and epilepsy, i.e. the action targets of the grass wu against epilepsy; S3 enrichment analysis of the epilepsy-acting pathway of the grass wu: the action targets of the grass wu against epilepsy obtained in S2 are screened through a KEGG database to obtain the number of KEGG pathways, which reflect multiple biological processes in the body, indicating that the grass wu may exert its anti-epilepsy effect by improving these biological processes;

[0012] S4 construction of a network of drug active ingredients-key targets-diseases-pathways: the action targets of the active ingredients of the grass wu obtained in S1, the gene targets of epilepsy diseases obtained in S2 and the pathways obtained in S3 are used to construct a network diagram of “drug active ingredients-key targets-diseases-pathways” through Cytoscape, which clearly shows the mutual relationship between the drug ingredients and their metabolites, the diseases and their genes and the pathways, facilitating the exploration of the action mechanism of the grass wu against epilepsy;

[0013] S5 enrichment analysis of the action targets of the grass wu against epilepsy to explore the action mechanism: the action targets of the grass wu against epilepsy obtained in S2 are subjected to GO analysis of cellular components, biological processes and molecular functions, a protein interaction network is constructed, and the action mechanism of the grass wu against epilepsy is obtained;

[0014] S6 molecular docking of the active ingredients of the grass wu and the anti-epilepsy targets: the protein receptors in the action targets of the grass wu against epilepsy obtained in S2 are collected for crystal structures through an RCSB PDB database, the proteins are then pretreated to obtain protein active sites, the action targets of the active ingredients of the grass wu in the action targets of the grass wu against epilepsy obtained in S2 are pretreated to obtain pretreated ligands, the protein active sites are subjected to molecular docking with the pretreated ligands, and the action mechanism of the grass wu against epilepsy is verified.

[0015] Preferably, in the S1 screening of the active ingredient targets of the grass wu, the main active ingredient targets of the grass wu are predicted through searching of a traditional Chinese medicine systematic pharmacology database and analysis platform TCMSP, and the active ingredient target proteins are uniformly converted into gene names by using an Uniprot database.

[0016] Preferably, in the S2 screening of key targets of antiepileptic effects of the Aconitum, the targets of antiepileptic effects of the Aconitum are six, i.e., muscarinic acetylcholine receptor M1, alpha-2A adrenergic receptor, muscarinic acetylcholine receptor M2, delta opioid receptor, mu opioid receptor and tumor necrosis factor, and the active ingredients of the Aconitum are N-dimethyl-1-phenylmethylamine, 2-(3,4-dimethoxyphenyl)-N-methylethylamine, izatrien and palmitic acid.

[0017] Preferably, in the S3 pathway enrichment analysis of the Aconitum, the KEGG pathways include: neural active ligand-receptor interaction, calcium signaling pathway, PI3K-Akt signaling pathway, regulation of actin cytoskeleton, pathway of neurodegenerative multiple diseases, cholinergic synapse, sphingolipid signaling pathway and cGMP-PKG signaling pathway.

[0018] Preferably, the S5 enrichment analysis of the targets of antiepileptic effects of the Aconitum includes the following analysis in the exploration of the mechanism:

[0019] GO enrichment analysis: the targets of antiepileptic effects of the Aconitum obtained by S2 are screened through the DAVID database, and the screening results are subjected to GO analysis to obtain the signal pathways of biological processes, cell components and molecular functions participated by the Aconitum;

[0020] Construction of PPI network: the targets of antiepileptic effects of the Aconitum obtained by S2 are introduced into the STRING database through venny to construct a protein-protein interaction relationship diagram to obtain protein genes participating in mutual regulation.

[0021] Preferably, in the construction of the PPI network, the protein genes participating in mutual regulation are: muscarinic acetylcholine receptor M1, alpha-2A adrenergic receptor, muscarinic acetylcholine receptor M2, delta opioid receptor, mu opioid receptor and tumor necrosis factor.

[0022] Preferably, the mu opioid receptor has the best participation degree.

