Pseudolaric Acid B Combats Drug‐Resistant Candida albicans Infection via Dual‐Action Mechanisms of Direct Antifungals and Vaginal Microbiota Restoration
Tianmeng Shao, Yunshan Zhang, Binqing Xue, Weihua Chu
TL;DR
Pseudolaric acid B treats drug-resistant yeast infections by killing the fungus and restoring healthy vaginal bacteria.
Contribution
PAB is shown to combat Candida albicans through dual mechanisms: direct antifungal action and microbiota restoration.
Findings
PAB inhibits biofilm formation and reduces ergosterol biosynthesis by downregulating ERG11.
PAB restores vaginal microbiota and reduces inflammation in a mouse model of vaginitis.
PAB shows superior therapeutic efficacy compared to conventional antibiotics in treating Candida infections.
Abstract
Candidal vaginitis is a prevalent inflammatory condition of the reproductive tract that has a substantial impact on women's health. Conventional antibiotic therapy frequently results in recurrence due to the non‐selective elimination of vaginal microbiota and subsequent dysbiosis. In order to overcome this limitation, the focus was directed towards traditional Chinese medicine, which has antimicrobial properties and it was here that pseudolaric acid B (PAB) was identified as a promising active monomer through the utilisation of virtual screening and bioinformatics approaches. In vitro experiments confirmed that PAB inhibits biofilm formation, reduces ergosterol biosynthesis by downregulating the key gene ERG11, and enhances fungal cell membrane permeability, ultimately resulting in fungal cell death. In vivo studies in a mouse model of Candida albicans ‐induced vaginitis demonstrated…
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Taxonomy
TopicsBiological Activity of Diterpenoids and Biflavonoids · Medicinal plant effects and applications · Synthesis and bioactivity of alkaloids
Introduction
1
The rising tide of antimicrobial resistance (AMR) represents one of the most pressing challenges to global public health in the 21st century. Fungal infections, particularly those caused by drug‐resistant Candida species, contribute significantly to this burden, as their treatment is increasingly hampered by the limited arsenal of antifungal agents and their propensity to induce dysbiosis—a disruption of the protective host microbiota. Consequently, there is an urgent need to develop novel therapeutic strategies that not only overcome pathogen resistance but also actively preserve or restore the host's native microbial ecology to prevent recurrence. Candida albicans is among the most prevalent and extensively studied fungal pathogens. As a commensal microorganism, it naturally resides within the human microbiota, colonising surfaces such as the skin, mucous membranes and genital tract (Kashem and Kaplan 2016). Under normal conditions, C. albicans coexists with the host without causing harm. Conventional antifungal therapies, such as fluconazole, primarily employ a single‐target, ‘bug‐and‐drug’ paradigm. While effective initially, this approach often lacks selectivity, indiscriminately impairing commensal flora alongside pathogens and fails to address the dysbiotic microenvironment that perpetuates recurrence. This underscores the limitations of a pathogen‐centric view and highlights the necessity for a paradigm shift towards ‘ecologically informed’ antimicrobial agents. Such next‐generation therapeutics would be characterised by dual or multi‐mechanistic actions: directly eradicating the pathogen while simultaneously fostering a restoration of the host's microecological homeostasis, thereby creating a resilient state that is resistant to reinfection (Grela et al. 2019; Hsieh et al. 2018). The urgency of this paradigm shift is underscored by the sophisticated resistance mechanisms evolved by Candida species, including modification or overexpression of drug targets—such as amino acid substitutions in ERG11, which diminish the binding affinity of azole drugs to lanosterol demethylase (Lee et al. 2021) and activation of cellular stress response pathways that enhance fungal adaptability and survival under drug pressure (Robbins et al. 2017). In response to these challenges, novel antifungal strategies are being explored. These include microbial‐derived compounds (e.g., the macrolide antibiotic mandelamide (Deng et al. 2025)) and probiotic‐based nanozyme hydrogels, such as rGO@FeS2/Lactobacillus@HA (Wei et al. 2023), and animal‐derived agents such as Als3‐specific immunoglobulin Y antibodies targeting a key adhesin of C. albicans (Lee et al. 2025). Particularly noteworthy are plant‐derived metabolites, which represent a rich and historically validated reservoir of antifungal agents. Compounds such as eugenol from clove oil (Gao, Guo, et al. 2025; Gao, Wang, et al. 2025), allicin from garlic and berberine from Coptis chinensis have exhibited broad‐spectrum antifungal activity (Zainal et al. 2021; Liu et al. 2020), often acting through multi‐target mechanisms that reduce the likelihood of resistance development. In this context, natural products, especially those derived from traditional medicine systems with a long history of managing infectious diseases, represent a fertile ground for discovering such multi‐faceted therapeutic agents. These compounds have often evolved to interact with biological systems in a complex manner, offering a rich source of chemical scaffolds that may inherently possess the desired multi‐target or host‐modulating activities. We therefore turned to Traditional Chinese Medicine (TCM) to identify lead compounds that embody this dual‐mechanism philosophy.
Pseudolaric acid B (PAB; C_23_H_28_O_8_) is a tricyclic diterpenoid acid originally isolated from the root and stem bark of Pseudolarix kaempferi Gordon (Peng et al. 2025). It exhibits a broad spectrum of biological activities. For instance, PAB has been shown to inhibit tumour growth and metastasis by suppressing the PI3K/AKT, ERK1/2 and mitochondria‐mediated apoptosis pathways (Wang et al. 2017). In models of atherosclerosis, PAB reduces lipid levels and deposition within plaques, while also decreasing the proliferation of macrophages in lesion areas (Li et al. 2018). Additionally, PAB significantly alleviates inflammatory responses and reduces fungal load in rats with Fusarium‐induced fungal keratitis (Liu et al. 2024).
The ERG11 gene encodes sterol 14α‐demethylase (CYP51), a key enzyme that catalyses the conversion of lanosterol to ergosterol—an essential component of the fungal cell membrane. This enzyme is not only critical for ergosterol biosynthesis but also serves as a primary target of azole antifungals (Urbanek et al. 2022; Wu et al. 2018).
