Ethanolic extract of Withania somnifera (EEWS) augments the activity of fluconazole against drug-resistant vulvovaginal candidiasis

2.1 Materials

The essential reagents used in this study comprised FLZ, acquired from Santa Cruz Biotechnology (Dallas, TX, USA), Sabouraud Dextrose Agar (SDA) and broth (SDB) from HiMedia (Mumbai, India), and estradiol valerate, procured from MedChem Express (St. Louis, USA)

2.2 Candida albicans

For this study, FLZ-resistant C. albicans (ATCC 96901) was obtained from the American Type Culture Collection (ATCC) (Manassas, Virginia, USA). The strain was preserved and sub-cultured on SDA to maintain its growth and viability for subsequent analyses. C. albicans identification was confirmed through the germ tube formation assay and subsequent growth on CHROMagar Candida medium, which produced characteristic colony coloration.

2.3 Preparation of ethanolic extract of W. somnifera (EEWS)

W. somnifera, Ashwagandha, root powder was purchased from the iHerb company (Ghaziabad, India). Approximately 50–100 g of the powdered root was placed in a Soxhlet apparatus, and 95% ethanol was used as the extraction solvent. The extraction was carried out for 6–8 hours, after which the solution was cooled and filtered to remove plant debris. The solvent is evaporated using a water bath at 40–50°C to obtain a concentrated extract. Vacuum drying was performed on the final product. Extraction of W. somnifera roots with ethanol produced about 10.2% dried EEWS relative to the starting plant material. Yield percentage was calculated to assess extraction efficiency and ensure reproducibility. The extract was assigned a unique lot number and stored in airtight containers at −20 °C to preserve its phytoconstituents.

2.4 Total flavonoid content (TFC) analysis in EEWS

The total flavonoid content in EEWS was evaluated by the Aluminum chloride calorimetry method as described in our earlier method (Institute 2008). First, a standard curve of quercetin was prepared by dissolving it in methanol at concentrations ranging from 0 to 100 µg/mL. A total of 0.5 mL of EEWS was combined with 0.16 mL of 5% NaNO2, 0.16 mL of 10% AlCl3, and 1 mL of 1 M NaOH. The mixture was stirred well, and the absorbance was recorded at 510 nm using a spectrophotometer. TFC was determined with reference to a quercetin standard curve and expressed as mg quercetin equivalents per g of EEWS.

2.5 Total phenolic content (TPC) in EEWS

The Folin–Ciocalteu assay was employed to determine the TPC in the methanolic extract, with a gallic acid standard curve ranging from 1.4 to 1000 μg/mL, following established methodology (Institute 2008). In this analysis, 1.6 mL of either EEWS or gallic acid solution was reacted with 0.2 mL of a diluted Folin–Ciocalteu reagent (5-fold diluted) and 0.2 mL of 10% sodium carbonate. The mixture was incubated for 30 minutes at room temperature in the dark to prevent light interference. Absorbance at 765 nm was recorded, and the TPC was calculated and expressed as milligrams of gallic acid equivalents per g of EEWS (mg GA/g EEWS)

2.6 Total antioxidant activity of EEWS

To evaluate 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity (FRSA), a slightly modified method was adopted (Institute 2008). The EEWS extract and the reference antioxidant, ascorbic acid, were prepared in dimethyl sulfoxide (DMSO), which served as a blank for calibration. In a 96-well plate, 20 μL of each test solution was combined with 180 μL of freshly prepared 100 mM DPPH solution. The reaction mixtures were incubated at room temperature in the dark for 30 minutes. Absorbance at 517 nm was recorded using a Biotech microplate spectrophotometer. IC50 values, representing the concentration required to inhibit 50% of DPPH radicals, were calculated for EEWS (10–500 μg/mL) and ascorbic acid (1–20 μg/mL).

