Preliminary screening of anti-caries active compounds of Caesalpinia sappan

Preliminary screening of anti-caries active compounds of Caesalpinia sappan | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Preliminary screening of anti-caries active compounds of Caesalpinia sappan Yongliang Jia, Dongdong Zhang, Hongxing Lu, Yanhong Li, Yuehu wang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7620115/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 14 Apr, 2026 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Dental caries represents a major global oral health issue, primarily caused by Streptococcus mutans ( S. mutans ). While previous studies have indicated that Caesalpinia sappan ( CS ) possesses anti-caries activity, its specific anti-caries chemical constituents remain largely unexplored. This study aimed to identify the potential anti-caries constituents of CS using an integrated approach combining network pharmacology, ultra-Performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS), bioassay-guided isolation targeting S. mutans , and molecular docking. We identified a total of 35 active ingredients in CS , sharing 79 potential therapeutic targets associated with dental caries, including AKT1, EGFR, BCL2, PTGS2, MMP9, ERBB2, and HSP90AA1. Key pathways implicated included nitrogen metabolism and calcium signaling. Bioassay-guided isolation yielded 33 compounds in the fifth fraction from CS . However, only brazilin overlapped with the network pharmacology-predicted key ingredients. Brazilin effectively inhibited biofilm formation and acid production by S. mutans and demonstrated strong binding affinity to EGFR, BCL2, and MMP9 in molecular docking analysis, with docking scores all below -7 kcal/mol. Collectively, these findings not only elucidate the anti-caries chemical constituents of CS and their potential mechanisms of action but also provide novel insights into the bioactive constituents and mechanisms underlying natural plant-derived medicines for the prevention and treatment of dental caries. Biological sciences/Biochemistry Health sciences/Diseases Biological sciences/Drug discovery Biological sciences/Microbiology Dental caries Caesalpinia sappan Streptococcus mutans Network pharmacology Molecular docking Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 1. Introduction According to the Global Burden of Disease Study 2021, dental caries ranks as one of the most prevalent oral diseases globally [ 1 ] . As dental caries progresses, it leads to defects in dental hard tissues, impairing masticatory function. Moreover, untreated dental caries can progress to oral diseases such as pulpitis and apical periodontitis, significantly impacting oral health [ 2 ] . Beyond the oral cavity, severe dental caries has been associated with an elevated risk of systemic conditions, including cardiovascular and gastrointestinal diseases, thereby affecting overall systemic health [ 3 ] . Dental caries represents an infectious disease that causes chronic and progressive destruction of the hard tissues of the teeth, mainly by various factors such as bacteria. Currently, Streptococcus mutans ( S. mutans ) is still considered the main cariogenic microorganism. S. mutans has exceptional acid-producing and acid-resistant properties. It can produce acidic metabolites through glycolysis, resulting in the formation of an acidic environment within the dental plaque [ 4 ] . S. mutans can survive in an acidic environment and maintain its glycolysis ability, and ultimately the long-term presence of acidic metabolites causes demineralization of tooth enamel, which ultimately leads to the development of dental caries [ 5 ] . Furthermore, S. mutans can also synthesize large amounts of extracellular polysaccharide (EPS) utilizing sucrose substrate. These EPS assemble into structurally complex biofilms that enhance bacterial adhesion and provide protection for embedded microorganisms [ 5 , 6 ] . Currently, two types of anti-caries agents are used clinically to effectively prevent the initiation and progression of dental caries. The first is fluoride, which remains the most effective anti-caries agent in global public health [ 7 ] . Fluoride inhibits S. mutans growth and promotes enamel remineralization [ 8 ] . However, excessive fluoride exposure may cause adverse effects of fluorosis, fluoride-resistant microorganisms, and disruption of oral microecological balance [ 9 – 11 ] . The alternative agent is chlorhexidine (CHX), which is a highly effective cationic surface-active disinfectant with a broad spectrum of antibacterial effects. However, long-term use can cause noticeable staining of the tooth surface and taste alterations, and even cause dysbiosis of the commensal oral flora [ 12 ] . These limitations underscore the need for novel anti-caries therapeutics. In recent years, with the continuous development of natural plant medicine research, it has garnered significant scientific interest. Natural plants represent promising anti-caries candidates owing to their wide sources, favorable safety profiles, and minimal induction of microbial resistance. Numerous natural plant extracts demonstrate inhibitory activity against common oral pathogens, such as Galla chinensis, Rhodiola, and Arnebia root, which have been used to prevent dental caries [ 13 – 15 ] . Their primary mechanisms involve: (1) inhibition of the growth, acid production, and biofilm formation of S. mutans and (2) preventing demineralization or promoting remineralization of hard tooth tissues. Our group's previous study investigated the inhibitory effect of six common natural plant extracts from Yunnan Province, China, on common dominant bacteria in the oral cavity, and found that the extract of Caesalpinia sappan (CSE) had a better inhibitory effect on S. mutans [ 16 ] . Nevertheless, the specific bioactive constituents responsible for CS need to be further explored. Network pharmacology is an emerging interdisciplinary approach integrating pharmacology, bioinformatics, and systems biology. This methodology enables systematic construction of drug-target-disease interaction networks to elucidate bioactive compounds and their mechanisms of action [ 17 ] . Complementary to this, molecular docking serves as a pivotal drug discovery tool that computationally predicts binding affinities and conformational orientations between candidate molecules and target proteins [ 18 , 19 ] . This study aims to elucidate the anti-caries compounds and molecular mechanisms using integrated network pharmacology and molecular docking, and further screen the possible anti-caries active components in CS through bioassay-guided isolation based on the inhibitory activity of S. mutans , in order to provide a foundation for dental caries-preventive studies of CS , as well as contribute to the public health endeavor of caries prevention and treatment. 2. Materials and methods 2.1 Network pharmacology analysis 2.1.1 Data acquisition We obtained the compounds for CS from TCMSP databases ( https://www.tcmsp-e.com/tcmsp.php ) and HERB databases ( http://herb.ac.cn/ ), obtained the compounds’s Simplified Molecular Input Line Entry Specification (SMILES) from Pubchem database ( https://pubchem.ncbi.nlm.nih.gov/ ), and obtained the related targets from Swiss Target Prediction database ( http://www.swisstargetprediction.ch/ ) [ 20 , 21 ] . We limited our search to the “Homo sapiens” species and probability “0” to find the targets of compounds. We obtained the target genes for dental caries from the GeneCards ( https://www.genecards.org/ ), TTD ( https://db.idrblab.net/ttd/ ), and OMIM ( https://omim.org/ ) database using “dental caries” and “caries” as search keywords. We used the online Venn platform ( http://bioinformatics.psb.ugent.be/webtools/Venn/ ) to visualize the results. 2.1.2 PPI network construction In order to explore the core targets from coincident targets of CS and dental caries, the coincident targets were input into the STRING 12.0 database ( https://cn.string-db.org/ ). The biological was set to “Homo sapience”, the meaning of network edges was set to “confidence”, the minimum required interaction score was set to “medium confidence (0.400)”. Then, the Cytoscape version 3.10.0 was used to construct the Protein-Protein Interaction (PPI) network among targets. In this network analysis, the degree value is used to reflect the interaction intensity between targets. 2.1.3 GO and pathway enrichment analyses The targets were input into Metascape database ( https://metascape.org/ ) to identify the term enrichment and analyze the share genes. The species was set as “Homo sapience” and the statistical significance of enrichment was set as “P value cut off = 0.01”. We limited the minimum number of pathways of 3, showing only the top 20. Then, the online Weishengxin platform ( http://www.bioinformatics.com.cn/ ) was used to visualize the analytical results. This analysis included Gene Ontology (GO) enrichment for Biological Process (BP), Molecular Function (MF), Cellular Component (CC), and KEGG metabolic pathways. 2.1.4 Construction of the target-drug-pathways network The Cytoscape version 3.10.0 was used to constructed the interaction network of target-drug-pathways. 2.2 Bioassay-guided isolation of compounds from CS against S. mutans 2.2.1 Chemicals and reagents The heartwood of CS was purchased from Luosiwan market (Kunming, China). Ethyl alcohol (EtOH), petroleum ether (PE), ethyl acetate (EtOAc), n -butanol was purchased from Chuandong Chemical Co., LTD (Chongqing, China). S. mutans UA159 was provided by the Yunnan Key Laboratory of Stomatology (China). Crystal violet (CV) and sucrose were purchased from Yuanye (Shanghai, China) and Solarbio (Beijing, China). Brain heart infusion (BHI) broth and agar were from Bacton, Dickinson and Company. 2.2.2 Plant materials and extracts preparation The CS powders were ultrasonically extracted for 30 min with different concertation EtOH (0–90%) at 60°C. Gradient EtOH crude extract were obtained by concentrated the solvent. The 70% EtOH extract were suspended in water and then successively extracted with petroleum ether, ethyl acetate, and n -butanol to yield the petroleum ether-soluble, ethyl acetate-soluble, and n -butanol-soluble extracts, and the remaining water phase part. Then, the EtOAc extract were subjected to silica gel column chromatography (PE-EtOAc, 1:0→0:1) to yield fractions 1–10 (Fr. 1–Fr. 10). 2.2.3 Bacterial strains culture conditions S. mutans UA 159 was recovered and inoculated on BHI agar plates, and single bacterial colony was added into BHI broth grown in anaerobic chamber (10% H 2 , 5% CO 2 , and 85% N 2 ) at 37°C. BHI broth was replaced with BHI supplemented with 1% (w/v) sucrose (BHIS) in biofilm assays and glycolytic pH drop assay. 2.2.4 Determination of the minimum inhibitory concentration (MIC) The antimicrobial activity of different extracts was evaluated by MIC [ 22 ] . Different extracts mother solutions were diluted with BHI medium to different concentrations by the doubled dilution method. S. mutans was cultured to mid-log phase and diluted with BHI medium according to the volume ratio of 1:100, and then anaerobically cocultured with different extracts at 37°C for 24 h, the lowest concentration with no bacterial growth was defined as the MIC. 2.2.5 Biofilm formation assays The effect of different extracts on S. mutans was analyzed using crystal violet (CV) staining [ 23 ] . Briefly, S. mutans was diluted using BHIS medium and co-cultured anaerobically with different extracts at 37°C for 24 h. The biofilm was rinsed three times with sterile water. The bacterial cells were fixed with 4% (w/v) paraformaldehyde for 10 min and then stained with 0.1% (w/v) CV solution for 10 min in each well. The solution was removed and rinsed repeatedly and gently with sterile water. The dye was solubilized by adding 33% (v/v) acetic acid solvent to the well plates for 10 min. Finally, the 100 µL of solution per well was pipetted into a new 96-well plate and the optical density (OD) was read at 575 nm using a Microplate reader. 2.2.6 Glycolytic pH drop assay The effect of different extracts on acid production for S. mutans biofilm was assessed by determining the pH of the medium [ 24 ] . S. mutans was co-cultured with different extracts in 24-well plates, the initial pH was adjusted to 7.0 and anaerobically incubated at 37°C for 24 h. The decrease in pH of each well was measured using a pH meter (Mettler Toledo, Switzerland). 