Time-dependent antibacterial effects of Aloe vera and Zataria multiflora plant essential oils compared to calcium hydroxide in teeth infected with Enterococcus faecalis

ORIGINAL ARTICLE

Endodontics

Time-dependent antibacterial effects of Aloe vera andZataria multiflora plant essential oils compared to calciumhydroxide in teeth infected with Enterococcus faecalisAbbas Abbaszadegan1, Safoora Sahebi1, Ahmad Gholami2, Alireza Delroba3, Amin Kiani3, Aida Iraji4

& Paul Vincent Abbott5

1 Department of Endodontics, Faculty of Dentistry, Shiraz University of Medical Sciences, Shiraz, Iran

2 Pharmaceutical Sciences Research Center and Department of Pharmaceutical Biotechnology, School of Pharmacy, Shiraz University of Medical

Sciences, Shiraz, Iran

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

4 Central Laboratory, Shiraz University of Medical Sciences, Shiraz, Iran

5 School of Dentistry, University of Western Australia, Perth, Western Australia, Australia

Keywords

Aloe vera, antimicrobial activity, calcium

hydroxide, root canal treatment, Zataria

multiflora.

Correspondence

Dr Ahmad Gholami, School of Pharmacy,

Shiraz University of Medical Sciences,

Akbarabad Ring Way, Shiraz, Iran.

Tel: +98-91-7314-0518

Fax: +98-71-1626-3192

Email: [email protected]

Received 20 February 2014; accepted 2 July

2014.

doi: 10.1111/jicd.12123

AbstractAim: In the present in vitro study, we investigated the time-related antimicro-

bial efficacy of Aloe vera and Zataria multiflora (Z. multiflora) plant essential

oils compared to calcium hydroxide ([Ca[OH]2) to eliminate Enterococcus fae-

calis (E. faecalis) from root canals.

Methods: A new strain of E. faecalis (Enterococcus spp. AGH04) was isolated

from a previously root-filled tooth with persistent apical periodontitis. The 16S

rRNA sequence was analyzed and deposited in GeneBank under accession

number KF465681. A total of 108 extracted human single-rooted teeth were

contaminated with this bacterial strain and treated with Aloe vera essential oil,

Z. multiflora essential oil, and Ca(OH)2 for 1, 7, and 14 days. Gas chromatog-

raphy–mass spectrometry (GC–MS) was used to determine the chemical com-

position of the oils. The percentage reduction from initial c.f.u./mL counts

were calculated and analyzed.

Results: Carvacrol, thymol, and linalool were the main constituents of both

essential oils. The c.f.u./mL count reductions significantly increased for all three

medicaments when the contact time was extended. A statistically-significant dif-

ference was observed between the medicaments after 1 and 7 days, but there

was no significant difference after 14 days.

Conclusions: Both medicinal herbs showed equal antimicrobial efficiency

against E. faecalis, comparable to Ca(OH)2 for the prolonged contact time of

14 days.

Introduction

The aim of root canal treatment is either prevention or

the treatment of periapical pathosis caused by microor-

ganisms.1 Achieving this goal is highly related to the

degree of reduction of the intracanal bacterial popula-

tion.2 Although complete elimination of bacteria is chal-

lenging and might not always be successful, it is generally

achieved by chemomechanical root canal preparation with

the aid of antimicrobial irrigation solutions and intracanal

dressings between treatment visits.3 Applying an interap-

pointment antimicrobial dressing in infected root canals

is recommended, as instrumentation and irrigation do

not reliably eradicate all of the bacteria.4

Enterococcus faecalis (E. faecalis) is one of the most

resistant bacteria found in infected root canals, especially

ª 2014 Wiley Publishing Asia Pty Ltd 1

Journal of Investigative and Clinical Dentistry (2014), 5, 1–9

in cases with persistent apical periodontitis.5 Its ability to

invade dentinal tubules and survive harsh canal

conditions, together with its adaptability to lethal chal-

lenges, are the key factors that make this organism the

most persistent pathogen in root canal treatment.6 There-

fore, efforts to eliminate this bacterium might equate with

achieving successful disinfection.