2.7 To measure the anti-Candida activity of FLZ and EEWS and their MIC values

The antifungal efficacy of FLZ, EEWS, and their combinations was evaluated using the agar well diffusion method. C. albicans cultures were grown on SDA plates, and wells (8 mm diameter) were created using a sterile cork borer. A 50 µL aliquot of each test compound—FLZ (100 µg), EEWS (500 µg), FLZ (100 µg) + EEWS (250 µg), and FLZ (100 µg) + EEWS (500 µg)—was carefully added into the wells. Plates were incubated at 37 °C for 24 h, and the inhibition zones were measured in millimeters to assess antifungal activity.

The MICs of FLZ and EEWS were determined using the broth macrodilution method following the Clinical and Laboratory Standards Institute (CLSI, 2008) protocol. The study followed CLSI guidelines issued in Wayne (NCLLS) (Khan et al., 2025). Stock solutions of FLZ and EEWS were prepared in sterile DMSO and serially diluted in SDB to obtain the desired concentration ranges. Two-fold serial dilutions were prepared as follows:

• FLZ: 0.125–128 µg/mL (10 dilutions)

• EEWS: 2–1000 µg/mL

Each test tube contained 3 mL of SDB with the respective concentration and was inoculated with 100 µL of C. albicans suspension (1 × 10⁶ CFU/mL). Tubes containing medium and inoculum without test compounds served as growth controls, while those containing medium only served as sterility controls. All assays were performed in triplicate. The MIC was defined as the lowest concentration that produced ≥50% inhibition of visible fungal growth relative to the control after incubation at 37 °C for 24 h.

2.8 Determination of synergy between EEWS and FLZ

To examine the synergistic relationship between EEWS and FLZ, the fractional inhibitory concentration index (FICI) was used to quantify their combined inhibitory efficacy. The concentration ranges for both agents were consistent with those previously established for determining MICs. The FICI value was calculated as per the equation shown here: ∑FIC = FIC-FLZ​ + FIC-EEWS ​=MIC_FLZ^Comb/MIC_FLZ^alone​​​ + MIC_EEWS^Comb/MIC_EEWS^alone​​.

The activity of FLZ, EEWS, or the FLZ and EEWS combination against C. albicans was also observed by analyzing the treated cells under the light microscope at 400X magnification. In a 6-well culture plate, C. albicans was grown in SDB and treated with FLZ at 8 µg/mL, EEWS (500 µg/mL), or FLZ-8 +EEWS-250 and FLZ-8 + EEWS-500 for 24 h. The cells were gently washed to remove debris and observed under a light microscope for analysis.

2.9 Assessment of FLZ, EEWS, or FLZ + EEWS on pre-established C. albicans biofilms

FLZ, EEWS, or their combination was tested on C. albicans preformed biofilms as reported earlier (Allemailem 2021). C. albicans was grown to a concentration of 1 × 10⁶ CFUs/mL, introduced into 96-well plates, and incubated at 37°C for 24 hours. After replacing the medium with SDB containing FLZ (8–64 µg/mL), EEWS (125–1000 µg/mL), or FLZ (8 µg/mL) + EEWS (125–500 µg/mL), a subsequent incubation period of 24 h was carried out for the plates. Washing with phosphate buffered saline (PBS), drying in air for 20 minutes, and staining with 0.1% crystal violet for 20 minutes were performed on the wells. After washing and drying, the biofilm was solubilized with 100 µL of 95% ethanol, and optical density (OD) was measured at 595 nm. The % biofilm formation was expressed relative to the untreated control, which was considered as 100% biofilm formation.

2.10 Mice

The King Saud University (KSU) animal facility in Riyadh, Saudi Arabia, provided 12- to 14-week-old female Swiss mice for this study. The methods for administering injections and performing euthanasia complied with the ethical standards set forth by the Animal Ethics Committee of the Deanship of Scientific Research, Qassim University, Buraydah [Ethical number: 24-94-03; June 9, 2024]. The mice were accommodated in meticulously maintained, pathogen-free, and sanitary environments, providing ideal conditions for the conduct of the study. The study adhered to the ARRIVE guidelines, ensuring proper design, conduct, and transparent reporting of animal use to promote reproducibility and welfare.

