Effective Material Basis and Mechanism Analysis of Compound Banmao Capsule against Tumors Using Integrative Network Pharmacology and Molecular Docking

Purpose Compound banmao capsule (CBC), a well-known traditional Chinese medical material, is known to inhibit various tumors. However, its material basis and pharmacological mechanisms remain to be elucidated. This study aimed to investigate the effective material basis and mechanisms of action of CBC against tumors. Methods Active compounds of CBC were identified using public database and reports to build a network. The corresponding targets of active compounds were retrieved from online databases, and the antitumor targets were identified by GeneCards database. The antitumor hub targets were generated via protein-protein interaction analysis using String, and key compounds and targets from the integrative network were detected by molecular docking and ADMET. Top targets in hepatocellular carcinoma were confirmed by quantitative real-time PCR (qPCR). Finally, the multivariate biological network was built to identify the integrating mechanisms of action of CBC against tumor cells. Results A total of 128 compounds and 436 targets of CBC were identified successfully. Based on the generated multivariate biological network analysis, 25 key compounds, nine hub targets, and two pathways were further explored. Effective material bases of cantharidin, baicalein, scutellarin, sesamin, and quercetin were verified by integrative network analysis. PTGS2, ESR1, and TP53 were identified as hub targets via multivariate biological network analysis and confirmed using qPCR. Furthermore, VEGF and estrogen signaling pathways seem to play a role in the antitumor activity of CBC. Thus, breast cancer may be a potential clinical indication of CBC. Conclusion This study successfully identified the material basis of CBC and its synergistic mechanisms of action against tumor cells.


Introduction
Studies have shown that the five-year survival rate of cancer generally rise when tumor survival trends in global areas are evaluated. However, the survival rate of malignant tumors, such as liver cancer, is still low [1]. At present, antitumor therapy is mainly based on radiotherapy and chemotherapy, which are linked to severe adverse reactions [2]. us, exploring new antitumor drugs with fewer side effects is urgently needed. Traditional Chinese medicine (TCM) is popular, plays a role in the modern healthcare system, and can provide potential Fel Ursi (XD), Glycyrrhizae Radix Et Rhizoma (GC), Corni Fructus (SZY), Ligustri Lucidi Fructus (NZZ), and Sparganii Rhizoma (SL). CBC has been used to clinically treat primary liver tumor, lung tumor, rectal tumor, and malignant lymphoma [5]. Most notably, it differs from other antitumor drugs as CBC not only can directly inhibit tumor proliferation but also enhance immunity of patients. Modern antitumor mechanisms of CBC mainly involve proapoptosis, protein phosphorylation, and immunoregulation [6][7][8]. However, the specific pharmacodynamic material and molecular mechanisms underlying tumor treatment of CBC still remain unclear.
Regarding the integrative network pharmacology, a multicompound, multitarget, and multipathway approach has been used in TCM, especially in antitumor research [9,10]. Here, we attempt to identify effective material basis and molecular mechanisms of CBC using network pharmacology and molecular docking. Hub targets aim to be verified by quantitative real-time PCR (qPCR) in human hepatocellular carcinoma cells (SMMC-7721 cell), providing a theoretical basis for further research.

Identification of Candidate Compounds of CBC.
All compounds of 11 TCM herbs of CBC were retrieved from the encyclopedia of TCM (ETCM) database (http://www. nrc.ac.cn:9090/ETCM/) and literature reports. ETCM is an encyclopedia of TCM, which includes comprehensive and standardized information for the commonly used herbs and formulas of TCM as well as their ingredients [11].

Screening for Bioactive Compounds of CBC.
A quantitative metrics, known as the quantitative estimate of drug likeness (QED), was used to assess drug likeness. Estimated values ranged from 0 (all properties are unfavorable) to 1 (all properties are favorable) [12]. ETCM classified all 7274 ingredients into three groups according to their QED scores: good (QED > 0.67), moderate (0.49 ≤ QED ≤ 0.67), and weak (QED < 0.49) [11]. Compounds of CBC evaluated as good (QED > 0.67) were collected as bioactive compounds. Likewise, active compounds that did not fulfill the criteria for QED but have been reported in the available literature were recruited separately.

