Hydroxychavicol (Fig. 1) was isolated in the pure form from the chloroform extraction of the aqueous leaf extract of P. betle L., (Piperaceae) as described previously 12 . Amphotericin B was purchased from Sigma Chemical Co. (St. Louis, MO), and terbinafine was obtained as kind gift from Lupin Laboratories, Pune, India.

Propidium iodide (Sigma), a small cationic, nucleic acid-binding fluorochrome largely excluded by intact cell membranes was used to stain the yeast cells 14 . Sodium deoxycholate (Sigma), an anionic detergent, was used to facilitate diffusion of propidium iodide into the yeast cell membranes which were damaged by the antifungal agent 15 .

A total of 124 fungal strains were tested for their susceptibility to hydroxychavicol. These strains comprised of Candida albicans (ATCC 90028, ATCC 10231 and 23 clinical isolates), Candida glabrata (ATCC 90030 and 7 clinical isolates), Candida krusei (ATCC 6258 and 3 clinical isolates), Candida parapsilosis (ATCC 22019 and 5 clinical isolates), Candida tropicalis (ATCC 750 and 11 clinical isolates), Cryptococcus neoformans (ATCC 204092 and 2 clinical isolates), Aspergillus flavus (MTCC 1973, MTCC 2799 and 10 clinical isolates), Aspergillus fumigatus (MTCC 1811 and 17 clinical isolates), Aspergillus niger (ATCC 16404 and 6 clinical isolates), Aspergillus parasiticus (MTCC 2796), Epidermophyton floccosum (MTCC 613 and 1 clinical isolate), Microsporum canis (MTCC 2820 and 3 clinical isolates), Microsporum gypsium (MTCC 2819 and 2 clinical isolates), Trichophyton mentagrophytes (ATCC 9533 and 7 clinical isolates), and Trichophyton rubrum (MTCC 296 and 9 clinical isolates). Reference strains were procured from the American Type Culture Collection (ATCC, Manassas, VA, USA), and Microbial Type Culture Collection (MTCC, Chandigarh, India). The clinical isolates were obtained from the Department of Microbiology, Acharya Shri Chander College of Medical Sciences, Sidhra, Jammu, India.

Suspensions of the yeasts and Aspergillus species were prepared in sterile normal saline (0.85%) containing 0.05% polysorbate 20 (NST) from 24 h (48 h for C. neoformans) and 7-day-old cultures "respectively" grown on potato dextrose agar (Difco Laboratories, Detroit, Mich) at 35°C 16 17 . A stock inoculums suspension of each dermatophytes was prepared from fresh, mature (7-day-old) cultures grown on potato dextrose agar with 2% in-house rice flour slants at 28°C. The densities of these suspensions were adjusted with a spectrophotometer (Multiskan spectrum, Thermo electron, Vantaa, Finland) at a wavelength of 530 nm to a transmittance of 65 to 70% to yield an initial inoculum of 1 × 10⁶ to 5 × 10⁶ cfu/ml 18 . All adjusted suspensions were quantified by plating on Sabouraud dextrose agar (SDA; Difco Laboratories) plates.

The MIC was performed by broth microdilution methods as per the guidelines of Clinical and Laboratory Standard Institute (formerly, the National Committee for Clinical Laboratory Standards) 16 17 , with RPMI 1640 medium containing L-glutamine, without sodium bicarbonate and buffered to pH 7.0 with 0.165 M morpholinepropanesulfonic acid (RPMI) (both from Sigma). Stock solution of hydroxychavicol was prepared in 100% dimethyl sulfoxide (DMSO; Sigma) and twofold serial dilutions were prepared in media in amounts of 100 μl per well in 96-well U-bottom microtiter plates (Tarson, Mumbai, India). The above-mentioned fungal suspensions were further diluted in media, and a 100 μl volume of this diluted inoculum was added to each well of the plate, resulting in a final inoculum of 0.5 × 10⁴ to 2.5 × 10⁴ cfu/ml 19 for yeasts and 0.4 × 10⁴ to 5 × 10⁴ cfu/ml for dermatophytes and Aspergillus species. The final concentration of hydroxychavicol ranged from 3.90 to 2000 μg/ml. The medium without the agents was used as a growth control and the blank control used contained only the medium. Amphotericin B and terbinafine served as the standard drug controls. The microtiter plates were incubated at 28°C for 7 days for dermatophytes 18 , and at 35°C for 48 h for Candida species (72 h for C. neoformans) and Aspergillus species 16 17 . The plates were read visually, and the MIC was defined as the lowest concentration of the antifungal agents that prevented visible growth with respect to the growth control.

