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Essential oil of Algerian Eucalyptus citriodora: Chemical composition, antifungal activity
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ORIGINAL ARTICLE/ARTICLE ORIGINAL
Essential oil of Algerian Eucalyptus citriodora: Chemical composition, antifungal activity Huile essentielle d’Eucalyptus citriodora : composition chimique ´ antifongique et activite H. Tolba a,*, H. Moghrani a, A. Benelmouffok b, D. Kellou b, R. Maachi a a
Laboratory of reaction engineering, department of process engineering, faculty of mechanical engineering and process engineering, USTHB, PO Box 32 El Alia, Bab Ezzouar, 16111 Algiers, Algeria b Laboratory of Mycology, Pasteur Institute, Algiers, Algeria Received 13 April 2015; received in revised form 11 October 2015; accepted 12 October 2015
KEYWORDS Eucalyptus Citriodora; Essential oil; Dermatophytes; Antifungal activity
Summary Objective. — Essential oil of Eucalyptus citriodora is a natural product which has been attributed for various medicinal uses. In the present investigation, E. citriodora essential oil was used to evaluate its antifungal effect against medically important dermatophytes. Material and methods. — Essential oil from the Algerian E. citriodora leaves was analyzed by GC and GC/MS. The antifungal effect of E. citriodora essential oil was evaluated against four dermatophytes: Microsporum canis, Microsporum gypseum, Trichophyton mentagrophytes, Trichophyton rubrum using disc diffusion method, disc volatilization method, and agar dilution method. Results. — The chemical composition of the oil revealed the presence of 22 compounds accounting for 95.27% of the oil. The dominant compounds were citronellal (69.77%), citronellol (10.63%) and isopulegol (4.66%). The disc diffusion method, MIC and MFC determination, indicated that E. citriodora essential oil had a higher antifungal potential against the tested strains with inhibition zone diameter which varied from (12 to 90 mm) and MIC and MFC values ranged from (0.6 to 5 mL/mL and 1.25 to 5 mL/mL) respectively. The M. gypseum was the most resistant to the oil.
* Corresponding author. E-mail address: [email protected] (H. Tolba). http://dx.doi.org/10.1016/j.mycmed.2015.10.009 1156-5233/# 2015 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: Tolba H, et al. Essential oil of Algerian Eucalyptus citriodora: Chemical composition, antifungal activity. Journal De Mycologie Médicale (2015), http://dx.doi.org/10.1016/j.mycmed.2015.10.009
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H. Tolba et al. Conclusion. — The results of the present study indicated that E. citriodora essential oil may be used as a new antifungal agent recommended by the pharmaceutical industries. # 2015 Elsevier Masson SAS. All rights reserved.
MOTS CLÉS Eucalyptus Citriodora ; Huile essentielle ; Dermatophytes ; Activité antifongique ; Composition chimique
Re ´sume ´ Objectif. — L’huile essentielle d’Eucalyptus citriodora est un produit naturel auquel a été attribué différentes utilisations médicinales. Dans la présente étude, l’huile essentielle de E. citriodora a été utilisée pour évaluer son effet antifongique contre les dermatophytes médicalement importants. Mate´riel et me´thode. — L’huile essentielle des feuilles de l’E. citriodora algérien a été analysée par GC et GC/MS. L’effet antifongique d’huile essentielle de l’E. citriodora a été évalué contre quatre dermatophytes : Microsporum canis, Microsporum gypseum, Trichophyton mentagrophytes, Trichophyton rubrum en utilisant la méthode de diffusion, la méthode de la volatilisation et la méthode de dilution. Re ´sultats. — La composition chimique de l’huile a révélé la présence de 22 composés qui représente 95,27 % de l’huile. Les composés dominants étaient le citronellal (69,77 %), le citronellol (10,63 %) et isopulégol (4,66 %). La méthode de diffusion ainsi que la détermination de la CMI et de la CMF ont indiqué que l’huile essentielle de l’E. citriodora a un potentiel antifongique élevé contre les souches testées avec un diamètre d’inhibition, qui variait de (12 à 90 mm) et des CMI et CMF qui variaient de (0,6 à 5 mL/mL et 1,25 à 5 mL/mL) respectivement. Le M. gypseum était le plus résistant à l’huile. Conclusion. — Les résultats de notre étude indiquent que l’huile essentielle de l’E. citriodora peut être utilisée comme un nouvel agent antifongique recommandé par les industries pharmaceutiques. # 2015 Elsevier Masson SAS. Tous droits réservés.