Prediction of Drug Targets for CBC and Network
Construction. Targets of active compounds of CBC were retrieved from ETCM. Other active compounds not included in ETCM were drawn via ChemDraw 19.0 software and imported into TargetNet database (http://targetnet. scbdd.com/), a web service for predicting potential drugtarget interaction profiling via multitarget SAR models, for predicting targets [13]. Targets were filtered by accuracy (AUC) ≥ 0.7 and probability (Prob) ≥ 0.9. Targets were corrected and transformed using UniProt database (https:// www.uniprot.org/) and defined as Homo sapiens. Subsequently, correction targets were imported into Cytoscape 3.7.0 software to construct a network of herb-compoundstargets.

Collection of Gene Targets Associated with Tumors.
e terms "anti-tumor" and "anti-cancer" were used as search terms in the GeneCards database (https://www. genecards.org/) [14], a searchable and integrative database that provides comprehensive and user-friendly information on all annotated and predicted human genes. e targets generated by the intersection between the tumor-related targets and predicted targets of CBC were identified as tumor-targets of CBC.

A Protein-Protein Interaction (PPI) Network Construction and Hub Targets Screening.
e tumor-targets of CBC were submitted to STRING 11.0 database (https:// string-db.org/) [15], which stores information about protein interactions. Only proteins with the confidence score higher than 0.9 were selected. e protein information was imported into Cytoscape to construct the protein-protein interaction (PPI) network and merged with the herbcompounds-targets network. Finally, the hub targets and core compounds were screened by the values of degree and betweenness for constructing the core compound-target network. e hub targets were applied for enrichment and antitumor location analysis.

Network Construction of Nature and Flavor, Channel
Tropism, and Tumor Location of CBC. Herbal drug properties in CBC were extracted from the Pharmacopoeia of the People's Republic of China according to the Four Properties (warm, cold, hot, and cool) and Five Flavors (pungent, sweet, sour, bitter, and salty) [16]. e nature and flavor (Chinese name, Xing wei), including Four Properties and Five Flavors, also called drug properties in herbal drug, which were clinically used according to clinical treatment experience of presented clinical symptom in patients in TCM theories. e channel tropism (Chinese name, Gui jing) is a theory of positioning and orientation of a certain organ, meridian, or specific clinical indication location to reveal the effect of TCM. e nature and flavour and channel tropism of CBC were collected to construct a network via Cytoscape to analyze drug properties and clinical indications. Meanwhile, hub targets filtered by PPI network were uploaded to the Comparative Toxicogenomics Database (http://ctdbase.org/) to collect diseases' information. Diseases marked with "marker/mechanism" and defined as cancer were selected and imported into Cytoscape to explore the cluster site of CBC clinical indication.

Gene Ontology and Pathway Enrichment Analysis of CBC.
To evaluate the role of targets by bioinformatic annotation, hub targets were imported into Enrichr database (http://amp.pharm.mssm.edu/Enrichr/) [17], which currently contains a large collection of diverse gene set libraries available for analysis. Hub targets were utilized by Enrichr high-throughput functional annotation bioinformatics to perform functional annotation, enrichment analysis, and gene ontology (GO) terms based on Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, with P < 0.05. Terms and pathways were sorted based on combined scores, and target-pathway network was constructed via Cytoscape to display the tightly connection between targets and tumor-related pathways.

Integrative Mechanism Analysis of CBC in Tumor
Treatment. To further explore potential mechanisms of CBC against tumor, networks of the core compound-target and target-pathway were merged to construct the herb-compound-target-pathway integrative network. e major tumor pathways were screened; among them, linked compounds and targets were identified as key compounds and targets subsequently. e integrative network was further verification of key compounds and targets, to integrate characteristics of multicompound, multitarget, and multipathway of CBC. Finally, the multivariate biological network was merged with the herb-compound-targetpathway, drug properties, and antitumor location network for comprehensive integration.

