Abstract
The genus Dendrobium is a rich source of bioactive secondary metabolites, particularly bibenzyl derivatives with substantial pharmacological activities, but Dendrobium stuartii remains an underexplored species. Therefore, this study aimed to investigate the drug discovery potential of D. stuartii through an integrated in silico and experimental method. Four key compounds were prioritized through literature analysis, and the interactions with the epidermal growth factor receptor (EGFR) were evaluated using molecular docking. The chemical profile of the acetone extract was characterized using Liquid Chromatography–High Resolution Mass Spectrometry (LC-HRMS). Additionally, biological activities were assessed through antibacterial, antibiofilm, and anti-inflammatory assays. Batatasin III had the strongest predicted binding affinity toward EGFR, and the LC-HRMS analysis confirmed the presence of bibenzyl derivative 3,4ʹ-dihydroxy-5,5ʹ-dimethoxybibenzyl (gigantol). The extract provided antibacterial activity, specifically against S. aureus and P. acnes, inhibited biofilm formation during the mid-phase, and produced substantial anti-inflammatory activity, as evidenced by significant inhibition of protein denaturation. The results suggested D. stuartii as a promising underexplored source of bioactive compounds with potential applications in anticancer and anti-infective drug development.
Introduction
The genus Dendrobium (Orchidaceae) is among the largest and most diverse genera within the orchid family, comprising more than 1,500 species distributed across Asia and Oceania. 1 Numerous Dendrobium species have been widely used in traditional medicine systems, particularly in Chinese, Thai, and Indonesian systems, because of their health-promoting and therapeutic properties. 2 Phytochemical investigations show that Dendrobium species are rich sources of structurally diverse secondary metabolites, including alkaloids, flavonoids, polysaccharides, and bibenzyl derivatives. Many of the metabolites have significant pharmacological activities such as antioxidant, anti-inflammatory, anticancer, and hepatoprotective effects. 3 In recent years, increasing scientific attention has focused on identifying natural product-derived lead compounds for drug discovery. Natural products continue to play a crucial role in modern pharmacotherapy due to their chemical diversity, biological relevance, and evolutionary optimization for interactions with biological targets. 4 In this context, Dendrobium species have been identified as promising candidates for the discovery of novel bioactive compounds with potential applications in oncology and inflammatory diseases. 5 Several studies reported that bibenzyl and phenanthrene derivatives isolated from Dendrobium showed potent anticancer activity by modulating key cellular pathways, including apoptosis, autophagy, and oxidative stress regulation. 6
Among the bioactive constituents of Dendrobium, bibenzyl compounds such as moscatilin, gigantol, lusianthridin, and Batatasin III have been repeatedly reported with anticancer, antioxidant, and anti-inflammatory activities. 7 Moscatilin can inhibit cancer cell proliferation and induce apoptosis in various in vitro and in vivo models. 8 Similarly, gigantol has been shown to suppress tumor growth and metastasis by regulating oncogenic signaling pathways. 9 These results suggest that bibenzyl derivatives from Dendrobium may serve as promising lead compounds for targeted cancer treatment. However, most existing studies have focused on isolated biological activities or single molecular targets, and comprehensive investigations integrating in silico target prediction with experimental pharmacological validation remain limited. Dendrobium stuartii is a relatively underexplored species within the genus, and the phytochemical composition and pharmacological potential have not been comprehensively characterized. Preliminary reports suggest that this species contains bioactive metabolites comparable to those found in well-studied Dendrobium species, showing the potential as a source of novel drug leads. Systematic studies combining compound prioritization, molecular docking, chemical profiling, and biological evaluation of D. stuartii are currently lacking.
