Mechanism of Action of Toddalia asiatica (L.) Lam. in the Treatment of Endometriosis Based on Network Pharmacology

Endometriosis (EMs) is a prevalent gynecological disorder with unclear pathogenesis, in which abnormal cell proliferation, inflammation, and immune dysregulation play central roles. Traditional Chinese medicine, including Toddalia asiatica (L.) Lam., has shown therapeutic potential, yet its mechanisms of action remain poorly elucidated.

To elucidate the mechanisms of Toddalia asiatica against EMs, a network pharmacology strategy was implemented. The active compounds, collected from TCMSP, PubChem, and published literature, underwent filtration based on SwissADME criteria. Potential targets of these compounds were identified via SwissTargetPrediction, and targets associated with EMs were retrieved from GeneCards. The interplay among compounds, targets, and the disease was visualized through a network constructed in Cytoscape. Protein–protein interaction networks were generated using STRING, and functional enrichment analyses (GO and KEGG) were carried out with Metascape. The binding affinities of key compounds to their targets were further validated by molecular docking simulations performed with AutoDock Vina.

Our analysis identified 40 active compounds from Toddalia asiatica, with 158 corresponding targets predicted. Subsequent cross-referencing with disease databases revealed 40 of these targets were EMs-related genes. Network analysis identified ERBB2, EGFR, mTOR, AKT1, and HIF1A as central hub nodes. KEGG enrichment analysis further showed that these targets were significantly enriched in critical pathways including EGFR, PI3K–Akt, and PD-1/PD-L1 signaling. Finally, molecular docking simulations confirmed that the core compounds—toddacoumalone, magnoflorine, and corytuberine—exhibited strong binding affinities to the respective key targets.

The therapeutic effects of Toddalia asiatica against EMs are likely mediated by the modulation of cell proliferation, energy metabolism, inflammation, estrogen signaling, and the immune microenvironment, primarily through the EGFR, PI3K-Akt, and PD-1/PD-L1 pathways. Our findings provide a systematic network pharmacology foundation for its use and highlight promising targets for future experimental validation. EGFR Signaling; Endometriosis; Immune Microenvironment; Molecular Docking; Network Pharmacology; PI3K–Akt Pathway; Toddalia asiatica EMs: Endometriosis TCMSP: Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform PPI: Protein-Protein Interaction AKT1: Serine/threonine-protein kinase AKT mTOR: Serine/threonine-protein kinase mTOR EGFR: Epidermal growth factor receptor erbB1 ERBB2: Receptor tyrosine-protein kinase erbB-2 HIFI1A: Hypoxia-inducible factor 1 alpha GO: Gene Ontology BP: Biological Process CC: Cellular Component MF: Molecular Function KEGG: Kyoto Encyclopedia of Genes and Genomes VEGF: Vascular Endothelial Growth Factor Ang-1: Angiopoietin-1 Endometriosis (EMs) is a gynecological condition characterized as the presence and proliferation of ectopic endometrial tissue. With a prevalence of approximately 5–10% [1], its hallmark symptoms—chronic pain, pelvic masses, and infertility—significantly impair patient well-being [2]. The clinical burden of EMs is substantial, often diminishing quality of life and reproductive prospects. Conventional management relies on hormonal suppression and surgical intervention. Nonetheless, limitations including symptom recurrence post-therapy, breakthrough bleeding, and fertility concerns are associated with these options [3,4]. Therefore, identifying novel strategies that are safer, more efficacious, and less prone to recurrence represents a critical unmet need [5-8]. Within the framework of traditional Chinese medicine (TCM), EMs falls under the categories of “dysmenorrhea,” “abdominal mass,” and “infertility,” with its fundamental pathogenesis described as “Qi stagnation and blood stasis”. Correspondingly, treatment strategies emphasize activating blood circulation, removing stasis, and regulating menstruation. An increasing number of studies indicate that TCM formulas achieve their effects through multi-component, multi-target, and multi-pathway regulation, influencing inflammation, immune response, endocrine function, and oxidative stress [9-11]. This holistic paradigm may offer comparative advantages over single-target Western medicines in terms of comprehensive symptom management, recurrence reduction, and fertility protection. To bridge TCM theory with modern systems biology, platforms such as the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) have been developed. TCMSP facilitates the systematic identification of active compounds, potential targets, and key pathways of herbal medicines, thereby elucidating their integrative mechanisms [11-13]. Toddalia asiatica (L.) Lam. (abbreviated as T. asiatica), a plant of the Rutaceae family, is a herb used in traditional Chinese medicine. Valued for its properties in promoting blood circulation, removing stasis, regulating menstruation, and relieving pain, its root or stem bark serves as the medicinal part. Consequently, it finds wide application in managing dysmenorrhea, amenorrhea, metrorrhagia, rheumatic pain, and traumatic injuries [14]. Given its traditional use in treating gynecological disorders such as dysmenorrhea and amenorrhea, the ethanol extract of T. asiatica has been modernly shown to possess anti-inflammatory, analgesic, and other multifaceted bioactivities [15,16]. These properties align with the key pathological features of EMs, suggesting a therapeutic potential that extends beyond its current clinical application in cancer. To scientifically validate and decipher this potential, we applied network pharmacology and molecular docking. This study aims to identify the specific active compounds and molecular targets through which T. asiatica may act against EMs, and to map out its integrated mechanism, bridging traditional use with contemporary mechanistic understanding. Screening of Toddalia asiatica-related targets The initial dataset of chemical constituents for T. asiatica was compiled from two sources: the TCMSP database [17] (https://www.tcmsp-e.com/#/database) and a targeted literature search. The two-dimensional chemical structures of these constituents were constructed using ChemDraw (version 21.0.0). To identify compounds with drug-like properties, the structural files were submitted to the SwissADME web tool (http://www.swissadme.ch/) for pharmacokinetic profiling and filtering. Subsequently, the potential protein targets of the filtered active compounds were predicted using the SwissTargetPrediction server (http://www.swisstargetprediction.ch/). Finally, the predicted targets were normalized, and redundant entries were removed by querying