J Conserv Dent. 2016 Jan-Feb; 19(1): 101–105.

doi: 10.4103/0972-0707.173212

PMCID: PMC4760003

In vitro comparison of antimicrobial effect of sodium

hypochlorite solution and Zataria multiflora essential oil as
irrigants in root canals contaminated with Candida albicans
Mahdi Sedigh-Shams, Parisa Badiee,1 Alireza Adl,2 Milad Dadollahi Sarab,3 Abbas
Abbaszadegan, andMohammadreza Nabavizadeh4

Author information ► Article notes ► Copyright and License information ►

This article has been cited by other articles in PMC.

Abstract
Go to:

INTRODUCTION
Microorganisms and their products are the most important factors for the progression of pulp
and periapical diseases.[1] Although bacteria are frequently known to cause root canal
infections, recent studies have shown that fungi also cause root canal infections.[2] Fungi
have been detected in root canals with primary and secondary endodontic infections,[3]
abscesses or cellulitis of endodontic origin,[4] and periapical lesions in asymptomatic
teeth.[5] Candida albicans is the most commonly fungus isolated from the oral cavity and
root canals.[6]
Use of irrigants to thoroughly clean root canal systems during instrumentation is central to
successful endodontic treatment.[7] Irrigation is complementary to instrumentation and
facilitates the removal of pulp tissues and/or microorganisms.[7] In addition to providing
mechanical flushing action, an irrigant should exert microbicidal effects without damaging
periradicular tissues.[7]
Sodium hypochlorite (NaOCl) is the most popular irrigant used during instrumentation. It is a
nonspecific proteolytic agent with wide-ranging activities against endodontic
microorganisms.[7] Many in vitro and in vivo studies have shown its superiority in
eliminating C. albicans.[8] Despite the favorable qualities of NaOCl, it has significant
clinical disadvantages such as unpleasant odor and taste, cytotoxicity,[7] deteriorative effects
on the mechanical and chemical properties/composition of the dentine[9] and negative
effects on the mechanical properties, and cutting efficiency of nickel-titanium
instruments.[10]
Side effects of synthetic drugs have prompted researchers to search for herbal alternatives.
Pharmacological studies have acknowledged the value of medicinal plants as potential
sources of bioactive compounds.[11] Some natural plant extracts have antimicrobial and
therapeutic properties, suggesting their potential use as endodontic irrigants or intracanal
medications.[12,13,14] In addition to antimicrobial effects, herbal irrigants are safe and
nontoxic to host tissues.[13]
Zataria multiflora is a thyme-like plant belonging to the Lamiaceae family that is endemic to
Iran, Pakistan, and Afghanistan.[14] In Iran, this plant is called Avishan Shirazi and is known
to have antiseptic, anesthetic, and antispasmodic uses.[15] The essential oil (EO) of this plant
mainly contains phenolic compounds such as carvacrol, thymol, and eugenol.[14,15] In
vitro studies have shown that this EO has excellent activity
against Candida species.[16,17] Z. multiflora EO prevents the progression of candidiasis to
the viscera in mice.[18] In dentistry, Z. multiflora EO decreases C. albicans count in denture
stomatitis[19] and on the surface of removable orthodontic appliances.[20]
However, limited data are available on the antifungal activity of Z. multiflora EO in a dental
model. Therefore, this study aimed to compare the antifungal efficacy of Z. multiflora as a
root canal irrigant with that of NaOCl against C. albicans.
Go to:

MATERIALS AND METHODS

Biological process
Z. multiflora EO used in this study was purchased from Barij Essence Co., (Barij Essence,
Kashan, Iran). According to the certification of analysis for EO from Barij Essence Co., this
EO is soluble in 80% ethanol. Therefore, 80% ethanol was used for the initial dilution of Z.
multiflora EO.
C. albicans was collected from the oral cavity of a patient and was cultured after
confirmation by using API methods. Next, 1:20 dilution of C. albicans was prepared using
0.5 McFarland standard, and the subsequent 1:50 dilution was prepared using RPMI-1640
(Sigma, Germany).
Z. multiflora EO and NaOCl were serially diluted according to CLSI M27-A2 to determine
the minimum fungicidal concentration (MFC). To ensure that ethanol used for the initial
dilution of Z. multiflora EO did not add to its antifungal activity, minimum inhibitory
concentration (MIC) of ethanol was also determined.

Irrigants
Two concentrations of Z. multiflora EO (MFC and twice the MFC) and MFC of NaOCl were
used as the irrigants.

Antifungal evaluation
C. albicans seeded on Petri dishes containing sabouraud dextrose agar (SDA) was used as
the test microbe. C. albicans suspensions in standard saline solution were prepared by
spectrophotometry (λ = 530 nm). This procedure yielded a yeast stock suspension in RPMI
containing 1 × 106 to 5 × 106 cells/ml.