In this study, we aimed to screen TCM‐derived monomeric compounds targeting ergosterol, a crucial component of the fungal cell membrane, using an integrated approach combining bioinformatics, in vitro assays and in vivo validation. We further elucidated the underlying mechanisms of action of the most promising candidate.
Materials and Methods
2
Identification and Screening of Bioactive Constituents From Chinese Medicinal Herbs
2.1
An extensive literature review was conducted to identify Chinese herbal medicines with documented antimicrobial properties. Based on this review, potential bioactive constituents were selected from the following plant species: Coptis chinensis Franch, Phellodendron amurense Rupr, Houttuynia cordata Thunb, Phellinus igniarius, Scutellariae Radix, Sophorae Flavescentis Radix, Andrographis Herba, Stephania Root (Radix Stephaniae Tetrandrae), Pseudolaricis Bark (Cortex Pseudolaricis), Paris Rhizome (Rhizoma Paridis), Dioscorea Rhizome (Dioscoreae Hypoglaucae Rhizoma), Cynanchum Root (Radix Cynanchi), Japanese Yam Rhizome (Rhizoma Dioscoreae Nipponicae), Veratrum nigrum , Notopterygii Root and Rhizome and Clove Flower (Xia 2019; Wang et al. 2022). The bioactive components of these herbs were initially screened using the Traditional Chinese Medicine Systems Pharmacology (TCMSP) and HERB databases.
Prediction of Molecular Druggability and Pharmacokinetic Properties
2.2
Molecular structures and canonical SMILES codes of the candidate active components were retrieved from the PubChem database, with three‐dimensional structures downloaded in. SDF format. File format conversion to. PDB and. MOL was carried out using Open Babel software. Pharmacophore modelling and pharmacokinetic profiling were performed using SwissADME, and Discovery Studio. Druggability screening criteria comprised Lipinski's Rule of Five, Ghose Filter, Veber Rule, Egan Rule and Muegge Rules. Pharmacokinetic parameters including blood–brain barrier (BBB) penetration and gastrointestinal (GI) absorption were also evaluated. Additionally, toxicity profiles of the screened molecules were predicted, covering hepatotoxicity, Ames mutagenicity, developmental toxicity potential, FDA rodent carcinogenicity and weight‐of‐evidence carcinogenicity to comprehensively assess mutagenic and carcinogenic risks.
Analysis of Ergosterol Metabolic Pathways and Candidate Molecular Target Screening
2.3
The metabolic pathways associated with ergosterol biosynthesis in Candida albicans were analysed using the KEGG database (Skariyachan et al. 2020). This investigation identified the ERG11‐mediated signalling pathway as playing a critical role in ergosterol synthesis, establishing ERG11 as a key molecular target for antifungal intervention (Figure S5).
Molecular Docking Study
2.4
The ERG11 protein was selected as the receptor for molecular docking. Its crystal structure was retrieved from the Protein Data Bank (PDB) and processed using PyMOL 3.1 to define the active pocket, remove water molecules and add hydrogen atoms. The screened drug molecules served as ligands; their three‐dimensional structures were obtained from the PubChem database and energy‐minimised using Chem3D 18.0 before being exported in. PDB format. Semi‐flexible docking was performed using AutoDock Vina, with optimised grid box dimensions and genetic algorithm parameters (Xiong et al. 2024). The binding affinity (in kcal/mol) was calculated for each complex, with more negative values indicating stronger binding. The docking results were visualised using PyMOL and Discovery Studio to analyse binding modes, hydrogen bond interactions and ligand positioning within the binding pocket.
Molecular Dynamics Simulation
2.5
The lowest‐binding‐energy conformation obtained from molecular docking was selected as the initial structure for molecular dynamics (MD) simulations. All simulations were performed using GROMACS software to assess the stability and interaction dynamics of the protein–ligand complex in an explicit aqueous solvent. The topology of the receptor was constructed using the CHARMM36 force field (Huang et al. 2017), while that of the ligand was generated with the sobtop tool. A cubic periodic boundary box was defined to encapsulate the system. The TIP4P water model was used to solvate the box, and chloride or sodium ions were added to neutralise the overall system charge. Energy minimization was followed by pre‐equilibration under NVT (constant number of particles, volume and temperature) and NPT (constant number of particles, pressure and temperature) ensembles. Finally, production MD simulations were conducted for further analysis.
Microorganisms
2.6
Candida albicans (ATCC 10231, ATCC 14053) was obtained from the American Type Culture Collection. Lactobacillus crispatus CPU2401 was a clinical isolate. All five strains— C. parapsilosis clinical resistant strain No. 1 (* C. parapsilosis‐R1), C. orthopsilosis, C. tropicalis clinical resistant strain No. 1 ( C. tropicalis‐R1), C. parapsilosis clinical resistant strain No. 2 ( C. parapsilosis‐R2) and C. tropicalis clinical resistant strain No. 2 ( C. tropicalis‐*R2)—were clinically isolated drug‐resistant isolates. The strains were cultured in YPD medium (1% yeast extract, 2% peptone and 2% glucose) at 37°C with shaking at 180 rpm for 12 h. Fungal cultures were stored at 4°C for short‐term preservation.
Animal Welfare and Ethics
2.7
Female specific‐pathogen‐free (SPF) Balb/c mice (5 weeks old) were supplied by Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co. Ltd. All animal procedures were performed in compliance with national and institutional guidelines for the care and use of laboratory animals. The experimental protocol was approved by the Committee for the Care and Use of Laboratory Animals at China Pharmaceutical University (Ethics Approval No.: YSL‐202507032).
Antifungal Susceptibility Testing
2.8
The susceptibility of C. albicans to the test compound was evaluated using the Oxford cup method (Duan et al. 2025). Briefly, 100 μL of a C. albicans suspension was spread evenly onto agar plates. Wells were formed in the agar using a sterile Oxford cup, into which 100 μL of the test compound was added. The plates were incubated at 37°C for 12 h, after which the presence and size of inhibition zones were measured to determine the antimicrobial activity of PAB at various concentrations.