Molecular targeting and immunotherapy are widely recommended methods in cancer treatment. 10 The combination with chemotherapy can make the treatment more effective. The epidermal growth factor receptor (EGFR) is a cell surface protein that regulates cell proliferation and metastasis. 11 The amplification of the EGFR gene is associated with several human malignancies, suggesting that EGFR targeting tends to enhance therapeutic outcomes 12 while mutations in the EGFR kinase domain are key drivers of adenocarcinoma. 13 Tyrosine kinase inhibitors (TKIs) are among the selective active targets in the EGFR protein. 14 Several next-generation TKIs targeting triple-mutant kinase domains have been developed and have shown promising results in preclinical studies. 15 There are numerous reports on the anticancer potential of natural sources, including synthetic derivatives. Some natural compounds have various therapeutic potential for inhibiting or inducing ferroptosis. 16 Dendrobium species are reported to inhibit tumor growth in lung cancer, 17 liver cancer, 18 and breast cancer. 19
Prior results showed that the primary compounds of Dendrobium were evaluated for activity toward EGFR using molecular docking. Based on the background information, this study aimed to explore the pharmacological potential of D. stuartii through an integrated method combining in silico target-based screening and experimental evaluation of antibacterial, antibiofilm, and anti-inflammatory activities. In silico predictions were correlated with experimental pharmacological data to comprehensively assess D. stuartii as a promising source of bioactive compounds and support the potential application in the development of novel therapeutic agents. Previous investigations of Dendrobium species predominantly focused on isolated pharmacological activities or individual phytochemical reports. However, this study integrates systematic literature-based compound prioritization, molecular docking against EGFR, Liquid Chromatography–High Resolution Mass Spectrometry (LC-HRMS)-based chemical profiling, and in vitro antibacterial, antibiofilm, and anti-inflammatory assays in a single workflow. The results serve as the first report on the combination of EGFR-targeted in silico screening with experimental bioactivity evaluation of D. stuartii, thereby positioning the species as a promising, underexplored source of lead compounds for drug development. The overall workflow of the study is presented in Figure 1.
Study Concept Workflow.
Materials and Methods
Literature Mining and Compound Prioritization
Systematic literature mining was conducted to identify major bioactive compounds reported from Dendrobium species. Scientific databases, including Google Scholar, PubMed, ScienceDirect, Wiley Online Library, and Elsevier, were searched for relevant articles published up to 2023. The search strategy focused on keywords related to Dendrobium species, phytochemical composition, and pharmacological activities. A total of 33 peer-reviewed articles covering 33 Dendrobium species were selected. Information on plant parts, secondary metabolite classes, identified compounds, and reported biological activities was extracted and tabulated. Based on frequency of occurrence and pharmacological relevance, four major compounds, namely moscatilin, gigantol, lusianthridin, and Batatasin III, were prioritized for subsequent molecular docking analysis. The study selection process is summarized in Figure 2.
PRISMA Flowchart Representing the Study Selection and Identification Process.
Molecular Docking
Protein Preparation
Molecular docking was performed to evaluate the interaction between selected Dendrobium-derived compounds and EGFR. The three-dimensional crystal structure of EGFR was retrieved from the Protein Data Bank (ID: 5UG8). The protein structure was prepared by removing water molecules and co-crystalized ligands, then adding hydrogen atoms. The binding site was defined based on the coordinates of the native ligand, focusing on key residues within the Adenosine Triphosphate (ATP)-binding pocket. 20
Ligand Preparation
The chemical structures of moscatilin, gigantol, lusianthridin, and Batatasin III were obtained from public chemical databases and prepared using ChemAxon software. Energy minimization was performed, and multiple conformers were generated for each ligand to ensure conformational flexibility during docking. 21
Docking Procedure and Validation
Docking simulations were conducted using Protein-Ligand ANT System (PLANTS) software with default parameters. The docking protocol was validated by redocking the native ligand into the EGFR binding site, and the root-mean-square deviation (RMSD) between the docked and crystallographic poses was calculated. An RMSD value below 2.0 Å was considered acceptable. Docking scores were used to rank ligand binding affinity, with more negative values showing stronger predicted interaction. Protein-ligand interactions were visualized and analyzed using Discovery Studio Visualizer.22,23
Plant Material and Extraction Procedure
The D. stuartii plant material was thoroughly washed with distilled water to remove surface contaminants. The samples were sliced into small pieces and dried in a hot-air oven at 40 °C for 3 h, and then the dried material was ground into a fine powder. Approximately 1.5 kg of the powdered sample was macerated in acetone at room temperature. The solvent was replaced every 24 h, and the mixture was intermittently agitated every 6 h to enhance extraction efficiency. After completion of the extraction process, the solvent was filtered and concentrated under reduced pressure.