Collection and preparation of samples

Sixty single-canal mandibular premolars were collected, disinfected with NaOCl for 24 h,
and stored in normal saline solution to prevent dehydration. The number of canals was
verified using periapical radiographs, and the samples were visually inspected to ensure the
absence of any defect such as external resorption, crack, and caries. All the samples were
decoronated at the CEJ level by using a high-speed diamond fissure bur (Tiz Kavan, Iran).
This produced a root length of 12-15 mm.
After determination of working length, the canals were enlarged using number 15 and
number 20 K-files, and saline solution was used as the irrigant. To prevent the leakage of
microorganisms and irrigants, the apex was sealed using Z-100 composite resin (3M, St Paul,
MN, USA) and the roots were sealed externally, except for the cervical opening, by using
nail polish. The teeth were then placed on acrylic blocks. All the specimens were sterilized at
121°C for 10 min at 15 psi and were stored aseptically at 100% humidity and 30°C until use.
The root canals were infected using 10-20 μl of C. albicans suspension (depending on root
canal diameter) with an insulin syringe. The samples were incubated at 37°C in a humid
atmosphere for 72 h. Next, microbial samples were collected using sterile paper points before
instrumentation. When C. albicansgrowth after 72 h reached 2 × 105 colony-forming units
(CFU)/ml, the samples were divided into four groups (n = 15) according to the irrigants used:
Group 1, teeth irrigated using MFC of Z. multiflora EO; Group 2, teeth irrigated using twice
the MFC of Z. multiflora EO; Group 3, teeth irrigated using MFC of NaOCl (Pak Shoo
Chemical, Iran); and Group 4, teeth irrigated using distilled water (DW, negative control).
The root canals were instrumented using S1, S2, F1, F2, and F3 ProTaper rotary files
(Dentsply Maillefer, Switzerland). Each root canal was irrigated with 2 ml of experimental
irrigants between instrumentations (a total of 10 ml for each canal) by using a 27-gauge
needle. At the end of root canal preparation, root specimens from Groups 1 and 2 were
irrigated with 2 ml of sterile DW to remove the remaining Z. multiflora EO. Root canals
from Group 3 were irrigated with 2 ml of 4% sterile sodium thiosulfate solution to neutralize
the remaining NaOCl. Time required for cleaning and shaping each tooth was 12-14 min.
Postinstrumentation sampling was performed immediately after completing root canal
preparation. Three sterile paper points were used for each initial and postinstrumentation
sampling. The paper points were extended until the working length, were left in the canal for
1 min, and were transferred to Eppendorf tubes containing 0.5 ml sterile saline solution. The
tubes were shaken for 30 s (Vortex AP 56; Phoenix, Araraquara, SP, Brazil), and 0.1 ml
aliquots were plated on Petri plates containing SDA. The SDA plates were incubated at 37°C
± 1°C for 48 h. Fungal growth was verified, and the number of CFU of C. albicanswas
counted.

Statistical analysis
Percentage reduction in fungal colonies was calculated using the formula (S1 − S2)/S1 × 100,
where S1 is the number of fungal colonies before irrigation, and S2 is the number of fungal
colonies after irrigation. Kruskal-Wallis and Mann-Whitney tests were used to compare
differences in colony counts between the groups. SPSS version 13 (SPSS Inc., Chicago, IL,
USA) was used for data analysis. Data were expressed as median (mean ± standard
deviation).
Go to:

RESULTS
MFC of Z. multiflora EO was 1:1024 (1 mg/ml) and that of 5% NaOCl was 1:16 (3 mg/ml).
Statistically significant difference was observed among the groups (P < 0.001). Percentage
reduction in fungal colonies from S1 to S2 and results of pairwise comparison of groups are
shown in Table 1 and Figure 1.

Table 1

Mean ± SD and the percentage of reduction of fungi

Figure 1

Percentage reduction in fungal colonies

Z. multiflora EO at twice the MFC and NaOCl at MFC showed the highest antifungal
efficacy that was significantly higher than the antifungal efficacies of Z. multiflora EO at
MFC and DW. Antifungal efficacy of Z. multiflora EO at twice of MFC was significantly
higher than MFC.
Go to:

