Equivalent component group of Angong Niuhuang Pills for improving ischemic stroke injury and its use

Abstract

The present invention discloses an equivalent component group of Angong Niuhuang Wan for improving ischemic stroke damage and its use. The present invention claims a composition consisting of 28, 39 or 64 compounds, and the use of the composition as an equivalent component group of Angong Niuhuang Wan for improving ischemic stroke damage. The zebrafish cerebral ischemia model and the mouse middle cerebral artery occlusion (MCAO) model prove that the equivalent component group has a significant improvement effect on ischemic stroke damage, and the improvement effect is not significantly different from the original drug of Angong Niuhuang Wan, showing excellent equivalence. Therefore, the composition can be used as an equivalent component group of Angong Niuhuang Wan for improving ischemic stroke damage, and then used to prepare a drug that is equivalent to Angong Niuhuang Wan for improving ischemic stroke damage, or used to prepare a composition for controlling the quality of Angong Niuhuang Wan.

Description

Technical Field

[0001] The invention belongs to the field of medicine and relates to the discovery and application of equivalent component groups in classic Chinese medicine prescriptions, and specifically to equivalent component groups of Angong Niuhuang Pills for improving ischemic stroke damage and uses thereof. Background Art

[0002] Stroke is divided into ischemic stroke and hemorrhagic stroke. The most common type in clinical practice is ischemic stroke caused by cerebral artery occlusion or infarction. The pathogenesis of ischemic stroke is extremely complex and is related to multiple internal and external factors such as excitatory amino acid toxicity, calcium overload, reactive oxygen damage, energy metabolism disorders, neuroinflammation, and cell apoptosis. Various factors are mutually causal and jointly lead to the occurrence of the disease.

[0003] Angong Niuhuang Pills are a compound preparation of 11 Chinese herbs, including bezoar, concentrated powder of buffalo horns, musk or artificial musk, pearl, cinnabar, realgar, coptis root, scutellaria, gardenia, curcuma and borneol. It has the effects of clearing away heat and detoxifying, calming the nerves and opening the mind. It is clinically used to treat various brain diseases, high fever, inflammation, etc. It has good clinical application value in the treatment of craniocerebral injury, cerebral infarction, cerebral hemorrhage, pediatric fever, cancer fever, viral encephalitis, pneumonia, etc. Although Angong Niuhuang Pills have clear therapeutic effects, due to the complexity and diversity of its formula, its "effective ingredient group", that is, which ingredients work together to exert the efficacy, has always been a key scientific issue that is difficult to clarify.

[0004] "Equivalent ingredient group" is a research hypothesis for the effective ingredient group of traditional Chinese medicine, that is, although traditional Chinese medicine contains many ingredients, not all ingredients are effective for specific diseases / effects. There is naturally a group of chemical ingredients with clear composition, clear content ratio, and equivalent efficacy to the original traditional Chinese medicine. They interact with the key nodes / targets / pathways of the body and coordinate the efficacy as a whole. For complex traditional Chinese medicine compound preparations such as Angong Niuhuang Pills, a thinking mode of selecting efficacy based on disease symptoms and determining ingredients (groups) based on efficacy is established, so that "ingredients-effects-disease symptoms" are organically connected, providing a basis for establishing a quality control model for traditional Chinese medicine with dose-effect correlation. At the same time, based on equivalent substitution, a composite ingredient group with clear ingredients and easy-to-control quality can be used to perform equivalent substitution for the clinical treatment use of traditional Chinese medicine. This is of great significance to the modernization and internationalization of traditional Chinese medicine.

[0005] At present, among the hundreds of chemical components contained in Angong Niuhuang Pills, it is still unclear whether there is a certain combination of components and which combination of components can equivalently replace the effect of Angong Niuhuang Pills in improving ischemic stroke damage. Based on this research and discovery, the present invention is specially proposed. Summary of the invention

[0006] The purpose of the present invention is to overcome the deficiencies of the prior art and provide an equivalent component group of Angong Niuhuang Pills for improving ischemic stroke damage and its use.

[0007] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0008] A composition consisting of the following 28 compounds:

[0009] (+)-Borneol, baicalin, isoborneol, cholic acid, berberine, taurocholic acid, geniposide, glycocholic acid, deoxycholic acid, wogonin, coptisine, palmatine, musk ketone, baicalein, argentin, β-caryophyllene, wogonin, chenodeoxycholic acid, melaleucalyptol A, curcumin, rutin, ursolic acid, ursodeoxycholic acid, isoquercetin, demethoxycurcumin, bisdemethoxycurcumin, eucalyptol, and tauroursodeoxycholic acid.

[0010] A composition consisting of the following 39 compounds:

[0011] (+)-Borneol, baicalin, isoborneol, cholic acid, berberine, taurocholic acid, geniposide, glycocholic acid, deoxycholic acid, wogonin, coptisine, palmatine, taurodeoxycholic acid, melastosin, musk ketone, 3-O-feruloylquinic acid, epiberberine, geniposide, norwogonin, baicalein, argentin, jatrorrhizine, β-caryophyllene, wogonin, chenodeoxycholic acid, melastosin A, α-curcumene, curcumin, rutin, chlorogenic acid, ursolic acid, ursodeoxycholic acid, isoquercetin, neochlorogenic acid, demethoxycurcumin, lithocholic acid, bisdemethoxycurcumin, eucalyptol, and tauroursodeoxycholic acid.

[0012] A composition consisting of the following 64 compounds:

[0013] (+)-Borneol, baicalin, isoborneol, cholic acid, berberine, taurocholic acid, geniposide, glycocholic acid, deoxycholic acid, wogonin, coptisine, palmatine, taurodeoxycholic acid, melaleucaside, musk ketone, 3-O-feruloylquinic acid, epiberberine, geniposide, norbaicalin, glycodeoxycholic acid, crocin-I, magnoliavine, 5-O-feruloylquinic acid, fangfang pine, baicalein, 7-keto-3α,12α-dihydroxycholanic acid, 4-O-feruloylquinic acid, fennel, taurochenodeoxycholic acid, jatrorrhizine, β-caryophyllene ene, grandisin, chrysin-7-O-β-glucuronide, gardenia jasminoides B, glycochenodeoxycholic acid, baicalein, chenodeoxycholic acid, melaleucain A, scutellariae flavonoids II, α-curcumene, curcumin, demethyleneberberine, rutin, chlorogenic acid, synthetic camphor, ursolic acid, ursodeoxycholic acid, scutellariae flavonoids I, 8-oxidoberine, isoquercetin, neochlorogenic acid, baicalein, demethoxycurcumin, terpinolene, geniposide, lithocholic acid, bisdemethoxycurcumin, camphene, eucalyptol, α-caryophyllene, tauroursodeoxycholic acid, α-pinene, limonene and pinolene.

[0014] The above-mentioned composition is used as an equivalent component group of Angong Niuhuang Pills for improving ischemic stroke damage.

[0015] The composition is used for preparing a drug that is equivalent to and replaces Angong Niuhuang Pills to improve ischemic stroke damage.

[0016] Furthermore, the drug uses the above-mentioned composition as an active ingredient and is prepared into a pharmaceutically acceptable dosage form through a pharmaceutically acceptable carrier or excipient.

[0017] Furthermore, the carrier or auxiliary material is solid, liquid or semi-solid.

[0018] Furthermore, the dosage forms include tablets, capsules, pills, and injections.

[0019] The composition is used for preparing a composition for controlling the quality of Angong Niuhuang Pills, and the composition contains standard products of each compound in the composition.

[0020] Furthermore, the quality of Angong Niuhuang Pills is the quality based on the efficacy of Angong Niuhuang Pills in improving ischemic stroke damage.

[0021] Beneficial effects:

[0022] The present invention provides an equivalent component group of Angong Niuhuang Pills for improving ischemic stroke damage, which is composed of 28, 39 or 64 chemical components. Zebrafish cerebral ischemia model and mouse MCAO model prove that the equivalent component group has a significant improvement effect on ischemic stroke damage, and the improvement effect is not significantly different from that of the original drug of Angong Niuhuang Pills, showing excellent equivalence.

