EXCLI Journal 2016;15:280-289 – ISSN 1611-2156 Received: October 02, 2014, accepted: December 13, 2014, published: April 19, 2016 Original article: SENSITIVITY OF CLINICAL ISOLATES OF CANDIDA TO ESSENTIAL OILS FROM BURSERACEAE FAMILY Miloš Nikolića, Marija Smiljkovića, Tatjana Markovićb, Ana Ćirića, Jasmina Glamočlijaa, c a,* Dejan Marković , Marina Soković a Institute for Biological Research “Siniša Stanković”, University of Belgrade, Bulevar Despota Stefana 142, 11000, Belgrade, Serbia b Institute for Medicinal Plant Research “Josif Pančić”, Tadeuša Košćuška 2, 11000 Belgrade, Serbia c Faculty of Dental Medicine, Department of Pediatric and Preventive Dentistry, University of Belgrade, dr Subotića 8, 11000 Belgrade, Serbia * Corresponding author: Marina Soković PhD, Institute for Biological Research “Siniša Stanković”, Bulevar Despota Stefana 142, 11000 Belgrade, Serbia Phone: +381 11 207 84 19, Fax: +381 11 2 761 433, e-mail: [email protected] http://dx.doi.org/10.17179/excli2014-621 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/). ABSTRACT The aim of this study was to investigate the chemical composition and antifungal activity of four commercial essential oils from the Burseraceae family - two Boswellia carterii Flueck oils, Canarium luzonicum (Blume) A. Gray oil, and Commiphora myrrha (Nees) Engl oil, against most common Candida spp. recovered from the hu- man oral cavity. The essential oil samples were analyzed by GC-FID and GC/MS. The analysis showed that ma- jor essential oils’ components were –pinene (23.04 % and 31.84 %), limonene (45.62 %) and curzerene (34.65 %), respectively. Minimum inhibitory (MIC) and minimum fungicidal (MFC) concentrations were deter- mined using a microdilution standardized technique. All tested Candida spp. clinical isolates and ATCC strains showed susceptibility to tested essential oils in a dose dependent manner. The strongest antifungal activity was shown by essential oil of B. carterii, sample 2; the average MIC values ranged from 1.25 to 1.34 mg/ml, and MFC values ranged from 2.50 to 3.75 mg/ml, depending on the fungus. This study supports the possible use of essential oils from the Bursecaceae family in reduction and elimination of Candida spp. populations in patients with oral cavity fungal infections. Keywords: Candida spp., susceptibility, essential oils, Burseraceae, oral candidosis INTRODUCTION ta, C. krusei, C. tropicalis and C. dublinien- Among the hundreds of microbial species sis are generally less frequent, although there from the oral cavity, members of the genus are indications that the incidence of non- Candida are representative of several yeast albicans species recovered from cases of oral species considered to be commensal oral mi- candidosis is increasing (Samaranayake, crobiota. Among them, Candida albicans is 1991; Scully et al., 1994). the most commonly isolated from the human Under certain circumstances Candida oral cavity (Back-Brito et al., 2009), while spp. commensals become pathogens and the non-albicans species, such as C. glabra- cause mucous membrane infections ranging 280 EXCLI Journal 2016;15:280-289 – ISSN 1611-2156 Received: October 02, 2014, accepted: December 13, 2014, published: April 19, 2016 from pseudomembranous candidodis and intended for use in oral hygiene mainte- denture-induced stomatitis (Lam et al., 2012; nance, and prevention and management of Gonsalves et al., 2007; Da Costa et al., 2006; oral infections. Ben-Aryeh et al., 1980), to life-threatening In addition, mindful of increased re- systemic diseases (Samaranayake and Yaa- sistance of Candida spp. to synthetic anti- cob, 1990), particularly in immunocompro- fungal drugs (Pina-Vaz et al., 2004; Zomoro- mised patients with AIDS, cancer and diabe- dian et al., 2011), testing the susceptibilities tes mellitus (Seneviratne et al., 2008; Ed- of clinical isolates to natural products such mond et al., 1999). as essential oils, has contributed to develop- Oral candidosis is an opportunistic infec- ment of novel antifungal treatments intended tion usually accompanied by various symp- for use at target sites. Our approach also toms including burning, painful sensation, supports the idea that apart from classical change of taste and swallowing difficulty, antifungal treatments, combination therapy but it can be also asymptomatic. It could be and preventive therapy represent emerging treated with various synthetic antifungal strategies for treating invasive fungal infec- agents, though they possess some disad- tions (Rüping et al., 2008). vantages such as high toxicity to the host tis- The aim of this study was to investigate sues, emergence of drug-resistant species, the chemical composition and antifungal ac- and high cost (Runyoro