Inhibitory effect of calcium hydroxide combined with Nigella sativa against Enterococcus faecalis

Background: Calcium hydroxide is the gold standard medicament for root canal treatment. Enterococcus faecalis, the primary cause of intraradicular persistent endodontic infection, is often identified even after endodontic treatment. Thymoquinone, an active ingredient of Nigella sativa, has an antimicrobial effect on both gram-negative and positive bacteria, including E. faecalis. Purpose: This study aimed to evaluate the inhibitory effect of calcium hydroxide combined with Nigella sativa extract and determine the best ratio for the combined material. Methods: This is an experimental study comprised of six groups (n = 4 per group) based on the material and its ratio, namely; (1) calcium hydroxide; (2) Nigella sativa extract; and groups of the combination of calcium hydroxide and Nigella sativa extract with a ratio (3) 70:30, (4) 50:50, (5) 30:70, (6) 10:90. The inhibitory effect against E. faecalis was evaluated by the agar well diffusion method in Muller–Hinton agar. Observation of the inhibitory zone was performed on the first, third, and seventh days. The collected data were analysed by a one-way ANOVA and LSD post hoc test. Results: Calcium hydroxide has the highest inhibitory effect, and the combination of Nigella sativa extract with calcium hydroxide ratio 50:50 was second. The inhibitory zone of these two groups was significantly higher than in any other group (p<0.05). Conclusion: Nigella sativa extract combine with calcium hydroxide did not enhance calcium hydroxide’s antimicrobial property against E. faecalis. An equal amount of Nigella sativa and calcium hydroxide is the best combination ratio, with a stable effect for up to seven days.


INTRODUCTION
Deep caries or traumatic injury often become a pathway for bacteria to enter the pulp and resume infection to the periapical tissue, known as endodontic infection. The endodontic infection may form as primary, secondary, or persistent infection. 1 One bacterium commonly found in persistent infections is Enterococcus faecalis (E. faecalis). Virulence factors of E. faecalis increase the bacteria's survival in harsh environments as it adheres to and penetrates the dentinal tubules. Bacterial virulence is one of the primary factors of root canal treatment failure. 2 The persistent infection commonly results in failed tissue regeneration seen as an unhealed radiolucency in the apical area or as unrelieved symptoms such as pain or ongoing sinus tract problems. 3 In multiple visit endodontic treatments, the root canal system needs to be filled by an intracanal medicament to suppress the amount and inhibit the growth of bacteria, reduce pain, and provide long-term disinfection. Calcium hydroxide has been the most commonly used material in endodontics since 1920, with various indications from pulp capping, apexification, apexogenesis, and root canal disinfection. 4 The main mechanism of action of calcium hydroxide is due to the release of calcium and hydroxyl ions in the presence of water. The hydroxyl ion elevates the environment pH to an alkaline condition, which has a detrimental effect on bacteria. It reduces bacterial virulence and eventually reduces symptoms, assisting the healing process and tissue regeneration. 5  paste's viscosity, penetration ability, and bacterial virulence, which can survive alkaline and starvation conditions. 6,7 Nigella sativa is a herb that is widely used by people, especially in the Middle East. Nigella sativa seeds and oils have been used as an analgetic, antibacterial, anti-allergy, anti-cancer, and anti-inflammatory agent. Thymoquinone is an active ingredient in Nigella sativa, which can inhibit the growth of bacteria and function as an antimicrobial agent. Thymoquinone can damage the bacteria cell's integrity, resulting in the death of bacteria cells through necrosis and apoptosis. [8][9][10] Nigella sativa extract effectively eliminates both gram-negative and positive bacteria, including E. faecalis. A previous study reported that Nigella sativa is dose-dependent, in which 100% extract had better antibacterial action against Enterococcus than 25%, 50%, and 75%. 11 Other studies mentioned that Nigella sativa extract with low viscosity could penetrate and provide a good antibacterial effect, unlike the high viscosity extract. 12,13 The Nigella sativa's ability to eliminate E. faecalis and the low viscosity makes them of interest as an intracanal disinfection agent. It was hoped future research of the compound could synergically improve the antimicrobial efficacy of calcium hydroxide.
Based on previous studies on the high potency of Nigella sativa, particularly as an antimicrobial agent, we proposed using this agent as supplemental to the commonly used endodontic intracanal medicament, calcium hydroxide. As far as we know, this has not been previously studied. Therefore, the proper ratio in combining the materials has yet to be determined. Whether this combination can work synergistically also needs to be evaluated; one of the ways is by evaluating the inhibitory effect of both materials in different combinations. Considering calcium hydroxide is a time-dependent medicament, the evaluation was done on three spesific days (days one, three and seven) to evaluate the stability of the material. 14 Therefore, we investigated the efficacy of the Nigella sativa extract and its combination with calcium hydroxide in several ratios to evaluate the inhibitory effect against E. faecalis, and determined the best ratio for the combining material.