[0023] Compared with the prior art, the present application has the following beneficial effects:

[0024] The present application finds the application of the Aconitum in the preparation of antiepileptic drugs, develops a new use of the Aconitum, and also explores a new traditional Chinese medicine for the treatment of epilepsy.

[0025] The present application starts from the chemical structure of the active ingredients of the Aconitum, applies network pharmacology and molecular docking technology to construct a "drug active ingredient-key target-disease-pathway" network diagram of the Aconitum against epilepsy, reveals how the active ingredients of the Aconitum exert their efficacy through "multi-target, multi-pathway and multi-pathway" joint regulation, predicts the targets and mechanism of the Aconitum against epilepsy, and provides a certain theoretical reference for the research and development of the Aconitum as an antiepileptic drug and functional food.

[0026] The application networks and systematizes the research method of the disease affected by the medicine, compared with the traditional research method, the research and development range is reduced, the research and development purpose is accurate, so that the medicine research and development cost is saved. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 It is a Venn diagram of the intersection of kudou and epilepsy.

[0028] Figure 2 It is a KEGG pathway enrichment analysis diagram.

[0029] Figure 3 It is a kudou-active ingredient-target-epilepsy-pathway network relationship diagram.

[0030] Figure 4 It is a GO enrichment analysis.

[0031] Figure 5 It is a GO enrichment analysis bubble chart.

[0032] Figure 6 It is a target protein interaction network relationship diagram (PPI).

[0033] Figure 7 It is a kudou active ingredient and anti-epilepsy target molecule docking diagram. DETAILED DESCRIPTION

[0034] The technical solutions and technical effects of the embodiments of the application are further described in detail in combination with the drawings of the application.

[0035] The application of kudou in preparing an anti-epilepsy medicine.

[0036] Compared with the prior art, the application has the beneficial effects that:

[0037] The application finds the application of kudou in preparing an anti-epilepsy medicine, develops a new use of kudou, and also explores a new traditional Chinese medicine for epilepsy treatment.

[0038] Further, the active ingredients of kudou are: BDMA, N-Methylhomoveratrylamine, Izoteolin, palmitic acid.

[0039] Further, kudou plays an anti-epilepsy role through CHRM1, ADRA2A, CHRM2, OPRD1 and OPRM1 key related disease targets, and regulates the G-protein coupled receptor molecular signal pathway of the nerve active ligand-receptor pathway.

[0040] Further, the active ingredient of the grass wu is: Izoteolin.

[0041] The mechanism analysis method of the anti-epilepsy effect of the grass wu as described above comprises the following steps:

[0042] S1: Screening of the effective component target of the grass wu: the action target of the active ingredient of the grass wu is predicted through the traditional Chinese medicine systematic pharmacology database and analysis platform;

[0043] S2: Screening of the key target of the anti-epilepsy effect of the grass wu: the epilepsy disease gene target is obtained through the disease target database Disgenet, and then the action target of the active ingredient of the grass wu obtained through S1 is intersected with the epilepsy disease gene target to obtain the intersection target of the grass wu-epilepsy, i.e. the action target of the anti-epilepsy effect of the grass wu;

[0044] S3: Pathway enrichment analysis of the anti-epilepsy effect of the grass wu: the action target of the anti-epilepsy effect of the grass wu obtained through S2 is screened through the KEGG database to obtain the number of KEGG pathways, reflecting multiple biological processes in the body, indicating that the grass wu may exert its anti-epilepsy effect by improving these biological processes;

[0045] S4: Construction of the network of the active ingredient-key target-disease-pathway of the drug: the action target of the active ingredient of the grass wu obtained through S1, the epilepsy disease gene target obtained through S2 and the pathway obtained through S3 are constructed into the network diagram of the active ingredient-key target-disease-pathway of the drug through Cytoscape, which clearly shows the mutual relationship between the drug components and their metabolites, the disease and its genes and the pathway, facilitating the exploration of the action mechanism of the anti-epilepsy effect of the grass wu;

