Antidermatophytic Action of Resorcinol Derivatives: Ultrastructural Evidence of the Activity of Phenylethyl Resorcinol Against Microsporum Gypseum

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

Carlo Romagnoli 1, Anna Baldisserotto 2, Chiara B. Vicentini 2, Donatella Mares 2, Elisa Andreotti 1, Silvia Vertuani 2 and Stefano Manfredini 2,*

1 Department of Life Sciences, University of Modena and Reggio Emilia, Viale Caduti in Guerra 127, 41121 Modena, Italy; [email protected] (C.R.); [email protected] (E.A.) 2 Department of Life Sciences and Biotechnology, Master Course in Cosmetic Science and Technology, University of Ferrara, Via Fossato di Mortara 17-19, 44121 Ferrara, Italy; [email protected] (A.B.); [email protected] (C.B.V.); [email protected] (D.M.); [email protected] (S.V.) * Correspondence: [email protected]; Tel.: +39-0532-455294; Fax: +39-0532-455378

Academic Editor: Diego Muñoz-Torrero Received: 14 July 2016; Accepted: 27 September 2016; Published: 30 September 2016

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

1. Introduction 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].

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Resorcinol isis one one of theof the components components in the in well-known the well-known Castellani’s Castellani’s solution, solution, which was which developed was indeveloped 1905 by Aldoin 1905 Castellani, by Aldo an Castellani, Italian doctor an Italian [3]. The doctor traditional [3]. The Castellani’s traditional solution Castellani’s contains solution boric acid,contains phenol, boric fuchsine,acid, phenol, resorcinol, fuchsine, acetone, resorcinol, alcohol acetone, and water.alcohol Itsand antifungal water. Its activityantifungal is attributedactivity is toattributed fuchsine, to and fuchsine, its antibacterial and its antibacterial activity is ascribedactivity tois ethanol.ascribed However,to ethanol. the However, role of resorcinol the role of is alsoresorcinol intriguing. is also In intriguing. fact, in the In last fact, few in years, the last increasing few years, data increasing have indicated data have that theindicated compound that hasthe variouscompound biological has various activities, biological including activities, antimicrobial, including antiparasitic antimicrobial, and cytotoxic antiparasitic activities and [4, 5],cytotoxic such as antioxidantactivities [4,5], (anti-inﬂammatory) such as antioxidant and (anti-in antigenotoxicflammatory) activities and antigenotoxic [6]. activities [6]. We selectedselected threethree compoundscompounds inin thisthis class—(class—(±)phenylethylresorcinol±)phenylethylresorcinol ( (11),), 4-hexylresorcinol ( (2)) and 4-butylresorcinol (3) (Figure1 1)—based)—based onon theirtheir widespreadwidespread useuse inin dermatologydermatology andand investigatedinvestigated their effects against nine dermatophytes.dermatophytes. To To the best of our knowledge, these compounds are not currently used as anti-dermatophytes, and rather they are currently employed as skin-lighteningskin-lightening agents because they inhibit tyrosinase [[7,8].7,8].

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

The results of a preliminary screening were encouraging: plate tests and germination tests showedThe good results activity, of a preliminary with high inhibition screening rates were against encouraging: many fungi. plate testsThese and favourable germination data tests emphasized showed goodthe need activity, for a deeper with high investigation inhibition of ratesthe antifungal against many activities fungi. of these These substances, favourable including data emphasized determination 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 offor their the two IC50 compoundsvalues, which that reflect gave the effectivenessbest preliminary of a results. compound. These measurements were made for theSubsequently, two compounds the thatmechanism gave the of best action preliminary of the most results. active molecule, phenylethylresorcinol, againstSubsequently, Microsporum the gypseum mechanism (Bodin) of Guiart action & ofGrigorakis, the most was active investigated molecule, by phenylethylresorcinol, electron microscopy. 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 ThisM. gypseum fungus waswere chosen the lowest based among on itsIC all50 thevalues. fungi The tested, IC50 valuesindicating of the that selected this organism resorcinols is againsthighly M.sensitive gypseum to werethe treatment. the lowest among all the fungi tested, indicating that this organism is highly sensitive to theM. treatment. gypseum is a geophilic fungus. It is a component of the complex soil mycoflora and is frequentlyM. gypseum foundis in a geophilic soils rich fungus. in organic It is amatter. component It is ofdistributed the complex worldwide soil mycoflora and andinfects is frequently humans, foundparticularly in soils children rich in and organic rural matter.workers Itduring is distributed warm humid worldwide weather, and producing infects humans, circinate particularlyherpes and childrentinea capitis and rural[9,10]. workers during warm humid weather, producing circinate herpes and tinea capitis [9,10]. Lesions caused by M. gypseum frequently induce strong inflammatoryinflammatory reactions that mimic dermatitis. This This dermatitis dermatitis is is often often treated treated with with steroids to reduce inflammation,inflammation, resulting in an atypical appearance as tinea. It 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 [[11].11]. The available literature literature on on M.M. gypseum gypseum isis fairly fairly sparse. sparse. In Inthe the current current study, study, we weinvestigated investigated the thefungus fungus using using scanning scanning electron electron microscopy microscopy and tr andansmission transmission electron electron microscopy microscopy (SEM (SEMand TEM, and TEM,respectively). respectively). 2. Results 2. Results 2.1. Antifungal Activity Table1 1 shows shows the the growth growth inhibition inhibition induced induced by theby the three three tested tested resorcinol resorcinol derivatives derivatives against against nine dermatophytes.nine dermatophytes. Notably, Notably, 4-butylresorcinol 4-butylresorcino showedl onlyshowed low inhibitiononly low against inhibition three dermatophytesagainst three (dermatophytesM. gypseum, T. mentagrophytes(M. gypseum, T.and mentagrophytesE. floccosum ),and even E. atfloccosum the two), highesteven at doses.the two highest doses.