LC-HRMS Analysis
Chemical profiling of the D. stuartii acetone extract was performed using LC-HRMS. Chromatographic separation was achieved using a reversed-phase column maintained at 30 °C. The mobile phase consisted of acetonitrile and 5 mM acetate buffer, delivered at a flow rate of 1.5 mL/min under a gradient elution program. Mass spectrometric analysis was conducted in negative ionization mode with optimized parameters, including gas temperature of 300 °C, gas flow of 11 L/min, nebulizer pressure of 40 psi, and capillary voltage of 3,500 V. Compound identification was performed based on accurate mass measurements and database matching (e.g., METLIN, PubChem). The identification level is considered putative (level 2–3 confidence) due to the absence of MS/MS fragmentation data and reference standards. 24
Antibacterial Assay
The antibacterial activity of D. stuartii extract was evaluated using the disk diffusion method against S. aureus, P. acnes, E. coli, and P. aeruginosa. Bacterial suspensions were standardized to McFarland 0.5 and inoculated onto nutrient agar plates. Sterile paper disks impregnated with extract concentrations of 5%, 10%, 15%, 20%, and 25% (w/v) were placed on the agar surface. Chloramphenicol and tetracycline were used as positive controls, while 10% dimethyl sulfoxide (DMSO) served as a negative control. Plates were incubated at 37 °C for 24 hours, inhibition zones were measured using a digital caliper, and all assays were performed in triplicate. 25
Antibiofilm Assay
Antibiofilm activity was assessed using a microbroth dilution method in 96-well microplates against S. aureus and E. coli. Bacterial cultures were adjusted to McFarland 0.5 (1.5 × 10⁸ CFU/mL) and inoculated into brain heart infusion medium. The extract was tested at concentrations of 1%, 0.5%, 0.25%, and 0.125% (w/v). Chloramphenicol (1%) and DMSO (1%) were used as positive and negative controls, respectively. Plates were incubated at 37 °C for 24, 48, and 72 h to represent the mid-phase, maturation phase, and eradication phase of biofilm development. Biofilm inhibition was quantified using standard optical density measurements after staining. 26
In Vitro Anti-inflammatory Assay (Protein Denaturation)
Anti-inflammatory activity was evaluated using a protein denaturation assay. Test solutions of the extract were prepared at concentrations of 25, 50, 75, 100, and 125 ppm. Each solution was mixed with albumin solution and adjusted to physiological pH, then the mixture was incubated at 37 °C for 15 min, and heated to denature the protein. After cooling, absorbance was measured at 660 nm using a UV–Vis spectrophotometer. Diclofenac sodium (100 ppm) was used as a positive control due to the well-established anti-inflammatory activity, mediated by inhibition of protein denaturation and cyclooxygenase pathways. Dose-response data were analyzed using nonlinear regression with a four-parameter logistic model to determine the half-maximal effective concentration (IC50). 23
Data Analysis
All experiments were performed in triplicate (n = 3), and the results are expressed as mean ± standard deviation. Statistical analysis was conducted using a t-test, and a p < .05 was considered statistically significant. Dose-response data were analyzed using a nonlinear regression model to determine IC50 values.
Results
Phytochemical Distribution of Dendrobium Species
A systematic literature analysis of 33 eligible studies covering 33 Dendrobium species identified 85 major compounds of secondary metabolites across Dendrobium species. The distribution of phytochemical classes identified from Dendrobium species is shown in Figure 3. D. nobile and D. officinale were the most widely studied types of Dendrobium, with various biological activities. The stem is the most extensively studied plant part in Dendrobium species, which contains bibenzyl, with bibenzyl derivatives representing the predominant class of bioactive compounds. D. nobile and D. officinale are among the most extensively investigated Dendrobium species due to their rich phytochemical diversity and broad pharmacological potential. Previous studies have reported the presence of bibenzyls, alkaloids, polysaccharides, and phenolic glycosides in these species, contributing to antioxidant, immunomodulatory, and anticancer activities. Detailed information regarding Dendrobium species, major compounds, plant parts, and pharmacological activities is presented in Table 1.60,61
Phytochemical Compound of Dendrobium.