2.2.7 UPLC-MS/MS analysis of the extract In order to further explore the composition of active components, Fr. 5 was analyzed by ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) [ 25 ] . The Fr. 5 powder was redissolved in methanol with a concentration of 4 mg/mL by ultrasonication, centrifuge at 13,000 rpm/min for 10 min and take the supernatant for analysis, storage time should not exceed 24 h. UltiMate 3000 UHPLC and Thremo Hypersil gold C18 (2.1 mm×100 mm, 1.9 µm) were used in liquid phase separation, the liquid phase was 0.1% formic acid/methanol (B)-0.1% formic acid/water (A), the gradient elution procedure of the solution, as detailed in Table 1 . Chromatographic condition: 0 − 20 min, flow rate was 0.3 mL/min. Table 1 Mobile phase gradient elution procedure Time (min) A B 0 90 10 1 90 10 13 0 100 17 0 100 17.1 90 10 20 90 10 The mass spectrometer was a Q-Exactive (Thermo Fisher Scientific, CA, USA) HESI source with an ion source temperature of 310°C, a capillary temperature of 320°C, a sheath gas flow rate of 30 units, an auxiliary gas flow rate of 10 units, and a spray voltage of 2.8 kV for the negative ion mode. The analysis was performed using data-dependent scanning analysis (DDA) with the loop count set to 10, and the HCD energies were step-wise normalized collision energies set to 10, 28, and 35 eV. The primary mass spectrometry was scanned over a range of 100–1500 m/z , with the resolution set to 70000, the AGC target set to 3E6, and the injection time set to 200 ms. The resolution of the secondary mass spectrum was set to 17500, the AGC target was set to 1E5, and the injection time was set to 50 ms. 2.3 Molecular Docking We used molecular docking to test the reliability of potential targets for dental caries treatment. The PDB files of the targets were obtained from PDB database ( https://www.rcsb.org/ ) and the SDF files of the compound 2D structure were obtained from Pubchem database ( https://pubchem.ncbi.nlm.nih.gov/ ). We used the AutoDock Vina software to perform the molecular docking, and the PyMol software to visualize the results. 2.4 Statistical analysis Statistical analysis was performed using GraphPad Prism 9.5.1 (California, USA). T-test was used for comparisons between two groups. One-way ANOVA was performed to assess variance among multiple groups, and Tukey’s multiple comparison test was used for multiple comparisons. All significant differences were identified at 0.05. 3. Results 3.1 Network pharmacology analysis 3.1.1 Active ingredients of CS After excluding active ingredients without predicted targets, a total of 35 active ingredients were obtained (Table 2 ) Table 2 Information on the main active ingredients of the CS Code number Ingredient ID Ingredient name Ingredient formula C1 HBIN001020 1,3,4,5-tetracaffeoylquinic acid C 43 H 36 O 18 C2 HBIN002875 1-octacosanol C 28 H 58 O C3 HBIN007845 3,9-dihydroxy-8-methoxydibenzo [b, d] pyran-6-one C 14 H 10 O 5 C4 HBIN008435 3-deoxysappanchalcone C 16 H 14 O 4 C5 HBIN008436 3'-deoxysappanol C 16 H 16 O 5 C6 HBIN008437 3-deoxysappanone b C 16 H 14 O 5 C7 HBIN008792 3'-methoxy-4',5,7-trihydroxyflavone C 16 H 12 O 6 C8 HBIN012903 7,3′,4′-trihydroxy-3-benzyl-2 H -chromene C 16 H 14 O 4 C9 HBIN013224 7-hydroxy-3-(4'-hydroxybenzylidene)-chroman-4-one C 16 H 12 O 4 C10 HBIN018719 bonducellin C 17 H 14 O 4 C11 HBIN018818 brazilein C 16 H 12 O 5 C12 HBIN018819 brazilin C 16 H 14 O 5 C13 HBIN024649 (e)-2-nonenal C 9 H 16 O C14 HBIN025418 episappanol C 16 H 16 O 6 C15 HBIN027030 gallic acid C 7 H 6 O 5 C16 HBIN028989 hematein C 16 H 12 O 6 C17 HBIN034745 menthol C 10 H 20 O C18 HBIN035418 (−)-methyl selina-3,11-dien-14-oate C 16 H 24 O 2 C19 HBIN036718 neoprotosappanin C 32 H 26 O 10 C20 HBIN038026 oleic acid C 18 H 34 O 2 C21 HBIN038089 ombuin C 17 H 14 O 7 C22 HBIN039794 phytol C 20 H 40 O C23 HBIN040939 protosappanin A C 15 H 12 O 5 C24 HBIN040941 protosappanin A dimethyl acetal C 17 H 18 O 6 C25 HBIN040942 protosappanin B C 16 H 16 O 6 C26 HBIN040948 protostemonine C 23 H 31 NO 6 C27 HBIN041495 quercetin C 15 H 10 O 7 C28 HBIN042158 rhamnetin C 16 H 12 O 7 C29 HBIN043129 sappanchalcone C 16 H 14 O 5 C30 HBIN043131 sappanol C 16 H 16 O 6 C31 HBIN043133 sappanone b C 16 H 14 O 6 C32 HBIN043148 sarcosine C 3 H 7 NO 2 C33 HBIN044730 stearic acid C 18 H 36 O 2 C34 HBIN045541 Taraxerol C 30 H 50 O C35 HBIN046012 tetraacetylbrazilin C 24 H 22 O 9 3.1.2 Potential targets prediction Using a probability threshold > 0 as the selection criterion, 348 putative targets were obtained for the 35 active compounds, and 2568 dental caries targets were retrieved. Through Venn mapping, 79 common targets of CS and dental caries were obtained (Fig. 1 ). 3.1.3 PPI network construction and analysis The interaction among 79 overlapping targets were analyzed by Protein-protein interaction (PPI) network. The number of edges in PPI network is 444, and the degree value of average nodes is 11.53. In addition, the degree values of 34 targets were exceeded the average value (Fig. 2 ), identifying AKT1, EGFR, BCL2, PTGS2, MMP9, ERBB2, and HSP90AA1 as topologically significant hub nodes. These high-degree targets represent potential key mediators of CS ’s anti-caries mechanisms. 3.1.4 GO function and KEGG pathway enrichment Functional enrichment analysis identified 881 significant GO terms and 113 KEGG pathways. The results show that the most significant biological processes were associated with cellular response to nitrogen compound and cellular response to hormone stimulus, the most significant cellular component was associated with extracellular matrix, and the most significant molecular function was related to protein kinase activity (Fig. 3 A). Moreover, KEGG pathway enrichment analysis demonstrated that the overlapped targets were mainly enriched in the nitrogen metabolism, pathways in cancer, prostate cancer, microRNAs in cancer, calcium signaling pathway (Fig. 3 B). 3.1.5 Target-drug-pathways (T-D-P) network in dental caries construction and analysis The final interaction network was constructed using the previously obtained information of 79 overlapping targets, 17 KEGG pathways involved in the anti-caries mechanism of CS . The results of the T-D-P network revealed five hub pathways with degree centrality exceeding the network average (12.53): prostate cancer, pathways in cancer, calcium signaling pathway, microRNAs in cancer, and nitrogen metabolism. Concurrently, the 11 active compounds were prioritized based on the degree value of average nodes (7.86), including C7, C4, C3, C1, C6, C15, C8, C27, C35, C19, and C12 (Fig. 4 ). 3.2 Bioassay-guided isolation of active ingredients from CS against S. mutans 3.2.1 Effect of ethanol extracts on biofilm formation and acid production in S. mutans According to the results, the MIC of the 0% ethanol extract was 1.25 mg/mL, while extracts prepared with 30–90% ethanol exhibited a lower MIC of 0.625 mg/mL. The effect of ethanol extracts on S. mutans biofilm formation is shown in Fig. 5 A. The crystal violet staining experiment revealed that compared to the control group, the biomass of S. mutans biofilms could be effectively decreased by 70–90% ethanol extracts at 1/2 MIC concentration (89–93% reduction). Furthermore, as shown in Fig. 5 B, all ethanol extracts at 1/2 MIC concentration could inhibit acid production by S. mutans compared to the control. 3.2.2 Effect of EtOAc, n-Butanol, and water extracts on biofilm formation and acid production in S. mutans EtOAc, n -butanol, and water extracts were obtained by further fractionating the 70% ethanol extract with various solvents. A PE fraction was not obtained in significant yield. The MIC value of the EtOAc extract was 0.625 mg/mL, the MIC value of the n -butanol extract was 0.3125 mg/mL, and the MIC value of the water extract was more than 5 mg/mL. CV staining revealed that only the EtOAc extract significantly inhibited S. mutans biofilm formation (Fig. 6 A, B). Furthermore, assessment of acid production inhibition showed that only the EtOAc extract significantly inhibited S. mutans acid production (Fig. 6 C, D), consistent with the biofilm formation results. 3.2.3 Effect of different fractions on biofilm formation and acid production in S. mutans The EtOAc extract was further fractionated to yield 10 fractions (Fr. 1-Fr. 10), of which the MIC valus of Fr. 5 was 0.625 mg/mL, Fr. 8, Fr. 9 and Fr. 10 were 2.5 mg/mL, and the other fractions were more than 5 mg/mL. The inhibition of biofilm formation of S. mutans by different fractions was investigated by CV staining and it was found that Fr. 5, Fr. 8, and Fr. 10 were effective in inhibiting the biofilm formation at the 1/2 MIC and 1/4 MIC, and Fr. 9 could significantly inhibit the biofilm formation at only 1/2 MIC (Fig. 7 A). However, in acid production, Fr. 5 and Fr. 10 could effectively inhibit the acid production by S. mutans , Fr. 8 and Fr. 9 could significantly inhibit the acid production at only 1/2 MIC (Fig. 7 C). Interestingly, Fr. 2 and Fr. 6 inhibit the acid production at 2.5 mg/mL (Fig. 7 D). These results suggest that different components may influence the cariogenicity of S. mutans through distinct mechanisms. 3.2.4 The ingredients analysis of F5 by UPLC-MS/MS Due to its superior inhibitory activity against S. mutans biofilm formation and acid production at lower concentrations compared to other fractions, F5 was selected for compositional analysis. According to the retention time, the high-resolution accurate molecular weight and MS/MS fragment information of each chemical component were obtained by UPLC-MS detection. A total of 33 constituents were tentatively identified in F5. The total ion chromatogram (TIC) in negative ion mode is presented in Fig. 8 . Based on relative peak abundance, the major compounds were tentatively identified as brazilin, sappanol, and 3-deoxysappanone B (Table 3 ). Comparison with the network pharmacology-predicted anti-caries components in CS revealed brazilin as the sole overlapping constituent. This suggests brazilin is a key anti-caries component in CS (Fig. 9 ). Table 3 List of ingredients detected from Fr. 5. No RT (min) Molecular formula Measured mass [M − H] ⁻ ( m/z ) Putative Identification Peak area (%) 1 6.594 C 16 H 14 O 5 285.07718 brazilin 23.31 2 3.361 C 16 H 16 O 6 303.0878 sappanol 20.73 3 4.02 C 16 H 14 O 5 285.07712 3-deoxysappanone B 18.26 4 5.604 C 16 H 12 O 5 283.06155 brazilein 9.03 5 5.805 C 16 H 14 O 6 301.07199 hematoxylin 7.80 6 5.709 C 15 H 12 O 5 271.06149 protosappanin A 1.90 7 7.47 C 15 H 12 O 5 271.06149 3',4',7-trihydroxyflavanone 1.38 8 7.006 C 15 H 10 O 7 301.03577 quercetin 0.77 9 3.388 C 7 H 6 O 3 137.02348 2,5-dihydroxybenzaldehyde 0.74 10 7.256 C 16 H 14 O 4 269.08221 10,11-dihydroxydracaenone C 0.73 11 1.929 C 8 H 8 O 4 167.03436 6-methoxysalicylic acid 0.49 12 8.798 C 17 H 14 O 7 329.06702 ombuin 0.32 13 12.04 C 18 H 32 O 4 293.21265 9-HpODE 0.30 14 7.242 C 16 H 12 O 7 315.05148 isorhamnetin 0.29 15 5.682 C 15 H 12 O 7 303.05133 taxifolin 0.28 16 4.703 C 8 H 8 O 2 135.04424 2-methylbenzoic acid 0.14 17 8.513 C 16 H 12 O 6 299.05634 tectorigenin 0.10 18 5.187 C 9 H 8 O 3 163.03932 2-hydroxycinnamic acid 0.07 19 6.903 C 7 H 6 O 3 137.02338 3-hydroxybenzoic acid 0.06 20 3.837 C 8 H 8 O 4 167.03421 isovanillic acid 0.06 21 2.24 C 8 H 10 O 3 153.05489 3,4-dihydroxyphenylethanol 0.06 22 15.953 C 14 H 30 O 4 293.17972 myristyl sulfate 0.05 23 18.608 C 30 H 48 O 3 455.35391 ursolic acid 0.05 24 3.877 C 10 H 8 O 4 191.03423 7,8-dihydroxy-4-methylcoumarin 0.05 25 5.807 C 8 H 8 O 3 151.03908 2-hydroxyphenylacetic acid 0.04 26 11.255 C 16 H 22 O 4 277.14481 dibutyl phthalate 0.02 27 5.307 C 9 H 10 O 5 197.04507 ethyl gallate 0.02 28 6.145 C 11 H 12 O 4 207.0659 sinapoyl aldehyde 0.02 29 3.728 C 9 H 6 O 4 177.01862 7,8-dihydroxycoumarin 0.02 30 1.103 C 16 H 12 O 6 299.05649 hematein 0.02 31 3.567 C 9 H 10 O 3 165.05493 3-hydroxy-1-(4-hydroxyphenyl) propan-1-one 0.01 32 9.279 C 15 H 20 O 3 247.13405 atractylenolide III 0.01 33 9.477 C 14 H 22 O 2 221.15451 2,5-di-tert-butylhydroquinone 0.01 3.2.5 Effect of brazilin on biofilm formation and acid production in S. mutans The aim of this study was to assess the effect of brazilin on S. mutans biofilm formation and acid production. The results demonstrated that brazilin at 1/2 MIC significantly inhibited biofilm formation, reducing total biomass by approximately 36% compared to the control (Fig. 10 A, B). Furthermore, brazilin effectively reduced acid production, as evidenced by a higher final pH compared to the control (Fig. 10 C). 3.3 Validation of key targets using molecular docking Molecular docking analysis was performed between brazilin and seven key targets (AKT1, EGFR, BCL2, PTGS2, MMP9, ERBB2, and HSP90AA1). Generally, lower docking scores indicate stronger predicted binding affinity, with docking scores < -7 kcal/mol typically considered indicative of strong binding. The interaction of brazilin and keys targets was shown in Fig. 11 , the results showed docking scores were − 6.8, -8.3, -7.8, -9.3, -8.5, -6.3, -7.9 kcal/mol respectively, indicating that brazilin demonstrated stronger predicted binding affinity towards EGFR, BCL2, PTGS2, MMP9, and HSP90AA1. 