Calcium hydroxide (Ca[OH]2) is perhaps the most

frequently used intracanal medicament in endodontics.7 It

has positive features, such as antimicrobial activity, the

ability to induce hard tissue formation, and it can prevent

inflammatory root resorption.7,8 However, it can change

the strength of dentine and weaken it, even after short-

term use.9 In addition, this medicament has low capacity

to penetrate dentinal tubules and it does not have sufficient

effectiveness against all species of endodontic microorgan-

isms and their endotoxins.10–12 It has also been proposed

that the increase in pH, which might be a result of using

alkaline medicaments, such as Ca(OH)2, can enhance the

bacterial attachment to collagen fibers of dentine and can

help protect them from the disinfection processes.13

Interest in the use of herbal remedies in endodontics has

recently grown. Medicinal herbs are supposed to be

effective in the treatment of infectious diseases, are biocom-

patible, and mitigate the side-effects of synthetic antimicro-

bials.14 The antimicrobial activity of some natural products

against E. faecalis has been evaluated in recent years for

herbs, such as Arctium lappa,15 Morinda citrifolia,16 Tripha-

la, green tea polyphenols,17 liquorice,18 Syzygium aromati-

cum, Ocimum sanctum, and Cinnamomum zeylanicum.19

Zataria multiflora (Z. multiflora) is a natural thyme-like

plant with similar chemical and pharmacological proper-

ties to the well-known medicinal herb Thymus vulgaris.20

It is known to have favorable antimicrobial activity, anti-

oxidant, and anti-inflammatory effects.20 The essential oils

of this plant contain significant amounts of oxygenated

monoterpenes, including thymol and carvacrol, which

have well-known antimicrobial characteristics.21 P-Cym-

ene is another component of the oil. When this ingredi-

ent is combined with carvacrol, synergistic antimicrobial

efficacy increases.22 The antibacterial efficacy of Z. multifl-

ora, as an irrigant in contaminated teeth with E. faecalis

has been previously demonstrated by Ravanshad et al.23

However, its effectiveness has not been evaluated as an in-

tracanal medicament.

Aloe vera (Aloe barbadensis miller) is a cactus-like plant

that belongs to the Liliaceal family.24 It is a natural medica-

ment with a long history of usage in medicine and nutri-

tion. It is also used in dentistry in cases of dry socket and

acute mouth lesions.25 It has been reported to be beneficial

for controlling inflammation when used in periodontal sur-

gery sites and around dental implants.25 The antimicrobial

properties of Aloe vera against various species of microor-

ganisms, including E. faecalis, have also been reported.26

Aloe vera has potent antibacterial, antiviral, and antifungal

activities.27,28 In in vitro disc diffusion studies by Alemdar

et al. and Athiban et al., the adequate effectiveness of Aloe

vera against E. faecalis was verified.29,30 In contrast, Valera

et al., Bhardwaj et al., and Vinothkumar et al., failed to

report acceptable efficacy against E. faecalis in infected root

canals when used as an endodontic irrigant.31–33

Due to the shortcomings of Ca(OH)2 as an intracanal

dressing, several studies have been performed to find suit-

able, cost-effective, non-toxic, and efficient alternatives for

this medicament. The purpose of the present in vitro study

was to compare the time-related antimicrobial efficacy of

Aloe vera and Z. multiflora with Ca(OH)2 as intracanal

medicaments in human infected teeth with E. faecalis.