Introduction Fungal strains are potential public health threats all over the world. They are widespread due to environment factors [28]. Dermatophytes are the major cause of superficial mycoses. They have the ability to invade skin, hair, nail and cause dermatophytosis, and can be contagious fungal disease [22]. The incidence of dermatophytic infections has increased considerably during decades [11]. Dermatophytes are responsible for serious human pathogenic disorders in various parts of the world, routinely concern people who are otherwise healthy and individuals with compromised immune systems are particularly susceptible [15]. Throughout the world, there has been an increasing incidence of fungal infections, and because of drug resistance and toxicity associated with long-term treatment with antifungal drugs, it is an interest to develop new natural therapeutic alternative with acceptable antifungal effect especially among the medicinal plants. Many plants species are known for their antimicrobial properties and their wide uses in traditional medicine [13]. Previous works have focused on the therapeutic properties of the natural product. This investigation is due to their potential ressources of essential oil. Eucalyptus citriodora essential oil is a natural product that exhibits various biological properties. It is one of the most widely used species in modern cosmetics, food and pharmaceutical industries. E. citriodora essential oil is known to possess a wide spectrum of biological activities including toxicity against a wide range of microbes, including bacteria, fungi including yeasts [6,10,12,14,18,19,25], analgesic and anti-inflammatory
effects [9,23], antioxidant activity [24] and antimosquito properties [16]. To the best of our knowledge, the present study might be the first report on the antidermatophytic activity of E. citriodora growing in Algeria. Moreover, no systematic studies comparing the antimicrobial activity in both liquid and vapour phases of the E. citriodora oil are available. In the present study, the essential oil was extracted from the E. citriodora leaves growing in Algeria via steam distillation. We selected E. citriodora essential oil to investigate its chemical composition, its effectiveness in vitro against four dermatophytes using disc diffusion method, disc volatilization method, and agar dilution method.
Material and methods Plant material Fresh E. citriodora leaves were collected from the National Institute of Agronomy (El Harrach located 15 km of Algiers) in September 2014. Plant samples were identified by the herbarium, at the National Institute of Agronomy (Algiers, Algeria). The leaves were removed from the stalk and shade dried.
Essential oil extraction E. citriodora essential oil was isolated by process of conventional steam distillation apparatus [17]. The vapor produced by the steam generator crosses the plant, charged with essential oil and then passes through the condenser to a
Please cite this article in press as: Tolba H, et al. Essential oil of Algerian Eucalyptus citriodora: Chemical composition, antifungal activity. Journal De Mycologie Médicale (2015), http://dx.doi.org/10.1016/j.mycmed.2015.10.009
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Chemical composition and antifungal activity of Eucalyptus citriodora essential oil receiving Florentine flask. The obtained essential oil was dried over anhydrous sodium sulphate and, after filtration, stored at 4 8C until tested.
Gas chromatographic (GC) and gas chromatographic/mass spectrometry (GC/MS) analysis The essential oil was analysed by gas chromatography coupled to mass spectrometry (GC—MS) (Hewlett-Packard computerized system, comprising a 6890-gas chromatograph coupled to a 5973A mass spectrometer), equipped with a HP5MSTM fused silica capillary column (30 m, 0.25 mm i.d., 0.25 mm film thickness). GC—MS spectra were obtained using the following conditions: carrier gas, helium; flow rate, 0.5 ml/min; mode, split (50:1); injection volume, 1 mL; injection temperature, 250 8C; oven temperature programme, 60 8C for 8 min, then increased at 2 8C/min to 250 8C and held at 250 8C for 15 min; ionization mode, electronic impact at 70 eV. The relative percentages of the components were electronically calculated from GC—FID peak areas. Retention index (RI) of all the constituents was determined via the Kovats method by co-injection of the samples with a solution containing the homologous series of n-alkanes (C8-C24) on the HP5MSTM column. Identification of the components was made by visual interpretation, comparing their retention indices and mass spectra with data published in the literature [1] and by matching their recorded mass spectra with reference spectra in the computer library (National Institute of Standards and Technology [NIST] and Wiley libraries). The quantification amounted to computing the percentage contribution of each compound to the total amount present.
Antifungal activity Fungal pathogens and culture conditions In the present study, four clinical isolates fungal strains were used to assess the efficacy of E. citriodora essential oil including: Microsporum canis, Microsporum gypseum, Trichophyton mentagrophytes, Trichophyton rubrum, which were procured from Pasteur Institute, Alger, Algeria. Fungal pathogens were grown on Sabouraud dextrose agar (SDA) for 7—14 days at 27 8C, after which time spores were prepared by gently scraping the culture surfaces using a sterile glass rond after macerating in sterile saline (0.9%) solution. The final spore suspension was adjusted to 105—106 CFU/mL. Disc diffusion method The antifungal activity of E. citriodora essential oil was assessed by disc diffusion method using SDA in Petri dishes (90 mm diameter). The sterile discs (6 mm diameter) were impregnated with 3 different concentrations (10, 20, 30 mL/ disc) of the oil and placed on the surface of the media previously inoculated with fungal suspension (106 CFU/ mL). The tested plates were incubated at 27 8C for 7 days and the inhibition diameters were measured. All tests were performed in triplicate [5]. Disc volatilization method This method describes the diffusion of the essential oil in vapour phase. Three different volumes (10, 20 and 30 mL per
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disc) of the essential oil were pipetted on the paper disc (diameter 6 mm) and placed on the inside surface of the upper lid of Petri dishes. The lids were immediately closed with the plates containing the solid media SDA previously inoculated with microbial suspension (106 CFU/mL) and sealed with parafilm to prevent leakage of the EO vapour. Plates were inoculated at 27 8C during 7 days, and the diameter of the inhibition zone was measured [27]. Tests were carried out in triplicate.