4. Discussion Dental caries remains a global public health concern, adversely impacting both oral and systemic health while imposing significant economic burdens. Natural plant medicine shows considerable promise for dental caries management. In this study, we investigate the underlying targets and molecular mechanisms of CS in dental caries through a network pharmacology approach. Our analysis identified key targets (AKT1, EGFR, BCL2, PTGS2, MMP9, ERBB2, and HSP90AA1) and pathways (nitrogen compound metabolic process, calcium signaling pathway) potentially modulated by CS . Given that S. mutans is the primary microorganism responsible for the occurrence of dental caries, we subsequently isolated the active component(s) of CS through bioassay-guided fractionation targeting S. mutans . This approach coupled with network pharmacology led to the identification of brazilin, which effectively inhibited S. mutans acid production and biofilm formation. Consequently, brazilin was identified as an active compound within CS contributing to dental caries prevention. Furthermore, molecular docking analysis suggests that its anti-caries effects may be mediated through potential interactions with EGFR, BCL2, PTGS2, MMP9, HSP90AA1. Gene ontology enrichment analysis indicated that the molecular function of protein kinase activity is related to CS against dental caries. The activities of numerous protein kinases are critically involved in caries pathogenesis, primarily by modulating S. mutans virulence factors. Specifically, serine/threonine protein kinases (STPKs) and histidine kinase (HK) are involved in two-component system (TCS) signaling of S. mutans and can regulate the expression of metabolism-related genes in the cell wall by sensing environmental signals and activating response regulators [ 26 , 27 ] . KEGG has been used in biological big data analysis, for example, for uncovering pathway through the KEGG mapping procedure [ 28 ] . Concurrently, nitrogen metabolism and calcium signaling are two pathways enriched related to dental caries in KEGG analysis. Nitrogen metabolism is essential for the growth and survival of bacteria, and the bacteria can provide the essential materials by metabolizing nitrogen-containing compounds [ 29 , 30 ] . Furthermore, the first gene of the ciaRH operon in S. mutans is a calcium sensing signal peptide that allows the CiaRH system to regulate the cariogenicity of S. mutans by modulating the expression of its own operon in response to calcium ions in the oral environment [ 31 ] . CS contains a variety of active compounds with obvious antibacterial effects [ 32 ] . Our integrated analyses suggest that CS may interfere with nitrogen metabolism and the calcium signaling pathway to prevent dental caries. Traditional separation methods often face challenges in efficiently isolating compounds with specific biological activities. Bioassay-guided isolation addresses this limitation by integrating activity assessment throughout the separation process, thereby increasing the probability of obtaining bioactive compounds and enhancing their practical utility [ 33 ] . The cariogenicity of S. mutans is primarily related to the formation of dental plaque biofilm and the production of acidic metabolites. Therefore, the inhibitory effects of different extracts on S. mutans biofilm formation and acid production were determined during the isolation process. Initial extraction employed ethanol as the solvent, and the results indicated that 70–90% ethanol extracts exhibited significant inhibitory effect on S. mutans . Given that lower solvent concentrations can better preserve compound activity [ 34 ] . Therefore, the 70% ethanol extract was selected for further fractionation. Further, UPLC-MS/MS analysis of the Fr. 5 identified brazilin as the predominant compound. Combined with the potential active compounds screened in the network pharmacology analysis, only brazilin overlapped with the Fr. 5 of UPLC-MS/MS analysis, and brazilin was the main compound obtained the heartwood of CS [ 35 ] . Brazilin was identified as the key compound within CS responsible for dental caries prevention. Furthermore, its demonstrated efficacy in inhibiting S. mutans biofilm formation and acid production substantiates its anti-caries potential. Analysis of potential targets identified AKT1, EGFR, BCL2, PTGS2, MMP9, ERBB2, and HSP90AA1 as potential key targets. Molecular docking analysis further assessed the interactions between brazilin and these proteins. Lower docking scores indicate stronger predicted binding affinity. Notably, the docking scores for EGFR, BCL2, PTGS2, MMP9, and HSP90AA1 were all below − 7 kcal/mol, suggesting strong binding potential. EGFR plays a critical role in proliferation, differentiation, development, and promoting downstream signaling, the expression of EGFR is up-regulated in dental caries, which may be involved in the extracellular matrix (ECM) degradation process [ 36 , 37 ] . Interestingly, brazilin has been shown to reduce ECM synthesis, suggesting a potential counteracting mechanism [ 38 ] . BCL2 is involved in apoptosis through multiple pathways, and has been reported to be overexpressed in teeth with advanced dental caries [ 39 , 40 ] . A study showed that brazilin can suppress the expression of BCL2 [ 41 ] . PTGS2, also known as cyclooxygenase-2 (COX-2), is an inducible enzyme associated with prostaglandin synthesis and also plays an important role in inflammatory responses, cell proliferation, and apoptosis [ 42 , 43 ] . However, PTGS2 is mainly related to dental pulp inflammation in dental caries research, the AgI/II protein produced by S. mutans is capable of binding to receptors on the host cell surface, which is responsible for producing a variety of inflammatory mediators and causing the occurrence of dental pulp inflammation [ 44 , 45 ] . MMP9 is a protein of the matrix metalloproteinase family that plays an essential role in the proteolysis of the ECM, and may be involved in the destruction of the dentin organic matrix during caries [ 46 ] . A study showed that brazilin can suppress MMP9 [ 41 ] . HSP90AA1 encodes heat shock protein 90α (Hsp90α), involved in cell cycle control, signal transduction, and other physiological processes [ 47 ] . However, it also promotes inflammation induced by Porphyromonas gingivalis lipopolysaccharide via autophagy regulation [ 48 ] . It is hypothesized that HSP90AA1 might similarly influence caries-associated bacteria through autophagy. Based on the stronger evidence linking their functions to caries pathogenesis and the direct experimental evidence of brazilin's modulation, EGFR, BCL2, and MMP9 emerge as the most plausible primary targets for brazilin's anti-caries effects. Finally, this study provides insights into the potential of CS for dental caries prevention and treatment and proposes potential mechanism. However, our findings require further experimental validation. An additional limitation of this study is that although only S. mutans was selected for the activity screening, considering its important role in the occurrence and development of dental caries. Nevertheless, brazilin, identified as a key active component of CS , demonstrated significant potential anti-caries effects against this key cariogenic bacterium. 5. Conclusion In conclusion, we propose that CS exerts its anti-caries effects potentially through modulation of seven key targets (AKT1, EGFR, BCL2, PTGS2, MMP9, ERBB2, and HSP90AA1) and two pathways (nitrogen metabolism and calcium signaling pathway). Furthermore, brazilin was identified as the active compound within CS responsible for caries prevention and treatment. Molecular docking analysis revealed EGFR, BCL2, and MMP9 as its core interacting targets, with spontaneous binding predicted for each. Therefore, brazilin represents a promising candidate compound for dental caries prevention, likely acting through multi-target and multi-pathway mechanisms. Declarations Author contributions Yongliang Jia: conceptualization, performed the experiments, data curation, formal analysis, visualization, validation, writing– ori ginal draft. Yongliang Jia, Hongxing Lu: performed the experiments, data curation, formal analysis, visualization. Dongdong Zhang: conceptualization, supervision, writing– review & editing. Yanhong Li, Yuehu Wang: conceptualization, funding acquisition, project administration, supervision, validation, writing– review & editing. Conflicts of interest The author(s) declare no competing interests. Data availability statement Data available on request due to privacy or ethical restrictions. If someone wants to request the data from this study, please contact the Yanhong Li. Funding This work was supported by the National Natural Science Foundation of China (grant number 82160179), the "Xingdian Talent Support Plan" of Yunnan Province-Medical and Health Talents Special Project (grant number XDYC-YLWS-2023-0047), the Major Science and Technology Projects in Yunnan Province (grant number 202302AA310038), the Degree and Graduate Education Innovation Fund Project of Kunming Medical University (grant number 2025B007), and the National Key Clinical Specialty Development Project of Pediatric Dentistry Division (grant number 20230610). References Bernabe E. et al. Trends in the global, regional, and national burden of oral conditions from 1990 to 2021: a systematic analysis for the Global Burden of Disease Study 2021. The Lancet . 405(10482), 897-910 (2025). Pitts N B. et al. Dental caries. Nature Reviews Disease Primers . 25, 3, 17030; 10.1038/nrdp.2017.30 IF: 60.6 Q1 (2017). Wang X H, Xu Z Q, Bian Z, Meng L Y. 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Cite Share Download PDF Status: Published Journal Publication published 14 Apr, 2026 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 30 Oct, 2025 Reviews received at journal 27 Oct, 2025 Reviews received at journal 25 Oct, 2025 Reviewers agreed at journal 07 Oct, 2025 Reviewers agreed at journal 07 Oct, 2025 Reviewers invited by journal 07 Oct, 2025 Editor assigned by journal 07 Oct, 2025 Editor invited by journal 26 Sep, 2025 Submission checks completed at journal 22 Sep, 2025 First submitted to journal 22 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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14:47:11","extension":"html","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":164651,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7620115/v1/7542521095b4bc8d2b18ccba.html"},{"id":94118744,"identity":"41a7777e-a5e3-447e-9f36-a3e6ee0958cc","added_by":"auto","created_at":"2025-10-22 14:47:10","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":50743,"visible":true,"origin":"","legend":"\u003cp\u003eVenn diagram of the intersection of the \u003cem\u003eCS\u003c/em\u003etargets and dental caries targets.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7620115/v1/16c69b4d569d1a57e6305462.png"},{"id":94120142,"identity":"cb7603ed-757d-4dc3-b244-71516eeb0efa","added_by":"auto","created_at":"2025-10-22 14:55:10","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":500139,"visible":true,"origin":"","legend":"\u003cp\u003ePPI network of the 79 potential therapeutic targets. Each node represents a relevant gene, and the edge thickness indicates the strength of the data support.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7620115/v1/ab6fa520f9f71329afb8ea39.png"},{"id":94118749,"identity":"1cdfa457-8de1-430c-b4ab-7a7cbd27c306","added_by":"auto","created_at":"2025-10-22 14:47:10","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":146468,"visible":true,"origin":"","legend":"\u003cp\u003eGO enrichment and KEGG pathway enrichment analysis. (A) GO enrichment analysis: the top 10 Biological Process (BP), Cellular Component (CC) and Molecular Function (MF); (B) Bubble chart of the KEGG pathways.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7620115/v1/ae1e5a6b7f913e72a6ce9c98.png"},{"id":94118746,"identity":"75888efe-d933-48b1-b053-2f3b63c40c05","added_by":"auto","created_at":"2025-10-22 14:47:10","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":770327,"visible":true,"origin":"","legend":"\u003cp\u003eT-D-P network. The diamonds nodes indicate metabolic pathways, the triangle nodes indicate active ingredients of \u003cem\u003eCS\u003c/em\u003e, the blue oval nodes indicate targets. The lines indicate interactions.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7620115/v1/154790aa779e53e8ea2fdaa2.png"},{"id":94120729,"identity":"ef1aa906-e5c6-4ab0-a0d5-bbdc0ef2fccc","added_by":"auto","created_at":"2025-10-22 15:03:10","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":49207,"visible":true,"origin":"","legend":"\u003cp\u003eDifferent ethanol extracts on biofilm formation and acid production in \u003cem\u003eS. mutans\u003c/em\u003e. (A) Quantitative analysis by crystal violet staining. (B) \u003cem\u003eS. mutans\u003c/em\u003emedium pH measurement.