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Table 1. Percentage of growth inhibition induced by the three tested resorcinol derivatives against nine dermatophytes after seven days of treatment at 5, 10, 20, 50, 100 and 200 µg/mL. Each value is the mean of three measurements (all standard deviation values, not reported, were within 10%).

Compound 5 µg/mL 10 µg/mL 20 µg/mL 50 µg/mL 100 µg/mL 200 µg/mL M. gypseum 4-Hexylresorcinol 5.5 21.12 71.5 88.32 93.46 94.57 Phenylethylresorcinol 20.99 34.16 54.73 75.31 98.51 99.50 4-Butylresorcinol 25.00 33.33 M. canis 4-Hexylresorcinol 17.5 32.5 82.5 85 92.68 97.5 Phenylethylresorcinol + + 10 47.5 94.74 95 4-Butylresorcinol + + T. violaceum 4-Hexylresorcinol 12.46 21.45 37.02 59.21 63.16 66.09 Phenylethylresorcinol 0.97 3.38 5.80 31.40 68.92 100 4-Butylresorcinol + + A. cajetani 4-Hexylresorcinol 18.11 43.31 69.29 74.02 78.13 82.29 Phenylethylresorcinol + 13.77 39.86 67.39 84.56 100 4-Butylresorcinol + + T. mentagrophytes 4-Hexylresorcinol 31.48 43.52 81.08 82.41 87.04 88.89 Phenylethylresorcinol 5.88 10.92 36.13 93.28 99.16 100 4-Butylresorcinol 16.67 45 E. ﬂoccosum 4-Hexylresorcinol 5.73 20.31 50.00 69.27 78.13 86.40 Phenylethylresorcinol 22.42 24.66 36.32 47.09 66.28 85.82 4-Butylresorcinol 12.61 19.82 N. gypsea 4-Hexylresorcinol 13.61 19.73 53.74 76.19 83.67 85.47 Phenylethylresorcinol 11.56 16.76 31.21 50.87 82.66 97.78 4-Butylresorcinol + 21.43 T. rubrum 4-Hexylresorcinol 28.95 38.16 59.65 73.68 78.95 81.90 Phenylethylresorcinol 18.41 26.37 39.30 65.17 70.12 81.33 4-Butylresorcinol + 5.86 T. tonsurans 4-Hexylresorcinol 4.6875 18.75 75 85.94 92.19 93.10 Phenylethylresorcinol 12.5 14.0625 37.5 89.0625 100 100 4-Butylresorcinol + + + the substance showed an hormone-like effect with a fungal growth higher than that of the controls.

Both 4-hexylresorcinol and phenylethylresorcinol generally showed very good antifungal activity against the nine dermatophytes at all concentrations. Both substances exhibited nearly 50% inhibition of almost all fungi, even at a concentration of 50 µg/mL; however, against M. gypseum, both exceeded 50% inhibition at a concentration of 20 µg/mL. Only phenylethylresorcinol achieved 100% inhibition at the highest concentration against three dermatophytes (T. violaceum, A. cajetani and T. mentagrophytes), whereas against T. tonsurans, it caused 100% inhibition at concentrations as low as 100 µg/mL. Based on these results, we decided to more thoroughly investigate phenylethylresorcinol. This compound may be of potential use in antifungal therapy because of its increased activity against the nine dermatophytes tested, shown in Table2, in comparison with that of the well-known antifungal agent ﬂuconazole. The results clearly show that ﬂuconazole was less active than phenylethyl-resorcinol. Molecules 2016, 21, 1306 4 of 12

Table 2. Percent growth inhibition induced by phenylethylresorcinol on nine dermatophytes after seven days of treatment at 5, 10, 20, 50, 100 and 200 µg/mL. The percent growth inhibition induced by ﬂuconazole is shown for comparison purposes. Each value is the mean of three measurements (all standard deviation values, which are not reported, were within 10%).

