Effect of Thymol Incorporation on Anti-bacterial Activity of the Locally Prepared Zinc Oxide-Guaiacol Root Canal Sealer : An in Vitro Study

فادهلأا : فدهت  ام يف لومياثلا تارولب جمد قيرط نع ةديدج ةغيص دادعإ ىلإ ةساردلا كنزلا ديسكأ( رذجلا ةانق ةدادس ةد ةرارضملا لوكااياوغ لوانيجولا كانزلا دياسكأ( ىالع مئااقلا رذاجلا ةاانق دادسب جئاتنلا ةنراقمو ايريتكبلل دارملا طاشنلا مييقتو اًيلضم ااوملا رط وئا ق  ومعلا ةفااضإ : ( ةفلتخم زيكارت ثلاثب لومياثلا تارولب 2 ، ٪ 5 و ، ٪ 10 ةدارملا ةيلاعفلا نيسضتل ٪  كانزلا دياسكأ (رذجلا ةانق ةدادسل ايريتكبلل –  : لوكااياوغ مادختسا مت 40  مجم عبرأ ىلإ يرتب قابطا ميسقت مت رابآ ةسمخ اهب ةضيفص لك ، سكوكوريتنا راجأ طسو نم يرتب ةضيفص تاعو  بسح  تاراتف ( ةنارح 1 ، موي 2  ، 3  ، 7  عام رااجلا رااشتنا رابتخا مادختساب ، عون لكل مايأ 0.5  م مادختاساب اًيئااًحإ ااهليلضتو ةرااكعلل داننرافكم رييااع نكنادراابتخاو ، اااجتنا يدااحأ اافونا راابتخاو ، يفاصولا اااًحلإا .  ئجوتنلا كانزلا دياسكأ( تااعومجم : –  اجمدملا لوكااياوغ اب ة يااثل لوم ( ةسوردملا تازيكرتلاب 2 ، ٪ 5 ٪ و ، 10 مواي( دانع ةناارضلا تاراتف عايمج يف ترمتسا ةيريتكب ومن طيبثت قطانم جتنت ٪ 1  ، 2  ، 3  ، 7  ماايأ دنع يونعم قرفبو p≤0.01 لا كانزلا دياسكأ ىالع ةامئاقلا داداسلا ةداامب ةنراقم دانع يوانعم عاافخنا ااانن لوانيجو p≤0.01 ةايلاعفلا ياف  ملا ةدار  لل ةنارح ةرتف يف جمدلا تاعومجم عيمج يف ايريتكب 7  لومياثلا جمد ةعومجم ، مايأ 5 عايمجل ةايريتكب ومن طيبثت قطانم ربكأ تجتنا ٪ ةنارضلا تارتف تجججتنتسلاا لا ديسكأ( نم دادسلا تابيكرت نأ ىلإ ةساردلا اذن تًلخ : كنز –  ةاسوردم زايكارت ةثلاث عم ةررضملا لوكاياوغ م لب ن تارو  لرفأ ميثارجلل ةدارم ةيلاعف اهل لومياثلا  جمدلا ةبيكرت ، لونيجولا كنزلا ديسكأ( رذج ةانق ةدادس يف ةدوجوملا كلت نم 5 لوميااث ٪ انريغ نم ايريتكبلل ةدارملا ةيلاعفلا يف لرفلا ين .  ABSTRACT Aims: This study aimes to prepare a new formula by thymol crystals incorporation into locally prepared zinc oxide-guaiacol root canal sealer and evaluating the antibacterial activity and comparing the results with zinc oxide eugenol-based root canal sealer. Materials and Methods: The addition of thymol crystals at three different concentrations (2%, 5%, and 10%) to improve the antibacterial activity of zinc oxide-guaiacol endodontic sealer was done: 40 Petri plates of Enterococcus agar media were used, each plate with five wells. Petri plates divided into four groups incubation intervals (1, 2, 3, and 7 days) for each type, using agar diffusion test (ADT) with 0.5 McFarland turbidity standards and statistically analyzed using descriptive statistics, a one-way ANOVA test, and Duncanʼs Multiple Range Test. Results: The thymol incorporated zinc oxideguaiacol sealer groups at studied concentrations (2%, 5%, and 10% ) produced bacterial growth inhibition zones persisted at all incubation time intervals at (1, 2, 3, and 7 days) and high significantly at p≤0.01 than that of zinc oxide eugenol based sealer. There is a significant reduction at p≤0.01 in antibacterial efficacy in all incorporation groups at 7 days incubation period, 5% thymol incorporation group with significant largest bacterial growth inhibition zones for all incubation periods. Conclusions: This study concluded that the prepared zinc oxide guaiacol sealer formulas with three studied thymol crystals concentrations having better antibacterial efficacy than that of zinc oxide eugenol root canal sealer, 5% thymol incorporation formula is the best in antibacterial activity than others.