2.11 Establishing a vulvovaginal candidiasis model in mice

Hormonal priming of each mouse was achieved by subcutaneous injection of 500 µg Estradiol dipropionate in 100 µL of sunflower oil. After a period of 3 days, which allowed the hormone to exert its physiological effects, each mouse was intravaginally inoculated with 2 × 10⁶ colony-forming units (CFUs) of Candida albicans. The procedure was conducted as previously described in the literature, ensuring consistency with established protocols for studying VVC (Yadava et al., 2011).

2.12 To examine the role of FLZ in treating VVC

Seventy-two hours after infection, four groups of mice were formed, with ten mice assigned to each group (n = 10). The infected control group received the vehicle (0.5% carboxymethyl cellulose in distilled water) orally once daily for seven days. FLZ was dissolved in 0.5% carboxymethyl cellulose (CMC) in distilled water, a safe, inert vehicle commonly used for oral drug delivery in mice. The treatment groups received FLZ orally at doses of 12.5, 25, and 50 mg/kg once every 24 h for one week to assess dose-dependent therapeutic efficacy against VVC.

2.13 To evaluate the therapeutic potential of EEWS against VVC

For in vivo administration, EEWS were freshly prepared each day immediately before dosing to maintain stability and consistency of the formulations. The EEWS was similarly suspended in the same 0.5% CMC vehicle to ensure uniform dispersion of the extract and accurate dose delivery through oral gavage. The suspension was continuously stirred during administration to avoid sedimentation and maintain homogeneity of the test compound. The therapeutic potential of EEWS was assessed in the treatment of VVC. Daily oral doses of EEWS at 50, 100, and 200 mg/kg were administered as part of the treatment regimen over one week. The experimental design included four groups of infected mice, with ten individuals per group (n = 10):

• 1.

  Infected control

• 2.

  EEWS-50 mg/kg

• 3.

  EEWS-100 mg/kg

• 4.

  EEWS-200 mg/kg

At eight days post-infection, three mice from each experimental group were humanely sacrificed to assess the therapeutic efficacy of EEWS (Khan et al., 2021). Vaginal tissues were carefully excised using sterile surgical instruments under aseptic conditions to prevent contamination. The collected tissues were immediately rinsed in sterile PBS to remove adherent debris and blood. Each tissue sample was weighed and homogenized in cold PBS using a sterile glass homogenizer to obtain a uniform suspension. Serial ten-fold dilutions of the homogenates were prepared in PBS, and 100 µL aliquots from appropriate dilutions were plated in triplicate onto SDA plates. Plates were incubated at 37°C for 48 h to allow for colony development. Following incubation, colonies were counted and expressed as CFUs per g of vaginal tissue. This quantitative estimation of fungal burden enabled precise evaluation of the antifungal potential of EEWS therapy in reducing C. albicans load.

In parallel, the tissues from three mice were also processed for additional analyses in related experiments, including cytokine profiling and histopathological assessment (as described in later sections). However, for the present assay, emphasis was placed on fungal burden reduction as the primary marker of antifungal efficacy. This systematic evaluation allowed us to determine the dose-dependent effectiveness of EEWS and its potential role in mitigating resistant Candida infection in the murine VVC model.

2.14 Dual Administration of EEWS and FLZ for VVC

To assess the synergistic potential of EEWS and FLZ in treating VVC, mice were treated with EEWS (100 mg/kg) and FLZ (20 or 40 mg/kg) in separate formulations at the same time for a week. Each group contained ten mice. By the eighth day post-treatment administration, vaginal tissues were collected from three mice per group to quantify fungal burden.

• 1.

  Infected control

• 2.

  EEWS-100 mg/kg

• 3.

  FLZ-20 mg/kg

• 4.

  FLZ-40 mg/kg

• 5.

  EEWS-100 mg/kg and FLZ-20 mg/kg

• 6.

  EEWS-100 mg/kg and FLZ-40 mg/kg

2.15 Assessing the combined efficacy and safety of EEWS and FLZ for the treatment of VVC

Vaginal tissues from three mice per group were collected on day 8 post-treatment, homogenized in cold PBS, and plated on SDA after serial dilution. The fungal population was quantified by counting CFUs post 48 h of incubation. Additionally, blood samples were analyzed for blood urea nitrogen (BUN) and creatinine to assess potential renal toxicity caused by the treatment (Allemailem 2021).

