Antifungal Activity of the Essential Oil of Echinops kebericho Mesfin: An In Vitro Study

Background Echinops kebericho is an endemic medicinal plant in Ethiopia widely used in the treatment of infectious and noninfectious diseases. Essential oils are known for their antibacterial, antifungal, antiviral, insecticidal, and antioxidant properties. This study evaluated the antifungal activity of essential oil from E. kebericho against four common pathogenic fungi and two standard strains. Methods The essential oil was obtained by hydrodistillation. The antifungal screening was done by agar well diffusion method. Minimal inhibitory concentrations (MICs) were determined by broth microdilution. Minimal fungicidal concentrations (MFCs) were determined by subculturing fungal strains with no visible growth onto a Sabouraud dextrose agar (SDA) plate. Results Candida albicans and Cryptococcus neoformans were highly sensitive while Aspergillus flavus did not show sensitivity up to 1 mg/ml of essential oil; MICs ranged from 0.083 mg/ml to 0.208 mg/ml. Concentration and fungal species showed significant dose-dependent associations (p < 0.0001) with antifungal activity. The MICs of essential oil were comparable to those of the standard drug (fluconazole) against C. glabrata and C. krusei. The lowest MFC of the essential oil was observed against Candida parapsilosis (0.145 mg/ml) while the highest MFC was against Candida krusei (0.667 mg/ml). Conclusion Echinops kebericho essential oil showed noteworthy antifungal activity against Cryptococcus neoformans, Candida albicans, and Candida glabrata and could be a potential candidate for further antifungal drug development.


Background
Fungi are eukaryotic organisms that cause a range of clinical infections including skin, hair, nail, mucosal, subcutaneous, and invasive infections. e majority of fungi cause opportunistic infections in immune-compromised people or individuals taking immune suppressive drugs [1,2]. ese include people with HIV/ AIDS, primary immune deficiency, cancer chemotherapy, hematologic and solid organ transplantation, and prematurity and those on immune-modulatory medications [3]. More than 90% of reported fungal-associated deaths are caused by species belonging to three genera: Cryptococcus, Candida, and Aspergillus [4]. e burden of fungal infections is alarming in the world: more than 10 million people have mucosal candidiasis and more than 150 million people have serious fungal diseases, which have a major impact on their lives or are fatal [5]. Mortality associated with a fungal disease is more than 1.6 million, similar to that of tuberculosis and over 3-fold more than malaria [6]. e major concern of fungal infections is the high rate of mortality associated with invasive fungal infections, which often exceeds 50% despite the availability of antifungal drugs [4].
is is due to the emergence of widespread drug resistance against antifungal agents that limits treatment options [7]. Antifungal treatment options are highly limited, as there are few chemical classes represented by existing antifungal drugs [4]. To date, there are three different classes of antifungal agents, polyenes, azoles, and echinocandins [8]. Drug toxicity is the major limitation of polyenes and the older generation of azole antifungal agents. New antifungal agents (e.g., echinocandins and second-generation triazoles) in the past decade have transformed the management of invasive mycoses. ese newer antifungal agents have also important limitations such as in pharmacokinetics, drugdrug interactions, and unusual toxicities associated with long-term use [9].
Echinops kebericho is an endemic medicinal plant widely used in Ethiopia. It is used to treat different infectious and noninfectious diseases such as fever, headache, stomachache, malaria, and cough in the community [10]. Extracts and essential oils of the roots of E. kebericho have been assessed for their antimicrobial [11], anthelminthic [12], and molluscicidal activities [13].
Essential oils are known for their antibacterial, antifungal, antiviral, insecticidal, and antioxidant properties [14]. ey are widely used in traditional medicine and the food industry. e main constituents of essential oils are terpenes, terpenoids, aromatic and aliphatic constituents, and some hydrocarbons, which also exhibit antimicrobial effects [15]. Studies have shown that essential oils target the cell wall membrane, thereby disturbing ATP production and pH homeostasis [16]. A study conducted by Ameya et al. on the crude methanol and ethanol extracts of E. kebericho demonstrated activity against clinical isolates of Candida albicans and Aspergillus flavus [11]. ough the research was done on the crude extract against the two strains, the effect on the other fungal strains is unknown, and there has been no study on the essential oil of E. kebericho. e aim of this study, therefore, was to evaluate the antifungal activity of the essential oil of E. kebericho M. against Candida albicans, Candida glabrata, Cryptococcus neoformans, and Aspergillus flavus.