INTRODUCTION
The main aim of chemomechanical endodontic management is the elimination or severe reduction of microbe's existent in the root canal system (1) . Sealers are principally designated based on their sealing capacity, adhesive properties, biocompatibility, and antimicrobial ability (2) .
The antimicrobial action of root canal sealers may help to eradicate residual microbes unaffected by chemomechanical planning of the root canal structure, stop the development of microbes, and help the healing procedure of apical and periapical tissues. Many studies have considered the agar diffusion test (ADT) is one of the most frequently used methods to estimate the antimicrobial activity of dental materials (3) .
Generally, zinc oxide-guaiacol endodontic sealer has setting time, film thickness, solubility, and flowability fulfilled the requirement of ANSI/ADA specification No. 57/2008 for dental root canal sealing materials. It has enhanced apical seal and biocompatibility related to that of ZOE, Its antibacterial activity against E. faecalis continued overall incubation times periods (1day, 2days, 3days, and 7days) but lower to that of ZOE based sealer. The antimicrobial action of zinc oxide-guaiacol endodontic sealer is produced through the main components of the material without introducing the definite antibacterial agent (4) .
The locally prepared root canal sealer powder/liquid formula depends on the main reaction of zinc oxide and guaiacol as a base /acid interaction with adding the other ingredients to improve materials properties: Zinc oxide 62%, natural rosin 20%, locally made egg-shell hydroxyapatite 2%, bismuth sub-carbonate 15%, zinc acetate 1%, guaiacol 85%, and olive oil 15% (5) .
Today, the widespread of herbal or natural products due to their antimicrobial action, biocompatibility, lack of bacterial resistance, antioxidant, anti-inflammatory, easy obtainability, and low-cost properties (1) . Many natural happening antimicrobials have gained consideration among examiners due to their safety and nonhazardous status, such as thymol that displays the potential to obstruct the dental infection (6) . The most frequently stated procedure of antibacterial action of thymol involves the disturbance of bacterial membrane effect in bacterial lysis and escape of intracellular substances causing in death (7) . The thymol looks as the major active antibacterial ingredient of thyme oil, which employed its antimicrobial accomplishment (8,9) .
Thymol (2-isopropyl-5-methylphenol) is a white crystal-like material that provides thyme its durable flavor, pleasurable aromatic perfume, and durable antiseptic features. It considers as a safe natural antibacterial agent with many dental, medical, and food industries (10) .
The present study aimed to compare and evaluate the effect of (2%, 5%, and 10% ) thymol crystals incorporation on the antibacterial activity of the locally prepared zinc oxide-guaiacol root canal sealer at different incubation periods

Thymol incorporation
At the beginning, the thymol crystals (BDH, England) were grinded with an electrical grind-er (Geepas, China), as well as all other elements of the original zinc oxide -guaiacol root canal sealer each one separately and to standardize particle size; the materials were sieved by using a sieve represented by double layers of muslin tissue (11) , then incorporated in zinc oxide-guaiacol endodontic sealer in three formulas (Table 1).  (4) .

Initialization of Enterococcus selective media
Forty-two grams of agar Enterococcus media (HiMedia, India) were added to 1 liter of distilled water in a glass flask with continuous mixing by glass road till totally melted in water.
The mixture was heated up till initial blistering without over warming. The flask was detached from the heater, and the pH of the media was identified by pH meter (pH 7). The media were left to come to be warm and emptied in one-use Petri dishes in a sterile condition in the hood with the existence of a gas burner. The plates were chilled and kept in the refrigerator (Concord, France) until used.

