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JAC Advance Access originally published online on March 21, 2007
Journal of Antimicrobial Chemotherapy 2007 59(5):934-940; doi:10.1093/jac/dkm066
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© The Author 2007. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Chemical composition, toxicological aspects and antifungal activity of essential oil from Lippia sidoides Cham.

R. O. S. Fontenelle1, S. M. Morais1,2, E. H. S. Brito1, M. R. Kerntopf1, R. S. N. Brilhante3, R. A. Cordeiro3, A. R. Tomé1, M. G. R. Queiroz4, N. R. F. Nascimento1, J. J. C. Sidrim3 and M. F. G. Rocha1,3,*

1 Post-Graduation Program in Veterinary Sciences, Veterinary Faculty, State University of Ceará, Fortaleza, Ceará, Brazil 2 Department of Chemistry, State University of Ceará, Fortaleza, Ceará, Brazil 3 Medical Mycology Specialized Center, Faculty of Medicine, Department of Pathology and Legal Medicine, Federal University of Ceará, Fortaleza, Ceará, Brazil 4 Department of Clinical Biochemistry, School of Pharmacy, Federal University of Ceará, Fortaleza, Ceará, Brazil

* Corresponding author. Tel: +55-85-3295-1736; Fax: +55-85-3295-1736. E-mail: rocha{at}rapix.com.br

Received 10 November 2006; returned 11 December 2006; revised 26 January 2007; accepted 8 February 2007


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Objectives: The aims of this study were to test the essential oil from Lippia sidoides Cham. for antifungal activity, in vitro, against Candida spp. and Microsporum canis, to evaluate its acute and subchronic toxicological effects, in vivo, and to determine its chemical constituents.

Methods: The antifungal activity, in vitro, was initially evaluated by the agar-well diffusion technique, and the MIC and minimum fungicidal concentration (MFC) were determined by the broth microdilution method. The acute and subchronic toxicological effects were determined in mice and rats, respectively. The chemical composition of the essential oil was determined by gas chromatography coupled to mass spectroscopy.

Results: The essential oil obtained from L. sidoides was effective against all tested strains by the agar-well diffusion method. The MICs of L. sidoides essential oil for strains of M. canis ranged from 4 to 70 mg/L and the MFCs ranged from 9 to 150 mg/L. The MICs for strains of Candida spp. ranged from 620 to 2500 mg/L and the MFCs ranged from 1250 to 5000 mg/L. The main constituents of L. sidoides essential oil were thymol (59.65%), E-caryophyllene (10.60%) and p-cymene (9.08%). The acute administration of the essential oil up to 3 g/kg by the oral route to mice was devoid of overt toxicity. The 30 day oral administration of L. sidoides oil (117.95 mg/kg/day) to rats did not induce any significant histopathological, haematological or serum biochemical alterations.

Conclusions: The essential oil from L. sidoides may be a promising source in the search for new antifungal drugs due to its efficacy and low toxicity.

Keywords: L. sidoides , dermatophytes , yeasts , antifungal activity


   Introduction
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Mycosis constitutes a common health problem, especially in tropical and subtropical developing countries; dermatophytes, Malassezia spp. and Candida spp. being the most frequent pathogens in humans and animals.1–7 In recent years, there has been an increasing search for new antifungal compounds due to the lack of efficacy, side effects and or resistance associated with some of the existing drugs.8–11 Much attention has been paid to plant-derived antifungal compounds,12 based on the knowledge that plants have their own defence systems against fungal pathogens.13

Natural products obtained from many plants have been attracting scientific interest.9–16 More recently, the antifungal properties of allicin and ajoene isolated from garlic (Allium sativum) were demonstrated.14,15 In traditional medicine, many essential oils have been claimed to be effective against fungal pathogens, although most of them are not clinically available. Many authors have reported that essential oils are one of the most promising groups of natural compounds from which a new prototype of antifungal agents may be developed.11,14–21 Therefore, research in this field may lead to the development of effective drugs against pathogenic fungi.11,14

Widely spread in North-East Brazilian flora, Lippia species are known to be a natural topical antiseptic. Previous studies have reported that the essential oil of Lippia sidoides Cham. shows antimicrobial activity in vitro as well as larvicidal effect against Aedes aegyptii.22,23

The aims of this study were to test the essential oil from L. sidoides Cham. for in vitro antifungal activity against Candida spp. and Microsporum canis, to evaluate its acute and subchronic toxicological effects, in vivo, and to determine its chemical constituents.