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7620115/v1/9aa82052e85be421bdbfb16e.png"},{"id":94120145,"identity":"94919825-e694-4762-8967-2ce914b5088c","added_by":"auto","created_at":"2025-10-22 14:55:10","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":49366,"visible":true,"origin":"","legend":"\u003cp\u003eEtoAc, \u003cem\u003en\u003c/em\u003e-butanol, and water extracts on biofilm formation and acid production in \u003cem\u003eS. mutans\u003c/em\u003e. (A, B) Quantitative analysis by crystal violet staining. (C, D) \u003cem\u003eS. mutans\u003c/em\u003e medium pH measurement.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7620115/v1/9a079a8bb8d23f50038e71f2.png"},{"id":94120146,"identity":"4dbf4932-0cd7-49f7-ba47-65c1701e8056","added_by":"auto","created_at":"2025-10-22 14:55:10","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":56165,"visible":true,"origin":"","legend":"\u003cp\u003eDifferent fractions on biofilm formation and acid production in \u003cem\u003eS. mutans\u003c/em\u003e. (A) Quantitative analysis by crystal violet staining. (B) \u003cem\u003eS. mutans\u003c/em\u003e medium pH measurement.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7620115/v1/a7b633308552ec092d792ac9.png"},{"id":94118754,"identity":"15cb2dee-7ed4-454f-82d5-bddb305157c8","added_by":"auto","created_at":"2025-10-22 14:47:10","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":66796,"visible":true,"origin":"","legend":"\u003cp\u003eThe total ion current map of Fr. 5 in the negative ion mode.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-7620115/v1/83bfddd1a03b02e54638f831.png"},{"id":94121478,"identity":"af55f433-cc16-4bc2-a946-c8fded5a239e","added_by":"auto","created_at":"2025-10-22 15:11:11","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":24585,"visible":true,"origin":"","legend":"\u003cp\u003eThe chemical structural formula of brazilin.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-7620115/v1/96d421c03e2094cbe9aa943c.png"},{"id":94120733,"identity":"27126e75-b609-4a79-9424-d32f28ae03fc","added_by":"auto","created_at":"2025-10-22 15:03:11","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":98034,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of brazilin on biofilm formation and acid production in \u003cem\u003eS. mutans\u003c/em\u003e. (A) Crystal violet staining. (B) Quantitative analysis by crystal violet staining. (C) \u003cem\u003eS. mutans\u003c/em\u003e medium pH measurement.\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-7620115/v1/81e6e88af9fde0f0a994b8a3.png"},{"id":94120150,"identity":"341d0bd8-1abc-4073-878b-a117aaf54681","added_by":"auto","created_at":"2025-10-22 14:55:11","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":284338,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic diagram of the interaction of brazilin and keys targets. (A) Brazilin-AKT1. (B) Brazilin-EGFR. (C) Brazilin-BCL2. (D) Brazilin-PTGS2. (E) Brazilin-MMP9. (F) Brazilin-ERBB2. (G) Brazilin-HSP90AA1.\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-7620115/v1/7f4097a9ac86086abfd23823.png"},{"id":107350919,"identity":"192dc958-d8b1-4576-8494-f3d2441d22a6","added_by":"auto","created_at":"2026-04-20 16:07:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2598786,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7620115/v1/71417a90-c72a-43ba-a1e0-b10e56d7bd38.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Preliminary screening of anti-caries active compounds of Caesalpinia sappan","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAccording to the Global Burden of Disease Study 2021, dental caries ranks as one of the most prevalent oral diseases globally\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. As dental caries progresses, it leads to defects in dental hard tissues, impairing masticatory function. Moreover, untreated dental caries can progress to oral diseases such as pulpitis and apical periodontitis, significantly impacting oral health\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. Beyond the oral cavity, severe dental caries has been associated with an elevated risk of systemic conditions, including cardiovascular and gastrointestinal diseases, thereby affecting overall systemic health\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eDental caries represents an infectious disease that causes chronic and progressive destruction of the hard tissues of the teeth, mainly by various factors such as bacteria. Currently, \u003cem\u003eStreptococcus mutans\u003c/em\u003e (\u003cem\u003eS. mutans\u003c/em\u003e) is still considered the main cariogenic microorganism. \u003cem\u003eS. mutans\u003c/em\u003e has exceptional acid-producing and acid-resistant properties. It can produce acidic metabolites through glycolysis, resulting in the formation of an acidic environment within the dental plaque\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. \u003cem\u003eS. mutans\u003c/em\u003e can survive in an acidic environment and maintain its glycolysis ability, and ultimately the long-term presence of acidic metabolites causes demineralization of tooth enamel, which ultimately leads to the development of dental caries\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Furthermore, \u003cem\u003eS. mutans\u003c/em\u003e can also synthesize large amounts of extracellular polysaccharide (EPS) utilizing sucrose substrate. These EPS assemble into structurally complex biofilms that enhance bacterial adhesion and provide protection for embedded microorganisms\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eCurrently, two types of anti-caries agents are used clinically to effectively prevent the initiation and progression of dental caries. The first is fluoride, which remains the most effective anti-caries agent in global public health\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. Fluoride inhibits \u003cem\u003eS. mutans\u003c/em\u003e growth and promotes enamel remineralization\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. However, excessive fluoride exposure may cause adverse effects of fluorosis, fluoride-resistant microorganisms, and disruption of oral microecological balance\u003csup\u003e[\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. The alternative agent is chlorhexidine (CHX), which is a highly effective cationic surface-active disinfectant with a broad spectrum of antibacterial effects. However, long-term use can cause noticeable staining of the tooth surface and taste alterations, and even cause dysbiosis of the commensal oral flora\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. These limitations underscore the need for novel anti-caries therapeutics.\u003c/p\u003e\u003cp\u003eIn recent years, with the continuous development of natural plant medicine research, it has garnered significant scientific interest. Natural plants represent promising anti-caries candidates owing to their wide sources, favorable safety profiles, and minimal induction of microbial resistance. Numerous natural plant extracts demonstrate inhibitory activity against common oral pathogens, such as Galla chinensis, Rhodiola, and Arnebia root, which have been used to prevent dental caries\u003csup\u003e[\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. Their primary mechanisms involve: (1) inhibition of the growth, acid production, and biofilm formation of \u003cem\u003eS. mutans\u003c/em\u003e and (2) preventing demineralization or promoting remineralization of hard tooth tissues. Our group's previous study investigated the inhibitory effect of six common natural plant extracts from Yunnan Province, China, on common dominant bacteria in the oral cavity, and found that the extract of \u003cem\u003eCaesalpinia sappan\u003c/em\u003e (CSE) had a better inhibitory effect on \u003cem\u003eS. mutans\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. Nevertheless, the specific bioactive constituents responsible for \u003cem\u003eCS\u003c/em\u003e need to be further explored.\u003c/p\u003e\u003cp\u003eNetwork pharmacology is an emerging interdisciplinary approach integrating pharmacology, bioinformatics, and systems biology. This methodology enables systematic construction of drug-target-disease interaction networks to elucidate bioactive compounds and their mechanisms of action\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. Complementary to this, molecular docking serves as a pivotal drug discovery tool that computationally predicts binding affinities and conformational orientations between candidate molecules and target proteins\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThis study aims to elucidate the anti-caries compounds and molecular mechanisms using integrated network pharmacology and molecular docking, and further screen the possible anti-caries active components in \u003cem\u003eCS\u003c/em\u003e through bioassay-guided isolation based on the inhibitory activity of \u003cem\u003eS. mutans\u003c/em\u003e, in order to provide a foundation for dental caries-preventive studies of \u003cem\u003eCS\u003c/em\u003e, as well as contribute to the public health endeavor of caries prevention and treatment.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Network pharmacology analysis\u003c/h2\u003e\u003cdiv id=\"Sec4\" class=\"Section3\"\u003e\u003ch2\u003e2.1.1 Data acquisition\u003c/h2\u003e\u003cp\u003eWe obtained the compounds for \u003cem\u003eCS\u003c/em\u003e from TCMSP databases (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.tcmsp-e.com/tcmsp.php\u003c/span\u003e\u003cspan address=\"https://www.tcmsp-e.com/tcmsp.php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and HERB databases (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://herb.ac.cn/\u003c/span\u003e\u003cspan address=\"http://herb.ac.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), obtained the compounds\u0026rsquo;s Simplified Molecular Input Line Entry Specification (SMILES) from Pubchem database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubchem.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://pubchem.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and obtained the related targets from Swiss Target Prediction database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.swisstargetprediction.ch/\u003c/span\u003e\u003cspan address=\"http://www.swisstargetprediction.ch/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. We limited our search to the \u0026ldquo;Homo sapiens\u0026rdquo; species and probability \u0026ldquo;0\u0026rdquo; to find the targets of compounds. We obtained the target genes for dental caries from the GeneCards (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.genecards.org/\u003c/span\u003e\u003cspan address=\"https://www.genecards.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), TTD (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://db.idrblab.net/ttd/\u003c/span\u003e\u003cspan address=\"https://db.idrblab.net/ttd/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and OMIM (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://omim.org/\u003c/span\u003e\u003cspan address=\"https://omim.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) database using \u0026ldquo;dental caries\u0026rdquo; and \u0026ldquo;caries\u0026rdquo; as search keywords. We used the online Venn platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioinformatics.psb.ugent.be/webtools/Venn/\u003c/span\u003e\u003cspan address=\"http://bioinformatics.psb.ugent.be/webtools/Venn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to visualize the results.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section3\"\u003e\u003ch2\u003e2.1.2 PPI network construction\u003c/h2\u003e\u003cp\u003eIn order to explore the core targets from coincident targets of \u003cem\u003eCS\u003c/em\u003e and dental caries, the coincident targets were input into the STRING 12.0 database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cn.string-db.org/\u003c/span\u003e\u003cspan address=\"https://cn.string-db.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The biological was set to \u0026ldquo;Homo sapience\u0026rdquo;, the meaning of network edges was set to \u0026ldquo;confidence\u0026rdquo;, the minimum required interaction score was set to \u0026ldquo;medium confidence (0.400)\u0026rdquo;. Then, the Cytoscape version 3.10.0 was used to construct the Protein-Protein Interaction (PPI) network among targets. In this network analysis, the degree value is used to reflect the interaction intensity between targets.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\u003ch2\u003e2.1.3 GO and pathway enrichment analyses\u003c/h2\u003e\u003cp\u003eThe targets were input into Metascape database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://metascape.org/\u003c/span\u003e\u003cspan address=\"https://metascape.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to identify the term enrichment and analyze the share genes. The species was set as \u0026ldquo;Homo sapience\u0026rdquo; and the statistical significance of enrichment was set as \u0026ldquo;P value cut off =\u0026thinsp;0.01\u0026rdquo;. We limited the minimum number of pathways of 3, showing only the top 20. Then, the online Weishengxin platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.bioinformatics.com.cn/\u003c/span\u003e\u003cspan address=\"http://www.bioinformatics.com.