Abstract Febrile seizures (FS) are the most common type of seizures for children. As a commonly used representative cold formula for resuscitation, Zixue Powder (ZP) has shown great efficacy for the treatment of FS in clinic, while its active ingredients and underlying mechanism remain largely unclear. This study aimed to preliminarily elucidate the material basis of ZP and the potential mechanism for the treatment of FS through ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS), network pharmacology, and molecular docking. UPLC-Q-TOF-MS was firstly applied to characterize the ingredients in ZP, followed by network pharmacology to explore the potential bioactive ingredients and pathways of ZP against FS. Furthermore, molecular docking technique was employed to verify the binding affinity between the screened active ingredients and targets. As a result, 75 ingredients were identified, containing flavonoids, chromogenic ketones, triterpenes and their saponins, organic acids, etc. Through the current study, we focused on 13 potential active ingredients and 14 key potential anti-FS targets of ZP, such as IL6, STAT3, TNF, and MMP9. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis showed that inflammatory response, EGFR tyrosine kinase inhibitor resistance, AGE-RAGE signaling pathway in diabetic complications, and neuroactive ligand-receptor interaction were the main anti-FS signaling pathways. Licochalcones A and B, 26-deoxycimicifugoside, and hederagenin were screened as the main potential active ingredients by molecular docking. In conclusion, this study provides an effective in-depth investigation of the chemical composition, potential bioactive components, and possible anti-FS mechanism of ZP, which lays the foundation for pharmacodynamic studies and clinical applications of ZP. Keywords: Zixue powder, Febrile seizures, UPLC-Q-TOF-MS, Network pharmacology, Molecular docking 1. Introduction Febrile seizures (FS) are the most common convulsive disease for infants and children aged 6 months to 5 years, affecting 2–5% of children worldwide [[37][1], [38][2], [39][3]]. Studies have shown that recurrent FS may increase the risk of temporal lobe epilepsy, psychiatric disease, and even death [[40]4,[41]5]. The occurrence of FS has been linked to inflammation, familial history, and deficiencies of certain elements (iron, selenium, and zinc) [[42][6], [43][7], [44][8], [45][9]]. In clinical practice, anticonvulsant drugs, such as phenobarbital, diazepam, and valproic acid, have been used to treat FS [[46]10]. According to traditional Chinese medicine (TCM) theory, FS belong to the category of “acute convulsion”, and the curing principle for FS is to relieve spasm, clear heat, eliminate phlegm, relieve shock, and calm wind [[47]11], which has been proved to be effective for FS [[48]12]. Zixue Powder (ZP) is a classical Chinese herbal formula for FS in children, composed of 16 TCMs, including Gypsum Fibrosum (Shigao), Gypsum Rubrum (Beihanshuishi), Talcum (Huashi), Magnetitum (Cishi), Scrophulariae Radix (Xuanshen, XS), Cimicifugae Rhizoma (Shengma, SM), Glycyrrhizae Radix et Rhizoma (Gancao, GC), Caryophylli Flos (Dingxiang, DX), Aucklandiae Radix (Muxiang, MX), Aquilariae Lignum Resinatum (Chenxiang, CX), Natrii Sulfas (Mangxiao), Nitre (Xiaoshi), Powerdered Buffalo Horn Extract (Shuiniujiaonongsuofen), Saigae Tataricae Cornu (Lingyangjiaofen), artificial Mosk (Rengongshexiang), and Cinnabaris (Zhusha) [[49]13]. ZP can clear heat and induce resuscitation, relieve spasmolysis and tranquilize based on TCM theory. Modern pharmacological studies have demonstrated that ZP has antipyretic, sedative, anti-inflammatory, and anticonvulsant effects [[50]14,[51]15]. At present, ZP has been frequently used in the clinical treatment of children with FS, septic shock, etc [[52]16]. However, no comprehensive studies have been conducted to explore the chemical constituents in ZP and the underlying mechanisms for anti-FS. Network pharmacology is an integrated approach that blends the disciplines of systems