Introduction

Pelargonium genus as perennial small shrubs compromises more than 250 species [1], P. graveolens (Pelargonium roseum) is highly valued by industries for producing geranium essential oil [2]. According to BS ISO 4371-2012, P. graveolens essential oil from different geographical origins should have geraniol (5–20%), citronellol (18–43%), citronellyl formate (4–12%), geranyl formate (1–8%), and linalool (2–11%) as the main components [3]. The chemical profile of P. graveolens essential oil is affected from its geographical origin. The pharmacological effects of P. graveolens essential oil such as antioxidant [4, 5], antimicrobial [6,7,8,9], anti-inflammatory [10, 11] and wound healings [12] activities is well documented. Furthermore, evaluation the antifungal activities of P. graveolens essential oil have been the subject of different experimental studies [13,14,15,16]. The antifungal activities of P. graveolens essential oil were confirmed against Trichophyton spp. [15], and Candida albicans [13, 14, 16]. Although, there are one report on anti-dermatophyte effects of P. graveolens essential oil against Trichophyton spp. [15], but there is no investigation to compare the anti-dermatophyte effects of P. graveolens essential oils against different kinds of dermatophytes (Microsporum canis, M. gypseum, Trichophyton rubrum, T. mentagrophytes, and T. schoenleinii).

Dermatophytes (Trichophyton, Microsperum, Epidermophyton) are one of major cause of superficial fungal infections with an estimate lifetime risk of 20–25% [17]. The use of topical and systemic drugs (Azoles/allylamines, terbinafine) is used for treatment of dermatophytes that are associated with adverse effects and appearance of resistant microbial strains [18]. Different kinds of essential oils have been evaluated against dermatophytes. Prunus armeniaca, Prunus dulcis var. amara, Olea europaea, Mentha piperita [19], Artemisia sieberi [20], Juniperus communis ssp. alpina, J. oxycedrus ssp. oxycedrus and J. turbinate [21] were effective against dermatophytes.

Therefore, the subject of this study was to evaluate the anti-dermatophyte effects of nine different P. graveolens essential oils from Iran, Germany and India against dermatophytes.

Materials and methods

P. graveolens essential oils

Nine different P. graveolens essential oils with identified chemical profiles as Table 1 were used in this study. Essential oils were prepared by Barijessence Pharmaceutical Company (Kashan, Iran). The chemical compositions of essential oil were determined by Quality Control of BarijEssence Pharmaceutical Company, Kashan, Iran.

Table 1 The chemical composition of P. graveolens essential oils
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Dermatophytes strains

Microsporum canis PTCC (Persian Type Culture Collection) 5069, Microsporum gypseum PTCC 5070, Trichophyton rubrum PTCC 5143, Trichophyton mentagrophytes PTCC 5054, and Trichophyton schoenleinii PTCC 5221 were used in this study. For preparing the dermatophytes, they were cultured on Sabauraud dextrose agar and the cultures were incubated at 22.5 ± 2.5 °C for 14 days. After growth of dermatophytes, suspensions of dermatophytes were prepared in normal saline with 0.05% polysorbate-80. This suspension was used for further antifungal evaluations.

Anti-dermatophyte activity of P. graveolens essential oils by mycelium growth inhibition method

P. graveolens essential oils (200 ppm) were incorporated into Sabauraud dextrose agar. Sabauraud dextrose agar without essential oils was used as controls. Then, the fungal patches were prepared by spreading the dermatophyte suspensions on the surface of Sabauraud dextrose agar and were incubated for 7 days at 25 °C in aerobic condition. The surface of cultured media (6 mm) were punched and put on the surface of Sabauraud dextrose agar with or without P. graveolens essential oils. The plates were incubated at the same conditions for 7 days. The mycelium growth inhibitions were measured for each plate and the growth inhibition percent were calculated from comparison between the growth in Sabauraud dextrose agar with or without essential oils. The experiment were replicated five times and the means of replicates were used in calculations [22].

Anti-dermatophytes evaluations by micro-broth dilution assay

For determining the Minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) of P. graveolens essential oils, each essential oil were dissolved in dimethyl sulfoxide (DMSO) and serially diluted in RPMI broth media (8–0.015 μl/ml) in 96 micro-titer plates. Then, the adjusted dermatophyte suspensions (103–104 CFU/ml) in RPMI by spectrophotometric method was added to the wells of plates containing essential oil’s serial dilutions, and was incubated at 22.5 ± 2.5 °C for 5 days. The lowest concentration of essential oil with visible fungal growth inhibition was defined as Minimal Inhibitory Concentration (MIC) and the first dilution with no growth after culturing on Sabauraud dextrose Agar was Minimal Fungicidal Concentration (MFC).The experiments were replicated three times [22].