Material and methods

Preparation of the specimens

The present study was approved by the Ethics Committee

of Shiraz University of Medical Sciences. A total of 108

extracted, intact, human single-rooted teeth of similar

lengths were collected. The teeth had one straight canal

with mature apices and there were no fractures or caries

in the roots. The specimens were stored in distilled water

to avoid dehydration. The teeth were decoronated 2 mm

below the cemento–enamel junction using a high-speed

hand piece and fissure burs (SS White Burs Inc., Lake-

wood, NJ, USA) under water coolant to create root speci-

mens that were 13–15 mm long. The working length

(WL) of each canal was measured by inserting a #15 K-file

(Dentsply Maillefer, Ballaigues, Switzerland) into the canal

until it was evident at the apical foramen. The WL was

calculated to be 1 mm less than the length attained by this

initial file. Root canal instrumentation was performed

using Protaper rotary instruments (Dentsply Maillefer

Tulsa, OK, USA) up to size F3, according to the manufac-

turer’s instructions. The canals were irrigated with 2 mL

of 2.5% sodium hypochlorite (NaOCl) (Vista Dental

Products, Racine, WI, USA) after each file. The specimens

were placed in an ultrasonic bath of 17% ethylenediamine-

tetraacetic acid (EDTA) (Vista Dental Products, Racine,

WI, USA) for 5 min, followed by 2.5% NaOCl irrigation

to remove the smear layer. In order to avoid bacterial

leakage from the canals, the apical foramen of each canal

was sealed with composite resin, and the external root sur-

faces (except the coronal access opening) were covered

with epoxy adhesive. The experimental specimens (n = 10

in each group) were randomly divided into nine 24-well

cell culture microplates (Corning, Corning, NY, USA) and

six control microplates (n = 3). Specimens were fixed to

the wells with acrylic resin, and then sterilized with ethyl-

2 ª 2014 Wiley Publishing Asia Pty Ltd

Antibacterial efficacy of medicinal herbs A. Abbaszadegan et al.

ene oxide (Acecil, Campinas, S~ao Paulo, Brazil). The

efficiency of the sterilization process was checked by filling

the root canals with brain–heart infusion (BHI) (Himedia

Laboratories, Mumbai, India) medium. The specimens

were then kept in an incubator (Mart Microbiology BV,

JB Drachten, the Netherlands) for 48 h at 95% relative

humidity (RH) and 37°C. Subsequently, microbial assess-

ment was performed for each root canal.

Isolation of the test microorganism

The microorganism used in this study was a new strain of

E. faecalis, which had been isolated from a patient under-

going root canal retreatment of a single-rooted tooth that

had been previously root filled, but had persistent apical

periodontitis. The sample was taken from the root canal

after the tooth was isolated with rubber dam and disinfec-

ted with 30% H2O2 and 2.5% NaOCl. Then 5% sodium

thiosulphate was employed to neutralize the disinfectants.

An access cavity was prepared, and the root filling was

removed mechanically using Gates–Glidden burs and

hand files without the use of solvents. After WL determi-

nation, the root canal was irrigated with saline, and sterile

paper points were used, 1 mm short of the apex, to soak

up the entire contents of the canal. The paper points were

then transferred into a tube containing sterile BHI med-

ium and dispersed within a vortex for 1 min. The result-

ing solution was serially diluted 10-fold in BHI broth.

Aliquots of 100 lL from the suspension were spread on

BHI agar plates (enriched with 5% defibrinated sheep

blood) and incubated at 37°C for 24 h. Individual colo-

nies of the isolate were identified by Gram stain, catalase

reaction, colony morphology, and by the pattern of car-

bohydrate fermentation.34

In order to verify and determine the species, the

sequence of a small subunit of rRNA was studied using

molecular markers. Genomic DNA of bacterial strains was

prepared according to the protocol proposed by Ghasemi

et al.35 DNA fragments of approximately 800 base pairs

(bp) were amplified from the genomic DNA of bacterial

strains with polymerase chain reaction (PCR) by using

universal primers against the 16S rRNA genes. PCR

amplifications were determined by 1% (w/v) agarose gel

electrophoresis in Tris–borate–EDTA buffer.

PCR products were purified from agarose gel with the

CoreBio PCR purification kit (cat no. GE-100, CoreBio,

Seoul, Korea), and used as templates in sequencing reac-

tions by CinnaGen (CinnaGen, Tehran Iran). The 16S

rRNA sequence was analyzed by using the BLAST online

program and annotations of bacterial strain, and then

deposited in GeneBank (National Institutes of Health

genetic sequence database, USA) under accession number

KF465681.

Contamination of the specimens with E. faecalis

The isolated 48-h colonies of E. faecalis, which were

grown on BHI agar plates, were suspended in 5 mL BHI

broth media. The cell suspension was spectrophotometri-

cally adjusted to match the turbidity of 6 9 108 c.f.u./mL

(equivalent to two McFarland standards).

Under laminar flow, the sterile BHI broth in the root

canals was replaced by bacterial inoculums using sterile

pipettes (except for the negative control groups). The

roots were maintained in an incubator at 37°C and 95%

RH for 21 days. Half of the contaminated media in each

canal was replaced with fresh BHI every 2 days for the

purpose of continuous bacterial feeding. In order to con-

firm the purity of the bacterial culture, Gram staining and

catalase reaction tests were performed on the last day.

