EVALUATION OF ANTICANDIDAL EFFECTS OF ESSENTIAL OILS OF COMMERCIAL LAVENDER ( Lavandula angustifolia Miller) IN COMBINATION WITH KETOCONAZOLE AGAINTS SOME Candida Berkhout STRAINS

: Application of combination studies with essential oils and standard antifungal drugs may reduce adverse effects of synthetic drugs and serve as alternative approach against various pathologies including candidiasis. The aim of the present study was to determine the interaction of two commercial lavender ( Lavandula angustifolia Miller) essential oils in combination with ketoconazole against clinical and standard strains of the human pathogens Candida albicans ATCC 10231, C. krusei NRRL Y-7179 and C. glabrata ATCC 66032. The chemical compositions of the investigated essential oils were confirmed both by gas chromatography/flame ionization detector (GC/FID) and gas chromatography/mass spectrometry (GC/MS) methods. Anticandidal activities of the essential oils were screened in vitro by the microdilution method. The resulting interaction of combining lavender essential oils and ketoconazole was tested using the checkerboard method. The results showed that the interaction between essential oils and ketoconazole revealed additive and indifferent effects against the tested strains. In conclusion, the effects observed by the combination of lavender essential oils and ketoconazole may be encouraging to be used against pathogenic Candida strains.


Introduction
Infectious diseases have recently increased the prolonged use of various antifungal drugs, contributing to the development of drug resistance against some species of the fungal genus Candida Berkhout, such as the azoleresistant Candida albicans (Robin) Berkhout strains along with C. krusei. In addition, many cases have been attributed to Candida infections particularly in persons with hematologic malignencies and transplant recipients patients (Pfaller et al. 2008).
The antifungal effect of azoles is due to their actions in inhibition of ergosterol biosynthesis on fungal cell membranes (Casalinuovo et al. 2004, Sardi et al. 2013. Currently used antifungal drugs may have the risk of toxicity, may result drug-drug interactions and lack of fungicidal efficacy. Furthermore, resistance to these drugs often develops rapidly (Metzger & Hofmann, 1997, Pinto et al. 2009). e2 G. Göger et al. Plants are known to produce a great variety of compounds. Particularly, aromatic plants have been used in folk medicine as antimicrobial agents since ancient times. (Hosseinzadeh et al. 2015). Essential oils (EOs) of many plants are known to possess a large number of pharmacologically active compounds and have antimicrobial activity. EOs show a wide-spectrum antimicrobial activity, including disruption of cellmembrane structures, leading to an alteration in membrane permeability and a consequent leakage of cell contents (Cox et al. 2000, Chen et al. 2013). For instance, recent studies on antifungal activities of some EOs have shown disruption of the fungal membrane (Pinto et al. 2006, Khan et al. 2010, Samber et al. 2015. With this in mind, combination of antifungal therapies with natural sources may be achieved to overcome the resistance against Candida species. Specifically, EOs are known to be used in therapeutic preparations for centuries (Hosseinzadeh et al. 2015 (Shin 2003). In more recent studies, Ocimum sanctum EO and its combination with two azoles, fluconazole and ketoconazole, were studied against resistant isolates (Amber et al. 2010). Matricaria recutita EO was combined with fluconazole against C. albicans (Göger et al. 2018). It thus appears that, although proved to have antimicrobial activities when tested alone, studies on combination of lavender EOs with antifungal drug are rather limited. In one recent study, nystatin was tested in combination with Lavandula angustifolia EO against C. albicans ATCC 10231 and an indifference effect was observed for the combination (De Rapper et al. 2016) This study was performed with the aim of identification of chemical compositions of two EOs of lavender (L. angustifolia Miller) by GC/FID and GC/MS analytical methods and evaluate their anticandidal activities with ketoconazole by microdilution and checkerboard methods against C. albicans ATCC 10231, C. krusei NRRL Y-7179 and C. glabrata ATCC 66032 strains.