[0046] S5: Enrichment analysis of the action target of the anti-epilepsy effect of the grass wu to explore the action mechanism: the action target of the anti-epilepsy effect of the grass wu obtained through S2 is subjected to GO analysis of cellular components, biological processes and molecular functions, a protein interaction network is constructed, and the action mechanism of the anti-epilepsy effect of the grass wu is obtained;

[0047] S6: Molecular docking of the active ingredient and the anti-epilepsy target of the grass wu: the protein receptors in the action target of the anti-epilepsy effect of the grass wu obtained through S2 are collected through the RCSB PDB database to obtain the crystal structure corresponding to the protein, the protein is pretreated to obtain the active site of the protein, the action target of the active ingredient of the grass wu in the action target of the anti-epilepsy effect of the grass wu obtained through S2 is pretreated to obtain a pretreated ligand, and the active site of the protein is subjected to molecular docking with the pretreated ligand to verify the action mechanism of the anti-epilepsy effect of the grass wu.

[0048] Specifically, the traditional Chinese medicine grass wu is screened for components through the TCMSP database, and the component protein targets are converted into gene targets using the uniprot database. Then, the epilepsy disease gene targets are screened through the Disgenet database. Thus, the gene targets of the components of grass wu and the gene targets of epilepsy disease are obtained through the Venny software to obtain 6 intersection gene targets, from which the component list is deduced to obtain 4 active components. At the same time, the intersection targets are converted into KEGG ID numbers through the KEGG database to obtain related pathways. According to the obtained component genes, disease genes and pathways, a grass wu-component-epilepsy-pathway network diagram is drawn according to the Cytoscape software, and the mechanism of grass wu against epilepsy is visually observed. In order to explore the biological processes, cellular components, molecular functions and other information of the 6 genes, David database is used for further GO enrichment analysis. At the same time, in order to explore the mutual relationship of the 6 intersection genes, String database is used to construct protein interaction network (PPI), and the mechanism of grass wu against epilepsy is further explored, and the constructed grass wu-component-epilepsy-pathway network diagram is also further verified. In order to further verify whether the 6 disease gene targets and the 4 active component targets have effects, molecular docking is carried out, which shows good docking effect, and verifies the components and mechanism of grass wu against epilepsy.

[0049] The present application starts from the chemical structure of the effective components of grass wu, and constructs a "drug active component-key target-disease-pathway" network diagram of grass wu against epilepsy by applying network pharmacology and molecular docking technology, reveals how the active components of grass wu play their efficacy through "multi-target, multi-pathway and multi-pathway" joint regulation, predicts the targets and mechanism of grass wu against epilepsy, and provides certain theoretical reference for the research and development of grass wu as an anti-epilepsy drug and functional food.

[0050] The present application networks the research method of drug action on disease, and systematizes the research method compared with the traditional research method, which reduces the research and development range, and accurately the research and development purpose, thereby saving the drug research and development cost. At the same time, the efficiency of screening and predicting the drug active components is greatly improved.

[0051] Further, in the S1 grass wu effective component target screening, the main effective component targets of grass wu are predicted through the traditional Chinese medicine systematic pharmacology database and analysis platform TCMSP, and the effective component target proteins are uniformly converted into gene names by using the Uniprot database.

[0052] Further, in the S2 screening of key targets of antiepileptic of S. wallichii, the targets of S. wallichii for antiepileptic are six, namely muscarine acetylcholine receptor M1, alpha-2A adrenergic receptor, muscarine acetylcholine receptor M2, delta opioid receptor, Mu opioid receptor, and tumor necrosis factor, and the active ingredients of S. wallichii are N-dimethyl-1-phenylmethylamine, 2-(3,4-dimethoxyphenyl)-N-methyl ethylamine, isazotelin, and palmitic acid.

[0053] Further, in the S3 pathway enrichment analysis of S. wallichii for antiepileptic, the KEGG pathway includes: neuroactive ligand-receptor interaction, calcium signaling pathway, PI3K-Akt signaling pathway, regulation of actin cytoskeleton, pathway of neurodegenerative multiple diseases, cholinergic synapse, sphingolipid signaling pathway, and cGMP-PKG signaling pathway.