Study Design and Setting.
is was an in vitro experimental study. e extraction of the plant essential oil was carried out at the Pharmacognosy Laboratory at Addis Ababa University. Determination of in vitro activity was done at the Mycology Unit of the Microbiology Laboratory at Mbarara University of Science and Technology. Clinical fungal strains of C. albicans, C. glabrata, C. neoformans, and A. flavus and quality control strains, C. parapsilosis ATCC 22019 and C. krusei ATCC 6258, were used for this study.

Plant Collection and Extraction.
Fresh tubers of E. kebericho were collected from areas around Addis Ababa, Ethiopia. A taxonomist identified the plant and a voucher number of 001TB/19 was given and deposited at Addis Ababa University (AAU) Herbarium. e tubers were separated from the rest of the plant parts and washed. After air-drying in shade, the tuber material was pounded using a mortar and pestle to a fine powder; then 500 gm of the powder was dissolved in water in a 1 : 5 (weight-by-volume) ratio in a round-bottom flask and hydrodistilled for 3 hours using a Clevenger apparatus [17]. e volume of the essential oil obtained in the receiver was measured and stored in the refrigerator until used.

Preparation of Essential Oil for Antifungal Activity
Assay. Essential oil (0.1 gram) was dissolved in 8 ml of 1% DMSO and filled up to a total solution of 10 ml with DMSO. A twofold dilution was prepared from this stock solution, which was filled in each well [18]. e EO of 10 mg/ml to 0.038 mg/ml concentration was used for the antifungal test.

Well Diffusion Assay.
Well diffusion assay for Candida spp. and Cryptococcus spp. was conducted according to M44-A Standard [19,20]. Sabouraud's dextrose agar (SDA) media for yeast and potato dextrose agar were used for the subculturing of the A. flavus [20,21]. Mueller-Hinton agar supplemented with 2% glucose and 0.5 μg/ml methylene blue dye medium was used for testing of Candida spp. and Cryptococcus spp. [20]. Nonsupplemented Mueller-Hinton agar was used for testing of the A. flavus [21]. Fluconazole (0.25 mg/ml) was used as a positive control for Candida spp. and Cryptococcus spp. [22]. Amphotericin B (0.1 mg/ml) was used as a positive control for A. flavus [21]. For the negative control, 1% DMSO was used [11]. After the inoculation of the organisms, 8 mm wells were created and filled with 100 μl of different concentrations of essential oil. Positive control (100 μl) and negative control were also added [2]. All the plates were prepared in triplicate and incubated at 35°C for 24 hours for Candida spp. and 48 hours for Cryptococcus spp. At the end of the incubation period, the diameter of the zone of inhibition in each of the inoculated plates was measured and the diameter of the well was subtracted to determine the antifungal activities of the essential oil [2,11,18].