Materials
Two commercial Lavender EOs were purchased from Sevil (Turkey) and Caesar & Lorethz GmbH (Hilden, Germany) companies and were labeled as L1 and L2, respectively. The standard powders of ketoconazole and resazurin were obtained from Sigma-Aldrich.
Anticandidal activities of the EOs of L1 and L2 were tested against standard (C. albicans ATCC 10231, C. krusei NRRL Y-7179 and C. glabrata ATCC 66032) and clinical (C. albicans, C. krusei and C. glabrata) isolates provided from Akdeniz University, Faculty of Medicine, Antalya.

GC /FID analysis
The EOs were analyzed by capillary GC using the Shimadzu GC 2010 system. The column and analysis conditions used for GC/MS were also applied for GC. FID detector temperature was 250°C. In order to get the same elution order with GC/MS, injections were done by using the same column and operational parameters.

GC/MS analysis
The GC/MS analysis was realized with the Shimadzu GC 2010 system. CP-Sil 5 CB column (25 m x 0.25 mm Ø, 0.25 μm film thickness) was used with helium as the carrier gas (1 mL/min). GC oven temperature was kept at 60°C for 10 min and programmed to 260°C at a rate of 4°C/min, and kept constant at 260°C for 5 min. Split ratio was adjusted to 50:1. The injector temperature was at 250°C. MS were recorded at 70 eV, where the mass range was from m/z 35 to 450.

Identification of the volatile components
Mass spectrometry (MS) was used to characterize the volatiles by comparing their relative retention times with those of authentic samples or by comparison of their relative retention index (RRI) to series of n-alkanes. Computer matching against commercial (Wiley GC/MS Library, MassFinder 3 Library) (McLafferty & Stauffer 1989, Koenig et al. 2004) and in-house "Başer Library of Essential Oil Constituents" built up by genuine compounds and components of known oils, as well as MS literature data (Joulain & Koenig 1998, ESO (1998)) were also used for the identification.

Determination of minimum inhibitory concentration (MIC)
Anticandidal activities were performed according to the adapted guidelines of Clinical and Laboratory Standards Institute (CLSI) M27-A2 standard method. Minimum inhibitory concentrations (MIC) of EOs of L1 and L2 and ketoconazole were determined using the standard and clinically isolated Candida isolates (see above).
Stock solutions of EOs and ketoconazole were dissolved in DMSO. EOs were diluted two-fold initially, with a final concentration range 2560 -5 µg/mL, whereas the ketoconazole was diluted from 64 to 0.125 µg/mL. Candida cultures with a final inoculum size of 1x10 3 colonies forming units (CFU/mL) were prepared. A 10 µL of the suspensions at 10 3 CFU/mL were inoculated into a two-fold diluted solutions of the tested EOs and ketoconazole in 96 well microplates. The microplates were incubated at 35°C for 48 h. Microbial growth was visualized by adding 20 µL resazurin (0.01%). MIC was defined as the lowest concentration, which did not result in any visible growth compared with the control plates. Microplates free of antifungal standards were used as the growth controls. Negative controls were used to confirm sterility and the effects of the solvents. Assays were undertaken in triplicate.

Combination of Lavender EOs and ketoconazole by the checkerboard method
The antifungal interaction between ketoconazole and EOs of L1 and L2 were studied by the checkerboard method using a 96-well plate using an 8-by-8 well configuration (Van Vuuren et al. 2009). EOs of L1 and L2 and ketoconazole were prepared as serial dilutions using the same solvents (medium) in the MIC test. 50 µL aliquots of each EOs were added to the wells of a 96-well plate in a vertical orientation and 50 µL aliquots of each ketoconazole dilution were added in a horizontal orientation so that the plate contained various concentration combinations of the EOs and ketoconazole. Growth controls were applied in wells free of antifungal standards. Negative control was also applied to confirm sterility. Each well was inoculated with 100 µL (5 x 10 3 CFU per well) of one of the six Candida suspensions and incubated at 35°C for 48 hours. Following the incubation, 20 µL resazurin was added to the wells and the wells were left at 35°C for 2 h. Growth in wells was indicated by the color change from blue to pink.
In vitro interaction between Lavender EOs and ketoconazole were determined by using the fractional inhibitory concentrations (FIC). The FIC is expressed as the interaction of two compounds, where the concentration of each test compound in combination is expressed as a fraction of the concentration that would produce the same effect when used singularly (Vuuren et