[0054] Further, the S5 enrichment analysis of the targets of S. wallichii for antiepileptic includes the following analysis:

[0055] GO enrichment analysis: The targets of S. wallichii for antiepileptic obtained by S2 are screened by DAVID database, and the screening results are analyzed by GO to obtain the signal pathways of biological processes, cell components and molecular functions participated by S. wallichii;

[0056] Construction of PPI network: The targets of S. wallichii for antiepileptic obtained by S2 are introduced into STRING database by venny to construct a protein-protein interaction relationship diagram, and the protein genes involved in mutual regulation are obtained.

[0057] Further, in the construction of PPI network, the protein genes involved in mutual regulation are: muscarine acetylcholine receptor M1, alpha-2A adrenergic receptor, muscarine acetylcholine receptor M2, delta opioid receptor, Mu opioid receptor, and tumor necrosis factor.

[0058] Further, the Mu opioid receptor has the best participation.

[0059] Further, the S6 aconite active ingredient and antiepileptic target molecule docking is specifically: collect the crystal structures corresponding to the six proteins through the RCSB PDB database. Through the Protein Preparation Wizard module of Schrodinger software, the collected protein crystals are pretreated as follows: ①protein preprocess; ②regenerate states of native ligand; ③H-bond assignment optimization; ④protein energy minimization; ⑤remove waters. Then pretreat the ligand: pretreat the 2Dsdf structure file of the four compounds through the LigPrep module in Schrodinger to generate all 3D chiral conformations thereof. Then identify the active site: first, use the SiteMap module of Schrodinger to predict the best binding site, then use the ReceptorGrid Generation module of Schrodinger to make the best Enclosing box and perfectly wrap the predicted binding site, so as to obtain the active sites of the six proteins. Then molecular docking. The four ligand compounds that have been processed are respectively docked with the active sites of the six proteins (using the highest precision XP docking), and the lower the score, the lower the free energy of the compound binding to the protein, and the more stable the binding. Finally, MM-GBSA analysis. Through the use of MM-GBSA to calculate and analyze the four ligand compounds and the active sites of the six proteins, MM-GBSAdG Bind can approximately represent the free energy of the protein and small molecule binding, and the lower the binding free energy, the more stable the ligand compound and the protein binding

[0060] For the convenience of understanding, the present application is further illustrated by the following examples:

[0061] Example 1:

[0062] S1 aconite effective ingredient target screening: taking "Aconitum flavum Hand.-Mazz." as the keyword, searching in the Traditional Chinese Medicine System Pharmacology Database and Analysis Platform TCMSP, taking drug likeness (DL) ≥0.02 and oral bioavailability (OB) ≥18% as the screening conditions, predicting the main effective ingredient targets of aconite. And input the ingredient targets into Uniprot database (https: / / www.uniprot.org / ) with the screening condition of Human, and convert the effective ingredient targets into gene names; obtain 51 aconite chemical ingredients as shown in Table 1:

[0063] Table 1

[0064]

[0065]

[0066] Screening of key targets for the anti-epileptic effect of Aconitum carmichaelii (S2): Using the Disgenet disease gene search platform (https: / / www.disgenet.org / home / ), with "Epilepsy" as the keyword and a score > 0.3 as the screening criterion, target genes related to epilepsy were searched, yielding 114 disease targets related to epilepsy, as shown in Table 2. Using the venny 2.1.0 online platform (https: / / bioinfogp.cnb.csic.es / tools / venny), the targets of Aconitum carmichaelii and epilepsy targets were imported into the software for intersection analysis, yielding 6 interactive targets, as shown in Table 3. Figure 1 As shown, these are the muscarinic acetylcholine receptor M1 (CHRM1), α-2A adrenergic receptor (ADRA2A), muscarinic acetylcholine receptor M2 (CHRM2), delta-type opioid receptor (OPRD1), mu-type opioid receptor (OPRM1), and tumor necrosis factor (TNF). Figure 1 Among them, CHRM1, ADRA2A, CHRM2, OPRD1, and OPRM1 are all G-protein-coupled receptors, and their corresponding active ingredients are N-dimethyl-1-phenylmethylamine (BDMA), 2-(3,4-dimethoxyphenyl)-N-methylethylamine (N-Methylhomoveratrylamine), Izoteolin, and palmitic acid. BDMA is the active ingredient, with ID number MOL004753. 2-(3,4-dimethoxyphenyl)-N-methylethylamine (N-Methylhomoveratrylamine) is a metabolite of MOL004754, Izoteolin is a metabolite of MOL004763, and palmitic acid is a metabolite of MOL000069 (Table 3).