Preparation of the medicaments

All preparations were performed under strict aseptic con-

ditions. Ca(OH)2 powder (Henry Schein Company, Mel-

ville, NY, USA) was mixed with sterile saline

(Darupakhsh, Tehran, Iran) in a 6:4 powder/saline ratio

to obtain a paste-like consistency.

Z. multiflora plants were collected from highlands

around Shiraz, Iran, and Aloe vera leaves were collected

from northern areas of the Persian Gulf. The plant species

were identified and authenticated by Dr. Mahmoud Reza

Moein, a plant taxonomist, according to the morphologi-

cal description and previously-collected, known samples.

The voucher specimen of each plant was deposited at the

Herbarium of the Department of Pharmacognosy, Faculty

of Pharmacy, Shiraz University of Medical Sciences, Shi-

raz, Iran. Air-drying of the plants was performed after

washing and storing them in a shady place for 20 days at

room temperature.

In order to prepare the applicable form of the

Z. multiflora, the plants were grounded in a blender to

produce a fine powder. The essential oil was obtained

from 300 g of the powder by steam distillation using a cle-

venger-type apparatus (yield 1.5%). The organic layer was

separated, concentrated under reduced pressure, dried

over anhydrous sodium sulfate, and stored in sealed vials

at low temperature (4°C). The oil was finally mixed with

hydroxyethyl cellulose (Merck, Darmstadt, Germany) as a

thickening agent at a ratio of 2:1 (v/w).

The Aloe vera essential oil was prepared using the fol-

lowing procedure. The dried plant materials of Aloe vera

were crushed to small pieces and powdered in an electric

grinder. Three hundred grams of powder were subjected

to hydrodistillation for 4 h using a clevenger-type appara-

tus to produce essential oil (yield 1.3%). The distillate

was extracted with n-hexane. As with Z. multiflora, the


A. Abbaszadegan et al. Antibacterial efficacy of medicinal herbs

organic layer was separated and concentrated. Hydroxy-

ethyl cellulose was employed as a thickening agent in the

same way as used for the Z. multiflora essential oil.

It is notable that hydroxyethyl cellulose is a highly-inert

agent that is non-ionic and water soluble, and has been

used in several studies for gel formation.36,37

GC–MS of the essential oils

GC–MS analyses of the essential oils were run on an Agi-

lent 7000 mass spectrometer coupled to Agilent 7890A

series gas chromatograph in electron impact mode

(Agilent Technologies, Santa Clara, CA, USA). A fused silica

DB1 column (30 m, 0.32 mm diameter, 0.25-mm film

thickness) was directly coupled to the mass spectrometer.

Helium was the carrier gas, which was used at a flow

rate of 1.2 mL/min and a split ratio equal to 1/40. The

injector was operated at 250°C. The oven temperature was

programmed to increase from 60 to 280°C at a rate of

3°C per min and finally held for 4 min. The transfer line

temperature was 280°C. The quadruple mass spectrometer

was scanned over the 46–650 amu with an ionizing volt-

age of 70 eV and an ionization current of 150 mA.

The constituents were identified by comparison of

Kov�ats retention indices, referring to compounds known

from the literature database, and also by comparing their

mass spectra with the Wiley Library or with the published

mass spectra. Relative percentage amounts were calculated

from the total area under the peaks by the apparatus soft-

ware.

Initial microbial sampling from the root canals

The initial microbial assessment was carried out after

21 days using three sterile paper points (Gapadent Co.,

Ltd., Tianjin, China), which were placed in each canal for

1 min. Under laminar flow, the contaminated paper

points were transferred into tubes containing 1 mL BHI.

The tubes were vortexed for 1 min, and the resulting

solution was serially diluted 10-fold in the BHI broth.

Aliquots of 100 lL from the suspension were spread on

BHI agar plates and subsequently incubated at 37°C for

24 h. After this period, bacterial growth was measured by

the c.f.u./mL counts of E. faecalis and confirmed by col-

ony morphology and Gram stain.

Repreparation of the specimens

The root canals were reprepared using 5 mL of sterile sal-

ine solution, and then filled with 17% EDTA for 3 min.

Final irrigation with sterile saline solution was performed.

The root canals were then dried with sterile paper points

and filled with the intracanal medicament.