   Materials and methods
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Plant material and essential oil extraction

Plant samples were collected in Horizonte city (3°33'46'' latitude S, 41°05'42'' longitude W), North-East Brazil. Taxonomic identification was confirmed by experts at the Prisco Bezerra Herbarium (Federal University of Ceará, Brazil), where a voucher sample was deposited with a reference number 25 149. L. sidoides essential oil was extracted from the leaves by the steam distillation method in a Clevenger apparatus, as described by Craveiro et al.24

Gas chromatography/mass spectral analysis

The chemical composition of the essential oil was determined by gas chromatography coupled to mass spectroscopy performed on a Hewlett–Packard 5971 GC/MS instrument in a polydimethylsiloxano-DB-5 (30 mm x 0.25 µm film thickness)-fused silica capillary column; the carrier gas was helium (1 mL/min). The column temperature ranged from 35°C to 180°C at 4°C/min, then from 180°C to 280°C at 20°C/min; mass spectra were obtained by electronic impact at 70 V. The identification of the constituents was performed by a computer-based library search, with retention indices and visual interpretation of the mass spectra.23

Fungal strains

The strains were obtained from the fungal collection of the Medical Mycology Specialized Centre (CEMM, Federal University of Ceará, Brazil), where they were maintained in saline (0.9% NaCl) at 28°C. At the time of the analysis, an aliquot of each suspension was taken and inoculated onto potato dextrose agar (Difco, Detroit, MI, USA), and then incubated at 28°C for 2–10 days. A total of 10 strains of M. canis, 5 strains of Candida albicans and 3 strains of Candida tropicalis were included in this study. Both M. canis and Candida spp. strains were isolated from dogs and cats. In addition, Candida parapsilosis (ATCC 22 019) and Candida krusei (ATCC 6528) strains were used for quality control.

Inoculum preparation for antifungal susceptibility tests

For the agar-well diffusion method, based on Tepe et al.25 and Gurgel et al.,12 stock inocula were prepared on day 2 and day 10 for Candida spp. and M. canis, respectively, grown on potato dextrose agar (Difco) at 28°C. Potato dextrose agar was added to the agar slant and the cultures were gently swabbed to dislodge the conidia. The suspension of conidia with blastoconidia of Candida spp. or that of hyphal fragments of M. canis was transferred to a sterile tube and adjusted by turbidimetry to obtain an inoculum of ~106 or 105 cfu/mL, respectively. The optical densities of the suspensions were spectrophotometrically determined at 530 nm.

For the broth microdilution method, the standardized inocula for Candida spp. (2.5–5 x 103 cfu/mL) and M. canis (5 x 104 cfu/mL) were also prepared by turbidimetry. Stock inocula were prepared on day 2 and day 10 for Candida spp. and M. canis, respectively, grown on potato dextrose agar at 28°C. Sterile saline solution (0.9%) was added to the agar slant and the cultures were gently swabbed to dislodge the conidia from the hyphal mat and from the blastoconidia for M. canis26 and Candida spp.,4 respectively. The suspension of conidia with hyphal fragments of M. canis and the blastoconidia suspension of Candida spp. were transferred to sterile tubes and the volume of both suspensions adjusted to 4 mL with sterile saline solution. The resulting suspension was allowed to settle for 5 min, at 28°C, and its density was read at 530 nm and then adjusted to 95% transmittance. The suspensions were diluted to 1:2000 for Candida spp. and 1:500 for M. canis [both with RPMI 1640 medium with L-glutamine and without sodium bicarbonate (Sigma Chemical Co., St Louis, MO, USA), buffered at pH 7.0 with 0.165 M MOPS (Sigma Chemical Co.)] to obtain inocula of ~2.5–5 x 103 and 5 x 104 cfu/mL, respectively.

Agar-well diffusion susceptibility test

The antifungal activity of essential oils from L. sidoides was evaluated against C. albicans (n = 5), C. tropicalis (n = 3) and M. canis (n = 10) by the agar-well diffusion method.12,25 Petri dishes with a diameter of 15 cm were prepared with potato dextrose agar (Difco). The wells (6 mm in diameter) were then cut from the agar and 0.100 mL of essential oil or drugs was delivered to them. The oil was weighed and dissolved in mineral oil to obtain the test concentrations of 25, 50, 75 and 100 mg/mL. Stock solutions of griseofulvin (1 mg/mL; Sigma Chemical Co.) and amphotericin B (5 mg/L; Sigma Chemical Co.) were prepared in distilled water and tested as positive controls for M. canis and Candida spp., respectively. Each fungal suspension was inoculated onto the surface of the agar. After incubation, for 3–5 days for Candida spp. and 5–8 days for M. canis, at 28–35°C, all dishes were examined for zones of growth inhibition and the diameters of these zones were measured in millimetres. Each experiment was repeated at least twice.

