Tibetan medicine flood connection quality evaluation method based on network pharmacology and molecular docking technology and application of Tibetan medicine flood connection quality evaluation method

Abstract

The invention provides a Tibetan medicine flood connection quality evaluation method based on network pharmacology and a molecular docking technology and application, and belongs to the technical field of biology. The quality evaluation method of the two core primitives is established according to the component difference characteristics of the Lanlian core primitives (Lagotis brevituba and Lagotis uniflora). Compared with the existing standard, the quality evaluation method has the advantages that the iridoid glycoside active ingredients are introduced into a quantitative control system, and differentiated detection indexes are designed for different primitives, so that the problem of insufficient accuracy caused by one-step cutting of the traditional quality control method is solved. Through verification, the method is high in precision, good in repeatability (RSD is smaller than 2.0%) and accurate in quantification (the recovery rate ranges from 95% to 105%), the quality of the lagotis medicinal materials can be accurately evaluated and effectively controlled, and a key technical support is provided for authenticity identification and grading of the lagotis medicinal materials and quality standardization of related preparations.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, and particularly relates to a Tibetan medicine Honglian quality evaluation method based on network pharmacology and molecular docking technology and application. BACKGROUND

[0002] Honglian is a clinically common Tibetan medicine with high medicinal value and wide application scenarios. It has clear medicinal effects, can clear heat and detoxify, and can also regulate blood and menstruation. It is commonly used in the treatment of systemic fever, nephritis, pulmonary disease, hypertension, atherosclerosis, irregular menstruation, and poisoning of various toxic substances and "heat" syndrome, and is particularly good at treating heat-induced liver disease. It is an important medicinal resource for treating acute and chronic hepatitis, and provides important support for local medical health and pharmaceutical industry development.

[0003] At present, the original of Honglian is different in the current national drug standards. The 2020 edition of Chinese Pharmacopoeia takes "Honglian" as the medicinal material name, and collects the dry whole plant from Lagotis brevituba Maxim. (also known as short tube rabbit ear grass); while the Ministry of Health of the People's Republic of China Pharmaceutical Standards (Ministry of Tibetan Medicine) takes "rabbit ear grass" as the medicinal material name, and collects the dry whole plant from Lagotis brevituba Maxim. or Lagotis integra W.W.Smith. Through previous research, it was found that the Honglian medicinal materials actually used in the market channel, Tibetan hospitals and related pharmaceutical enterprises are derived from various plants of the genus Lagotis, among which Lagotis brevituba and Lagotis integra are the two most commonly used original plants of Honglian medicinal materials. However, studies have shown that the compositions of Honglian medicinal materials from these two original plants are significantly different, which has brought great challenges to the quality control of Honglian medicinal materials. Moreover, the current standards cannot effectively evaluate the content and quality of medicinal materials, which seriously affects the reliability of Honglian medicinal materials in clinical application and the stability in pharmaceutical product development, and restricts the healthy development of Honglian related pharmaceutical industry. Therefore, it is urgent to establish a more scientific and reasonable quality control method.

[0004] In addition, with the continuous development of modern medical research technology, network pharmacology and molecular docking technology provide new ideas and means for the quality control and mechanism research of traditional Chinese medicine. In 2007, the British pharmacologist Hopkins proposed the concept of "network pharmacology", and since then, the technology has been widely used in traditional Chinese medicine, biology and other fields. In 2021, the World Federation of Chinese Medicine Societies issued the "Network Pharmacology Evaluation Guidelines", which further standardized the research process and credibility. Network pharmacology emphasizes the analysis of the molecular mechanism of drug treatment from the perspective of the whole biological network, and has been widely used in the field of traditional Chinese medicine, such as active compound discovery, overall disease mechanism interpretation, and complex rule analysis of traditional Chinese medicine formula. Molecular docking is a method based on computer simulation structure, which predicts the interaction between ligand and receptor at the molecular level through docking means, evaluates the stability of the docking system or assesses the structure-activity relationship, and can preliminarily verify the results of network pharmacology. Therefore, it is crucial to establish a more scientific and reasonable quality control method for Honglian based on network pharmacology. SUMMARY

[0005] The purpose of the present application is to provide a Tibetan medicine Honglian quality evaluation method and application based on network pharmacology and molecular docking technology, which successfully selects MMP9 and LGALS3 as the key action targets of Honglian in treating hepatitis through active ingredient-disease target correlation analysis, and further evaluates the quality of Tibetan medicine Honglian.