The MFC was determined by plating a 100 μl volume on SDA from the wells showing no visible growth. The plates were incubated as described above in MIC. The minimum concentration of hydroxychavicol that showed ≥ 99.9% reduction of the original inoculums was recorded as the MFC 19 .

Time-kill curve studies were performed as described by Ernst et al 20 , using RPMI. C. albicans ATCC 90028 and C. glabrata ATCC 90030 were used as the test strains in this study. One milliliter of the adjusted inoculum suspension (≈ 5 × 10⁶ cfu/ml) was added to nine ml of RPMI with or without hydroxychavicol, providing the starting inoculum of ≈ 5 × 10⁵ cfu/ml. The range of hydroxychavicol concentrations tested was one to eight times the MICs for test strains i.e. 250 to 2000 μg/ml for C. albicans and 31.5 to 250 μg/ml for C. glabrata. The culture flasks were incubated with agitation at 35°C. At predetermined time points (0, 0.5, 1, 2, 4, 6, 8, 10, 12, and 24 h following the addition of hydroxychavicol), a 100 μl aliquot was removed from each culture flask and serially diluted in sterile normal saline containing 0.1% polysorbate 80 (Sigma) for the inactivation of hydroxychavicol. A 20 μl aliquot was plated onto a Sabouraud dextrose agar with lecithin and polysorbate 80 (BBL, Becton Dickinson and Company, Cockeysville, MD) plate for colony count determination. When the colony counts were expected to be less than 1000 cfu/ml, samples of 20 μl or 100 μl were taken directly from the test solution and plated or subcultured without dilution. Plates were then incubated at 35°C for 24 to 48 h. The lower limit of accurate and reproducible quantification was 50 cfu/ml for each of the strains.

The PAFE of hydroxychavicol was performed in RPMI by the method described by Craig and Gudmundsson 21 . C. albicans ATCC 90028, C. tropicalis ATCC 750, C. glabrata ATCC 90030 and C. parapsilosis ATCC 22019 were used as the test strains in this study. One milliliter of the adjusted inoculum suspension (≈ 5 × 10⁷ cfu/ml) was added to nine ml of RPMI with or without hydroxychavicol, providing the starting inoculum of ≈ 5 × 10⁶cfu/ml. The hydroxychavicol concentrations ranged from one to four times the MIC. After exposures to the hydroxychavicol for 2 h, samples were diluted to 1: 1,000 in prewarmed medium to effectively remove the hydroxychavicol. The diluted cultures were then incubated with agitation (200 rpm) at 35°C and sampling was done after 0, 2, 4, 6, 8, 10, 12, 16 and 24 h for colony counts. The colony counts were determined as described above in time-kill curve studies. The PAFE was calculated by the following equation: PAFE = T-C, where T represents the time required for the count in the test culture to increase 1 log₁₀ cfu/ml above the count observed immediately after drug (hydroxychavicol) removal and C represents the time required for the count of the untreated control flask to increase by 1 log₁₀ cfu/ml.

The first step mutant frequency of reference strains of C. albicans ATCC 90028, C. tropicalis ATCC 750, C. glabrata ATCC 90030, C. parapsilosis ATCC 22019, A. flavus MTCC 2799 and A. fumigatus MTCC 1811 were selected, using previously described method 22 . A fungal suspension containing 10⁹ cfu (100 μl) was plated on SDA containing hydroxychavicol at concentrations equal to two, four and eight times the MIC. Mutation frequency was calculated by counting the total number of colonies appearing after 48 h of incubation at 35°C on the hydroxychavicol containing plate and by dividing the number by the total number of cfu plated.