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to visualize the analytical results. This analysis included Gene Ontology (GO) enrichment for Biological Process (BP), Molecular Function (MF), Cellular Component (CC), and KEGG metabolic pathways.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\u003ch2\u003e2.1.4 Construction of the target-drug-pathways network\u003c/h2\u003e\u003cp\u003eThe Cytoscape version 3.10.0 was used to constructed the interaction network of target-drug-pathways.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Bioassay-guided isolation of compounds from \u003cem\u003eCS\u003c/em\u003e against \u003cem\u003eS. mutans\u003c/em\u003e\u003c/h2\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003e2.2.1 Chemicals and reagents\u003c/h2\u003e\u003cp\u003eThe heartwood of \u003cem\u003eCS\u003c/em\u003e was purchased from Luosiwan market (Kunming, China). Ethyl alcohol (EtOH), petroleum ether (PE), ethyl acetate (EtOAc), \u003cem\u003en\u003c/em\u003e-butanol was purchased from Chuandong Chemical Co., LTD (Chongqing, China). \u003cem\u003eS. mutans\u003c/em\u003e UA159 was provided by the\u003c/p\u003e\u003cp\u003eYunnan Key Laboratory of Stomatology (China). Crystal violet (CV) and sucrose were purchased from Yuanye (Shanghai, China) and Solarbio (Beijing, China). Brain heart infusion (BHI) broth and agar were from Bacton, Dickinson and Company.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section3\"\u003e\u003ch2\u003e2.2.2 Plant materials and extracts preparation\u003c/h2\u003e\u003cp\u003eThe \u003cem\u003eCS\u003c/em\u003e powders were ultrasonically extracted for 30 min with different concertation EtOH (0\u0026ndash;90%) at 60\u0026deg;C. Gradient EtOH crude extract were obtained by concentrated the solvent. The 70% EtOH extract were suspended in water and then successively extracted with petroleum ether, ethyl acetate, and \u003cem\u003en\u003c/em\u003e-butanol to yield the petroleum ether-soluble, ethyl acetate-soluble, and \u003cem\u003en\u003c/em\u003e-butanol-soluble extracts, and the remaining water phase part. Then, the EtOAc extract were subjected to silica gel column chromatography (PE-EtOAc, 1:0\u0026rarr;0:1) to yield fractions 1\u0026ndash;10 (Fr. 1\u0026ndash;Fr. 10).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section3\"\u003e\u003ch2\u003e2.2.3 Bacterial strains culture conditions\u003c/h2\u003e\u003cp\u003e\u003cem\u003eS. mutans\u003c/em\u003e UA 159 was recovered and inoculated on BHI agar plates, and single bacterial colony was added into BHI broth grown in anaerobic chamber (10% H\u003csub\u003e2\u003c/sub\u003e, 5% CO\u003csub\u003e2\u003c/sub\u003e, and 85% N\u003csub\u003e2\u003c/sub\u003e) at 37\u0026deg;C. BHI broth was replaced with BHI supplemented with 1% (w/v) sucrose (BHIS) in biofilm assays and glycolytic pH drop assay.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\u003ch2\u003e2.2.4 Determination of the minimum inhibitory concentration (MIC)\u003c/h2\u003e\u003cp\u003eThe antimicrobial activity of different extracts was evaluated by MIC\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. Different extracts mother solutions were diluted with BHI medium to different concentrations by the doubled dilution method. \u003cem\u003eS. mutans\u003c/em\u003e was cultured to mid-log phase and diluted with BHI medium according to the volume ratio of 1:100, and then anaerobically cocultured with different extracts at 37\u0026deg;C for 24 h, the lowest concentration with no bacterial growth was defined as the MIC.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\u003ch2\u003e2.2.5 Biofilm formation assays\u003c/h2\u003e\u003cp\u003eThe effect of different extracts on \u003cem\u003eS. mutans\u003c/em\u003e was analyzed using crystal violet (CV) staining\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. Briefly, \u003cem\u003eS. mutans\u003c/em\u003e was diluted using BHIS medium and co-cultured anaerobically with different extracts at 37\u0026deg;C for 24 h. The biofilm was rinsed three times with sterile water. The bacterial cells were fixed with 4% (w/v) paraformaldehyde for 10 min and then stained with 0.1% (w/v) CV solution for 10 min in each well. The solution was removed and rinsed repeatedly and gently with sterile water. The dye was solubilized by adding 33% (v/v) acetic acid solvent to the well plates for 10 min. Finally, the 100 \u0026micro;L of solution per well was pipetted into a new 96-well plate and the optical density (OD) was read at 575 nm using a Microplate reader.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section3\"\u003e\u003ch2\u003e2.2.6 Glycolytic pH drop assay\u003c/h2\u003e\u003cp\u003eThe effect of different extracts on acid production for \u003cem\u003eS. mutans\u003c/em\u003e biofilm was assessed by determining the pH of the medium\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. \u003cem\u003eS. mutans\u003c/em\u003e was co-cultured with different extracts in 24-well plates, the initial pH was adjusted to 7.0 and anaerobically incubated at 37\u0026deg;C for 24 h. The decrease in pH of each well was measured using a pH meter (Mettler Toledo, Switzerland).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section3\"\u003e\u003ch2\u003e2.2.7 UPLC-MS/MS analysis of the extract\u003c/h2\u003e\u003cp\u003eIn order to further explore the composition of active components, Fr. 5 was analyzed by ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS)\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. The Fr. 5 powder was redissolved in methanol with a concentration of 4 mg/mL by ultrasonication, centrifuge at 13,000 rpm/min for 10 min and take the supernatant for analysis, storage time should not exceed 24 h. UltiMate 3000 UHPLC and Thremo Hypersil gold C18 (2.1 mm\u0026times;100 mm, 1.9 \u0026micro;m) were used in liquid phase separation, the liquid phase was 0.1% formic acid/methanol (B)-0.1% formic acid/water (A), the gradient elution procedure of the solution, as detailed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Chromatographic condition: 0\u0026thinsp;\u0026minus;\u0026thinsp;20 min, flow rate was 0.3 mL/min.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eMobile phase gradient elution procedure\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTime (min)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eA\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eB\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e90\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e90\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e17.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e90\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e90\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe mass spectrometer was a Q-Exactive (Thermo Fisher Scientific, CA, USA) HESI source with an ion source temperature of 310\u0026deg;C, a capillary temperature of 320\u0026deg;C, a sheath gas flow rate of 30 units, an auxiliary gas flow rate of 10 units, and a spray voltage of 2.8 kV for the negative ion mode.\u003c/p\u003e\u003cp\u003eThe analysis was performed using data-dependent scanning analysis (DDA) with the loop count set to 10, and the HCD energies were step-wise normalized collision energies set to 10, 28, and 35 eV. The primary mass spectrometry was scanned over a range of 100\u0026ndash;1500 \u003cem\u003em/z\u003c/em\u003e, with the resolution set to 70000, the AGC target set to 3E6, and the injection time set to 200 ms. The resolution of the secondary mass spectrum was set to 17500, the AGC target was set to 1E5, and the injection time was set to 50 ms.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Molecular Docking\u003c/h2\u003e\u003cp\u003eWe used molecular docking to test the reliability of potential targets for dental caries treatment. The PDB files of the targets were obtained from PDB database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.rcsb.org/\u003c/span\u003e\u003cspan address=\"https://www.rcsb.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and the SDF files of the compound 2D structure were obtained from Pubchem database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubchem.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://pubchem.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). We used the AutoDock Vina software to perform the molecular docking, and the PyMol software to visualize the results.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Statistical analysis\u003c/h2\u003e\u003cp\u003eStatistical analysis was performed using GraphPad Prism 9.5.1 (California, USA). T-test was used for comparisons between two groups. One-way ANOVA was performed to assess variance among multiple groups, and Tukey\u0026rsquo;s multiple comparison test was used for multiple comparisons. All significant differences were identified at 0.05.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1 Network pharmacology analysis\u003c/h2\u003e\n \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e\n \u003ch2\u003e3.1.1 Active ingredients of \u003cem\u003eCS\u003c/em\u003e\u003c/h2\u003e\n \u003cp\u003eAfter excluding active ingredients without predicted targets, a total of 35 active ingredients were obtained (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e)\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eInformation on the main active ingredients of the \u003cem\u003eCS\u003c/em\u003e\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCode number\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eIngredient ID\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eIngredient name\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eIngredient formula\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHBIN001020\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1,3,4,5-tetracaffeoylquinic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e43\u003c/sub\u003eH\u003csub\u003e36\u003c/sub\u003eO\u003csub\u003e18\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHBIN002875\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1-octacosanol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e28\u003c/sub\u003eH\u003csub\u003e58\u003c/sub\u003eO\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHBIN007845\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3,9-dihydroxy-8-methoxydibenzo [b, d] pyran-6-one\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e14\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHBIN008435\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3-deoxysappanchalcone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e14\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHBIN008436\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u0026apos;-deoxysappanol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e16\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHBIN008437\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3-deoxysappanone b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e14\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHBIN008792\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u0026apos;-methoxy-4\u0026apos;,5,7-trihydroxyflavone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHBIN012903\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7,3\u0026prime;,4\u0026prime;-trihydroxy-3-benzyl-2\u003cem\u003eH\u003c/em\u003e-chromene\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e14\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHBIN013224\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7-hydroxy-3-(4\u0026apos;-hydroxybenzylidene)-chroman-4-one\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHBIN018719\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ebonducellin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e17\u003c/sub\u003eH\u003csub\u003e14\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHBIN018818\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ebrazilein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHBIN018819\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ebrazilin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e14\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHBIN024649\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(e)-2-nonenal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e9\u003c/sub\u003eH\u003csub\u003e16\u003c/sub\u003eO\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHBIN025418\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eepisappanol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e16\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHBIN027030\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003egallic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e7\u003c/sub\u003eH\u003csub\u003e6\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHBIN028989\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ehematein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHBIN034745\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ementhol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e10\u003c/sub\u003eH\u003csub\u003e20\u003c/sub\u003eO\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHBIN035418\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(\u0026minus;)-methyl