Antifungal Activity, Toxicity and Chemical Composition of the Essential Oil of Coriandrum sativum L. Fruits

The aims of this study were to test the antifungal activity, toxicity and chemical composition of essential oil from C. sativum L. fruits. The essential oil, obtained by hydro-distillation, was analyzed by gas chromatography/mass spectroscopy. Linalool was the main constituent (58.22%). The oil was considered bioactive, showing an LC50 value of 23 µg/mL in the Artemia salina lethality test. The antifungal activity was evaluated against Microsporum canis and Candida spp. by the agar-well diffusion method and the minimum inhibitory concentration (MIC) and the minimum fungicidal concentration (MFC) were established by the broth microdilution method. The essential oil induced growth inhibition zones of 28 ± 5.42 and 9.25 ± 0.5 for M. canis and Candida spp. respectively. The MICs and MFCs for M. canis strains ranged from 78 to 620 and 150 to 1,250 µg/mL, and the MICs and MFCs for Candida spp strains ranged from 310 to 620 and 620 to 1,250 µg/mL, respectively. C. sativum essential oil is active in vitro against M. canis and Candida spp. demonstrating good antifungal activity.


Introduction
Dermatophytosis is one of the most frequent skin diseases of pets and livestock. Contagion among animal communities, high treatment cost, difficulties of control measures and public health consequences of animal ringworm are all factors urging the study of these fungi [1]. Fungal disease agents are widespread and can be isolated from a wide range of sick animals or asymptomatic carriers, which can represent important reservoirs for people in close contact with them. This situation should be considered as an important risk factor for those with impaired immune systems and anyone working with or handling animals. The incidence of dermatophytosis caused by Microsporum canis is increasing in human patients in many places around the World, including several Brazilian cities, and it is often the predominant fungus seen in dermatological clinics [2].
Yeasts of the Candida genera can be found as commensal microorganisms in animals and are considered one of the most important species in veterinary medicine. Strains of Candida spp. isolated from dogs showed high resistance to azole antifungal agents [3]. Although effective antimicrobials have been developed over the years, there has been increased development of antimicrobial drug resistance to currently available antimicrobials [4]. Many essential oils and plant extracts used in therapy have advantages over antibiotics, although the latter are more effective [5].
The essential oil from leaves of C. sativum showed antimicrobial activity against both Gram positive and Gram negative bacteria. This plant is known not to be toxic because it has been consumed for centuries without showing any signs of toxicity [6].
Due to known antibacterial activity of the leaf essential oil of C. sativum, the aim of this study was to evaluate the antifungal activity of the essential oil from fruits by the agar diffusion and microdilution methods, and to determine its main chemical constituents. Also, to evaluate the potential use of C. sativum essential oil as a phytotherapic product, the toxicity was investigated using the Artemia salina lethality test.

