We recently initiated a systematic study [1
] that utilizes a multi-target drug discovery approach to discover new molecules with antifungal activities among drugs already used for other dermatological applications. The objective of this study is to discover novel antifungals with low toxicity, as proven by their long history of dermatological use. In particular, our attention was drawn to resorcinols, which are naturally occurring phenolic compounds that are mainly synthesized by plants; these molecules are widely used in the dermatology and cosmetic fields as skin lighteners and have a very good safety profile.
Resorcinol has a long history of therapeutic use for its keratolytic properties [2
] and has been included, usually with sulphur, in topical preparations for the treatment of acne and seborrhoeic skin conditions, although other treatments are generally preferred [2
Resorcinol is one of the components in the well-known Castellani’s solution, which was developed in 1905 by Aldo Castellani, an Italian doctor [3
]. The traditional Castellani’s solution contains boric acid, phenol, fuchsine, resorcinol, acetone, alcohol and water. Its antifungal activity is attributed to fuchsine, and its antibacterial activity is ascribed to ethanol. However, the role of resorcinol is also intriguing. In fact, in the last few years, increasing data have indicated that the compound has various biological activities, including antimicrobial, antiparasitic and cytotoxic activities [4
], such as antioxidant (anti-inflammatory) and antigenotoxic activities [6
We selected three compounds in this class—(±)phenylethylresorcinol (1
), 4-hexylresorcinol (2
) and 4-butylresorcinol (3
) (Figure 1
)—based on their widespread use in dermatology and investigated their effects against nine dermatophytes. To the best of our knowledge, these compounds are not currently used as anti-dermatophytes, and rather they are currently employed as skin-lightening agents because they inhibit tyrosinase [7
The results of a preliminary screening were encouraging: plate tests and germination tests showed good activity, with high inhibition rates against many fungi. These favourable data emphasized the need for a deeper investigation of the antifungal activities of these substances, including determination of their IC50 values, which reflect the effectiveness of a compound. These measurements were made for the two compounds that gave the best preliminary results.
Subsequently, the mechanism of action of the most active molecule, phenylethylresorcinol, against Microsporum gypseum (Bodin) Guiart & Grigorakis, was investigated by electron microscopy. This fungus was chosen based on its IC50 values. The IC50 values of the selected resorcinols against M. gypseum were the lowest among all the fungi tested, indicating that this organism is highly sensitive to the treatment.
is a geophilic fungus. It is a component of the complex soil mycoflora and is frequently found in soils rich in organic matter. It is distributed worldwide and infects humans, particularly children and rural workers during warm humid weather, producing circinate herpes and tinea capitis [9
Lesions caused by M. gypseum
frequently induce strong inflammatory reactions that mimic dermatitis. This dermatitis is often treated with steroids to reduce inflammation, resulting in an atypical appearance as tinea. It must also be noted that the risks associated with modern lifestyle activities, e.g., cosmetic tattooing and pet keeping, have increased interest in the discovery of novel multi-target drugs against M. gypseum
The available literature on M. gypseum is fairly sparse. In the current study, we investigated the fungus using scanning electron microscopy and transmission electron microscopy (SEM and TEM, respectively).
3. Experimental Section
The following seven fungal strains investigated in this study were purchased from the Centraal Bureau voor Schimmelcultures (CBS; Baarn, The Netherlands): Arthroderma cajetani Ajello, strain CBS 495.70; Epidermophyton floccosum (Hartz) Langeron and Milochevitch, strain CBS 358.93; Trichophyton violaceum Malmsten, strain CBS 459.61; Trichophyton tonsurans Malmsten, strain CBS 483.76; Trichophyton mentagrophytes (Robin) Blanchard, strain CBS 160.66; Microsporum canis Bodin, strain CBS 4727; and Nannizzia gypsea (Nann.) Weitzman, McGinnis, A.A. Padhye and Ajello, strain CBS 286.63. The remaining two strains were purchased from the Institute of Hygiene and Epidemiology-Mycology Laboratory (IHME; Brussels, Belgium): Trichophyton rubrum (Castellani) Sabouraud, strain IHME 4321 and Microsporum gypseum (Bodin) Guiart & Grigorakis, strain IHME 3999. All dermatophytes were maintained at 4 °C as agar slants on Sabouraud dextrose agar (SDA; Difco Laboratories, Inc., Detroit, MI, USA).
The tested substances—4-butylresorcinol (4-butyl-1,3-benzenediol, Acteosome), 4-hexyl-resorcinol (4-hexyl-1,3-dihydroxybenzene, Synovea) and racemic (±)phenylethylresorcinol (4-(1-phenylethyl)-1,3-benzenediol, Symwhite 377)—were purchased from Sigma-Aldrich SRL (Milano, Italy) and Symrise GmbH & Co. KG (Holzminden, Germany). Resazurin, used for fixation, and solvents were also purchased from Sigma-Aldrich SRL.