2.16 Assessment of cytokine profiles in vaginal tissue inflammation

To preserve cytokine integrity, vaginal tissues from three mice were promptly collected, processed, and then homogenized and lysed in ice-cold lysis buffer. To inhibit protease activity, the buffer was supplemented with a cocktail such as Roche’s complete™ protease Inhibitor. At 4°C, the homogenized tissue suspensions were centrifuged at 5000 rpm for 15 minutes. The supernatant containing soluble cytokines was carefully collected to measure IL-1β, TNF-α, and IL-6.

2.17 Microscopic histopathological investigation of vaginal tissues

In accordance with prior descriptions, mouse vaginal tissues were harvested and preserved in 10% neutral-buffered formalin to ensure detailed cellular integrity (Yadava et al., 2011). Once fixed, the tissues were embedded in paraffin and sectioned into 5 μm slices using a microtome for uniform thickness. Hematoxylin and Eosin (H & E) staining was employed for histological visualization. The sections were deparaffinized and rehydrated through graded alcohols, then stained with hematoxylin for 45s to highlight nuclear structures. After a rinse, the tissue was counterstained with eosin for 45s to differentiate the cytoplasm and connective tissue. Once thoroughly rinsed, the slides were mounted in dibutylphthalate polystyrene xylene (DPX) to prevent tissue section degradation. Histological examination under a Leica light microscope (USA) revealed any pathological alterations or inflammatory responses within the tissue samples.

2.18 Statistics

All statistical analyses were conducted using GraphPad Prism (version 6.0, La Jolla, CA, USA). Data are expressed as mean ± standard deviation (SD). Group differences were analyzed using one-way analysis of variance (ANOVA) followed by Bonferroni post hoc tests. A value of p < 0.05 was considered statistically significant. For clarity in figures and tables, statistical significance was denoted as follows: *p < 0.05, **p < 0.01, and ***p < 0.001.

3.1 Antioxidant potential, phenolic, and flavonoid profiles of EEWS

Studies have previously established that alcoholic extracts of W. somnifera roots exhibit superior antioxidant activity compared to those extracted with other solvents (Sobel and Sobel 2018). Consequently, the FRSA of EEWS was analyzed. EEWS showed an IC₅₀ of 124.4 ± 5.4 μg/mL. Additionally, TPC was quantified using the Folin-Ciocalteu method, a widely employed assay for measuring phenolics, and the results were expressed as gallic acid equivalents (GAE) to standardize the measurements. TFC was determined using a calibration curve constructed with quercetin as the standard. The results were expressed as milligrams of quercetin equivalents (mg QE) per g of EEWS (mg QE/g EEWS). The TPC was calculated to be 98.2 ± 4.4 mg GAE/g. Similarly, the TFC was found to be 20.8 mg QE/g.

3.2 Evaluation of Candida albicans sensitivity to FLZ, EEWS, and their combination

The SDA plate containing C. albicans and treated with the vehicle control exhibited no visible zone of inhibition, indicating an absence of antifungal activity (Fig. 1a). Wells containing 50 and 100 µg of FLZ did not produce a well-defined zone of inhibition; however, a slight fungistatic effect was observed surrounding the well with 100 µg of FLZ, suggesting partial inhibition of fungal growth (Figs. 1a and b). In contrast, the wells containing 250 and 500 µg of EEWS displayed a distinct zone of inhibition measuring 16 and 18 mm in diameter Figs. 1(a), (c) and (d) demonstrating its antifungal effect. Notably, when FLZ (50 µg) was combined with EEWS at concentrations of 250 µg and 500 µg, a marked synergistic interaction was observed (Figs. 1a, e). The combination resulted in significantly larger inhibition zones of 21 mm and 24 mm Figs. 1(a), (e) and (f) respectively, indicating enhanced antifungal activity as compared to the individual treatments. A similar trend was observed on Candida CHROMagar, which can be used to visually differentiate C. albicans from potential non-albicans species (Fig. 1b). FLZ showed no anti-Candida activity Figs. 1(a) and (b) whereas EEWS exhibited impressive activity, with zones of inhibition measuring 22 mm and 25 mm in diameter (Figs. 1b, c and d). Most interestingly, a combination of FLZ with EEWS-250 and EEWS-50 was highly effective in inhibiting C. albicans, as there were 28 and 29 mm of inhibition zones (Figs. 1b, e and f).