Compound 5 µg/mL 10 µg/mL 20 µg/mL 50 µg/mL 100 µg/mL 200 µg/mL M. gypseum Fluconazole 12.34 34.54 55.12 76.01 80.23 85.40 Phenylethyl resorcinol 19.65 35.48 58.12 74.07 97.98 99.02 M. canis Fluconazole 17.12 26.48 39.95 61.87 80.43 88.51 Phenylethyl resorcinol + + 11.32 46.80 93.54 96.11 T. violaceum Fluconazole 36.76 67.65 83.78 81.33 97.08 94.65 Phenylethylresorcinol 1.13 5.43 6.02 27.58 69.05 100 A. cajetani Fluconazole 0.0 0.0 0.0 0.0 0.0 8.67 Phenylethylresorcinol + 13.77 39.86 67.39 84.56 100 T. mentagrophytes Fluconazole 62.46 79.67 100 100 100 100 Phenylethylresorcinol 7.31 11.96 35.56 94.00 98.00 100 E. ﬂoccosum Fluconazole 98.00 100 100 100 100 100 Phenylethylresorcinol 21.79 25.11 39.87 46.87 68.53 87.67 N. gypsea Fluconazole 0.0 0.0 0.0 0.0 1.09 5.78 Phenylethylresorcinol 13.43 18.99 35.11 50.95 83.74 98.89 T. rubrum Fluconazole 13.13 25.66 40.44 58.76 68.68 76.06 Phenylethylresorcinol 17.15 28.55 39.92 64.69 72.34 84.48 T. tonsurans Fluconazole 27.77 48.56 44.12 72.33 71.33 79.90 Phenylethylresorcinol 13.17 16.44 35.57 89.74 100 100 + the substance showed an hormone-like effect with a fungal growth higher than that of the controls.

In addition, the two resorcinol derivatives identiﬁed as the most active in the previous screening were also evaluated for their potency, as reported in Table3.

Table 3. IC50 values of 4-hexylresorcinol and phenylethylresorcinol against nine dermatophytes. Each value is the mean of three measurements (all standard deviation values, which are not reported, were within 10%).

4-Hexylresorcinol Phenylethylresorcinol Dermatophyte IC50 µg/mL M. gypseum 18.77 11.42 M. canis 14.02 43.47 T. violaceum 48.39 54.60 A. cajetani 16.22 30.87 T. mentagrophytes 12.97 24.68 E. ﬂoccosum 29.42 37.40 N. gypsea 24.72 33.64 T. rubrum 16.22 31.42 T. tonsurans 20.09 23.24 Molecules 2016, 21, 1306 5 of 12

The IC50 values were low for almost all tested fungi. T. violaceum was the least inhibited with phenylethyl resorcinol, whereas the most inhibited fungi was M. gypseum (11.42 µg/mL).

2.2. Inhibition of Spore Germination by Resazurin Assay As M. gypseum was the most sensitive fungus to the three substances, it was tested for spore germination inhibition. As shown in Table4, the results of the test conﬁrmed the excellent inhibitory capacities of phenylethylresorcinol and 4-hexylresorcinol, as evidenced by the blue staining of the cuvettes, which indicated the absence of viable spores (Figure2).

Molecules 2016, 21, 1306 5 of 12 Table 4. Resazurin assay. Percentage inhibition of spore germination after treatment with three resorcinolscapacities at doses of phenylethylr of 100 andesorcinol 200 µg/mL. and 4-hexylresorcinol, as evidenced by the blue staining of the cuvettes, which indicated the absence of viable spores (Figure 2).

TableCompound 4. Resazurin assay. Percentage Concentration inhibition of spore % germination Inhibition after of Spore treatment Germination with three resorcinols at doses of 100 and 200100 μg/mL.µg/mL 100 4-Hexylresorcinol Compound 200Concentrationµg/mL % Inhibition of Spore Germination 100 100 100µg/mL μg/mL 100 100 Phenylethylresorcinol4-Hexylresorcinol 200 200µg/mL μg/mL 100 100 100 μg/mL 100 Phenylethylresorcinol 100 200µg/mL μg/mL 100 13.5 ± 0.8 4-Butylresorcinol 100 μg/mL 13.5 ± 0.8 4-Butylresorcinol 200 µg/mL 80.2 ± 1.1 200 μg/mL 80.2 ± 1.1