ADMET Absorption Level Analysis and Molecular
Docking Simulation. Effective material basis of CBC was verified by ADMET absorption level and molecular docking. ADMET absorption level of compounds was obtained using the ETCM database. Levels of "0, 1, 2, and 3" meant "good, moderate, low, and very low," respectively. Key compounds with good ADMET absorption levels were selected for the integrative network analysis [18].
Molecular docking was performed to simulate structural stability between key compounds and targets using iGEMDOCK 2.1 software, a commonly used tool for molecular recognition, which evaluates and improves scoring functions [19]. e iGEMDOCK energy functions consist of electrostatic, steric, and hydrogen bonding potentials, which were used to recognize complexes. e key compounds in herb-compound-target-pathway network were downloaded using PubChem database (https://pubchem.ncbi.nlm.nih. gov/), energy minimized via Chem3D Table 1 lists the primer sequences. Gene expression of PTGS2, TP53, ESR1, ABCB1, and TOP2A was investigated. e samples were exposed to predenaturation at 95°C for 30 s, followed by 39 cycles of denaturation at 95°C for 5 s, and at 60°C for 30 s, and annealing at 95°C for 10 s. e dissolution curve conditions were 65°C for 0.05 s and 95°C for 0.5 s using 10 μL TB Green Premix EX Taq II, 0.8 μL 10 μmol/L forward primer, 0.8 μL 10 μmol/L reverse primer, and 2 μL cDNA. Water was added to achieve a total volume of 6 μL. GAPDH was used as the internal control, and data were analyzed using the 2 −ΔΔCt method. e experiment was repeated three times.

Statistical Analysis.
Statistical analysis was performed using Prism 8.0.2 software. Data were expressed as means ± SD and analyzed using Student's t-test. Differences between groups were considered to be statistically significant if P < 0.05.

Herb-Compound-Target Network Construction and
Targets of CBC against Tumors. In this study, 436 targets were screened against compounds from CBC, which corresponded to an average of almost three targets per compound.
e herb-compound-target network was constructed via Cytoscape software, where degree value changes with color and size variation ( Figure 2). In order to Evidence-Based Complementary and Alternative Medicine       Evidence-Based Complementary and Alternative Medicine identify correlation between each herb in CBC, a heat map was applied to reveal common number of targets. EZ correlated with 242 targets of CBC, while banmao was connected to 49 targets (Figure 3). Alternatively, by searching "anti-tumor" and "anti-cancer" in the GeneCards database, 1848 and 2024 targets were obtained, respectively. e 2920 common targets were selected (Figure 1(c)). Subsequently, the intersection of 207 CBC tumor targets was filtered out between disease targets and CBC targets (Figure 1(d)).

PPI and Core Compound-Target Network Construction.
Tumor-targets of CBC were brought to the String database to perform a PPI network with the highest confidence (0.9). A total of 158 nodes and 619 edges were obtained in the PPI network ( Figure 4). e PPI network was merged with the herb-compounds-targets network to build the core compound-target network based on topological parameters with average degree value > 18.43 and average betweenness > 0.007. en, 39 putative hub targets and 29 core compounds were identified ( Figure 5), suggesting that the hub targets and compounds had multiple beneficial biological functions for treating tumors at the molecular level.

Drug Properties and Organism Location of Antitumor
Analysis. Based on Pharmacopoeia of the People's Republic of China, the 11 herbs of CBC were categorized according to the Four Properties and Five Flavors and imported into Cytoscape ( Figure 6). Based on topological analysis, the pungent (degree � 5), bitter (degree � 5), and sweet (degree � 4) presented a higher degree value in nature and flavor. In addition, the liver-meridian (degree � 7), kidneymeridian (degree � 6), and spleen-meridian (degree � 6) had a higher degree value in channel tropism.
A total of 96 diseases containing 13 categories of tumors were collected by enrichment analysis of 39 hub targets. Detail mapping relations were visualized via Cytoscape  Evidence-Based Complementary and Alternative Medicine      hepatocellular carcinoma (degree � 14) presented higher degree values. In general, the antitumor activity of CBC may have a superior efficacy in liver, kidney, and breast tumors.