Result of Tabulating Data.
Medicinal plants produce several major classes of bioactive compounds, including flavonoids, alkaloids, and stilbenoids. In the stilbenoid group, bibenzyls represent a structurally complex subdivision with diverse biological activities. During phytochemical surveys, bibenzyl derivatives are among the most frequently reported compound classes, with moscatilin and gigantol identified as the most prevalent constituents. The frequency of occurrence of the major compounds identified from Dendrobium species is presented in Figure 4.
Most Common Compound of Dendrobium.
Molecular docking was performed using the PLANTS software to evaluate the interaction of four major bibenzyl- related compounds, including moscatilin, gigantol, lusianthridin, and Batatasin III, with EGFR. The active site was determined through redocking of the native ligand. Based on the screening, the coordinates X: −13.7156, Y: 15.3976, Z: −26.7021, with a radius of 11.7701, were obtained. Docking validation through redocking of the native ligand produced an RMSD of 1.47 Å, which was significantly less than 2 Å, confirming the reliability of the docking protocol. The coordinates of this method can be used for the docking of samples, and the results of redocking are shown in Figure 5.
Visualization of Docking Validation (Red Native and Blue Redocking Native Ligand).
Docking analysis was performed to evaluate ligand-EGFR interactions. The residues engaged in binding are summarized in Table 2. When compared with native ligands, the scores obtained were Batatasin III (79%), gigantol (71%), lusianthridin (70%), and moscatilin (68%). Among the tested compounds, Batatasin III had the lowest docking score, suggesting suitable binding affinity for EGFR and a potential interaction with the ATP-binding site. A higher percentage value corresponds to a more negative docking score, showing stronger ligand-EGFR binding affinity. 62 The interaction profile showed that ligand-EGFR binding was primarily stabilized through hydrogen bonding and hydrophobic interactions, including key amino acid residues in the EGFR active site. Docking results only are insufficient to confirm biological activity and therefore require experimental validation.
Docking and Residue EGFR Are Involved in the Interaction.
Based on Figure 6, two types of interactions were obtained between the ligand and EGFR. The pink color shows a hydrogen bond donor, and the green color represents a hydrogen bond acceptor. The interactions are hydrogen and hydrophobic bonds, with the results of the residue interaction analysis showing moscatilin (8), gigantol (7), lusianthridin (6), and Batatasin III (6) (5). Hydrogen bonding was found in ligands moscatilin (6), gigantol (2), lusianthridin (4), and Batatasin III (4) (2). The consideration of hydrogen bonds is crucial because this particular bond type has the strongest interactions with EGFR and distances below 3.8 Å.
Visualization of the Interaction Between Ligand and EGFR.
LC-HRMS analysis identified nine major compounds, specifically the detection of 3,4′-Dihydroxy-5,5′-dimethoxybibenzyl, which confirmed the presence of bioactive bibenzyl compounds 63 in D. stuartii. The identified compounds spanned a wide retention time range, reflecting diverse polarities and structural complexities. The identified compounds and their LC-HRMS characteristics are summarized in Table 3.
LC-HRMS Data Obtained from D. stuartii Detailing the Identities of Nine Compounds as Major Compounds.
Antibacterial Activity
The antibacterial activity of D. stuartii was evaluated against E. coli, S. aureus, P. aeruginosa, and P. acnes, with the extract showing concentration-dependent antibacterial activity. At lower concentrations (5%–20%), inhibition zones were generally less than 10 mm for all tested bacteria. At the highest concentration (25%), significant inhibition zones were observed, particularly against S. aureus and P. acnes. However, inhibitory activity against P. aeruginosa was limited across all concentrations. The positive control antibiotics produced the largest inhibition zones, confirming assay validity. The antibacterial activity observed in this study represents preliminary screening results, as minimum inhibitory concentration (MIC) and minimum bactericidal concentration were not determined. The antibacterial activity of D. stuartii extract against the tested bacterial strains is shown in Figure 7.
Antibacterial Activity of D. stuartii.
