In the present study, human blood and buccal epithelial cells were collected from healthy individuals after procuring a written informed consent. The experimental protocol and the use of human blood and HBECs were assessed and approved by the Institutional Ethical Committee, Alagappa University, Karaikudi (IEC Ref No. IEC/AU/2018/5). All the methods were carried out in accordance with relevant guidelines.

Piperine (PubChem CID: 638024) and thymol (PubChem CID: l6989) were commercially procured from HiMedia and Alfa Aesar, respectively. Stock solutions of both the phytocompounds were prepared with the concentration of 50 mg/ml in methanol. The highest volume of compound used in the test group was taken as the volume of methanol for vehicle control in each assay.

The impact of individual effects of piperine and thymol on the growth of C. albicans reference and clinical strains was determined by microbroth dilution assay as per the CLSI guideline (Wayne, 2008). The determination of the biofilm inhibitory concentration was performed as described earlier (Priya and Pandian, 2020). Briefly, in a 24-well microtiter plate (MTP), 1 ml of spider broth was added with increasing concentrations of individual phytochemicals in the range of 2–1,024 µg/ml. The medium added with methanol served as vehicle control. To this, 1% of overnight culture of C. albicans was inoculated. Wells containing medium alone served as blank. The plates were incubated at 37°C for 48 h at static conditions. Following incubation, the planktonic part was discarded and the loosely bound biofilm cells were detached by carefully rinsing the wells with sterile PBS. Plates were air dried. Biofilm cells bound to the surface of the MTP were stained with 0.4% crystal violet and incubated at room temperature for 5 min, and the excess unbound stain was decanted. Plates were then destained with 10% glacial acetic acid solution and read at 595 nm in a multifunctional spectrophotometer. Inhibition of biofilm formation was calculated in percentage using the following formula:

MBIC was considered as the minimum concentration required to inhibit a minimum of 85% or more of biofilm when compared to the control biofilm.

The interaction between two phytochemicals piperine and thymol in exhibiting increased antibiofilm activity at lesser concentrations than their individual effect was evaluated through two-dimensional checkerboard assay for C. albicans reference and clinical strains. A total of 25 different antibiofilm combinations were analysed between phytochemicals in a decreasing concentration ranging from biofilm inhibitory concentration (BIC) to 1/16 BIC. The assay was performed in a 48-well MTP. In the first column, piperine was added to the spider medium at the BIC concentration, and then 2-fold dilutions were made for the subsequent plate columns. In a similar way, BIC of thymol was added to the first row and 2-fold dilutions were made for the subsequent plate rows. The last drug concentration in the row and column of the plate was set to 0 µg/ml, to examine the activity of individual phytochemicals at various test concentrations in combination. Appropriate control, vehicle control and negative controls were maintained in parallel. Plates were incubated at 37°C for 48 h in static conditions. At the end of the incubation period, the plates were processed to analyse the antibiofilm activity with the similar procedure detailed in the previous methods section. The percentage of biofilm inhibition by individual and combinations of piperine and thymol was calculated, and a heatmap was generated. The fractional inhibitory concentration (FIC) was calculated individually, and the FIC index (FICI) was calculated by summing up the FIC of both the phytocompounds as the following:

FIC_A =BIC of A in combination with B/BIC of A alone

FIC_B =BIC of B in combination with A/BIC of B alone

The combinations were said to be synergistic when the FICI is ≤ 0.5. Combinations with the FICI in the range of greater than 0.5 to less than or equal to 4 is said to be indifferent. When FICI is greater than 4, the phytochemicals in the combination were known to exhibit antagonistic effect.

Checkerboard assay was performed with YEPD broth and incubated at 37°C for 24 h. Followed by incubation, culture was mixed well and 5 µl from each well was spotted on the agar plate which was incubated for 37°C for 24 h. Similarly, C. albicans grown in the absence and presence of the identified synergistic combinations were grown and CFU analysis was performed.

