Plant and components
Dry pericarps of the fruits of S. saponaria were collected on the campus of the State University of Maringá, Paraná, Brazil (UEM). The plant was identified by staff members of the UEM Department of Botany, and an exsiccate was deposited in the Herbarium of this institution (HUM 11710). The dried pericarps of the fruits (450.0 g) of S. saponaria were ground and extracted with EtOH:H2O (9:1) at room temperature, by a process of dynamic maceration with constant mechanical stirring. The extraction was carried out in an amber flask, maintained at ambient temperature, for six consecutive days, for 6 h per day. The extract was concentrated under low pressure in a rotary evaporator, at a temperature of 40°C. After elimination of the solvent, the extract was frozen in liquid nitrogen and lyophilized in a Martin Christ Alpha 1-2 freeze dryer. The lyophilized extract was stored in a closed plastic flask and kept frozen.
The HE of the pericarp (50.15 g) was chromatographed in a column (ϕi = 4.0 cm) of silica gel 60 (Merck, Darmstadt, Germany), and eluted with solvents of increasing polarity including hexane, dichloromethane, ethyl acetate, and methanol (Merck, Darmstadt, Germany). The solvents were evaporated at a temperature of 40°C, frozen in liquid nitrogen, and lyophilized in a Martin Christ Alpha 1-2 freeze dryer. The lyophilized dichloromethane, hexane, ethyl acetate, and methanol fractions were stored in closed containers and kept frozen. The methanol extract was suspended in H2O and extracted with n-butanol, which after evaporation gave a solid residue (28.9 g) (BUTE), which was also lyophilized. The structures were established with the use of spectroscopic methods (1H and 13C NMR, HSQC, HMBC, and ESI/MS) and by comparing them with literature data [2, 4].
For the experiments on in vitro susceptibility, 56 vaginal isolates obtained from patients with VVC were tested, including 46 isolates of C. albicans and 10 of C. glabrata, which are part of a bank of yeasts at the Medical Mycology Laboratory/UEM. In this yeast bank, aliquots of the yeasts are stored after their identification in 10% glycerinated water at -20°C. The yeasts used in this study were isolated and identified in 2008, by classical methods [9–11] and also by rDNA sequencing . Prior to each experiment, the isolates were reactivated in Sabouraud Dextrose Broth (SDB) (Difco, Detroit, USA) at 25°C for 24/48 h, seeded on Sabouraud Dextrose Agar (SDA) (Difco, Detroit, USA) with chloramphenicol (2.0 mg/mL), and incubated again under the previous conditions. A new subculture was made in CHROMágar Candida® (Probac, France) to assure the purity of the isolates.
Stock solutions of fluconazole (FLU) (5000 mg/mL; Pfizer Inc., NY, USA) and itraconazole (ITRA) (1000 mg/mL; Janssen Pharmaceutica, Titusville, NJ, USA) were prepared. From the first prepared solution, new stock solutions of FLU and ITRA were prepared at a concentration of 10 times that of the final test concentration, and diluted in bicarbonate-free RPMI-1640 with L-glutamine, supplemented with 2% dextrose and buffered to pH 7.0 with 0.165 M morpholinopropanesulfonic acid (MOPS) (Sigma, Steinheim, Germany). The lyophilized HE and BUTE extracts were dissolved in sterile distilled water to obtain a 10 mg/mL solution of each extract.
Determination of minimum inhibitory (MIC) and fungicidal concentrations (MFC)
The tests of susceptibility of Candida spp. to FLU and ITRA were carried out according to the broth microdilution method recommended by the CLSI (Clinical Laboratory Standards Institute, 2002), and of susceptibility to HE and BUTE according to the same document, with adaptations for natural products [13, 14].
A suspension of yeast compatible with 1.0 to 5.0 × 106 colony-forming units per mL (CFU/mL) was prepared in sterile saline, adjusting the cell density by means of a spectrophotometer (Spectronic 70, Bausch & Lomb, USA) at 530 nm with 90 ± 2% transmittance. From this suspension, new dilutions were made: 1:50 in sterile saline, and then 1:20 in RPMI (Sigma, Steinheim, Germany), thus obtaining the desired final inoculum of 0.5 to 2.5 × 103 CFU/mL. The tests were carried out in sterilized plastic microplates (TPP Zellkultur Test Plate 96F, Switzerland) containing 96 wells arranged in 8 rows labeled A to H, each row with 12 wells numbered 1 to 12. Each row (A-H) corresponded to one isolate, and each well received 100 μL of the measured inoculum, except for the 12th well which was the negative control. Aliquots of 100 μL of RPMI (Gibco, NY, USA) were distributed from columns 2 to 11. Aliquots of 100 μL of FLU, ITRA, HE, or BUTE, prepared as previously described, were added to the columns of the microplates, and from column 2 a 2-fold serial dilution was made up to the 10th well (diluitions between 0.125 and 64.0 μg/mL for FLU, 0.03 and 16.0 μg/mL for ITRA, and 9.0 and 5000.0 μg/mL for HE and BUTE).