Broth Microdilution.
Broth microdilution was used to determine the minimum inhibitory concentration (MIC). Sabouraud's dextrose agar (SDA) media were used to subculture yeast following the recommendation of 2 Evidence-Based Complementary and Alternative Medicine international guidelines [23]; for A. flavus, potato dextrose agar (PDA) media were used [21,24]. e essential oil was prepared in serial dilutions in dimethyl sulphoxide (DMSO). 100 mg of E. kebericho essential oil was measured and DMSO was added to 1 ml total solution forming 100 mg/ml. Serial dilution was made in twofold dilution (1.56 mg/ ml-100 mg/ml). Each serially diluted essential oil was further diluted in a ratio of 1 : 50 in RPMI media (100 μl of stock solution was added to 4.9 ml of the RPMI), giving dilutions in the range of 0.03125 to 2 mg/ml. 100 μl from each dilution was filled in the 96-well plates in accordance with CLSI protocol. e essential oil in the well was further diluted when the inoculum was added to the well to obtain dilutions in the range of 0.0156 mg/ml-1 mg/ml [23]. Fluconazole and amphotericin B were used as standard drugs for yeasts and Aspergillus, respectively. Twofold serial dilutions ranging from 0.25 to 128 μg/ml for fluconazole and 0.031 to 16 μg/ml for amphotericin B were used as a positive control; 1% DMSO was used as a negative control. Quality was controlled by using quality control strains: C. parapsilosis (ATCC 90018) and C. krusei (ATCC 6258) [25]. e MIC was the lowest concentration that inhibited the visible growth of the fungi.

Inoculum Preparation.
e inoculum was prepared according to CLSI guidelines [23,24]: C. albicans, C. glabrata, C. neoformans, C. parapsilosis ATCC 22019, and C. krusei ATCC 6258 were subcultured on SDA media, while A. flavus was subcultured on PDA media. e inoculum was prepared from 24-hour-old colonies of Candida species. Approximately 1 mm diameter was suspended in 5 ml of saline and adjusted to 0.5 McFarland Standard. For Cryptococcus, the 72-hour-old colonies were suspended in 5 ml of saline and adjusted to 0.5 McFarland standards. e prepared suspension was vortexed for 15 minutes and the cell density was adjusted to 0.5 McFarland standards. is suspension was mixed for 15 seconds with a vortex, diluted to 1 : 50, and further diluted to 1 : 20 with RPMI medium to obtain the twofold test inoculum. e (twofold) inoculum was diluted when the wells were inoculated [23]. For A. flavus, the spores were harvested from 7-day-old cultures on PDA.
e saline was poured into the PDA slant and mixed with a sterile swab. After 10 minutes of sedimentation, the suspension was transferred to a sterile test tube and adjusted to the 0.5 McFarland standards. is suspension was further diluted to 1 : 50 (100 μl of 0.5 McFarland Standard was added to 4.9 ml RPMI media); 100 μl of the diluted was then added to the well [24]. e minimal fungicidal concentrations (MFCs) were determined by subculturing fungal isolates from the wells with no visible growth onto SDA plates. e inoculated plates were incubated for 3 days at 35°C. MFC was the lowest concentration of drug that yielded a negative subculture [23,26].

Statistical Analysis.
e results were graphically presented as mean ± SD. Linear regression was used to determine the relationship between the concentration of the essential oil as an independent variable and zone of inhibition (response) as a dependent variable. Two-way analysis of variance was used to compare the effect of treatment using EO on the test microorganisms and to examine the difference in MIC of essential oil at different concentrations and fluconazole for each test strain. e independent variables were fungal strain and treatment (essential oil, fluconazole, and negative control). p value <0.05 was considered statistically significant. Statistical analysis was done using GraphPad Prism 5.01 software.

Ethical Considerations.
e research protocol was approved by the Research Ethics Committee (REC) of Mbarara University of Science Technology (approval number 05/01-19).

Results
e percentage yield of the essential oil was 0.18% w/v of dried weight.

Agar Well Diffusion Assay.
In agar well diffusion, wells filled with 100 μl of different concentrations of EO produced inhibition zones ranging from 3 mm to 17 mm. e largest diameter was against C. neoformans and the lowest was against C. krusei ATCC 6258. e zone of inhibition of controls varied from 10 ± 2 mm to 22 ± 2 mm. e lowest diameter was produced by A. flavus and the widest was produced by C. albicans (Table 1). Two-way analysis of variance showed that both concentration and fungal species had significant effects (p < 0.0001) on the zone of inhibition (Table 1); for concentration, F � 39.08, df � 9, p < 0.0001; and for fungal species, F � 32.41, df � 4, p < 0.0001.