Figure 2. Colours of the cuvettes after the resazurin assay. Legend: 1—negative control; 2—positive Figure 2. control;Colours 3—phenylethylresorcinol of the cuvettes (200 after μg/mL); the resazurin 4—phenylethylresorcinol assay. Legend: (100 μg/mL);1—negative 5—4- control; 2—positivehexylresorcinol control; 3—phenylethylresorcinol (200 μg/mL); 6—4-hexylresorcinol (200 (200µg/mL); μg/mL);4 —phenylethylresorcinol7—4-butyl-resorcinol (200 μg/mL); (100 µg/mL); 5—4-hexylresorcinol8—4-butylresorcinol (200 (100µg/mL); μg/mL). 6—4-hexylresorcinol (200 µg/mL); 7—4-butyl-resorcinol (200 µg/mL); 8—4-butylresorcinol (100 µg/mL). In contrast, 4-butylresorcinol inhibited spores germination at different stages, as indicated by the pinkish colour of the test cuvettes. Given these results, we chose the most active compound In contrast,(phenylethylresorcinol) 4-butylresorcinol and the inhibited most sensitive spores fungus germination (M. gypseum at different) for electron stages, microscopic as indicated by the pinkishevaluations. colour of the test cuvettes. Given these results, we chose the most active compound (phenylethylresorcinol)2.3. SEM Observations and the most sensitive fungus (M. gypseum) for electron microscopic evaluations.

2.3. SEM ObservationsThe control mycelium of M. gypseum, (Figure 3A) showed a typical morphology, exhibiting lengthened hyphae of constant diameter with a rounded or lightly tapering apex and smooth external The controlsurface. No mycelium microconidia of orM. macroconidia gypseum, (Figurewere visible3A) (Figure showed 3B) in a the typical mycelium, morphology, as is expected exhibiting for a young culture of 24 h. lengthened hyphaeIn samples of constant treated with diameter 20 μg/mL with phenylethylresorcinol, a rounded or lightly the appearance tapering of apexthe mycelium and smooth was external surface. Nosimilar microconidia to that of the or controls, macroconidia except for were the numerous visible (Figuremicroconidia3B) in(Figure the mycelium,3C). In contrast, as isat 100 expected for a young cultureμg/mL, ofthe 24 microconidia h. were no longer visible, although the mycelium contained abundant In samplesmacroconidia treated (Figure with 3D) 20showingµg/mL the typical phenylethylresorcinol, “cigar shape” with characteristic the appearance knobs on the of theoutermycelium surface (Figure 3E). was similar toThese that alterations of the controls, on M. gypseum except treated for with the phenylethylresorci numerous microconidianol are very interesting (Figure3 becauseC). In contrast, at 100 µg/mL,they potentially the microconidia reflect the development were no longer of defence visible, strategies. although As reported the mycelium in the literature contained on other abundant macroconidiadermatophytes (Figure3 [12],D) showing an increase the in typicalthe number “cigar of reproductive shape” with structures characteristic represents knobsa defensive on the outer surface (Figureresponse3E). by the microorganism. The production of microconidia or macroconidia is a form of asexual reproduction that occurs in an attempt to overcome nutrient starvation [13].

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Figure 3. (A) Control M. gypseum: detail of straight apex with smooth wall. SEM. Scale bar: 1 μm; (B) Figure 3. (A) Control M. gypseum: detail of straight apex with smooth wall. SEM. Scale bar: 1 µm; Same sample: mycelium without visible microconidia. SEM. Scale bar: 20 μm; (C) M. gypseum treated (B) Same sample: mycelium without visible microconidia. SEM. Scale bar: 20 µm; (C) M. gypseum with 20 μg/mL phenylethylresorcinol: microconidia are visible. SEM. Scale bar: 10 μm; (D) M. gypseum treated with 20 µg/mL phenylethylresorcinol: microconidia are visible. SEM. Scale bar: 10 µm; treated with 100 μg/mL phenylethylresorcinol: numerous macroconidia in the mycelium. SEM. Scale (D) M. gypseum treated with 100 µg/mL phenylethylresorcinol: numerous macroconidia in the bar: 20 μm; (E) Same sample: typical cigar-shaped macroconidium with knobs on the surface. SEM. mycelium. SEM. Scale bar: 20 µm; (E) Same sample: typical cigar-shaped macroconidium with knobsScale bar: on the2 μm. surface. SEM. Scale bar: 2 µm.