the UniProt knowledgebase (https://www.uniprot.org). Screening of potential therapeutic targets for EMs Potential therapeutic targets for endometriosis (EMs) were retrieved from the GeneCards database (https://www.genecards.org/). In this database, a target’s relevance score quantitatively reflects its association with the disease, with a higher score indicating a stronger association. To focus on the most relevant targets, a score greater than 1 was set as the screening threshold. Construction of PPI Network for Toddalia asiatica (L.) Lam. Components and Endometriosis Targets The common therapeutic targets of Toddalia asiatica and EMs were identified by intersecting their respective target sets using the Jvenn online tool (https://jvenn.toulouse.inrae.fr/app/example.html), which also provided a visual Venn diagram. The resulting intersection targets were imported into the STRING (http://version10.string-db.org/cgi/input.pl) database to generate a PPI network. The network was constructed under the following parameters: organism, Homo sapiens; minimum required interaction score, “highest confidence” (>0.9); all other settings, default. This PPI network was subsequently analyzed in Cytoscape 3.10.0 with the MCODE plugin to perform topological analysis, identify key functional protein clusters, and explore their potential biological roles. Construction of the "Toddalia asiatica(L.) Lam. -Active Ingredient-Shared Targets-EMs" network A network diagram integrating Toddalia asiatica, its active constituents, their intersection targets with EMs, and the disease was established in Cytoscape 3.10.0. Topological analysis of this network was then performed using the software’s built-in tool, with parameters such as Degree, Betweenness Centrality, and Closeness Centrality being calculated. These topological indices were employed to filter the network nodes, aiming to identify core therapeutic targets and principal efficacious compounds, thereby facilitating the interpretation of their specific roles in the intervention of EMs by T. asiatica. Functional and Pathway Enrichment Analysis of Toddalia asiatica(L.) Lam. Components and EMs Targets Functional enrichment analysis, including GO and KEGG pathways, was performed on the common targets using the Metascape platform (https://metascape.org/gp/index.html#/main/step1). A p-value threshold of < 0.01 was applied to determine statistical significance. The enrichment results were graphically represented using the Weishengxin online tool (http://www.bioinformatics.com.cn) [18]. Molecular Docking Validation Based on degree centrality within the network, the top five hub targets were prioritized for molecular docking. The UniProt database (https://www.uniprot.org) was queried to obtain their respective PDB IDs, and the experimental 3D structures were downloaded from the RCSB PDB (https://www.rcsb.org/). Prior to docking, protein structures were prepared with PyMOL (https://www.pymol.org/pymol.html) and AutoDock Tools (https://autodock.scripps.edu/); this involved removing water molecules, adding hydrogens, and converting files to PDBQT format. Docking simulations were then carried out with AutoDock Vina to assess the binding affinities of key T. asiatica compounds to these targets. Interactions with calculated binding energies < −5.0 kcal/mol were deemed to represent strong binding. Acquisition of Targets for Active Components of Toddalia asiatica(L.) Lam. Initially, 120 chemical components were extracted from Toddalia asiatica (L.) Lam. (Supplementary Table 1). Following the screening of ADME (Absorption, Distribution, Metabolism, and Excretion) properties, a total of 41 pharmacologically active components were identified (Table 1), including 5,7-dimethoxycoumarin, bufotalin, 8-formyllimettin, and 5,7,8-trimethoxycoumarin. Additionally, 158 potential molecular targets were predicted (Supplementary Table 2). Table 1: Active Components of Toddalia asiatica (L.) Lam. This table summarizes the pharmacologically active components of Toddalia asiatica (L.) Lam. obtained after ADME property screening, serving as a core basis for subsequent target prediction and mechanism analysis of the herb. Acquisition of EMs-Related Targets A total of 1,476 therapeutic targets for EMs were retrieved from the GeneCards database. Subsequently, a screening threshold of Score > 1 was applied to identify potential targets,yielding a final set of 1,379 candidate therapeutic targets for EMs (Supplementary Table 3). Construction of the PPI Network for Toddalia asiatica (L.) Lam. Components and EMs Targets Intersection analysis was conducted between the screened active component targets of Toddalia asiatica (L.) Lam. and the potential therapeutic targets of EMs. A Venn diagram was generated using the jvenn tool (Figure 1), which revealed 40 common targets in total. These common targets were then imported into the STRING platform for PPI network construction (Figure 2 & Supplementary Table 4). Based on network topological analysis and supported by relevant literature, the top five core targets with the highest significance were identified (Table 2 & Figure 3), namely Serine/threonine-protein kinase AKT (AKT1), Serine/threonine-protein kinase mTOR (mTOR), Epidermal growth factor receptor erbB1 (EGFR), Receptor tyrosine-protein kinase erbB-2 (ERBB2), and Hypoxia-inducible factor 1 alpha (HIF1A). Figure 1: Venn Diagram of Toddalia asiatica (L.) Lam. Component Targets and EMs Targets. This figure is designed to systematically screen for overlapping associated targets between the active component targets of Toddalia asiatica (L.) Lam. and the potential therapeutic targets of EMs via intersection analysis. It ultimately identifies a total of 40 overlapping targets between the two sets, providing a foundational basis for the subsequent exploration of core molecular nodes through which Toddalia asiatica (L.) Lam. exerts its regulatory effects on EMs. Figure 2: PPI Network Diagram of Toddalia asiatica (L.) Lam. and Ems. Using the 40 common targets identified in Figure 1 as the basis, this figure further dissects the PPI relationships among these targets and uncovers the functional regulatory network potentially formed by them in the pathological process of EMs. Table 2: Top 5 Core Targets of Toddalia asiatica (L.) Lam. for EMs Intervention. This table focuses on the 5 key core targets, each of which is accompanied by standardized identifiers, including gene symbols, full names, and UniProt IDs. Figure 3: Hub Targets Diagram of Toddalia asiatica (L.) Lam. for EMs Intervention. This figure represents the result of in-depth mining of the PPI network in Figure 2, and is intended to screen for hub targets that play critical regulatory roles in the EMs pathological network from the 40 common targets, so as to focus the research priorities.