[0006] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions:

[0007] The present application provides a Tibetan medicine Honglian quality evaluation method based on network pharmacology and molecular docking technology, comprising the following steps:

[0008] (1) Disease target prediction of effective compounds

[0009] According to the disease target prediction of effective compounds in Tibetan medicine Honglian, the disease target of the effective compound is obtained;

[0010] (2) Compound-disease intersection target

[0011] Taking disease as the research object, the disease-related target is screened, and the disease-related target of the effective compound in step (1) is mapped to each other to obtain the potential therapeutic target of Honglian in treating disease;

[0012] (3) Protein interaction network construction

[0013] The potential therapeutic target of step (2) is introduced into the database for protein interaction network construction, and the key target is screened out;

[0014] (4) GO enrichment analysis and gene pathway annotation analysis of the potential therapeutic target of step (2) are performed;

[0015] (5) Molecular docking verification of key compounds and target points

[0016] The active compounds for evaluating the quality of Honglian are screened by carrying out molecular docking verification analysis on the iridoid glycosides and phenylpropanoid compounds in the Tibetan medicine Honglian and the screened key target points, and the quality of Honglian is evaluated.

[0017] Preferably, the Tibetan medicine Honglian is selected from the group consisting of Lagotis glaucescens and Lagotis brevituba.

[0018] Preferably, in step (2), the disease includes liver cancer, and the potential therapeutic target point includes one or more of ACE, IL2, SELP, MMP9, MMP13, LGALS3, MGMT, APP, LGALS1, LGALS9 and ADA.

[0019] Preferably, in step (3), the key target point includes MMP9 and / or LGALS3.

[0020] Preferably, in step (5), the iridoid glycosides include catalpol and aucubin; and the active compounds for evaluating the quality of Honglian include one or more of catalpol, aucubin, echinacoside, plantainoside, impatiensin B and verbascoside.

[0021] Preferably, in step (5), the condition for evaluating the quality of the Tibetan medicine Honglian includes:

[0022] When the Tibetan medicine Honglian is Lagotis brevituba: the total amount of catalpol and aucubin is greater than or equal to 1.00% by mass, and the total amount of echinacoside and verbascoside is greater than or equal to 1.50% by mass.

[0023] When the Tibetan medicine Honglian is Lagotis glaucescens: the total amount of catalpol and aucubin is greater than or equal to 2.50% by mass; and the total amount of plantainoside and impatiensin B is greater than or equal to 4.50% by mass.

[0024] The application further provides an application of the above-mentioned Tibetan medicine Honglian quality evaluation method based on network pharmacology and molecular docking technology in quality evaluation of the Tibetan medicine Honglian.

[0025] Compared with the prior art, the application has the following beneficial effects:

[0026] (1) The application successfully screens MMP9 and LGALS3 as two key role targets of Honglian in treating hepatitis through active ingredient-disease target correlation analysis based on network pharmacology and molecular docking technology. Meanwhile, GO function annotation and KEGG pathway enrichment analysis results show that the above key targets can play a therapeutic role through regulating cell signal transduction, zinc ion-mediated biological effects, interleukin-17 inflammatory pathways, and vascular relaxation regulation, etc. multiple core biological processes and pathways, clearly explaining the correspondence between the traditional efficacy of Honglian and its modern pharmacological effects from the molecular network level, and providing new experimental evidence and theoretical support for the scientific treatment of complex diseases by Tibetan medicine.

[0027] (2) The application simulates and verifies the interaction between the key targets and the active ingredients by molecular docking technology, and the results show that the six active ingredients (catalpol, onjisaponin, echinacoside, plantain glycoside, globularia glycoside B, and clarkia glycoside B) with high content in Honglian can spontaneously and efficiently bind to MMP9 and LGALS3 targets, and the binding conformation is stable and the affinity is excellent, which reveals the activity of the related active ingredients in Honglian in treating hepatitis from the molecular level, and suggests that the action may be through the synergistic effect of multiple components and multiple targets, and also provides a clear targeting basis for the screening of quality markers of Honglian.