For each isolate tested, negative controls (only RPMI) and positive (RPMI plus inoculum, with no antifungals added) were included, for growth and for the possible action of the diluent of the extracts or drugs (only butanol, ethanol, or polyethyleneglycol 400 with the inoculum). On each microplate a strain of Candida parapsilosis (ATCC 22019) was included as the reference yeast. The plates thus set up were incubated in an oven at 35°C with daily monitoring. After 48 h they were read for FLU and ITRA in a microplate reader (Asys Hitech GmbH, Eugendorf, Austria) and after 72 h for the extracts, by visual comparison of the reflection in a mirror.
The MIC for FLU/ITRA was determined as the lowest concentration of the drug that was capable of inhibiting 50% of the growth of each yeast, with reference to its respective positive control . The criteria for definition of susceptibility/resistance to FLU/ITRA were those established by CSLI . For HE and BUTE, the MIC was considered to be the smallest concentration of the extract that was capable of inhibiting 100% of the inoculum compared to its respective positive control . The MIC50 and MIC90 for the drugs and extracts were defined as the MICs capable of inhibiting 50% and 90% of the isolates, respectively .
To determine the MFC, subcultures from all wells showing growth inhibition were made by seeding 5.0 μL in SDA at 25°C. After 48 h, the CFU were counted to determine the viability. All the assays for determination of the MIC and MFC were carried out in duplicate, independently. The MFC for HE and BUTE was considered as the lowest concentration that impeded the growth of 100% of the inoculum. The MFC50 and MFC90 for the extracts were defined as the MFCs capable of inhibiting 50% and 90% of the isolates, respectively.
Experimental vaginal infection
A rat model was used as previously described , with some adaptations. The experiments were carried out with three yeast isolates, which were selected according to the results of the in vitro tests: C. albicans susceptible to FLU and ITRA (SCA), C. albicans resistant to ITRA (RCA), and C. glabrata resistant to both antifungals (RCG).
The experiments were carried out with groups of five rats for each isolate, in duplicate and on two different days. Non-oophorectomized Wistar rats (Rattus norvegicus) weighing from 200 to 300 g and 70 days old (from the UEM Central Animal Facility) were used. The rats received subcutaneous injections of estradiol valerate (Sigma, Steinheim, Germany) at a concentration of 0.2 mg/week/rat. Six days after the first injection of the hormone, the animals were inoculated intravaginally with 108 yeast cells/mL of each isolate tested, prepared in 0.1 mL of sterile saline and counted in a Neubauer chamber.
For the treatment, HE and BUTE were administered intravaginally (0.1 mL at 1%, 2.5%, and 5.0% in distilled water) at 1, 24, and 48 h after the induction of the vaginal infection. Rats receiving FLU (3 doses of 100 μg intravaginally over the same time periods as the extracts) or distilled water served as positive and negative treatment controls, respectively. The kinetics of the Candida vaginal infection in the treated and control rats was monitored in each animal by means of the number of CFU/mL in the vaginal fluid at 24 and 48 h after induction of the infection, and on days 5, 7, 14, and 21. The animal experimentation carried out in this investigation was approved by the UEM Committee on Ethical Conduct in Animal Use (Protocol No. 013/2006, Opinion No. 050/2006).
Toxicity in cervical cells
Cells from the HeLa human cervical line were previously cultured in Eagle's minimum essential medium (MEM, PPA Laboratories, Germany) supplemented with 10% fetal bovine serum (FBS, Laborclin, Brazil), 0.1 mM non-essential amino acids, and 1 mM sodium pyruvate, at 37°C in a humid oven with 5% CO2. In the exponential growth stage, the cells were diluted in the same medium and plated in volumes of 0.2 mL of a suspension of 2.5 × 105 cells per well in a 24-well plate (Corning Glass, New York, USA), and incubated in the same conditions overnight to allow them to form a cell monolayer. The culture medium was replaced by serial dilutions of the HE or BUTE extracts at 1%, 2.5%, 5%, and 10% concentrations, in triplicate. The control wells contained only cells and culture medium. The microplate was incubated again at 37 °C for 24 h, and the extracts were replaced by a trypsin-EDTA solution to undo cell adhesion, followed by addition of 0.2 ml of PBS with 50% trypan blue. Live and dead cells in each well were counted with the aid of light microscopy.
Scanning electron microscopy (SEM)
The vaginal epithelium of rats infected with CAS, CGR, and CAR before and after treatment with HE and BUTE was observed by SEM. After 48 h of infection and also at the end of treatment, the rats to be analyzed by SEM were killed with an overdose of anesthetics (Ketamine and Xylazine, Parke-Davis Co, Morris Plains, NJ, USA). The vagina was removed, washed, fixed in a solution of 2.5% glutaraldehyde in 0.1 M cacodylate buffer (Sigma Chemical, St. Louis, MO, USA), and dehydrated in an ascending ethanol series. The critical point was obtained in a Balzers CPD-010 (Balzers Instruments, Balzers, Liechtenstein) with carbonic gas. Metallization in gold was carried out in a Balzers SCD-030 (Balzers Instruments, Balzers, Liechtenstein). The vagina, uterine cervix, and tissue sections of all rats were observed and photographed with a JEOL-JSM 6360 LV scanning electron microscope (JEOL Ltd, Tokyo, Japan) at the Electron Microscopy Center, Federal University of Paraná/Curitiba/Brazil.
The results were analyzed using Student's t test and Tukey's test for multiple comparisons of the different in vivo experimental treatment situations. The significance level was set at 5%. The tests were carried out by means of the program Graph Pad Prismâ version 3.0 (Graph Pad Software Inc.).