Minimum Inhibitory Concentration.
e evaluation of the MIC of E. kebericho essential oil showed notable activity against all fungal strains tested except for A. flavus. Candida parapsilosis (ATCC 22019) and Candida krusei (ATCC 6258) which were used as quality controls had MICs in the recommended ranges. e MIC ranged from 0.083 mg/ml to 0.208 mg/ml. Candida albicans and Cryptococcus neoformans were highly sensitive, showing comparable activity to C. parapsilosis. Aspergillus flavus did not show sensitivity up to 1 mg/ml of the essential oil. ere were no significant differences in MIC of essential oil and fluconazole against yeast (p value >0.05).
e essential oil showed activity comparable to that of the standard drug (fluconazole) against C. glabrata and C. krusei (Figure 1).
All the strains tested were susceptible to the standard drug (fluconazole for Candida species and Cryptococcus neoformans; amphotericin B for A. flavus). Linear regression showed that there was a positive relationship between concentration and zone of inhibition (Figure 2). e slopes for all, except A. flavus, were positive. e essential oil did not show a zone of inhibition against the isolate of A. flavus. e negative control (DMSO) did not show a zone of inhibition.

Minimum Fungicidal Concentration.
e essential oil showed the lowest MFC against C. parapsilosis and the highest MFC against C. krusei (Figure 3). e essential oil, however, did not show a fungicidal effect against A. flavus and C. albicans in the range of concentrations tested.