[0023] The present invention provides a certain reference for clarifying the material chemical basis of the efficacy of Angong Niuhuang Pills, and promotes the research on the material basis of the efficacy and the improvement of the quality standard of Angong Niuhuang Pills. Specifically: (1) the composition of these 28, 39 or 64 ingredients can be prepared into a pharmaceutical composition that is equivalent to Angong Niuhuang Pills and can improve ischemic stroke damage; (2) the composition of these 28, 39 or 64 ingredients can be prepared into a marker composition for controlling the quality of Angong Niuhuang Pills, so as to achieve the stability, repeatability and controllability of the chemical composition in monitoring the stability, repeatability and controllability of the anti-ischemic stroke activity of Angong Niuhuang Pills. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a Venn diagram of the targets of the ingredients of Angong Niuhuang Wan and the targets of ischemic stroke;

[0025] Figure 2 This is the network relationship diagram of "traditional Chinese medicine-ingredient-disease target";

[0026] Figure 3 It is the protein-protein interaction (PPI) network (ranked in the top 100 by degree value);

[0027] Figure 4 The top 30 signaling pathways obtained by enrichment of 214 key target KEGG pathways;

[0028] Figure 5 The top 10 entries obtained by GO enrichment of 214 key targets;

[0029] Figure 6 The motion trajectory diagram of Angong Niuhuang Wan and its candidate component groups regulating the zebrafish cerebral ischemia model (the lines in the figure represent the motion trajectory of the zebrafish, the red line represents fast movement, the green line represents medium speed movement, and the black line represents slow movement) (A), the statistical analysis diagram of the motion trajectory of the zebrafish (B), the fluorescence intensity diagram of zebrafish brain cell apoptosis (the red circle in the figure represents the apoptotic area) (C), and the statistical analysis diagram of zebrafish brain cell apoptosis (D);

[0030] Figure 7 Angong Niuhuang Wan and its equivalent components regulate the behavioral scores of MCAO mice (A), TTC staining of brain tissue sections (B), statistical diagram of cerebral infarction volume (C), levels of inflammatory factors IL-1β, IL-6 and TNF-α in serum (DF), inflammatory cell activation in brain tissue sections (G), oxidative stress injury (H), vascular integrity (I) and cell apoptosis (J);

[0031] Figure 8Angong Niuhuang Wan and its equivalent component groups regulate the gene expression of vascular endothelial growth factor FLT1 and VEGFAA in zebrafish cerebral ischemia model (A); the gene expression of inflammatory factors IL1B, IL6, and TNFB (B); the gene expression of apoptosis-related proteins CASP3, CASP7, CASP8, and CASP9 (C); and the gene expression of neurotrophic factors BDNF, GDNFA, MBP, and ATATL (D). DETAILED DESCRIPTION

[0032] The essential contents of the present invention are described in detail below in conjunction with the embodiments, but the protection scope of the present invention is not limited thereto.

[0033] 1. Experimental Materials, Reagents, and Animals

[0034] The experimental materials, reagents and animals used in the present invention are all commercially available.

[0035] The sample information of 8 batches of Angong Niuhuang Pills is shown in Table 1.

[0036] Table 1 Angong Niuhuang Pills Sample Information

[0037]

[0038] The standard information of some chemical components in Angong Niuhuang Pills is shown in Table 2.

[0039] Table 2 Sample information of chemical component standards in Angong Niuhuang Pills

[0040]

[0041]

[0042]

[0043]

[0044] Acetonitrile and methanol (MS grade) were purchased from Merck Chemical Technology (Shanghai) Co., Ltd., and anhydrous ethanol and ethyl acetate (HPLC grade) were purchased from Yonghua Chemical Technology Co., Ltd., Suzhou, China. Ponatinib (lot number: 114513) was purchased from Med ChemExpress (Monmouth Junction, NJ, USA), and acridine orange was purchased from Shanghai MacLean Biochemical Technology Co., Ltd. (Shanghai, China). Sodium chloride (analytical grade) was purchased from Jiangsu Qiangsheng Functional Chemical Co., Ltd., potassium chloride (analytical grade) and anhydrous calcium chloride (analytical grade) were purchased from Nanjing Chemical Reagent Co., Ltd., anhydrous magnesium sulfate was purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd., edaravone injection (batch number: 2203203) was purchased from Sinopharm Guorui Pharmaceutical Co., Ltd., dimethyl sulfoxide (DMSO) and 2,3,5-triphenyltetrazolium chloride (TTC) were purchased from Sigma-Aldrich, USA, mouse IL-6 ELISA kit (batch number: EHC007) was purchased from Shenzhen Xinbosheng Biotechnology Co., Ltd., mouse IL-1β and TNF-α ELISA kits were purchased from Wuhan Huamei Bioengineering Co., Ltd., in situ nick end labeling (Td T mediated dUTPnick end labeling, TUNEL) apoptosis detection kit was purchased from Yisheng Biotechnology Co., Ltd., Iba-1 antibody (ab178847), CD31 (ab9498) and donkey anti-rabbit IgG H&L (Alexa 488) antibody (ab150073) was purchased from Abcam, DAPI (BD5010) was purchased from Bioworld Technology, RNA extraction reagent Trizol, reverse transcription kit Q RT SuperMix for qPCR (+g DNA wiper) and real-time fluorescence quantitative kit (ChamQ SYBR Color qPCR Master Mix) were purchased from Nanjing Novozyme Biotechnology Co., Ltd. The primers used in the experiment were synthesized by Beijing Qingke Biotechnology Co., Ltd. (Nanjing). Deionized water was prepared by distilled water through the Milli-Q system (Millipore, Bedford, MA, USA).

[0045] Wild-type AB zebrafish were purchased from Nanjing Yaoshunyu Biotechnology Co., Ltd. Adult zebrafish were kept in a light- and temperature-controlled culture facility with a water temperature range of 28±0.5℃, a normal pH range of 7.2-7.6, a conductivity of 500-550ms / cm, a dissolved oxygen level of not less than 6.0mg / L, a daily photoperiod of 14h light cycle and 10h dark cycle, and Artemia larvae were fed twice a day. Before the experiment, 1-2 pairs of adult zebrafish were placed in a spawning tank, with a partition separating the male and female fish. Light stimulation was started at 9:00 the next morning, and the partition was removed. Zebrafish embryos were collected 2 hours later and stored in E3 culture medium (5mmol / LNaCl, 0.17mmol / LKCl, 0.33mmol / L CaCl2, 0.33mmol / LMgSO4) at 28°C. The E3 culture medium was replaced every day and dead embryos were removed. Zebrafish 2 days after fertilization (2dpf) were used for drug administration experiments. Zebrafish breeding meets the requirements of international AAALAC certification.

[0046] Adult male C57BL / 6J mice, 20-22 g, 6-8 weeks old, were purchased from the Center for Comparative Medicine, Yangzhou University, Nanjing. The mouse breeding environment conditions were strictly implemented in accordance with the standards for experimental animal breeding, with a 12:12 hour light-dark cycle, a suitable temperature of 22±2°C, and a constant relative humidity of 55±5%. All mice were free to drink water and eat, and they could be used in formal experiments after one week of adaptation. All experimental operations strictly followed the "Regulations on the Management of Experimental Animals" and the "Implementation Measures for the Management of Experimental Animals in Jiangsu Province", and met the requirements of the Experimental Animal Welfare Ethics Committee of the Pharmaceutical Animal Experiment Center of China Pharmaceutical University.