selina-3,11-dien-14-oate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e24\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHBIN036718\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eneoprotosappanin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e32\u003c/sub\u003eH\u003csub\u003e26\u003c/sub\u003eO\u003csub\u003e10\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHBIN038026\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eoleic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e34\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHBIN038089\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eombuin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e17\u003c/sub\u003eH\u003csub\u003e14\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHBIN039794\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ephytol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e20\u003c/sub\u003eH\u003csub\u003e40\u003c/sub\u003eO\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHBIN040939\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eprotosappanin A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHBIN040941\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eprotosappanin A dimethyl acetal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e17\u003c/sub\u003eH\u003csub\u003e18\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHBIN040942\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eprotosappanin B\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e16\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHBIN040948\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eprotostemonine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e23\u003c/sub\u003eH\u003csub\u003e31\u003c/sub\u003eNO\u003csub\u003e6\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHBIN041495\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003equercetin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHBIN042158\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003erhamnetin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHBIN043129\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003esappanchalcone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e14\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHBIN043131\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003esappanol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e16\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHBIN043133\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003esappanone b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e14\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHBIN043148\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003esarcosine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e3\u003c/sub\u003eH\u003csub\u003e7\u003c/sub\u003eNO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHBIN044730\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003estearic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e36\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHBIN045541\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTaraxerol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e30\u003c/sub\u003eH\u003csub\u003e50\u003c/sub\u003eO\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHBIN046012\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003etetraacetylbrazilin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e24\u003c/sub\u003eH\u003csub\u003e22\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec21\" class=\"Section3\"\u003e\n \u003ch2\u003e3.1.2 Potential targets prediction\u003c/h2\u003e\n \u003cp\u003eUsing a probability threshold\u0026thinsp;\u0026gt;\u0026thinsp;0 as the selection criterion, 348 putative targets were obtained for the 35 active compounds, and 2568 dental caries targets were retrieved. Through Venn mapping, 79 common targets of \u003cem\u003eCS\u003c/em\u003e and dental caries were obtained (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec22\" class=\"Section3\"\u003e\n \u003ch2\u003e3.1.3 PPI network construction and analysis\u003c/h2\u003e\n \u003cp\u003eThe interaction among 79 overlapping targets were analyzed by Protein-protein interaction (PPI) network. The number of edges in PPI network is 444, and the degree value of average nodes is 11.53. In addition, the degree values of 34 targets were exceeded the average value (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e), identifying AKT1, EGFR, BCL2, PTGS2, MMP9, ERBB2, and HSP90AA1 as topologically significant hub nodes. These high-degree targets represent potential key mediators of \u003cem\u003eCS\u003c/em\u003e\u0026rsquo;s anti-caries mechanisms.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\n \u003ch2\u003e3.1.4 GO function and KEGG pathway enrichment\u003c/h2\u003e\n \u003cp\u003eFunctional enrichment analysis identified 881 significant GO terms and 113 KEGG pathways. The results show that the most significant biological processes were associated with cellular response to nitrogen compound and cellular response to hormone stimulus, the most significant cellular component was associated with extracellular matrix, and the most significant molecular function was related to protein kinase activity (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). Moreover, KEGG pathway enrichment analysis demonstrated that the overlapped targets were mainly enriched in the nitrogen metabolism, pathways in cancer, prostate cancer, microRNAs in cancer, calcium signaling pathway (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec24\" class=\"Section3\"\u003e\n \u003ch2\u003e3.1.5 Target-drug-pathways (T-D-P) network in dental caries construction and analysis\u003c/h2\u003e\n \u003cp\u003eThe final interaction network was constructed using the previously obtained information of 79 overlapping targets, 17 KEGG pathways involved in the anti-caries mechanism of \u003cem\u003eCS\u003c/em\u003e. The results of the T-D-P network revealed five hub pathways with degree centrality exceeding the network average (12.53): prostate cancer, pathways in cancer, calcium signaling pathway, microRNAs in cancer, and nitrogen metabolism. Concurrently, the 11 active compounds were prioritized based on the degree value of average nodes (7.86), including C7, C4, C3, C1, C6, C15, C8, C27, C35, C19, and C12 (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2 Bioassay-guided isolation of active ingredients from \u003cem\u003eCS\u003c/em\u003e against \u003cem\u003eS. mutans\u003c/em\u003e\u003c/h2\u003e\u003cspan\u003e\n \u003cp\u003e\u003cstrong\u003e3.2.1 Effect of ethanol extracts on biofilm formation and acid production in\u003c/strong\u003e \u003cstrong\u003eS. mutans\u003c/strong\u003e According to the results, the MIC of the 0% ethanol extract was 1.25 mg/mL, while extracts prepared with 30\u0026ndash;90% ethanol exhibited a lower MIC of 0.625 mg/mL. The effect of ethanol extracts on \u003cem\u003eS. mutans\u003c/em\u003e biofilm formation is shown in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA. The crystal violet staining experiment revealed that compared to the control group, the biomass of \u003cem\u003eS. mutans\u003c/em\u003e biofilms could be effectively decreased by 70\u0026ndash;90% ethanol extracts at 1/2 MIC concentration (89\u0026ndash;93% reduction). Furthermore, as shown in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB, all ethanol extracts at 1/2 MIC concentration could inhibit acid production by \u003cem\u003eS. mutans\u003c/em\u003e compared to the control.\u003c/p\u003e\n \u003c/span\u003e\u003cspan\u003e\n \u003cp\u003e\u003cstrong\u003e3.2.2 Effect of EtOAc, n-Butanol, and water extracts on biofilm formation and acid production in\u003c/strong\u003e \u003cstrong\u003eS. mutans\u003c/strong\u003e\u003c/p\u003e\n \u003c/span\u003e\n \u003cp\u003eEtOAc, \u003cem\u003en\u003c/em\u003e-butanol, and water extracts were obtained by further fractionating the 70% ethanol extract with various solvents. A PE fraction was not obtained in significant yield. The MIC value of the EtOAc extract was 0.625 mg/mL, the MIC value of the \u003cem\u003en\u003c/em\u003e-butanol extract was 0.3125 mg/mL, and the MIC value of the water extract was more than 5 mg/mL. CV staining revealed that only the EtOAc extract significantly inhibited \u003cem\u003eS. mutans\u003c/em\u003e biofilm formation (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA, B). Furthermore, assessment of acid production inhibition showed that only the EtOAc extract significantly inhibited \u003cem\u003eS. mutans\u003c/em\u003e acid production (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC, D), consistent with the biofilm formation results.\u003c/p\u003e\n \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\n \u003ch2\u003e3.2.3 Effect of different fractions on biofilm formation and acid production in \u003cem\u003eS. mutans\u003c/em\u003e\u003c/h2\u003e\n \u003cp\u003eThe EtOAc extract was further fractionated to yield 10 fractions (Fr. 1-Fr. 10), of which the MIC valus of Fr. 5 was 0.625 mg/mL, Fr. 8, Fr. 9 and Fr. 10 were 2.5 mg/mL, and the other fractions were more than 5 mg/mL. The inhibition of biofilm formation of \u003cem\u003eS. mutans\u003c/em\u003e by different fractions was investigated by CV staining and it was found that Fr. 5, Fr. 8, and Fr. 10 were effective in inhibiting the biofilm formation at the 1/2 MIC and 1/4 MIC, and Fr. 9 could significantly inhibit the biofilm formation at only 1/2 MIC (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eA). However, in acid production, Fr. 5 and Fr. 10 could effectively inhibit the acid production by \u003cem\u003eS. mutans\u003c/em\u003e, Fr. 8 and Fr. 9 could significantly inhibit the acid production at only 1/2 MIC (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eC). Interestingly, Fr. 2 and Fr. 6 inhibit the acid production at 2.5 mg/mL (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eD). These results suggest that different components may influence the cariogenicity of \u003cem\u003eS. mutans\u003c/em\u003e through distinct mechanisms.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e\n \u003ch2\u003e3.2.4 The ingredients analysis of F5 by UPLC-MS/MS\u003c/h2\u003e\n \u003cp\u003eDue to its superior inhibitory activity against \u003cem\u003eS. mutans\u003c/em\u003e biofilm formation and acid production at lower concentrations compared to other fractions, F5 was selected for compositional analysis. According to the retention time, the high-resolution accurate molecular weight and MS/MS fragment information of each chemical component were obtained by UPLC-MS detection. A total of 33 constituents were tentatively identified in F5. The total ion chromatogram (TIC) in negative ion mode is presented in Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e. Based on relative peak abundance, the major compounds were tentatively identified as brazilin, sappanol, and 3-deoxysappanone B (Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). Comparison with the network pharmacology-predicted anti-caries components in \u003cem\u003eCS\u003c/em\u003e revealed brazilin as the sole overlapping constituent. This suggests brazilin is a key anti-caries component in \u003cem\u003eCS\u003c/em\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eList of ingredients detected from Fr. 5.