Growth Curve and Time‐Kill Assay
2.9
The growth kinetics of Candida albicans ATCC 10231, ATCC 14053 and five drug‐resistant clinical strains in response to PAB treatment were assessed by turbidimetry through measuring the optical density (OD) at 600 nm over time (Kantroo et al. 2024). C. albicans was inoculated at a density of 2 × 10^5^ CFU/mL into media containing PAB. The cultures were incubated at 37°C with shaking for 12 h. Aliquots were taken at 0, 2, 4, 8 and 12 h, transferred to a 96‐well plate, and the OD600 was measured. Growth curves were generated by plotting OD600 against incubation time.
In Vitro Biofilm Formation Assay
2.10
Biofilm formation was assessed using a 24‐well pyrogen‐free polystyrene plate. To each well were added the herbal monomer PAB at its minimum inhibitory concentration (MIC) and dimethyl sulfoxide (DMSO) (1%) as the vehicle control. Each well was inoculated with C. albicans at a density of 1 × 10^3^ CFU/mL and incubated at 37°C for 24 h. After incubation, the culture medium was carefully discarded, and the adhered biofilm was stained with 0.1% crystal violet (CV) for 15 min at room temperature. Excess stain was removed, and the bound dye was solubilised with 33% acetic acid. The optical density (OD) of the solution was measured at 580 nm to quantify biofilm formation (Sumlu et al. 2024).
Effects of PAB on Fungal Morphology and Cell Membrane
2.11
The effect of PAB on the cellular morphology of C. albicans was evaluated using scanning electron microscopy (SEM) according to established methods (Kantroo et al. 2024). Briefly, fungal cells were cultured to the logarithmic growth phase, harvested by centrifugation at 3500 ×g and 4°C for 10 min and washed twice with phosphate‐buffered saline (PBS). The pellet was resuspended to a density of 2 × 10^7^ CFU/mL in PBS. Subsequently, PAB was added to the cell suspension and incubated at 37°C for 4 h. An equal volume of electron microscopy fixative (containing 2.5% glutaraldehyde) was then added, and the cells were fixed at 4°C for 24 h. After fixation, the samples were dehydrated through a graded ethanol series (30%, 50%, 70%, 90% and 100%), critical‐point dried, sputter‐coated with gold and imaged using a scanning electron microscope.
Co‐Cultivation Experiment
2.12
Candida albicans and L. crispatus were cultured individually or co‐cultured in the presence or absence of 15.625 μg/mL pseudolaric acid B. Following overnight incubation, cultures were serially diluted (10^−2^ to 10^−8^) and spread‐plated for CFU enumeration and comparative analysis.
Quantification of Ergosterol
2.13
Single colonies were inoculated into six separates fresh YEPD broth flasks containing PAB at 1×, 2× and 4× MIC, fluconazole, DMSO and control, and incubated at 30°C for 16 h. Mid‐exponential‐phase cells were then harvested by washing and centrifugation (5000 rpm, 5 min), and the net wet weight of each pellet was determined. Subsequently, 3 mL of 60% (w/v) KOH, 4 mL methanol and 1 mL pyrogallol (0.5%, in methanol) were added to the pellet, vortex‐mixed and refluxed at 80°C for 2 h. After cooling to room temperature, 3 mL n‐heptane was added to extract sterols. The supernatant was scanned from 200 to 400 nm with a UV–Vis spectrophotometer. Ergosterol and 24(28)‐dihydroergosterol were identified by the characteristic four‐peak spectrum, with ergosterol plus 24(28)‐DHE showing absorbance at 281.5 nm and 24(28)‐DHE alone at 230 nm (Kantroo et al. 2024).
Effect of PAB on Fungal Cell Membrane Permeability
2.14
The membrane permeability of fungal cells was evaluated using a propidium iodide (PI) uptake assay, as previously described (Duan et al. 2025). Briefly, C. albicans cells in the logarithmic growth phase were collected, washed twice with sterile phosphate‐buffered saline (PBS) and resuspended to a final density of 2 × 10^7^ CFU/mL. The cell suspension was treated with PAB at concentrations of 1×, 2×, 4×, 8×, 16× MIC and fluconazole (62.5 μg/mL) and incubated at 37°C for 30 min. Subsequently, PI was added to a final concentration of 1 μg/mL, and the mixture was incubated in the dark for an additional 30 min. Fluorescence intensity was measured using an EnVision Multimode Microplate Reader at excitation and emission wavelengths of 535 and 615 nm, respectively.
Quantitative Polymerase Chain Reaction (qPCR) for
C. albicans
2.15
Based on molecular docking results suggesting potential inhibition of ERG11 by PAB, its effect on the expression of ERG11—a key gene involved in ergosterol biosynthesis—was evaluated using qRT‐PCR. Fungal genomic DNA was extracted with a commercial kit according to the manufacturer's instructions, and its concentration and purity were determined. Quantitative real‐time PCR was then performed to analyse the expression levels of ERG11, using ITS as the reference gene (Chand et al. 2024).
Animal Experiments
2.16
After 7 days of acclimatisation with ad libitum feeding, estrus synchronisation was induced in female Balb/c mice through daily intraperitoneal injections of β‐estradiol (14–16 μg/kg, dissolved in olive oil) at a volume of 100 μL per mouse for three consecutive days (Esposito et al. 2018). On Day 4, all mice except those in the control group were intravaginally inoculated with 20 μL of a C. albicans suspension (2 × 10^7^ CFU/mL) to establish fungal vaginitis. Throughout the 4‐day modelling period, mice were anaesthetised daily using 2.5%–3.5% (v/v) isoflurane. Control mice received 20 μL of sterile saline instead of fungal inoculum. To promote fungal adhesion, mice were maintained in a supine position for 1–2 min after each inoculation. The mice were randomly divided into five groups (n = 5 per group): Control (healthy, no infection), Model ( C. albicans infection, no treatment), Vehicle control (infection + DMSO), Positive control (infection + fluconazole, 2 mg/mL) and PAB treatment (infection + PAB, 15.6 μg/mL). Starting from Day 8 post‐inoculation, therapeutic agents or vehicle were administered intravaginally once daily for 7 days. The model group received saline in place of treatment. Twenty‐four hours after the final treatment, all mice were euthanized. Vaginal tissues were photographed macroscopically, collected and either fixed in 10% neutral formalin for paraffin embedding and sectioning (3–4 μm) or stored at −80°C for subsequent analysis. Histopathological evaluation was performed using haematoxylin and eosin (H&E) and periodic acid–Schiff (PAS) staining (Lu et al. 2023).
