The standard strain of Candida albicans ATCC 90028 provided by Institute of Microbial Technology, Chandigarh, India. Yeast extract Peptone Dextrose (YPD) agar medium was used to maintain culture and the culture was stored at 4˚C. PMS and all the media components purchased from HiMedia, India. A single isolated colony of C. albicans from YPD agar plate was used to inoculate 50 ml YPD broth in a 250 ml conical flask. The flasks were incubated for 24 h in an orbital shaking incubator (100 rpm) at 30˚C [10] .

The effect of PMS on the growth of C. albicans in planktonic form was studied by using the standard broth microdilution methodology based on the Clinical Laboratory Standards Institute guidelines (Pfaller et al., 1994). Various concentrations of PMS ranging from 0.062 to 2 mg/ml were prepared in RPMI-1640 medium in 96 well plates (Costar, Corning Inc. USA). Cell suspension (1 × 10³ cells ml⁻¹) was added to 100 μl of RPMI-1640 medium in each well. The plates were incubated at 35˚C for 48 h and growth was analyzed by measuring absorbance at 620 nm using a microplate reader (Multiskan EX, Thermo Electron Corp. USA). The lowest concentration of PMS which caused a 50% reduction in the absorbance compared to the control was considered as the minimum inhibitory concentration (MIC) for the growth of C. albicans [10] .

To determine the MFC, Candida cells from the wells containing MIC and above the MIC were used. Aliquots of 10 μl from these wells were spread on YPD agar. These plates were incubated for 48 h at 30˚C and observed for the presence of colonies. No appearance of colonies on the agar plates was noted as fungicidal effect. The lowest concentration of the test molecule in the microplate well from which an aliquot showing no growth was considered as the MFC [11] .

The effect of Capric and Caprylic acid on the adherence of C. albicans to a solid surface (i.e. polystyrene) was studied using a microplate-based assay. Different series of concentrations were prepared in Phosphate buffered saline (PBS) and of 50 μl of aliquots were added in to wells of microtiter plates. The final volume of the assay system in each well was kept at 100 μl. The plate is incubated at 37˚C for 90 min at 100 rpm in an orbital shaking incubator to allow adherence of cells. The adherence of cells were observed in each well by relative metabolic activity (RMA) using the XTT-assay [10] .

Biofilm formation was done on tissue culture treated 96-well polystyrene plates. 100 μl of Cell suspensions of 1 × 10⁷ cells ml⁻¹ were added into each well and allowed to adhere on solid surface at 37˚C for 90 min at 100 rpm. After adhesion wells were washed with PBS two times. RPMI-1640 medium (200 μl) along with various concentrations of PMS were incubated for 48 h at 37˚C. Developed biofilms were observed under an inverted light microscope (Metzer, India). XTT metabolic assay was used Quantitation of Biofilm growth. Photographs were taken with a Labomed microphotography system (Labomed, India) at 200 X magnification [12] .

To determine the activity of the PMS against mature C. albicans biofilms, 24 h old biofilm was used. Wells were washed with PBS to remove nonadhered planktonic cells. Various concentrations of PMS prepared in RPMI 1640 were added to wells. Plates were incubated for 48 h and then washed with PBS. Wells were observed for the presence or absence of biofilm using an inverted light microscope (Metzer, India). XTT metabolic assay was used to analyze the biofilm growth [12] .

Biofilm growth was quantified using XTT metabolic assay. XTT solution was prepared by mixing 1 mg/ml of XTT salt in PBS and stored at 20˚C. Menadione (Sigma Aldrich, India) solution was prepared in acetone (4 μM) and added to the XTT salt solution. The wells containing biofilms were washed with PBS to remove non-adhered cells and incubated with 100 μl of XTT-menadione solution in dark, at 37˚C for 5 h. Color formation by the water soluble formazan product was measured at 450 nm using a microplate reader (Multiskan EX, Thermo Electron Corp. USA) [12] .

Serum-induced Y-H transition in C. albicans was studied in a microplate-based assay. A series of concentrations of PMS was prepared in deionized distilled water with 20% serum. 1 × 10⁶ cells ml⁻¹ Cells were inoculated in test and control wells. The final volume of the assay was 200 μl. After incubation for 2 h at 37˚C in an orbital shaker at 200 rpm, formation of germ tubes were observed microscopically. Numbers of yeast cells and hyphae were counted by using an inverted microscope (Metzer, India). Photographs were taken with a Labomed microphotography system at 200 X magnification. Inhibition of 50% hyphae formation was compared with control and considered as MIC for morphogenesis. [10] .

Adhesion of Candida albicans cells on polystyrene discs (1 × 10⁷ cells/ml) were carried out in 12-well plates at 37˚C at 50 rpm for 90 min. Samples were fixed in 2.5% of glutaraldehyde in 0.1 mol phosphate buffer (pH 7.2) for 24 h at 4˚C. Samples were post fixed in 2% aqueous solution of osmium tetraoxide for 4 h, and then dehydrated in a series of graded alcohols. The samples were mounted over stubs and gold coating was performed using an automated gold coater. Images were obtained by JOEL 6360 (Japan) scanning electron microscope [12] .

Hemolytic activity was determined by using human red blood cells [13] . Suspension of RBC was diluted 1:10 in PBS. Aliquotes of 100 μL from this suspension was added in to 100 μL of a different concentration of test molecules in the same buffer in Eppendorff tubes. 1% Triton X 100 was used for total hemolysis. After incubation of 1 h at 37˚C it was centrifuged for 10 min at 2000 rpm at 20˚C. 150 μL from supernatant was transferred to a flat-bottomed microtiter plate, and O.D. is taken at 450 nm. All the experiments were done in triplicates.