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Fig. 1.

Anti-fungal activity of FLZ, EEWS, or FLZ and EEWS combination against C. albicans on (A) Sabouraud dextrose agar, and (B) Candida CHROMagar. VC: Vehicle control; (a) 50 µg FLZ, (b) 100 µg FLZ, (c) 250 µg EEWS, (d) 500 µg EEWS, (e) 50 µg FLZ + 250 µg EEWS, and (f) 50 µg FLZ + 500 µg EEWS. All experiments were independently repeated three times, and each assay was performed in triplicate.

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Assessment of FLZ’s antifungal activity against C. albicans using the macrodilution method yielded an MIC of 64 µg/mL for the current isolate. The MIC of FLZ for C. albicans was 64 µg/mL, meeting the threshold for resistance as defined by CLSI guidelines. In contrast, the MIC for EEWS was determined to be 500 µg/mL against the same isolate. When FLZ and EEWS were combined, their MIC values were notably reduced, with FLZ decreasing to 8 µg/mL and EEWS to 125 µg/mL. This reduction reflects a synergistic interaction between the two agents, as evidenced by a mean FIC index of 0.375, indicating enhanced antifungal efficacy when used in combination.

Untreated C. albicans showed uniform growth in SDB, whereas the presence of FLZ at 8 µg/mL showed reduced C. albicans proliferation (Fig. 2). The lower proliferation observed at 8 µg/mL FLZ reflects its fungistatic nature, which inhibits ergosterol synthesis, thereby suppressing fungal cell growth and replication without causing complete cell death. EEWS, at a concentration of 500 µg/mL, showed enhanced antifungal activity against C. albicans relative to FLZ (Fig. 2). Interestingly, the combinations of FLZ and EEWS were highly effective, as the FLZ-8 + EEWS-250 combination inhibited C. albicans proliferation by more than 90%, and the FLZ-8 + EEWS-500 combination almost killed all cells (Fig. 2).

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Fig. 2.

Microscopic assessment of the antifungal effects of FLZ, EEWS, and their combination against Candida albicans. The impact of treatments involving FLZ, EEWS, or their combination (FLZ-8 µg/mL and EEWS at 250 or 500 µg/mL) on the proliferation and morphology of C. albicans was evaluated to determine their individual and synergistic antifungal effects. Yeast cells were cultured in Sabouraud Dextrose Broth (SDB) for 24 h and visualized under a light microscope at 400X magnification.

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3.3 Combination of FLZ and EEWS achieves superior biofilm clearance

The study investigated the effectiveness of FLZ at 8-64 µg/mL in reducing an established C. albicans biofilm. When applied alone, even at 64 µg/mL, FLZ led to only a modest 25% reduction in biofilm mass compared with the vehicle control. However, the reduction was statistically significant (Fig. 3a) (p<0.001). This indicates that, while FLZ has some biofilm-inhibitory properties, its efficacy as a sole treatment against mature C. albicans biofilms remains limited.

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Fig. 3.

The effect of (a) FLZ, (b) EEWS, or (c) EEWS + FLZ on C. albicans biofilm formation. The % Biofilm formation was expressed relative to the untreated control, which was considered as 100% biofilm formation. Results from three independent experiments are shown as mean ± SD, indicating biofilm formation inhibition after treatment. *p<0.05, **p<0.01, ** *p<0.001, relative to Vehicle treatment; •••p<0.001 relative to FLZ-8 or EEWS-125 µg/ml; ÷ ÷ ÷p<0.001 relative to FLZ-8 or EEWS-250 µg/ml; ≠≠≠p<0.001 relative to FLZ-8 or EEWS-500 µg/ml.