Vaginal Lavage
2.17
Prior to euthanasia, vaginal lavage was performed by rinsing the mouse vaginal tract with sterile phosphate‐buffered saline (PBS). The lavage fluid was collected and first used for fungal load quantification through plate count enumeration. Specifically, aliquots were inoculated onto YPD agar plates supplemented with 50 μg/mL chloramphenicol and incubated to determine viable fungal counts (Peters et al. 2014; Willems et al. 2017). Subsequently, 5 μL of the lavage fluid was utilised for microscopic analysis: Diff‐Quick staining (Gilbert et al. 2013) and methylene blue staining were employed to evaluate the abundance of exfoliated vaginal epithelial cells and the presence of C. albicans . The remaining fluid was stored at −80°C for subsequent 16S rRNA high‐throughput sequencing to characterise compositional changes in the vaginal bacterial communities across experimental groups (Lu et al. 2023).
Cytokines and SOD Detection by ELISA
2.18
Blood samples collected from mice after treatment were centrifuged at 3000 rpm/min at 4°C for 10 min, and serum was collected. Cytokines interleukin‐1β (IL‐1β), interleukin‐4 (IL‐4), interleukin‐6 (IL‐6) and tumour necrosis factor‐α (TNF‐α) were measured using corresponding commercial kits (Shanghai, China). Following the manufacturer's protocol, samples were diluted in buffer and adjusted to concentrations within the linear range of the standard curve, using control group serum as the reference (Liu et al. 2021; Yang et al. 2018).
Quantitative Real‐Time PCR (qRT‐PCR)
2.19
Total RNA was extracted from vaginal tissue using TRIzol reagent (Vazyme, Nanjing, China), and its concentration and purity were measured. One‐step quantitative real‐time PCR was performed to analyse the expression levels of TNF‐α, IL‐1β and IL‐6 genes, with GAPDH gene serving as the endogenous reference gene. The reaction conditions consisted of reverse transcription at 50°C for 3 min, initial denaturation at 95°C for 30 s, followed by 40 cycles of denaturation at 95°C for 10 s and annealing/extension at 60°C for 30 s. A melting curve analysis was subsequently carried out at 95°C for 15 s, 60°C for 60 s and 95°C for 15 s. Relative gene expression levels were calculated using the 2−ΔΔCT method, with data processed in Microsoft Excel and visualised using GraphPad Prism 10. Primer sequences are provided in Table S1.
Statistical Analysis
2.20
All data are presented as mean ± standard error of the mean (SEM). Statistical analyses were performed using GraphPad Prism software. For comparisons among multiple groups, one‐way ANOVA was applied, followed by appropriate post hoc tests. Unpaired Student's t‐test was used for comparisons between two groups. For data that did not follow a normal distribution, the nonparametric Kruskal–Walli's test was employed, with Dunn's test used for post hoc pairwise comparisons. A p value of < 0.05 was considered statistically significant.
Results
3
Screening of Lead Compounds From Traditional Chinese Medicine and Drug Targets
3.1
This study initially selected 16 commonly used Chinese medicinal herbs based on their broad‐spectrum antimicrobial activity. A total of 1093 chemical constituents were retrieved from the TCMSP and HERB databases. Preliminary screening using thresholds of oral bioavailability (OB) ≥ 30% and drug‐likeness (DL) ≥ 0.18 yielded 343 active molecules. Subsequently, systematic drug‐likeness evaluation, pharmacokinetic profiling and toxicity prediction were performed using SwissADME and Discovery Studio, ultimately identifying PAB and 4‐[2‐[(1S,4aR,5S,6S,8aS)‐6‐hydroxy‐5‐(hydroxymethyl)‐5,8a‐dimethyl‐2‐methylidene‐3,4,4a,6,7,8‐hexahydro‐1H‐naphthalen‐1‐yl]‐2‐oxoethyl]‐2H‐furan‐5‐one (referred to as Compound A) as candidate antifungal lead compounds (See Appendix S1 for detailed data).
Through KEGG pathway analysis of C. albicans ergosterol biosynthesis, the rate‐limiting enzyme in the downstream module—sterol 14α‐demethylase (encoded by ERG11)—was identified as the target. This enzyme catalyses a three‐step oxidative reaction, removing the 14α‐methyl group (C‐32) from the substrate lanosterol/eburicol in the form of formic acid, yielding 14‐demethyl eburicol and 4,4‐dimethyl‐5α‐cholesta‐8,14,24‐trien‐3β‐ol, thereby affecting the synthesis of the final ergosterol product. Structural and functional annotations from UniProt further confirmed ERG11 as an ideal drug target.
Using the DEPTH server, potential binding pockets of ERG11 were predicted. Energy‐minimised PAB and Compound A were docked as ligands via AutoDock Vina for semi‐flexible molecular docking. Results showed that PAB‐ERG11 exhibited the lowest binding free energy (−9.46 kcal/mol) (Figure 1A), followed by Compound A‐ERG11 (−9.36 kcal/mol) (Figure 1B). Visualisation in Discovery Studio revealed that PAB deeply inserts into the enzyme's active cavity, forming stable hydrogen bonds and hydrophobic interactions with key residues.
Molecular docking and molecular dynamics simulations. Molecular docking results of PAB and ERG11. (B) Molecular docking results of A and ERG11. (C) Root means square deviation (RMSD) values of the PAB‐ERG11 complex during simulation. (D) Root means square fluctuation (RMSF) values for residues in the PAB‐ERG11 complex. (E) Radius of gyration (Rg) of the PAB‐ERG11 complex. (F) Solvent‐accessible surface area (SASA) of the PAB‐ERG11 complex throughout the simulation. (G) Number of hydrogen bonds within the PAB‐ERG11 complex during the simulation. (H) Gibbs free energy landscape of the PAB‐ERG11 complex constructed via principal component analysis (PC1) of the molecular dynamic's trajectory. The colour scale is in kcal/mol, with dark blue indicating low‐energy, highly occupied conformational regions.