[0028] (3) The application establishes a quality evaluation method for two kinds of raw materials of Honglian (short-tube rabbit ear grass and whole-rabbit ear grass) according to their different component characteristics. For whole-rabbit ear grass, four active ingredients, including iridoid glycosides (catalpol and onjisaponin) and phenylpropanoid glycosides (plantain glycoside and globularia glycoside B), are used as detection indexes for quality evaluation of whole-rabbit ear grass. For short-tube rabbit ear grass, four active ingredients, including iridoid glycosides (catalpol and onjisaponin) and phenylpropanoid glycosides (clarkia glycoside B and echinacoside), are used as detection indexes for quality evaluation of short-tube rabbit ear grass. Compared with the existing standard, the quality evaluation method includes iridoid glycoside active ingredients in the quantitative control system, and designs different detection indexes for different raw materials, solving the problem of insufficient accuracy caused by the "one-size-fits-all" of traditional quality control methods. The method has been verified to have high precision, good repeatability (RSD < 2.0%), and accurate quantification (recovery rate 95%-105%), and can realize accurate evaluation and effective control of the quality of Honglian medicinal materials, providing key technical support for the authenticity identification, grade division of Honglian medicinal materials, and quality standardization of related preparations. BRIEF DESCRIPTION OF DRAWINGS

[0029] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings in the following description only constitute some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained from these drawings without creative labor.

[0030] Figure 1 A compound-target relationship diagram in the embodiments of the present application;

[0031] Figure 2 A disease-drug Venn diagram in the embodiments of the present application;

[0032] Figure 3 A PPI network diagram in the embodiments of the present application;

[0033] Figure 4 A GO function analysis result diagram in the embodiments of the present application;

[0034] Figure 5 A KEGG pathway enrichment analysis result in the embodiments of the present application;

[0035] Figure 6 A MMP9 visualization diagram in the embodiments of the present application, wherein A is aucubin; B is catalpol; C is scullcapin; D is plantain; E is gypsophila oldhami glycoside B; and F is verbascoside;

[0036] Figure 7 A LGALS3 visualization diagram in the embodiments of the present application, wherein A is aucubin; B is catalpol; C is scullcapin; D is plantain; E is gypsophila oldhami glycoside B; and F is verbascoside;

[0037] Figure 8 An HPLC content determination chromatogram of short-tube rabbit ear grass and full-margined rabbit ear grass in the embodiments of the present application, wherein 1 is catalpol; 2 is aucubin; S1 is a mixed solution of reference substances; S2 is short-tube rabbit ear grass; and S3 is full-margined rabbit ear grass;

[0038] Figure 9 An HPLC content determination chromatogram of short-tube rabbit ear grass in the embodiments of the present application, A is a mixed solution of reference substances; B is a test sample solution; 1 is scullcapin; and 2 is verbascoside;

[0039] Figure 10 An HPLC content determination chromatogram of full-margined rabbit ear grass in the embodiments of the present application, A is a mixed solution of reference substances; B is a test sample solution; 1 is plantain; and 2 is gypsophila oldhami glycoside B. DETAILED DESCRIPTION

[0040] The following detailed description of various exemplary embodiments of the application will not be considered limiting of the application, but rather a description of certain aspects, features and embodiments of the application.

[0041] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. Additionally, the use of "including," "comprising," "having," "containing," and variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. Unless otherwise indicated, the use of the terms "a" and "an" are meant to encompass the plural as well as the singular. The use of the term "comprising" is meant to encompass the terms "including," "containing," "having," and the like. The use of the term "about" is meant to encompass the term "comprising" and variations thereof. The use of the term "comprising" is meant to encompass the terms "consisting essentially of" and "consisting of." The use of the term "consisting essentially of" is meant to encompass the term "consisting of."

[0042] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the application pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. In case of conflict, the content of the present specification controls.

[0043] Various modifications and changes can be made to the specific embodiments described herein without departing from the scope or spirit of the application. Other embodiments will be apparent to those of ordinary skill in the art from the description and examples provided herein. The description and examples are illustrative only and are not intended to limit the scope of the application.

[0044] As used herein, the terms "comprise", "comprising", "include", "including", "have", "having" or variants thereof are open-ended, and include one or more steps, integers, compositions or elements of any integer number of steps, integers, compositions or elements.