Results and Discussion
The chemical analysis of the C. sativum is shown in Table 1. The main constituents of the essential oil of C. sativum were linalool (58.65%), geraniol (17.87%) and neryl acetate (12.22%). The brine shrimp lethality test of the essential oil showed an LC 50 of 23 µg·mL −1 , being considered bioactive, and the estimated LD 50 obtained by linear regression was 2,139.98 mg/kg. * Retention index. The identified constituents are listed in their order of elution from a nonpolar column; ** The % composition is the % peak area of the total essential oil composition.
The essential oil from C. sativum fruits was effective against all tested fungal strains in the agar-well diffusion susceptibility tests ( Table 2). The oil induced a significant growth inhibition zone (28 ± 5.42 mm) at a concentration of 10,000 µg/mL against M. canis strains (n = 4). For Candida strains (n = 4), the growth inhibition zone induced by the oil was 9.25 ± 0.5 mm, at a concentration of 10,000 µg/mL. The positive control, griseofulvine, induced a significant growth inhibition zone (55.25 ± 3.69 mm) against M. canis and amphotericin B induced a significant growth inhibition zone (10.25 ± 1.26 mm) against Candida spp.
The broth microdilution method showed that the MICs for M. canis strains (n = 5) ranged from 78 to 620 µg/mL and the MFCs ranged from 150 to 1,250 µg/mL. The MICs for Candida spp. strains (n = 5) ranged from 310 to 620 µg/mL and the MFCs varied from 620 to 1,250 µg/mL (Table 3).
Many essential oils have been advocated for use in complementary medicine for bacterial and fungal infections [7]. Previous studies have investigated the activity of essential oils against dermatophytes and yeasts [5][6][7]. In our ongoing search for new antimicrobial agents, the essential oil of C. sativum was tested against animal fungal strains and produced good results.
The antifungal activity of the essential oil of C. sativum fruits may be attributed to its main constituents, linalool (58.22%) and geraniol (17.87%). Previous studies of the chemical composition of C. sativum fruit essential oil carried out by Pino et al. [8] and Burt [9] reported linalool concentrations of 54.57% and 70%, respectively. Antimicrobial activity of linalool against several bacteria and fungi has also been reported [10][11][12]. Dorman and Deans [13] tested the antimicrobial activity of some essential oils and their main chemical constituents. Among them, linalool and geraniol were individually effective against 23 different bacterial strains.  Linalool and linalyl acetate are monoterpenoid compounds that are common in many essential oils of several aromatic species. A number of linalool and linalyl acetate producing species are used in traditional medicinal systems to relieve symptoms and cure a variety of ailments, both acute and chronic [14].
The essential oil from C. sativum leaves showed antimicrobial activity against Gram positive (Staphylococcus aureus, Bacillus spp.) and Gram negative bacteria (Escherichia coli, Salmonella typhi, Klebsiella pneumonia, Proteus mirabilis) and a pathogenic fungus (Candida albicans). The main constituents revealed by chemical analysis were 2(E)-decenal (15.9%), decanal (14.3%), 2(E)-decen-1-ol (14.2%) and n-decanol (13.6%) [6]. According to these authors, the MIC against C. albicans was 163 mg/mL and the oil from fruits in that study was 434 µg·mL −1 . The essential oil of C. sativum fruits exhibits higher antifungal activity against Candida spp. Begnami et al. [15] reported the antifungal activity of this plant against different Candida species. They suggested that the essential oil of this plant could be used as potential antimicrobial agents to treat or prevent Candida yeast infections. This is corroborated by our findings. Silva et al. [16] found an increasing incidence of drug-resistant pathogens and toxicity of existing antifungal compounds while studying the synergism of this essential oil with amphotericin B against Candida species. Their results suggest that the essential oil of C. sativum could be useful in designing new formulations for candidosis treatment.
In veterinary practice, dermatophytoses are among the most common infectious skin diseases in mammals worldwide. They are frequently observed in domestic animals, but also in captive and wild fauna [1]. Based on the results of this study, the essential oil obtained from fruits of C. sativum could be an alternative natural source to treat animal dermatophytoses.
In the evaluation of plant extract toxicity by the brine shrimp bioassay, an LC 50 value lower than 1,000 µg/mL is considered bioactive [17]. In this study, the essential oil from C. sativum fruits showed an LC 50 value of 23 µg/mL. This result corroborates the antifungal properties of the oil. Parra et al. [18] assessed the effect of acute treatment of A. salina larvae and mice with several extracts drawn from autochthonous plants in Cuba. The aim of their study was to develop a low-cost method applicable to countries where the use of medicines obtained from vegetable species is common and is an affordable way to fight diseases. They calculated LC 50 values for A. salina larvae and LD 50 values for mice and established significant correlations between both parameters, suggesting the use of A. salina larvae as a suitable, accurate and inexpensive alternative to pre-screening chemical toxicity with mammals [19]. The estimated LD 50 in mice for C. sativum essential oil was 2,139.98 mg/kg and this value indicates a low toxicity in accordance with Hedge and Sterner [20].