C. albicans was allowed to form biofilm in the absence and presence of piperine, thymol and their synergistic combinations in spider medium for 48 h at 37°C. Planktonic cells were removed, and loosely bound cells were removed by gentle wash with PBS. The adhered biofilm cells were resuspended in PBS. Serial dilutions were made and plated on YEPD agar plates. CFU/mL was calculated after incubation for 24 h at 37°C, and log reduction in biofilm cells compared to control was plotted as graph.

Biofilm architecture and filamentous morphology of C. albicans in the absence and presence of individual and synergistic combinations of piperine and thymol were microscopically evaluated. Briefly, C. albicans was allowed to form biofilm on a 1 × 1-cm glass surface for 48 h at 37°C. After incubation, the glass slides were carefully removed and processed as mentioned earlier. Crystal violet-stained glass slides were observed under the light microscope (Nikon Eclipse 80i, USA) at ×400 magnification.

Checkerboard assay was performed as mentioned previously. Instead of liquid medium, solid spider agar was prepared and 5% foetal bovine serum was supplemented after the agar reached ambient temperature to induce the filamentation. To this mixture, various concentrations of piperine and thymol were added, mixed well and poured on glass plates. After proper solidification, 5 µl of C. albicans overnight culture was spotted on the centre of the medium. Plates were incubated at 37°C for 5–7 days. Variation in the filamentous morphology was observed and documented using the gel documentation system (Bio-Rad Gel Doc™ XR⁺).

The ability of the phytochemicals to control C. albicans phenotypic switching between yeast to hyphal and vice versa was appraised by the following methods.

C. albicans cells were briefly exposed to the phytochemicals for 1 h at 37°C with gentle shaking. After incubation, the compounds were removed by three rounds of centrifugation. Subsequently, phytochemical-exposed C. albicans cells were used as inoculum and introduced into RPMI medium which was further incubated for 2 h. Later, cells were observed under the microscope.

Sensitivity of the C. albicans cells treated with individual and synergistic combinations of piperine and thymol to antifungals such as miconazole, itraconazole, ketoconazole and nystatin was evaluated by disc diffusion assay. Briefly, C. albicans cells were grown in the absence and presence of individual and combination of phytochemicals overnight in YEPD medium at 37°C. The cultures were inoculated on the YEPD agar plates, and antifungal discs were placed on the centre and incubated for 24 h at 37°C. The zone of clearance was noted.

The dynamics in the expression of candidate genes responsible for biofilm formation, hyphal regulation, adhesion between the control and synergistic combinations of piperine and thymol-treated C. albicans was assessed through qPCR analysis. RNA from control and phytocompound-treated C. albicans was extracted by the TRIzol method using commercial TRI reagent (Sigma-Aldrich, India). Extracted RNA was converted to cDNA using the high-capacity cDNA Reverse Transcription Kit (Applied Biosystems, United States), and qPCR analysis was performed with SYBR Green Master Mix (Applied Biosystems, United States) with the Applied Biosystems^® 7500 Real-Time PCR system. A list of candidate genes, primer details and functions is provided in Table 1 . Alterations in the gene expression were calculated by the ^ΔΔCT method.

The toxic effect of phytocompounds and their combinations were analysed on human red blood cells and human buccal epithelial cells (HBECs).

Human erythrocytes were collected from healthy volunteers in a tube containing EDTA as anticoagulant. Cells were harvested by centrifugation for 10 min at 2,000 rpm and subsequently washed in PBS thrice. Cells were diluted to get a suspension of 1:10 in PBS. Test compounds were added to 1 ml of this suspension and incubated for 1 h at 37°C. Positive control was maintained with 1% Triton X-100. After incubation period, the tubes were centrifuged for 10 min at 2000 rpm. The supernatant was transferred to MTP, and the absorbance was measured spectroscopically at 450 nm. The percentage of haemolysis was calculated by the following formula (Ahmad et al., 2010)