[0047] 2. Experimental Methods

[0048] 1 Quantitative analysis of chemical components of Angong Niuhuang Wan

[0049] 1.1 Quantitative analysis of chemical components of Angong Niuhuang Wan based on UHPLC-QQQ MS

[0050] Preparation of test solution: Cut Angong Niuhuang Pills into pieces, accurately weigh 0.25 g, place in a 25 mL stoppered conical flask, accurately add 10 mL 75% methanol solution, seal, ultrasonically treat for 50 min, weigh after leaving at room temperature, make up the lost weight with 75% methanol, shake well, centrifuge at 4°C 13000 rpm for 10 min, take the supernatant and store it at 4°C for later use.

[0051] Preparation of reference solution: Accurately weigh 1.00 mg of each reference substance, dissolve it in methanol, make up to volume in a 1 mL volumetric flask, mix well, and prepare a reference stock solution with a final concentration of 1 mg / mL. Store at -80 °C until analysis.

[0052] Chromatographic conditions: A Shimadzu LC-30A ultra-high performance liquid chromatograph was used, equipped with a vacuum degasser, binary pump, autosampler, column oven, and diode array detector. The chromatographic column is an InfinityLab Poroshell 120EC-C18 column (2.1×100mm, 1.9μm); column temperature: 30°C; injection volume: 1μL; flow rate: 0.4mL / min; equilibrium time: 5min; mobile phase A: 0.1% formic acid solution; mobile phase B: acetonitrile; elution gradient: 0-4min, 5-12%B; 4-14min, 12-17%B; 14-17min, 17%B; 17-22min, 17-30%B; 22-35min, 30-52%B; 35-37min, 52-100%B; 37-38min, 100%B, 38-40min, 100-5%B; 40-45min, 5%B.

[0053] Mass spectrometry conditions: Shimadzu LCMS-8050 triple quadrupole mass spectrometer was used for detection with an electrospray ion source. Ion source temperature, 300°C, nebulizer gas flow rate: 3 L / min; heating block temperature, 400°C, heating gas flow rate, 10.0 L / min; desolvation tube temperature, 250°C; drying gas flow rate: 10.0 L / min; data acquisition mode: multiple reaction detection mode; positive and negative ion scanning, scanning range: m / z 50-1500. Labsolutions workstation LCMS RealtimeAnalysis function was used for data acquisition.

[0054] Data analysis: The collected data were processed using the Labsolutions workstation LCMS Postrun and Browse functions.

[0055] 1.2 Quantitative analysis of chemical components of Angong Niuhuang Wan based on GC-QQQ MS

[0056] Preparation of test solution: Cut Angong Niuhuang Pills into pieces, weigh 1.50g accurately, place in a 250mL round-bottom flask, add 100mL distilled water, and extract for 4h according to the Chinese Pharmacopoeia Volatile Oil Determination Method (General Rule 2204 Method A). Collect the volatile oil, remove the remaining water with anhydrous sodium sulfate, and finally dissolve in 2mL ethyl acetate. Add n-tridecane as an internal standard and directly analyze the solution.

[0057] Preparation of reference solution: Accurately weigh 1.00 mg of each reference substance, dissolve it in anhydrous ethanol, dilute to volume in a 1 mL volumetric flask, mix well, and prepare a reference stock solution with a final concentration of 1 mg / mL. All working solutions were stored at -20 °C until analysis.

[0058] Chromatographic conditions: Agilent 7890B / 7000D triple quadrupole gas chromatography-mass spectrometer was used, and chromatographic separation was performed using an Agilent DB-5MS UI capillary column (30m×0.25mm id, 0.25μm film); injection volume: 1μL; split ratio: 10:1; carrier gas: high-purity He (≥99.99%); flow rate: 1mL / min; injection port temperature: 250℃; transfer line temperature: 250℃. Program temperature rise: column initial temperature 60℃, increased to 76℃ at 8℃ / min, increased to 82℃ at 2℃ / min, increased to 130℃ at 15℃ / min, increased to 230℃ at 20℃ / min, and maintained for 2min; post-operation temperature: 280℃; post-operation time: 2min.

[0059] Mass spectrometry conditions: ion source temperature: 230°C; quadrupole temperature: 150°C; solvent extension time: 4.5 min; quenching gas: high-purity He (≥99.99%); quenching gas flow rate: 2.25 mL / min; collision gas: high-purity N2 (≥99.99%); collision gas flow rate: 1.5 mL / min; electron energy: +70 eV; scanning mode: dynamic multiple reaction detection mode. Data acquisition was performed using Agilent MassHunter Workstation GC / MS Data Acquisition Software version 10.0.368.

[0060] Data analysis: The acquired data were processed using Agilent MassHunter Workstation Qualitative Analysis Software Version 10.0 and Agilent MassHunter Workstation Qualitative Analysis for QQQ Software Version 10.0.

[0061] 1.3 Methodological Review

[0062] Standard curve, limit of quantitation and limit of detection

[0063] The mother solution of the reference substance of each compound was taken and diluted into a series of gradient concentrations, and an equal amount of internal standard was added. The injection volume of each mixed reference solution was 1 μL. The peak area ratio of each test compound and the internal standard compound was used to fit the linear regression equation to obtain the standard curve of each component. The mixed reference solution was further diluted with solvent and analyzed. The concentration with a signal-to-noise ratio of 10 was defined as the quantitative limit, and the concentration with a signal-to-noise ratio of 3 was defined as the detection limit.

[0064] Intra-day precision: Select three mixed reference solutions of different concentrations at high, medium and low positions in the concentration gradient, and inject each mixed reference solution 3 times in one day with an injection volume of 1 μL. Record the peak area ratio of each test compound to the internal standard compound, and calculate the relative standard deviation (RSD) among the three injections.

[0065] Inter-day precision: Select three mixed reference solutions of different concentrations at high, medium and low positions in the concentration gradient, and inject each mixed reference solution three times a day for three consecutive days with an injection volume of 1 μL. Record the peak area ratio of each test compound to the internal standard compound, and calculate the RSD among the nine injections.

[0066] Repeatability: Take 6 portions of Angong Niuhuang Pills from the same batch and accurately weigh them respectively. Prepare 6 test solutions in parallel with an injection volume of 1 μL. Record the peak area ratio of each test compound to the internal standard compound and calculate the RSD among the 6 injections.

[0067] Stability: The test solution was injected and analyzed at 0h, 2h, 4h, 8h, 12h and 24h, and the peak area ratio of each test compound to the internal standard compound was recorded, and the RSD among the 6 injections was calculated.

[0068] Accuracy: The recovery rate experiment was used for investigation. Take 6 portions of the same batch of Angong Niuhuang Pills and accurately weigh them. Add a known amount of mixed reference solution. Prepare 6 portions of test bath solution in parallel for sampling and detection. The injection volume is 1 μL. Record the peak area ratio of each test compound to the internal standard compound, and bring it into the standard curve to calculate the content of each test compound. Calculate the recovery rate of the sample according to the formula, and calculate the RSD between the 6 needles. Recovery rate % = (actual measured content - sample content) / added reference amount × 100%.

[0069] 1.4 Content determination

[0070] According to the established analytical method, the components in 8 batches of Angong Niuhuang Pills were determined with an injection volume of 1 μL. The peak area ratio of each analyte to the internal standard compound was recorded and the content of each compound was calculated by substituting it into the standard curve.

[0071] 2 Screening of potential components of Angong Niuhuang Pills in improving ischemic stroke injury based on network pharmacology

[0072] 2.1 Screening of the targets of the chemical components of Angong Niuhuang Pills

[0073] 89 chemical components accurately quantified in Angong Niuhuang Pills were selected as candidate components for exploring the targets of Angong Niuhuang Pills. Six online databases were used, including the Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP, https: / / tcmsp-e.com / tcmsp.php), the Gene and Chemical Interaction Prediction Database (STITCH, http: / / stitch.embl.de / ), the Comparative Toxicogenomics Database (CTD, http: / / ctdbase.org / ), the Therapeutic Target Database (TTD, https: / / db.idrblab.net / ttd / ), the Drug Database (DrugBank, https: / / go.drugbank.com / ), and the Medical Database (pubmed, https: / / pubmed.ncbi.nlm.nih.gov / ), and the English name or CAS number of the chemical component was used as the search keyword to search and record the collected targets. The collected targets were converted into unified gene names (Genename) through the International Protein Database (Uniprot, https: / / www.uniprot.org / ), the species origin was limited to Homo sapiens, and duplicate targets and targets with non-human species origin were deleted, and these were used as the component targets of Angong Niuhuang Pills.