MIC and MFC determination The oil was prepared in 0.2% sterile agar suspension [20]. The MIC of the oil was determined by the agar dilution method. The oil was mixed with SDA medium and was poured into Petri dishes to obtain a final concentration ranging from 10 mL/mL to 0.15 mL/mL. Sterile absorbent disk was impregnated with the fungal suspension (106 CFU/mL) and placed on the surface of the media. After incubation for 7 days at 27 8C, the MIC was red as the highest dilution (lowest concentration) of oil where no visible growth of microorganisms was noticeable. MFC was indicated by the disc that showed no growth after subculturing on Sabouraud dextrose agar medium and was red after 7 days.
Statistical analysis All data were reported as means standard deviation of two replicates. ANOVA was undertaken to our results to determine differences. To determine significant differences between the levels of the major factor, Tukey’s post hoc multicomparison test was applied between means. ANOVAs were made with the subsequent factors: fungal strains (4 levels) and quantity of EO used. Statistical data analysis was undertaken using XLStat software (XLStat, Paris, France). P values < 0.05 were reported as significant.
Results Chemical composition Steam distillation of E. citriodora leaves yielded a paleyellow colored oil (yield: 2.26%, w/w, dry weight basis). Table 1 provides both qualitative and quantitative analyses for the E. citriodora essential oil volatile profiles. A total of 22 compounds which represented 95.27% of the oil, were identified. The E. citriodora oil is generally monoterpenoid in the nature. As shown in this table, E. citriodora essential oil contained mainly oxygenated monoterpenoids (91.8%), monoterpene hydrocarbons (0.76%), sesquiterpene hydrocarbons (1.51%), oxygenated sesquiterpenes (0.29%) and other non-terpenoid compounds (0.91%). The dominant compounds of the essential oil was citronellal (69.77%) followed by citronellol (10.63%) and isopulegol (4.66%). The remaining constituents amount to less than 2%.
Antifungal activity Disc diffusion method The results from the antifungal potential of E. citriodora essential oil determined via the disc diffusion method are
Please cite this article in press as: Tolba H, et al. Essential oil of Algerian Eucalyptus citriodora: Chemical composition, antifungal activity. Journal De Mycologie Médicale (2015), http://dx.doi.org/10.1016/j.mycmed.2015.10.009
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H. Tolba et al. Table 1 Chemical composition of Eucalyptus citriodora essential oil. Composition chimique de l’huile essentielle de l’Eucalyptus citriodora. N8
Compound a
Monoterpene Hydrocarbons a-pinene b-pinene Limonene Oxygenated Monoterpenes 1,8 cineole 4 cis-Rose oxide 5 6 trans-Rose oxide Isopulegol 7 Citronellal 8 9 NeoIsoisopulegoI a-Terpineol 10 Citronellol 11 Geranial 12 13 Isopulegyl acetate p-Menthane-3,8-diol 14 Citronellyl acetate 15 Jasmone <(Z)-> 16 Sesquiterpene Hydrocarbons b-Caryophyllene 17 a-Humulene 18 BicycIogermacrene 19 Oxygenated Sesquiterpenes Spathulenol 20 Caryophyllene oxide 21 Other Compounds 22 2,6-Dimethyl-5-heptenal Total identified components Not identified 1 2 3
RI b
Percentage %
929 973 1029
0.08 0.43 0.25
1035 1112 1128 1152 1167 1176 1199 1242 1262 1276 1350 1355 1398
0.14 0.27 0.13 4.66 69.77 0.24 0.33 10.63 0.32 0.16 2.76 2.16 0.23
1415 1450 1493
1.34 0.10 0.07
1574 1583
0.07 0.22
1055
0.91 95.27 4.73
10, 20, 30 mL) of the oil. The highest inhibition zone value was observed against Microsporum canis (64 mm), Trichophyton mentagrophytes (64 mm), Trichophyton rubrum (39 mm) by using 10 mL of the oil, such that it inhibits growth absolutely of the three dermatophytes at 20 and 30 mL of the oil, while Microsporum gypseum was the most resistant to the oil with inhibition zone from (10,20 mL) of the oil was 12 mm and 29.5 mm at 30 mL of the oil. Disc volatilization method The antifungal activity of E. citriodora essential oil was also evaluated in the vapour phase. The zone of inhibition resulting from the exposure of the oil vapour is given in Table 2. As observed in the liquid phase, the zone of inhibition due to the oil vapour also increased with increasing concentration of the oil. Further, Microsporum canis, Trichophyton mentagrophytes were completely inhibited by the oil vapours at 20 and 30 mL, while Trichophyton rubrum and Microsporum gypseum formed a 39-mm and 24-mm inhibition zone, respectively, from the vapours of 30 mL E. citriodora essential oil. Microsporum gypseum was more resistant to the oil vapour at 10 and 20 mL. For all the tested strains, the inhibition zone resulting from exposure to E. citriodora essential oil vapours was less than that resulting from the same concentration of the oil in liquid phase.
MIC and MFC determination
a
Essential oil compounds sorted by chemical families and percentages on non-polar HP5MSTM capillary column. b Retention indices relative to C8—C24 n-alkanes calculated on non-polar HP5MSTM capillary column.