HBECs were collected from healthy individuals with good oral hygiene by gently rubbing the inner mucosal surface of the cheeks with a sterile swab which was subsequently swirled and suspended in sterile PBS. HBEC suspension was used immediately after collection. The cell suspension from different individuals was pooled together and centrifuged at 3,000 rpm for 10 min. The supernatant was discarded, and the pellet was resuspended in PBS. Cells were counted using the Automated Cell Counter (Countess II FL, Invitrogen, United States) and adjusted to 5 × 10⁵ cells/ml. HBECs were then incubated with different concentrations of individual and combinations of piperine and thymol for 20 min at 37°C. Hydrogen peroxide was used as positive control; after incubation, cells were stained with crystal violet and observed under the microscope (Nikon Eclipse TsR2, Japan) (Priya and Pandian, 2020).

All the experiments were carried out in at least three biological replicates with at least two technical replicates, and values are presented as mean ± standard deviation (SD). To analyse the significant difference between the value of control and treated samples, one-way analysis of variance (ANOVA) and Duncan’s post hoc test were performed with the significant p-value of <0.05 by the SPSS statistical software package version 17.0 (Chicago, IL, United States).

The biofilm inhibitory potential of piperine against C. albicans was reported in our previous study (Priya and Pandian, 2020). Piperine at 32 µg/ml inhibited the biofilm formation of the reference strain of C. albicans without affecting the viability ( Figure 1A ). From the present investigation, the antibiofilm potential of thymol was identified. At 32 µg/ml, thymol inhibited up to 90% of surface adherence and biofilm formation of the C. albicans reference strain without affecting the growth. A concentration higher than 32 µg/ml was found to be growth inhibitory in a dose-dependent manner ( Figure 1B ). Thus, 32 µg/ml was determined to be MBIC for both the phytochemicals piperine and thymol for the reference strain. MBIC was found to be slightly different for the clinical isolates. For CI-1, 128 µg/ml of piperine and thymol was determined as MBIC. For CI-2, 64 and 32 µg/ml of piperine and thymol, respectively, were identified as MBIC. The MBIC of piperine and thymol for CI-3 was identified as 32 µg/ml, whereas for CI-4, 32 and 64 µg/ml of piperine and thymol, respectively, was considered as MBIC ( Supplementary Figure 1 ).

The interaction between piperine and thymol in exerting antibiofilm activity against C. albicans was analysed through checkerboard assay. The interaction was found to be synergistic at the following four different combinations of piperine and thymol in µg/ml: 8 + 8, 8 + 4, 8 + 2 and 4 + 8. The antibiofilm activity was found in other combinations. However, the FICI was found to be higher than 0.5 ( Table 2 ). Thus, the aforementioned four combinations of piperine and thymol were determined to the synergistic combinations with antibiofilm activity against C. albicans ( Figure 2A ). When compared to the individual concentrations of piperine and thymol in synergistic combinations, the antibiofilm efficacy of the synergistic combinations was found to be higher ( Figure 2B ). The heatmap displaying the synergistic antibiofilm efficacy of piperine and thymol for clinical strains of C. albicans is provided in Supplementary Figure 2 .

The effect of combinations of piperine and thymol on the growth of C. albicans was assessed through spot assay and CFU analysis. Individual and combinations of piperine and thymol were not found to exhibit any growth inhibitory effect on C. albicans as observed through the spot assay ( Figure 3A ). Control and synergistic combinations of piperine and thymol-treated C. albicans were spread plated in order to identify alterations in the CFU, if any. No significant change in CFU/mL was observed between the control and synergistic combinations treated, which validates that the combinatorial antibiofilm effect of piperine and thymol is not due to growth inhibition but rather to the phytochemical target only of the biofilm formation and virulence of the pathogen ( Figure 3B ).

Biofilm cells of C. albicans in the absence and presence of individual and synergistic combinations of piperine and thymol were subjected to CFU analysis. It is observed that the phytocompounds reduced the number of adhered biofilm cells drastically. A log reduction of more than 2 in biofilm cells was observed in treatment with phytocompounds at MBIC and synergistic combinations ( Figure 4 ).