The hemolysis percentage was calculated by following formula:

% of hemolysis = [(A₄₅₀ of test compound treated Sample − A₄₅₀ of buffer treated sample)/(A₄₅₀ of 1% Triton X 100 treated sample − A₄₅₀ of buffer treated sample)] × 100

C. albicans yeast cells from the log phase were harvested, washed and starved for nutrients for 1 h. cells (10³ cfu/ml) were inoculated to each flask containing 50 ml YPD broth and MIC of PMS. Flask without treatment of PMS was considered as Control. All the flasks were incubated at 30˚C for 3 h. Cells were centrifuged at 1000 rpm for 5 min. washed and fixed in 1 ml of 70% ethanol by incubating at room temperature on a rotary shaker (REMI, India) for 30 min. Cells were harvested by centrifugation (Genei, Bangalore, India) at 1000 rpm for 5 min, washed twice with 1 ml of 50 mM Tris (pH 7.8). Washed cells were resuspended in 500 μl of 50 mM Tris containing 10 μg RNase A and incubated for 2 h at 37˚C. After incubation samples were subjected to protease treatment (5 mg/ml pepsin in 0.05 M HCl) for 1 h at 25˚C to reduce cellular clumps. Cells were harvested by centrifugation and resuspended in 500 μl FACS-buffer (200 mM Tris/HCl pH 7.5; 200 mM NaCl; 78 mM MgCl₂). 15 μl of propidium iodide (1 mg/ml) was added to each tube and incubated for 30 min and stored at 4˚C. Before analysis, cells were sonicated for 10 s and analyzed using a FACS Calibur cytometer (Becton-Dickinson, San Jose, Calif.) and analyzed with Cell Quest 3.0 software [14] .

The expression of hyphal genes during serum induced morphogenesis was measured by using Real Time PCR. C. albicans yeast phase cells (1 × 10⁶ cells/ml) were inoculated and incubated for 90 min in 20% serum containing PMS (0.062 mg/ml). Total RNA was isolated by using RNeasy^® Mini Kit (QIAGEN, USA) and converted to cDNA by using SuperScript^® III for first strand synthesis (Invitrogen, Life technologies, USA). PCR reactions were conducted by using KAPA SYBR^® Fast qPCR Kit Master mix (2x) (BIOSYSTEMS, South Africa) in 96 well PCR plates with preliminary denaturation for 3 min at 95˚C. This was followed by 32 amplifications cycles of denaturation at 95˚C for 30 s, annealing at 60˚C for 20 s and primer extension at 72˚C for 30 s (CFX 96 Real time System, Bio-Rad, USA). Primers were purchased from Eurofins Genomics India Pvt. Ltd. (Table 1). Actin was used as an internal control and the transcript levels of selected genes were calculated using the formula 2^-ΔΔCT [15] .

Values mentioned are the mean with standard deviations, obtained from three different observations. Values in the control and treatment groups were compared using Student’s t-test. A value of p < 0.05 was considered statistically significant [10] .

PMS inhibited planktonic growth of Candida albicans ATCC 90028 in a concentration dependent fashion (Figure 1). More than 90% (p < 0.05) of the growth was inhibited at 0.062 mg/ml. 99% of planktonic growth was inhibited by PMS after 48 hrs of exposure at 0.25 mg/ml (Table 1). PMS caused significant inhibition of developing as well as mature biofilm formation (Figure 1, Figure 2). MIC values for developing biofilm and mature biofilm were 0.25 mg/ml and 0.5 mg/ml respectively. >70% of 48 h old mature biofilm growth was eradicated at 0.5 mg/ml (Figure 1). MIC values were established for Adhesion for PMS (Table 2). Adhesion of C. albicans ATCC 90,028 to polystyrene plate surface was inhibited significantly (p < 0.05) by PMS at >16 µg /ml (Figure 1). MIC50 for inhibition of adhesion was 16 µg /ml (Table 2). After treatment of PMS (0.031 mg/ml) on C. albicans caused cell cycle arrest in S phase (Figure 3).

Significant (p < 0.05) inhibition of serum induced yeast to hyphal form transition was observed in presence of PMS in a concentration dependent manner. More than fifty percentage inhibition of germ tube induction was found at 0.062 mg/ml (Figure 4).

Since PMS has shown inhibition of serum induced hyphal induction in C. albicans, we analyzed changes in expression levels of the genes involved in Ras1- cAMP-Efg1, Cek1-MAPK pathways with quantitative real-time PCR. It was found that the expression of Ras1 which is a master regulator was downregulated up to 1.14 fold (p < 0.05) by treatment with PMS. Cdc35 and Pde2, the upstream components of cAMP dependent PKA pathway were upregulated by 1.09 and 1.46 fold respectively. Similarly expression of its downstream components Bcy1, Efg1 and Tec1 were upregulated by 2.75, 1.12, 1.65 fold. Ece1 and Hwp1 downstream of Efg1 were significantly downregulated by 1.56 and 11.16 fold respectively (Figure 5). Expression of Hst7, Cek1 and Cph1 involved in Cek1-MAPK pathways were upregulated by 1.44, 1.43 and 2.57 fold in presence of PMS respectively (Figure 5). Negative regulators of hyphal induction, Nrg1, Mig1 and Tup1 were upregulated by 1.53, 2.96 fold and 17.95 fold respectively (Figure 5).