biology and bioinformatics and network science to investigate the underlying molecular mechanisms between drugs and therapeutic targets, which has been frequently used in the research of TCM [[53]17]. Meanwhile, molecular docking can be used to screen active ingredients by predicting the binding affinity between ingredients and proteins, which is a method widely used in drug discovery [[54]18,[55]19]. The combination of network pharmacology and molecular docking allows for greater efficiency on screening active ingredients and identifying their potential targets in TCM [[56]20]. In this research, we identified the possible active compounds and the potential molecular action mechanisms of ZP against FS. A fast and effective method was developed for the chemical characterization of ZP based on ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS), and a strategy combined with network pharmacology and molecular docking was further used to explore the underlying action mechanism of ZP against FS. This study provides a reference for clinical practice and further research on ZP. 2. Materials and methods 2.1. Materials and reagents Reference standards including agarotetrol, isoferulic acid, and harpagide were obtained from National Institutes for Food and Drug Control (Beijing, China). Liquiritigenin, isoliquiritigenin, angoroside C, cimicifugine, glycyrrhizic acid, liquiritin, liquiritin apioside, isoliquiritin, trans-cinnamic acid, harpagoside, sucrose, formononetin, and licochalcone A were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). The purities of all the reference substances were higher than 98 %. ZP samples were provided by Tianjin Hongrentang Pharmaceutical Co., Ltd. (Tianjin, China). LC-MS grade methanol was purchased from Sigma-Aldrich (St. Louis, MO, USA). Formic acid was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Water used for UPLC-Q-TOF-MS analysis was purified by Milli-Q water purification system (Millipore, Billerica, MA, USA). 2.2. Preparation of reference and sample solutions Sixteen reference compounds were accurately weighed and respectively dissolved in methanol as stock solutions except for sucrose dissolved in water. An appropriate amount of each reference stock solution was accurately transferred in a 10 mL volumetric flask and diluted with 75 % aqueous methanol (v/v) to obtain the mixed solution at the concentrations of 0.0521 mg/mL harpagide, 0.0500 mg/mL liquiritin, 0.0501 mg/mL cimicifugin, 0.0555 mg/mL isoliquiritin, 0.0501 mg/mL agarotetrol, 0.0581 mg/mL apioside liquiritin, 0.0500 mg/mL angoroside C, 0.0503 mg/mL liquiritigenin, 0.0500 mg/mL isoferulic acid, 0.0501 mg/mL isoliquiritigenin, 0.0502 mg/mL harpagoside, 0.0511 mg/mL glycyrrhizic acid, 0.0500 mg/mL trans-cinnamic acid, 0.0304 mg/mL licoricechalcone A, 0.0487 mg/mL sucrose, and 0.0560 mg/mL formononetin. 1.0 g of ZP was accurately weighed and transferred into a conical flask and ultrasonically extracted at 60 °C by 75 % aqueous methanol for 30 min, then cooled to ambient temperature and diluted to scale by adding 75 % aqueous methanol. The extracted solution was centrifuged at 12700 rpm for 10 min to obtain the supernatant. All the standard and sample solutions were stored at 4 °C when not in use. 2.3. UPLC-Q-TOF-MS analysis Chemical analysis was conducted on an Acquity UPLC I Class system (Waters Corporation, Milford, MA, USA) equipped with XEVO G2-XS quadrupole time-of-flight mass spectrometry (Waters Corp.). UPLC analysis was performed on the Agilent ZORBAX SB-C18 column (4.6 × 100 mm, 1.8 μm, Agilent, Santa Clara, CA, USA) using a flow rate of 0.5 mL/min at 45 °C for chromatographic separation. The mobile phase consisted of methanol (A) and 0.1 % formic acid (B), using a gradient elution of 10–52 % A for 0–8 min, 52–58 % A for 8–12 min, and 58–95 % A for 12–20 min. The injection volume was 2 μL. The mass spectrometric detection was operated on Waters Xevo G2-XS QTOF mass spectrometer equipped with ESI source in both positive and negative modes from m/z 100–1500 Da. Detailed