Experimental groups

The microplates containing the roots were randomly

divided into the following experimental groups based on

the medicaments used and their intracanal contact time:

(a) groups 1–3 (n = 10 in each group): Ca (OH)2 for 1,

7, and 14 days;

(b) groups 4–6 (n = 10 in each group): Z. multiflora

essential oil for 1, 7, and 14 days;

(c) groups 7–9 (n = 10): Aloe vera essential oil for 1, 7,

and 14 days;

(d) groups 10–12 as positive controls (n = 3 in each

group): sterile saline for 1, 7, and 14 days;

(e) groups 13–15 as negative controls (n = 3 in each

group): no bacterial contamination for 1, 7, and

14 days

Ca(OH)2 was inserted into the root canals using a size 30

spiral filler (Dentsply Maillefer, Ballaigues, Switzerland).

The Z. multiflora and Aloe vera essential oils were inserted

into the root canals using sterile endodontic pressure syrin-

ges. Each medicament was applied into the root canals until

some of the medicament extruded from the access cavity.

After the excess material was removed, sterile cotton

pellets were placed in the access cavities, and the speci-

mens were incubated in a microaerophilic environment at

37°C for the defined contact time, based on the experi-

mental groups.

Microbial sampling from the root canals at the defined

contact time

Medicaments were removed from the root canal at the

defined contact time for each experimental group using

#30 K-files and irrigation with 5 mL sterile saline. The

specimens in groups 1–3 were irrigated by 1 mL of 0.5%

citric acid (Merck, Darmstadt, Germany) followed by

2 mL sterile saline to neutralize the Ca(OH)2.38 The other

experimental groups (groups 4–9) were irrigated again

with 2 mL sterile saline. Microbial assessments were per-

formed after the allocated time of incubation with medi-

caments using three sterile paper points. Bacterial growth

was measured by the c.f.u./mL counts of E. faecalis.

Statistical analysis

The results were first subjected to logarithmic transforma-

tion and expressed as log10(x + 10). The percentage of

reduction from the initial c.f.u./mL count (c.f.u.i) for each

sample in each defined contact time (c.f.u.t) was then cal-

culated as x = log10 c.f.u.t – log10 c.f.u.i/log10c.f.u.i 9 100. The percentages of reductions were then

compared and analyzed. Due to the violation of normal-

ity assumption, non-parametric Kruskal–Wallis and



Mann–Whitney tests were employed. The analyses were

performed using SPSS 15.0 software (SPSS, Chicago, IL,

USA). The significance level was set at P < 0.05 and was

adjusted based on the number of comparisons.

Results

Isolation of the test microorganism

The isolated microorganism was verified as catalase-nega-

tive, Gram-positive, and facultative anaerobic cocci with

b-hemolysis ability. The result of gel electrophoresis (Fig-

ure 1) showed a band at approximately 800 bp under UV

light. The analyses indicated that the isolate was 98%

consistent with E. faecalis. This strain is now confirmed

as Enterococcus spp. AGH04.

GC–MS of the essential oils

The solvent extraction of Aloe vera yielded a green oily

extract, while the oil isolated by hydrodistillation of

Z. multiflora was found to be a yellow liquid with a

strong aromatic odor. The detailed chemical compositions

of the volatile oils of Aloe vera and Z. multiflora are listed

in Tables 1 and 2, respectively. Carvacrol (50.78%), thy-

mol (16.94%), and linalool (12.68%) were main constitu-

ents of the Aloe vera essential oil, and thymol (32.52%),

carvacrol (32.17%), and linalool (10.89%) were the main

components of the Z. multiflora essential oil.

Microbial sampling from the root canals at the defined

contact time

The bacterial contamination of the root canals with

E. faecalis was confirmed in all teeth at the initial

sampling, which was carried out 21 days after incubation.

One-way ANOVA test showed no statistically-significant dif-

ference between c.f.u./mL counts of all groups at the ini-

tial sampling step (P = 0.672). The specimens in the

positive control groups (groups 10–12) showed similar

c.f.u./mL counts for all three sampling stages with no sta-

tistical differences (P = 0.43). The specimens in the nega-

tive control groups (groups 13–15) confirmed the absence

of microbial growth at all three contact times.

