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issn=09720707;year=2014;volume=17;issue=5;spage=4
49;epage=453;aulast=Nabavizadeh;type=3
ORIGINAL ARTICLE
Year : 2014 | Volume : 17 | Issue : 5 | Page : 449-453

Chemical constituent and antimicrobial effect of essential oil from Myrtus
communis leaves on microorganisms involved in persistent endodontic
infection compared to two common endodontic irrigants: An in vitro study

Mohammadreza Nabavizadeh1, Abbas Abbaszadegan1, Ahmad Gholami2, Reza Sheikhiani3, Mehdi
Shokouhi1, Mahdi Sedigh Shams1,Younes Ghasemi2
1

Department of Endodontics, School of Dentistry, Shiraz University of Medical Sciences, Shiraz, Iran

2

Department of Pharmaceutical Biotechnology, School of Pharmacy; Pharmaceutical Research Center, Shiraz

University of Medical Sciences, Shiraz, Iran
3

Undergraduate Student, Student Research Center Committee, School of Dentistry, Shiraz University of Medical

Sciences, Shiraz, Iran
Click here for correspondence address and email

Date of Submission
Date of Decision
Date of Acceptance
Date of Web Publication

18-Mar-2014
22-Jun-2014
01-Jul-2014
1-Sep-2014

Abstract
Introduction: Persistent infections of human root canals play a fundamental role in the failure of endodontic
treatment. The purpose of this study is to determine the chemical composition of Myrtus communis (M.
communis) essential oil and to assess its antimicrobial activity against Enterococcus faecalis, Staphylococcus
aureus and Candida albicans compared to that of sodium hypochlorite (NaOCl) and chlorhexidine (CHX).
Materials and Methods: Gas chromatography-mass spectrometry (GC-MS) was used to determine the
chemical composition of essential oil from M. communis leaves. A micro-dilution susceptibility assay and disk
diffusion methods were utilized to evaluate the antimicrobial activity [minimum inhibitory concentration (MIC)
and minimum lethal dose concentration] of the tested solutions against selected microorganisms.

isobutyl-isobutyrate (0. reduce inflammation. Shams MS. Recent evidences support that essential oil from M. .Results: GC-MS analyses revealed that M.62%). 8-Cineole (28.in/text.[4] Effective endodontic antimicrobial agents should be active against persistent pathogens while being compatible to peri-apical tissues. Sheikhiani R. eugenol (1.3%) as the major compounds and Geraniol (1. some specific organisms including gram-positive facultative bacteria. there is no study in literature evaluating its antimicrobial effectiveness on microorganisms involved in persistent endodontic infections compared to NaOCl and CHX.6%). Sheikhiani R.org. α-Pinene (17. Chlorhexidine had the lowest MIC value among all medicaments tested.3%).jcd. there has been a growing trend to use natural products with minimal cytotoxicity and phytochemicals in endodontic practice during the past years.[2] Fungi and some strains of staphylococci can also be found in persistent infections. but it had more MIC value against C. M. non-toxic.[7] have made the clinicians to still continue their efforts to find a more compatible. chlorhexidin. sodium hypochlorite How to cite this article: Nabavizadeh M.55%). faecalis. antibacterial. communis contained 1. cost-effective. communis essential oil in different studies. since there is considerable variability in the detailed composition of M. Conclusion: M.032-32 μg/mL was an effective antimicrobial agent against persistent endodontic microorganisms. especially E. Abbaszadegan A.17:449-53. Keywords: Antibacterial activity. [1]. and Geranylacetate (6. Chemical constituent and antimicrobial effect of essential oil from Myrtus communis leaves on microorganisms involved in persistent endodontic infection compared to two common endodontic irrigants: An in vitro study.[10]. improve the healing outcome. and provide clinical advantages to the patients such as pain reduction. communis) is an ever green small tree with a wide distribution in Iran and other tropical regions of the world. Iran and compare the antimicrobial activity of this oil with NaOCl and CHX. Chemical constituent and antimicrobial effect of essential oil from Myrtus communis leaves on microorganisms involved in persistent endodontic infection compared to two common endodontic irrigants: An in vitro study. J Conserv Dent [serial online] 2014 [cited 2015 Jan 24]. and antioxidant activities. Shokouhi M. [8]. [5] Reports from the disadvantages of commonly used irrigants together with their cytotoxicity and possible caustic effects in case of inadvertently extrusion to the peri-radicular region [6]. Ghasemi Y. Myrtus communis. root canal irrigant. Linalool (17. communis essential oil with the minimum inhibitory concentration in the range of 0. Myrtus communis (M. and efficient alternatives. As a result. [3]. Gholami A. Gholami A.8%). this study aimed to determine its chemical composition derived from the leaves collected from the southern regions of Fars province. may still survive after careful chemomechanical preparation of the root canal system and cause persistent intraradicular infections. Furthermore. Shokouhi M. communis leaves has exhibited satisfactory antifungal. and methyl chavicol (0.5%) as minor components. communis oil had less MIC values than NaOCl against both bacteria. Abbaszadegan A. Shams MS.[11] To the best of our knowledge. Ghasemi Y. However.17:449-53 How to cite this URL: Nabavizadeh M.[9]. α-Humulene (1.5%). Available from: http://www. albicans.8%). J Conserv Dent 2014.asp? 2014/17/5/449/139836 Introduction The success of endodontic treatments highly relates to the effectiveness of antimicrobial agents to eliminate the living microorganisms in infected root canals.