Adhesion of C. albicans cells to the glass surface was impaired by the influence of phytochemicals. Yeast cells entangled within the hyphal mesh were observed in the control whereas scarcely dispersed yeast cells without hyphal extension were observed in the C. albicans treated with MBIC of piperine and thymol. The individual effect of the concentrations in synergistic combinations was also analysed. Piperine at 8-µg/ml concentration resulted in few hyphal elongations. Although the number of adhered cells was lesser than the control, other individual concentrations in synergistic combinations of piperine and thymol resulted in hyphal morphogenesis, whereas the synergistic combinations of piperine and thymol significantly impeded the biofilm formation and hyphal elongation ( Figure 5 ).

Since the filamentous morphology of C. albicans was inhibited by the synergistic combinations of piperine and thymol in the microscopic observation, synergistic antihyphal activity was appraised. Compared to the individual MBIC of phytochemicals, a combination of piperine and thymol at a concentration of 8 + 8 µg/ml ensued in a synergistic antihyphal activity. The filamentous structure around the entire surface of the C. albicans colony was observed in control. Piperine at MBIC completely inhibited the hyphal growth. Synergistic antihyphal activity was observed at 8 + 8 µg/ml of piperine and thymol with an FIC index of 0.5. Certain other combinations were found to exhibit antihyphal activity, but the FICI was greater than 0.5 ( Figure 6 ).

Phenotypic switching in C. albicans is one of the major virulence determinants. Under hyphal-inducing conditions, the ability of the phytochemicals piperine and thymol in inhibiting the hyphal protrusion and relapsing the hyphal form to yeast morphology was assessed. FBS favourably enhanced the filamentous morphology in control, whereas the MBIC of piperine and thymol completely suppressed the filamentation. Similarly, only yeast cells were observed in the synergistic combinations ( Figure 7 ). Contrarily, synergistic combinations of piperine and thymol limited and reversed the hyphal extension ( Figure 8 ).

A brief exposure of C. albicans to individual and synergistic combinations of piperine and thymol limited the extension of filamentous morphology in hyphal inducing conditions. Also, yeast cell morphology was observed which could be due to the reversal of extended hyphae to yeast cells ( Figure 9 ).

The antifungal susceptibility of C. albicans treated with individual and synergistic combinations of piperine and thymol was found to be increased. The zone of inhibition in C. albicans exposed to synergistic combinations of piperine and thymol was comparatively equal or higher than the inhibition in C. albicans exposed to MBIC of individual phytochemicals ( Table 3 ).

Proficiency to inhibit biofilm formation and hyphal morphogenesis and to control the phenotypic switching between yeast and hyphal forms by the synergistic combination of piperine and thymol against C. albicans was validated through real-time PCR. Negative regulators of filamentation growth such as nrg1 and tup1 were upregulated whereas the transcriptional activator of filamentous growth, ume6, was downregulated. Filamentation and adhesion-related genes such as efg1, cph1, eap1, ras1, als1 and ece1 were found to be downregulated, which further corroborates the in vitro proficiency of piperine and thymol such as antibiofilm, antihyphal and control of phenotypic switch in C. albicans ( Figure 10 ).

Phytocompounds piperine, thymol and their synergistic combinations were subjected to toxicity analysis on human red blood cells and HBECs. No toxic effect was observed at the tested concentrations in both erythrocytes and HBECs. In erythrocytes, the positive control Triton X-100 completely lysed the red blood cells as the treatment with phytocompounds did not lyse any detectable number of erythrocytes ( Figure 11A ). Similarly, no morphological alterations were observed in HBECs treated with phytocompounds whereas positive control hydrogen peroxide instigated alterations in the normal morphology of the cells ( Figure 11B ). These results demonstrate that the phytocompounds alone or in combination did not instigate any adverse effects to human erythrocytes or HBECs and thus can be considered safe for human application.