[0074] 2.2 Screening of ischemic stroke targets

[0075] We used five online databases, including DisGeNET (https: / / www.disgenet.org / ), Therapeutic Target Database (TTD, https: / / db.idrblab.net / ttd / ), Online Mendelian Inheritance in Man (OMIM, https: / / omim.org / ), Drug Bank (https: / / go.drugbank.com / ) and Gene Cards (https: / / www.genecards.org / ), as well as published online medical literature, to retrieve targets related to ischemic stroke. We then used the Uniprot database to screen the targets and delete duplicate targets, which were then used as disease targets.

[0076] 2.3 Construction of the “TCM-ingredient-disease target” network of Angong Niuhuang Pills

[0077] The potential targets of the chemical components of Angong Niuhuang Pills were mapped to the targets of ischemic stroke to obtain intersection targets. The intersection target component correspondence table and the intersection target component attribute relationship table were imported into Cytoscape software, and the "TCM-component-disease target" compound network was constructed using Cytoscape3.9.1 software. The network topology analysis was performed using the Network Analysis plug-in to calculate the degree (Degree), betweenness centrality (BC) and closeness centrality (CC) of the nodes. The size of the node was set proportional to the degree.

[0078] 2.4PPI analysis

[0079] The obtained common targets were imported into the STRING database to obtain information about the protein interaction network. The interval targets with the lowest interaction score greater than 0.9 were selected, and the targets without connection relationships were hidden. The analysis results were imported into the Cytoscape software to visualize the relationship between the targets and calculate the Degree, BC, and CC of the intersection targets.

[0080] 2.5 KEGG signaling pathway and GO enrichment analysis

[0081] In order to study the potential biological functions of the targets, the key targets were imported into the DAVID database (https: / / david.ncifcrf.gov / home.jsp) for KEGG pathway and GO enrichment analysis. KEGG enrichment analysis can find signal pathways involved in biological processes, and GO enrichment analysis is used to understand biological processes (BP), cell components (CC), and molecular functions (MF). Finally, the results were imported into the bioinformatics data analysis platform (http: / / www.bioinformatics.com.cn / ) to draw bubble charts.

[0082] 3 Proposal of candidate component groups

[0083] According to the content of each component in Angong Niuhuang Pills and its degree parameter value in network topology analysis in network pharmacology, the candidate component group was designed.

[0084] 4 Discovery of the equivalent component group of Angong Niuhuang Pills against ischemic stroke based on the zebrafish cerebral ischemia model

[0085] 4.1 Solution preparation

[0086] Preparation of E3 culture medium: accurately weigh 17532mg NaCl, 769.5mg KCl, 2197.8mg CaCl2 and 2383.3mg MgSO4 and place them in a 1L glass bottle, add 1L pure water, and dissolve by ultrasonic to obtain 60× E3 culture medium mother solution, which is stored in a refrigerator at 4°C. When using, accurately pipette 16.67mL E3 culture medium (60×), add pure water to 1L to obtain 1× E3 culture medium, which is used for daily embryo culture and solvent preparation.

[0087] Preparation of Ponatinib: Accurately weigh 1.02 mg of Ponatinib, add DMSO to dissolve to 1 mg / mL as the mother solution, and dilute it to 1 μg / mL with E3 culture medium before use. Prepare and use immediately.

[0088] Preparation of acridine orange staining solution: Accurately weigh 1.43 mg of acridine orange powder, add DMSO to dissolve to 2.5 mg / mL as the mother solution, and dilute to 2.5 μg / mL with E3 culture medium when using. Protect from light throughout the process and use it immediately after preparation.

[0089] Preparation of Angong Niuhuang Wan solution: accurately weigh 1.0054g of Angong Niuhuang Wan (S8 batch), place in a 100mL conical flask, add 40mL of 75% methanol, ultrasonically treat for 50min, stand at room temperature, weigh again, make up the lost weight with 75% methanol, mix, centrifuge at 4°C, 13000rpm to take the supernatant, evaporate the solvent in a solvent evaporation workstation for standby use. Accurately weigh 1.0038g of Angong Niuhuang Wan, extract volatile oil by volatile oil extraction, and remove excess water with anhydrous sodium sulfate. Dissolve the obtained extract powder and volatile oil with DMSO, add E3 culture medium to dissolve into mother liquor, store at -20°C for standby use, dilute to the required concentration when used, and the DMSO content does not exceed 0.2%.

[0090] Preparation of candidate component group solutions: Based on the quantitative analysis results of Angong Niuhuang Pills, calculate the content of chemical components in 1.0046g of Angong Niuhuang Pills, accurately weigh, dissolve with DMSO to form a mother liquor, mix into each candidate component group solution according to the designed candidate component group, and dilute with E3 culture medium to the required concentration when used. The DMSO content shall not exceed 0.2%.

[0091] 4.2 Construction of zebrafish cerebral ischemia model and animal grouping

[0092] 2dpf normal wild-type AB zebrafish were selected and placed in a six-well plate, with 30 young fish in each well. They were divided into 8 groups, including control group, model group, Angong Niuhuang Wan group, candidate component group I, candidate component group II, candidate component group III, candidate component group IV and candidate component group V. When administering, the E3 culture medium in the well was aspirated, the control group was given 3mL of 0.1% DMSO solution, the model group was given 3mL of 1μg / mL ponatinib solution, the Angong Niuhuang Wan group was given 3mL of 500μg / mL Angong Niuhuang Wan solution and 1μg / mL ponatinib solution, and the candidate component group was given 3mL of a standard mixed solution prepared in proportion according to the quantitative results of Angong Niuhuang Wan (the dosage of each chemical component in the candidate component group was consistent with that in the Angong Niuhuang Wan group, the same below) and 1μg / mL ponatinib solution.

[0093] 4.3 Assessment of movement disorders in zebrafish

[0094] After each group of zebrafish was incubated in a 28°C incubator for 24 hours, 12 zebrafish were selected from each group and placed in a 96-well plate, with 1 fish placed in each well, and 200 μL of E3 culture medium was added. After the 96-well plate was placed in a 28°C incubator for 10 minutes, the movement trajectory of the zebrafish was recorded. The entire experiment lasted 60 minutes, including 3 light / dark cycles (bright 10 minutes, dark 10 minutes). The total movement distance of the zebrafish in the light / dark cycle was recorded and analyzed by a zebrafish viewpoint behavior analyzer (ZebralabV3, viewpoint LifeSciences Co., Ltd.).

[0095] 4.4 Zebrafish brain cell apoptosis detection

[0096] After incubating zebrafish in each group in a 28°C incubator for 24 h, 3 mL of 2.5 μg / mL acridine orange solution was added to each well for staining at room temperature in the dark for 30 min, and then rinsed three times with E3 culture medium. Twenty zebrafish in each group were fixed on a glass slide with 3% methylcellulose and placed under a stereo microscope to observe the fluorescence intensity of the zebrafish brain, and photos were taken to evaluate the apoptosis of brain cells. Image J software was used to quantitatively analyze the fluorescence intensity of the zebrafish brain.