[0045] The following materials were used in the examples below:

[0046] Japan Shimadzu LC-20AD high performance liquid chromatograph, PDA detector; Agilent 1260 high performance liquid chromatograph, DAD detector; Waters X-brige C 18 (4.6 x 250 mm, 5 μm); Waters Atlantis T3 C 18 (4.6 x 250 mm, 5 μm); Sartorious BP211D electronic balance; Milli-Q ultrapure water machine.

[0047] Hemiphroside B (99.6% by content) was a control sample isolated and purified by Chengdu Pus Biotechnology Co., Ltd.; Plantain (batch number: 111914-201604, 90.2% by content), Verbascoside (batch number: 111530-201512, 96.7% by content), Arbutin (batch number: 111670-201907, 91.8% by content), Aucubin (batch number: 111761-200601, dried for 24 h using P2O5 before use, 100.0% by content), and Catalpol (batch number: 111808-202112, 98.8% by content) were purchased from China Institute for Drug Control. Methanol and acetonitrile were chromatographically pure (Fisher Company); water was Milli-Q purified water.

[0048] 16 batches of whole-leaf rabbit ear grass and 13 batches of short-cylinder rabbit ear grass were identified by Professor Jiang Guihua of Chengdu University of Traditional Chinese Medicine as medicinal materials with accurate origins. The specific sample information is shown in Table 1.

[0049] Table 1 Sample information

[0050]

[0051]

[0052] Example

[0053] The embodiment of the application provides a quality detection method for Tibetan medicine Honglian, and the specific steps are as follows:

[0054] Exploring the mechanism of Tibetan medicine Honglian in treating hepatitis based on network pharmacology and molecular docking

[0055] (1) Effective compound disease target prediction

[0056] The SMILES (Simplified Molecular Input Line Entry System) formula of 8-Epiloganic acid, 10-DC, 10-PC, Arborescosidic acid, Aucubin, Catalpol, Echinacoside, Gardoside, Geniposidic acid, Hemiphroside B, Mussaenosidic acid, Plantamajoside and Verbascoside was exported from the PubChem database. The active target Uniport was obtained by checking the target in the SwissTarget Prediction database with homo gene as the selection standard, and the gene name is shown in Table 2.

[0057] The Cytoscape3.9.1 software was used to construct the compound and potential target view, and the Network analysis analysis was performed, and the results are shown in Figure 1 .

[0058] Table 2: Predicted targets of 13 compounds

[0059]

[0060]

[0061] (2) Compound and disease intersection target

[0062] Taking Hepatitis as the disease research object, the disease targets were searched in the Disgenet database with Homo gene as the condition, and 656 disease targets were obtained. The obtained disease targets were mapped with the compound targets obtained in step (1) (as shown in Figure 2 ), and 11 potential action targets of Hepatitis treated by Honglian were obtained, including ACE, IL2, SELP, MMP9, MMP13, LGALS3, MGMT, APP, LGALS1, LGALS9 and ADA.

[0063] (3) Protein-protein interaction (PPI) network construction

[0064] The 11 common targets in step (2) were introduced into the STRING database, Homo gene was selected, and the protein-protein interaction network was constructed. The single target was removed, and the STRING data was introduced into

[0065] Cytoscape3.9.1 for network construction, and the results are shown in Figure 3 .

[0066] Figure 3 It is shown that after the Hepatitis-Compound common target is analyzed by the protein-protein interaction network construction, the key targets are mainly MMP9 and LGALS3, and the Degree values of the 11 targets are MMP9 (6), LGALS3 (5), ACE (4), SELP (4), IL2 (4), APP (2), ADA (2), LGALS9 (2), MGMT (1), LGALS1 (1) and MMP13 (1).

[0067] (4) GO enrichment analysis and gene pathway annotation analysis

[0068] The 11 target genes were input into DAVID, OFFICE GENE SYMBOL was selected, Homo Sapiens was selected as the background, and GO (Genen Ontology) analysis was performed (results are shown in Table 3 and Figure 4 Table 4 and Figure 5 KEGG (Kyoto Encyclopedia Genes and Genomes) target gene pathway annotation analysis (results are shown in Table 4 and

[0069] Table 3 GO function analysis results process

[0070]

[0071]

[0072] Table 3 and Figure 4 show that the most important process involved in molecular function (Molecular Function, MF) is zinc ion binding (related to zinc ion binding), the most important process involved in biological process (Biological Process, BP) is positive regulation of apoptotic process (any process that activates or increases the frequency, rate or degree of cell death through the process of apoptosis), and the most important process involved in cellular component (Cellular Component, CC) is extracellular exosome.