3.3. Growth Inhibition
Antifungal activity was determined as follows. Each test substance was dissolved in dimethyl sulfoxide (DMSO), and a suitable dilution was aseptically mixed with sterile SDA medium at 45 °C to obtain final concentrations of 5, 10, 20, 50, 100 and 200 µg/mL. The DMSO concentration in the final solution was adjusted to 0.1%. Controls were also prepared with equivalent concentrations (0.1% v/v) of DMSO. For the experiments, cultures were obtained by transplanting mycelium disks (10 mm in diameter) from a single mother culture in the stationary phase. They were incubated at 26 ± 1 °C on SDA on thin sheets of cellophane until the logarithmic growth phase. Subsequently, the cultures were transferred to Petri dishes with media containing 5, 10, 20, 50, 100 and 200 µg/mL of the three substances and incubated under growth conditions. The fungal growth was evaluated daily by measuring the colony diameters (in millimetres) for seven days beginning at the onset of treatment.
The percentage inhibition of growth was determined as the average of three different experiments. IC50 values were obtained for only the two substances that were active against the nine dermatophytes—phenylethylresorcinol and 4-hexylresorcinol—at concentrations of 5, 10, 20, 50, 100 and 200 µg/mL. The IC50 values were calculated as the average of three different experiments, and they indicate the concentration of the substance needed to inhibit the growth of the fungus by half.
3.4. Spore Germination Assay
3.4.1. Evaluation of Spore Germination Inhibition
The efficacies of the three resorcinol derivatives tested were evaluated by resazurin assays using an optimized incubation time and spore density, as described by Romagnoli et al. [1
]. A stock solution of each substance was prepared in DMSO. Then, phenylethylresorcinol, 4-hexylresorcinol and 4-butylresorcinol were added to test vials in duplicate at concentrations of 100 and 200 μg/mL. The test vials contained the substances to be tested, 105
spore/mL (determined via previous evaluation), and 100 μL of resazurin stock solution in Sabouraud dextrose broth. The vials were covered, gently rotated horizontally to mix the contents and incubated in the dark at 24 °C for 120 h. Duplicate negative control vials containing 10 mL of medium and 100 μL of resazurin stock solution and duplicate positive control vials containing 10 mL of medium, 105
spore/mL and 100 μL of resazurin stock solution were also processed. Fluorometric measurements and visual inspections were also conducted to evaluate spore germination. Fluorescence data were expressed as the percentage of resazurin reduced as a function of incubation time. After 24 h, the percentage of resazurin reduction was determined fluorometrically by measuring the fluorescence at 578 nm. The absorbance was read with a DU®
530 Life Science UV/Vis spectrophotometer (Beckman CoulterTM
, Brea, CA, USA) using a single-cell module.
The colours of the wells were also visually recorded (Figure 2
). Blue was interpreted to indicate the absence of metabolic activity (no spore germination), whereas fluorescent pink was interpreted to indicate the presence of metabolic activity (spore germination). Purple was interpreted as a trailing result, reflecting the presence of some metabolic activity; however, prolonging the incubation time caused the purple colour to change to pink.
3.4.2. TEM and SEM
The youngest hyphae of M. gypseum were chosen from untreated mycelia and from mycelia treated for 24 h with 20, 100 and 200 µg/mL phenylethylresorcinol for TEM and SEM analyses. Samples were fixed with 6% glutaraldehyde (GA) in 0.1 M sodium cacodylate buffer, pH 6.8, for 6 h at 4 °C. After rinsing in the same buffer solution, the samples for TEM were post-fixed for 15 h with 1% osmium tetroxide (OsO4) in the same buffer, dehydrated in a graded series of alcohol and embedded in Epon-Araldite resin. Sections were cut with an Ultratome III (LKB Instruments, Mount Waverley, Australia) stained with uranyl acetate and lead citrate and observed with an H-800 electron microscope at 100 kV (Hitachi, Altavilla Vicentina (VI), Italy),provided by the Electron Microscopy Center of Ferrara University). For SEM, samples were fixed with 6% GA in 0.1 M sodium cacodylate buffer, pH 6.8, for 6 h at 4 °C, briefly (1 h) post-fixed with 1% OsO4 in the same buffer, dehydrated in a graded series of alcohol, critical point-dried and gold-coated using an S 150 sputter coater (Edwards SpA, Cinisello Balsamo, Italy). SEM observations were collected using an EVO 40 instrument (Zeiss, Oberkochen, Germany) provided by the Electron Microscopy Center of Ferrara University).