At the lower doses tested, the substances only exhibited fungistatic activities, concordant with M. gypseum the literatureThese alterations on the onantifungal activitiestreated of with resorcinol phenylethylresorcinol derivatives agai arenst very phytopathogens interesting because [6]. theyConversely, potentially at the reﬂect highest the development concentration of defencetested (200 strategies. μg/mL), As the reported mycelium in the presented literature onnotable other dermatophytesmorphological abnormalities. [12], an increase This in concentration the number ofprobably reproductive overwhelms structures the defensive represents capabilities a defensive of responsethe fungus, by inducing the microorganism. the rupture The or productionbursting of of its microconidia defensive structures. or macroconidia At low ismagnification, a form of asexual the reproductionmycelium appears that occurs highly in compact an attempt (Figure to overcome 4A), with nutrienthyphae fused starvation together [13]. to form a singular woven structureAt the that lower is occasionally doses tested, interrupted the substances by onlyareas exhibited with the fungistaticappearance activities, of "stretch concordant marks". withNeither the literaturemicro- nor on macroconidia the antifungal are activities visible, ofbut resorcinol in addition derivatives to crushed against and very phytopathogens suppressed hyphae, [6]. Conversely, strange µ atformations the highest with concentration the appearance tested of burs (200t balloonsg/mL), can the be mycelium observed presented(Figure 4B). notable morphological abnormalities.At the highest This resolution concentration (Figure probably 4C), in overwhelms the layers underlying the defensive the cave capabilities formations, of the a fungus,type of inducingtrabecula thecan rupturebe observed or bursting that seems of its to defensive be derived structures. from the At fusion low magniﬁcation, of hyphae. The the presence mycelium of appearsmaterial highlywith a compact“spongy” (Figure consiste4A),ncy with in some hyphae of these fused abnormal together tobo formdies exhibiting a singular wovenseveral structureopenings thatis noteworthy. is occasionally interrupted by areas with the appearance of “stretch marks”. Neither micro- nor macroconidiaIn Figure are4D visible,one of butthese in bodies addition has to on crushed its ou andter surface very suppressed the typical hyphae, roughness strange or blebbing formations of withmacroconidia. the appearance The highest of burst concentrat balloonsion can of be phenylethylresorcinol observed (Figure4B). (200 μg/mL) may have exerted a fungicidalAt the action highest by resolutionblocking fungal (Figure defence4C), in strategies. the layers underlying the cave formations, a type of trabecula can be observed that seems to be derived from the fusion of hyphae. The presence of material with a “spongy” consistency in some of these abnormal bodies exhibiting several openings is noteworthy. In Figure4D one of these bodies has on its outer surface the typical roughness or blebbing of macroconidia. The highest concentration of phenylethylresorcinol (200 µg/mL) may have exerted a fungicidal action by blocking fungal defence strategies.

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Figure 4. M. gypseum treated with 200 μg/mL phenylethylresorcinol. (A) Compact mycelium with 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 hyphae fused together and some stretch marks. SEM. Scale bar: 10 µm; (B) Strange formations Figurecompact 4. mycelium M. gypseum with treated the appearance with 200 μofg/mL burst phenylethylresorcinol. balloons. SEM. Scale bar: (A )2 Compactμm; (C) Cave mycelium formations, with in the compact mycelium with the appearance of burst balloons. SEM. Scale bar: 2 µm; (C) Cave hyphaeprobably fused derived together from andhyphae some fusion, stretch with marks. spongy SEM. material Scale bar: visible 10 μ inside.m; (B) SEM.Strange Scale formations bar: 2 μm; in (theD) formations, probably derived from hyphae fusion, with spongy material visible inside. SEM. Scale bar: compactHigh magnification mycelium with of theone appearance of the exploded of burst balloons.structures, SEM. showing Scale bar: the 2 μtypicalm; (C) Caveroughness formations, of a µ D 2 m;probablymacroconidia ( ) High derived magniﬁcation on thefrom external hyphae ofsurface. fusion, one of SEM. with the Scalespongy exploded bar: material 1 structures,μm. visible inside. showing SEM. the Scale typical bar: 2 roughness μm; (D) of a macroconidiaHigh magnification on the external of one surface.of the SEM.exploded Scale structures, bar: 1 µm. showing the typical roughness of a 2.4. TEMmacroconidia Observations on the external surface. SEM. Scale bar: 1 μm. 2.4. TEMFigure Observations 5A shows a typical structure of the M. gypseum control, with glycogen rosettes close to 2.4.the TEMplasmalemma. Observations The cell wall is normally structured with two typical layers visible: the outer, Figure5A shows a typical structure of the M. gypseum control, with glycogen rosettes close to non-electron-transparent, mannoproteic layer and the inner, translucent layer composed primarily of the plasmalemma.Figure 5A shows The cell a typical wall structure is normally of the structured M. gypseum with control, two with typical glycogen layers rosettes visible: close the to outer, theglucan plasmalemma. and chitin (Figure The cell 5A,B). wall is normally structured with two typical layers visible: the outer, non-electron-transparent, mannoproteic layer and the inner, translucent layer composed primarily of non-electron-transparent, mannoproteic layer and the inner, translucent layer composed primarily of glucanglucan and chitinand chitin (Figure (Figure5A,B). 5A,B).