The MIC and MFC values of E. citriodora essential oil are shown in Table 3. The obtained data indicated that the MIC and MFC values of the oil varied from 0.6 to 5 mL/mL and 0.125—5 mL/mL, respectively, the lowest MIC and MFC values were for Microsporum canis and Trichophyton rubrum (0.6— 1.25 mL/mL) followed by Trichophyton mentagrophytes (1.25—1.25 mL/mL). The highest MIC and MFC values were for Microsporum gypseum (5 mL/mL—5 mL/mL). E. citriodora essential oil showed fungicidal effect against the tested strains.
Discussion summarized in Table 2. Based on the obtained results, E. citriodora essential oil successfully inhibited the growth of all dermatophytes tested in the present study. The zone of inhibition increased with the increasing concentration (i.e
In our study, E. citriodora essential oil showed potential antidermatophytic effect against the tested fungal strains in liquid and vapour phases. Several works have focused on the
Table 2 Antifungal activity of Eucalyptus citriodora essential oil. ´ antifongique de l’huile essentielle de l’Eucalyptus citriodora. Activite Fungal strains
Disc diffusion assay
Vapour diffusion assay
Diameter of inhibition zone (mm)
Diameter of inhibition zone (mm)
10 mL Microsporum canis Microsporum gypseum Trichophyton mentagrophytes Trichophyton rubrum
20 mL a
64 0.0 12 0.7 b 65 1.41 a 39 0.0 c
30 mL a
90 0.0 12 0.7 b 90 0.0 a 90 0.0 a
10 mL a
90 0.0 29.5 3.5 b 90 0.0 a 90 0.0 a
20 mL d
90 0.0 Ra 48.5 2.12 c 24 1.41 b
30 mL a
90 0.0 Rb 90 0.0 a 33 1.41 c
90 0.0 a 24.5 3.5 b 90 0.0 a 37.5 2.42 c
R: resistant. Means within the same column followed by the same small letter are not significantly different (P > 0.05) according to Tukey’s test Posthoc multicomparison. a Diameter of inhibition zone (mm) including disc diameter of 6 mm; DIZ values are presented as mean (mm) standard deviation of triplicates.
Please cite this article in press as: Tolba H, et al. Essential oil of Algerian Eucalyptus citriodora: Chemical composition, antifungal activity. Journal De Mycologie Médicale (2015), http://dx.doi.org/10.1016/j.mycmed.2015.10.009
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Chemical composition and antifungal activity of Eucalyptus citriodora essential oil Table 3 Determination of minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of Eucalyptus citriodora essential oil. ´ termination de la concentration minimale inhibitrice De (CMI) et la concentration minimale fongicide (CMF) de l’huile essentielle de l’Eucalyptus citriodora. Strains
MIC (mL/mL)
MFC (mL/mL)
Microsporum canis Microsporum gypseum Trichophyton mentagrophytes Trichophyton rubrum
0.6 5 1.25 0.6
1.25 5 1.25 1.25
functional of Eucalyptus essential oil such as antimicrobial, antifungal activities [3]. The antimicrobial effects against a wide range of microorganisms have been considered in several studies [4,27]. While a few studies have been conducted to investigate the antidermatophytic activity of Eucalyptus species, such as Eucalyptus camaldulensis methanolic extracts were studied for their antidermatophyte activity in comparison with griseofulvin [7], Eucalyptus globulus, Eucalyptus maculate and Eucalyptus viminalis leaf extracts against Trichophyton mentagrophytes [26]. E. citriodora essential oil is known for its antifungal potential [2,8,12]. In our study, E. citriodora essential oil showed a strong antifungal activity against all tested strains, it exhibited completely Trichophyton mentagrophytes, Microsporum canis and Trichophyton rubrum at 30 mL of the oil. Similar results were shown by Shahi et al. [21] who reported that the oil of E. citriodora was effective as an antifungal agent against Trichophyton mentagrophytes, trichophyton rubrum, and Microsporum nanum. In our study, the MIC values of the oil in the case of Trichophyton rubrum and Trichophyton mentagrophytes were (0.6—0.125 mL/mL) respectively. Shahi et al. [21] have found 0.4 mL/mL as minimum effective concentration for the two strains. While Inouye et al. have studied the activity of E. citriodora essential oil vapor against Trichophyton mentagrophytes using Box vapor assay and showed that the MFD value was equal to 3 mg/mL air. However, there is no information about the antifungal activity of E. citriodora essential oil in vapour phase. To the best of our knowledge, the antifungal activity of E. citriodora essential oil has never been reported prior this study. The antifungal activity of essential oils is related to their chemical composition. In our study, the essential oil of E. citriodora could be due to the two major oxygenate monoterpenes (citronellal and citronellol). Ramezani et al. [19] studied the fungicidal effect of the monoterpene, citronellal, against Rhizoctonia solani, Helminthosporium oryzae and reported that citronellal alone was found to be more effective than eucalypt oils. Lee et al. [12] studied the antifungal activity and have shown that the inhibition rates of citronellol against Phytophthora cactorum were close to 100%. While, no attempt was focused on the antidermatophytic activity of the major compounds citronellal and citronellol. In total, the present study concludes that E. citriodora essential oil exhibited a higher antifungal potential against the four dermatophytes; then, it could be used as a source of
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natural antifungal agent to control human infections fungal pathogens. It is important to develop a new botanical drug formulated from E. citriodora essential oil for fungal infections as topical treatment.