[0097] 5 Evaluation of the anti-ischemic stroke activity of the equivalent components of Angong Niuhuang Pills based on the MCAO mouse model

[0098] 5.1 Solution preparation

[0099] Preparation of Angong Niuhuang Pills: According to the clinical dosage of Angong Niuhuang Pills, the dosage of mice is 780 mg / kg. Weigh 2.0036 g of Angong Niuhuang Pills (batch S8) accurately, place in a 100 mL conical flask, add 80 mL of 75% methanol, ultrasonically treat for 50 min, place at room temperature, weigh again, make up the lost weight with 75% methanol, mix, centrifuge at 4 ° C, 13000 rpm to take the supernatant, evaporate the solvent in a solvent evaporation workstation for use. Weigh 2.0038 g of Angong Niuhuang Pills accurately, extract volatile oil by volatile oil extraction, and remove residual water with anhydrous sodium sulfate. Dissolve the obtained extract powder and volatile oil with DMSO and add CMCNa to 25.64 mL, and the DMSO content does not exceed 1%.

[0100] Preparation of the solution of equivalent component groups of Angong Niuhuang Pills: Based on the quantitative results of Angong Niuhuang Pills, calculate the content of each chemical component of the equivalent component group in 2.0037g of Angong Niuhuang Pills, accurately weigh them, use DMSO as a co-solvent, and dissolve them in 25.64ml CMCNa.

[0101] Preparation of edaravone solution: Take an appropriate amount of edaravone injection and add normal saline to prepare a 1 mg / mL solution.

[0102] 5.2 Establishment of the MCAO model in mice and its experimental grouping

[0103] C57BL / 6J mice (about 20-22 g) were anesthetized with 1.5% isoflurane and fixed in the supine position. The right common carotid artery, external carotid artery and internal carotid artery were exposed and separated. The distal end of the external carotid artery was ligated, and the proximal end was cut. A thread plug was inserted from the cut and pushed to the intersection of the common carotid artery and the external carotid artery. The external carotid artery was then severed, and the thread plug was carefully reversed and pushed into the internal carotid artery. The thread plug was fixed and the common carotid artery was ligated at the same time. After 1 hour of ischemia, the thread plug was removed, the cut was ligated, the common carotid artery was released to restore blood supply, and the skin was sutured and the mouse was placed on a heating pad to keep warm until awake. The sham-operated mice underwent the same operation, but no thread plug was inserted.

[0104] After the MCAO mice were modeled, they were randomly divided into 4 groups: model group, Angong Niuhuang Wan group, equivalent component group, and edaravone group. The Angong Niuhuang Wan group and the equivalent component group were given medication once at the time of ischemia and 4 hours after reperfusion, the edaravone group was given medication once before reperfusion, and the sham operation group and the model group were given an equal amount of normal saline.

[0105] 5.3 Mouse behavioral scoring

[0106] After 24 hours of reperfusion, all mice were scored with the Zea-Longa neurological function score, and the scoring criteria were as follows: 0 points indicated no obvious motor deficits; 1 point indicated that the left forepaw could not be freely extended; 2 points indicated spontaneous circling; 3 points indicated falling to the left; 4 points indicated inability to move autonomously or even loss of consciousness. The higher the neurological score, the more severe the neurological deficit of the animal.

[0107] 5.4 Evaluation of cerebral infarction volume in mice by TTC staining

[0108] After 24 hours of reperfusion, the mice were euthanized, the brains were quickly removed and frozen at -20°C for 20 minutes, and the brain tissue was cut into 6 coronal slices of uniform thickness, about 2 mm thick. The slices were placed in a six-well plate containing 1% TTC dye solution and stained in a 37°C water bath in the dark for 10 minutes. After staining, the TTC dye solution was discarded and the slices were placed in 4% paraformaldehyde. After fixation for 24 hours, the photos were taken and the mouse brain infarction volume was calculated using Image J software. The calculation formula is: infarction volume (%) = (right hemisphere volume - left hemisphere non-infarction area volume) / right hemisphere volume × 100%.

[0109] 5.5ELISA kit to detect IL-6, IL-1β and TNF-α levels

[0110] 24h after reperfusion, mouse blood was collected in EP tubes. After standing for 30min, centrifuged at 3000rpm for 15min at room temperature. Serum was collected and the contents of IL-6, IL-1β and TNF-α were determined according to the instructions of the ELISA kit. A linear regression equation was drawn based on the concentration and absorbance of the standard, and the concentration of IL-6, IL-1β or TNF-α in serum was calculated.

[0111] 5.6 Immunofluorescence staining

[0112] 24 hours after reperfusion, the mice were euthanized, and the brains were quickly removed and fixed in 4% paraformaldehyde solution, embedded in paraffin and cut into 5 μm thick sections. The sections were then permeabilized with 0.3% Triton X-100 for 10 min and blocked with goat serum at room temperature for 2 h. After blocking, the primary antibodies Anti-Iba1 (1:100) or Anti-CD31 (1:100) were added and incubated overnight at 4°C. After washing with PBS, the sections were incubated with donkey anti-rabbit IgG H&L (Alexa Fluor 488) antibody at room temperature for 1.5 h. After washing with PBS, the cells were stained with DAPI (1:1000) for 10 min, and the images were observed and photographed under a laser confocal microscope.

[0113] 5.7 Superoxide anion (DHE) staining

[0114] After 24 hours of reperfusion, the mice were euthanized, and the brains were quickly removed and fixed in 4% paraformaldehyde solution, embedded in paraffin and cut into 5 μm thick sections. Then, 0.3% Triton X-100 was used for permeabilization for 10 minutes, and goat serum was used for blocking at room temperature for 30 minutes. After blocking, DHE dye (1:1000) was added and incubated at 37°C in the dark for 90 minutes. After washing with PBS, the brains were stained with DAPI (1:1000) for 10 minutes, and the images were observed with a laser confocal microscope and photographed and recorded.

[0115] 5.8 TUNEL staining

[0116] After 24 hours of reperfusion, the mice were euthanized, and the brains were quickly removed and fixed in 4% paraformaldehyde solution, embedded in paraffin, and cut into 5 μm thick sections. The staining was performed according to the instructions of the TUNEL cell apoptosis detection kit, and the cell nuclei were labeled with DAPI working solution. The images were observed under a laser confocal scanning microscope and photographed and recorded.

[0117] 6 Study on the mechanism of Angong Niuhuang Pills and its equivalent components in improving ischemic stroke injury

[0118] 6.1 Animal grouping

[0119] The solution preparation was the same as 4.1. 2dpf normal wild-type AB zebrafish were selected and placed in a six-well plate, with 30 fish per well. They were divided into 4 groups: blank control group, model group, Angong Niuhuang Wan group and equivalent component group. The blank control group was given 3mL of 0.1% DMSO solution, the model group was given 3mL of 1μg / mL ponatinib, the Angong Niuhuang Wan group was given 3mL of 500μg / mL Angong Niuhuang Wan solution and 1μg / mL ponatinib solution, and the equivalent component group was given 3mL of a standard mixed solution prepared in equal proportions according to the quantitative results of Angong Niuhuang Wan and 1μg / mL ponatinib solution.

[0120] 6.2 Real-time quantitative PCR (RT-qPCR)

[0121] Collection of zebrafish tissue samples: 24 hours after administration, wash with E3 culture medium 2-3 times, transfer the zebrafish to a 1.5 mL EP tube, aspirate the liquid, add 500 μL Trizol lysis solution, homogenize with a tissue grinder, and let stand for 1-2 minutes.

[0122] RNA extraction: Add 200μL DEPC water, shake upside down for 15s, let stand, and centrifuge at 4℃, 12000rpm for 15min. Pipette 500μL supernatant and transfer to a new 1.5mL EP tube. Add an equal amount of isopropanol and mix by inversion 4-5 times, and let stand at -20℃ for 2h. Centrifuge at 4℃, 12000rpm for 10min. The precipitate at the bottom of the tube after centrifugation is the RNA clump. Discard the liquid and add 1mL 75% ethanol (prepared with DEPC water in advance and precooled), mix by inversion, centrifuge at 4℃, 5500rpm for 10min, discard the ethanol, turn it upside down to dry until the edge of the RNA clump is transparent, and add an appropriate amount of DEPC water to dissolve the RNA sample.

[0123] RNA concentration and purity identification: Nano-100 micro-spectrophotometer was used to detect the concentration and purity of RNA samples.