[0067] Table 2

[0068]

[0069]

[0070] Table 3

[0071]

[0072] S3 S3 antiepileptic pathway enrichment analysis: The 6 intersection targets collected by S3 epilepsy disease were imported into the KEGG database through the venny2.1.0 online platform, the website is https: / / www.kegg.jp / kegg / pathway.html, and the KEGG MAPPER was connected. The top pathways include: neural active ligand-receptor interaction, calcium signaling pathway, PI3K-Akt signaling pathway, regulation of actin cytoskeleton, pathway of neurodegenerative multiple diseases, cholinergic synapse, sphingolipid signaling pathway, cGMP-PKG signaling pathway, reflecting multiple biological processes in vivo, and at the same time indicating that S3 may play its antiepileptic effect by improving these biological processes, such as Figure 2

[0073] S4 Drug active ingredients-key target-disease-pathway network construction: The active target of S4 and the target and pathway of epilepsy disease were imported into Cytoscape3.7.1 software to construct a network interaction diagram, as shown in Figure 3 The picture can directly show the interaction between the active ingredients of S4 and the targets of epilepsy. The yellow inverted triangle in the left center position represents epilepsy, the two circles in the center position are epilepsy gene targets (green circles), the red circle in the center represents the active ingredients of S4 (red diamonds), the purple diamonds represent metabolites, and the blue circles on the right represent related pathways (blue triangles). Each edge represents the interaction between target-disease, drug-active compound, and active compound-target;

[0074] S5 S3 antiepileptic target enrichment analysis to explore the mechanism of action:

[0075] GO enrichment analysis: The intersection targets of S3 and epilepsy obtained by Venny2.1.0 were imported into DAVID6.8 database (https: / / david.ncifcrf.gov), and the BP entries were obtained by statistical analysis according to P<0.01, 29, CC entries 17, and MF entries 7, as shown in Figure 4 ​As shown in the results of GO analysis, the top 5 P-value selected data showed that the biological processes in which grass wu was involved mainly included: G protein-coupled receptor signaling pathway, denylate cyclase inhibits G protein-coupled acetylcholine receptor signaling pathway, G protein-coupled receptor signaling pathway, opioid receptor signaling pathway, and phospholipase c activates G protein-coupled acetylcholine receptor signaling pathway. The cell components mainly included: presynaptic membrane components, axon terminals, plasma membrane components, presynaptic membranes, and postsynaptic density membrane components. The molecular functions mainly were g protein-coupled acetylcholine receptors, opioid receptor activity protein-coupled serotonin receptors, neuropeptide binding, and g protein-coupled receptors, such as Figure 5 As shown in the results of GO analysis, the top 5 P-value selected data showed that the biological processes in which grass wu was involved mainly included: G protein-coupled receptor signaling pathway, denylate cyclase inhibits G protein-coupled acetylcholine receptor signaling pathway, G protein-coupled receptor signaling pathway, opioid receptor signaling pathway, and phospholipase c activates G protein-coupled acetylcholine receptor signaling pathway. The cell components mainly included: presynaptic membrane components, axon terminals, plasma membrane components, presynaptic membranes, and postsynaptic density membrane components. The molecular functions mainly were g protein-coupled acetylcholine receptors, opioid receptor activity protein-coupled serotonin receptors, neuropeptide binding, and g protein-coupled receptors, such as

[0076] Construction of PPI network: The 6 intersection targets collected from grass wu-epilepsy were imported into the STRING database through the venny2.1.0 online platform, https: / / string-db.org / cgi / input.pl, with the species set as Homo sapiens, to construct a protein-protein interaction relationship diagram, as shown in Figure 6 The results showed that the protein kinase OPRM1 had a high degree of participation, in addition, CHRM2, CHRM1, ADRA2A, OPRD1, TNF, and the like also participated in mutual regulation.