[0073] Table 4 KEGG pathway enrichment analysis results

[0074]

[0075] Table 4 and Figure 5 KEGG gene pathway annotation analysis results show that a total of 4 gene pathways have significant meaning, Coronavirus disease - COVID-19 (new coronavirus), IL-17 signaling pathway (interleukin 17 signaling pathway, IL-17 family is a subset of cytokines composed of IL-17A-F, which plays a crucial role in both acute and chronic inflammatory responses), Chagas disease (Chagas disease), and Relaxin signaling pathway (relaxin signaling pathway, which plays a series of pleiotropic effects, including vasodilation, anti-fibrosis and angiogenesis).

[0076] (5) Molecular docking verification of key compounds and target points

[0077] Molecular docking verification analysis was performed on six compounds, including representative components and stable components in different major components, such as iridoid glycosides (catalpol, amentoflavone), and phenylpropanoids (scopoletin, plantain glycoside, spadix glycoside B, and verbascoside), and the screened key target points.

[0078] Molecular docking was performed on the key target points MMP9 and LGALS3 with large degree values in PPI analysis and the key compounds catalpol, amentoflavone, scopoletin, plantain glycoside, spadix glycoside B, and verbascoside. The three-dimensional structure of the active ingredients was obtained from the database PubChem and saved as an SDF file. The active ingredients were converted to an MOL2 file using ChemBio 3D 14.0.0.117, and then processed by AutoDockTools 1.5.6 and converted to a PDBQT file.

[0079] The crystal structure of the screened key target protein was obtained from the RCSB PDB database and saved as a PDB file. The water molecules and original ligands in the crystal of the key target protein were removed using PyMOL 2.5.4 software, and the processed key target protein was saved as a PDB file. Hydrogen and charge were added to the processed active ingredients and key target protein using AutoDockTools 1.5.6 software, and docking simulation was performed. The docking results were visualized using PyMOL 2.5.4 software. The binding energy results and visualized graphs of the core compounds and core target points are shown in Table 5 and Figure 6 、 7 .

[0080] The results of molecular interaction analysis are often expressed in terms of binding energy. If the binding energy is <0 kJ / mol, it indicates that the ligand and the receptor can spontaneously bind. If the binding energy is ≤-5 kJ / mol, it indicates that the compound and the target point can bind well. The smaller the binding energy, the greater the docking, and the more stable the binding between the compound and the target point, indicating that the possibility of molecular binding interaction is greater. Table 5 and Figure 6 、 7 show that the binding ability of these six components to the target point LGALS3 is relatively strong, and the binding stability is good. Except for scopoletin and MMP9, which have a binding energy greater than-5 kJ / mol and can spontaneously bind, the binding interaction is slightly weak. The binding ability of the other components to the target point MMP9 is relatively strong, and the binding stability is good.

[0081] Table 5 Binding energy (kJ / mol) of core compounds and core target points

[0082]

[0083] (6) Study on HPLC quality control method of Honglian (short-tube rabbit ear grass, whole-rim rabbit ear grass)

[0084] According to the network pharmacology analysis of 13 active compounds in Honglian and hepatitis disease-related targets, combined with the molecular verification results of key targets MMP9 and LGALS3 related compounds in PPI network with larger Degree value, the chemical composition and content of short-tube rabbit ear grass and whole-rim rabbit ear grass were investigated, and the content determination method and limit of short-tube rabbit ear grass were developed with catalpol, aucubin, verbascoside and echinacoside; the content determination method and limit of whole-rim rabbit ear grass were developed with catalpol, aucubin, sonchus and adenoside. The content determination conditions of iridoid glycosides in short-tube rabbit ear grass and whole-rim rabbit ear grass were consistent, so the method was studied together; the content determination conditions of phenylpropanoid components echinacoside and verbascoside in short-tube rabbit ear grass and sonchus and adenoside B in whole-rim rabbit ear grass were inconsistent, so the method was studied separately.