[0124] RNA reverse transcription into cDNA: Add a total of 1 μg of RNA and 4 μL of 5×HiScript IIQrtSuperMix II to each sample tube, and add RNase-free ddH2O water to the volume to 20 μL. Use a pipette to gently blow and mix, and place in a fluorescent quantitative PCR instrument. Set the reverse transcription program to 50℃, 15min, 85℃, 2min. Store the generated cDNA in a -20℃ refrigerator.

[0125] RT-PCR: The RT-PCR reaction system was prepared according to Table 3 and the assay was performed using a LightCycler 480 real-time fluorescence quantitative PCR instrument. The assay procedure was: pre-denaturation (95°C, 30 s), annealing (56°C, 30 s), extension (72°C, 60 s), and 40 cycles. The relative gene level of mRNA was normalized using Actb as the internal reference and the calculation formula was 2- ΔΔCt The primer sequences are shown in Table 4.

[0126] Table 3 RT-PCR reaction system

[0127]

[0128] Table 4 RT-qPCR primer sequences

[0129]

[0130] 7. Statistical analysis

[0131] The experimental data were processed using GraphPad Prism 9.3.1 software, and all values ​​were expressed as mean ± standard error (mean ± SEM). One-way ANOVA analysis and Dunnett-t test were used for comparison between two or more groups, and p < 0.05 indicated statistically significant differences.

[0132] 3. Experimental Results

[0133] 1 Quantitative analysis of chemical components of Angong Niuhuang Wan

[0134] Based on the previous qualitative characterization of the chemical components of Angong Niuhuang Pills by the research team, we selected accurately identified ingredients with high content or good activity reported in the literature. A total of 68 representative non-volatile compounds and 21 volatile compounds were selected for quantitative analysis.

[0135] UHPLC-QQQ MS method validation: 68 analytes with different detection coefficients (R 2 >0.9925) showed a good linear relationship. The detection limit and quantification limit were 0.002-17.42 ng / mL and 0.01-34.85 ng / mL, respectively. The RSDs of repeatability, intra-day precision and inter-day precision were 1.44-9.75%, 0.01-9.95% and 1.53-9.75%, respectively. At 4°C, all samples remained stable within 24 hours with an RSD range of 1.28-9.88%. The sample recovery was 74.63-126.93% with an RSD of 0.05-15.13%. Among them, the sample recovery of 64 compounds was 80.72-118.62%, and the RSDs of the remaining compounds, baicalein, taurochenodeoxycholic acid and ursolic acid, were slightly higher, which may be due to their low content and unstable ionization efficiency. The results showed that the established quantitative method was accurate, reproducible, and reliable, and could be used for the simultaneous quantitative determination of 68 selected compounds in Angong Niuhuang Wan.

[0136] GC-QQQ MS method validation: 21 analytes showed good linearity within their linear range, with correlation coefficients ranging from R 2 The values ​​ranged from 0.9918 to 0.9997. The limits of detection and quantification were 0.60 to 150.40 ng / mL and 6.00 to 451.20 ng / mL, respectively, indicating that the method had sufficient sensitivity for the quantitative analysis of the main volatile components of Angong Niuhuang Pills. The intra-day precision and inter-day precision RSD values ​​of all compounds were in the range of 0.04 to 5.84% and 0.48 to 6.83%, respectively, the repeatability RSD values ​​were between 1.13 and 9.00%, the stability RSD values ​​were in the range of 1.39 to 6.70%, the total recovery was in the range of 90.80 to 109.83%, and the RSD value was less than 8.68%. The results showed that the established quantitative method was sensitive, rapid, accurate and reproducible for the determination of representative volatile components in Angong Niuhuang Pills.

[0137] 2 Screening of the target of Angong Niuhuang Pills in the treatment of ischemic stroke

[0138] A total of 2474 component targets were collected by integrating TCMSP, STITCH, CTD, TTD, Drug Bank and pubmed databases, and a total of 1391 ischemic stroke targets were obtained by integrating DisGeNET, TTD, OMIM, Drug Bank and Gene Cards. The intersection of the two obtained 720 potential targets for Angong Niuhuang Wan to regulate ischemic stroke. The results are as follows Figure 1 These 720 targets were then mapped to the 89 components of Angong Niuhuang Wan, and 78 potential active components of Angong Niuhuang Wan in regulating ischemic stroke were obtained.

[0139] 3. Construction of the “TCM-ingredient-disease target” network

[0140] In order to visualize the relationship between the ingredients of Angong Niuhuang Wan and the disease targets, the "TCM-ingredient-disease target" compound network was constructed using Cytoscape 3.9.1 software. The results are shown in Figure 2. Figure 2 As shown in the figure, 798 nodes (78 chemical components and 720 potential targets) and 4694 interactions were obtained. The blue target in the middle is the potential target of Angong Niuhuang Wan in regulating ischemic stroke. The degree, betweenness centrality (BC) and closeness centrality (CC) of each node were calculated. In network pharmacology, it is generally believed that compounds with higher degree have more extensive pharmacological effects. The results of sorting the compounds from large to small according to degree are shown in Table 5. The top compounds are baicalin, curcumin, baicalein, geniposide and ursodeoxycholic acid. The degree results can be used as reference standards for subsequent candidate component group screening.

[0141] Table 5 Network topology analysis of the active ingredients of Angong Niuhuang Wan in regulating ischemic stroke

[0142]

[0143]

[0144]

[0145]

[0146] 4PPI analysis

[0147] In order to clarify the connection between targets, 720 potential targets of Angong Niuhuang Pills for the treatment of ischemic stroke were uploaded to the STRING database, with a total of 636 nodes. Figure 3When the values ​​of Degree, BC and CC were all greater than their medians (Degree>8, BC>0.000705, CC>0.318089), 214 key targets of Angong Niuhuang Wan in regulating ischemic stroke were obtained. The top ten targets were SRC, TP53, STAT3, MAPK3, PIK3R1, MAPK1, RELA, PIK3CA, JUN and AKT1, indicating that these targets played an important role in the efficacy of Angong Niuhuang Wan in improving ischemic stroke damage.

[0148] 5KEGG signaling pathway and GO enrichment analysis

[0149] The key targets screened were subjected to KEGG signal pathway enrichment analysis, and a total of 190 potential signal pathways for Angong Niuhuang Wan to improve ischemic stroke damage were obtained (p<0.05). According to the p value, the top 30 signal pathways are as follows: Figure 4 As shown, these include TNF, IL-17, MAPK and toll-like receptor signaling pathways related to inflammation, cell apoptosis related to neuronal apoptosis, VEGF signaling pathway related to angiogenesis, neural factor signaling pathway related to neural development, and others such as HIF-1, PI3K-AKT, FoxO and NF-κB signaling pathways, etc., suggesting that Angong Niuhuang Wan may exert its pharmacological effect against ischemic brain injury through these pathways.

[0150] The key targets screened were then subjected to GO enrichment analysis, and 1127 biological process (BP) entries, 129 cellular component (CC) entries, and 180 molecular function (MF) entries were obtained from the David database (p<0.05). According to the p-value sorting, the top 10 BP, CC, and MF entries are as follows: Figure 5 As shown, they are respectively related to the positive regulation of inflammatory response, hypoxia response, apoptosis process, neuronal death, MAPK cascade reaction and cell proliferation, suggesting that Angong Niuhuang Wan may improve ischemic stroke injury by regulating related biological processes such as inflammation, oxidative stress damage and apoptosis.

[0151] 6 Proposal of candidate component groups

[0152] Each edge in the "traditional Chinese medicine-ingredient-target" compound network represents the interaction between the chemical ingredient and the target. The degree of a node represents the number of edges connected to the node. The larger the degree, the more nodes are connected to it. Therefore, it is generally believed that compounds with higher degree have better pharmacological effects.