The effect of hydroxychavicol on C. albicans ATCC 90028 biofilm formation was examined by the microbroth dilution method, similar to MIC assays for planktonic cells 16 as described above. The fungal suspension was prepared from the overnight culture grown in yeast nitrogen base (Difco Laboratories) medium supplemented with 100 mM glucose 23 , and the cells were harvested in the late exponential growth phase, washed twice with sterile phosphate-buffered saline (PBS; pH 7.2; Ca²⁺ and Mg²⁺ free [Hi Media]) and the turbidity of the suspension was adjusted to 4 McFarland standard (≈ 5 × 10⁷ cfu/ml). The suspension was diluted in RPMI to obtain ≈ 5 × 10⁶ cfu/ml as the final inoculums. Twofold serial dilutions of hydroxychavicol were prepared in RPMI in the wells of a 96-well flat-bottom polystyrene microtiter plate (NUNC, Roskilde, Denmark) containing the same media in a volume of 100 μl per well. A 100 μl of above-mentioned suspension was added to each well; the final concentrations of hydroxychavicol ranged from 1.95 to 2000 μg/ml. Amphotericin B (at a final concentration range from 0.0156 to 16 μg/ml) was used as control drug. Following incubation at 35°C for 48 h, absorbance at 490 nm was recorded to assess culture growth. The culture supernatants from each well were then decanted, and planktonic cells were removed by washing the wells with sterile PBS. Biofilm formation was quantified by tetrazolium salt (XTT) reduction assay (see below).

The effect of hydroxychavicol was also examined on preformed C. albicans ATCC 90028 biofilm by the method as described previously 24 . Biofilms were prepared by inoculating the wells of a polystyrene microtiter plate in a manner similar to that described above. After incubation at 35°C for 48 h, the culture supernatant from each well was decanted, and the planktonic cells were removed by washing the wells with PBS. Two fold serial dilutions of hydroxychavicol were prepared in RPMI, and 200 μl of each dilution was added to the biofilm in the wells. The plate was further incubated at 35°C for 48 h. The final concentrations of hydroxychavicol ranged from 1.95 to 2000 μg/ml. Amphotericin B (at a final concentration range from 0.0156 to 16 μg/ml) was used as control drug. After the completion of incubation, the plates were decanted and washed three times with 200 μl of sterile PBS to remove loosely adherent cells. Biofilm reduction was quantified by XTT-reduction assay described below.

XTT (tetrazolium salt 2, 3-bis (2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide) reduction assay was performed by the method as described by Jin et al., 23 . The XTT (Sigma) solution was prepared in PBS (1 mg/ml), filter-sterilized through a 0.22-μm-pore-size filter (Millipore, Bangalore, India) and stored at -80°C until required. Menadione (Sigma) solution (0.4 mM prepared in acetone) was filtered and mixed with XTT solution at a ratio of 1 to 5 by volume before the assay. 200 μl of PBS and 12 μl of the XTT-Menadione solution were added to each of the washed wells and the plate was incubated in the dark for 2 h at 35°C. Following incubation, 100 μl of the solution was transferred to a fresh microtiter plate and, the color change in the solution was measured spectrophometrically using a microtitre plate reader (Multiskan spectrum, Thermo electron, Vantaa, Finland) at 490 nm.