Antibiofilm Activity
Antibiofilm activity was assessed against S. aureus and E. coli at different stages of biofilm development. The D. stuartii extract showed significant inhibition of biofilm formation during the mid-phase of development. The inhibitory effect was concentration-dependent, with higher extract concentrations achieving greater biofilm suppression. Reduced activity was observed during the maturation and eradication phase, showing phase-specific antibiofilm effect. The antibiofilm assay provides qualitative to semi-quantitative insight, and further standardized assays are required for precise quantification. The antibiofilm activity of D. stuartii extract during different phases of biofilm development is presented in Figure 8.
Antibiofilm Activity of D. stuartii.
Anti-inflammatory Activity
Anti-inflammatory activity was evaluated using a protein denaturation assay. The D. stuartii extract showed a concentration-dependent inhibition of heat-induced protein denaturation. Higher extract concentrations had a greater protective effect against protein aggregation. The observed activity was comparable to the positive control at higher concentrations, showing significant anti-inflammatory potential. The protein denaturation assay represents a preliminary model of anti-inflammatory activity and does not fully reflect complex inflammatory pathways in vivo. The percentage inhibition and IC50 values obtained from the protein denaturation assay are presented in Table 4.
Result of % Inhibition.
Discussion
Phytochemical Context and Reliability of Compound Annotation
The phytochemical landscape of the genus Dendrobium is characterized by extensive chemical diversity, particularly enriched in bibenzyl derivatives, which provide a strong foundation for the pharmacological potential. These compounds are known to modulate key oncogenic pathways, 6 including those associated with EGFR signaling. In this study, molecular docking further supports the relevance of modulation ability by showing suitable binding interactions between Batatasin III and EGFR. Beyond anticancer potential, the extract has antibacterial, antibiofilm, and anti-inflammatory activities, showing a multi-target pharmacological profile. Among the compounds, bibenzyl derivatives are consistently identified as dominant and pharmacologically relevant constituents, particularly in stem-derived extracts. The high recurrence of compounds such as moscatilin and gigantol across multiple species supports the chemotaxonomic significance and justifies the prioritization in this study. Specifically, moscatilin has attracted substantial attention due to the broad biological activity profile, with anticancer effects being the most extensively investigated. Previous experimental studies reported that moscatilin effectively suppressed breast cancer cell proliferation while promoting cell apoptosis. 19 These effects are mediated by modulation of key signaling pathways included in tumor growth, cell cycle regulation, and programmed cell death, as well as by the activation of autophagic mechanisms. Collectively, these results position moscatilin as a multifunctional bioactive compound with relevance to cancer chemoprevention and treatment. Antioxidant effects were consistently validated using standard radical scavenging assays, including 2,2-Diphenyl 1-picrylhydrazyl (DPPH) and 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), showing robust redox-modulating capacity. 64 Anticancer investigations have predominantly focused on breast and lung cancer models, reflecting the clinical relevance and high global burden of the malignancies.
Polysaccharides are among the most important antioxidant fractions in Dendrobium. These macromolecules exert protective effects by upregulating the activity of key antioxidant enzymes, such as superoxide dismutase and catalase, and increasing intracellular glutathione level. 65 The multi-target antioxidant mechanism supports the potential use of Dendrobium polysaccharides as adjunctive agents for the treatment of oxidative stress-related disorders, but careful interpretation of LC-HRMS data is essential. The chemical profile of D. stuartii was dominated by phenolic and bibenzyl-related compounds, including 3,4’-dihydroxy-5,5’- dimethoxybibenzyl (gigantol), widely reported in Dendrobium species. This refined interpretation corresponds with known phytochemical patterns and provides a more credible basis for pharmacological correlation.