GO and Pathway Enrichment Analysis.
irty-nine hub targets were imported into Enrichr database enrichment analysis. e top 20 KEGG pathways (P < 0.05) were visualized by bubble plot (Figure 7(a)), and target-pathway network was built using Cytoscape, obtaining 32 nodes of targets (Figure 7(b)). e top 10 GO enrichment analyses were performed individually (P < 0.05), including cellular component (CC), molecular function (MF), and biological processes (BP), which were ranked by a combined score (Figure 7(c)).
In this study, 132 pathways were enriched (P < 0.05), suggesting that the putative targets were highly connected with the regulation of prostate cancer, pancreatic cancer, estrogen signaling pathway, vascular endothelial growth factor (VEGF) signaling pathway, small cell lung cancer, and mitophagy. Alternatively, 290 GO terms were obtained (P < 0.05), including estrogen metabolic process, prostaglandin metabolic process, cellular response to angiotensin, estrogen biosynthetic process, and prostaglandin biosynthetic process, suggesting that CBC may influence cancer-related pathways, mitophagy, angiogenesis, and metabolism of estrogen and prostaglandin for tumor treatment.

Multivariate Biological Network Build and Verification
Analysis.
e herb-compound-target-pathway network was constructed using the target-pathway network and core compound-target network (Figure 8). e integrative network comprised 11 herbs, 26 key compounds, 13 hub targets, and five pathways, suggesting that the characteristics of multicompound-target-pathway of CBC, and each herbal drug was indispensable in tumor treatment. Among them, VEGF signaling pathway, estrogen signaling pathway, small cell lung cancer, pancreatic cancer, and prostate cancer were selected as mainly tumor-related pathways. Further integration of the multivariate biological network was merged based on VEGF and estrogen signaling pathways, involving nine targets, which are linked with 10   tumors, five urogenital tumors, and breast neoplasms ( Figure 9). To verify the accuracy of our prediction, 26 core compounds were examined by ADMET absorption level, suggesting that 65% of the compounds presented a good or moderate level (Figure 10(a)). Among them, 46% of the compounds showed a good level, indicating our prediction achieved great accuracy. Subsequently, 13 hub targets were docked with core compounds. e targets PTGS2 (−109.75), HSP90AA1 (−98.21), and AKT1 (−96.38) presented the most stable structures (Figure 10(b)). Gancaonin A (−108. 53 Figure 9: Multivariate-biological-network of compound banmao capsule (CBC). e left side stands for nature and flavor, channel tropism, and disease and disease categories, while the right side stands for herbs, compounds, targets, and pathways.  Evidence-Based Complementary and Alternative Medicine (−102.37) showed a great potential against tumor targets by molecular docking and may be the main effective material basis of CBC. e ADMET absorption level of scutellarin was very low, but it still showed a great stable structure (Figure 10(c)).

Results of qPCR for Liver Tumor Target Genes.
e top five target genes PTGS2, TP53, ESR1, ABCB1, and TOP2A with a high degree of connectivity were identified in hepatocellular carcinoma of the antitumor location network. e effect of CBC on the expression of mRNA in SMMC-7721 cells was explored by qPCR ( Figure 10(d)). In this study, the mRNA expression levels of PTGS2, TOP2A, ESR1, and ABCB1 were decreased in the LCBC group (P < 0.01), while PTGS2 was increased in the HCBC group (P < 0.05), and TP53 showed to be significantly increased in both doses (P < 0.01). Our results indicate that PTGS2, TP53, ESR1,  Evidence-Based Complementary and Alternative Medicine ABCB1, and TOP2A may be the potential targets of CBC in the treatment of liver tumors.

Discussion
CBC is a traditional antitumor drug of Miao ethnic in Guizhou Province of China, although their effective material basis and mechanisms of action remain unclear [37]. In this study, CBC's bioactive compounds and antitumor targets were identified. A PPI network was constructed to calculate hub targets, which were enriched to explore potential synergistic effects of CBC's components. Finally, key compounds and targets were validated using molecular docking and qPCR. CBC is clinically used as blood-activating, stasis-resolving, and detoxicating in TCM. Based on the Pharmacopoeia of the People's Republic of China, the 11 herbs of CBC were categorized according to the five properties and five flavors, reflecting the body's effect of "inclination" of the ups and downs of Yin and Yang and the "semi-interior phase" change during drug intervention [38]. To further explore the drug efficacy of complex compounds, nature and flavour analysis was applied. Our results indicated that CBC belonged to the pungent, bitter, and sweet categories. Pungent compounds have the potential action of "bloodactivating" and "stasis-resolving" and may have potential in treating liver diseases [39]. Herbs with bitter taste can be used for "discharging" and "down-bearing," referred to as "heat-clearing" and "detoxicating" herbs. Herbs with sweet taste may be used for "supplementation, moderation, and harmonization," referred to as "tonifying and replenishing" herbs [40]. However, sweet-tasting herbs with "spleenstrengthening" functions are used more frequently than herbs with a bitter taste for "clearing heat." ere is a close relationship between recurrence and metastasis of breast cancer and liver. Herbs for "nourishing the yin-blood," "emolliating and soothing the liver," and "smoothing the meridians" are key in the treatment of breast cancer [40]. Our results indicated that CBC may have a multiaction "blood-activating," "stasis-resolving," "heat-clearing," and "detoxicating," and thus, it is potentially useful against breast and liver cancers.
To further explore the synergistic effects of CBC, the identification of the effective material basis and molecular mechanisms of CBC against tumors was analyzed.