Disclosure of interest The author declares that he has no competing interest.
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ORIGINAL ARTICLE/ARTICLE ORIGINAL
Essential oil of Algerian Eucalyptus citriodora: Chemical composition, antifungal activity Huile essentielle d’Eucalyptus citriodora : composition chimique ´ antifongique et activite H. Tolba a,*, H. Moghrani a, A. Benelmouffok b, D. Kellou b, R. Maachi a a
Laboratory of reaction engineering, department of process engineering, faculty of mechanical engineering and process engineering, USTHB, PO Box 32 El Alia, Bab Ezzouar, 16111 Algiers, Algeria b Laboratory of Mycology, Pasteur Institute, Algiers, Algeria Received 13 April 2015; received in revised form 11 October 2015; accepted 12 October 2015
KEYWORDS Eucalyptus Citriodora; Essential oil; Dermatophytes; Antifungal activity
Summary Objective. — Essential oil of Eucalyptus citriodora is a natural product which has been attributed for various medicinal uses. In the present investigation, E. citriodora essential oil was used to evaluate its antifungal effect against medically important dermatophytes. Material and methods. — Essential oil from the Algerian E. citriodora leaves was analyzed by GC and GC/MS. The antifungal effect of E. citriodora essential oil was evaluated against four dermatophytes: Microsporum canis, Microsporum gypseum, Trichophyton mentagrophytes, Trichophyton rubrum using disc diffusion method, disc volatilization method, and agar dilution method. Results. — The chemical composition of the oil revealed the presence of 22 compounds accounting for 95.27% of the oil. The dominant compounds were citronellal (69.77%), citronellol (10.63%) and isopulegol (4.66%). The disc diffusion method, MIC and MFC determination, indicated that E. citriodora essential oil had a higher antifungal potential against the tested strains with inhibition zone diameter which varied from (12 to 90 mm) and MIC and MFC values ranged from (0.6 to 5 mL/mL and 1.25 to 5 mL/mL) respectively. The M. gypseum was the most resistant to the oil.
* Corresponding author. E-mail address: [email protected] (H. Tolba). http://dx.doi.org/10.1016/j.mycmed.2015.10.009 1156-5233/# 2015 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: Tolba H, et al. Essential oil of Algerian Eucalyptus citriodora: Chemical composition, antifungal activity. Journal De Mycologie Médicale (2015), http://dx.doi.org/10.1016/j.mycmed.2015.10.009
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H. Tolba et al. Conclusion. — The results of the present study indicated that E. citriodora essential oil may be used as a new antifungal agent recommended by the pharmaceutical industries. # 2015 Elsevier Masson SAS. All rights reserved.
MOTS CLÉS Eucalyptus Citriodora ; Huile essentielle ; Dermatophytes ; Activité antifongique ; Composition chimique
Re ´sume ´ Objectif. — L’huile essentielle d’Eucalyptus citriodora est un produit naturel auquel a été attribué différentes utilisations médicinales. Dans la présente étude, l’huile essentielle de E. citriodora a été utilisée pour évaluer son effet antifongique contre les dermatophytes médicalement importants. Mate´riel et me´thode. — L’huile essentielle des feuilles de l’E. citriodora algérien a été analysée par GC et GC/MS. L’effet antifongique d’huile essentielle de l’E. citriodora a été évalué contre quatre dermatophytes : Microsporum canis, Microsporum gypseum, Trichophyton mentagrophytes, Trichophyton rubrum en utilisant la méthode de diffusion, la méthode de la volatilisation et la méthode de dilution. Re ´sultats. — La composition chimique de l’huile a révélé la présence de 22 composés qui représente 95,27 % de l’huile. Les composés dominants étaient le citronellal (69,77 %), le citronellol (10,63 %) et isopulégol (4,66 %). La méthode de diffusion ainsi que la détermination de la CMI et de la CMF ont indiqué que l’huile essentielle de l’E. citriodora a un potentiel antifongique élevé contre les souches testées avec un diamètre d’inhibition, qui variait de (12 à 90 mm) et des CMI et CMF qui variaient de (0,6 à 5 mL/mL et 1,25 à 5 mL/mL) respectivement. Le M. gypseum était le plus résistant à l’huile. Conclusion. — Les résultats de notre étude indiquent que l’huile essentielle de l’E. citriodora peut être utilisée comme un nouvel agent antifongique recommandé par les industries pharmaceutiques. # 2015 Elsevier Masson SAS. Tous droits réservés.