DISCUSSION
In recent years, use of plant and herbal alternatives in medicine and dentistry has increased
because of their innate properties, wide availability, safety, and low adverse effects.[14] Z.
multiflora EO has excellent antibacterial and antifungal activities in vitro,[11,16] especially
against Candida.[19,20]
The present study used extracted human teeth to test the antifungal efficacy of Z.
multiflora EO at MFC against C. albicans. To date, no study has evaluated the antifungal
efficacy of Z. multiflora EO against C. albicans obtained from human root canals. Most
studies have used agar diffusion test. Although agar diffusion method is very common, many
factors other than the antibacterial activity of test materials might affect its reliability and
reproducibility. These include molecular size and chemical formulation of the agar medium,
solubility and diffusion of test material through the agar medium, interaction between test
materials and the gel, agar viscosity, storage conditions of agar plates, and incubation
time.[21]
Unlike agar diffusion test, in the method used in the present study, dentin as an important
modifier for antibacterial activity of irrigants[21] comes into account, and the time of
exposure of microorganisms to irrigants is similar to the clinical situation. In addition,
interfering factors such as molecular weight and density of agar medium, which affect the
antimicrobial activity of test materials, are not applicable to the method.[21]
In the present study, a biofilm of C. albicans was allowed to form on the dentinal wall for 72
h. Studies by Lamfon et al.[22] and Baillie et al.[23] have confirmed that 72 h is sufficient
for the formation of a mature biofilm of C. albicans on the surface of root canals.
Because Z. multiflora EO is not water soluble (according to the recommendations of the
manufacturer), 80% ethanol was used for the initial dilution. To evaluate whether 80%
ethanol did not add to the antifungal effect of Z. multiflora EO against C. albicans, MIC of
80% ethanol was determined. The MIC of ethanol was much higher than the concentration
used to dilute Z. multiflora EO in the present study, indicating that it did not add to the
antifungal properties of Z. multiflora EO.
Percentage reduction in fungal colony count by the MFC of Z. multiflora EO (0.5 mg/ml)
was 99.7%. However, percentage reduction in fungal colony count by twice the MFC of Z.
multiflora EO (1 mg/ml) and MFC of NaOCl was 100%, which was statistically significant
compared with other irrigants. The finding that Z. multiflora EO at twice the MFC was as
effective as NaOCl at MFC in reducing the fungal count necessitates further studies for
comparing different concentrations of Z. multiflora EO with clinical concentrations of
NaOCl (0.5-5%).
Antifungal efficacy of Z. multiflora EO can be attributed to its high phenolic contents,
particularly carvacrol and thymol that have antibacterial properties.[14] Antifungal efficacy
of MFC of Z. multifloraEO was lower than MFC of NaOCl. However, considering the
disadvantages associated with NaOCl, especially its cytotoxicity, MFC of Z. multiflora EO
has the potential to be used as a root canal irrigant.
Although no study has been performed on the cytotoxicity of Z. multiflora EO on the
periapical tissues, some studies have shown that EOs from this plant protect lymphocytes and
hepatocytes from the adverse effects of chemotherapy and radiotherapy.[24,25] Therefore, it
can be assumed that in contrast to NaOCl, which has high cytotoxicity,[8] Z. multiflora EO
has no or low cytotoxicity. Nevertheless, further studies are needed to investigate the
cytotoxicity of Z. multiflora EO before its clinical application as a root canal irrigant.
Go to:

CONCLUSION
Z. multiflora EO has acceptable antifungal effect against C. albicans. Considering the
disadvantages associated with NaOCl, Z. multiflora EO has the potential to be used as a root
canal irrigant.

Financial support and sponsorship

Nil.

Conflicts of interest
There are no conflicts of interest.
Go to:

Acknowledgment
The present article was extracted from thesis written by M. Dadollahi and was financially
supported by Shiraz University of Medical Sciences, international branch (Grant #8692009).
Go to:

REFERENCES
1. Tronstad L, Sunde PT. The evolving new understanding of endodontic infections. Endod
Topics. 2003;6:57–77.

2. Siqueira JF, Jr, Rôças IN, Lopes HP, Magalhães FA, de Uzeda M. Elimination of Candida
albicansinfection of the radicular dentin by intracanal medications. J Endod. 2003;29:501–
4. [PubMed]
3. Dumani A, Yoldas O, Yilmaz S, Koksal F, Kayar B, Akcimen B, et al. Polymerase chain
reaction of enterococcus faecalis and candida albicans in apical periodontitis from Turkish
patients. J Clin Exp Dent. 2012;4:e34–9. [PMC free article] [PubMed]

4. Baumgartner JC, Watts CM, Xia T. Occurrence of Candida albicans in infections of

endodontic origin. J Endod. 2000;26:695–8. [PubMed]

5. Nair P, Sjögren U, Krey G, Kahnberg KE, Sundqvist G. Intraradicular bacteria and fungi in
root-filled, asymptomatic human teeth with therapy-resistant periapical lesions: A long-
term light and electron microscopic follow-up study. J Endod. 1990;16:580–8. [PubMed]

6. Ballal V, Kundabala M, Acharya S, Ballal M. Antimicrobial action of calcium hydroxide,

chlorhexidine and their combination on endodontic pathogens. Aust Dent J. 2007;52:118–
21. [PubMed]