Results and discussion

Essential oils are favorable natural compounds that can be used as natural alternative treatment for management of dermatophytosis. Citronellol (0.2–48.3%), geraniol (3.4–95.6%), citronellyl formate (0.3–16.2%), geranyl formate (1.6–10.8%), and linalool (0.7–13.4%) were the main components of 9 samples of P. graveolens essential oils (Table 1). The highest and lowest amounts of geraniol (95.6%) and citronellol (0.2%) were found in P. graveolens essential oil E20 from India. Trans-caryophyllene was the second main component of this essential oil (3.5%), followed by geranyl formate (1.6%), and linalool (0.7%). The highest amounts of citronellol were in P. graveolens essential oil E21 (48.3%), E9 (48.0%), E11 (45.6%), and E6 (41%).

Citronellol was the first main component of 8 samples of P. graveolens essential oils, while geraniol was the second main component of 2 samples of P. graveolens essential oils (E4 and E14). Linalool was the second main component of one sample P. graveolens essential oil. Citronellyl formate was found in 5 samples of P. graveolens essential oil as the second main component.

The chemical profiles and main components of P. graveolens essential oil were changed from one sample to others. The chemical profile of P. graveolens essential oils were not according to standard P. graveolens essential oil (BS ISO 4371-2012) [3].

The anti-dermatophyte effects of P. graveolens essential oils against dermatophytes by mycelium growth inhibition test exhibited that P. graveolens essential oil E20 had the highest mycelium growth inhibition percent against dermatophytes (30.9–53.3%), followed by E4 (17.8–43.6%) and E14 (19.9–43.6%), respectively. T. rubrum had the higher mycelium inhibition percent (22.5–53.3%) by P. graveolens essential oils, followed by M. gypseum (17–45.4%), and T. mentagrophytes (16.5–43.6%). M. canis and T. schoenleinii had the less mycelium inhibition percent by P. graveolens essential oils (Table 2).

Table 2 Mycelium growth inhibition of dermatophytes by 200 ppm P. graveolens essential oils
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In micro-broth dilution assay, the MIC and MFC values for E20 were in the ranges of 0.03–0.125 and 0.06–0.25 μl/ml, respectively. E14 (MIC, MFC values of 0.06–0.125, 0.125–0.25 μl/ml), E4 and E21 (MIC, MFC values of 0.06–0.25, 0.125–0.5 μl/ml) showed anti-dermatophyte activity, followed by E11, E9 (MIC, MFC values of 0.125–0.25, 0.125–0.5 μl/ml), E3 (MIC, MFC values of 0.125–0.25, 0.25–0.5) μl/ml), E6 (MIC, MFC values of 0.125–0.5, 0.25–0.5 μl/ml). E22 (MIC, MFC values of 0.125–0.5, 0.25–1 μl/ml) had the higher MIC and MFC values against dermatophytes than the others (Table 3).

Table 3 The antifungal activity of P. graveolens essential oils against dermatophytes
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As the results of anti-dermatophyte screening exhibited that E20 with high amount of geraniol had the highest activity against dermatophytes, followed by E4, E14. Indeed the P. graveolens essential oils with high amount of citronellol and geraniol as the first and second main components had the higher anti-dermatophyte effects. The essential oil with high amounts of citronellol or citronellyl formate showed anti-dermatophytes effects lower than the essential oils with citronellol and geraniol as the main components.

The results of anti-dermatophyte effects of P. graveolens essential oils have not been in according to their anti-candidal effects. There was no significant difference in anti-candidal activities of six samples of P. graveolens essential oils with citronellol (7.7–43.7%) and geraniol (19.3–48.5%) against clinical isolates of C. albicans in regard to inhibition zone diameters and the MIC and MFC values [23].

The results of this study confirmed that corresponding components responsible for anti-dermatophyte activity of P. graveolens essential oils were geraniol and citronellol. The mechanism of action for geraniol and citronellol is interfering with cell membrane of dermatophytes and decreasing the ergosterol content of cell by inhibition of ergosterol biosynthesis [24, 25], while the anti-candidal activity against C. albicans is not mediated by ergosterol biosynthesis [26].

Conclusions

Therefore, for preparing anti-dermatophyte products containing P. graveolens essential oils, the use of essential oil with main components of geraniol (30–35%) and citronellol (21%) are recommended. The third main component of essential oil should be linalool or citronellyl formate in higher percentage of 5%. More studies are required for comparing the efficacy of P. graveolens essential oils in animal studies.