[0085] 1) HPLC quality control method of iridoid glycosides

[0086] A. Preparation of reference solution

[0087] Take about 10 mg of catalpol and aucubin reference substances, accurately weigh, respectively, in 10 mL volumetric flask, add mobile phase to dissolve and constant volume to the mark, to obtain each reference substance stock solution. Take the appropriate amount of each reference substance stock solution into the same 10 mL volumetric flask, add mobile phase to prepare the above-mentioned mixed solution with the concentration of 80 μg·mL -1 of each component.

[0088] B. Chromatographic conditions and system suitability

[0089] Chromatographic column: Waters Atlantis T3 C 18 (4.6×250mm, 5μm); mobile phase: acetonitrile: 0.1% phosphoric acid solution (1:99); flow rate: 1.0 mL·min -1 ; detection wavelength: 210 nm; column temperature: 30℃; injection volume: 10 μL.

[0090] C. Preparation of test solution

[0091] Take about 0.1 g of sample powder (pass through No. 4 sieve) of S2 and S3, respectively, accurately weigh, place in a conical flask with a plug, accurately add 50 mL of methanol, weigh, ultrasonic treat (power 250 W, frequency 33 kHz) for 30 min, cool, re-weigh, make up the weight loss with methanol, shake well, filter, accurately take 10 mL of the filtrate, evaporate to dryness, dissolve with mobile phase and transfer to a 5 mL volumetric flask, shake well, filter, take the filtrate, and you get it.

[0092] D. System suitability test

[0093] Detect according to the chromatographic conditions of step 2), and calculate the theoretical plate number of catalpol peak to be greater than 12000 and the separation degree to be greater than 1.5. The chromatograms of the reference substance and the sample are shown in Figure 8 .

[0094] 2) HPLC content determination of phenylpropanoid glycosides (base: S2)

[0095] A. Preparation of reference substance solution

[0096] Accurately weigh about 10 mg of echinacoside and verbascoside reference substances, respectively, place in a 10 mL volumetric flask, dissolve with 50% methanol and dilute to the mark, and you get the reference substance stock solution. Take an appropriate amount of each reference substance stock solution into the same 10 mL volumetric flask, and prepare a mixed solution with the concentration of the above two components being 25 μg·mL -1 .

[0097] B. Preparation of test solution

[0098] Accurately weigh 0.1 g of S2 powder (pass through No. 4 sieve), place in a conical flask with a plug, accurately add 50 mL of 50% methanol, weigh, ultrasonic treat (power 250 W, frequency 33 kHz) for 30 min, cool, re-weigh, make up the weight loss with 50% methanol, shake well, filter, and you get the filtrate.

[0099] C. System suitability test

[0100] Chromatographic column: Hypersil GOLD C 18 (4.6 mm x 250 mm, 5 μm); mobile phase: acetonitrile-water solution (16:84); flow rate: 1.0 mL·min -1 ; detection wavelength: 330 nm; column temperature: 30 °C; injection volume: 10 μL.

[0101] Under the above conditions, the results are shown in Figure 9 . Calculate the theoretical plate number of echinacoside peak to be greater than 10000 and the separation degree to be greater than 1.5.

[0102] 3) Determination of phenylpropanoid glycosides (base: Rabdosia japonica)

[0103] A. Preparation of reference solution

[0104] Each 10 mg of plantain glycoside and globoside B was accurately weighed and placed in a 10 mL volumetric flask, dissolved with 70% methanol and diluted to the mark to obtain the reference stock solution. An appropriate amount of each reference stock solution was taken into a 10 mL volumetric flask, and 70% methanol was added to prepare a mixed solution with the above two components at concentrations of 100 μg·mL -1 and 90 μg·mL -1 , respectively.

[0105] B. Preparation of test solution

[0106] About 0.15 g of QY-1 to QY-16 Rabdosia japonica powder (passed through a No. 4 sieve) was accurately weighed and placed in a stoppered conical flask. 50 mL of 70% methanol was accurately added, the weight was determined, and ultrasonic treatment (power 250 W, frequency 33 kHz) was performed for 30 min. The flask was cooled and weighed again. The lost weight was made up with 70% methanol, shaken well, filtered, and the filtrate was collected.