Broth microdilution method

The MIC and minimum fungicidal concentration (MFC) for Candida spp. were determined by the broth microdilution method, in accordance with the CLSI (formerly NCCLS) guidelines (M27-A2).27 The broth microdilution assay for M. canis was performed as described previously,26,28,29 based on the M38-A document,30 in accordance with the CLSI.

The essential oil of L. sidoides was prepared in 100% mineral oil. Amphotericin B (Sigma Chemical Co.) and griseofulvin (Sigma Chemical Co.) were prepared in distilled water. For the susceptibility analysis, the essential oil was diluted in mineral oil and tested in a concentration range between 0.002 and 5 mg/mL.

The microdilution assay was performed in 96-well microdilution plates. Growth and sterile control wells were included for each isolate tested. The microplates were incubated at 37°C and read visually after 2 days for Candida spp. and 5 days for M. canis. All isolates were run in duplicate and repeated at least twice. The MIC was defined as the lowest oil concentration that caused 80% inhibition of visible fungal growth. The results were read visually as recommended by the CLSI. The MFC was determined by subculturing 100 µL of solution from wells without turbidity, on potato dextrose, at 28°C. The MFCs were determined as the lowest concentration resulting in no growth on the subculture after 2 days for Candida spp. and 5 days for M. canis.

Animals

Wistar rats (Rattus norvegicus; 180–200 g) and Swiss mice (Mus musculus; 25–30 g), of both sexes, were housed in temperature-controlled rooms and were given food and water ad libitum until used. All the protocols that included animals were approved by the Ethics Committee in research of the State University of Ceará, Fortaleza, Ceará, Brazil. The animals were used as recommended by the guide for the care and use of laboratory animals from the National Academy Press (USA; 1996), which fulfils the principles for animal use in Brazil.

Acute and subchronic toxicity

For the acute toxicity analysis, the essential oil was administered to the mice (n = 10 mice per group) orally or intraperitoneally (ip) at doses ranging from 100 to 3000 mg/kg. The results obtained were compared with those for the control animals [3% (v/v) Tween 80 in saline]. The LD50 was calculated by the probit method by using SPSS 7.0 for Windows. The animals were observed for an additional period of 1 h and the general effects were noted in a table modified from Malone and Robichaud.31

To investigate the subchronic toxicity of the essential oil of L. sidoides, after 30 days of oral administration to rats, the following parameters were evaluated: haematological, histopathological and serum biochemistry. The rats were separated into two groups (n = 10 per group) and treated with L. sidoides essential oil (117.95 mg/kg/day) or 3% (v/v) Tween 80 in saline by oral gavage. Blood samples were collected by puncture in the infraorbital plexus on day 0 (1 day before starting essential oil or vehicle administration) and then on day 15 and day 30. The serum concentrations of urea, creatinine, glutamic-oxalacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) were determined by using commercial kits (Labtest, Lagoa Santa, MG, Brazil). The blood samples collected on day 0 and day 30 were used for determining red cell and leucocyte counts and for haemoglobin, haematocrit and biochemical parameter analysis. The values obtained were compared within and between the groups. Additionally, at the end of the experimental period (30 days), histopathological analysis of heart, lungs, liver, kidneys and spleen was performed by optical microscopy.

Statistical analysis

The antifungal activity evaluated by the agar-well diffusion method was expressed as mean ± SD of the diameter of the growth inhibition zones (mm). The antifungal activity of the essential oils was analysed by linear correlation for individual analysis and the two-tailed paired Student's t-test was used to evaluate differences between the data of essential oils and the controls. The LD50 was calculated at 95% confidence intervals, using SPSS 7.0 for Windows. The data obtained from subchronic toxicological studies were expressed as mean ± 95% confidence intervals and data range. The differences within and between the groups were evaluated by the analysis of variance method, followed by the correction of Tukey–Kramer with the significance level set at 5%.


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The chemical analysis of the L. sidoides is shown in Table 1. The major constituents of the essential oil of L. sidoides were thymol (59.65%), E-caryophyllene (10.60%) and p-cymene (9.08%).


