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issn=09720707;year=2014;volume=17;issue=5;spage=4
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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

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.

Results: GC-MS analyses revealed that M. communis contained 1, 8-Cineole (28.62%), -Pinene (17.8%),
Linalool (17.55%), and Geranylacetate (6.3%) as the major compounds and Geraniol (1.6%), -Humulene
(1.5%), eugenol (1.3%), isobutyl-isobutyrate (0.8%), and methyl chavicol (0.5%) as minor components.
Chlorhexidine had the lowest MIC value among all medicaments tested. M. communis oil had less MIC values
than NaOCl against both bacteria, but it had more MIC value against C. albicans.
Conclusion: M. communis essential oil with the minimum inhibitory concentration in the range of 0.032-32
g/mL was an effective antimicrobial agent against persistent endodontic microorganisms.

Keywords: Antibacterial activity; chlorhexidin; Myrtus communis; root canal irrigant;

sodium hypochlorite
How to cite this article:
Nabavizadeh M, Abbaszadegan A, Gholami A, Sheikhiani R, Shokouhi M, Shams MS, Ghasemi Y. 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 2014;17:449-53
How to cite this URL:
Nabavizadeh M, Abbaszadegan A, Gholami A, Sheikhiani R, Shokouhi M, Shams MS, Ghasemi Y. 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];17:449-53. Available from: http://www.jcd.org.in/text.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. However, some specific organisms including gram-positive
facultative bacteria, especially E. faecalis, may still survive after careful chemomechanical preparation of the
root canal system and cause persistent intraradicular infections. [1],[2] Fungi and some strains of staphylococci
can also be found in persistent infections. [3],[4]
Effective endodontic antimicrobial agents should be active against persistent pathogens while being
compatible to peri-apical tissues, reduce inflammation, improve the healing outcome, and provide clinical
advantages to the patients such as pain reduction. [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],[7] have made the clinicians to still
continue their efforts to find a more compatible, cost-effective, non-toxic, and efficient alternatives. As a result,
there has been a growing trend to use natural products with minimal cytotoxicity and phytochemicals in
endodontic practice during the past years.
Myrtus communis (M. communis) is an ever green small tree with a wide distribution in Iran and other tropical
regions of the world. Recent evidences support that essential oil from M. communis leaves has exhibited
satisfactory antifungal, antibacterial, and antioxidant activities. [8],[9],[10],[11] To the best of our knowledge, there is
no study in literature evaluating its antimicrobial effectiveness on microorganisms involved in persistent
endodontic infections compared to NaOCl and CHX. Furthermore, since there is considerable variability in the
detailed composition of M. communis essential oil in different studies, this study aimed to determine its
chemical composition derived from the leaves collected from the southern regions of Fars province, Iran and
compare the antimicrobial activity of this oil with NaOCl and CHX.

Materials and methods

Collection of plant materials
The fresh leaves of M. communis plants were collected from the southern regions of Iran, around Noorabad in
Fars province in June 2013. A voucher specimen was prepared, identified by Dr. M Moein, a pharmacognosist,
and deposited at the herbarium of the department of pharmacognosy, faculty of pharmacy, Shiraz University of
Medical Sciences, Shiraz, Iran under the specific number 687.
Isolation of the essential oil As the aim of this in vitro study was two-fold, we first collected and identified the
plant materials. Then, 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. Second, we evaluated the antimicrobial efficacy of the oil against some
persistent endodontic pathogens (i.e. Staphylococcus aureus, E. faecalis, and Candida albicans) and
compared it to that of sodium hypochlorite (NaOCl) and chlorhexidine (CHX). The abstract of methodology
used in this study.

The collected leaves were dried in a dark room at 24C for 1 week. 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
0.9%). The obtained essential oil was isolated from water, kept in sealed vials, and stored in a refrigerator at
4C until starting the experiments.
GC-MS analysis
A gas chromatography (GC) apparatus (Agilent 7890A; USA) with capillary column (DB-1MS; 30 m 0.250
mm, 0.2 L injection) was used to analyze the M. communis essential oil. The carrier gas in this assay was
Helium (1.2 mL/min). The program of GC oven was set as follows: initial temperature at 70C for 5 min,
programmed rate at 3C/min up to 280C for 4 min and temperature of injector at 250C. An electron ionization
system with ionization energy of 70 eV was applied for the mass spectrometry (MS) detector (7000 Triple
Quad). The temperature of MS transfer line was also adjusted to 280C. In order to calculate Kovats indices
(KI) for the detected compounds, n-alkanes were employed. 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. [12]
Antimicrobial activity
Experimental solutions
According to the yield of the essential oil, concentration of 32 g/mL of the M. communis essential oil was
obtained and involved in antimicrobial assessments; also, 5% NaOCl (Sigma-Aldrich Co.; St. Louis, MO) and
2% CHX (Sigma-Aldrich Co.; St. Louis, MO) were selected for further comparisons in this study. All
experimental solutions were freshly prepared just before the commencement of the experiments.
Microbial strains
S. aureus (PTCC 1189), E. faecalis (PTCC 1237), and C. albicans (PTCC 5027) were used in this study as
test organisms.