[0107] C. Column: SPOLAR C 18 (4.6 x 250 mm, 5 μm); mobile phase: acetonitrile-water solution (15:85); flow rate: 1.0 mL·min -1 ; detection wavelength: 325 nm; column temperature: 30 °C; injection volume: 10 μL.

[0108] The detection was performed according to the above chromatographic conditions, and the chromatograms of the reference and sample are shown in Figure 10 . The theoretical plate number was greater than 10,000 and the separation degree was greater than 1.5, calculated based on the peak of plantain glycoside.

[0109] (7) Methodology investigation

[0110] 1) The above mixed reference stock solution was diluted to different concentrations to obtain a reference mixture solution containing all reference substances. The sample was injected and analyzed according to the above chromatographic conditions, and the peak area was recorded. The standard curve was drawn with the peak area (Y) as the vertical coordinate and the mass concentration (μg·mL -1 ) as the horizontal coordinate (X). The linear relationship of the six components was good (r≥0.9995). The regression equation, linear range and correlation coefficient of the standard curve of each component are shown in Table 6.

[0111] Table 6 Standard curve equation, linear range and correlation coefficient of each measured component

[0112]

[0113]

[0114] 2) Precision test

[0115] 10 μL of the above-mentioned reference mixture solution was precisely sucked and continuously injected for 6 times, and the peak area was recorded. The RSD of the peak area of each reference was less than 1.0%, indicating that the precision of the instrument was good.

[0116] 3) Reproducibility test

[0117] 12 portions of DG-1 and QY-1 sample powders were respectively taken, and the sample solutions were prepared according to the above-mentioned method, and were determined according to the corresponding chromatographic method, the peak area was recorded, and the content was calculated. The RSD of the peak area of each reference was less than 1.0%, and the result showed that the reproducibility was good.

[0118] 4) Stability test

[0119] The sample solution was taken, and was injected for determination at 0, 2, 4, 8, 12 and 24 h. The RSD of the peak area of each reference was less than 0.5%, and the result showed that the sample solution was stable within 24 h.

[0120] 5) Determination of detection limit and quantitative limit

[0121] The above-mentioned mixed reference solution was diluted with the corresponding solvent, and was injected for determination. The signal-to-noise ratio of 3:1 in the chromatogram was the detection limit, and the signal-to-noise ratio of 10:1 was the quantitative limit. The results showed that the detection limits of verbascoside and acteoside were 2.8 and 3.2 μg·mL -1 , respectively, and the quantitative limits were 9.1 and 10.5 μg·mL -1 , respectively. The detection limits of plantainoside and globoside B were 12.2 and 13.1 μg·mL -1 , respectively, and the quantitative limits were 32.1 and 33.5 μg·mL -1 , respectively. The detection limits of catalpol and aphylline were 1.6 and 1.8 μg·mL -1 , respectively, and the quantitative limits were 4.1 and 4.4 μg·mL -1 .

[0122] 6) Sample recovery test

[0123] Take the known content of DG-1 and QY-1 sample powder each 12, precision weighing, respectively, in the conical flask with plug, respectively, precision add Echinacoside, Verbascoside, Plantainoside, Globularia B, Catalpol and Aucubin control product appropriate amount. According to the corresponding method, respectively, prepare test solution, respectively, sample analysis, calculate the recovery rate, Echinacoside recovery rate in 96.4% ~ 99.0%, the average is 97.5%, RSD is 0.90%, Verbascoside recovery rate in 99.0% ~ 101.9%, the average is 100.3%, RSD is 1.20%, Plantainoside recovery rate in 97.9% ~ 100.4%, the average is 99.6%, RSD is 1.94%, Globularia B recovery rate in 99.3% ~ 102.0%, the average is 100.6%, RSD is 1.00%, Catalpol recovery rate in 100.8% ~ 103.5%, the average is 101.9%, RSD is 0.95%, Aucubin recovery rate in 99.2% ~ 101.5%, the average is 100.3%, RSD is 0.82%. The results show that the method of sample recovery test results is good.

[0124] Respectively, take the different sample powder in table 1, pass through 4 screen, take the upper book method to prepare test solution and determine, record the peak area of 8 kinds of ingredients, calculate the content, the results are shown in table 7.