[0153] Based on the content and network pharmacological regulation prediction analysis of the chemical components of Angong Niuhuang Wan, five groups of candidate components were proposed: candidate component group I: components with a content greater than 100 μg / g and Degree ≥ 10; candidate component group II: aromatic drug components with a content greater than 100 μg / g and Degree ≥ 10; candidate component group III: non-aromatic drug components with a content greater than 100 μg / g and Degree ≥ 10; candidate component group IV: components with a content greater than 500 μg / g and Degree ≥ 50; candidate component group V: components with a content greater than 1000 μg / g and Degree ≥ 75. The detailed information of each candidate component group is shown in Tables 6 to 10.

[0154] Table 6 Candidate component group I compound composition

[0155]

[0156]

[0157]

[0158] Table 7 Candidate component group II compound composition

[0159]

[0160]

[0161] Table 8 Candidate component group III compound composition

[0162]

[0163]

[0164] Table 9 Candidate component group IV compound composition

[0165]

[0166]

[0167]

[0168] Table 10 Candidate component group V compound composition

[0169]

[0170]

[0171] 7 Discovery of equivalent component groups of Angong Niuhuang Wan in improving ischemic stroke injury based on zebrafish cerebral ischemia model

[0172] A zebrafish cerebral ischemia model induced by Ponatinib was established, and Angong Niuhuang Pills and candidate component group IV were administered. It was found that Angong Niuhuang Pills and candidate component groups I, IV, and V could significantly alleviate Ponatinib-induced movement disorders in zebrafish; candidate component group II showed a slight improvement, but its efficacy was not as good as Angong Niuhuang Pills; the efficacy of candidate component group III was weakened after the aromatic drug components were removed, indicating that aromatic drugs play an important role in the treatment of ischemic stroke by Angong Niuhuang Pills ( Figure 6 AB). At the same time, Angong Niuhuang Wan and the five candidate ingredient groups can reduce brain cell apoptosis and have obvious neuroprotective effects ( Figure 6 CD).

[0173] For the evaluation of bioactivity equivalence, the bioassay results were converted into efficacy values, and the bioactivity equivalence between the candidate component group and Angong Niuhuang Wan was evaluated by calculating the 90% confidence interval (CI). Compared with the original prescription, if the 90% CI of the relative efficacy was within 70-143%, the candidate component group was considered to be equivalent to Angong Niuhuang Wan. Table 11 lists the results of the bioactivity equivalence evaluation of the five candidate component groups, among which the 90% CI of the relative efficacy of candidate component groups I, IV, and V for zebrafish ischemic stroke injury were all within 70-143%, which can be regarded as equivalent to Angong Niuhuang Wan. The three groups of components were attributed to seven kinds of animal and plant Chinese medicines, including musk, borneol, yellow silk turmeric, scutellaria, coptis, gardenia and bezoar, with only differences in quantity. Therefore, candidate component group V (a total of 28 components) was selected as the equivalent component group of Angong Niuhuang Wan for subsequent experiments.

[0174] Table 11 Results of bioequivalence evaluation of candidate ingredient groups in zebrafish model

[0175]

[0176] 8 Evaluation of the efficacy of Angong Niuhuang Pills and its equivalent components in improving ischemic stroke injury based on the MCAO mouse model

[0177] The MCAO model of mice was established, and Angong Niuhuang Pills and its equivalent components were administered orally. It was found that the neurological function scores of mice in the model group were significantly increased compared with those in the sham operation group. Treatment with Angong Niuhuang Pills, its equivalent components and edaravone can significantly improve the neurological damage caused by ischemia-reperfusion in mice ( Figure 7 A). Brain tissue sections were taken for TTC staining. The brain tissue sections of the sham operation group were uniformly red, and the brain tissue sections of the ischemic side of the mice in the model group had obvious white infarction areas, with an average infarction volume of 46.82%. After treatment with Angong Niuhuang Pills, equivalent component groups and edaravone, the brain infarction volume of mice was significantly reduced, with average infarction volumes of 28.14%, 29.33% and 21.82%, respectively ( Figure 7BC). The levels of inflammatory factors IL-1β, IL-6 and TNF-α were detected in the mouse serum. It was found that compared with the sham operation group, the expression level of inflammatory factors in the serum of the model group mice was significantly increased. Administration of Angong Niuhuang Pills, equivalent component groups and edaravone significantly reduced the release of proinflammatory factors caused by ischemia-reperfusion ( Figure 7 DF). According to the results of the biological activity equivalence evaluation and analysis, the 90% CI of the relative efficacy of the equivalent component group on the degree of neurological damage, cerebral infarction volume, and inflammatory factor levels in MCAO mice were all within the specified range, and it can be regarded as equivalent to Angong Niuhuang Wan (Table 12).

[0178] Table 12 Results of bioequivalence evaluation of candidate ingredient groups in the mouse MCAO model

[0179]

[0180] Immunofluorescence staining was used to observe the expression of Iba-1 protein in the peri-infarction area of ​​MCAO mice. Compared with the sham operation group, the microglia in the peri-infarction area of ​​the model group were significantly activated, the cell bodies were enlarged, the processes were shortened, and they were round or rod-shaped, and the fluorescence intensity was stronger. After administration of Angong Niuhuang Pills, equivalent component groups, and edaravone, the morphology of microglia showed a trend toward normal state, and the efficacy of the equivalent component group and Angong Niuhuang Pills was equivalent ( Figure 7 G). Immunofluorescence staining was used to observe the DHE fluorescence intensity in the peri-infarction area of ​​MCAO mice. Compared with the sham operation group, the fluorescence intensity of the model group was enhanced, indicating that the production of ROS increased significantly. After treatment with Angong Niuhuang Pills, equivalent component groups and edaravone, the red fluorescence decreased, and the accumulation of ROS was reduced, indicating that Angong Niuhuang Pills, equivalent component groups and edaravone can protect brain tissue from oxidative stress damage, and the equivalent component group has the same efficacy as Angong Niuhuang Pills ( Figure 7 H). Immunofluorescence staining was used to observe the expression of CD31 protein in the peri-infarction area of ​​MCAO mice. Compared with the sham operation group, the blood vessels in the model group were damaged and had no complete structure. After treatment with Angong Niuhuang Pills, equivalent component groups and edaravone, the integrity of the vascular structure was improved, indicating that Angong Niuhuang Pills, equivalent component groups and edaravone can improve the vascular damage caused by cerebral ischemia-reperfusion in mice and promote angiogenesis. The equivalent component group has the same efficacy as Angong Niuhuang Pills ( Figure 7 I).

[0181] TUNEL staining showed that compared with the sham operation group, the number of green fluorescent labeled apoptotic cells in the brain tissue sections of the model group increased significantly, and the number of apoptotic cells decreased significantly after the administration of Angong Niuhuang Pills, equivalent component groups and edaravone, indicating that Angong Niuhuang Pills and its equivalent component groups have the effect of inhibiting cell apoptosis, and the equivalent component group has the same efficacy as Angong Niuhuang Pills ( Figure 7 J).

[0182] 9. Study on the mechanism of Angong Niuhuang Pills and its equivalent components in improving ischemic stroke injury

[0183] 9.1 Angong Niuhuang Pills and Equivalent Components Upregulate the Expression of Vascular Endothelial Growth Factor

[0184] Vascular endothelial growth factor (VEGF) is a major factor that promotes angiogenesis and enhances vascular permeability, and plays a variety of roles in the process of vascular development. VEGFR1 (encoded by the gene flt1) is a receptor for VEGF, and inhibition of VEGFR1 expression can lead to apoptosis of vascular endothelium. VEGFA (encoded by the gene vegfaa) is a ligand for the VEGF receptor and plays an important role in angiogenesis and development. Figure 8 As shown in A, after ponatinib modeling, the gene expression of flt1 and vegfaa was significantly reduced, and Angong Niuhuang Pills and its equivalent component groups could upregulate the gene expression of flt1 and vegfaa (p<0.01&p<0.05), indicating that Angong Niuhuang Pills and its equivalent component groups may improve ischemic stroke injury by upregulating the expression of vascular endothelial growth factor.