Bacterial isolation
The oral Enterococcus faecalis were insulated from the periapical lesion of a tooth, after indicated tooth isolation and access opening.
Expand the root canal till file (size 25). Before any sterilization of the canal, a sterilized paper point of (size 20) was introduced cautiously into the canal for 1 minute. It was pulled cautiously and positioned into a sterilized vial having (5 ml) of sterilized brain heart infusion broth (Neogen culture media, UK) and vortexes to diffuse the sticking bacteria. It was then positioned at 37°C in an incubator for 30 minutes.
All microbiological effort was completed in a laminar air current chamber (locally made, Iraq).

Subculturing of Bacteria
The suspension was marked on the superficial part of Enterococcus's selective media by a cotton swab. The only microorganism that matures on this media is E. faecalis. The E. faecalis seemed as reddish-pink colonies on the superficial part of the media. Then incubation of the media anaerobically at 37°C by an anaerobic candle jar for 18 hours (12) .

Bacterial dilution
The brain heart infusion broth (Neogen culture media, UK) of 4.5 ml was organized in screw plugged vials to revitalize and dilute the bacteria to the desired dilution. Successive dilution was prepared by drawing 0.5 ml from the stock broth and supplementing to 4.5 ml of a freshly organized broth. The dilutions (1*10 -3 ) were completed until a detached colony in the Petri dish was shaped.

Antibacterial study
The antibacterial activity of trial sealer materials and ZOE based root canal sealers were experienced against E. faecalis (13) , by agar diffusion test (agar-well technique) (3) .

RESULTS
The mean of inhibition zone diameters and standard deviations of tested sealers are demonstrated in Table (2). The result concluded that there is an increase in bacterial growth inhibi-tion zones after thymol powder incorporations for all groups at all incubation periods (1 day, 2 days, 3 days, and 7 days) compared to that of zinc oxide-guaiacol root canal sealer.     About 40-60% of root canals stay positive for bacterial existence after this treatment. (14) .

Thymol Incorporation on Antibacterial Activity of Root Canal Sealer
Enterococcus faecalis is the microbe which is commonly correlated to the etiology of persistent periradicular lesions. It owns numerous virulence factors such as (gelatinase production, Enterococcus surface protein (Esp), aggregation substance (AS) and biofilm for-mation) (15) , that give to its ability to persist the effects of conventional root canal treatment.
Also, this Gram-positive facultative anaerobe can attack dentinal tubules and bind to collagen. For these details, E. faecalis was the microbe designated in our research to assess the antimicrobial features of the endodontic materials experienced (3) .
The agar diffusion test has been broadly bility and diffusion properties of both the experiment material and media (3) . Therefore, the plates were saved for two hours at room temperature (permit the diffusion) as proposed by Poggio (3) .
The results of the present study revealed that all sealers showed antibacterial activity substantiated by the formation of growth inhibition zones against E. faecalis at all incubation time intervals. The antimicrobial ability of endodontic sealers is detected by its composition, which also affects its physical, chemical, and biological action (16) .
Zinc oxide eugenol based sealer created significantly larger inhibitory zones at 1 day, 2 days, 3 days against E. faecalis as compared with zinc oxide-guaiacol sealer before incorporations which was in agreement to the similar inhibitory action of zinc oxide eugenol based sealers by Kothari and Langalia (17) .
The greatest challenge in adjusting materials to modify their biological, antimicrobial, and/or mechanical features is gaining a product that offerings stability over time, has homogeneous integration of the additives, and can be easily manipulated (16) . The integration of antimicrobial agents into endodontic root canal sealers raises this capability (18) .
In this study, thymol crystals were incorporated as an antibacterial agent with three different concentrations (2%, 5%, and 10%) into zinc oxide-guaiacol sealer to improve antibacterial activity. existence of thymol (19) . Declining in antibacterial action after 10% thymol integration may be related to aggregation that occurs after mixing the ingredients of powder, this associated to the type of the thymol self-aggregating structure formed which depends on thermodynamic and geometric restrictions associated with the hydrophilic-hydrophobic balance within the molecule especially in high concentration. This aggregation decreases the available surface area leading to a decrease in the reactivity (20).

CONCLUSIONS
The thymol crystals incorporation appears to produce better antibacterial activity against E. faecalis than that of zinc oxide eugenol and zinc oxide-guaiacol endodontic sealer at all incubation periods for studied concentrations.
The 5% thymol crystals incorporation indicated for better antibacterial activity against E. faecalis with appropriate other properties.