[0077] S6 Docking of active ingredients of A. bracteatum and anti-epilepsy target molecules: Select the active ingredients of A. bracteatum BDMA, N-Methylhomoveratrylamine, Izoteolin, palmitic acid and the key target CHRM1, ADRA2A, CHRM2, OPRD1, OPRM1, TNF of epilepsy treatment for molecular docking. The XP docking results are based on the reference value of XP G score. When the value is less than -6, it means that the protein and ligand have stable binding performance. The MM-GBSA analysis results are based on the reference value of MM-GBSAdG Bind. When it is lower than -30 kcal / mol, it means that the binding free energy is low, and the protein and ligand are stable. Comprehensive analysis of XP docking and MM-GBSA results, the docking score of N-Methylhomoveratrylamine and TNF is -3.651, and the MM-GBSA result is -31.22 kcal / mol. The binding free energy is low, the docking score is low, and it indicates that the compound is combined with TNF more stably. The docking of Izoteolin and CHRM2 performs best, with a docking score of -4.802 and a MM-GBSA result of -36.40 kcal / mol. The binding free energy is low, the docking score is low, and it indicates that Izoteolin is combined with CHRM2 more stably. The docking score of Izoteolin and OPRM1 is -5.955, and the MM-GBSA result is -38.82 kcal / mol. The binding free energy is low, the docking score is low, and it indicates that the compound is combined with OPRM1 more stably. The docking score of Izoteolin and ADRA2A is -8.602, and the MM-GBSA result is -49.65 kcal / mol. The binding free energy and docking score are low, and it indicates that the compound is combined with ADRA2A stably. The docking of BDMA and CHRM1 performs best, with a docking score of -10.786 and a MM-GBSA result of -33.73 kcal / mol. The binding free energy is low, the docking score is very low, and it indicates that BDMA is combined with CHRM1 very stably. The docking score of Izoteolin and OPRD1 is -6.060, and the MM-GBSA result is -45.36 kcal / mol. The docking score and binding free energy are low, and it indicates that Izoteolin is combined with OPRD1 stably, as shown in Figure 7 .

[0078] Through the mutual verification of the above disease network framework and molecular docking, it is shown that A. bracteatum can resist epilepsy, and A. bracteatum is combined with CHRM1, ADRA2A, CHRM2, OPRD1, OPRM1, TNF key related disease targets to regulate the G-protein coupled receptor molecular signal pathway of the nerve active ligand-receptor pathway to play an anti-epilepsy role.

[0079] The above disclosure is merely the preferred embodiments of the present application, and of course cannot be used to limit the scope of the present application, and those skilled in the art can understand that all or part of the processes of the above embodiments can be implemented, and equivalent changes made according to the claims of the present application, still belong to the scope covered by the present application.