of the Toddalia asiatica (L.) Lam. -Active Components-Common Targets-EMs Network The "Toddalia asiatica (L.) Lam-active component-common target-EMs" network was constructed using Cytoscape 3.10.0 (Figure 4). Network topological parameters, involving 201 nodes and 384 edges, were calculated using the built-in Network Analyzer tool of Cytoscape (Supplementary Table 5). Based on the degree values, the top 5 core components were identified: toddacoumalone, magnoflorine, corydine, toddaculine, and 5,7-dimethoxy-2H-1-benzopyran-2-one (5,7-dimethoxycoumarin). Furthermore, topological analysis revealed that node size was proportional to the degree value. Node shapes in the network represented different entity types: circle for Toddalia asiatica (L.) Lam, octagon for EMs, hexagon for active components, and diamond for common targets. Notably, toddacoumalone exhibited the highest degree value and betweenness centrality, indicating that it is a key component of Toddalia asiatica (L.) Lam in the treatment of EMs (Table 3). Figure 4: Network Diagram of Toddalia asiatica (L.) Lam.-Active Components-Common Targets-Ems. This figure was constructed using the bioinformatics visualization software Cytoscape 3.10.0. Within the network, node size is positively correlated with degree value (i.e., nodes with higher degree values appear larger in the figure). Different types of entities are distinguished by nodes of distinct shapes, with specific rules as follows: Circle: Represents Toddalia asiatica (L.) Lam; Octagon: Represents endometriosis (EMs); Hexagon: Represents active components; Diamond: Represents common targets. Table 3: Characteristic Parameters of Network Nodes for Main Active Components of Toddalia asiatica (L.) Lam. This table presents the results of topological parameter calculation for Fig. 4, conducted using the built-in Network Analyzer tool of Cytoscape. Based on degree value and betweenness centrality—an indicator for measuring the "bridging role" of nodes in the network, where a higher value denotes a more critical role of the node in connecting other nodes—the top 5 core active components were identified. These components are as follows: toddacoumalone, magnoflorine, corydine, toddaculine, and 5,7-dimethoxy-2H-1-benzopyran-2-one. Enrichment Analysis of Target Functions and Pathways Signal pathway analysis of the potential therapeutic targets of Toddalia asiatica (L.) Lam. for EMs was performed using the Metascape database platform, and the results were visualized via the WeShengXin platform, a domestic bioinformatics visualization tool. A total of 791 terms were obtained from the Gene Ontology (GO) enrichment analysis, including 691 biological process (BP) terms, 54 cellular component (CC) terms, and 46 molecular function (MF) terms (Supplementary Table 6). The analysis indicated that the potential targets of Toddalia asiatica (L.) Lam are mainly enriched in the following biological processes: cellular response to organonitrogen compounds, response to inorganic substances, protein autophosphorylation, and cellular response to growth factor stimuli. The main cellular components involved include the extracellular matrix, membrane side, membrane raft, chromosomal region, and neuronal cell body. Additionally, the related targets exhibited significant enrichment in the following molecular functions: protein kinase activity, kinase binding, protein tyrosine kinase activity, monooxygenase activity, and insulin receptor substrate binding (Figure 5). Figure 5: Results of GO enrichment analysis. This figure presents the results of pathway analysis for potential therapeutic targets of Toddalia asiatica (L.) Lam in the treatment of EMs, which were visualized using WeShengXin, a domestic bioinformatics visualization tool, based on data from the Metascape database platform. In parallel, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis identified a total of 106 terms (Supplementary Table 7), with key signaling pathways including: Pathways in cancer, EGFR tyrosine kinase inhibitor resistance, Endocrine resistance, Proteoglycans in cancer, and PI3K-Akt signaling pathways (Figure 6). Figure 6: Top 20 KEGG pathways sorted by P-value in enrichment analysis. This figure illustrates the results of KEGG pathway enrichment analysis on the potential therapeutic targets of Toddalia asiatica (L.) Lam in treating EMs. Based on the top 20 pathways ranked by P-value and further integration with relevant literature, we inferred that Toddalia asiatica (L.) Lam. may exert its therapeutic effects on EMs by modulating the following mechanisms: core cancer pathways and therapeutic resistance; the PI3K-Akt signaling network; cell migration and microenvironment remodeling; immune escape regulation; and hormone and endocrine regulation. Molecular Docking The top 5 active components of Toddalia asiatica (L.) Lam with the highest degree values in the "Toddalia asiatica (L.) Lam - active component - common target - EMs" network were selected as ligands. Correspondingly, the top 5 core targets with the highest degree values were chosen as receptors for molecular docking. The binding energies between the key active components and core targets were calculated using AutoDock Vina, and the detailed docking results are presented in figure 7. Figure 7: Heatmap of molecular docking binding energies. This figure displays the molecular docking results between the core active components of Toddalia asiatica (L.) Lam and key targets related to endometriosis (EMs). Specifically, the top 5 active components of T. asiatica with the highest degree values in the "T. asiatica (L.) Lam–active component–common target–EMs" network were selected as ligands, while the top 5 core targets with the highest degree values were chosen as receptors for molecular docking. The binding energies between these main active components and key targets were calculated using AutoDock Vina, and the docking results are summarized in figure 