mass spectrometric parameters were set as follows: the capillary voltage was set at 3.00 kV (ESI^+) and −2.5 kV (ESI^–); the ion source temperature was 120 °C; the sample cone voltage was 40 V; the cone gas flow was set at 50 L/h; the desolvation temperature was set at 500 °C; the desolvation gas flow was 800 L/h. The low collision energy was 6 eV and the high collision energy was 10∼45 eV. 2.4. Network pharmacology analysis 2.4.1. Target genes of the identified compounds related to FS The structures of compounds in ZP were downloaded from PubChem ([57]https://pubchem.ncbi.nlm.nih.gov/) and saved in SDF format, which were submitted to SwissTargetPrediction database ([58]http://www.swisstargetprediction.ch/) and Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP, [59]https://www.tcmsp-e.com/) for the targets prediction of the focused compounds with the species “Homo sapiens”. All the proteins obtained previously were converted to the official symbol formats for genes using UniProt database ([60]https://www.uniprot.org/). Meanwhile, the FS-related targets were obtained from GeneCards database ([61]https://www.genecards.org/) and Online Mendelian Inheritance in Man database (OMIM, [62]https://www.omim.org/). Then, the common targets between the predicted targets of chemical compounds and FS were obtained by Venny 2.1 platform ([63]https://bioinfogp.cnb.csic.es/tools/venny/index.html), which were considered as potential therapeutic targets. To further illustrate the mechanism, Cytoscape 3.9.1 software was used for the visualization of the interactions among TCMs, ingredients, and targets in ZP. The potential bioactive compounds were screened according to the degree values, which were used as key parameters to reflect the importance of nodes. 2.4.2. Gene Ontology (GO) and Kyoto Encyclopedia of genes and genomes (KEGG) pathway enrichment analysis The common genes were imported into DAVID database ([64]https://david.ncifcrf.gov/) to perform GO and KEGG pathway enrichment analysis with species restricted to “Homo sapiens”. The results of biological process (BP), cellular component (CC), and molecular function (MF) of GO enrichment analysis, and KEGG pathway analysis were downloaded and saved as TXT, respectively. Go terms and KEGG pathways were visualized by Bioinformatics ([65]http://www.bioinformatics.com.cn/) with P-value <0.05. 2.4.3. Construction of protein-protein interaction (PPI) network Targets of the common genes were imported into STRING ([66]https://cn.string-db.org/) database. The species was limited to “Homo sapiens” in the operation interface, and the confidence score was set up to 0.70 (higher confidence). Cytoscape 3.9.1 software was used to construct the PPI network, and the core targets were screened by two algorithms of maximal clique centrality (MCC) and degree value, which could improve accuracy of the results. 2.5. Molecular docking verification The crystal structures of the targets were acquired from the RCSB Protein Data Bank ([67]https://www.rcsb.org/), whose detailed information was displayed in [68]Table 1. The tested compound's structure files (mol2 format) were downloaded from TCMSP database, and then converted into a 3D structure using Chem 3D 19.0. Then, the 3D structures of target proteins and compounds were imported into Discovery Studio 2020 software. The test compounds as ligands were prepared to constrain the optimal conformation. The target proteins were prepared by removing water molecules, adding hydrogen, fixing the missing residues or rings, and defining the active center. The tested compound was then docked to the target by CDocker module, from which the CDocker interaction energy scores were ranked to predict the interaction capacity. Based on the CDocker interaction energy value, the interaction between the ligand and the receptor was quantified, with the lower energy indicating the stronger interaction. Table 1. Detailed information on the crystal structures of the targets. No. Full name of the protein Abbreviation PDB ID Resolution (Å) References