7. Becker TD, Woollard GW. Endodontic irrigation. Gen Dent. 2001;49:272–6. [PubMed]

8. Mohammadi Z. Sodium hypochlorite in endodontics: An update review. Int Dent

J. 2008;58:329–41.[PubMed]

9. Marending M, Luder HU, Brunner TJ, Knecht S, Stark WJ, Zehnder M. Effect of sodium
hypochlorite on human root dentine — Mechanical, chemical and structural evaluation. Int
Endod J. 2007;40:786–93.[PubMed]

10. Busslinger A, Sener B, Barbakow F. Effects of sodium hypochlorite on nickel-titanium

Lightspeed instruments. Int Endod J. 1998;31:290–4. [PubMed]

11. Prusti A, Mishra SR, Sahoo S, Mishra SK. Antibacterial activity of some Indian medicinal
plants. Ethnobotanical leaflet. 2008;12:227–30.

12. Nabavizadeh M, Abbaszadegan A, Gholami A, Sheikhiani R, Shokouhi M, Shams MS, et

al. 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 vitrostudy. J Conserv Dent. 2014;17:449. [PMC
free article] [PubMed]

13. Vinothkumar TS, Rubin MI, Balaji L, Kandaswamy D. In vitro evaluation of five different
herbal extracts as an antimicrobial endodontic irrigant using real time quantitative
polymerase chain reaction. J Conserv Dent. 2013;16:167. [PMC free article] [PubMed]
14. Abbaszadegan A, Sahebi S, Gholami A, Delroba A, Kiani A, Iraji A, et al. Time-dependent
antibacterial effects of Aloe vera and Zataria multiflora plant essential oils compared to
calcium hydroxide in teeth infected with Enterococcus faecalis. J Investig Clin
Dent. 2014 doi: 10.1111/jicd.12123. [PubMed]

15. Hosseinzadeh H, Ramezani M, Salmani G. Antinociceptive, anti-inflammatory and acute

toxicity effects of Zataria multiflora Boiss extracts in mice and rats. J
Ethnopharmacol. 2000;73:379–85. [PubMed]

16. Najari R, Alizadeh A, Samani RB. Antimicrobial activity and essential oil composition
of Zataria multiflora Boiss in two regions of Iran. Int J Biosci. 2014;4:147–52.

17. Saei-Dehkordi SS, Tajik H, Moradi M, Khalighi-Sigaroodi F. Chemical composition of

essential oils in Zataria multiflora Boiss. from different parts of Iran and their radical
scavenging and antimicrobial activity. Food Chem Toxicol. 2010;48:1562–7. [PubMed]

18. Khosravi AR, Shokri H, Tootian Z, Alizadeh M, Yahyaraeyat R. Comparative efficacies

of Zataria multiflora essential oil and itraconazole against disseminated Candida
albicans infection in BALB/c mice. Braz J Microbiol. 2009;40:439–45. [PMC free
article] [PubMed]

19. Amanlou M, Beitollahi JM, Abdollahzadeh S, Tohidast-Ekrad Z. Miconazole gel compared

with Zataria multiflora Boiss. gel in the treatment of denture stomatitis. Phytother
Res. 2006;20:966–9.[PubMed]

20. Oshagh M, Dashliborun YN, Saravi ME, Bazargani A. Evaluation of chlorhexidine

and Zataria multiflora essential oil in removing Streptococcus Viridans and Candida from
the surface of removable orthodontic appliances: A randomized clinical trial. J Mazandaran
Univ Med Sci. 2014;23:192–9.

21. Luddin N, Ahmed HM. The antibacterial activity of sodium hypochlorite and
chlorhexidine against Enterococcus faecalis: A review on agar diffusion and direct contact
methods. J Conserv Dent. 2013;16:9–16. [PMC free article] [PubMed]

22. Lamfon H, Porter SR, McCullough M, Pratten J. Formation of Candida albicans biofilms
on non-shedding oral surfaces. Eur J Oral Sci. 2003;111:465–71. [PubMed]

23. Baillie GS, Douglas LJ. Role of dimorphism in the development of Candida
albicans biofilms. J Med Microbiol. 1999;48:671–9. [PubMed]
24. Hosseinimehr SJ, Mahmoudzadeh A, Ahmadi A, Ashrafi SA, Shafaghati N, Hedayati N.
The radioprotective effect of Zataria multiflora against genotoxicity induced by? Irradiation
in human blood lymphocytes. Cancer Biother Radiopharm. 2011;26:325–9. [PubMed]

25. Shokri H, Asadi F, Bahonar AR, Khosravi AR. The Role of Zataria multiflora essence
(Iranian herb) on innate immunity of animal model. Iran J Immunol. 2006;3:164–
8. [PubMed]