Discussion
e present study investigated the antifungal activity of the essential oil of E. kebericho on highly pathogenic invasive fungal strains, C. albicans, C. glabrata, C. neoformans, and A. flavus and quality control strains C. parapsilosis and C. krusei.
e study demonstrated noteworthy antifungal activity of the essential oil of E. kebericho against the fungal strains: C. neoformans was highly sensitive to the essential oil of E. kebericho, followed by C. albicans, while C. glabrata was moderately sensitive. Aspergillus flavus, however, was not sensitive at all the tested concentrations. e zone of inhibition diameter decreased as the concentration of the essential oil decreased, showing a dose-dependent effect. e dose-dependent effect was similar to a previous study conducted with peppermint oil against fungal strains [27]. Fungicidal effects were observed against C. glabrata,   strain. e straight line shows the line of best fit. e slope was significantly nonzero for all the strains, except C. albicans (p � 0.0091 for C. krusei; p � 0.0193 for C. glabrata; p � 0.0075 for C. parapsilosis; p � 0.0117 for C. neoformans; and p � 0.0626 for C. albicans). e R 2 for C. krusei was 0.6452, for C. glabrata was 0.5660, for C. parapsilosis was 0.6633, for C. neoformans was 0.6210, and for C. albicans was 0.4115. Values shown are means ± SD; n � 3 replicates. 4 Evidence-Based Complementary and Alternative Medicine C. neoformans, C. krusei ATCC 6258, and C. parapsilosis ATCC 22019. Screening of medicinal plants for antifungal activity is important for novel drug discovery [28]. In many countries, fungal diseases have been treated using herbal remedies as many pathogenic fungi are becoming commonly resistant to numerous conventional antifungal drugs [29]. is study is the first of its kind to investigate the antifungal activity of essential oil from E. kebericho, although a previous study showed noteworthy antifungal activity using methanolic and ethanolic crude extracts of E. kebericho against C. albicans and A. flavus [11]. e activity of E. kebericho essential oil against the fungal strains studied varied with the fungal strain: better activity was observed against C. neoformans than the other strains. In a previous study, the essential oil of Lavandula viridis L. and Zingiberaceae species (Z. officinale and Z. cassumunar) showed noteworthy activity against C. neoformans. For C. krusei [30], a comparable result was also observed with the essential oil of Cinnamomum zeylanicum Blume [31]. Higher MIC was observed for C. krusei on control (fluconazole) and available antifungals [32]. A relatively higher MIC was also seen with C. glabrata, although it was less than that seen with C. Krusei.
is was in agreement with a study conducted with the essential oil of Artemisia sieberi and Origanum vulgare [33]. Candida krusei is a multidrug-resistant fungal pathogen showing resistance with amphotericin B or fluconazole [34]. A study conducted on the essential oil of yme, Summer savory, and Clove against A. flavus, however, showed good antifungal activity against A. flavus [35]. is difference could be due to differences in the chemical constituents of essential oil between the plants, geographical variations, and/or genetic differences of the isolates used in the study [33]. e essential oil of E. kebericho possessed fungicidal activity against C. glabrata, C. parapsilosis, C. krusei, and C. neoformans, a finding also seen in a study conducted on ymus x viciosoi, Angelica major, and Artemisia sieberi essential oil [33,36,37]. E. kebericho essential oil, however, showed MFC >1 mg/ml against C. albicans. In a recent study conducted on Pistacia vera L., the essential oil showed fungicidal effect against C. albicans at a concentration greater than 1 mg/ml, a finding which is in agreement with the present study [38]. In another study conducted by Devkatte et al. [39] on essential oils as potential inhibitors of C. albicans growth, out of 38 herbal oils, 9 oils (orange oil, rosemary oil, bergamot oil, clary sage oil, juniper oil, ginger oil, rose oil, Citronella oil, and Eucalyptus oil) also showed higher MFCs [39].
An analysis of the chemical composition of the essential oil of E. kebericho by Hymete et al. revealed sesquiterpenoid compounds as the main component [17]. ese compounds could be responsible for the antifungal activity because of their highly lipophilic nature, their low molecular weights, and their capability of disrupting the cell membrane, causing cell death or inhibiting sporulation and germination [40,41].
Two basic methods for the assessment of antimicrobial activities of essential oils are commonly used: the agar diffusion method (paper disc or well) and the broth dilution method. Although the use of microdilution in determining the activity of essential oil is the best way, agar diffusion could be utilized as a screening method, or in combination with a microdilution assay [2,42]. In the present study, we used the microdilution method for the determination of MIC and MFC [2,43]. Most of the antimicrobial molecules in plant extracts are significantly nonpolar and do not diffuse well in the aqueous agar matrix used in agar diffusion. e minimal inhibitory concentration (MIC) method remains the preferred one in the determination of the antimicrobial activity of plant extracts [44].
is study had a number of limitations. First, the study was in vitro and did not reflect the actual therapeutic effect that may arise from variations in pharmacokinetic and pharmacodynamic in vivo models. Second, the volatility of the essential oils could have reduced the activity. ird, the chemical composition of the essential was not studied though it has previously been studied. However, this study provides a broader insight for further in vivo studies that would also include specific experiments involving the isolated and identified active components of the essential oil of E. kebericho.

Conclusion and Recommendations
In the current study, the essential oil of E. kebericho showed noteworthy activity against C. neoformans and C. albicans. e activity was dose-dependent and fungicidal against C. glabrata, C. neoformans, C. parapsilosis, and C. krusei. Further studies are warranted to evaluate the effectiveness of the essential oil in vivo, elaborate the mechanism of action, and isolate and identify the most active component(s). e activity of the combination of the essential oil with conventional antifungal drugs should also be investigated.   Data Availability e datasets used and/or analyzed during the current study are available from the corresponding author on request.

Conflicts of Interest
e authors declare that they have no conflicts of interest.

Authors' Contributions
TBB, SD, and PEA conceived and developed the research idea and designed the project; TBB and SD collected the plant material and extracted the essential oil; TBB conducted the experiment; PEA mentored and supervised the study process, analyzed data, and provided critical revisions to the manuscript. All authors read and approved the final manuscript.