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRT (min)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMolecular formula\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMeasured mass [M\u0026thinsp;\u0026minus;\u0026thinsp;H] ⁻ (\u003cem\u003em/z\u003c/em\u003e)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePutative Identification\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePeak area (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6.594\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e14\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e285.07718\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ebrazilin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e23.31\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.361\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e16\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e303.0878\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003esappanol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e20.73\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e14\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e285.07712\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3-deoxysappanone B\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e18.26\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.604\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e283.06155\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ebrazilein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9.03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.805\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e14\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e301.07199\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ehematoxylin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7.80\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.709\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e271.06149\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eprotosappanin A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.90\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7.47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e271.06149\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u0026apos;,4\u0026apos;,7-trihydroxyflavanone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.38\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7.006\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e301.03577\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003equercetin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.77\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.388\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e7\u003c/sub\u003eH\u003csub\u003e6\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e137.02348\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2,5-dihydroxybenzaldehyde\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.74\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7.256\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e14\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e269.08221\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10,11-dihydroxydracaenone C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.73\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.929\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e8\u003c/sub\u003eH\u003csub\u003e8\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e167.03436\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6-methoxysalicylic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.49\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.798\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e17\u003c/sub\u003eH\u003csub\u003e14\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e329.06702\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eombuin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.32\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e12.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e32\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e293.21265\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9-HpODE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.30\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7.242\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e315.05148\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eisorhamnetin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.29\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.682\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e303.05133\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003etaxifolin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.28\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.703\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e8\u003c/sub\u003eH\u003csub\u003e8\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e135.04424\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2-methylbenzoic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.14\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.513\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e299.05634\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003etectorigenin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.187\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e9\u003c/sub\u003eH\u003csub\u003e8\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e163.03932\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2-hydroxycinnamic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6.903\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e7\u003c/sub\u003eH\u003csub\u003e6\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e137.02338\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3-hydroxybenzoic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.837\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e8\u003c/sub\u003eH\u003csub\u003e8\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e167.03421\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eisovanillic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e8\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e153.05489\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3,4-dihydroxyphenylethanol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e15.953\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e14\u003c/sub\u003eH\u003csub\u003e30\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e293.17972\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003emyristyl sulfate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e18.608\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e30\u003c/sub\u003eH\u003csub\u003e48\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e455.35391\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eursolic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.877\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e10\u003c/sub\u003eH\u003csub\u003e8\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e191.03423\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7,8-dihydroxy-4-methylcoumarin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.807\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e8\u003c/sub\u003eH\u003csub\u003e8\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e151.03908\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2-hydroxyphenylacetic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e11.255\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e22\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e277.14481\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003edibutyl phthalate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.307\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e9\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e197.04507\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eethyl gallate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6.145\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e11\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e207.0659\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003esinapoyl aldehyde\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.728\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e9\u003c/sub\u003eH\u003csub\u003e6\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e177.01862\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7,8-dihydroxycoumarin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.103\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e299.05649\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ehematein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.567\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e9\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e165.05493\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3-hydroxy-1-(4-hydroxyphenyl) propan-1-one\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9.279\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e20\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e247.13405\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eatractylenolide III\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9.477\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e14\u003c/sub\u003eH\u003csub\u003e22\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e221.15451\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2,5-di-tert-butylhydroquinone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec28\" class=\"Section3\"\u003e\n \u003ch2\u003e3.2.5 Effect of brazilin on biofilm formation and acid production in \u003cem\u003eS. mutans\u003c/em\u003e\u003c/h2\u003e\n \u003cp\u003eThe aim of this study was to assess the effect of brazilin on \u003cem\u003eS. mutans\u003c/em\u003e biofilm formation and acid production. The results demonstrated that brazilin at 1/2 MIC significantly inhibited biofilm formation, reducing total biomass by approximately 36% compared to the control (Fig. \u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003eA, B). Furthermore, brazilin effectively reduced acid production, as evidenced by a higher final pH compared to the control (Fig. \u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003eC).\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec29\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3 Validation of key targets using molecular docking\u003c/h2\u003e\n \u003cp\u003eMolecular docking analysis was performed between brazilin and seven key targets (AKT1, EGFR, BCL2, PTGS2, MMP9, ERBB2, and HSP90AA1). Generally, lower docking scores indicate stronger predicted binding affinity, with docking scores \u0026lt; -7 kcal/mol typically considered indicative of strong binding. The interaction of brazilin and keys targets was shown in Fig. \u003cspan class=\"InternalRef\"\u003e11\u003c/span\u003e, the results showed docking scores were \u0026minus;\u0026thinsp;6.8, -8.3, -7.8, -9.3, -8.5, -6.3, -7.9 kcal/mol respectively, indicating that brazilin demonstrated stronger predicted binding affinity towards EGFR, BCL2, PTGS2, MMP9, and HSP90AA1.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eDental caries remains a global public health concern, adversely impacting both oral and systemic health while imposing significant economic burdens. Natural plant medicine shows considerable promise for dental caries management. In this study, we investigate the underlying targets and molecular mechanisms of \u003cem\u003eCS\u003c/em\u003e in dental caries through a network pharmacology approach. Our analysis identified key targets (AKT1, EGFR, BCL2, PTGS2, MMP9, ERBB2, and HSP90AA1) and pathways (nitrogen compound metabolic process, calcium signaling pathway) potentially modulated by \u003cem\u003eCS\u003c/em\u003e. Given that \u003cem\u003eS. mutans\u003c/em\u003e is the primary microorganism responsible for the occurrence of dental caries, we subsequently isolated the active component(s) of \u003cem\u003eCS\u003c/em\u003e through bioassay-guided fractionation targeting \u003cem\u003eS. mutans\u003c/em\u003e. This approach coupled with network pharmacology led to the identification of brazilin, which effectively inhibited \u003cem\u003eS. mutans\u003c/em\u003e acid production and biofilm formation. Consequently, brazilin was identified as an active compound within \u003cem\u003eCS\u003c/em\u003e contributing to dental caries prevention. Furthermore, molecular docking analysis suggests that its anti-caries effects may be mediated through potential interactions with EGFR, BCL2, PTGS2, MMP9, HSP90AA1.\u003c/p\u003e\u003cp\u003eGene ontology enrichment analysis indicated that the molecular function of protein kinase activity is related to \u003cem\u003eCS\u003c/em\u003e against dental caries. The activities of numerous protein kinases are critically involved in caries pathogenesis, primarily by modulating \u003cem\u003eS. mutans\u003c/em\u003e virulence factors. Specifically, serine/threonine protein kinases (STPKs) and histidine kinase (HK) are involved in two-component system (TCS) signaling of \u003cem\u003eS. mutans\u003c/em\u003e and can regulate the expression of metabolism-related genes in the cell wall by sensing environmental signals and activating response regulators\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. KEGG has been used in biological big data analysis, for example, for uncovering pathway through the KEGG mapping procedure \u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. Concurrently, nitrogen metabolism and calcium signaling are two pathways enriched related to dental caries in KEGG analysis. Nitrogen metabolism is essential for the growth and survival of bacteria, and the bacteria can provide the essential materials by metabolizing nitrogen-containing compounds\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. Furthermore, the first gene of the \u003cem\u003eciaRH\u003c/em\u003e operon in \u003cem\u003eS. mutans\u003c/em\u003e is a calcium sensing signal peptide that allows the CiaRH system to regulate the cariogenicity of \u003cem\u003eS. mutans\u003c/em\u003e by modulating the expression of its own operon in response to calcium ions in the oral environment\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. \u003cem\u003eCS\u003c/em\u003e contains a variety of active compounds with obvious antibacterial effects\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. Our integrated analyses suggest that \u003cem\u003eCS\u003c/em\u003e may interfere with nitrogen metabolism and the calcium signaling pathway to prevent dental caries.