MATERIALS AND METHODS
This is an experimental study with a posttest-only control design, consisted of six groups (n = 4 per group), paired numeric analytic with 95% confidence interval) according to the sample components and ratio: (1) calcium hydroxide; (2) Nigella sativa extract; a combination of calcium hydroxide with Nigella sativa (3) ratio 70:30; (4) ratio 50:50; (5) ratio 30:70; (6) ratio 10:90. To ensure the bacteria itself was not affected by the distilled water used as a solvent for the calcium hydroxide a group by using only distilled water was used as negative control. The extraction of Nigella sativa was carried out by maceration using methanol as a solvent at the Department of Chemistry, Faculty of Sciences, Institut Teknologi Sepuluh Nopember, Surabaya, Indonesia. 15 Calcium hydroxide powder (Merck, Darmstadt, Germany) was mixed with distilled water with the w/p ratio of 0.8 to provide the best consistency as intracanal medicament paste to have acceptable flowability. The total crude extract (100% concentration) and calcium hydroxide were weighed on an analytical scale to obtain the exact weight for each group according to its ratio (Table 1).   The E. faecalis ATCC 2912 was reidentified and cultured to achieve a pure suspension of E. faecalis. The concentration of the bacteria was standardised by spectrophotometry based on the McFarland standard at 0.5. The antibacterial test was carried out by preparing a bacterial suspension followed by the well diffusion test. 15 Mueller-Hinton agar plates were prepared, and the bacteria suspension was plated in each agar plate. Each agar plate was divided into four, and then a well was made with a sterilised perforator. Afterwards, each sample group was placed into the well. The agar plates with the tested material were then incubated at 37 o C for the evaluation interval. All work was conducted on a workbench and in a sterile protocol.
Observation of the inhibitory zone was performed on the first, third, and seventh days. Digital callipers were used to measure the diameter of the inhibition zone around the well. The measurement was repeated three times for each inhibition zone, and the average value was taken. Collected data were analysed using a one-way analysis of variance (ANOVA) test (p<0.05) to compare the inhibitory zones between groups, and a post hoc least significant difference (LSD) test was performed to determine which specific groups were statistically significant from others (p<0.05).

RESULTS
The inhibition zone of Nigella sativa extract, calcium hydroxide, and its combinations are depicted in Figure 1. Descriptively, all samples showed an inhibitory area surrounding the sample-containing well, except for the negative control group.
The mean value of each group and the one-way ANOVA test are presented in Table 2. In all three intervals evaluated, calcium hydroxide has higher antibacterial efficacy than the others. The combination of Nigella sativa extract with a calcium hydroxide ratio 50:50 was second after calcium hydroxide. On all observation days, the mean value of an inhibitory zone for Nigella sativa extract was the lowest of all medicament groups. The inhibitory zone for all groups did not show notable differences until the seventh day of observation. Comparative analysis of the study groups by the post hoc LSD test (p<0.05) showed significant differences between tested groups. Intergroup comparisons between groups on each day by the post hoc analysis using the LSD test are described in Table 3. On the first day and third day of observation, the inhibitory zone of calcium hydroxide was significantly higher compared to all other tested groups. However, on the third, the inhibitory zone did not differ significantly between the calcium hydroxide group and the combination group of Nigella sativa and calcium hydroxide. In all three evaluations, a consistent significant difference between different combination ratios of Nigella sativa and calcium hydroxide extract groups compared to Nigella sativa alone was only observed in the 50:50 ratio group. The 50:50 ratio had a higher inhibitory zone than Nigella sativa alone, but less than the calcium hydroxide group. The addition of Nigella sativa extract to calcium hydroxide did not increase the inhibitory zone of calcium hydroxide.