Introduction Fungal strains are potential public health threats all over the world. They are widespread due to environment factors [28]. Dermatophytes are the major cause of superficial mycoses. They have the ability to invade skin, hair, nail and cause dermatophytosis, and can be contagious fungal disease [22]. The incidence of dermatophytic infections has increased considerably during decades [11]. Dermatophytes are responsible for serious human pathogenic disorders in various parts of the world, routinely concern people who are otherwise healthy and individuals with compromised immune systems are particularly susceptible [15]. Throughout the world, there has been an increasing incidence of fungal infections, and because of drug resistance and toxicity associated with long-term treatment with antifungal drugs, it is an interest to develop new natural therapeutic alternative with acceptable antifungal effect especially among the medicinal plants. Many plants species are known for their antimicrobial properties and their wide uses in traditional medicine [13]. Previous works have focused on the therapeutic properties of the natural product. This investigation is due to their potential ressources of essential oil. Eucalyptus citriodora essential oil is a natural product that exhibits various biological properties. It is one of the most widely used species in modern cosmetics, food and pharmaceutical industries. E. citriodora essential oil is known to possess a wide spectrum of biological activities including toxicity against a wide range of microbes, including bacteria, fungi including yeasts [6,10,12,14,18,19,25], analgesic and anti-inflammatory
effects [9,23], antioxidant activity [24] and antimosquito properties [16]. To the best of our knowledge, the present study might be the first report on the antidermatophytic activity of E. citriodora growing in Algeria. Moreover, no systematic studies comparing the antimicrobial activity in both liquid and vapour phases of the E. citriodora oil are available. In the present study, the essential oil was extracted from the E. citriodora leaves growing in Algeria via steam distillation. We selected E. citriodora essential oil to investigate its chemical composition, its effectiveness in vitro against four dermatophytes using disc diffusion method, disc volatilization method, and agar dilution method.
Material and methods Plant material Fresh E. citriodora leaves were collected from the National Institute of Agronomy (El Harrach located 15 km of Algiers) in September 2014. Plant samples were identified by the herbarium, at the National Institute of Agronomy (Algiers, Algeria). The leaves were removed from the stalk and shade dried.
Essential oil extraction E. citriodora essential oil was isolated by process of conventional steam distillation apparatus [17]. The vapor produced by the steam generator crosses the plant, charged with essential oil and then passes through the condenser to a
Please cite this article in press as: Tolba H, et al. Essential oil of Algerian Eucalyptus citriodora: Chemical composition, antifungal activity. Journal De Mycologie Médicale (2015), http://dx.doi.org/10.1016/j.mycmed.2015.10.009
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Chemical composition and antifungal activity of Eucalyptus citriodora essential oil receiving Florentine flask. The obtained essential oil was dried over anhydrous sodium sulphate and, after filtration, stored at 4 8C until tested.
Gas chromatographic (GC) and gas chromatographic/mass spectrometry (GC/MS) analysis The essential oil was analysed by gas chromatography coupled to mass spectrometry (GC—MS) (Hewlett-Packard computerized system, comprising a 6890-gas chromatograph coupled to a 5973A mass spectrometer), equipped with a HP5MSTM fused silica capillary column (30 m, 0.25 mm i.d., 0.25 mm film thickness). GC—MS spectra were obtained using the following conditions: carrier gas, helium; flow rate, 0.5 ml/min; mode, split (50:1); injection volume, 1 mL; injection temperature, 250 8C; oven temperature programme, 60 8C for 8 min, then increased at 2 8C/min to 250 8C and held at 250 8C for 15 min; ionization mode, electronic impact at 70 eV. The relative percentages of the components were electronically calculated from GC—FID peak areas. Retention index (RI) of all the constituents was determined via the Kovats method by co-injection of the samples with a solution containing the homologous series of n-alkanes (C8-C24) on the HP5MSTM column. Identification of the components was made by visual interpretation, comparing their retention indices and mass spectra with data published in the literature [1] and by matching their recorded mass spectra with reference spectra in the computer library (National Institute of Standards and Technology [NIST] and Wiley libraries). The quantification amounted to computing the percentage contribution of each compound to the total amount present.
Antifungal activity Fungal pathogens and culture conditions In the present study, four clinical isolates fungal strains were used to assess the efficacy of E. citriodora essential oil including: Microsporum canis, Microsporum gypseum, Trichophyton mentagrophytes, Trichophyton rubrum, which were procured from Pasteur Institute, Alger, Algeria. Fungal pathogens were grown on Sabouraud dextrose agar (SDA) for 7—14 days at 27 8C, after which time spores were prepared by gently scraping the culture surfaces using a sterile glass rond after macerating in sterile saline (0.9%) solution. The final spore suspension was adjusted to 105—106 CFU/mL. Disc diffusion method The antifungal activity of E. citriodora essential oil was assessed by disc diffusion method using SDA in Petri dishes (90 mm diameter). The sterile discs (6 mm diameter) were impregnated with 3 different concentrations (10, 20, 30 mL/ disc) of the oil and placed on the surface of the media previously inoculated with fungal suspension (106 CFU/ mL). The tested plates were incubated at 27 8C for 7 days and the inhibition diameters were measured. All tests were performed in triplicate [5]. Disc volatilization method This method describes the diffusion of the essential oil in vapour phase. Three different volumes (10, 20 and 30 mL per
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disc) of the essential oil were pipetted on the paper disc (diameter 6 mm) and placed on the inside surface of the upper lid of Petri dishes. The lids were immediately closed with the plates containing the solid media SDA previously inoculated with microbial suspension (106 CFU/mL) and sealed with parafilm to prevent leakage of the EO vapour. Plates were inoculated at 27 8C during 7 days, and the diameter of the inhibition zone was measured [27]. Tests were carried out in triplicate.
