Effect of Piperine on Growth and Biofilm of C. albicans

Impact of piperine on the survival of C. albicans was assessed using microbroth dilution method according to the guidelines of Clinical and Laboratory Standards Institute (Clinical and Laboratory Standards Institute [CLSI], 2008). Briefly, 1% of overnight C. albicans culture was used to inoculate 1 mL of YEPD broth. Test groups were supplemented with varying concentrations of piperine, ranging from 2 to 1,024 μg mL¹. YEPD medium alone served as negative control. The plate was incubated for 24 h at 37°C. Following incubation, cell density was evaluated by measuring absorbance at 600 nm in multifunctional spectrophotometer (Spectra Max 3, Molecular Devices, United States). For visible assessment of the growth effects, 5 μL of cells from each well was spotted on YEPD agar plate and incubated at 37°C for 24 h, and the image was photographed.

Metabolic viability of C. albicans control and piperine-treated cells was ascertained through a dye-based assay (Repp et al., 2007). Stock solution of 10 mg mL^–1 of Alamar blue [Resazurin (7-Hydroxy-3H-phenoxazin-3-one 10-oxide)] was prepared in 1 × phosphate buffered saline (PBS) and stored at 4°C until use. Control and piperine-treated C. albicans cells were cultured as previously mentioned. Past incubation, cells were harvested by centrifugation at 8,000 rpm for 10 min and washed twice with PBS. Cell pellets resuspended with 1 mL of 1 × PBS were added with 100 μg of Alamar blue and incubated in dark at 37°C for 18–24 h. Alamar blue added to PBS alone served as negative control. Subsequently, samples were centrifuged, and fluorescent intensity of the supernatant was observed at 560 and 590 nm of excitation and emission wavelengths, respectively.

The effect of piperine on the biofilm formation of C. albicans was performed in 24-well MTP (Muthamil et al., 2018). Briefly, overnight culture of C. albicans (1%) was used to inoculate 1 mL of Spider broth in the absence (untreated control) and presence (treated) of test phytochemical piperine with the increasing concentrations, from 2 to 1,024 μg mL^–1. Appropriate vehicle and negative controls were maintained parallelly. The plates were incubated statically for 48 h at 37°C. Planktonic cells were aspirated and the biofilm cells that are adhered to the surface of wells were rinsed with sterile distilled water to detach loosely bound cells and allowed to air dry. Successively, the wells were stained with 0.4% crystal violet stain for 10 min at room temperature. Surplus stain that are not bound were removed and washed with sterile distilled water. Stain bound to the biofilm cells was resuspended with 1mL of 15% glacial acetic acid. Absorbance was read at 570 nm using multifunctional spectrophotometer. A minimum of 80% inhibition in biofilm formation was considered as MBIC (Subramenium et al., 2018). The percentage of biofilm inhibition was determined by means of the following formula:

To assess the effect of piperine (at BIC-32 μg mL^–1) on growth of C. albicans, growth curve analysis was performed in the absence and presence of piperine and amphotericin B (antifungal, positive control for growth inhibition) over a 24-hour period with 1-hour time interval, spectroscopically. Growth curve was plotted as absorbance at 600 nm against time interval.