Effective Material Basis of CBC.
Twenty-six active compounds from 11 herbs were filtered as material basis of CBC by the herb-compound-target-pathway network analysis. In addition, the core compounds were verified by ADMET absorption level. Our results indicated that 65% of the core compounds showed a great absorption level, suggesting that our previous prediction was accurate. Among them, CTD, the core material basis of CBC and the most critical component in banmao, can efficiently inhibit the activity of mammalian and plant protein phosphatase 2A (PP2A) in the treatment of tumors [41], and CTD may elevate leukocyte and enhance immunity [42].
Similarly, other compounds with great absorption levels, such as baicalein, sesamin, quercetin, and kaempferol, indicated a better interaction with hub targets by molecular docking. Besides, scutellarin may positively interact with the studied targets. Flavonoids of BZL, including scutellarin and baicalein from CBC, have been found to significantly treat liver tumors [43,44]. e antitumor effects of sesamin have been mainly attributed to apoptotic, inflammatory, metastatic, and autophagocytic activities [45,46]. Quercetin exhibits anticancer properties, especially in cancers located in the digestive and urogenital systems [47,48]. Kaempferol was able to treat various tumors in the liver, skin, and colon through apoptosis, cell cycle arrest, and epithelial-mesenchymal transition [49]. In summary, baicalein, scutellarin, sesamin, quercetin, and kaempferol may be the core components of CBC, which could influence the apoptotic, inflammatory, metastatic, and autophagic activity in tumor cells.

Antitumor Location of CBC and qPCR Verification of Liver Tumor Targets.
e "channel tropism" is the core characteristic of TCM herbal property theory, which classifies drugs acting in the "body's viscera and meridians" to illustrate the selectivity of the drug's effects on a certain part of the body, thereby providing a basis for clinical dialectical medication [50]. Our results indicated that CBC belonged to "liver-meridian" and "kidney-meridian," confirming its indications against tumors in the digestive and urogenital systems, based on TCM theories. Based on the antitumor location network analysis of CBC, digestive and urogenital systems tumors were mainly enriched in disease categories, especially in liver and breast cancer in disease terms. e hub targets associated with hepatocellular carcinoma in antitumor location network of CBC were further verified by qPCR. e mRNA expression of PTGS2, TP53, TOP2A, ESR1, and ABCB1 was significantly changed by CBC in SMMC-7721 cells, suggesting that these hub targets were crucial for the activity of CBC on hepatocellular carcinoma. e mRNA expression level of PTGS2 indicated a contrary trend in both doses, suggesting a dynamic regulation in the treatment of tumors with CBC. e mechanistic studies implicate PTGS2 is overexpressed in liver cancer for promoting angiogenesis and inflammation [51]. Studies have shown that EZ from CBC was able to inhibit hepatocellular carcinoma by inhibiting PTGS2 in SMMC-7721 cell [52]. TP53, a tumor suppressor gene, mediates DNA repair, apoptosis, cell cycle arrest, autophagy, and metabolic processes [53]. TP53 can be both an activator or inhibitor of autophagy [54]. Studies have shown that CTD may treat tumors by upregulating TP53 [55]. CTD was also able to inhibit breast cancer cells by suppressing autophagy and inducing apoptosis [56]. In addition, ESR1 may play a role in the development and prognosis of liver cancer [57]. e ABCB1 are considered to be a prime factor for inducting multidrug resistance in liver cancer treatment [58]. However, the effect of CBC on ESR1, TOP2A, and ABCB1 remains unclear. In this study, we Evidence-Based Complementary and Alternative Medicine concluded that CBC may dynamically regulate PTGS2, TP53, TOP2A, ESR1, and ABCB1 targets by inhibiting SMMC-7721 cells.

