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.

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%), α-Pinene (17.8%), Linalool (17.55%), and Geranylacetate (6.3%) as the major compounds and Geraniol (1.6%), α-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 μg/mL was an effective antimicrobial agent against persistent endodontic microorganisms.

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.[12] Fungi and some strains of staphylococci can also be found in persistent infections.[34]

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[67] 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.[891011] 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

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 chromatography-mass 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.

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

The collected leaves were dried in a dark room at 24°C 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 4°C 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 μL 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 70°C for 5 min, programmed rate at 3°C/min up to 280°C for 4 min and temperature of injector at 250°C. 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 280°C. 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 μg/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 37°C. 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 37°C 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 × 106 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 37°C 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 37°C.

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. communis contained 1, 8-cineole (28.62%), α-pinene (17.8%), linalool (17.55%), and geranyl acetate (6.3%) as the major compounds and geraniol (1.6%), α-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

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 μg/mL), and minimum bactericidal concentration (MBC/MFC in μg/mL) mean

DISCUSSION

As it can be found on Table 1, the main constituents of the essential oil were 1, 8-cineole (28.62%), α-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.[11141516] However, the sequence of abundant components reported in the literature is divergent. For instance, some studies[1117] have considered 1,8-cineole or limonene as the second major components while α-pinene was reported as the major constituent in Zomorodian et al, Rasooli et al., and Messaoud et al., investigations.[81018] 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, α-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-α (TNFα) and interleukin-1 β (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.[234]

The growth of all tested microorganisms was inhibited by the essential oil of M. communis oil at the concentration range of 0.32–32 μg/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 μl/mL.

Our study results demonstrated that M. communis essential oil was more potent in lower concentration on S. aureus than on C. albicans and E. 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 μL/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 μg/mL was an effective antimicrobial agent against persistent endodontic microorganisms.