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In parallel, EEWS was evaluated for its potential to disrupt C. albicans biofilms across a range of doses. The effect of EEWS on biofilm inhibition was dose-dependent, with the most pronounced reduction occurring at 1000 µg/mL, where it achieved a significant 66% biofilm reduction compared to vehicle control (Fig. 3b) (p<0.001). Although EEWS alone showed greater biofilm reduction than FLZ, it still failed to eradicate the biofilm completely.

Recognizing the limitations of each treatment independently, the study examined the combined effects of FLZ and EEWS. The combination therapy was tested by pairing a fixed low dose of FLZ (8 µg/mL) with increasing concentrations of EEWS (125, 250, and 500 µg/mL). This combination proved substantially more effective than either treatment alone. Specifically, FLZ (8 µg/mL) in combination with EEWS at 125, 250, and 500 µg/mL reduced biofilm mass by 40.3%, 64.6%, and 89.4%, respectively, relative to the vehicle control (Fig. 3c) (p<0.001). This enhanced inhibition reflects a synergistic interaction between EEWS and FLZ, significantly strengthening their collective anti-biofilm efficacy.

3.4 EEWS and FLZ dual therapy exhibits synergy in treating VVC

FLZ’s therapeutic efficacy was evaluated by analyzing vaginal fungal loads in mice infected with C. albicans. Treatment with FLZ at 10, 20, and 40 mg/kg did not significantly reduce fungal load compared with the untreated control, indicating limited effectiveness at these concentrations (Fig. 4a). Even at 40 mg/kg, FLZ-treated mice exhibited 163,365 ± 15,865 CFUs, which was not statistically different from 242,699 ± 46,975 CFUs observed in untreated animals (p>0.05). These findings suggest that FLZ alone, even at higher doses, is insufficient to eradicate VVC in this model. The antifungal activity of EEWS was next examined by quantifying fungal burden in vaginal tissues of infected mice. EEWS produced a clear dose-dependent reduction in fungal load (Fig. 4b). The highest dose (200 mg/kg) markedly reduced the fungal burden from 230,025 ± 42,998 to 34,365 ± 16,188 CFUs/g of vaginal tissue (p<0.001). A moderate dose (100 mg/kg) also produced a significant reduction to 83,230 ± 18,841 CFUs/g (p<0.01). In comparison, the lowest dose (50 mg/kg) showed minimal effect, indicating that higher EEWS concentrations are necessary for meaningful antifungal activity. To determine whether EEWS enhances the therapeutic potential of FLZ, combined treatments were evaluated in infected mice. FLZ alone at 20 or 40 mg/kg showed minimal efficacy (Fig. 4a), but its combination with EEWS (100 mg/kg) dramatically reduced fungal burden (Fig. 4c). Specifically, FLZ (20 mg/kg) combined with EEWS (100 mg/kg) lowered fungal counts to 19,831 ± 11,019 CFUs, compared to 185,495 ± 17,571 CFUs in mice treated with FLZ alone. Likewise, coadministration of FLZ (40 mg/kg) and EEWS (100 mg/kg) nearly eradicated infection, reducing fungal load to 219 ± 201 CFUs from 146,721 ± 16,966 CFUs observed with FLZ monotherapy (p<0.001). Collectively, these results demonstrate a clear synergistic interaction between EEWS and FLZ, supporting their combined use as a potent therapeutic strategy for VVC.

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Fig. 4.

EWSS combined with FLZ effectively cleared C. albicans from the vaginal tissues. (a) Fungal burden in mice treated with FLZ at 10, 20, and 40 mg/kg. (b) Dose-dependent antifungal activity of EEWS (50, 100, and 200 mg/kg). (c) Synergistic reduction in fungal load following combined administration of FLZ (20 or 40 mg/kg) with EEWS (100 mg/kg). The untreated group represents infected but untreated mice. Data are expressed as mean ± SD (n = 10 mice per group; 3 sacrificed for CFU determination). **p<0.01 and ***p<0.001 relative to infected control; ≠≠≠p<0.001 in relation to FLZ-20 or FLZ-40 mg/kg.