The PAB‐ERG11 complex with the highest affinity was selected as the initial conformation for molecular dynamics (MD) simulations on the GROMACS platform to evaluate thermodynamic stability and binding mode reliability. This preliminary prediction lays the foundation for subsequent structural optimisation and in vitro/in vivo validation. Figure 1 depicts the solvated state of the PAB‐ERG11 complex in an aqueous environment. The MD simulations demonstrated structural stability until key observables converged: RMSD stabilised at ~0.2 nm, indicating structural equilibrium (Figure 1C). RMSF analysis revealed moderate flexibility in functional loop regions (Figure 1D). Radius of gyration (Rg) and solvent‐accessible surface area (SASA) remained constant, reflecting a compact and well‐timed conformation (Figure 1E,F). The number of hydrogen bonds remained stable, confirming the preservation of critical interaction networks (Figure 1G). Gibbs free energy plateaued in later stages, further verifying thermodynamic stability. Principal component analysis (PCA) showed convergence of PC1 projections over time, confirming stable collective motions and validating the simulation's reliability (Figure 1H).
PAB Is an Effective Antifungal Agent
3.2
The susceptibility of C. albicans to PAB was evaluated using the Oxford cup method. As illustrated in Figure 2A, the inhibition zones enlarged dose‐dependently with increasing PAB concentrations. To determine whether PAB also exerts antifungal activity against drug‐resistant Candida sp. isolates (Figure S1), we tested five clinically isolated fluconazole‐resistant strains together with the reference strain C. albicans ATCC 14053; all showed appreciable, albeit variable, levels of growth inhibition. To further assess the antifungal dynamics, fungal growth curves were monitored over time under various PAB treatments (Figure 2B). PAB administration resulted in noticeable inhibition after 4 h of co‐incubation, with robust suppression observed by 12 h, in clear contrast to the control group.
In vitro antibacterial efficacy of PAB. (A) Antifungal effect of PAB on Candida albicans . (B) Growth curves of C. albicans at different PAB concentrations. (C) Microscopic images of Candida albicans after PAB treatment under oil immersion microscopy and scanning electron microscopy. (D) Biofilm formation capacity of fungi after PAB or 1% DMSO treatment. (E) Expression levels of the ERG11 gene in C. albicans under various treatments. (F) PAB inhibits the co‐culture of C. albicans and Lactobacillus crispatus . (G) Changes in ergosterol content of C. albicans across different treatment groups. (H) Changes in cell membrane permeability of C. albicans after PAB treatment.
Scanning electron microscopy (SEM) further revealed that although control cells displayed smooth and intact membranes, PAB‐treated cells exhibited pronounced morphological alterations, including irregular wrinkling and membrane depression, suggesting substantial disruption of cell membrane integrity (Figure 2C).
Given the association between biofilm formation and the pathogenicity of C. albicans , we examined the impact of PAB on biofilm formation after overnight culture. Both PAB and the DMSO vehicle significantly reduced biofilm biomass—by 1.6‐fold and 1‐fold, respectively—relative to the untreated control (Figure 2D). Lactobacillus crispatus , a dominant vaginal commensal, produces lactic acid to maintain a low pH environment that inhibits pathogen colonisation. In vitro, PAB exhibited pronounced inhibitory activity against C. albicans . Using a co‐culture system of L. crispatus and C. albicans , we observed that PAB suppressed C. albicans growth without affecting L. crispatus viability (Figure 2F).
Additionally, qRT‐PCR analysis demonstrated that PAB treatment significantly downregulated the expression of ERG11 (Figure 2E), a key gene involved in ergosterol biosynthesis. This suppression led to reduced ergosterol production, which may enhance the fungal cells' susceptibility to PAB. We measured the ergosterol content in C. albicans after treatment with PAB and found that its inhibitory effect on ergosterol synthesis was dose‐dependent (Figure 2G). These results imply that PAB exerts its antifungal effect through a mechanism that extends beyond those of conventional antibiotics. Taken together, the downregulation of ERG11, the concomitant reduction in ergosterol and the resulting membrane damage collectively suggest that the antifungal mechanism of PAB involves the inhibition of ergosterol biosynthesis, potentially via targeting Erg11.
While the growth curve (Figure 2B) showed a prolonged lag phase rather than complete cessation of growth at the highest concentration in vitro, this observation aligns with the proposed mechanism of membrane‐targeting action. The initial, severe inhibition of ergosterol biosynthesis disrupts fundamental cellular processes, leading to a period of stasis (the lag phase) and culminating in the profound membrane damage observed via SEM and PI uptake (Figure 2C,H). This pattern is distinct from a transient, non‐lethal stress response. Furthermore, the potent inhibition of biofilm formation at sub‐inhibitory concentrations (Figure 2D) underscores a critical anti‐virulence effect. Therefore, the integrated in vitro profile—comprising growth suppression, biofilm prevention and irreversible membrane disruption—provides a coherent mechanistic basis for the excellent therapeutic efficacy observed in vivo, where sustained local drug exposure and host‐microbiome interactions would prevent the eventual growth recovery seen in static batch culture.
In Vivo Efficacy of PAB in a Mouse Model of Candidal Vaginitis
3.3
To further assess the localised antifungal efficacy of PAB, we established a mouse model of C. albicans ‐induced fungal vaginitis and evaluated therapeutic outcomes using fluconazole as a clinical reference control. As depicted in Figure 3A, successful infection was confirmed in the animal model. Analysis of vaginal lavage fluid revealed that PAB treatment resulted in a pronounced reduction in fungal burden, exhibiting efficacy comparable to that of fluconazole (Figure 3B). Body weight measurements remained stable throughout the experiment, with no significant differences observed before and after modelling (Figure 3C).
*Characterisation of Vaginal Lavage Fluid and Body Weight in Mice. (A) Mouse model of C. albicans ‐induced invasive fungal vaginitis. (B) C. albicans cell viability in vaginal washings after treatment. (C) Changes in mouse body weight. (D and E) Diff‐Quick staining and scoring (D) and methylene blue staining and scoring (E) of vaginal washings. Data are expressed as mean ± standard deviation (n = 5). p < 0.05.