7. (Note: The numbers in the squares represent the molecular docking binding energies between the horizontal label and vertical label corresponding to each square, with the unit of kcal/mol.) The docking analysis revealed that the core targets AKT1, EGFR, mTOR, ERBB2, and HIF1A formed stable binding interactions with the five key components, with all binding energies lower than –5 kcal/mol. Notably, EGFR exhibited strong binding affinity toward toddacoumalone, magnoflorine, corydine, and toddaculine, whereas HIF1A showed high binding affinity with toddacoumalone and toddaculine. The top four docking complexes with the most favorable binding energies were further visualized using PyMOL, as illustrated in figure 8. Figure 8: Representative molecular docking diagrams. This figure displays the top 4 docking complexes with the most favorable binding energies were selected and visualized using PyMol. Among them, Panel A represents the binding of EGFR to toddaculine, Panel B represents the binding of HIF1A to toddaculine, Panel C represents the binding of EGFR to toddacoumalone, and Panel D represents the binding of EGFR to corydine. Endometriosis (EMs) is a common gynecological disorder characterized by progressive dysmenorrhea, menstrual irregularities, chronic pelvic pain, and impaired fertility. Although pathologically benign, EMs displays several malignancy-like features, including tissue invasion, uncontrolled cell proliferation, local infiltration, distant metastasis, and high recurrence rates. For these reasons, EMs is often regarded as a benign disease with malignant biological behaviors [19]. Current mainstream treatments for EMs in Western medicine mainly consist of conservative hormonal therapy and surgical resection for large endometriotic cysts. Hormonal drugs can effectively relieve pain associated with EMs; however, symptoms frequently recur after drug discontinuation [20-22]. Moreover, long-term hormonal interventions carry risks of adverse effects and potential impacts on fertility [23]. In comparison, surgical treatment can directly remove visible lesions, but complete eradication of ectopic tissues remains difficult, and intraoperative damage to adjacent organs may occur. Accumulating evidence has confirmed that resection of ovarian endometriotic lesions significantly impairs ovarian reserve [24], which is particularly distressing for patients with fertility needs. Additionally, the 5-year postoperative recurrence rate of EMs-related pain can reach 40%–50% [25]. Traditional Chinese Medicine (TCM) offers unique advantages in the management of EMs, such as alleviating clinical symptoms, improving pregnancy rates, reducing adverse reactions, and lowering recurrence rates [26]. Toddalia asiatica (L.) Lam. is a representative traditional ethnic medicinal herb, whose primary chemical constituents include coumarins, alkaloids, terpenoids, flavonoids, and phenolic acids. Among these components, coumarins exert notable anti-inflammatory and analgesic effects, whereas alkaloids display antitumor activities [27,28]. In this study, 41 bioactive components of Toddalia asiatica (L.) Lam. were screened, corresponding to 158 therapeutic targets, among which 40 common targets overlapped between the herb and EMs. The key active components of Toddalia asiatica (L.) Lam. potentially responsible for its anti-EMs effects were preliminarily identified as toddacoumalone, magnoflorine, corydine, and others. Previous studies have demonstrated that toddacoumalone significantly inhibits PDE4, thereby attenuating systemic inflammatory responses [29,30]. Magnoflorine exerts anti-inflammatory and immunosuppressive effects by regulating the MAPK, NF-κB, and PI3K/Akt signaling pathways. Meanwhile, it can suppress tumor cell proliferation via the miR-410-3p/HMGB1/NF-κB axis [31-33]. Molecular docking validation further revealed that the active components (including toddacoumalone and magnoflorine) displayed strong binding affinities with the core targets ERBB2, EGFR, mTOR, AKT1, and HIF1A. Among these interactions, EGFR showed the strongest binding affinity for toddacoumalone, followed by HIF1A. AKT1 (protein kinase B, PKB) is a pivotal serine/threonine kinase and a central transducer in cell survival signaling [34]. It regulates cell survival and apoptosis-related proteins through phosphorylation. Importantly, abnormal overactivation of AKT1 is frequently observed in various malignant tumors, including breast, cervical, ovarian, lung, and colorectal cancers [35-39]. EGFR belongs to the HER family of transmembrane glycoproteins and possesses intrinsic tyrosine kinase activity. It primarily mediates the RAS/RAF/MEK/ERK and PI3K/AKT signaling pathways, which are essential for cell cycle progression and apoptosis [40,41]. EGFR also modulates tumor angiogenesis by regulating the expression of vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1). Notably, upregulated EGFR expression has been detected in numerous malignant tissues, including breast, lung, pancreatic cancer, and glioblastoma [42-46]. mTOR (mammalian target of rapamycin) is an indispensable serine/threonine protein kinase that plays a central role in regulating cell growth, survival, metabolism, and immune responses. It participates in protein synthesis, nutrient sensing, growth factor signaling, and cell migration [47]. Mounting evidence indicates that dysregulation of the mTOR pathway is closely associated with inflammation, infections, metabolic disorders, cancer, and autoimmune diseases [48-51]. ERBB2 (also known as HER2) is a critical oncogene. The encoded transmembrane glycoprotein regulates cell growth, proliferation, differentiation, and apoptosis [52], and is closely implicated in the initiation and progression of various malignancies, including breast, ovarian, and upper gastrointestinal adenocarcinomas [53-55]. HIF1A (hypoxia-inducible factor-1α) participates in multiple essential biological processes, including