faecalis (PTCC 1237). Shiraz. An electron ionization system with ionization energy of 70 eV was applied for the mass spectrometry (MS) detector (7000 Triple Quad). All experimental solutions were freshly prepared just before the commencement of the experiments. kept in sealed vials. The carrier gas in this assay was Helium (1. [12] Antimicrobial activity Experimental solutions According to the yield of the essential oil.. we isolated the essential oil of the plant and subjected it to the gas chromatographymass spectrometry (GC-MS) analysis to define the chemical compositions of the oil and also to have better understanding of its bioactivity. and deposited at the herbarium of the department of pharmacognosy. Louis.250 mm. The abstract of methodology used in this study. and Candida albicans) and compared it to that of sodium hypochlorite (NaOCl) and chlorhexidine (CHX). Then. 30 m × 0. communis plants were collected from the southern regions of Iran. The program of GC oven was set as follows: initial temperature at 70°C for 5 min. The hydrodistillation method was applied for 800 g of dried plant material together with doubly water for 4 h using a clevenger-type apparatus (yield 0. The collected leaves were dried in a dark room at 24°C for 1 week. The obtained essential oil was isolated from water. . GC-MS analysis A gas chromatography (GC) apparatus (Agilent 7890A. 5% NaOCl (Sigma-Aldrich Co.9%). we first collected and identified the plant materials. Iran under the specific number 687. USA) with capillary column (DB-1MS. Shiraz University of Medical Sciences. faculty of pharmacy. a pharmacognosist. St. and C.2 μL injection) was used to analyze the M. 0. albicans (PTCC 5027) were used in this study as test organisms.. communis essential oil was obtained and involved in antimicrobial assessments. around Noorabad in Fars province in June 2013. communis essential oil.2 mL/min). The temperature of MS transfer line was also adjusted to 280°C. Isolation of the essential oil As the aim of this in vitro study was two-fold. programmed rate at 3°C/min up to 280°C for 4 min and temperature of injector at 250°C. faecalis. MO) were selected for further comparisons in this study. Microbial strains S. Staphylococcus aureus. A voucher specimen was prepared. identified by Dr. Louis. M Moein. MO) and 2% CHX (Sigma-Aldrich Co. E. we evaluated the antimicrobial efficacy of the oil against some persistent endodontic pathogens (i. aureus (PTCC 1189). St. E. In order to calculate Kovats indices (KI) for the detected compounds.Materials and methods Collection of plant materials The fresh leaves of M. and stored in a refrigerator at 4°C until starting the experiments.e. Second. also. Identification of the components was performed by comparing the relative retention time of components and mass spectra with those previously obtained from Wiley 275 library and Adams data for GC-MS. n-alkanes were employed. concentration of 32 μg/mL of the M.