\u003c/p\u003e\u003cp\u003eTraditional separation methods often face challenges in efficiently isolating compounds with specific biological activities. Bioassay-guided isolation addresses this limitation by integrating activity assessment throughout the separation process, thereby increasing the probability of obtaining bioactive compounds and enhancing their practical utility\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. The cariogenicity of \u003cem\u003eS. mutans\u003c/em\u003e is primarily related to the formation of dental plaque biofilm and the production of acidic metabolites. Therefore, the inhibitory effects of different extracts on \u003cem\u003eS. mutans\u003c/em\u003e biofilm formation and acid production were determined during the isolation process. Initial extraction employed ethanol as the solvent, and the results indicated that 70\u0026ndash;90% ethanol extracts exhibited significant inhibitory effect on \u003cem\u003eS. mutans\u003c/em\u003e. Given that lower solvent concentrations can better preserve compound activity\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. Therefore, the 70% ethanol extract was selected for further fractionation. Further, UPLC-MS/MS analysis of the Fr. 5 identified brazilin as the predominant compound. Combined with the potential active compounds screened in the network pharmacology analysis, only brazilin overlapped with the Fr. 5 of UPLC-MS/MS analysis, and brazilin was the main compound obtained the heartwood of \u003cem\u003eCS\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. Brazilin was identified as the key compound within \u003cem\u003eCS\u003c/em\u003e responsible for dental caries prevention. Furthermore, its demonstrated efficacy in inhibiting \u003cem\u003eS. mutans\u003c/em\u003e biofilm formation and acid production substantiates its anti-caries potential.\u003c/p\u003e\u003cp\u003eAnalysis of potential targets identified AKT1, EGFR, BCL2, PTGS2, MMP9, ERBB2, and HSP90AA1 as potential key targets. Molecular docking analysis further assessed the interactions between brazilin and these proteins. Lower docking scores indicate stronger predicted binding affinity. Notably, the docking scores for EGFR, BCL2, PTGS2, MMP9, and HSP90AA1 were all below \u0026minus;\u0026thinsp;7 kcal/mol, suggesting strong binding potential. EGFR plays a critical role in proliferation, differentiation, development, and promoting downstream signaling, the expression of EGFR is up-regulated in dental caries, which may be involved in the extracellular matrix (ECM) degradation process\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e. Interestingly, brazilin has been shown to reduce ECM synthesis, suggesting a potential counteracting mechanism\u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e. BCL2 is involved in apoptosis through multiple pathways, and has been reported to be overexpressed in teeth with advanced dental caries\u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e. A study showed that brazilin can suppress the expression of BCL2\u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e. PTGS2, also known as cyclooxygenase-2 (COX-2), is an inducible enzyme associated with prostaglandin synthesis and also plays an important role in inflammatory responses, cell proliferation, and apoptosis\u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e. However, PTGS2 is mainly related to dental pulp inflammation in dental caries research, the AgI/II protein produced by \u003cem\u003eS. mutans\u003c/em\u003e is capable of binding to receptors on the host cell surface, which is responsible for producing a variety of inflammatory mediators and causing the occurrence of dental pulp inflammation\u003csup\u003e[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]\u003c/sup\u003e. MMP9 is a protein of the matrix metalloproteinase family that plays an essential role in the proteolysis of the ECM, and may be involved in the destruction of the dentin organic matrix during caries\u003csup\u003e[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]\u003c/sup\u003e. A study showed that brazilin can suppress MMP9\u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e. HSP90AA1 encodes heat shock protein 90α (Hsp90α), involved in cell cycle control, signal transduction, and other physiological processes\u003csup\u003e[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]\u003c/sup\u003e. However, it also promotes inflammation induced by \u003cem\u003ePorphyromonas gingivalis\u003c/em\u003e lipopolysaccharide via autophagy regulation\u003csup\u003e[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]\u003c/sup\u003e. It is hypothesized that HSP90AA1 might similarly influence caries-associated bacteria through autophagy. Based on the stronger evidence linking their functions to caries pathogenesis and the direct experimental evidence of brazilin's modulation, EGFR, BCL2, and MMP9 emerge as the most plausible primary targets for brazilin's anti-caries effects.\u003c/p\u003e\u003cp\u003eFinally, this study provides insights into the potential of \u003cem\u003eCS\u003c/em\u003e for dental caries prevention and treatment and proposes potential mechanism. However, our findings require further experimental validation. An additional limitation of this study is that although only \u003cem\u003eS. mutans\u003c/em\u003e was selected for the activity screening, considering its important role in the occurrence and development of dental caries. Nevertheless, brazilin, identified as a key active component of \u003cem\u003eCS\u003c/em\u003e, demonstrated significant potential anti-caries effects against this key cariogenic bacterium.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn conclusion, we propose that \u003cem\u003eCS\u003c/em\u003e exerts its anti-caries effects potentially through modulation of seven key targets (AKT1, EGFR, BCL2, PTGS2, MMP9, ERBB2, and HSP90AA1) and two pathways (nitrogen metabolism and calcium signaling pathway). Furthermore, brazilin was identified as the active compound within \u003cem\u003eCS\u003c/em\u003e responsible for caries prevention and treatment. Molecular docking analysis revealed EGFR, BCL2, and MMP9 as its core interacting targets, with spontaneous binding predicted for each. Therefore, brazilin represents a promising candidate compound for dental caries prevention, likely acting through multi-target and multi-pathway mechanisms.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYongliang Jia: conceptualization, performed the experiments, data curation, formal analysis, visualization, validation, writing–\u0026nbsp;ori ginal draft.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eYongliang Jia, Hongxing Lu: performed the experiments, data curation, formal analysis, visualization.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDongdong Zhang: conceptualization, supervision, writing–\u0026nbsp;review \u0026amp; editing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eYanhong Li, Yuehu Wang: conceptualization, funding acquisition, project administration, supervision, validation, writing–\u0026nbsp;review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author(s) declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData available on request due to privacy or ethical restrictions. If someone wants to request the data from this study, please contact the Yanhong Li.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (grant number 82160179), the \"Xingdian Talent Support Plan\" of Yunnan Province-Medical and Health Talents Special Project (grant number XDYC-YLWS-2023-0047), the Major Science and Technology Projects in Yunnan Province (grant number 202302AA310038), the Degree and Graduate Education Innovation Fund Project of Kunming Medical University (grant number 2025B007), and the National Key Clinical Specialty Development Project of Pediatric Dentistry Division (grant number 20230610).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBernabe E. et al. 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HSP90AA1 promotes the inflammation in human gingival fibroblasts induced by Porphyromonas gingivalis lipopolysaccharide via regulating of autophagy. \u003cem\u003eBMC Oral Health\u003c/em\u003e. 22(1), 366; 10.1186/s12903-022-02304-0 (2022).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Dental caries, Caesalpinia sappan, Streptococcus mutans, Network pharmacology, Molecular docking","lastPublishedDoi":"10.21203/rs.3.rs-7620115/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7620115/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDental caries represents a major global oral health issue, primarily caused by \u003cem\u003eStreptococcus mutans\u003c/em\u003e (\u003cem\u003eS. mutans\u003c/em\u003e). While previous studies have indicated that \u003cem\u003eCaesalpinia sappan\u003c/em\u003e (\u003cem\u003eCS\u003c/em\u003e) possesses anti-caries activity, its specific anti-caries chemical constituents remain largely unexplored. This study aimed to identify the potential anti-caries constituents of \u003cem\u003eCS\u003c/em\u003e using an integrated approach combining network pharmacology, ultra-Performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS), bioassay-guided isolation targeting \u003cem\u003eS. mutans\u003c/em\u003e, and molecular docking. We identified a total of 35 active ingredients in \u003cem\u003eCS\u003c/em\u003e, sharing 79 potential therapeutic targets associated with dental caries, including AKT1, EGFR, BCL2, PTGS2, MMP9, ERBB2, and HSP90AA1. Key pathways implicated included nitrogen metabolism and calcium signaling. Bioassay-guided isolation yielded 33 compounds in the fifth fraction from \u003cem\u003eCS\u003c/em\u003e. However, only brazilin overlapped with the network pharmacology-predicted key ingredients. Brazilin effectively inhibited biofilm formation and acid production by \u003cem\u003eS. mutans\u003c/em\u003e and demonstrated strong binding affinity to EGFR, BCL2, and MMP9 in molecular docking analysis, with docking scores all below -7 kcal/mol. Collectively, these findings not only elucidate the anti-caries chemical constituents of \u003cem\u003eCS\u003c/em\u003e and their potential mechanisms of action but also provide novel insights into the bioactive constituents and mechanisms underlying natural plant-derived medicines for the prevention and treatment of dental caries.\u003c/p\u003e","manuscriptTitle":"Preliminary screening of anti-caries active compounds of Caesalpinia sappan","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-22 14:47:06","doi":"10.21203/rs.3.rs-7620115/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-30T05:25:33+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-27T13:16:44+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-25T05:14:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"317997929286381721675846712102793577291","date":"2025-10-07T10:16:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"268127561787561260126745423436188732707","date":"2025-10-07T10:05:27+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-07T05:56:08+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-07T05:49:19+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-09-26T15:41:53+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-22T23:52:25+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-09-22T11:40:12+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2011ea96-8f0a-4f3a-b2be-82905c2facde","owner":[],"postedDate":"October 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":56156651,"name":"Biological sciences/Biochemistry"},{"id":56156652,"name":"Health sciences/Diseases"},{"id":56156653,"name":"Biological sciences/Drug discovery"},{"id":56156654,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2026-04-20T16:04:26+00:00","versionOfRecord":{"articleIdentity":"rs-7620115","link":"https://doi.org/10.1038/s41598-026-47281-4","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-04-14 15:58:52","publishedOnDateReadable":"April 14th, 2026"},"versionCreatedAt":"2025-10-22 14:47:06","video":"","vorDoi":"10.1038/s41598-026-47281-4","vorDoiUrl":"https://doi.org/10.1038/s41598-026-47281-4","workflowStages":[]},"version":"v1","identity":"rs-7620115","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7620115","identity":"rs-7620115","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}