Diff‐Quick staining of vaginal exfoliated cells indicated a substantial increase in abnormal cellular morphology following infection. Notably, PAB administration reduced the abundance of these abnormal cells by 24.35‐fold (p < 0.05, Figure 3D). Furthermore, methylene blue staining demonstrated extensive colonisation by C. albicans in the model and DMSO control groups, evidenced by intense purple staining. In contrast, both PAB‐ and fluconazole‐treated groups showed markedly diminished fungal presence (Figure 3E).
Mice were euthanized and dissected for collection of vaginal and cervical tissues, which were subsequently subjected to quantitative analysis (Figure 4A,B). Compared to the control group, mice in the infection model group exhibited evident vaginal dilation and swelling. Such pathological changes were ameliorated following PAB treatment. Histological examination revealed marked cervical hyperplasia in C. albicans ‐infected mice, whereas tissues from PAB‐treated mice appeared similar to those of the controls. Histopathological evaluation using H&E and PAS staining further demonstrated the therapeutic effects of PAB. H&E‐stained sections (Figure 4C) showed that PAB significantly reduced vaginal keratinization and mucosal thickness compared to the model group, and its effect was superior to that of fluconazole. These results suggest that C. albicans infection causes severe damage to vaginal mucosal cells, while PAB facilitates structural recovery. PAS staining (Figure 4D) revealed extensive purple‐red hyphal structures of C. albicans pervading the mucosal layer in the model group. In contrast, both PAB and fluconazole treatments resulted in a substantial reduction or complete absence of visible hyphae. Collectively, these findings confirm that PAB effectively eliminates C. albicans colonisation and promotes restoration of normal tissue architecture in vivo.
*Histological Features of Fungal Vaginitis in Mice Vulvar and vaginal tissue characteristics in mice at the end of treatment. (B) Quantitative analysis of uterine length (left) and uterine diameter (right). (C and D) H&E‐stained (C) and PAS‐stained images of vaginal tissue with corresponding histological scores. Data are expressed as mean ± standard deviation (n = 3). *p < 0.05, **p < 0.01, ***p < 0.0001.
To further elucidate the anti‐inflammatory effects of PAB, we measured serum levels of key inflammatory cytokines—IL‐1β, IL‐4, IL‐6 and TNF‐α—in the vaginal infection mouse model (Figure 5A–D). Compared to the control group, mice in the model group showed significantly elevated levels of IL‐1β (1.17‐fold), IL‐6 (1.11‐fold) and TNF‐α (1.29‐fold), along with a slight reduction in IL‐4 (p < 0.05). In contrast, PAB treatment significantly suppressed the expression of IL‐1β, IL‐6 and TNF‐α, while upregulating IL‐4 levels. Consistent with protein‐level findings, RT‐qPCR analysis revealed markedly increased mRNA expression of IL‐1β, IL‐6 and TNF‐α genes in the model group (Figure 5E–G). PAB treatment effectively downregulated these pro‐inflammatory genes, restoring their expression to near‐normal levels. These results demonstrate that PAB not only reduces fungal load but also modulates host immune responses, alleviating infection‐associated inflammation.
*Inflammatory cytokine levels and expression in mice following different treatments (A–D) changes in serum levels of TNF‐α (A), IL‐1β (B), IL‐6 (C) and IL‐4 (D) in mice after different treatments. (E–G) Expression of TNF‐α (E), IL‐1β (F) and IL‐6 (G) genes in mouse vaginal tissue. (H) Changes in SOD content in mouse serum. *p < 0.05, **p < 0.01, ***p < 0.001, ***p < 0.0001.
Finally, we evaluated superoxide dismutase (SOD) activity, a key antioxidant enzyme, in the vaginal tissue homogenates. As shown in Figure 5H, both PAB and fluconazole treatments significantly restored SOD activity compared to the infected model group. Collectively, these results suggest that although fluconazole exhibits strong direct antifungal activity against C. albicans , PAB demonstrates a superior capacity in alleviating candidal vaginitis, likely owing to its combined antifungal, anti‐inflammatory and antioxidant properties.
Analysis of Vaginal Microbiota in Mouse Vaginal Lavage
3.4
Based on the above findings, we characterised the vaginal microbiota composition in mice by sequencing 16S rDNA from vaginal lavage fluids. At the phylum level (Figure 6A), the control group was predominantly colonised by Firmicutes (97.21%). In contrast, C. albicans infection induced a dramatic shift in microbial composition, marked by a sharp decline in Firmicutes (20%) and a pronounced increase in Proteobacteria (68.06%) and Bacteroidetes (30.85%). PAB treatment significantly restored the microbial balance, with Firmicutes rebounding to 92.82%, while Proteobacteria and Bacteroidetes were reduced to 2.39% and 0.70%, respectively. At the genus level (Figure 6B), the model group was dominated by the pathogenic taxon Escherichia_Shigella (65.19%). C. albicans infection disrupted the native microbial community (Figure 6C), resulting in increased bacterial diversity and a dysbiotic state consistent with clinical vaginitis profiles.
Improvement of vaginal microbiome in Candida albicans ‐induced vaginitis mice after PAB and fluconazole treatment. (A and B) Microbial differential analysis between mouse groups at the phylum level (A) and genus level (B). (C) Venn diagram analysis at the ASV level. (D and E) Microbial diversity analysis: (D) α‐diversity analysis using the Shannon index and (E) β‐diversity analysis via PCoA and NMDS. (F) Correlation network analysis at the genus level. (G) Heatmap showing correlations between group assignments and species abundance within the microbiota.
The α‐diversity Shannon index (Figure 6D) revealed a significant increase in microbial diversity following Candida albicans infection, while PAB treatment effectively restored microbial richness and evenness to levels comparable to those of the control group. β‐diversity analyses, including PCoA and NMDS (Figure 6E), further illustrated distinct clustering of microbial communities among groups. Notably, the PAB‐treated group exhibited a compositional profile much closer to that of the controls, suggesting a restorative effect on vaginal microbiota structure. These findings imply that PAB not only suppresses the growth of C. albicans but also facilitates the recovery of beneficial vaginal bacteria. To elucidate genus‐level interactions, Spearman correlation analysis was performed (|r| > 0.2, p < 0.05) and significant correlations were visualised via network analysis (Figure 6F). Escherichia_Shigella showed negative correlations with Enterococcus and Staphylococcus. Notably, Staphylococcus—a dominant genus under healthy conditions—contributes to a protective microenvironment through lactic acid production via glucose metabolism, thereby inhibiting pathogenic overgrowth (Rahman et al. 2023; Vrbanac et al. 2018). Meanwhile, a positive correlation was observed between the probiotic genus Muribaculum and Bacillus. Members of the Muribaculaceae family are known producers of short‐chain fatty acids, which play important roles in maintaining barrier integrity and modulating immune responses (Zhu et al. 2024).