All plates were then incubated at 37°C for 24 h. Determination of minimum bactericidal/fungicidal concentration The MBC and minimum fungicidal concentration (MFC) were determined by the following procedures. which showed no visible growth that were cultured respectively on tryptic soy agar and Sabouraud dextrose agar. [13] Briefly. In the case of bacterial experiments. NaOCl. Determination of minimum inhibitory concentration The MICs for the test solutions were determined by broth microdilution method according to the CLSI 2012 standard protocol. The media from wells with bacteria or fungi.0 (Chicago. to evaluate the antifungal effects.Disc diffusion assay Standardized routine disc susceptibility tests were performed to determine the rate of growth inhibition of test microorganisms against pure concentration of plant essential oil. Similarly. previously soaked with the experimental solutions. These experiments were also done in triplicate to remove any possible bias. amphotericin-B was considered as positive control. set to a final density of McFarland 0.5 standard using a spectrophotometer at wavelength of 625 nm.05 was considered as statistical significance. An antibiotic standard disc of ampicillin (Difco) was used as the positive control. Finally. and the sterile water as the negative control. Then. This occurred when no more than 4 colonies was seen in agar plates after 24 h incubation at 37°C. USA). Furthermore. The MBC and MFC values were defined as the lowest concentration. the bacterial strains were cultured overnight at 37°C on Mueller-Hinton broth. The inhibitory effects of the test solutions were determined by monitoring the absorbance at 625 nm in a microtiter plate reader (BioTek.5 × 10 6 CFU/mL yeasts (equal to 0. except that Mueller-Hinton agar was supplemented with 2% glucose to support C. and CHX were transferred to a 96-well microtiter plates containing RPMI-1640 media and a suspension of 1. Statistical analysis was performed by employing Kruskal-Wallis test using Statistical Package for the Social Sciences (SPSS software version 15. the inhibition zone diameter induced by each solution was measured after incubation period of 18 h. were put on the inoculated agar surfaces and incubated overnight at 37°C. P < 0. albicans growth. IL). and 2% CHX. the overnight cultured microbial broth was adjusted to an optical density equal to 0. NaOCl and CHX on MHB. Sterile filter paper discs (6 mm diameter). serial ten-fold dilutions of M. Then. the bacteria were cultured on plates containing Mueller-Hinton agar (Hi-media) with a sterilized cotton swap. [13] This experiment was performed in triplicate for each test solution. yielding a mortality of 98% of microorganisms in the initial inoculums. The antifungal assay was similar to the previously used disk diffusion method.5McFarland). 5% NaOCl. communis essential oil. they were transferred to a 96-well microtiter plates containing serial ten-fold dilutions of the essential oils. Ampicillin was used as positive control and sterile water as negative control. .5 McFarland standard by using a spectrophotometer apparatus at wavelength of 530 nm. Amphotericin-B was also used as positive control and sterile water as negative control.

were identified.55%).3%) as the major compounds and geraniol (1. linalool (17. α-humulene (1.3%). 8-cineole (28.5%). isobutyl-isobutyrate (0. The GC-MS analysis revealed that M. α-pinene (17. Table 1: Compositions of the essential oils obtained from leaves of M. which represented 99. communis are presented in [Table 1]. communis leaves. and methyl chavicol (0. communiscontained 1. and geranyl acetate (6. eugenol (1.19% of the total essential oil from M.8%).6%). communis Antimicrobial activity .Results Chemical composition of the essential oil The results obtained by GC-MS analysis of the essential oil of M. 31 constituents.5%) as the minor components.8%). Total.62%).