The results showed that the c.f.u./mL counts decreased

from the initial counts for all three tested medicaments

(Figure 2). The percentage reduction of log10 c.f.u./mL

counts significantly increased in all groups by extending

the contact time (P = 0.00). After the first day of incuba-

tion, the increase in log10 c.f.u./mL count reduction was

similar for Ca(OH)2 and the Z. multiflora essential oil,

but the Aloe vera essential oil had significantly lower effi-

ciency. Accordingly, the bacterial load reduction from 7

to 14 days of incubation was significant for Aloe vera, but

not significant for Ca(OH)2 and Z. multiflora. A statisti-

cally-significant difference was observed between the med-

icaments after 1 and 7 days of incubation, but there was

no significant difference between them after 14 days. The

results are summarized in Table 3.

Discussion

In the present study, root canals of human extracted teeth

were infected with E. faecalis to simulate the clinical condi-

tion of the teeth with infected root canal systems. This

microorganism was chosen because of its major role in

unfavorable outcomes of root canal treatment, and also its

resistance to Ca(OH)2-based medicaments.5,10,11 The

selected bacterium was a new strain of E. faecalis isolated

from a previously root-filled tooth with persistent apical

periodontitis. This isolate was verified and determined by

molecular and culture methods, and is now confirmed as

AGH04. In order to open the dentinal tubules and allow

E. faecalis to diffuse thoroughly into the dentine, the smear

Figure 1. Analysis of 16S rRNA fragments of Enterococcus spp.

obtained after polymerase chain reaction amplification and gel elec-

trophoresis. Lane 1, gene ladder; lanes 2 and 3, 16srRNA gene

sequence showing the length of the gene at approximately 800 base

pairs .



layer was removed and a 21-day incubation period was

chosen.38

The effectiveness of Ca(OH)2 against E. faecalis was

evaluated, and its possible time-dependent antimicrobial

activity was assessed and compared with two medicinal

herbs, Aloe vera and Z. multiflora. The evaluation was

based on the notion that the efficiency of the intracanal

dressings could probably be changed during their time

of contact in the root canal. Although the effectiveness

of Ca(OH)2 against E. faecalis is still debated in the liter-

ature, some investigations have reported its beneficial

activity.11

The exact time required for achieving optimal bacteri-

cidal effects of medicaments has not been fully under-

stood. Behnen et al. reported the elimination of

E. faecalis from infected dentine samples after 1 day of

exposure to Ca(OH)2, while Sj€ogren et al. and Shuping

et al. stated that Ca(OH)2 should remain in the root

canal for at least 7 days to be effective. Some studies

have recommended that the intracanal contact time

should be more than 14 days for Ca(OH)2-based medi-

caments when used in infected teeth.8,39–42 The results of

the current study revealed that Ca(OH)2 could not com-

pletely eradicate E. faecalis, even after 14 days of intraca-

nal medication. Although Ca(OH)2 significantly reduced

the number of bacteria from 1 to 7 days, there was not

a more significant reduction in bacterial population by

extending the contact time to 14 days. These results were

in line with the outcome of studies in which Ca(OH)2could not lead to a negative culture, even after long

exposure times.43

In the current study, both medicinal herbs presented

acceptable bactericidal effects against E. faecalis and were

comparable to Ca(OH)2, although the results were depen-

dent on the amount of time they remained inside the

root canal. After the first day of incubation, the effective-

ness of Ca(OH)2 was similar to the Z. multiflora essential

oil, but the Aloe vera essential oil had significantly lower

efficiency. By extending the contact time, the results for

the three medicaments were similar by 14 days. These

findings showed that Aloe vera had slow antimicrobial

Table 1. Composition of Aloe vera essential oil

Peak no. Compounds

Kov�ats retention

indices Area (%)

1 Terpinene (c) 1048 0.55358

2 Linalool 1080 12.68156

3 Vertocitral C (trans) 1086 1.518537

4 Unknown 1104 1.223972

5 Terpineol (a) 1169 1.000508

6 Thymol methyl ether 1223 0.680549

7 Thymol 1265 16.94769

8 Carvacrol 1274 50.7872

9 Thymol acetate 1340 1.361097

10 Caryophyllene-E 1410 0.868461

11 Spathulenol 1556 4.509904

12 Caryophyllene oxide 1561 6.551549

13 Docosane 2199 0.533266

14 Tricosane 2300 0.782123

Table 2. Composition of Zataria multiflora essential oil

Peak no. Compounds

Kov�ats

retention

indices Area (%)