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3.5 EEWS alleviated FLZ-induced renal toxicity

The investigation into the renal toxicity of FLZ and the mitigating potential of EEWS involved measuring BUN and creatinine levels (Figs. 5a and b). FLZ treatment (40 mg/kg) alone significantly increased BUN level from a baseline of 23.3 ± 3 mg/dl in infected control mice to 64 ± 12 mg/dl (p<0.001). This increase highlights the nephrotoxic potential of FLZ at this dose. When EEWS (100 mg/kg) was introduced alongside FLZ (40 mg/kg), BUN levels significantly declined to 34.33 ± 7.6 mg/dl (p<0.01), suggesting that EEWS moderates FLZ’s toxic effect on renal function (Fig. 5a). Creatinine levels also showed some elevation in FLZ-40 mg/kg-treated infected mice, but this was not significantly higher than in infected control mice (Fig. 5b). Mice treated with FLZ-40 mg/kg alone showed creatinine levels of 1.27 ± 0.2 mg/dl, compared to 0.866 mg/dl in infected control (p>0.001). However, co-treatment with EEWS at 100 mg/kg reduced creatinine to 1.06 ± 0.15 mg/dl (p > 0.05). These data imply that EEWS plays a protective role by moderating both BUN and creatinine levels, ultimately reducing renal toxicity. This suggests a promising therapeutic strategy where EEWS could be used to counteract the nephrotoxic effects of FLZ in clinical applications.

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Fig. 5.

The efficacy of combined treatment with Withania somnifera EEWS and FLZ on (a) BUN levels and (b) creatinine was analyzed in infected mice. The benchmark for statistical significance was set at p<0.05, with **p<0.01 and ***p<0.001 marking significant differences between the infected control group and the treatment groups. Results are reported as the mean ± S.D. from three distinct experiments.

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3.6 The EEWS and FLZ combination led to reduced inflammation markers in vaginal tissues

To evaluate VVC-induced inflammatory response, the IL-1β, IL-6, and TNF-α were quantified in vaginal tissue homogenate. Mice in the untreated infected group showed substantial increases in these pro-inflammatory cytokines compared to normal controls, indicating an intense inflammatory response due to infection. For example, IL-1β, which was recorded at 18 ± 6 pg/mL in normal mice, spiked to 548 ± 128 pg/mL in mice with VVC (p<0.001), showing a significant rise due to infection (Fig. 6a). EEWS-100 mg/kg treatment alone reduced IL-1β from 548 ± 128 pg/mL to 291 ± 66 pg/mL (p<0.01). Though the combined treatment with FLZ-40 mg/kg and EEWS-100 mg/kg further decreased IL-1β to 93 ± 46 pg/mL, which was significantly lower than that in infected controls or in the FLZ-40 mg/kg treatment (p<0.001), suggesting a potential anti-inflammatory effect. This suggests that EEWS combined with FLZ can help control inflammation in infected tissues by reducing elevated cytokine levels.

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Fig. 6.

Effect of fluconazole (FLZ), ethanolic extract of Withania somnifera (EEWS), and their combination on pro-inflammatory cytokine levels in vaginal tissues of mice with VVC. (a) Interleukin-1β (IL-1β), (b) Interleukin-6 (IL-6), and (c) Tumor necrosis factor-α (TNF-α) levels were quantified in vaginal tissue homogenates after treatment. Data are presented as mean ± SD (n = 10 mice per group; 3 used for cytokine estimation and 7 observed for recurrence evaluation). Statistical analysis was performed using one-way ANOVA followed by Bonferroni’s post-hoc test. Statistical significance is indicated as follows: ≠≠≠p< 0.001 (normal vs. infected control);** p < 0.01 (infected control vs. EEWS-100);*** p < 0.001 (infected control vs. FLZ-20 + EEWS-100 or FLZ-40 + EEWS-100); ** p < 0.01 (infected control vs. FLZ-20 + EEWS-100; TNF-α panel); •• p < 0.01 (FLZ-20 or FLZ-40 vs. FLZ-20 + EEWS-100 or FLZ-40 + EEWS-100; IL-1β and TNF-α panels); ••• p < 0.001 (FLZ-20 vs. FLZ-20 + EEWS-100; IL-6 panel).