Plant Material and Extraction of Essential Oils
The fruits used for extraction of the essential oil were harvested from plants cultivated in the Medicinal Plants Orchard of State University of Ceará, from commercial seeds produced by ISLA Sementes Ltda. (Porto Alegre, RS, Brazil). The C. sativum essential oil was extracted by the hydro-distillation method in a modified Clevenger apparatus, as described by Craveiro et al. [21].

Gas-Chromatography/Mass Spectral (GC-MS) Analysis
The chemical analysis of the essential oil constituents was performed on a Shimadzu QP-2010 instrument employing the following conditions: column: DB-5ms (Agilent, part No. 122-5532) coated fused silica capillary column (30 m × 0.25 mm × 0.25 µm); carrier gas: He (1 mL/min, in constant linear velocity mode); injector temperature was 250 °C, in split mode (1:100), and the detector temperature was 250 °C. The column temperature programming was 35 to 180 °C at 4 °C/min then 180 to 280 °C at 17 °C/min, and at 280 °C for 10 min; mass spectra: electron impact 70 eV. The injected sample volume was 1 µL. Compounds were identified by their GC retention times relative to know compounds and by comparison of their mass spectra with those present in the computer data bank (National Institute for Standard Technology-NIST-147, 198 compounds) and published spectra [22,23].

Brine Shrimp Lethality Bioassay
The essential oil of C. sativum was assayed using a modified test of lethality to A. salina [17]. The eggs of A. salina were incubated in a hatching chamber with seawater and kept at room temperature (average 27 °C) under artificial light around the clock. Larvae after 48 h were extracted and counted using a Pasteur pipette. A standard solution of 1,000 µg/mL was prepared with 100 mg of essential oil diluted in 1.0 mL of DMSO, and the volume was completed with seawater in a 100 mL volumetric flask. Concentrations of 900, 100, 10 and 1 µg/mL were prepared using standard solution. For each concentration, 10 brine shrimp larvae were used, placed in flasks that were filled with seawater to a total volume of 5 mL. Intermediate concentrations were made to calculate the LC 50 . For the control group, a solution was prepared with 100 µL of DMSO and 4.9 mL of seawater. After 24 h, the dead larvae were counted and the LC 50 value was estimated using the Origin 7.0 statistical program.

LD 50 Estimate Calculation for C. sativum Essential Oil
The LD 50 value was based on the comparative study of the assay of A. salina and the lethal dose (LD 50 ) value in mice, to determine acute oral toxicity of plant extracts [18]. The published LC 50 and LD 50 values of the extracts were correlated using the Origin 7.0 statistical program to obtain the linear regression equation Y = 169.57 + 85.67X (R = 0.86), where Y is the LD 50 value, X is the LC 50 value and R is the correlation coefficient. The LD 50 figure was expressed in mg/Kg.

Fungal Strains
A total of five strains of M. canis and five strains of Candida spp. were included in this study. Both M. canis and Candida spp. strains were isolated from symptomatic dogs and cats. The strains were stored in the fungal collection of the Specialized Medical Mycology Center-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 into potato dextrose agar (Difco, Detroit, MI, USA), and then incubated at 28 °C for 2-10 days.