1 Tricyclene 922 0.2016

2 Pinene (a)- 930 2.341159

3 Fenchene 943 0.071535

4 Octanone (3) 962 0.390193

5 Isolimonene 971 0.217858

6 Myrcene 981 0.468232

7 Phellandrene 999 0.071535

8 Terpinene (a) 1010 0.48449

9 Cymene (Para) 1013 3.589777

10 Cineol (1-8) 1021 0.650322

11 Terpinene (c) 1048 3.687325

12 Linalool oxide (cis) 1056 0.152826

13 Linalool oxide (trans) 1071 0.208103

14 Linalool 1085 10.88964

15 Terpinen-4-ol 1160 0.351174

16 Terpineol-a 1171 0.770631

17 Thymol, methyl ether 1215 0.952722

18 Carvacrol, methyl ether 1225 2.44521

19 Thymol 1271 32.5161

20 Carvacrol 1281 32.17468

21 Thymol acetate 1324 0.894193

22 Carvacrol acetate 1342 0.994993

23 Caryophyllene 1412 3.121545

24 Aromadendrene 1432 0.386942

25 Humulene 1445 0.143071

26 Macrocarpene (b) 1487 0.552774

27 Spathulenol 1556 0.650322

28 Caryophyllene oxide 1562 0.621057

Figure 2. Change in Enterococcus faecalis counts (log c.f.u./mL) by

extending the contact time. Bacterial counts in negative controls were

zero in all sampling stages.



activity against E. faecalis, which was enhanced signifi-

cantly from day 1 to 7 and 14 days of contact time. It

seems that the higher levels of monoterpenoid hydrocar-

bons that existed in the essential oil of Z. multiflora

increased the lipophilicity of the oil to incorporation into

the bacterial cell membrane and resulted in faster action

compared to Aloe vera.

There are limited studies evaluating the effectiveness of

Aloe vera as an intracanal medicament against E. faecalis.

The only report is by Bhardwaj et al.,44 in which the anti-

microbial activity of three natural extracts was compared

with 2% chlorhexidine gel and Ca(OH)2 after 1, 3, and

5 days of intracanal medication. In contrast to the current

study, they found that Ca(OH)2 had sufficient antibacte-

rial activity on the first day, and this decreased after

3 days, and then increased by 5 days. A gradual decrease

in the efficacy of Aloe vera was also seen by extending the

contact time to 5 days, even though the authors did not

present a reasonable explanation for this impaired

effect.44

The antimicrobial activity of Aloe vera and Z. multiflora

essential oils might be attributed to the presence of

thymol and carvacrol, which are natural monoterpenes

and act on the cell membrane of the organisms causing

cellular death.20 Several published studies have demon-

strated the excellent antimicrobial activities of thymol45,46

and carvacrol46,47 against E. faecalis. Moreover, in their in

vitro study, Ravanshad et al. reported that 1% and 2%

essential oils of Z. multiflora have the same effectiveness

as 2.5% NaOCl used as an irrigant to eliminate E. faecalis

from infected root canals.23

The existence of linalool might also play a significant

role in increasing the antimicrobial activity of the oils.48

This constituent has a pleasant taste and smell, and has

also been found to have anti-inflammatory properties.49

Ideally, further studies need to be performed using var-

ious forms of extracts or essential oils of Aloe vera and

Z. multiflora to investigate their antimicrobial activity,

their cytotoxicity, and their possible effects on dentine

and necrotic tissues.

Conclusions

Under the experimental conditions of the present study,

the antimicrobial efficiency of all tested medicaments was

similar after 14 days. Aloe vera and Z. multiflora essential

oils showed equal antimicrobial efficiency against E. fae-

calis in infected root canals and were comparable to Ca

(OH)2 when used for a prolonged contact time of

14 days.

Acknowledgments

This manuscript is based on the thesis of Dr Alireza Del-

roba and Dr Amin Kiani. The authors thank the vice

chancellery of Shiraz University of Medical Science for

supporting this research (grant # 4978), and Dr Shahram

Hamedani and Dr Mehrdad Vosooghi of the Research

Development Center, for their valuable comments that

improved the quality of the paper.

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Time-dependent antibacterial effects of Aloe vera and Zataria multiflora plant essential oils compared to calcium hydroxide in teeth infected with Enterococcus faecalis