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 Figurephenylethylresorcinol. 5. (A) M. gypseum Glycogen, control with which normal is no mitochondria,longer arranged nuclei in ro andsettes, ribosomes. is scattered Note throughout the glycogen the Figure 5. (A) M. gypseum control with normal mitochondria, nuclei and ribosomes. Note the glycogen rosettescytoplasm, near and the there plasmale is an increasedmma. TEM. number Scale of bar: large 1 vacuoles.μm; (B) TEM.M. gypseum Scale bar: treated 0.5 μ m.with 20 μg/mL µ B M. gypseum µ rosettesphenylethylresorcinol. near the plasmalemma. Glycogen, TEM. which Scaleis no longer bar: 1arrangedm; ( in) rosettes, is scatteredtreated throughout with 20 theg/mL μ phenylethylresorcinol.cytoplasm,In the samples and there treated Glycogen, is an with increased whichthe lowest number is no co longerofncentration large arrangedvacuoles. of TEM.phenylethylresorcinol in rosettes, Scale bar: isscattered 0.5 μm. (20 throughout g/mL), the cytoplasm,most evident and feature there is anthe increased increased number number of of large large vacuoles. vacuoles. TEM. Glycogen, Scale bar:which 0.5 isµ nom. longer visible as rosettes,In the samples is scattered treated throughout with the lowestthe cytoplasm concentration (Figure of phenylethylresorcinol5B), and some normally (20 μ g/mL),structured the mitochondria are visible. Inmost the evident samples feature treated is the withincreased the number lowest of concentration large vacuoles. of Glycogen, phenylethylresorcinol which is no longer (20 visibleµg/mL), as rosettes,At 100 μisg/mL, scattered many throughout smaller vacuoles the cytoplasm are present (Figure and contain5B), and non-electron-dense some normally structuredmaterials. the most evident feature is the increased number of large vacuoles. Glycogen, which is no longer mitochondriaGlycogen is distributed are visible. in the cytoplasm, accumulating near the vacuoles (Figure 6A). The TEM visible as rosettes, is scattered throughout the cytoplasm (Figure5B), and some normally structured imagesAt also100 showμg/mL, ultrastructural many smaller changes vacuoles reflecting are present the defensive and contain efforts non-electron-dense of the fungus. Notably, materials. the mitochondriaGlycogen areis distributed visible. in the cytoplasm, accumulating near the vacuoles (Figure 6A). The TEM Atimages 100 alsoµg/mL, show manyultrastructural smaller changes vacuoles reflecting are present the defensive and contain efforts non-electron-denseof the fungus. Notably, materials. the Glycogen is distributed in the cytoplasm, accumulating near the vacuoles (Figure6A). The TEM images also show ultrastructural changes reflecting the defensive efforts of the fungus. Notably, the number Molecules 2016, 21, 1306 8 of 12 of vacuolesMolecules increased,2016, 21, 1306 resulting from the activation of autophagic phenomena in filamentous8 of 12 fungi. Autophagy is primarily a pro-survival mechanism in which normal cellular components are absorbed number of vacuoles increased, resulting from the activation of autophagic phenomena in filamentous into vacuoles and digested as a major response to nutrient deprivation and stress. However, it can fungi. Autophagy is primarily a pro-survival mechanism in which normal cellular components are also beabsorbed induced into by vacuoles antifungal and compoundsdigested as a major [13,14 response]. Indeed, to thenutrient incorporation deprivation of and glycogen stress. However, into vacuoles, whichit wascan also seen be at induced the highest by antifungal concentration, compounds is a clear[13,14]. autophagic Indeed, the phenomenon incorporation thatof glycogen protects into against injuryvacuoles, [15]. which was seen at the highest concentration, is a clear autophagic phenomenon that Figureprotects6 Bagainst shows injury the [15]. possible formation of non-electron-transparent circular bodies in the cytoplasmFigure that are6B shows surrounded the possible by vacuoles formation and of subsequentlynon-electron-transparent appear to circular be segregated, bodies in as the shown in thecytoplasm previous that figure are (Figure surrounded6C). Theirby vacuoles incorporation and subsequently into the vacuoles appear to may be segregated, have the same as shown autophagic in purpose.the previous After treatment figure (Figure with 6C). phenylethylresorcinol Their incorporation into at the 200 vacuolesµg/mL, may the have most the evident same autophagic feature is the purpose. After treatment with phenylethylresorcinol at 200 μg/mL, the most evident feature is the large increase in the thickness of the cell wall, which appears to be at least twice as thick as that of the large increase in the thickness of the cell wall, which appears to be at least twice as thick as that of the control. This phenomenon can be seen in Figure6C, where several layers are evident. control. This phenomenon can be seen in Figure 6C, where several layers are evident.