cell proliferation, apoptosis, migration, invasion, angiogenesis, epithelial–mesenchymal transition (EMT), and inflammatory responses [56]. It is frequently overexpressed in tumors and ischemic diseases [57,58]. Notably, EMs shares several pathological hallmarks with tumors, such as abnormal proliferation and angiogenesis [19]. Aberrant activation or overexpression of AKT1, EGFR, mTOR, ERBB2, and HIF1A has been widely reported in EMs lesions [59-63]. These targets are also functionally interconnected within the PI3K/AKT/mTOR signaling axis, which acts as a core pathway in EMs pathogenesis [64,65]. GO and KEGG pathway enrichment analyses suggested that Toddalia asiatica (L.) Lam. may exert therapeutic effects on EMs by modulating cell proliferation, energy metabolism, inflammatory responses, estrogen receptor activity, and the immune microenvironment. Key pathways involved include the EGFR, PI3K/AKT, and PD-1/PD-L1 signaling pathways.’ It has been established that estrogen binds to estrogen receptors and activates downstream mediators shared with the EGFR pathway (e.g., MAPK). Phosphorylation of serine-118 further enhances EGFR signaling and promotes tumor invasion, metastasis, and angiogenesis [66]. In patients with EMs, PI3K/AKT phosphorylation is significantly elevated in endometrial tissues. Blocking this pathway can restore hormonal balance, reduce inflammation, inhibit angiogenesis, and induce autophagy in ectopic tissues, thereby delaying disease progression and relieving symptoms [67]. The PD-1/PD-L1 pathway is critical for maintaining peripheral immune tolerance by suppressing excessive activation of autoreactive T cells and preventing autoimmunity. However, persistent activation of PD-1/PD-L1 inhibits T-cell function, promotes tumor immune escape, and impairs antitumor immunity [68,69]. Additionally, the PD-1/PD-L1 axis indirectly maintains gut microbiota homeostasis by regulating the Th17/Treg balance. Abnormal activation of this pathway also contributes to the formation of a pro-inflammatory pelvic microenvironment in Ems [70,71]. This multi-dimensional immunoregulatory network suggests that the PD-1/PD-L1 pathway may serve as a promising therapeutic target connecting tumor immunity, autoimmunity, and microecological disorders. Notably, the active components of Toddalia asiatica (L.) Lam. identified in this study (e.g., toddacoumalone, magnoflorine) were verified to bind stably to key targets in these pathways (AKT1, EGFR, etc.) via molecular docking, further supporting the notion that Toddalia asiatica (L.) Lam. treats EMs through a multi-component, multi-target, and multi-pathway mode of action. In conclusion, Toddalia asiatica (L.) Lam. may exert therapeutic effects against EMs via a synergistic multi-target mechanism. Its key active components—including toddacoumalone, magnoflorine, corydine, toddaculine, and 5,7-dimethoxycoumarin—act on core targets (AKT1, EGFR, mTOR, ERBB2, HIF1A) and modulate crosstalk among critical signaling pathways, particularly the EGFR, PI3K-AKT, and PD-1/PD-L1 axes. The co-regulation of the PI3K-AKT/mTOR and EGFR pathways may enhance inhibitory effects on the pathological processes underlying EMs, including uncontrolled proliferation, abnormal angiogenesis, excessive inflammation, and immune microenvironment dysregulation, thereby achieving an integrated therapeutic effect. These bioinformatics predictions preliminarily clarify the potential mechanism of Toddalia asiatica (L.) Lam. in the treatment of EMs and provide a targeted theoretical foundation for future research. Priority can be given to validating high-potential components such as toddacoumalone, which exhibited the highest degree value in network analysis, through in vitro and in vivo experiments. This study has several limitations. First, the present work is based on network pharmacology predictions without in-depth experimental validation. Second, the active components were screened from public databases, and their pharmacokinetic profiles remain uncharacterized, which may affect clinical efficacy. Future studies will focus on verifying the binding affinities of key components, establishing EMs models to evaluate therapeutic effects, and exploring downstream signaling cascades to support the clinical translation of Toddalia asiatica (L.) Lam. for EMs management. Ethics approval and consent to participate Our analysis utilized publicly available summary statistics databases, such as TCMSP, Uniprot, and GeneCards. No new data were collected, and no new ethical approval was required. Consent for Publication Not applicable Availability of data and materials The chemical components of Toddalia asiatica (L.) Lam. were obtained from the TCMSP (https://www.tcmsp-e.com/#/database), as well as relevant literature searches. The therapeutic targets of EMs were derived from the GeneCards database (https://www.uniprot.org). All data analyzed in this research can be accessible freely through the corresponding websites. The authors declare that they have no competing interests. This study was supported by the Natural Science Research Project of Guangxi University of Chinese Medicine. (Grant No.2024MS052). Conceptualization: Yutao Geng, Junhong Gan, Wenyi Li, Jingfang Hu, Lu Zhong. Data curation: Yutao Geng, Junhong Gan. Funding acquisition: Lu Zhong. Investigation: Yutao Geng, Junhong Gan,Wenyi Li, Jingfang Hu, Lu Zhong. Methodology: Yutao Geng, Junhong Gan, Lu Zhong. Supervision: Wenyi Li, Lu Zhong. Writing – original draft: Yutao Geng, Junhong Gan, Mengqi Shen, Wenyi Li, Jingfang Hu, Lu Zhong. Writing – review & editing: Wenyi Li, Lu Zhong. All authors thank the patients and sequencers who provided samples and the publicly available databases. Supplementary Table 1: Initial Chemical Components of Toddalia asiatica (L.) Lam. This table systematically presents all initial chemical components of Toddalia asiatica (L.) Lam. (Feilongzhangxue in Chinese) obtained from the Traditional