The disruptive effect of hydroxychavicol on Candida albicans ATCC 90028 cell membranes was assessed by using hydroxychavicol-mediated propidium iodide uptake. One-milliliter volumes of ≈ 5 × 10⁷ cfu/ml cell suspensions of C. albicans in sterile MilliQ water were incubated with two to eight times the MIC (500 to 2000 μg/ml) of hydroxychavicol at 35°C for 60 min under agitation in the dark chamber. Fifteen minutes prior to the completion of incubation, 10 μl each of propidium iodide and sodium deoxycholate solution were added at a final concentration of 25 μg/ml and 2.5 mg/ml "respectively" 14 15 . Amphotericin B at eight times the MIC (4.0 μg/ml) was used as the positive control and, the cells without hydroxychavicol served as the negative (growth) control, treated in similar fashion. After incubation, 50 μl aliquot was transferred into fluorescence-activated cell sorting (FACS) tube (Becton Dickinson Biosciences, CA) containing 950 μl of sterile MilliQ water. Each tube was analyzed using a FACScan flow cytometer (BD-LSR; Becton Dickinson) with Cell Quest Pro software for data acquisition and analysis.

The MICs and MFCs of hydroxychavicol were evaluated in vitroagainst 58 strains of yeasts, 39 strains of Aspergillus species and 27 strains of dermatophytes and all values are listed in Table 1. Hydroxychavicol exhibited the MICs range between 15.62 to 500 μg/ml for yeasts, 125 to 500 μg/ml for Aspergillus species and 7.81 to 62.5 μg/ml for dermatophytes, where as the MFCs were found to be similar or two fold greater than the MICs. Among all the fungal species tested, dermatophytes were found to be the most susceptible species to hydroxychavicol.

The killing activities of hydroxychavicol for C. albicans ATCC 90028 and C. glabrata ATCC 90030 are shown in Fig. 2. Hydroxychavicol exhibited fungicidal activity against both Candida species and the reduction in the number of cfu per milliliter was greater than 3 log units (99.9%). The fungicidal endpoint for C. albicans was achieved after 10 and 1 h at 4 × MIC (4 × 250 μg/ml) and 8 × MIC (8 × 250 μg/ml) of hydroxychavicol (Fig. 2A). In C. glabrata, killing was observed at a lower concentration of hydroxychavicol due to its lower MIC. There was concentration dependent killing observed in case of C. glabrata, with two, four and eight times the MIC exhibited fungicidal activity in 10, 8 and 4 h "respectively".

Hydroxychavicol produced significant PAFE against all the Candida species tested (Table 2). Increase in the concentration of hydroxychavicol resulted in extended PAFE for all the Candida spp. tested. This increase in PAFE was more prominent for C. albicans and C. tropicalis, where a PAFE of >8 h was exhibited in these organisms at four times the MIC concentration of hydroxychavicol.

The frequencies of mutant selection of C. albicans, C. tropicalis, C. glabrata, C. parapsilosis, A. fumigatus, and A. flavus, are summarized in Table 3. Hydroxychavicol completely suppressed the emergence of mutants at two times its MIC for A. fumigatus and A. flavus, four times the MIC for C. albicans and C. tropicalis, and eight times the MIC for C. glabrata and C. parapsilosis "respectively". This concentration of hydroxychavicol at which no mutant was selected can be defined as the mutation prevention concentration.

Hydroxychavicol exhibited an inhibitory effect on the biofilm formation and reduction of preformed biofilm of C. albicans ATCC 90028. The 50% and 80% biofilm inhibition as well as biofilm reduction are represented in Fig. 3. The MBIC₅₀ and MBIC₈₀ values of hydroxychavicol were 125 μg/ml and 250 μg/ml, where as the MBRC₅₀ and MBRC₈₀values were 500 μg/ml and 1000 μg/ml. Reductions of preformed biofilms values were four fold greater than the concentration required to inhibit biofilm formation.

Exposing the cell suspension of C. albicans ATCC 90028 to two to eight times (500 to 2000 μg/ml) the MIC of hydroxychavicol for 60 min increased the cell permeability to the fluorescent nucleic acid stain, propidium iodide due to the disruption of membrane integrity. This resulted in the increase in fluorescence in comparison to untreated control (Fig. 4). This increase in fluorescence was proportional to the increase in the hydroxychavicol concentrations.