Figure 6. (A) M. gypseum treated with 100 μg/mL phenylethylresorcinol. Numerous small vacuoles µ Figurecontaining 6. (A) M. non-electron-transparent gypseum treated with material. 100 g/mL Note phenylethylresorcinol. the accumulation of glycogen Numerous near the small vacuoles. vacuoles containingTEM. Scale non-electron-transparent bar: 1 μm; (B) Same sample. material. Non-electron-transpare Note the accumulationnt body in of the glycogen cytoplasm near surrounded the vacuoles. TEM.by Scale two bar:vacuoles. 1 µm; TEM. (B) SameScale bar: sample. 1 μm; Non-electron-transparent (C) High magnification of the body cell wall in the of cytoplasmM. gypseum surroundedtreated by twowith vacuoles. 200 μg/mL TEM. phenylethylresorcinol. Scale bar: 1 µm; (C Th) Highe large magniﬁcation increase in the of thickness the cell wallof the of cellM. gypseumwall, whichtreated withcontains 200 µg/mL several phenylethylresorcinol. layers, is evident. TEM. Scale The bar: large 1 μ increasem; (D) Sample in the treated thickness with the of same. the cell Numerous wall, which containssmall several vacuoles layers, containing is evident. dark, TEM.irregularly Scale shaped bar: 1 glycogenµm; (D) Samplebodies. TEM. treated Scale with bar: the 1 μ same.m. Numerous small vacuoles containing dark, irregularly shaped glycogen bodies. TEM. Scale bar: 1 µm. At the same time, the cytoplasm is very rich in ribosomes and smooth endoplasmic reticulum, which indicates an increase in protein and lipid metabolism. Some vacuoles have a relatively fine Atelectron the same matrix, time, whereas the cytoplasm others display is very a matrix rich with in ribosomes darker, irregularly and smooth shaped endoplasmic areas that are reticulum, very whichopaque indicates to electrons an increase (Figure in 6D). protein Finally, and Figure lipid 7 metabolism.shows the presence Some of vacuolestransparent have and aempty relatively cells, ﬁne electrona clear matrix, sign of whereas cell death others induced display by the a highest matrix dose with of darker,the substance. irregularly shaped areas that are very opaque toAnother electrons prominent (Figure observation6D). Finally, is Figurerelated7 to shows the cell the wall, presence which is ofof transparentgreat importance and in empty fungus cells, a clearsurvival sign of [11,16]. cell death Cell inducedwall structure by the is highest highly dynamic, dose of the changing substance. constantly during cell division, Anothergrowth and prominent morphogenesis. observation It is generally is related accepted to the cell that wall, the whichpatternis of of cell great wall importance deposition inis fungusof primary importance in shaping the fungal cell and that interference or damage during the assembly survival [11,16]. Cell wall structure is highly dynamic, changing constantly during cell division, of this structure negatively impacts the morphogenesis or life of the fungus [16]. Mares et al. [13] growth and morphogenesis. It is generally accepted that the pattern of cell wall deposition is of interpreted this increase in cell wall thickness as premature and abnormal aging of the fungus. primary importanceThe cell wall inhas shaping not been the previously fungal celldescribed and that as a interferencetarget of resorcinols, or damage whereas during mitochondria, the assembly of thisnucleic structure acids, negatively proteins and, impacts in particular, the morphogenesis the endomembrane or life system of the fungusare frequently [16]. Maresreported et al.as [13] interpretedtargets of this these increase molecules in cell [4,17,18]. wall thickness as premature and abnormal aging of the fungus. The cell wall has not been previously described as a target of resorcinols, whereas mitochondria, nucleic acids, proteins and, in particular, the endomembrane system are frequently reported as targets of these molecules [4,17,18].

Molecules 2016, 21, 1306 9 of 12 Molecules 2016, 21, 1306 9 of 12

Figure 7. Cell death after treatment with 200 μg/mL phenylethylresorcinol; several cells are transparent Figure 7. Cell death after treatment with 200 µg/mL phenylethylresorcinol; several cells are transparent and empty. TEM. Scale bar: 1 μm. and empty. TEM. Scale bar: 1 µm.