Chinese Medicine Systems Pharmacology Platform (TCMSP) and relevant literatures, serving as the "original material library" for subsequent active component screening. The core information in the table includes, but is not limited to, basic attribute data of these 120 initial chemical components, such as their standard names, labels, molecular formulas, and CAS numbers. Supplementary Table 2: Predicted Potential Molecular Targets of Active Components. This table systematically documents all potential molecular targets predicted from the 41 active components listed in Table 1 of the main text, and serves as the "target library foundation" for subsequent "target-disease association analysis". The core information of this table includes the following key details for 158 potential targets: standard identifiers (e.g., gene symbols, full gene names), corresponding protein names of the targets and prediction confidence levels. Supplementary Table 3: Potential therapeutic targets for Ems. This table details the potential therapeutic targets for EMs. A total of 1,476 EMs-related therapeutic targets were first retrieved from the GeneCards database. To optimize target screening and focus on potentially relevant targets, a screening threshold (Score metric > 1) was applied; this filtering step resulted in a final set of 1,379 candidate therapeutic targets for EMs. Supplementary Table 4: Common Targets Identified by Intersection Analysis Between Screened Active Component Targets of Toddalia asiatica (L.) Lam. and Potential Therapeutic Targets of EMs (n=40). This table details the common targets obtained from intersection analysis. Specifically, this analysis was conducted between two target sets: the screened active component targets of Toddalia asiatica (L.) Lam. and the potential therapeutic targets of EMs. To visualize the overlap of these targets, a Venn diagram was generated using the jvenn tool (Figure 1); this intersection analysis ultimately identified a total of 40 common targets. Supplementary Table 5: Topological Parameters of the "Toddalia asiatica (L.) Lam.-Active Component-Common Target-Endometriosis (EMs)" Network. This table provides the topological parameters of the "Toddalia asiatica (L.) Lam.-active component-common target-EMs" network. This network was first constructed using Cytoscape 3.10.0 software (corresponding to Figure 4). Subsequently, the built-in Network Analyzer tool of Cytoscape was employed to calculate the network’s topological parameters, which include a total of 201 nodes and 384 edges. Supplementary Table 6: GO Enrichment Analysis Results of Potential Therapeutic Targets of Toddalia asiatica (L.) Lam for Ems. This table presents the results of GO enrichment analysis for the potential therapeutic targets of Toddalia asiatica (L.) Lam in treating (EMs. The analysis was conducted on the Metascape database platform, and the visualization of results was completed using WeShengXin (a domestic bioinformatics visualization tool). A total of 791 GO terms were identified, which are categorized into three ontologies as follows: 1. Biological Process (BP): 691 terms (focus on the biological processes involved in the therapeutic target-mediated effects); 2. Cellular Component (CC): 54 terms (focus on the cellular locations where the therapeutic targets exert functions); 3. Molecular Function (MF): 46 terms (focus on the molecular-level functions of the therapeutic targets). All detailed information of these GO terms (e.g., term ID, description, enrichment significance, gene count) is listed in this table. Supplementary Table 7: KEGG Pathway Enrichment Analysis Results of Potential Therapeutic Targets of Toddalia asiatica (L.) Lam for EMs (Focus on "Pathways in Cancer") This table presents the detailed results of KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis for the potential therapeutic targets of Toddalia asiatica (L.) Lam in treating endometriosis (EMs). A total of 106 KEGG pathway terms were identified through this analysis. This table typically includes comprehensive information for each relevant KEGG term (with a focus on "Pathways in Cancer"), such as pathway ID, pathway description, enrichment significance indicator, the number of potential therapeutic targets involved, and the list of corresponding targets. - Chapron C, Marcellin L, Borghese B, Santulli P (2019) Rethinking mechanisms, diagnosis and management of endometriosis. Nat Rev Endocrinol 15: 666-682. - Taylor HS, Kotlyar AM, Flores VA (2021) Endometriosis is a chronic systemic disease: clinical challenges and novel innovations. Lancet 397: 839-852. - Chinese Obstetricians and Gynecologists Association (2021) [Guideline for the diagnosis and treatment of endometriosis (Third edition)]. Zhonghua Fu Chan Ke Za Zhi 56: 812-824. - Muzii L, Tucci CD, Feliciantonio MD, Galati G, Donato VD, et al. (2018) Antimüllerian hormone is reduced in the presence of ovarian endometriomas: a systematic review and meta-analysis. Fertil Steril 110: 932-940. - Chen Y, Wei J, Zhang Y, Sun W, Li Z, et al. (2018) Anti-endometriosis Mechanism of Jiawei Foshou San Based on Network Pharmacology. Front Pharmacol 9: 811. - Lin Y, Wu L, Zhao R, Chung PW, Wang CC (2023) Chinese Herbal Medicine, Alternative or Complementary, for Endometriosis-Associated Pain: A Meta-Analysis. Am J Chin Med 51: 807-832. - Wang X, Shi Y, Xu L, Wang Z, Wang Y, et al. (2021) Traditional Chinese medicine prescription Guizhi Fuling Pills in the treatment of endometriosis. Int J Med Sci 18: 2401-2408. - Dong P, Ling L, Hu L (2021) Systematic review and meta-analysis of traditional Chinese medicine compound in treating infertility caused by endometriosis. Ann Palliat Med 10: 12631-12642. - Zheng W, Wu J, Gu J, Weng H, Wang J, et al. (2020) Modular characteristics and mechanism of action of herbs for endometriosis treatment in Chinese medicine: A data mining and network pharmacology-based identification. Front Pharmacol 11: 147. - Gu