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  		Table 1.. Chemical composition of Lippia sidoides Cham. essential oil

 
The essential oil from L. sidoides was effective against all tested strains in the agar-well diffusion susceptibility tests (Table 2). The L. sidoides oil induced a significant growth inhibition zone (36.8 ± 12.4 mm) at the lower concentration (25 mg/mL) against M. canis (n = 10). At concentrations ≥50 mg/mL, this essential oil totally inhibited M. canis (n = 10) grown in culture. For Candida strains (n = 8), the maximal inhibition of fungal growth induced by L. sidoides oil was 23.3 ± 1.8 mm, at the higher dose used (100 mg/mL). The positive control, griseofulvin, induced a significant growth inhibition zone (51.6 ± 6.7 mm) against M. canis (n = 10) and amphotericin B induced a significant growth inhibition zone (10.8 ± 1.5 mm) against Candida spp. (n = 8).


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  		Table 2.. Antifungal activity of the essential oil from Lippia sidoides Cham. against Microsporum canis and Candida spp. by the agar-well diffusion assay

 
By the broth microdilution method, it was seen that MICs for M. canis strains (n = 6) ranged from 4 to 70 mg/L and MFCs ranged from 9 to 150 mg/L. The MICs for Candida spp. strains (n = 6) ranged from 620 to 2500 mg/L and the MFCs ranged from 1250 to 5000 mg/L (Table 3).


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  		Table 3.. MIC and MFC of Lippia sidoides Cham. essential oil against Microsporum canis and Candida spp. in the broth microdilution method

 
The oral administration of the essential oil at doses ranging from 100 to 3000 mg/kg did not induce any remarkable alterations in the behaviour pattern of mice. The calculated LD50 (ip) for essential oil of L. sidoides was 117.95 (110.61–125.29) mg/kg.

The subchronic oral administration of L. sidoides essential oil (117.95 mg/kg/day) was devoid of overt toxicity. The body weight, which was not affected by the treatment, was 322.9 ± 18.96 g on day 1 and 328.3 ± 22.67 g on day 30 when compared with 331.1 ± 24.0 g versus 357.2 ± 21.2 g in vehicle-treated animals. Moreover, the serum biochemical parameters observed, i.e. creatinine, urea, GOT and GPT, were not significantly affected (Table 4). The histopathological evaluation of liver, kidneys, lungs, heart and spleen did not reveal any structural alterations in those organs obtained from L. sidoides essential oil-treated animals or in vehicle-treated animals. Similarly, the evaluations of red and white blood cells did not reveal any remarkable sign of haematological toxicity induced by L. sidoides essential oil (Table 5).


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  		Table 4.. Serum biochemical parameters during subchronic oral administration of Lippia sidoides Cham. essential oil

 


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  		Table 5.. Haematological parameters during subchronic oral administration of Lippia sidoides Cham. essential oil

 

   Discussion
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Plant essential oils are a potentially useful source of antimicrobial compounds.11,14 It is often quite difficult to compare the results obtained from different studies, because the compositions of the essential oils can vary greatly depending upon the geographical region, the variety, the age of the plant, the method of drying and the method of extraction of the oil.

In spite of the above-mentioned difficulties, essential oils from medicinal plants are excellent candidates for the development of remedies for many infectious diseases, including mycosis, due to the increasing development of antimicrobial resistance as well as the appearance of undesirable effects of some antifungal agents.11

Early reports on L. sidoides essential oil revealed its antimicrobial action. Lemos et al.22 reported the highest and broadest activity against bacteria and fungi, including yeasts, dermatophytes and non-dermatophyte fungi. The present study shows that the essential oil from L. sidoides is quite effective against M. canis, the most common species of dermatophytes that cause superficial fungal infection in cats and dogs worldwide.2,26 It induced a significant growth inhibition zone at the lower concentration (25 mg/mL), and at concentrations ≥50 mg/mL, this essential oil totally inhibited M. canis grown in culture. The positive control, griseofulvin, induced inhibition zones of 51.6 ± 6.7 mm in the agar-well diffusion method.

Concerning Candida spp., which are important yeasts involved in human and animal mycoses,4,6,10 L. sidoides essential oil induced significant growth inhibition zones varying from 9.8 ± 0.9 to 23.3 ± 1.8 mm. Amphotericin B induced inhibition zones of 10.8 ± 1.5 mm in the agar-well diffusion method.