Environmental factors such as air pollution and vehicle exhaust are recognized contributors to lung carcinogenesis through sustained oxidative stress and inflammation. 66 Bioactive compounds derived from Dendrobium may counteract these pathological triggers through combined antioxidant, anti-inflammatory, and antitumor mechanisms. Lung cancer represents the most intensively studied therapeutic target of Dendrobium-derived compounds. Gigantol has been reported to exhibit anticancer activity against several lung cancer models through modulation of proliferation and apoptotic pathways. 9 This molecular cascade helps to suppress tumor growth and metastatic potential. Available evidence suggests that gigantol has low toxicity, favorable solubility, and good bioavailability, supporting the candidacy for further drug development. 67 Precision oncology increasingly emphasizes the identification of well-defined molecular targets, particularly in cancers driven by aberrant kinase signaling. 68 The EGFR remains a central therapeutic target in non-small cell lung cancer, with objective response rates near 60% in EGFR-mutated cases. 69 Even though third-generation EGFR TKIs, such as osinertinib, represent the current standard of care, acquired resistance and disease progression remain significant clinical challenges. 70 Consequently, combination strategies including radiotherapy are frequently used to enhance treatment outcomes. 71
Molecular Docking Insights and Structure-activity Relationships
Molecular docking analysis showed that ligand-EGFR interactions are primarily stabilized through hydrogen bonding and hydrophobic interactions. Hydrogen bonds formed between donor groups (e.g., NH and OH) and electron-rich acceptor atoms (oxygen and nitrogen) play a crucial role in binding affinity and complex stability. The docking results obtained in this study are consistent with previous experimental evidence, supporting the anticancer relevance of the investigated bibenzyl derivatives. Moscatilin has been shown to inhibit proliferation and induce apoptosis in breast cancer models, both in vitro and in vivo, particularly in MDA-MB-231 cells.19,72 Gigantol has anticancer activity against hepatocellular carcinoma, while lusianthridin provides cytotoxic effects across multiple cancer cell lines, including H69AR, HeLa, HL-60, HepG2, and MOLT-3. 73 Batatasin III has been reported to suppress the proliferation and migration of H460 lung cancer cells.74,75 These results support a Structure–Activity Relationship (SAR) in which bibenzyl and related phenolic scaffolds play a central role in anticancer efficacy.
Structure-activity Relationship of Bibenzyl and Phenolic Compounds
The observed pharmacological activities can be rationalized based on the structural characteristics of bibenzyl and related phenolic compounds. These molecules primarily consist of two aromatic rings connected by an ethylene bridge and are variably substituted with hydroxyl and methoxy groups. 76 Hydroxyl groups play a critical role in mediating hydrogen bonding interactions with biological targets, contributing to binding affinity and antioxidant capacity. 77 This is consistent with the docking results, in which compounds such as Batatasin III show suitable predicted interactions with EGFR, probably due to multiple hydrogen bond donors. Meanwhile, methoxy substituents enhance lipophilicity and facilitate interaction with hydrophobic domains of proteins and biological membranes. 78 This dual hydrophilic-lipophilic balance may contribute to antibacterial and antibiofilm activities through membrane perturbation mechanisms. 79 Additionally, the conjugated aromatic system of bibenzyl derivatives facilitates stacking interactions with aromatic amino acid residues in protein binding pockets, thereby enhancing ligand-protein stability. 80 Hydroxyl groups contribute to hydrogen bonding, while methoxy substituents increase lipophilicity, enabling improved interaction with hydrophobic domains. 81 The structural features described collectively support a SAR in which substitution patterns modulate binding affinity and multi- target pharmacological activity.
Pharmacological Activities: Multi-target and Extract-based Effects
In addition to anticancer activity, Dendrobium-derived compounds have significant antibacterial and antibiofilm effects. The antibacterial, antibiofilm, and anti-inflammatory activities observed in this study should be interpreted as multi-target effects of a complex phytochemical mixture, rather than the action of a single compound. The presence of bibenzyl derivatives, such as gigantol, provides a plausible chemical basis for the observed antibacterial and anti-inflammatory activities. 82 These phenolic compounds are known to disrupt membrane integrity and inhibit essential bacterial enzymes, contributing to the antimicrobial effects. The antibacterial activity, particularly against S. aureus, may be attributed to phenolic compounds that disrupt membrane integrity and interfere with essential bacterial enzymes. 83 However, the disk diffusion method provides only preliminary screening data and does not reflect MIC or bactericidal activity. Flavonoids and isoflavonoids disrupt bacterial membrane permeability and inhibit essential enzymes such as Deoxyribonucleic Acid (DNA) gyrase and topoisomerase IV, particularly in S. aureus and E. coli. 84 The bibenzyl derivative 3,4′-dihydroxy-5,5′-dimethoxybibenzyl may contribute to the antimicrobial activity observed in this study. Similar to other phenolic compounds, its antibacterial effects may be associated with disruption of membrane integrity, interference with bacterial enzymes, and inhibition of biofilm formation. 85
Observations stated in this study are consistent with previous reports describing antimicrobial activity in several Dendrobium species. 86 Furthermore, the detected lipid and alkaloid compounds had additional detected lipid and alkaloid compounds may contribute to biofilm disruption and facilitate antibacterial activity. 87 The antibiofilm activity observed during the mid-phase of biofilm development suggests interference with early adhesion and extracellular matrix formation. This phase-specific activity is consistent with previous reports showing that phenolic compounds disrupt quorum sensing and inhibit biofilm maturation. However, the semi-quantitative nature of the assay limits precise interpretation.