X, Zhou H, Miao M, Hu D, Wang X, et al. (2024) Therapeutic potential of natural resources against endometriosis: Current advances and future perspectives. Drug Des Devel Ther 18: 3667-3696. - Wu Y, Zhu Y, Xie N, Wang H, Wang F, et al. (2022) A network pharmacology approach to explore active compounds and pharmacological mechanisms of a patented Chinese herbal medicine in the treatment of endometriosis. PLoS One 17: 0263614. - Li B, Tao W, Zheng C, Shar PA, Huang C, et al. (2014) Systems pharmacology-based approach for dissecting the addition and subtraction theory of traditional Chinese medicine: An example using xiao-chaihu-decoction and da-chaihu-decoction. Comput Biol Med 53: 19-29. - Zhang R, Zhu X, Bai H, Ning K (2019) Network pharmacology databases for traditional Chinese medicine: Review and assessment. Front Pharmacol 10: 123. - Zeng Z, Tian R, Feng J, Yang NA, Yuan L (2021) A systematic review on traditional medicine toddalia asiatica (L.) lam.: Chemistry and medicinal potential. Saudi Pharm J 29: 781-798. - Kimang’a A, Gikunju J, Kariuki D, Ogutu M (2016) Safety and Analgesic Properties of Ethanolic Extracts of Toddalia Asiatica (L) Lam. (Rutaceae) Used for Central and Peripheral Pain Management Among the East African Ethnic Communities. Ethiop J Health Sci 26: 55-64. - He H, Li N, Fan Y, Huang Q, Song J, et al. (2023) Six new coumarins from the roots of toddalia asiatica and their anti-inflammatory activities. Chin J Nat Med 21: 852-858. - Ru J, Li P, Wang J, Zhou W, Li B, et al. (2014) TCMSP: a database of systems pharmacology for drug discovery from herbal medicines. J Cheminformatics 6: 13. - Tang D, Chen M, Huang X, Zhang G, Zeng L, et al. (2023) SRplot: A free online platform for data visualization and graphing. PLoS One 18: 0294236. - Chen F, Zhu M, Li W (2024) Advances in research on malignant transformation of endometriosis-associated ovarian cancer. Front Oncol 14: 1475231. - Capezzuoli T, Rossi M, Torre FL, Vannuccini S, Petraglia F (2022) Hormonal drugs for the treatment of endometriosis. Curr Opin Pharmacol 67: 102311. - Kalaitzopoulos DR, Samartzis N, Kolovos GN, Mareti E, Samartzis EP, et al. (2021) Treatment of endometriosis: a review with comparison of 8 guidelines. BMC Womens Health 21: 397. - Vercellini P, Viganò P, Somigliana E, Fedele L (2014) Endometriosis: Pathogenesis and treatment. Nat Rev Endocrinol 10: 261-275. - Brichant G, Laraki I, Henry L, Munaut C, Nisolle M (2021) New therapeutics in endometriosis: A review of hormonal, non-hormonal, and non-coding RNA treatments. Int J Mol Sci 22: 10498. - Coccia ME, Nardone L, Rizzello F (2022) Endometriosis and Infertility: A Long-Life Approach to Preserve Reproductive Integrity. Int J Environ Res Public Health 19: 6162. - Guo SW (2009) Recurrence of endometriosis and its control. Hum Reprod Update 15: 441-461. - Meresman GF, Götte M, Laschke MW (2021) Plants as source of new therapies for endometriosis: a review of preclinical and clinical studies. Hum Reprod Update 27: 367-392. - Alagaraj P, Muthukrishnan S (2020) Toddalia asiatica L. - A Rich Source of Phytoconstituents with Potential Pharmacological Actions, an Appropriate Plant for Recent Global Arena. Cardiovasc Hematol Agents Med Chem 18: 104-110. - Hao XY, Peng L, Ye L, Huang NH, Shen YM (2004) [A study on anti-inflammatory and analgesic effects of alkaloids of Toddalia asiatica]. Zhong Xi Yi Jie He Xue Bao 2: 450-452. - Xu Y, Xue S, Zhang T, Jin X, Wang C, et al. (2022) Toddalolactone protects against osteoarthritis by ameliorating chondrocyte inflammation and suppressing osteoclastogenesis. Chin Med 17: 18. - Ni J, Zhao Y, Su J, Fang S, Li L, et al. (2020) Toddalolactone Protects Lipopolysaccharide-Induced Sepsis and Attenuates Lipopolysaccharide-Induced Inflammatory Response by Modulating HMGB1-NF-κB Translocation. Front Pharmacol 11: 109. - Xu T, Kuang T, Du H, Li Q, Feng T, et al. (2020) Magnoflorine: A review of its pharmacology, pharmacokinetics and toxicity. Pharmacol Res 152: 104632. - Guo S, Jiang K, Wu H, Yang C, Yang Y, et al. (2018) Magnoflorine Ameliorates Lipopolysaccharide-Induced Acute Lung Injury via Suppressing NF-κB and MAPK Activation. Front Pharmacol 9: 982. - Wang Y, Shang G, Wang W, Qiu E, Pei Y, et al. (2020) Magnoflorine inhibits the malignant phenotypes and increases cisplatin sensitivity of osteosarcoma cells via regulating miR-410-3p/HMGB1/NF-κB pathway. Life Sci 256: 117967. - Siddika T, Balasuriya N, Frederick MI, Rozik P, Heinemann IU, et al. (2022) Delivery of Active AKT1 to Human Cells. Cells 11: 3834. - McKenna M, Balasuriya N, Zhong S, Li SSC, O’Donoghue P (2020) Phospho-Form Specific Substrates of Protein Kinase B (AKT1). Front Bioeng Biotechnol 8: 619252. - Chen Y, Huang L, Dong Y, Tao C, Zhang R, et al. (2020) Effect of AKT1 (p. E17K) Hotspot Mutation on Malignant Tumorigenesis and Prognosis. Front Cell Dev Biol 8: 573599. - George B, Gui B, Raguraman R, Paul AM, Nakshatri H, et al. (2022) AKT1 Transcriptomic Landscape in Breast Cancer Cells. Cells 11: 2290. - Yeh YC, Ho HL, Wu YC, Pan CC, Wang YC, et al. (2020) AKT1 internal tandem duplications and point mutations are the genetic hallmarks of sclerosing pneumocytoma. Mod Pathol 33: 391-403. - Alves CP, Dey-Guha I, Kabraji S, Yeh AC, Talele NP, et al. (2018) AKT1low Quiescent Cancer Cells Promote Solid Tumor Growth. Mol Cancer Ther 17: 254-263. - Ouyang L, Chen Y, Wang XY, Lu RF, Zhang SY, et al. (2014) Polygonatum odoratum lectin induces apoptosis and autophagy via targeting EGFR-mediated Ras-Raf-MEK-ERK pathway in human MCF-7 breast cancer cells. Phytomedicine 21: 1658-1665. - Ganesan K, Xu C, Wu J, Du B, Liu Q, et al. (2024) Ononin inhibits triple-negative breast cancer lung metastasis by targeting the EGFR-mediated PI3K/Akt/mTOR pathway. Sci China Life Sci 67: 1849-1866. - Gong L, Yang S, Huang J, Li Y (2024) Modulation of gut microbiota in targeted cancer therapy:insights on the EGFR/VEGF/KRAS pathways. Cancer Biology & Medicine 21: 