3. Experimental Section 3. Experimental Section 3.1. Microorganisms 3.1. Microorganisms The following seven fungal strains investigated in this study were purchased from the Centraal BureauThe voor following Schimmelcultures seven fungal (CBS; strains Baarn, investigatedThe Netherlands): in this Arthroderma study were cajetani purchased Ajello, strain from CBS the Centraal495.70; Epidermophyton Bureau voor Schimmelcultures floccosum (Hartz) Langeron (CBS; Baarn, and The Milochevitch, Netherlands): strainArthroderma CBS 358.93; cajetani TrichophytonAjello, strainviolaceum CBS Malmsten, 495.70; Epidermophyton strain CBS floccosum459.61; Trichophyton(Hartz) Langeron tonsurans and Milochevitch,Malmsten, strain strain CBS CBS 483.76; 358.93; TrichophytonTrichophyton violaceum mentagrophytesMalmsten, (Robin) strain Blanchard, CBS 459.61; strainTrichophyton CBS 160.66; tonsurans MicrosporumMalmsten, canis strainBodin, CBS strain 483.76; CBS Trichophyton4727; and Nannizzia mentagrophytes gypsea (Nann.)(Robin) Weitzman, Blanchard, McGinnis, strain A.A. CBS Padhye 160.66; andMicrosporum Ajello, strain canis CBSBodin, 286.63. strain The CBSremaining 4727; two and strainsNannizzia were gypsea purchased(Nann.) from Weitzman, the Institute McGinnis, of Hygiene A.A. and Padhye Epidemiology-Mycology and Ajello, strain CBSLaboratory 286.63. (IHME; The remainingBrussels, Belgium): two strains Trichophyton were purchased rubrum (Castellani) from the Institute Sabouraud, of Hygienestrain IHME and Epidemiology-Mycology4321 and Microsporum gypseum Laboratory (Bodin) (IHME; Guiart Brussels,& Grigorakis, Belgium): strainTrichophyton IHME 3999. rubrumAll dermatophytes(Castellani) Sabouraud,were maintained strain IHMEat 4 °C 4321 as agar and Microsporumslants on Sabouraud gypseum (Bodin) dextrose Guiart agar &(SDA; Grigorakis, Difco Laboratories, strain IHME 3999.Inc., ◦ AllDetroit, dermatophytes MI, USA). were maintained at 4 C as agar slants on Sabouraud dextrose agar (SDA; Difco Laboratories, Inc., Detroit, MI, USA). 3.2. Chemicals 3.2. Chemicals The tested substances—4-butylresorcinol (4-butyl-1,3-benzenediol, Acteosome), 4-hexyl-resorcinol (4-hexyl-1,3-dihydroxybenzene,The tested substances—4-butylresorcinol Synovea) and (4-butyl-1,3-benzenediol, racemic (±)phenylethylresorcinol Acteosome), (4-(1-phenylethyl)- 4-hexyl-resorcinol ± (4-hexyl-1,3-dihydroxybenzene,1,3-benzenediol, Symwhite 377)—were Synovea) purchased and racemic from ( )phenylethylresorcinolSigma-Aldrich SRL (Milano, (4-(1-phenylethyl)- Italy) and 1,3-benzenediol,Symrise GmbH & Symwhite Co. KG 377)—were(Holzminden, purchased Germany). from Resazurin, Sigma-Aldrich used SRLfor fixation, (Milano, and Italy) solvents and Symrise were GmbHalso purchased & Co. KGfrom (Holzminden, Sigma-Aldrich Germany). SRL. Resazurin, used for fixation, and solvents were also purchased from Sigma-Aldrich SRL. 3.3. Growth Inhibition 3.3. Growth Inhibition Antifungal activity was determined as follows. Each test substance was dissolved in dimethyl 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 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 to obtain final concentrations of 5, 10, 20, 50, 100 and 200 µg/mL. The DMSO concentration in the solution was adjusted to 0.1%. Controls were also prepared with equivalent concentrations (0.1% v/v) final solution was adjusted to 0.1%. Controls were also prepared with equivalent concentrations of DMSO. For the experiments, cultures were obtained by transplanting mycelium disks (10 mm in (0.1% v/v) of DMSO. For the experiments, cultures were obtained by transplanting mycelium disks diameter) from a single mother culture in the stationary phase. They were incubated at 26 ± 1 °C on (10 mm in diameter) from a single mother culture in the stationary phase. They were incubated at SDA on thin sheets of cellophane until the logarithmic growth phase. Subsequently, the cultures were 26 ± 1 ◦C on SDA on thin sheets of cellophane until the logarithmic growth phase. Subsequently, transferred to Petri dishes with media containing 5, 10, 20, 50, 100 and 200 μg/mL of the three the cultures were transferred to Petri dishes with media containing 5, 10, 20, 50, 100 and 200 µg/mL of substances and incubated under growth conditions. The fungal growth was evaluated daily by the three substances and incubated under growth conditions. The fungal growth was evaluated daily measuring the colony diameters (in millimetres) for seven days beginning at the onset of treatment. 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.

Molecules 2016, 21, 1306 10 of 12

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 (Figure2). 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).

4. Conclusions In conclusion, select resorcinols already in use in the dermatology field for cosmetic applications were investigated and found to provide valuable activity against dermatophytic fungi. In particular, phenylethylresorcinol, widely used in the racemic form (under the trade name of Symwhite 377) in the dermo-cosmetic field as skin whitening, resulted of particular interest. These findings extend the Molecules 2016, 21, 1306 11 of 12 application of resorcinols in medicine, especially for the treatment of human infections from soil or pets. Although the study of their mechanisms of action cannot be considered conclusive and requires further chemical investigations and evaluations using several different fungi, our results are of interest regarding the development of agents endowed with specific activities towards this class of emerging human pathogens. This study is based on a relatively safe approach that begins with the selection of molecules whose use is already established in the dermo-cosmetic field.

Acknowledgments: We wish to thank Maria Rita Bovolenta and the staff of the Electron Microscopy Center of Ferrara University for their skilful technical assistance. We also thank the Ministry of Education and Research (PRIN, Grant 20082L3NFT_003) and Ambrosialab srl (Ferrara, Italy, Grant 2016) for the financial support provided. Author Contributions: S.M. proposed the subject; S.M. and C.R. designed and supervised the study; A.B., and E.A. carried out production and analysis; C.B.V., D.M. and S.V. contributed to the interpretation of the results; C.R., A.B., S.V. and S.M. wrote the manuscript. All the authors read and approved the final manuscript. Conflicts of Interest: The authors declare no conflict of interest.

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Sample Availability: Samples of the compounds are available from the authors.

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