In vitro comparison of antimicrobial effect of sodium hypochlorite solution and Zataria multiflora essential oil as irrigants in root canals contaminated with Candida albicans.

INTRODUCTION: This study compared the antifungal effect of Zataria multiflora essential oil (EO) with that of sodium hypochlorite (NaOCl) as an irrigant for root canals infected with Candida albicans.
MATERIALS AND METHODS: Sixty mandibular premolars were infected with C. albicans suspension. After 72 h of incubation, the samples were divided into four groups. Teeth in Group 1 were irrigated with minimum fungicidal concentration (MFC) of Z. multiflora EO, in Group 2 with twice the MFC of Z. multiflora, in Group 3 with MFC of NaOCl, and in Group 4 with distilled water (DW). Pre- and post-operative samples were cultured, and fungal colony count of each specimen was obtained. Data were analyzed using Kruskal-Wallis and Mann-Whitney tests (P < 0.05).
RESULTS: NaOCl at MFC and Z. multiflora EO at twice the MFC showed the highest antifungal efficacy, with no significant difference (P > 0.05). However, antifungal efficacies of these irrigants were significantly different from those of Z. multiflora EO at MFC and DW (P < 0.05).
CONCLUSION: Our results showed that Z. multiflora EO at twice the MFC had the same antifungal efficacy as NaOCl at MFC.

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.[121314] 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.[1415] In vitro studies have shown that this EO has excellent activity against Candida species.[1617] 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.

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. albicans growth 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. albicans was 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).

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.

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,[1116] especially against Candida.[1920]

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. multiflora EO 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.[2425] 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.

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.