1141-1155. - Wang F, Li C, Wu Q, Lu H (2020) EGFR exon 20 insertion mutations in non-small cell lung cancer. Transl Cancer Res TCR 9: 2982-2991. - Guangxi University of Chinese Medicine (2025) EGFR ligand shifts the role of EGFR from oncogene to tumour suppressor in EGFR-amplified glioblastoma by suppressing invasion through BIN3 upregulation-All Databases. Guangxi University of Chinese Medicine, China. - Fitzgerald TL, Lertpiriyapong K, Cocco L, Martelli AM, Libra M, et al. (2015) Roles of EGFR and KRAS and their downstream signaling pathways in pancreatic cancer and pancreatic cancer stem cells. Adv Biol Regul 59: 65-81. - Yeboah K, Kyei-Baafour E, Antwi DA, Asare-Anane H, Gyan B, et al. (2016) Circulating angiogenic factors in diabetes patients in a tertiary hospital in Ghana. J Diabetes Metab Disord 15: 44. - Hua H, Kong Q, Zhang H, Wang J, Luo T, et al. (2019) Targeting mTOR for cancer therapy. J Hematol Oncol 12: 71. - El Hiani Y, Egom EEA, Dong XP (2019) mTOR signalling: jack-of-all-trades 1. Biochem Cell Biol 97: 58-67. - Marafie SK, Al-Mulla F, Abubaker J (2024) mTOR: Its Critical Role in Metabolic Diseases, Cancer, and the Aging Process. Int J Mol Sci 25: 6141. - Mafi S, Mansoori B, Taeb S, Sadeghi H, Abbasi R, et al. (2021) mTOR-Mediated Regulation of Immune Responses in Cancer and Tumor Microenvironment. Front Immunol 12: 774103. - Assaf L, Eid AA, Nassif J (2022) Role of AMPK/mTOR, mitochondria, and ROS in the pathogenesis of endometriosis. Life Sci 306: 120805. - Groenendijk FH, Jong J, Putte EEF, Michaut M, Schlicker A, et al. (2016) ERBB2 Mutations Characterize a Subgroup of Muscle-invasive Bladder Cancers with Excellent Response to Neoadjuvant Chemotherapy. European Urology 69: 384-388. - Prat A, Bardia A, Curigliano G, Hammond MEH, Loibl S, et al. (2022) An Overview of Clinical Development of Agents for Metastatic or Advanced Breast Cancer Without ERBB2 Amplification (HER2-Low). JAMA Oncol 8: 1676. - Quaye L, Song H, Ramus SJ, Gentry-Maharaj A, Høgdall E, et al. (2009) Tagging single-nucleotide polymorphisms in candidate oncogenes and susceptibility to ovarian cancer. Br J Cancer 100: 993-1001. - Arrington AK, Davydova J, Vickers SM, Yamamoto M (2009) Anti-ERBB2 sh-RNA Suppress Both Cell Growth and Tumor Growth in ERBB2-Overexpressing Upper Gastrointestinal Adenocarcinomas. Journal of Gastrointestinal Surgery 13: 1754-1761. - Feng D, Gao J, Liu R, Liu W, Gao T, et al. (2024) CARM1 drives triple-negative breast cancer progression by coordinating with HIF1A. Protein Cell 15: 744-765. - Tiwari A, Tashiro K, Dixit A, Soni A, Vogel K, et al. (2020) Loss of HIF1A From Pancreatic Cancer Cells Increases Expression of PPP1R1B and Degradation of p53 to Promote Invasion and Metastasis. Gastroenterology 159: 1882-1897. - Yao H, Li J, Liu Z, Ouyang C, Qiu Y, et al. (2025) Ablation of endothelial Atg7 inhibits ischemia-induced angiogenesis by upregulating Stat1 that suppresses Hif1a expression. Autophagy 19: 1491-1511. - Badary DM, Abou-Taleb HA, Ibrahim M (2023) Hypoxia-inducible Factor-1α and mTOR as a Potential Therapeutic Target in Endometriosis: An Immunohistochemical Study. Appl Immunohistochem 31: 629-634. - Markov D, Poryazova E, Raycheva R, Markov G (2024) Expression of HIF-1alpha, Ki67, SMA and E-cadherin in endometriosis, endometrial and ovarian carcinoma. Folia medica 66: 97-103. - Poorasamy J, Garg D, Bharti J, Nambirajan A, Patil A, et al. (2022) Overexpression of ErbB-1 (EGFR) Protein in Eutopic Endome-Trium of Infertile Women with Severe Ovarian Endometriosis during the ‘Implantation Window’ of Menstrual Cycle. MEDICINE & PHARMACOLOGY. - Krakstad C, Berg HF, Lindemann K, Halle MK (2024) Frequency of ERBB2-Low Expression in Endometrial Cancer. JAMA Oncol 10: 1587-1588. - Kang N, Shan H, Wang J, Mei J, Jiang Y, et al. (2022) Calpain7 negatively regulates human endometrial stromal cell decidualization in EMs by promoting FoxO1 nuclear exclusion via hydrolyzing AKT1. Biol Reprod 106: 1112-1125. - Cao Y, Ye Q, Zhuang M, Xie S, Zhong R, et al. (2017) Ginsenoside Rg3 inhibits angiogenesis in a rat model of endometriosis through the VEGFR-2-mediated PI3K/Akt/mTOR signaling pathway. PLoS One 12: 0186520. - Cheng Y, Guo Q, Cheng Y, Wang D, Sun L, et al. (2024) Endostatin-expressing endometrial mesenchymal stem cells inhibit angiogenesis in endometriosis through the miRNA-21-5p/TIMP3/PI3K/Akt/mTOR pathway. Stem Cells Transl Med 14: 79. - Qu C, Wang C, Li H, Li Y, Han C, et al. (2022) Estrogen receptor variant ER-α36 facilitates estrogen signaling via EGFR in glioblastoma. Cell Biol Int 46: 1759-1774. - An M, Fu X, Meng X, Liu H, Ma Y, et al. (2024) PI3K/AKT signaling pathway associates with pyroptosis and inflammation in patients with endometriosis. J Reprod Immunol 162: 104213. - Suszczyk D, Skiba W, Zardzewialy W, Pawlowska A, Wlodarczyk K, et al. (2022) Clinical Value of the PD-1/PD-L1/PD-L2 Pathway in Patients Suffering from Endometriosis. IJMS 23: 11607. - Parvez A, Choudhary F, Mudgal P, Khan R, Qureshi KA, et al. (2023) PD-1 and PD-L1: Architects of immune symphony and immunotherapy breakthroughs in cancer treatment. Front Immunol 14: 1296341. - Li C, Lu Z, Bi K, Wang K, Xu Y, et al. (2019) CD4+/CD8+ mucosa-associated invariant T cells foster the development of endometriosis: a pilot study. Reprod Biol Endocrinol 17: 78. - Huang J, Liu D, Wang Y, Liu L, Li J, et al. (2022) Ginseng polysaccharides alter the gut microbiota and kynurenine/tryptophan ratio, potentiating the antitumour effect of antiprogrammed cell death 1/programmed cell death ligand 1 (anti-PD-1/PD-L1) immunotherapy. Gut 71: 734-745. Citation: Geng Y, Gan J, Li W, Hu J, Zhong L (2026) Mechanism of Action of Toddalia asiatica (L.) Lam. in the Treatment of Endometriosis Based on Network Pharmacology. HSOA J Altern Complement Integr Med 12: 695. Copyright: © 2026 Yutao Geng, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. © 2026, Copyrights Herald Scholarly Open Access. All Rights Reserved!