[0125] Table 7 sample determination results

[0126]

[0127]

[0128] From the above, the total amount of phenylpropanoid glycosides and iridoid glycosides in total is greater than that of short tube rabbit ear grass, so it is intended to develop limit. In Chinese Pharmacopoeia, Honglian (original: short tube rabbit ear grass) catalpol and aucubin, echinacoside and verbascoside are the same kind of ingredients, which have similar pharmacological effects. Therefore, the following standards are proposed:

[0129] Short tube rabbit ear grass: the total amount of catalpol (C 15 H 22 O 10 ) and aucubin (C 15 H 22 O9) is greater than or equal to 1.00%, and the total amount of echinacoside (C 35 H 46 O 20 ) and verbascoside (C 29 H 26 O 15 ) is greater than or equal to 1.50% (total amount is its mass).

[0130] Rabdosia japonica: total amount of rabdonol (C 15 H 22 O 10 ) and aucubin (C 15 H 22 O9) is greater than or equal to 2.50%. Total amount of plantain (C 29 H 36 O 16 ) and globularia (C 31 H 38 O 17 ) is greater than or equal to 4.50% (total amount is mass).

[0131] The above merely describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A method for evaluating the quality of Tibetan medicine *Coptis chinensis* based on network pharmacology and molecular docking technology, characterized in that... Includes the following steps: (1) Disease target prediction of effective compounds Based on the screening of effective compounds in Tibetan medicine Honglian, disease targets were obtained, and the disease targets related to the effective compounds were identified. (2) Intersecting targets of compounds and diseases Using diseases as the research object, disease-related targets are screened and mapped with the effective compound-related disease targets in step (1) to obtain potential therapeutic targets for treating diseases with Coptis chinensis. (3) Construction of protein interaction network The potential therapeutic targets in step (2) are imported into the database to construct a protein interaction network and screen out key targets. (4) Perform GO enrichment analysis and gene pathway annotation analysis on the potential therapeutic targets in step (2); (5) Molecular docking verification of key compounds and targets Molecular docking verification analysis was performed on iridoid glycosides and phenylpropanoids in Tibetan medicine Honglian and screened key targets to screen active compounds that can evaluate the quality of Honglian, and the quality of Honglian was evaluated.

2. The method for evaluating the quality of Tibetan medicine *Coptis chinensis* based on network pharmacology and molecular docking technology according to claim 1, characterized in that... The Tibetan medicine Honglian is selected from whole-edged rabbit ear grass or short-tubed rabbit ear grass.

3. The method for evaluating the quality of Tibetan medicine *Coptis chinensis* based on network pharmacology and molecular docking technology according to claim 1, characterized in that... In step (2), the disease is hepatitis, and the potential therapeutic targets include one or more of ACE, IL2, SELP, MMP9, MMP13, LGALS3, MGMT, APP, LGALS1, LGALS9, and ADA.

4. The method for evaluating the quality of Tibetan medicine *Coptis chinensis* based on network pharmacology and molecular docking technology according to claim 1, characterized in that... In step (3), the key targets include MMP9 and / or LGALS3.

5. The method for evaluating the quality of Tibetan medicine *Coptis chinensis* based on network pharmacology and molecular docking technology according to claim 1, characterized in that... In step (5), the iridoid glycosides include catalpol and aucubin; the active compounds that can evaluate the quality of Coptis chinensis include one or more of catalpol, aucubin, echinacoside, plantago asiatica glycoside, hygroscin B, and verbascoside.

6. The method for evaluating the quality of Tibetan medicine *Coptis chinensis* based on network pharmacology and molecular docking technology according to claim 1, characterized in that... In step (5), the conditions for quality evaluation of Tibetan medicine Honglian include: When the Tibetan medicine Honglian is *Cynanchum paniculatum*, it contains, by mass, a total amount of catalpol and aucubin greater than or equal to 1.00%, and a total amount of echinacoside and verbascoside greater than or equal to 1.50%. When the Tibetan medicine Honglian is *Cynanchum paniculatum*, it contains, by mass, a total amount of catalpol and aucubin greater than or equal to 2.50%; and a total amount of plantago asiatica glycoside and hygroscin B greater than or equal to 4.50%.

7. The application of the Tibetan medicine Honglian quality evaluation method based on network pharmacology and molecular docking technology as described in any one of claims 1 to 6 in the quality evaluation of Tibetan medicine Honglian.

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