Integrative Biological Network Analysis.
e herbcompound-target-pathway network indicated that potential mechanisms of CBC involved 13 targets and five pathways, including VEGF signaling pathway, estrogen signaling pathway, and three tumor-associated pathways. Of these 13 targets, AKT1, SRC, RAC1, and PTGS2 are involved in VEGF signaling pathway. VEGF can increase angiogenesis and vascular permeability, contributing to key aspects of tumorigenesis, including the function of cancer cells and tumor initiation [59]. More importantly, several herbs or compounds were able to inhibit the VEGF signaling pathway against tumors. CTD inhibited angiogenesis by downregulating VEGF in vivo and vitro [60], whereas astragaloside IV of HQ from CBC treated a glioma by inhibiting VEGF [61]. SZY from CBC treated osteosarcoma by downregulation of VEGF [62], and EZ and HQ from CBC activity against lung tumors were based on the inhibition of VEGF [63]. Our results indicated that the VEGF signaling pathway may be a critical pathway of the CBC's inhibition of angiogenesis. AKT1, also known as protein kinase B, activates the PI3K/AKT pathway during tumor growth [64]. Studies have shown that targeting AKT1 may impair angiogenesis [65]. BM and EZ from CBC may inhibit AKT1 [66,67]. In addition, increased SRC target activity was identified in several malignancies [68]. Activation of SRC may further stimulate PI3K/Akt pathway. Studies have shown that CTD and SZY from CBC may inhibit SRC in vivo and vitro [69,70], but the regulation of SRC of CBC still remains unclear. Furthermore, the overexpression of PTGS2 was able to activate VEGF for tumor growth and diffusion [71], and EZ from CBC could treat liver cancer by inhibiting PTGS2 and VEGF [52]. us, PTGS2 may be a potential target of CBC.
Of 13 targets, AKT1, ESR1, ESR2, HSP90AA1, PGR, PRKACA, and SRC are involved in estrogen signaling pathway, and AKT1 and SRC both involved in both VEGF and estrogen signaling pathways. Sustained estrogen exposure or deregulated estrogen signaling is associated with various types of tumor, including breast, endometrium, and prostate. Estrogen is a potential mediator of immunosuppression through modulation of protumors [72]. Studies have shown that san leng from CBC may treat breast cancer by regulating the estrogen receptor [73]. EZ from CBC may treat breast cancer through the inhibition of estrogenic activity [74,75]. e estrogen receptor signaling is mediated by estrogen receptors, ESR1 and ESR2, while the majority of breast cancers have disregulated estrogen receptor signaling [76]. ESR1 is able to mediate the biological effects of the steroid hormone estrogen, while its mutation in breast cancer is a common mechanism of hormonal therapy resistance [77]. With the inhibition of ESR1, the risk of hormone breast cancer would be diminished [78]. ESR2 has been used as a prognostic molecular biomarker [79].
In this study, effective material basis, antitumor location, and mechanism of CBC were integrated in a multivariate biological network (Figure 9), which combined TCM theories and molecular biological network, identifying the multicompound-target-pathway of CBC against tumors. Our results indicated that CBC displays is "blood-activating," "stasis-resolving," "heat-clearing," and "detoxicating," and this is associated with its pharmacological function in VEGF, estrogen signaling pathway, PTGS2, and ESR1. ese targets were further verified by qPCR. Furthermore, CBC's activity as antitumor against liver and breast tumors was confirmed by a literature review.

Conclusion
Effective material basis and potential molecular mechanisms of CBC against tumors have been here explored, based on TCM theories and biological network analysis. ese were further verified by qPCR and molecular docking. e potential therapeutic location and hub targets of CBC were identified by multivariate biological network integration. Future studies should focus on CBC's mechanisms of action by molecular experiments in vivo and in vitro. Additionally, these experiments would further validate our results as per the inherent limitations of network predictions.

Data Availability
e data used to support the findings of this study are included within the article.

Conflicts of Interest
e authors declare that they have no conflicts of interest.