Claims

1. A method for analyzing the anti-epileptic mechanism of Aconitum carmichaelii, characterized in that: Comprise the following steps: S1 screening of active ingredients of grass mallow target: screening by traditional Chinese medicine system pharmacology database and analysis platform, predicting the action target of active ingredients of grass mallow; S2 screening of key targets of grass mallow against epilepsy: screening by disease target database Disgenet, obtaining epilepsy disease gene targets, then intersecting the action targets of active ingredients of grass mallow obtained by S1 with the epilepsy disease gene targets to obtain grass mallow-epilepsy intersection targets, i.e. the action targets of grass mallow against epilepsy; In the S2 screening of key targets of grass mallow against epilepsy, the action targets of grass mallow against epilepsy are six, i.e. muscarine acetylcholine receptor M1, alpha-2A adrenergic receptor, muscarine acetylcholine receptor M2, delta opioid receptor, Mu opioid receptor, and tumor necrosis factor, and the corresponding active ingredients of grass mallow are N-dimethyl-1-phenylmethylamine, 2-(3,4-dimethoxyphenyl)-N-methylethylamine, isazotelin, and palmitic acid; S3 enrichment analysis of epilepsy pathways of grass mallow: screening the action targets of grass mallow against epilepsy obtained by S2 by KEGG database to obtain the number of KEGG pathways, reflecting multiple biological processes in vivo, indicating that grass mallow may exert its anti-epilepsy effect by improving these biological processes; S4 construction of drug active ingredient-key target-disease-pathway network: constructing the network diagram of "drug active ingredient-key target-disease-pathway" by Cytoscape with the action targets of active ingredients of grass mallow obtained by S1, the epilepsy disease gene targets obtained by S2, and the pathways obtained by S3, clearly showing the mutual relationship between drug components and their metabolites, diseases and their genes, and pathways, facilitating the exploration of the action mechanism of grass mallow against epilepsy; S5 enrichment analysis of action targets of grass mallow against epilepsy to explore the action mechanism: performing GO analysis of cell components, biological processes, and molecular functions of the action targets of grass mallow against epilepsy obtained by S2, constructing protein interaction network, and obtaining the in-depth action mechanism of grass mallow against epilepsy; S6 molecular docking of active ingredients of grass mallow and anti-epilepsy targets: collecting the crystal structure of protein corresponding to the protein receptor in the action targets of grass mallow against epilepsy obtained by S2 through RCSB PDB database, then preprocessing the protein to obtain protein active sites, preprocessing the action targets of active ingredients of grass mallow in the action targets of grass mallow against epilepsy obtained by S2 to obtain pretreated ligands, and performing molecular docking of the protein active sites and the pretreated ligands to verify the action mechanism of grass mallow against epilepsy.

2. The method according to claim 1, wherein the mechanism of the antiepileptic effect of the aconite is analyzed. In the S1 screening of active ingredients of grass mallow target, the main active ingredient targets of grass mallow are searched by TCMSP of traditional Chinese medicine system pharmacology database and analysis platform, and the active ingredient target proteins are uniformly converted into gene names by Uniprot database.

3. The method according to claim 1, wherein the antiepileptic mechanism of the aconite is analyzed. In the S3 enrichment analysis of epilepsy pathways of grass mallow, the KEGG pathways include: neuroactive ligand-receptor interaction, calcium signaling pathway, PI3K-Akt signaling pathway, regulation of actin cytoskeleton, pathway of neurodegenerative diseases, cholinergic synapse, sphingolipid signaling pathway, and cGMP-PKG signaling pathway.

4. The method according to claim 1, wherein the antiepileptic mechanism of the aconite is analyzed. The S5 grass Wu antiepileptic effect target enrichment analysis explores the mechanism of action, which includes the following analysis: GO enrichment analysis: through the DAVID database, the S2 obtained grass Wu antiepileptic effect target is screened, and the screening result is analyzed by GO, to obtain the biological process signal path, cell component and molecular function participated by grass Wu; Construction of PPI network: the S2 obtained grass Wu antiepileptic effect target is introduced into STRING database by venny to construct protein-protein interaction relationship diagram, and the protein gene participating in mutual regulation is obtained.

5. The method according to claim 4, wherein the antiepileptic mechanism of the aconite is analyzed by the method. In the construction of PPI network, the protein gene participating in mutual regulation is: muscarine acetylcholine receptor M1, alpha-2A adrenergic receptor, muscarine acetylcholine receptor M2, delta type opioid receptor, Mu type opioid receptor, tumor necrosis factor.

6. The method according to claim 5, wherein the antiepileptic mechanism of the aconite is analyzed by the method. The Mu type opioid receptor has the best participation degree.

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