Plant material and crude extract

Punica granatum fruits were collected in December 2007 in Mariná; Paraá; Brazil. The peel was separated manually (2183.8g) and extracted with a 90% (v/v) hydroalcoholic solution, by maceration at room temperature for 5 days in a dark room. The hydroalcoholic extracts were filtered, evaporated under vacuum at 40°, lyophilized, and kept in a freezer at −10°. This crude extract was assayed against four species of dermatophyte fungi and Gram-positive and Gram-negative bacteria.

Isolation of the active substance

First, 200μl of an aqueous solution of the crude extract (20μ) was submitted to liquid-liquid partition, and eluted with ethyl acetate and then with n-butanol; this procedure was repeatead four times with each solvent, resulting in three fractions: F1 (water), F2 (ethyl acetate), and F3 (n- butanol). The collected fractions were evaporated under vacuum and lyophilized in the same conditions as for the extract. Second, 0.5μ of the fraction with the best activity (F1) was dissolved in water, filtered through cotton wool and then placed in a Sephadex LH-20 column. The procedure was performed twice to maximize the yield. It was monitored by thin-layer chromatography (TLC), mobile phase n- butanol: acetic acid: water (40:10:50), and observed as a natural yellow substance. Finally, after antifungal tests, the active subfraction was placed in a Lobar (C-18) column and eluted with methanol:water (1:1), also monitored by TLC. The structure of the active compound was established with the use of spectroscopic methods (EI-MS, 1H NMR, 13C NMR, H-H COSY, HMBC, HMQC, and DEPT). The isolated substance was tested against Trichophyton rubrum.

Microorganisms and growth conditions

Dermatophyte species used for this investigation were Microsporum canis ATCC 32903, Microsporum gypseum ATCC 14683, Trichophyton mentagrophytes ATCC 1481 and Trichophyton rubrum ATCC 28189. Gram-negative bacteria Pseudomonas aeruginosa ATCC 27853 and Escherichia coli ATCC 25922 and Gram-positive bacteria Bacillus subtilis ATCC 6623 and Staphylococcus aureus ATCC 25923 were also investigated, because secondary infections may occur in dermatophytoses.

Microdilution broth assay

Antifungal and antibacterial assays were performed by microdilution method in sterile flat-bottom microplates according to CLSI [16],[17]. Each well contained appropriate test samples, culture medium, and approximately 105 cells for bacteria, and 104 spores in a total volume of 100μl. The plates were incubated at 37° and 24 h for bacteria and 28° during 72 h for dermatophytes. The MIC (Minimal Inhibitory Concentration) was defined as the lowest concentration of a compound at which the microorganism tested did not demonstrate visible growth. To determine the minimal fungicidal effect, 10μl of suspension from the MIC was spotted in Sabouraud agar and incubated for 24 to 72 h at 28°. The minimum fungicidal concentration (MFC) was defined as the lowest concentration that yielded negative subcultures or only one colony.

Conidial germination inhibition assay

Different concentrations of test samples in 90μl were prepared in 96-well flat-bottom micro-culture plates by the double dilution method. The wells were prepared in duplicate for each concentration. An inoculum of 10μl of spore suspension containing 2000-3000 spores was added to each well. Plates were incubated at 28° for 24 h and then examined for spore germination under an inverted microscope. For analysis, spores were considered germinated if they had a germ tube at least twice the length of the spore.

Disc diffusion method

Disc diffusion method and fluorescence microscopy were used to evaluate the hyphal growth inhibition. Plates with Sabouraud Dextrose Agar were centrally inoculated with T. rubrum and incubated at 28° for 3-5 days. Test discs were made with the extract, punicalagin, and Nystatin, with concentrations close to the MIC. These discs and one control disc (with 10μl of sterile water) were arranged around the colony on the plate, at a distance of 0.5°m, and incubated at 28° for 72 h. The hyphal growth inhibition was evaluated visually and photographed [18].

Fluorescence microscopy

Sub-inhibitory concentrations of the crude extract in 500μl of culture medium were prepared by the double-dilution method, in 24-well flat-bottom micro-culture plates, on which round cover slips were placed. The wells were prepared in duplicate for each concentration. The wells were inoculated with 50μl of spore suspension, containing 10,000-15,000 spores. The plates were incubated at 28° for 24 h. Then, the cover slips carefully removed were and washed in PBS, pH7.2, with light manual shaking. Next, the medium was carefully removed and cover slips with adhered cells were stained with Calcofluor White M2R (Sigma, St. Louis, MO, USA) and mounted on a slide with synthetic resin (Araldite 502). Slides were viewed by means of a Zeiss fluorescent microscope [19].

Cytotoxicity assay