Inoculum Preparation for Antifungal Susceptibility Tests
For the agar-well difusion method, based on Fontenelle et al. [5], 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 suspensions with blastoconidia of Candida spp. or suspension of hyphal fragments of M. canis were transferred to a sterile tube and adjusted by turbidimetry to obtain inocula of approximately 10 6 cfu/mL blastoconidia of Candida spp. and 10 5 cfu/mL hyphal fragments or conidia of M. canis. The optical densities of the suspensions were spectrophotometrically determined at 530 nm and then adjusted to 95% transmittance.
For the broth microdilution method, standardized inocula (2.5-5 × 10 3 cfu/mL for Candida spp. and 5 × 10 4 cfu/mL for M. canis) were also prepared by turbidimetry. Stock inocula were prepared on day 2 and day 10 for Candida spp. and M. canis cultures, respectively, grown on potato dextrose agar at 28 C. Sterile normal saline solution (0.9%; 3 mL) was added to the agar slant and the cultures were gently swabbed to dislodge the conidia from the hyphal mat for the M. canis [24] and the blastoconidia from Candida spp. [3]. The suspensions of conidia with hyphal fragments of M. canis and blastoconidia suspension of Candida spp. were transferred to a sterile tubes, and the volume of both suspensions adjusted to 4 mL with sterile saline solution. The resulting suspension were allowed to settle for 5 min at 28 C, and their density was read at 530 nm and the adjusted to 95% transmittance. The suspensions were diluted to 1:2,000 for Candida spp. and 1:500 for M. canis, both with RPMI 1640 medium (Roswell Park Memorial Institute-1640) with L-glutamine, without sodium bicarbonate (Sigma Chemical Co., St. Louis, MO, USA), buffered to pH 7.0 with 0.165 M morpholinopropanesulfonic acid (MOPS) (Sigma Chemical Co.), to obtain the inoculum size of approximately 2.5-5 × 10 3 cfu/mL for Candida spp. and 5 × 10 4 cfu/mL for M. canis.

Agar-Well Diffusion Susceptibility Test
The antifungal activity of essential oil from C. sativum was evaluated against Candida spp. (n = 4) and M. canis (n = 4), by the agar-well diffusion method according to Fontenelle et al. [5]. Petri dishes with 15 cm diameter were prepared with potato dextrose agar (Difco). The wells (6 mm in diameter) were then cut from the agar and 100 µL of essential oil was delivered into them. The oil was weighed and dissolved in DMSO to obtain the test concentration of 10,000 µg/mL. Stock solutions of griseofulvin (1,000 µg/mL; Sigma Chemical Co.) and amphotericin B (5 µg/mL; 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 on to the surface of the agar. After incubation, for 3-5 days for Candida spp. and 5-8 days for M. canis, at 28 °C, all dishes were examined for zones of growth inhibition and the diameters of these zones were measured in millimeters. Each experiment was repeated at least twice.

Broth Microdilution Method
The minimum inhibitory concentration (MIC) for Candida spp. was determined by the broth microdilution method, in accordance with the Clinical and Laboratory Standards Institute-CLSI (formerly NCCLS; M27-A2), [25]. The broth microdilution assay for M. canis was performed as described by Brilhante et al. [24], based on the M38-A document (CLSI; formerly NCCLS, 2002) [26]. The minimum fungicidal concentration (MFC) for both Candida spp. and M. canis were determined according Fontenelle et al. [5]. In addition, C. parapsilosis (ATCC 22019) and C. albicans (ATCC 1023) strains were used as quality controls for broth microdilution method.
The essential oil of C. sativum was prepared in DMSO. Amphotericin B (AMB) (Sigma Chemical Co.) and griseofulvine (Sigma Chemical Co.) were prepared in distilled water. For the susceptibility analysis, the essential oils were tested in concentrations ranging from 4 to 5,000 µg/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. The assays for all essential oils were run in duplicate and repeated at least twice. The MIC was defined as the lowest oil concentration that caused 100% inhibition of visible fungal growth. The results were read visually as recommended by 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.

Statistical Analysis
Antifungal activity was expressed as mean ± SD of the diameter of the growth inhibition zones (mm). The antifungal activity of the essential oils was analyzed by linear correlation for individual analysis and the two-tailed Student's t-test at 95% confidence intervals was used to evaluate differences between the essential oil and the controls. For the brine shrimp lethality bioassay, the LC 50 and the LC 50 values were estimated using the Origin 7.0 statistical program.

Conclusions
Owing to its broad spectrum of antifungal effect, in vitro, and low toxicity, the essential oil of C. sativum is a promising source in the search for new antifungal drugs. However, it is necessary to evaluate the acute toxicological effects and antifungal efficacy in vivo in order to be considered for a safe and effective antifungal agent.