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, 5% NaOCl, and 2% CHX.
In the case of bacterial experiments, the overnight cultured microbial broth was adjusted to an optical density
equal to 0.5 McFarland standard by using a spectrophotometer apparatus at wavelength of 530 nm. Then, the
bacteria were cultured on plates containing Mueller-Hinton agar (Hi-media) with a sterilized cotton swap.
Sterile filter paper discs (6 mm diameter), previously soaked with the experimental solutions, were put on the
inoculated agar surfaces and incubated overnight at 37C. An antibiotic standard disc of ampicillin (Difco) was
used as the positive control, and the sterile water as the negative control. Finally, the inhibition zone diameter
induced by each solution was measured after incubation period of 18 h. [13] This experiment was performed in
triplicate for each test solution.
The antifungal assay was similar to the previously used disk diffusion method, except that Mueller-Hinton agar
was supplemented with 2% glucose to support C. albicans growth. Furthermore, amphotericin-B was
considered as positive control. Statistical analysis was performed by employing Kruskal-Wallis test using
Statistical Package for the Social Sciences (SPSS software version 15.0 (Chicago, IL). P < 0.05 was
considered as statistical significance.
Determination of minimum inhibitory concentration
The MICs for the test solutions were determined by broth microdilution method according to the CLSI 2012
standard protocol. [13]
Briefly, the bacterial strains were cultured overnight at 37C on Mueller-Hinton broth; set to a final density of
McFarland 0.5 standard using a spectrophotometer at wavelength of 625 nm. Then, they were transferred to a
96-well microtiter plates containing serial ten-fold dilutions of the essential oils, NaOCl and CHX on MHB.
Ampicillin was used as positive control and sterile water as negative control.
Similarly, to evaluate the antifungal effects, serial ten-fold dilutions of M. communis essential oil, NaOCl, and
CHX were transferred to a 96-well microtiter plates containing RPMI-1640 media and a suspension of 1.5
10 6 CFU/mL yeasts (equal to 0.5McFarland). Amphotericin-B was also used as positive control and sterile
water as negative control.
All plates were then incubated at 37C for 24 h. The inhibitory effects of the test solutions were determined by
monitoring the absorbance at 625 nm in a microtiter plate reader (BioTek, USA). These experiments were also
done in triplicate to remove any possible bias.
Determination of minimum bactericidal/fungicidal concentration
The MBC and minimum fungicidal concentration (MFC) were determined by the following procedures. The
media from wells with bacteria or fungi, which showed no visible growth that were cultured respectively on
tryptic soy agar and Sabouraud dextrose agar. The MBC and MFC values were defined as the lowest
concentration, yielding a mortality of 98% of microorganisms in the initial inoculums. This occurred when no
more than 4 colonies was seen in agar plates after 24 h incubation at 37C.

Results
Chemical composition of the essential oil
The results obtained by GC-MS analysis of the essential oil of M. communis are presented in [Table 1]. Total,
31 constituents, which represented 99.19% of the total essential oil from M. communis leaves, were identified.
The GC-MS analysis revealed that M. communiscontained 1, 8-cineole (28.62%), -pinene (17.8%), linalool
(17.55%), and geranyl acetate (6.3%) as the major compounds and geraniol (1.6%), -humulene (1.5%),
eugenol (1.3%), isobutyl-isobutyrate (0.8%), and methyl chavicol (0.5%) as the minor components.

Table 1: Compositions of the essential oils obtained from leaves of M. communis

Antimicrobial activity

All three experimental solutions were found to be effective on the test microorganisms. M. communis essential
oil inhibited the growth of all tested microorganisms with the inhibition zone of 21-48 mm. The details are
presented in [Table 2]. Chlorhexidine had the least MIC values against all examined organisms with the
quantities similar to MBC values. M. communis essential oil was more potent than NaOCl with lower MIC on
both bacteria, but it had smaller MIC value against C. albicans. The details of disc diffusion test and MIC,
Minimal Bactericidal/Fungicidal Concentration (MBC/MFC) of the test solutions against the tested
microorganisms are summarized in [Table 2].