Results
In our present study, lavender EOs were analyzed by GC/FID and GC/MS methods and confirmed at the quality of Pharmacopoeia grade. The EOs are characterized by high amounts of linalyl acetate (30.5-42%) and linalool (35.9-46%) (Figures 1 and 2). The major constituents of EOs and their relative percentages are listed in Table 1   From this point of view, EOs of lavender tested for their anticandidal activities against Candida strains ( Table  2) were found to have a MIC range of 160-640 µg/mL compared to ketoconazole MIC range of 1-4 µg/mL. In particular, the inhibitory effect of L2 EO represented with a MIC value of 160 µg/mL was more pronounced than that of L1 EO when tested with standard and clinical C. krusei strains (Table2). The checkerboard results of EOs and ketoconazole combinations were listed in Tables 3 and 4. The L1 + ketoconazole combination against both clinical and standard C. albicans ATCC 10231, C. krusei NRRL Y-7179 and C. glabrata ATCC 66032 showed additive effects (FICI= 0.53 to 056) ( Table 3). The L2 + ketoconazole combination against C. albicans (FICI= 1.0) and C. glabrata (FICI= 0.56) clinical isolates also resulted in an additive activity pattern (Table 4).
In combination therapy, interactions between EOs and antifungals can display four possible types of effects as synergistic, additive, indifferent and antagonistic. Synergism is observed when the effect of the combined substances is greater than the sum of their individual effects. An additive effect is observed when the combined effect is equal to the sum of the individual effects. Indifference is observed when there is no interaction. Antagonism is observed when the effect of one or both compounds is less when they are applied together than when applied individually (Bassole et al. 2012, Yap et al. 2014).
The different interactions observed in antifungal activity between EOs of L1 and L2 can be explained by the chemical compositions EOS and the ratio of their components. Since EOs contain many chemical mixtures, differences can be observed in their antimicrobial effects and the type of effect mechanisms. Most of the antimicrobial activity effect for EOs was attributed to their chemical compositions derived from hydrocarbons, oxygenated terpenoids, particularly phenolic terpenes, phenylpropanoids and alcohols (Burt, 2004). L. angustifolia EO was also combined with the commercial antimicrobial drugs nystatin, chloramphenicol, ciprofloxacin and fusidic acid against S. aureus ATCC 6538, P. aeruginosa ATCC 27858 and C. albicans ATCC 10231. The best interaction was shown between L. angustifolia EO with chloramphenicol and tested against the pathogen P. aeruginosa (FICI= 0.29).
The mode of action of lavender EO on antimicrobial activity against multi-drug resistant Escherichia coli J53 R1 when used singly and in combination with piperacillin was demonstrated (Yap et al. 2014). The time-kill analysis showed synergistic interaction between lavender EO and piperacillin at 4 h. The results of this study indicate that lavender EO is able to disrupt membrane structures of E. coli and has the potential to inhibit bacterial quorum sensing.
Lavandula angustifolia essential oils in combination with ketoconazole against Candida strains e5 Trakya Univ J Nat Sci, Online First for 21(1): X-X, 2020 In conclusion, we reported here for the first time the anticandidal effects of L. angustifolia EOs with ketoconazole combination against the standard and clinical strains of the human pathogens C. albicans, C. glabrata and C. krusei in a comparative manner. As revealed by the results, the absence of antagonism may be promising for combination of lavender EOs and ketoconazole in anticandidal use. The effective dose of ketoconazole use in combination may be reduced, rapid drug resistance may be prevented or combination use may provide a solution to drug-drug resistance problems and can be considered a promising alternative pharmaceutical agent for topical application in surface infections. Therefore, lavender EOs should be combined with different antibiotic/antifungal drugs and tested against different pathogens.