Previous research has suggested that several essential oils show important in vitro antifungal activity, with varied MIC and MFC values, against dermatophytes, yeasts and other fungi.4,16,17,19,33,34 In this study, the MICs for M. canis strains ranged from 4 to 70 mg/L and the MFCs ranged from 9 to 150 mg/L. The MICs for C. albicans and C. tropicalis ranged from 620 to 2500 mg/L and the MFCs ranged from 1250 to 5000 mg/L. Candida spp. and M. canis strains used by Brito et al.4 and Brilhante et al.,26 respectively, as well as C. parapsilosis ATCC 22 019 and C. krusei ATCC 6528, were used as controls in MIC determinations and the results were within the recommended limits (Candida spp. MIC ≤1 mg/L for amphotericin B and M. canis MIC ≤1 mg/L for griseofulvin).

By the agar-well diffusion and broth microdilution methods, this study shows that the essential oil of L. sidoides causes fungicidal activity. As there is a good correlation between the MICs, MFCs and the agar-well diffusion values of the essential oil of L. sidoides, it may be concluded that the antifungal activity of essential oils could be preliminarily investigated by the agar-well diffusion test for rapid screening.

The antimicrobial activity of essential oils from Achillea setacea,17 Pimpinella anisum,21 Sesuvium portulacastrum,18 Melaleuca alternifolia,11,19,20 Juniperus spp.,16 Allium spp.14,15 and Thymus spp.32,33 is well known. The results obtained in the present research were very important to include the L. sidoides Cham. in this list of plants with antifungal activity.

Concerning Malassezia pachydermatis, which is the most common yeast in dermatitis and otitis externa in dogs,5 although it was not the aim of this research, the essential oil from L. sidoides Cham. was also tested for antifungal activity against this yeast, in vitro, by the agar-well diffusion method. Our preliminary data showed that essential oil from L. sidoides was effective in a dose-related way, being, at the lower dose used (25 mg/mL), as efficient (growth inhibition zone of 30.0 ± 10.0 mm; n = 10; data not shown) as the positive control itraconazole (29.7 ± 9.0 mm; n = 10; data not shown). Therefore, these data reinforce the potential antifungal activity of this essential oil.

To identify the composition of the oil from L. sidoides Cham., the oil derived from steam distillation was analysed by gas chromatography/mass spectroscopy. The main component was thymol (59.65%). The main constituents of essential oils, which show important antifungal activity, are phenolic compounds (terpenoids and phenylpropanoids), such as thymol, carvacrol or eugenol, of which antimicrobial activity is well documented.16 Therefore, the activity of the essential oil from L. sidoides against Candida spp., M. canis and M. pachydermatis may partly be explained by the high amounts of thymol, which was previously reported to be effective as an antifungal.32,33

Regarding pharmacokinetic studies with the essential oil from L. sidoides, this research was limited in this field. However, owing to its high liposolubility, it would appear that the absorption of this essential oil after oral or ip administration would not be impaired, as can be confirmed by the LD50 experimental protocol (LD50 = 117.95 mg/kg). Corroborating the methodology used for the evaluation of the acute and subchronic toxicity performed in this research, other studies have used a similar strategy for the toxicological study of essential oils.34,35 In addition, a study using thymol has shown that thymol sulphate and thymol glucuronide can be detected, after a single oral administration, for 24 h in urine and 41 h in plasma.36

The use of the essential oil from L. sidoides did not induce any significant acute toxicological alterations in the mice. The subchronic daily administration of L. sidoides essential oil for 30 days (oral administration) did not induce any remarkable alterations in the biochemical or haematological parameters analysed and nor was there any increase in the weight or structural pattern of the main organs, as revealed by the histopathological analysis. Although additional tests, such as reproductive toxicity analysis and cytotoxic and mutagenesis evaluation, must be performed, the present results show that L. sidoides essential oil is probably safe for acute use in vivo.

In this preclinical phase, the crude essential oil from L. sidoides Cham. administered by oral or ip route was evaluated to determine whether it induces any toxicity (physical, behavioural, biochemical, haematological or histopathological changes) after acute or subchronic experiments. Thus, the results obtained in this stage will certainly be helpful in future clinical studies, where specific tests will be performed to establish the safe profile of this essential oil for clinical use.

Owing to its broad spectrum of antifungal effect, in vitro, and low toxicity, the essential oil of L. sidoides Cham. is a promising source in the search for new antifungal drugs. However, specific pharmacological approaches will be needed in future clinical trials to validate its use as a phytotherapeutic product.


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None to declare.


   Acknowledgements
 
We are thankful for the financial support of FUNCAP (Ceará State Research Funding) and CNPq (National Council for Technological and Scientific Development, Brazil, Proc. CNPq: 478906/2004-8).


   References
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