[0185] 9.2 Angong Niuhuang Pills and Equivalent Components Inhibit the Expression of Inflammatory Factors

[0186] IL-1β, IL-6 and TNF-α are involved in the body's immune response and inflammatory response and are important pro-inflammatory factors. Figure 8 As shown in B, after ponatinib modeling, the gene expressions of il1b, il6 and tnfb were significantly increased, and administration of Angong Niuhuang Wan and equivalent component groups could significantly reduce the gene expressions of il1b, il6 and tnfb (p<0.05&p<0.0001&p<0.05), indicating that Angong Niuhuang Wan and equivalent component groups may improve ischemic stroke damage by blocking inflammatory response.

[0187] 9.3 Angong Niuhuang Pills and their equivalent components inhibit the expression of apoptosis-related genes

[0188] Caspase 8 and Caspase 9 are involved in the initiation of apoptosis, while Caspase 3 and Caspase 7 are involved in the execution of apoptosis, thereby inhibiting DNA repair and cleaving DNA chains. Figure 8 As shown in C, after ponatinib modeling, the expression of apoptosis-related genes casp3, casp7, casp8 and casp9 was significantly increased, and the administration of Angong Niuhuang Pills and equivalent component groups could significantly reduce the expression of casp3, casp7, casp8 and casp 9 genes (p<0.05&p<0.01&p<0.001&p<0.001), indicating that Angong Niuhuang Pills and equivalent component groups may improve ischemic stroke damage by blocking cell apoptosis.

[0189] 9.4 Angong Niuhuang Pills and Equivalent Components Upregulate the Expression of Neurotrophic Factor Genes

[0190] In addition to its well-known neurotrophic effects, brain-derived neurotrophic factor (BDNF) also plays an important role in brain plasticity and repair. The concentration of BDNF is related to the recovery after brain injury. Glial cell-derived nerve growth factor (GDNF) has a protective effect on various injuries to the central nervous system and nervous tissues by reducing apoptosis and necrosis. Myelin basic protein (MBP) is the main protein component of the central nervous system and the myelin sheath of certain neurons. Cytoskeleton microtubule protein (α-tubulin) is an important component of the cytoskeleton. Figure 8 As shown in D, after ponatinib modeling, the gene expressions of gdnfa, bdnf, mbp and atatl all showed a downward trend. Administration of Angong Niuhuang Wan and equivalent component groups could significantly increase the gene expressions of bdnf, gdnfa, mbp and atatl (p<0.05&p<0.01&p<0.01&p<0.01), indicating that Angong Niuhuang Wan and equivalent component groups may improve ischemic stroke damage by promoting neural repair.

[0191] It can be seen from the above examples that the present invention provides an equivalent component group for improving ischemic stroke injury with Angong Niuhuang, which is composed of 28, 39 or 64 chemical components. The zebrafish cerebral ischemia model and the mouse MCAO model prove that the equivalent component group has a significant improvement effect on ischemic stroke injury, and the improvement effect is not significantly different from that of the original drug of Angong Niuhuang Pills, showing excellent equivalence. The present invention provides a certain reference for clarifying the material and chemical basis of the efficacy of Angong Niuhuang Pills, and promotes the research on the material basis of the efficacy and quality standards of Angong Niuhuang Pills, specifically:

[0192] (1) The combination of these 28, 39 or 64 ingredients can be prepared into a pharmaceutical composition that is equivalent to Angong Niuhuang Pills in improving ischemic stroke damage;

[0193] (2) The combination of these 28, 39 or 64 components can be prepared into a marker composition for controlling the quality of Angong Niuhuang Pills, so as to monitor the stability, repeatability and controllability of the anti-ischemic stroke activity of Angong Niuhuang Pills by the stability, repeatability and controllability of the chemical components.

[0194] The purpose of the above-mentioned embodiments is to specifically introduce the essential content of the present invention, but those skilled in the art should know that the protection scope of the present invention should not be limited to the specific embodiments.

Claims

1. An equivalent composition representing the efficacy of Angong Niuhuang Pill in improving ischemic stroke damage, characterized in that: It is composed of the following 28 compounds: (+)-borneol, baicalin, isoborneol, cholic acid, berberine, taurocholic acid, geniposide, glycocholic acid, deoxycholic acid, baicalin, coptisine, palmatine, musk ketone, baicalein, aromatic curcumin, β-caryophyllene, baicalein, chenodeoxycholic acid, melaleucalyptol A, curcumin, rutin, ursolic acid, ursodeoxycholic acid, isoquercetin, demethoxycurcumin, bisdemethoxycurcumin, eucalyptol and tauroursodeoxycholic acid; and the content ratio of each compound is the same as that in Angong Niuhuang Pills.

2. An equivalent composition representing the efficacy of Angong Niuhuang Pill in improving ischemic stroke injury, characterized in that: It is composed of the following 39 compounds: (+)-borneol, baicalin, isoborneol, cholic acid, berberine, taurocholic acid, geniposide, glycocholic acid, deoxycholic acid, wogonin, coptisine, palmatine, taurodeoxycholic acid, melaleucaside, musk ketone, 3-O-feruloylquinic acid, epiberberine, geniposide, norwogonin, baicalein, aromatic turmeric, Jatrorrhizine, β-caryophyllene, baicalein, chenodeoxycholic acid, melaleucalyptol A, α-curcumene, curcumin, rutin, chlorogenic acid, ursolic acid, ursodeoxycholic acid, isoquercetin, neochlorogenic acid, demethoxycurcumin, lithocholic acid, bisdemethoxycurcumin, eucalyptol and tauroursodeoxycholic acid; and the content ratio of each compound is the same as that in Angong Niuhuang Pills.

3. An equivalent composition representing the efficacy of Angong Niuhuang Pill in improving ischemic stroke injury, characterized in that: The invention is composed of the following 64 compounds: (+)-borneol, baicalin, isoborneol, cholic acid, berberine, taurocholic acid, geniposide, glycocholic acid, deoxycholic acid, wogonin, coptisine, palmatine, taurodeoxycholic acid, melaleucoside, musk ketone, 3-O-feruloylquinic acid, epiberberine, geniposide, nor-wogonin, glycodeoxycholic acid, crocin-I, magnoliavine, 5-O-feruloylquinic acid, fangfang pine, baicalein, 7-keto-3α,12α-dihydroxycholanic acid, 4-O-feruloylquinic acid, zingiber officinale, taurochenodeoxycholic acid, jatrorrhizine, β-caryophyllene, grandisin, white Salicin-7-O-β-glucuronide, gardenia jasminoides B, glycochenodeoxycholic acid, baicalein, chenodeoxycholic acid, melaleucatin A, scutellaria baicalensis flavonoids II, α-curcumene, curcumin, demethyleneberberine, rutin, chlorogenic acid, synthetic camphor, ursolic acid, ursodeoxycholic acid, scutellaria baicalensis flavonoids I, 8-oxidoberine, isoquercetin, neochlorogenic acid, baicalein, demethoxycurcumin, terpinolene, geniposide, lithocholic acid, bisdemethoxycurcumin, camphene, eucalyptol, α-caryophyllene, tauroursodeoxycholic acid, α-pinene, limonene and terpinene; and the content ratio of each compound is the same as that in Angong Niuhuang Pills.

4. Use of the equivalent composition described in claim 1, 2 or 3 for preparing a drug that is equivalent to and replaces Angong Niuhuang Pills in improving ischemic stroke damage.

5. The use according to claim 4, characterized in that: The equivalent composition is used as an active ingredient and is prepared into a pharmaceutically acceptable dosage form through a pharmaceutically acceptable carrier or excipient.

6. The use according to claim 5, characterized in that: The carrier or auxiliary material is solid, liquid or semi-solid.

7. The use according to claim 5, characterized in that: The dosage forms include tablets, capsules, pills and injections.

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