the composition diversity of a plant is a pharmaceutical opportunity in which different therapeutic uses of the same plant species. and Messaoud et al. but it had smaller MIC value against C. The details of disc diffusion test and MIC. communis oil in current study. In another point of view. and linalool..62%). M. Fernandes et al. This reported diversity in the oil compositions is possibly due to the applied techniques and the different environmental growth conditions of the plant used. It was also found that humulene can induce important inhibitory influence on tumor necrosis factor-α (TNFα) and interleukin-1 β (IL1B). albicans.8cineole or limonene as the second major components while α-pinene was reported as the major constituent in Zomorodian et al. [22] showed that the extracts of M. 8-cineole (28. Ilcim et al. revealed that humulene can produce similar anti-inflammatory properties to dexamethasone. [11]. .. [8]. High monoterpene hydrocarbons such as 1. the main constituents of the essential oil were 1. these agents can be beneficial to decrease the post-operative pain in root canal treatments.. communis oil. communis essential oil inhibited the growth of all tested microorganisms and had an inhibition zone of 21-48 mm. communis oil. α-pinene (17/8%). the sequence of abundant components reported in the literature is divergent. minimum inhibitory concentration (MIC in μg/mL). Minimal Bactericidal/Fungicidal Concentration (MBC/MFC) of the test solutions against the tested microorganisms are summarized in [Table 2]. 8-cineole. communis oil. Akin et al. communis essential oil was more potent than NaOCl with lower MIC on both bacteria.53%).[18] In contrast with other reports. For instance.[10].[16] However. which can impact the results and make the comparisons difficult. [19] found that eucalyptol was the first major component of M.[14]. grown in different regions become possible. and humulene have also been found. The details are presented in [Table 2].Rasooli et al. and linalool (17. Chlorhexidine had the least MIC values against all examined organisms with the quantities similar to MBC values. investigations. The disc diffusion method revealed that M. These substances are anti-inflammatory agents and may have potential to reduce pain. which found cineole as the main ingredients of M. which were found as the main components of M. have been extensively proven as strong antimicrobial substances [20] and therefore might be responsible for the antibacterial activity of the experimented essential oil.[15]. and minimum bactericidal concentration (MBC/MFC in μg/mL) mean values Discussion As it can be found on [Table 1].. some studies [11]. In an animal study. These findings were consistent with the results of other studies. M.[17] have considered 1. eugenol.All three experimental solutions were found to be effective on the test microorganisms. α-pinene. communis essential oil inhibited the growth of all tested microorganisms with the inhibition zone of 21-48 mm. By analyzing the chemical constituents of M. Table 2: Disc diffusion (DD in mm). [21] Therefore.

Although MIC is the most reliable and easily interpreted method for comparison of different formulations. As with current study. determination of MIC together with the MBC is properly demonstrating the extent of antimicrobial activity. Zomorodian et al. for the statistical analysis.[3]. it also has drawbacks such as unpredictable interaction of medium components with one or more of the test medicaments and the instability of some essential constituent of the test agents. Teixeira FB.32-32 μg/mL. and C. Reza Sheikhiani. This manuscript is based on the thesis by Dr. communis essential oil showed less antimicrobial potency than NaOCl against C. [8] and Rasooli et al. Pinheiro ET. Sousa EL. References 1. Hamedani for his editorial assistance and Dr. Sh. We also assessed the antimicrobial efficiency of the test solutions by determining their MICs and MBCs against test organisms. faecalis. On the contrary to our findings. Conclusion M. Putting these together. .36:1-11.. faecalis was inhibited at a concentration value equal to 32 μl/mL. S. albicans based on MIC values. Souza-Filho FJ. it had smaller MFC value against this fungus.communis had an inhibition zone of 16-38 mm. the authors believe that M. [11] revealed that C. communis essential oil with the MIC in the range of 0. aureus when treated with M . Int Endod J 2003. [2]. a finding which is in line with the results of this study. where the host defense mechanisms are less active compared to systemic infections. In infected root canals. followed by M. aureus have also been found by Zomorodian et al. Although M. albicans andE.[10] These dissimilarities in the published results can be related to the different resistance levels between the strains used and the different concentration of M . Acknowledgments The authors thank the Vice-Chancellery of Shiraz University of Medical Science for supporting this research (Grant # 92-01-03-6123). The similar levels of sensitivity for S. Gomes BP. albicans was more sensitive than S. The authors would also like to thank Dr.[4] The growth of all tested microorganisms was inhibited by the essential oil of M.communis oil with the MBC value of 4 μL/mL. Comparison among the antimicrobial agents showed that CHX was efficient at the lowest concentrations for both bacteria.communis oil investigated. [23] The reason to select E. [8] found that the growth of E. albicans was based on the studies that linked these microorganisms to the refractory infections after endodontic treatments.032-32 μg/mL was an effective antimicrobial agent against persistent endodontic microorganisms. communis essential oil was more potent in lower concentration on S. aureus than on C. M. aureus. faecalis. Yadegarnia et al. Vosoughi from the Dental Research Development Center. communis oil and NaOCl. Microorganisms from canals of root-filled teeth with periapical lesions. communis have favorable potentials to be applied in endodontic treatments as a root canal irrigant or as an intracanal dressing between visits. communis oil at the concentration range of 0. Our study results demonstrated that M. Ferraz CC.

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