Antidermatophytic Action of Resorcinol Derivatives: Ultrastructural Evidence of the Activity of Phenylethyl Resorcinol against Microsporum gypseum

Abstract

In this work, we evaluated the antidermatophytic activities of three resorcinol derivatives that have a history of use in dermo-cosmetic applications to discover molecules with multiple dermatological activities (i.e., multi-target drugs), thereby reducing the cost and time necessary for new drug development. The antidermatophytic activities of the three skin lighteners were evaluated relative to the known antifungal drug fluconazole on nine dermatophytes responsible for the most common dermatomycoses: Microsporum gypseum, Microsporum canis, Trichophyton violaceum, Arthroderma cajetani, Trichophyton mentagrophytes, Epidermophyton floccosum, Nannizzia gypsea, Trichophyton rubrum and Trichophyton tonsurans. Among the three tested resorcinols, only two showed promising properties, with the ability to inhibit the growth of all tested dermatophytes; additionally, the IC₅₀ values of these two resorcinols against the nine dermatophytes confirmed their good antifungal activity, particularly for phenylethyl resorcinol against M. gypseum. Ultrastructural alterations exhibited by the fungus were observed using scanning electron microscopy and transmission electron microscopy and reflected a dose-dependent response to treatment with the activation of defence and self-preservation strategies.

Keywords: Microsporum gypseum; SEM; TEM; antifungal activity; dermatophytes; resorcinol derivatives.

Figure 1

Chemical structures of (±)-phenylethylresorcinol (1), 4-hexylresorcinol (2) and 4-butyl-resorcinol (3).

Figure 2

Colours of the cuvettes after the resazurin assay. Legend: 1—negative control; 2—positive control; 3—phenylethylresorcinol (200 μg/mL); 4—phenylethylresorcinol (100 μg/mL); 5—4-hexylresorcinol (200 μg/mL); 6—4-hexylresorcinol (200 μg/mL); 7—4-butyl-resorcinol (200 μg/mL); 8—4-butylresorcinol (100 μg/mL).

Figure 3

(A) Control M. gypseum: detail of straight apex with smooth wall. SEM. Scale bar: 1 µm; (B) Same sample: mycelium without visible microconidia. SEM. Scale bar: 20 μm; (C) M. gypseum treated with 20 μg/mL phenylethylresorcinol: microconidia are visible. SEM. Scale bar: 10 μm; (D) M. gypseum treated with 100 μg/mL phenylethylresorcinol: numerous macroconidia in the mycelium. SEM. Scale bar: 20 μm; (E) Same sample: typical cigar-shaped macroconidium with knobs on the surface. SEM. Scale bar: 2 μm.

Figure 4

M. gypseum treated with 200 μg/mL phenylethylresorcinol. (A) Compact mycelium with hyphae fused together and some stretch marks. SEM. Scale bar: 10 μm; (B) Strange formations in the compact mycelium with the appearance of burst balloons. SEM. Scale bar: 2 μm; (C) Cave formations, probably derived from hyphae fusion, with spongy material visible inside. SEM. Scale bar: 2 μm; (D) High magnification of one of the exploded structures, showing the typical roughness of a macroconidia on the external surface. SEM. Scale bar: 1 μm.

Figure 5

(A) M. gypseum control with normal mitochondria, nuclei and ribosomes. Note the glycogen rosettes near the plasmalemma. TEM. Scale bar: 1 μm; (B) M. gypseum treated with 20 μg/mL phenylethylresorcinol. Glycogen, which is no longer arranged in rosettes, is scattered throughout the cytoplasm, and there is an increased number of large vacuoles. TEM. Scale bar: 0.5 μm.

Figure 6

(A) M. gypseum treated with 100 μg/mL phenylethylresorcinol. Numerous small vacuoles containing non-electron-transparent material. Note the accumulation of glycogen near the vacuoles. TEM. Scale bar: 1 μm; (B) Same sample. Non-electron-transparent body in the cytoplasm surrounded by two vacuoles. TEM. Scale bar: 1 μm; (C) High magnification of the cell wall of M. gypseum treated with 200 μg/mL phenylethylresorcinol. The large increase in the thickness of the cell wall, which contains several layers, is evident. TEM. Scale bar: 1 μm; (D) Sample treated with the same. Numerous small vacuoles containing dark, irregularly shaped glycogen bodies. TEM. Scale bar: 1 μm.

Figure 7

Cell death after treatment with 200 μg/mL phenylethylresorcinol; several cells are transparent and empty. TEM. Scale bar: 1 μm.