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The IL-6 status was also measured in vaginal tissues. Infected mice showed a significant increase in IL-6 levels (652 ± 128 pg/mL) relative to controls (26 ± 9 pg/mL) (Fig. 6b). EEWS-100 mg/kg treatment reduced IL-6 level from 652 ± 128 pg/mL to 324 ± 109 pg/mL. FLZ treatment alone (20 or 40 mg/kg) did not significantly affect IL-6 levels in infected mice. In contrast, FLZ (40 mg/kg) and EEWS-100 mg/kg combination decreased IL-6 to 85 ± 29 pg/m, achieving a more significant reduction than FLZ-40 alone (p<0.001).

Mice with VVC demonstrated a notable elevation of TNF-α in their vaginal tissue (Fig. 6c). In the infected control mice, TNF-α levels were elevated to 416 ± 101 pg/mL, which was significantly higher than the 8.6 ± 3 pg/mL measured in normal mice (p<0.001). FLZ (20 mg/kg) and EEWS (100 mg/kg) combination lowered TNF-α levels to 133 ± 31 pg/mL. Likewise, an FLZ (40 mg/kg) and EEWS led to further reduction in TNF-α to 71 ± 22 pg/mL. In contrast, FLZ at both 20 and 40 mg/kg doses did not cause a significant reduction in TNF-α levels in vaginal tissue (Fig. 6c). However, EEWS (100 mg/kg) alone lowered TNF-α levels to 223 ± 56 pg/mL; however, it was not significantly lower than the levels seen in the untreated mice.

3.7 Administration of FLZ combined with EEWS significantly reduced the histopathological damage in vaginal tissues

To explore the therapeutic benefits of FLZ alone and in combination with EEWS for VVC treatment, a histological examination was performed on vaginal tissues from different groups of mice: untreated, treated with FLZ, treated with EEWS, or treated with both agents. Within the group serving as the normal control, a healthy lamina propria and a well-structured non-keratinized stratified squamous epithelium were evident in the vaginal tissues (Fig. 7a). However, in the infected, untreated mice, the vaginal tissues exhibited severe pathological changes, including epithelial necrosis, sloughing, and widespread ulceration (Fig. 7b).

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Fig. 7.

Histopathological evaluation of vaginal tissues showing the protective and restorative effects of Withania somnifera EEWS and FLZ treatment in mice with VVC. Vaginal tissues (n = 3 per group) were excised, fixed in 10% neutral-buffered formalin, processed, sectioned, and stained with hematoxylin and eosin (H&E) as described in the Methods section. Representative histological sections are shown for: (a) Healthy control, exhibiting normal epithelial architecture with intact stratified squamous epithelium (EP) and lamina propria (LP); (b) Infected control, showing extensive epithelial necrosis, ulceration, loss of keratohyaline granules (yellow star), and dense inflammatory cell infiltration (ICI) within the LP; (c) EEWS-treated group (100 mg/kg), displaying mild mucosal sloughing and partial restoration of epithelial structure with reduced inflammatory infiltration; (d) FLZ-treated group (40 mg/kg), revealing partial epithelial recovery but persistent mucosal sloughing and moderate leukocytic infiltration in the LP; and (e) Combination therapy (FLZ 40 mg/kg + EEWS 100 mg/kg), showing near-complete epithelial restoration with minimal inflammatory infiltration and well-organized LP, indicating marked tissue regeneration. All sections were examined under a light microscope at 40X magnification (H&E, 40X). Scale bar = 50 μm.

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Mice treated with either FLZ or EEWS showed partial recovery, with signs of moderate healing. While some tissue regeneration was observed, significant infiltration of inflammatory cells and scattered leukocytes remained (Figs. 7c and 7d). In contrast, the combination therapy with FLZ (40 mg/kg) and EEWS (100 mg/kg) resulted in more pronounced tissue regeneration. The vaginal tissues in these mice showed only mild epithelial necrosis and minimal inflammatory cell infiltration in the lamina propria, suggesting a more effective reversal of the infection-induced damage (Fig. 7e).