et al., 2006). There tivity of four commercial essential oils from is a number of case reports describing the the Burseraceae family against the most colonization and infection of immunocom- common Candida spp. recovered from the promised patients or denture wearers sub- human oral cavity. jected to long-term regimens of oral antifun- gal agents, from which drug less-responsive MATERIAL AND METHODS or resistant (Sheehan et al., 1999; Wingard et Essential oils al., 1991; Wingard, 1994 and 1995; Webb et Four commercial essential oils belonging al., 1998; Sanglard and Odds, 2002; Sullivan to the Burseraceae family were used in this et al., 2004), and even cross-resistant Can- experiment: 1) Boswellia carterii Flueck., dida spp. (Cross et al., 2000; Magill et al., sample 1 (EOBC1), purchased from an herb- 2006) have been recovered. In addition, re- al pharmacy in Rotterdam, Holland; 2) Bos- currences of the oral candidosis, which are wellia carterii Flueck., sample 2 (EOBC2), commonly observed in these patients, make purchased from Sensient Essential Oils the problem even greater (Taplin, 1976). Germany GmbH, Bremen, Germany; 3) Ca- Limitations of synthetic antifungal drugs narium luzonicum (Blume) A. Gray, (EOCL) have encouraged development of new classes also purchased from Sensient Essential Oils of antifungal medications based on potent Germany GmbH; and 4) Commiphora bioactive molecules of natural origin with myrrha (Nees) Engl. (EOCM), also pur- high therapeutic efficiency, low toxicity, chased from Sensient Essential Oils Germa- wide spectrum of activity and eco-friendly ny GmbH. nature (Saini et al., 2008; Rajeshkumar and Sundararaman, 2012). Since plant-derived Essential oil analyses procedure essential oils have a long tradition of use and The essential oil analyses procedure for cover a broad spectrum of biological activi- Gas Chromatography coupled with a Flame- ties, in various studies their efficacy has been Ionization Detector and Gas Chromato- tested over a wide range of oral bacteria and graphy/Mass Spectrometry analyses meets fungi (Hammer et al., 1999; Carvalhinho et standards ISO 7609:1985, ISO 11024-1:1998, al., 2012). Their natural origin, low cytotoxi- and 11024-2:1998, and they have been previ- ty and biodegradability made them potential ously reported by Nikolić et al. (2013). ingredients in novel antifungal medications 281 EXCLI Journal 2016;15:280-289 – ISSN 1611-2156 Received: October 02, 2014, accepted: December 13, 2014, published: April 19, 2016 GC-FID analysis was carried out using a sity of Belgrade, Serbia), were used in this GC Agilent Technologies 7890A apparatus, study. The clinical isolates were collected by equipped with the split-splitless injector and rubbing sterile cotton swabs over the oral automatic liquid sampler (ALS), attached to mucosa of randomly chosen patients at the HP-5 column (30 m x 0.32 mm, film thick- Faculty of Dental Medicine, University of ness 0.25 µm) and fitted with a flame- Belgrade, Serbia. For collecting clinical iso- ionization detector (FID). Operating condi- lates, used swabs were transferred to SD tions were: H2 was the carrier gas (1 ml/min/ broth medium which were then thoroughly 210 °C); injector and detector T were 250 °C mixed using a vortex mixer; 50 μl of suspen- and 280 °C, respectively, while the column T sions were subsequently inoculated on vari- was linearly programmed 40-260 °C at ous selective and non-selective media and 4 °C/min. Samples of essential oils were first incubated microaerobically for 48 h at 37 °C. dissolved in ethanol (approx. 1 %) and then The isolates were identified using biochemi- injected by ALS (1 µl, split-mode). Presence cal profiles with API 20C (bioMérieux of the oils’ compounds were calculated from France) and Chrom-Agar (Liofilchem sr.l. the peak areas attained in corresponding ar- Italy). ea-percent reports (results of the standard processing of chromatograms), without cor- Anticandidal activity rection factors. Minimum inhibitory concentrations The GC/MS was carried out on an HP (MIC) and minimum fungicidal concentra- G1800C Series II GCD analytical system tions (MFC) were established by the use of equipped with a column HP-5MS (30 m x microdilution standardized technique, EU- 0.25 mm, film thickness 0.25 µm). He was CAST (2002) with modification. In brief, the carrier gas (1 ml/min). Other chromato- with the use of sterile saline, fresh overnight graphic conditions were identical to those for yeast cultures were adjusted to a concentra- GC-FID. The transfer line was heated at tion 1.0 x 105 CFU/per well. The microplates 260 °C, while the mass spectra were record- were left at 37 °C for 24 h. Following the ed in EI mode (70 eV), ranging from 40 to addition