Supporting these findings, the species abundance clustering heatmap (Figure 6G) indicated a noticeable increase in the relative abundance of beneficial genera such as Cetobacterium, Staphylococcus, Dubosiella and Allobaculum after PAB treatment. Furthermore, a ternary phase diagram of species composition and distribution revealed that PAB intervention shifted the vaginal microbiota from a Proteobacteria‐dominated state to a Firmicutes‐dominant structure—a transition that was particularly pronounced in the model group (Figure S2). This suggests that PAB may promote ecological restoration at the phylum level by suppressing Proteobacteria and facilitating the recovery of Firmicutes. LEfSe analysis (Figure S3) identified taxa with significant differential abundance (LDA score > 4.0). The control group was enriched in Bacilli and Firmicutes, which include many beneficial vaginal probiotics. Bacilli, largely comprising lactobacilli, represent a core functional group that maintains an acidic environment and inhibits pathogen colonisation. In contrast, the model group showed marked enrichment of the Enterobacteriales‐Enterobacteriaceae*‐Escherichia/Shigella* branch—a group often associated with Gram‐negative pathogens. Importantly, PAB treatment led to increased abundance of Lactobacillaceae, underscoring its role in reestablishing a healthy microbiota. In conclusion, PAB administration effectively suppressed potentially pathogenic bacteria while restoring populations of beneficial vaginal microbes, thereby promoting a balanced microbial community and supporting therapeutic recovery.
Functional Prediction of the Vaginal Microbiota
3.5
To infer the functional consequences of the microbiota shifts induced by C. albicans infection and PAB treatment, we performed phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt2) analysis based on 16S rRNA gene sequences. As shown in Figure S4, the vaginal microbiota in the model group exhibited significant enrichment in KEGG pathways related to ‘Antimicrobial drug resistance’ and ‘Cellular community – prokaryotes’ (a category often associated with biofilm formation and bacterial colonisation), alongside pathways for ‘Glycan biosynthesis and metabolism’. Conversely, fundamental metabolic pathways such as ‘Energy metabolism’ and ‘Carbohydrate metabolism’ were notably diminished. Treatment with PAB substantially normalised this dysregulated functional profile. The intervention led to a marked downregulation of the resistance‐ and biofilm‐associated pathways, while concurrently restoring the activity of core microbial metabolic pathways towards a state resembling that of the healthy control group.
Discussion
4
Candidal vaginitis, resulting from infection with C. albicans , is a common gynaecological condition characterised by intense vulvar pruritus, erythema, swelling, burning pain and local inflammation. More importantly, it disrupts the ecological balance of the vaginal microbiome. In healthy individuals, the vaginal microbiota is predominantly composed of Lactobacillus species, which metabolise glycogen to produce lactic acid, thereby acidifying the vaginal environment and inhibiting the colonisation of pathogenic bacteria such as Gardnerella vaginalis and Prevotella spp. (France et al. 2022). Conventional antibiotic therapies often lack selectivity, impairing beneficial flora alongside pathogens and increasing the risk of recurrent infections (Melkumyan et al. 2015). The growing incidence of drug‐resistant fungal strains further diminishes the efficacy of existing clinical treatments, underscoring the urgent need for novel antimicrobial agents.
In this study, we screened traditional Chinese herbs with known antimicrobial properties to identify bioactive monomers with potential activity against ERG11. Our integrated computational approach, employing molecular docking and dynamics simulations, generated a testable mechanistic hypothesis by identifying PAB as a promising candidate based on its superior predicted binding affinity to Erg11. This in silico prediction is supported by convergent experimental evidence: PAB treatment specifically downregulated ERG11 gene expression, led to a dose‐dependent decrease in cellular ergosterol content and subsequently increased membrane permeability, culminating in cell death. While these results are consistent with and strengthen the hypothesis that PAB acts through the Erg11/ergosterol pathway, we acknowledge that definitive confirmation of direct target engagement remains to be established. Future studies employing direct biophysical techniques—such as surface plasmon resonance (SPR) or microscale thermophoresis (MST)—with purified Erg11 protein, alongside genetic approaches using ERG11‐mutant strains, will be necessary to unequivocally validate Erg11 as the molecular target of PAB.
Another major virulence trait of C. albicans is its ability to form biofilms on biotic and abiotic surfaces (Pereira et al. 2021), which utilise host nutrients for sustained growth and confer heightened resistance to antimicrobial agents. The resilience of these biofilms is a key factor contributing to the high recurrence rates of vaginal candidiasis (Alvendal et al. 2020). Our results demonstrate that PAB significantly inhibits biofilm formation, disrupting the structural integrity of C. albicans colonies within the vaginal environment. PAS staining of vaginal tissues from infected mice revealed that PAB treatment reduced hyphal development and degraded existing biofilm matrices. This disruption enhances drug penetration, improves antifungal efficacy and reduces the probability of recurrent infection.