MIC and MFC determination The oil was prepared in 0.2% sterile agar suspension [20]. The MIC of the oil was determined by the agar dilution method. The oil was mixed with SDA medium and was poured into Petri dishes to obtain a final concentration ranging from 10 mL/mL to 0.15 mL/mL. Sterile absorbent disk was impregnated with the fungal suspension (106 CFU/mL) and placed on the surface of the media. After incubation for 7 days at 27 8C, the MIC was red as the highest dilution (lowest concentration) of oil where no visible growth of microorganisms was noticeable. MFC was indicated by the disc that showed no growth after subculturing on Sabouraud dextrose agar medium and was red after 7 days.
Statistical analysis All data were reported as means standard deviation of two replicates. ANOVA was undertaken to our results to determine differences. To determine significant differences between the levels of the major factor, Tukey’s post hoc multicomparison test was applied between means. ANOVAs were made with the subsequent factors: fungal strains (4 levels) and quantity of EO used. Statistical data analysis was undertaken using XLStat software (XLStat, Paris, France). P values < 0.05 were reported as significant.
Results Chemical composition Steam distillation of E. citriodora leaves yielded a paleyellow colored oil (yield: 2.26%, w/w, dry weight basis). Table 1 provides both qualitative and quantitative analyses for the E. citriodora essential oil volatile profiles. A total of 22 compounds which represented 95.27% of the oil, were identified. The E. citriodora oil is generally monoterpenoid in the nature. As shown in this table, E. citriodora essential oil contained mainly oxygenated monoterpenoids (91.8%), monoterpene hydrocarbons (0.76%), sesquiterpene hydrocarbons (1.51%), oxygenated sesquiterpenes (0.29%) and other non-terpenoid compounds (0.91%). The dominant compounds of the essential oil was citronellal (69.77%) followed by citronellol (10.63%) and isopulegol (4.66%). The remaining constituents amount to less than 2%.
Antifungal activity Disc diffusion method The results from the antifungal potential of E. citriodora essential oil determined via the disc diffusion method are
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H. Tolba et al. Table 1 Chemical composition of Eucalyptus citriodora essential oil. Composition chimique de l’huile essentielle de l’Eucalyptus citriodora. N8
Compound a
Monoterpene Hydrocarbons a-pinene b-pinene Limonene Oxygenated Monoterpenes 1,8 cineole 4 cis-Rose oxide 5 6 trans-Rose oxide Isopulegol 7 Citronellal 8 9 NeoIsoisopulegoI a-Terpineol 10 Citronellol 11 Geranial 12 13 Isopulegyl acetate p-Menthane-3,8-diol 14 Citronellyl acetate 15 Jasmone <(Z)-> 16 Sesquiterpene Hydrocarbons b-Caryophyllene 17 a-Humulene 18 BicycIogermacrene 19 Oxygenated Sesquiterpenes Spathulenol 20 Caryophyllene oxide 21 Other Compounds 22 2,6-Dimethyl-5-heptenal Total identified components Not identified 1 2 3
RI b
Percentage %
929 973 1029
0.08 0.43 0.25
1035 1112 1128 1152 1167 1176 1199 1242 1262 1276 1350 1355 1398
0.14 0.27 0.13 4.66 69.77 0.24 0.33 10.63 0.32 0.16 2.76 2.16 0.23
1415 1450 1493
1.34 0.10 0.07
1574 1583
0.07 0.22
1055
0.91 95.27 4.73
10, 20, 30 mL) of the oil. The highest inhibition zone value was observed against Microsporum canis (64 mm), Trichophyton mentagrophytes (64 mm), Trichophyton rubrum (39 mm) by using 10 mL of the oil, such that it inhibits growth absolutely of the three dermatophytes at 20 and 30 mL of the oil, while Microsporum gypseum was the most resistant to the oil with inhibition zone from (10,20 mL) of the oil was 12 mm and 29.5 mm at 30 mL of the oil. Disc volatilization method The antifungal activity of E. citriodora essential oil was also evaluated in the vapour phase. The zone of inhibition resulting from the exposure of the oil vapour is given in Table 2. As observed in the liquid phase, the zone of inhibition due to the oil vapour also increased with increasing concentration of the oil. Further, Microsporum canis, Trichophyton mentagrophytes were completely inhibited by the oil vapours at 20 and 30 mL, while Trichophyton rubrum and Microsporum gypseum formed a 39-mm and 24-mm inhibition zone, respectively, from the vapours of 30 mL E. citriodora essential oil. Microsporum gypseum was more resistant to the oil vapour at 10 and 20 mL. For all the tested strains, the inhibition zone resulting from exposure to E. citriodora essential oil vapours was less than that resulting from the same concentration of the oil in liquid phase.
MIC and MFC determination
a
Essential oil compounds sorted by chemical families and percentages on non-polar HP5MSTM capillary column. b Retention indices relative to C8—C24 n-alkanes calculated on non-polar HP5MSTM capillary column.