The anti-inflammatory activity, evaluated using a protein denaturation model, reflects the ability of compounds to stabilize protein structure under stress conditions. This assay provides useful preliminary insight, without fully representing cyclooxygenase activity or immune modulation. Compounds that preserve protein structure can attenuate the release of inflammatory mediators. Daidzein has been reported to exhibit anti-inflammatory activity through modulation of inflammatory mediators. 88 These results show that the D. stuartii extract has preliminary pharmacological activity with a broad spectrum, and requires further validation in more advanced experimental models.
Integration with Molecular Docking: Predictive Insight Rather than Validation
Molecular docking analysis identified Batatasin III as the compound with the most suitable predicted binding affinity toward EGFR. This observation is consistent with the structural features, particularly the presence of hydroxyl groups capable of forming hydrogen bonds in the ATP-binding pocket. 89 However, molecular docking represents a predictive computational method and does not constitute experimental validation of anticancer activity. The absence of in vitro or in vivo anticancer assays limits the ability to directly correlate docking results with biological effects. The use of a single molecular target (EGFR) does not capture the complexity of cancer signaling networks. Therefore, the docking results should be interpreted as hypothesis-generating, emphasizing potential interactions that need further experimental investigation.
Conclusion
In conclusion, the D. stuartii acetone extract, dominated by 3,4’-Dihydroxy-5,5’-dimethoxybibenzyl, showed substantial antibacterial activity against S. aureus and P. acnes, inhibited the mid-phase of biofilm formation, and produced anti- inflammatory activities, with Batatasin III showing effective EGFR binding potential. Meanwhile, the limitations of this study included tentative conduction of LC-HRMS compound identification due to the absence of MS/MS confirmation. Biological assays were restricted to preliminary screening methods without advanced pharmacological validation. Molecular docking was performed on a single target without experimental anticancer validation. Several limitations should be acknowledged in the literature mining section, including potential publication bias, heterogeneity of phytochemical methods, and differences in biological assay protocols among included studies. Future studies should comprise compound isolation, structural confirmation, and mechanistic investigations.
Footnotes
Acknowledgements
The authors thank Lambung Mangkurat University for providing the opportunity and support to conduct this study through the 2025 Domestic Collaborative Research (PKDN) program.
Authors’ Contribution
All authors made substantial contributions to conception and design, acquisition of data, or analysis, and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; agreed to submit to the current journal; gave final approval of the version to be published; and agreed to be accountable for all aspects of the work. All the authors are eligible to be authors as per the International Committee of Medical Journal Editors’ requirements/guidelines.
Consent to Participate
Not applicable. This study did not involve human participants.
Consent for Publication
Not applicable.
Data Availability Statement
All the data is available with the authors and shall be provided upon request.
Declaration of Conflicting Interests
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Ethical Approval
This study does not involve experiments on animals or human subjects.
Funding
The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research was funded by Lambung Mangkurat University through the 2025 Domestic Collaborative Research (PKDN) program under derivative contract number 1803/UN8.2/PG/2025.
Informed Consent
Not applicable. No human participants were involved in this study.
Use of Artificial Intelligence-assisted Tools
The authors declare that they have not used artificial intelligence (AI) tools for writing and editing of the manuscript, and no images were manipulated using AI.