DISCUSSION
Our study revealed that the calcium hydroxide group has the highest inhibition zone. However, others reported that calcium hydroxide is ineffective against E. faecalis, a prevalent bacterium in persistent infection. 6,7 Controversial effectiveness of calcium hydroxide against E. faecalis is influenced mainly by the different methods used in the studies. Almost all studies that allow direct contact of calcium hydroxide to the bacteria resulted in high antimicrobial efficiency. It was found to be less effective in studies using root canal models or in vivo studies where the complexity of root canal morphology contributes significantly to the limitation of material to be in intimate contact with the bacteria. [16][17][18] This is mainly caused by the high viscosity of calcium hydroxide, limiting its ability to reach small, narrow areas and it has difficulty penetrating the dentine tubules. In this study, the calcium hydroxide was in close contact with the bacteria and could diffuse through the agar plate, resulting in an enhanced inhibitory effect.
Based on our study, the inhibition zones of all medicament groups were formed from day one and were still stable until day seven, showing that the samples were still active during all evaluations. In contrast, a study on the antibacterial activity of Nigella sativa in various germination phases against clinical bacterial strains found that the effectiveness of Nigella sativa extracts was highest on the fifth and eleventh days. However, between the fifth and eleventh days, the antibacterial inhibition of Nigella sativa extract decreased. 15 Another study also reported that the components of Nigella sativa extract such as tannins, saponins, phenols, and terpenoids concentrations decreased from the third until the seventh day of the observation. 17 However, Nigella sativa as a single component without the addition of calcium hydroxide had a lower inhibitory zone than those with the addition of calcium hydroxide. Thymoquinone is the bioactive constituent of Nigella sativa, which is reported to have an antimicrobial effect. [8][9][10] Other constituents with the same effect include thymol, monoterpene, and tannin. The mechanism of action of Nigella sativa depends on its ability to penetrate through the bacterial membrane and damage the integrity of the bacteria cells. Gram-negative bacteria have good permeability defences due to their outer membrane structure. Thus, the Nigella sativa extract is more effective against grampositive bacteria. 19,20 Calcium hydroxide can release stable hydroxyl ions for up to 14-21 days, maintaining the environment's alkalinity. Therefore, this intracanal medicament can be used for long-term interappointment dressings, which is in line with the results of this study. However, we did not observe any enhancement of calcium hydroxide's inhibitory effect when adding Nigella sativa extract. The combination did not seem to have a synergic effect from the inhibitory zone evaluation in all ratios evaluated because all combinations had a lower inhibitory effect. Also, the 50:50 ratio did not differ significantly compared to calcium hydroxide.
Looking at the inhibitory effect between the different combination ratios, calcium hydroxide combined with Nigella sativa extract in 50:50 ratio indicates a higher inhibitory effect than any other combination ratios or Nigella sativa extract alone. Compared to different ratios, this combination was the only combined material with a significantly higher inhibitory zone than Nigella sativa extract alone. However, compared to calcium hydroxide, this combination had a significantly lower inhibitory zone, confirming that the addition of Nigella sativa extract did not improve the inhibitory property of calcium hydroxide. The addition of the extract may impede the liberation of hydroxyl ions. The effectiveness of calcium hydroxide is strongly related to the dissociation of calcium hydroxide to hydroxyl and calcium ions. 21,22 In our present study, the addition of Nigella sativa extract decreased the inhibitory effect of calcium hydroxide. One possibility is that the addition of extract inhibits the hydroxyl ion release and contributes to a lower pH generated by the calcium hydroxide. This can be further investigated by evaluating the hydroxyl ion release and pH of the samples.
The method used in this study could affect penetration ability and contribute to the active component's success to diffuse through the agar plate. When preparing the samples, we noticed that the combination with a ratio of 50:50 formed a homogenous paste consistency and had better flowability than other ratios, affecting the agar plate's penetration ability. However, the conditions between in vitro and oral cavity will be different. Calcium hydroxide can maintain its pH level for sustained periods in the media compared to infected teeth. It will not be influenced by the buffering effect of dentinal fluid and hydroxyapatite content. This causes the antibacterial efficacy of calcium hydroxide to survive for a long time on the agar media. 20 Another limitation of this study is the simplified in vitro condition on agar media compared to the complex root canal system in vivo, which will affect the antimicrobial property. Therefore, further study will be needed before using the results clinically.
The evaluation methods or concentration could also be contributing to the results. An in vitro study reported a moderate ability of thymoquinone to reduce biofilm formation to Staphylococcus aureus and Staphylococcus epidermidis. In contrast, the thymoquinone concentration needs to be doubled for the same settings to have the same effect on E. faecalis. This showed that the effectiveness of Nigella sativa extract as an antibacterial agent depends on its concentration. 12 According to a study, the minimum concentration of thymoquinone to inhibit E. faecalis is 256 µg/ml. 13 In our present study, we use a total crude extract without evaluating different concentrations. This was based on a previous study reporting that 100% extract had better antibacterial action against Enterococcus than 25%, 50% and 75%. 11 However, this concentration was used for Nigella sativa without combining the extract with calcium hydroxide. This conclusion could be improved by a further study using different concentrations of Nigella sativa extract, such as by serial dilution of the extract. Dilution may provide a more aqueous vehicle for the dissociation of calcium hydroxide ions. Active component evaluation of the extract should also be conducted to confirm the concentration of thymoquinone on the extract, which could also be a contributing factor.
Nigella sativa extract and its combination with calcium hydroxide can provide antibacterial effects to Enterococcus faecalis as a common bacterium in persistent infections. The inhibitory effect of Nigella sativa extract and its combination with calcium hydroxide is not more significant than calcium hydroxide itself. In conclusion, Nigella sativa extract combined with calcium hydroxide did not improve calcium hydroxide antimicrobial properties against E. faecalis. A combination of 50:50 ratio provides a better inhibitory zone compared to other combinations.