The MIC and MFC values of E. citriodora essential oil are shown in Table 3. The obtained data indicated that the MIC and MFC values of the oil varied from 0.6 to 5 mL/mL and 0.125—5 mL/mL, respectively, the lowest MIC and MFC values were for Microsporum canis and Trichophyton rubrum (0.6— 1.25 mL/mL) followed by Trichophyton mentagrophytes (1.25—1.25 mL/mL). The highest MIC and MFC values were for Microsporum gypseum (5 mL/mL—5 mL/mL). E. citriodora essential oil showed fungicidal effect against the tested strains.
Discussion summarized in Table 2. Based on the obtained results, E. citriodora essential oil successfully inhibited the growth of all dermatophytes tested in the present study. The zone of inhibition increased with the increasing concentration (i.e
In our study, E. citriodora essential oil showed potential antidermatophytic effect against the tested fungal strains in liquid and vapour phases. Several works have focused on the
Table 2 Antifungal activity of Eucalyptus citriodora essential oil. ´ antifongique de l’huile essentielle de l’Eucalyptus citriodora. Activite Fungal strains
Disc diffusion assay
Vapour diffusion assay
Diameter of inhibition zone (mm)
Diameter of inhibition zone (mm)
10 mL Microsporum canis Microsporum gypseum Trichophyton mentagrophytes Trichophyton rubrum
20 mL a
64 0.0 12 0.7 b 65 1.41 a 39 0.0 c
30 mL a
90 0.0 12 0.7 b 90 0.0 a 90 0.0 a
10 mL a
90 0.0 29.5 3.5 b 90 0.0 a 90 0.0 a
20 mL d
90 0.0 Ra 48.5 2.12 c 24 1.41 b
30 mL a
90 0.0 Rb 90 0.0 a 33 1.41 c
90 0.0 a 24.5 3.5 b 90 0.0 a 37.5 2.42 c
R: resistant. Means within the same column followed by the same small letter are not significantly different (P > 0.05) according to Tukey’s test Posthoc multicomparison. a Diameter of inhibition zone (mm) including disc diameter of 6 mm; DIZ values are presented as mean (mm) standard deviation of triplicates.
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Chemical composition and antifungal activity of Eucalyptus citriodora essential oil Table 3 Determination of minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of Eucalyptus citriodora essential oil. ´ termination de la concentration minimale inhibitrice De (CMI) et la concentration minimale fongicide (CMF) de l’huile essentielle de l’Eucalyptus citriodora. Strains
MIC (mL/mL)
MFC (mL/mL)
Microsporum canis Microsporum gypseum Trichophyton mentagrophytes Trichophyton rubrum
0.6 5 1.25 0.6
1.25 5 1.25 1.25
functional of Eucalyptus essential oil such as antimicrobial, antifungal activities [3]. The antimicrobial effects against a wide range of microorganisms have been considered in several studies [4,27]. While a few studies have been conducted to investigate the antidermatophytic activity of Eucalyptus species, such as Eucalyptus camaldulensis methanolic extracts were studied for their antidermatophyte activity in comparison with griseofulvin [7], Eucalyptus globulus, Eucalyptus maculate and Eucalyptus viminalis leaf extracts against Trichophyton mentagrophytes [26]. E. citriodora essential oil is known for its antifungal potential [2,8,12]. In our study, E. citriodora essential oil showed a strong antifungal activity against all tested strains, it exhibited completely Trichophyton mentagrophytes, Microsporum canis and Trichophyton rubrum at 30 mL of the oil. Similar results were shown by Shahi et al. [21] who reported that the oil of E. citriodora was effective as an antifungal agent against Trichophyton mentagrophytes, trichophyton rubrum, and Microsporum nanum. In our study, the MIC values of the oil in the case of Trichophyton rubrum and Trichophyton mentagrophytes were (0.6—0.125 mL/mL) respectively. Shahi et al. [21] have found 0.4 mL/mL as minimum effective concentration for the two strains. While Inouye et al. have studied the activity of E. citriodora essential oil vapor against Trichophyton mentagrophytes using Box vapor assay and showed that the MFD value was equal to 3 mg/mL air. However, there is no information about the antifungal activity of E. citriodora essential oil in vapour phase. To the best of our knowledge, the antifungal activity of E. citriodora essential oil has never been reported prior this study. The antifungal activity of essential oils is related to their chemical composition. In our study, the essential oil of E. citriodora could be due to the two major oxygenate monoterpenes (citronellal and citronellol). Ramezani et al. [19] studied the fungicidal effect of the monoterpene, citronellal, against Rhizoctonia solani, Helminthosporium oryzae and reported that citronellal alone was found to be more effective than eucalypt oils. Lee et al. [12] studied the antifungal activity and have shown that the inhibition rates of citronellol against Phytophthora cactorum were close to 100%. While, no attempt was focused on the antidermatophytic activity of the major compounds citronellal and citronellol. In total, the present study concludes that E. citriodora essential oil exhibited a higher antifungal potential against the four dermatophytes; then, it could be used as a source of
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natural antifungal agent to control human infections fungal pathogens. It is important to develop a new botanical drug formulated from E. citriodora essential oil for fungal infections as topical treatment.
Disclosure of interest The author declares that he has no competing interest.
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