The presence of C. albicans is closely associated with altered inflammatory mediator expression and consequent tissue damage (Li et al. 2021). Informed by these mechanisms and supported by preliminary in vitro evidence, we employed a mouse vaginitis model to evaluate the anti‐inflammatory efficacy of PAB. Our results confirmed that fungal colonisation of vaginal epithelial cells triggers a robust innate immune response, leading to dysregulation of inflammatory markers. Following PAB treatment, we observed significant downregulation of key pro‐inflammatory cytokines—including TNF‐α, IL‐1β and IL‐6—along with the upregulation of the anti‐inflammatory cytokine IL‐4. These changes demonstrate the potent immunomodulatory effects of PAB. Furthermore, innate immune cells combat pathogens through the production of reactive oxygen species, such as superoxide anions, which are induced upon phagocyte exposure to C. albicans . While crucial for defence, these metabolites can also cause collateral tissue damage. SOD serves as a critical antioxidant enzyme that neutralises superoxide anions. Notably, PAB administration significantly enhanced SOD activity, indicating its role in alleviating oxidative stress and protecting host tissues. Together, these findings illustrate that PAB not only exerts direct antifungal action but also mitigates infection‐associated inflammation and oxidative damage, contributing to a comprehensive therapeutic response. Critically, the direct antifungal effect—evidenced by reduced fungal burden in vivo (Figure 3B) and hyphal clearance (Figure 4D)—provides the necessary foundation for breaking the infection cycle. The subsequent anti‐inflammatory and microbiota‐restorative effects then facilitate tissue healing and restore ecological resistance against recurrence, forming a comprehensive therapeutic strategy.
Furthermore, PAB contributes to the restoration of a healthy vaginal microbiota and facilitates recovery from inflammatory damage. As indicated by α‐ and β‐diversity analyses (Figure 5D–E), PAB treatment reduced the excessive microbial richness and diversity induced by infection, shifting the composition towards that of the control group. Specifically, Proteobacteria and Bacteroidetes—which were significantly increased in the model group—were effectively suppressed after PAB administration. Notably, overgrowth of Bacteroidetes, often regarded as opportunistic pathogens, is a hallmark of vaginitis and may be exacerbated by C. albicans infection (Gao, Guo, et al. 2025; Gao, Wang, et al. 2025). LEfSe analysis further revealed that PAB intervention led to a significant phylogenetic restructuring of the vaginal microbiota, characterised by the suppression of Enterobacteriales and promotion of Lactobacillaceae, thereby reestablishing a Lactobacillus‐dominant microbiome to counteract Candida‐induced dysbiosis. The observed restoration of the vaginal microbiota is likely a consequence of PAB's multi‐pronged mechanism. By effectively reducing the fungal burden and dampening the associated inflammatory response, PAB may create a permissive niche that facilitates the natural recovery of indigenous probiotics, such as lactobacilli. Concurrently, its direct suppression of pathogenic Proteobacteria further contributes to this ecological shift back to a Firmicutes‐dominated, healthy state. Furthermore, our functional prediction analysis provides a putative mechanistic link between the restored microbial structure and the therapeutic outcome. The observed enrichment of pathways for ‘Antimicrobial drug resistance’ and ‘Cellular community – prokaryotes’ in the model group aligns with the clinical challenge of recurrent and stubborn infections, suggesting a microbiota poised for pathogen persistence and enhanced defensive capabilities. The downregulation of these pathways following PAB treatment indicates that its action extends beyond simple pathogen clearance to reprogramming the functional potential of the vaginal ecosystem, making it less conducive to pathogen defence and colonisation. Importantly, the suppression of ‘Glycan biosynthesis and metabolism’ pathways post‐treatment may also be relevant, as glycans are key components of microbial cell walls and biofilm matrices (e.g., chitin and glucans in C. albicans ). This reduction could correlate with the observed inhibition of fungal biofilm formation (Figure 2D) and hyphal degradation in vivo (Figure 4D). Concurrently, the restoration of ‘Energy metabolism’ and ‘Carbohydrate metabolism’ pathways signifies a recovery of a metabolically active, homeostatic microbiota, which is fundamental for sustaining a resilient and protective community capable of producing inhibitory metabolites like lactic acid. Together, these data suggest that PAB facilitates a dual restoration—of both taxonomic composition and putative ecosystem function—thereby re‐establishing an ecological barrier against C. albicans recurrence. The emergence of antifungal resistance remains a major obstacle in treating vaginal infections. For instance, resistance mutations have already been reported for newer drugs such as eposin (Xie et al. 2017). Additionally, enhanced glycan synthesis—involving key components of the C. albicans cell wall, such as chitin, β‐1,3‐glucan and β‐1,6‐glucan (Gow and Lenardon 2023; Dickwella Widanage et al. 2025)—may promote structural integrity and biofilm stability, further complicating treatment efficacy. It is important to note the limitations of this study. The therapeutic effect of PAB was not validated in a recurrence model of vaginitis, and its long‐term impact on commensal microbiota and vaginal epithelial cells remains to be thoroughly evaluated.
Conclusion and Future Perspectives
5
In summary, this study identifies pseudolaric acid B (PAB) as a promising multi‐mechanism therapeutic candidate against drug‐resistant Candida albicans vaginitis. PAB exerts its efficacy through a combined action of direct antifungal activity—likely mediated via inhibition of the ergosterol biosynthesis pathway—coupled with immunomodulatory effects and, notably, the restoration of a healthy vaginal microbiota.
While our integrated approach provides compelling evidence implicating Erg11 as a potential target, further validation is warranted. To unequivocally establish the molecular target of PAB, future work will focus on direct biophysical binding assays (e.g., SPR or MST) using purified Erg11 protein, alongside genetic strategies such as the construction of ERG11 point‐mutation strains in C. albicans . Additionally, evaluating PAB in a recurrent infection model and assessing its long‐term impact on vaginal epithelial integrity and microbial ecology will be essential to fully elucidate its translational potential.
Author Contributions
Tianmeng Shao: investigation, methodology, writing – original draft, visualization. Yunshan Zhang: investigation, methodology, writing – original draft, visualization. Binqing Xue: investigation, methodology. Weihua Chu: conceptualization, supervision, writing – review and editing.
Funding
The authors have nothing to report.
Conflicts of Interest
The authors declare no conflicts of interest.
Supporting information
Data S1: mbt270331‐sup‐0001‐AppendixS1.xlsx.
Figure S1: Drug‐susceptibility profiles of PAB against a clinically isolated fluconazole‐resistant strain of Candida albicans and the reference strain ATCC 14053. Figure S2: LEfSe analysis of the murine vaginal microbiota. Figure S3: Ternary phase diagram analysis of the control, model and PAB groups. Figure S4: KEGG pathway prediction and analysis among the control, model and PAB groups. Figure S5: Ergosterol biosynthetic pathway in Candida albicans .
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