Table 2: Disc diffusion (DD in mm), minimum inhibitory concentration (MIC in g/mL),
and minimum bactericidal concentration (MBC/MFC in g/mL) mean values

Discussion
As it can be found on [Table 1], the main constituents of the essential oil were 1, 8-cineole (28.62%), -pinene
(17/8%), and linalool (17.53%). These findings were consistent with the results of other studies, which found
cineole as the main ingredients of M. communis oil. [11],[14],[15],[16] However, the sequence of abundant
components reported in the literature is divergent. For instance, some studies [11],[17] have considered 1,8cineole or limonene as the second major components while -pinene was reported as the major constituent in
Zomorodian et al,Rasooli et al., and Messaoud et al., investigations. [8],[10],[18] In contrast with other reports,
Akin et al., [19] found that eucalyptol was the first major component of M. communis oil. This reported diversity
in the oil compositions is possibly due to the applied techniques and the different environmental growth
conditions of the plant used, which can impact the results and make the comparisons difficult. In another point
of view, the composition diversity of a plant is a pharmaceutical opportunity in which different therapeutic uses
of the same plant species, grown in different regions become possible.
High monoterpene hydrocarbons such as 1, 8-cineole, -pinene, and linalool, which were found as the main
components of M. communis oil in current study, have been extensively proven as strong antimicrobial
substances [20] and therefore might be responsible for the antibacterial activity of the experimented essential
oil.
By analyzing the chemical constituents of M. communis oil, eugenol, and humulene have also been found.
These substances are anti-inflammatory agents and may have potential to reduce pain. In an animal study,
Fernandes et al. revealed that humulene can produce similar anti-inflammatory properties to dexamethasone.
It was also found that humulene can induce important inhibitory influence on tumor necrosis factor- (TNF)
and interleukin-1 (IL1B). [21] Therefore, these agents can be beneficial to decrease the post-operative pain in
root canal treatments.
The disc diffusion method revealed that M. communis essential oil inhibited the growth of all tested
microorganisms and had an inhibition zone of 21-48 mm. Ilcim et al., [22] showed that the extracts of M.

communis had an inhibition zone of 16-38 mm, a finding which is in line with the results of this study.
We also assessed the antimicrobial efficiency of the test solutions by determining their MICs and MBCs
against test organisms. In infected root canals, where the host defense mechanisms are less active compared
to systemic infections, determination of MIC together with the MBC is properly demonstrating the extent of
antimicrobial activity. Although MIC is the most reliable and easily interpreted method for comparison of
different formulations, 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. [23]
The reason to select E. faecalis, S. aureus, and C. albicans was based on the studies that linked these
microorganisms to the refractory infections after endodontic treatments. [2],[3],[4]
The growth of all tested microorganisms was inhibited by the essential oil of M. communis oil at the
concentration range of 0.32-32 g/mL. As with current study, Zomorodian et al. [8] found that the growth of E.
faecalis was inhibited at a concentration value equal to 32 l/mL.
Our study results demonstrated that M. communis essential oil was more potent in lower concentration on S.
aureus than on C. albicans andE. faecalis. On the contrary to our findings, Yadegarnia et al. [11] revealed
that C. albicans was more sensitive than S. aureus when treated with M .communis oil with the MBC value of
4 L/mL. The similar levels of sensitivity for S. aureus have also been found by Zomorodian et al., [8] and
Rasooli et al.[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 .communis oil investigated.
Comparison among the antimicrobial agents showed that CHX was efficient at the lowest concentrations for
both bacteria, followed by M. communis oil and NaOCl. Although M. communis essential oil showed less
antimicrobial potency than NaOCl against C. albicans based on MIC values, it had smaller MFC value against
this fungus. Putting these together, the authors believe that M. communis have favorable potentials to be
applied in endodontic treatments as a root canal irrigant or as an intracanal dressing between visits.

Conclusion
M. communis essential oil with the MIC in the range of 0.032-32 g/mL was an effective antimicrobial agent
against persistent endodontic microorganisms.

Acknowledgments
The authors thank the Vice-Chancellery of Shiraz University of Medical Science for supporting this research
(Grant # 92-01-03-6123). This manuscript is based on the thesis by Dr. Reza Sheikhiani. The authors would
also like to thank Dr. Sh. Hamedani for his editorial assistance and Dr. M. Vosoughi from the Dental Research
Development Center, for the statistical analysis.

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