Comparison of Antifungal Activity of Thymus Vulgaris Essential Oil and Triple Antibiotic Paste Against Candida Albicans Isolated from Root Canal (In Vitro Study)

Essential oil extract preparation

Dried thyme leaves (50 g) were hydrodistilled in a Clevenger-type apparatus for 3-4 h. The oils were maintained at 4 °C in sealed vials after drying over anhydrous sodium sulfate.¹¹

Patient selection and sample collection

Ten patients (their age ranged 25-45 years) who had pulpal necrosis, periapical lesions and needed endodontic treatment, were included in this study after signing an informed consent. All the selected teeth had one root. The teeth were symptomatic, and radiographic examination performed in the diagnostic department of a specialized dental clinic (Maysan) verified the diagnosis.

The selected teeth were isolated using a rubber dam. The working area was cleaned with 10% povidone-iodine solution. All coronal restorations and carious lesions were removed using a sterile carbide fissure bur, followed by field disinfection. Subsequently, an access opening to the root canal was created with a sterile diamond fissure bur, taking care not to go too deep inside the pulp chamber to preserve the microbiota. For the initial instrumentation, new and sterile K files of (15, 20) size were used, and a size #20 rotary file was used with no irrigants. Subsequently, normal saline was used to fill the canal, and the final file was agitated vertically to generate a microbiological suspension. After leaving the paper points inside the canal for one minute, they were transferred to the Brain Heart Infusion Broth to the laboratory for isolation and identification.¹²

Identification of candida albicans

GC-MS analysis of TVEO

Thyme Vulgaris essential oil^’s chemical compounds analysis was analyzed using a gas chromatograph mass spectrometer (model 5973N, Agilent USA) equipped with a capillary column type HP-5ms (30 m length, 0.25 μm thickness, 0.25 mm diameter) and helium, which was the carrier gas at a flow rate of 1 ml/min. The injector was operated at 280 °C. The oven temperature was programmed as follows: the starting temperature was 60 °C and held for 2 min. The temperature was increased at a rate of 10 °C/min to 300 °C and held for 5 min.¹⁶ This procedure was performed by the Ministry of Science and Technology, Department of Water and Environment/Environment Research Centre.

Test the sensitivity of Candida albicans to Thymus Vulgaris essential oil (TVEO) by Agar diffusion method

Dimethyl sulfoxide (DMSO) (LOBA Chemie, India) was used to dilute (TVEO) to various concentrations (25, 10, 8, 6, 4, and 2 mg/ml). The procedure involved the addition of 10% DMSO to the oil.

The Sabouraud dextrose agar (SDA) (OXOID, India) plates were inoculated with Candida albicans suspension that was produced as 0.5 McFarland using sterilized cotton swap. Six mm-diameter wells were created on the plate, and 100 μl of TVEO at the previously determined concentrations were placed in each well. Metronidazole (Gulphar, U.A.E.), ciprofloxacine (Pharma International, Jordan), and minocycline (Vulga XR, Jordan) each of these powders was made separately by grinding it into a fine powder by using a clean mortar and pestle. To achieve the desired 1:1:1 proportion, the three antibiotic powders were individually weighed using a digital scale, and then, to make a paste, one gram of powder was combined with one milliliter of sterile water.¹⁴ 10% DMSO and TAP, on the other hand, served as the negative and positive controls, respectively. The plates were then kept in an incubator for approximately 24 h at 37°C, after which a ruler was used to measure in millimeters the fungal growth inhibition zones around the wells (involving six mm of each well).¹⁷

Determination of MIC and MBC of (TVEO)

Minimum inhibitory concentration was measured by broth microdilution. μL each of the ten wells, numbered W1 to W10, received 100 μL of SDA broth in a 96-well microtiter plate (NUNC™ Brand products), 100 μL of TVEO at an 8 mg/ml concentration was added to W1, and W1 through W10 received a two-fold serial dilution. W11, which was the positive control, contained the inoculated media. In contrast, W12 contained uninoculated medium and acted as a negative control. 0.5 McFarland, a fungal suspension (20 μl), was used to inoculate all wells and incubated overnight at 37 °C. Following incubation, 30 μl of resazurin was added to each well (30 μl/well) and incubated for 2–4 h¹⁸ for the observation of color changes. After completion of incubation, rows with no color change (blue resazurin color remained unchanged) were scored as MFC, whereas the last blue well in the row was recorded as the MIC and the next pink well was recorded as the sub-MIC. The MFC was detected by sub-culturing 100 μl from the MIC well and the well before it on SDA plates. Results were obtained after 24 h of incubation at 37°C. After culturing, the concentration was considered to be MFC if it was equal to or higher than the MIC and there was no fungal growth on SDA.¹⁹

Statistical analysis

SPSS (Statistical Package for social Science) (version -22, Chicago, Illionis, USA) was used. Minimum, maximum, standard error, mean, and standard deviation, as well as the Levene test, Shapiro-Wilk test, and Tukey HSD with one-way analysis of variance (ANOVA) were used for data description, analysis, and presentation.

Level of significance when p value is less than 0.05.

Chemical composition of the essential oil

The essential oil yield was 1%. Freshly isolated essential oil is a golden liquid with a hot-burning taste. The thyme essential oil was analyzed using GC-MS, and 53 components were identified, representing 95.95% of the total detected constituents. The major constituents of the oil are thymol (35.48%), o-cymene (13.82%), γ-terpinene (13.55%), carvacrol (4.04), α-terpinene (3.11%), caryophyllene (2.99%), linalool (2.57%),3-Thujene (2%). Other ingredients were present in less than 2% of the samples (Table 1).

Identification of Candida albicans

Under a microscope, the tested isolate showed gram-positive, oval-shaped, or budding yeast cells. Candida colonies on the SDA appeared smooth, creamy, white, and had a yeasty smell. The results of the germ tube test were positive, suggestive of C. albicans.

Antifungal activity of TVEO against C. albicans

TVEO was effective against C. albicans at all concentrations and regions around the wells with no fungal growth, termed growth inhibition zones. The lack of an inhibitory zone suggests that the fungi were resistant to the drug under evaluation. Triple antibiotic paste, in contrast to DMSO, demonstrated antifungal activity against Candida albicans and created an inhibitory zone.

As TVEO concentration increased, the diameter of the growth-inhibitory zone also increased. The results of the ANOVA test showed that there was a highly significant difference between the concentrations of TVEO and TAP (p ≤ 0.01) (Table 2, Figure 1, and Figure 2).

Figure 1. Activity of Thymus vulgaris essential oil and Triple Antibiotic Paste (control) against Candida albicans.

Figure 2. Inhibition zone of TVEO in different concentrations.

A - 25 mg/ml, B - 10 mg/ml, C - 8 mg/ml, D - 6 mg/ml, E - 4 mg/ml, F - 2 mg/ml, T - triple antibiotic paste (positive control), N - dimethyl sulfoxide (negative control).

Since there was a significant difference between the extract concentrations and the control, each pair of TVEO concentrations was statistically compared using Tukey’s HSD test, and the results revealed a significant difference between them (p ≤ 0.05), while between 2 mg/ml and 4 mg/ml, 6 mg/ml and 8 mg/ml, and 8 mg/ml and 10 mg/ml was not significant (p > 0.05) (Table 3).

Determination of MIC and MFC of TVEO

MIC of TVEO was 0.015 mg/ml and MFC was 0.031 mg/ml.