Evaluation of Propolis as Intracanal Medicament against Enterococcus faecalis

The present study aimed to comparatively evaluate Propolis, Calcium hydroxide Ca(OH)2, Chlorhexidine gel 2% (CHX) intracanal medicaments, and a mixture of Propolis with Ca(OH)2 and CHX against Enterococcus faecalis. Material and methods: One hundred and eight single-rooted extracted human teeth were used; all teeth were cut 14 mm away from the apex and prepared until size 25 master apical file. Samples were sterilized and dipped in Brain Heart Infusion Broth (BHI) contaminated with E. faecalis for 21 days at 37 ̊C. Samples removed, divided into six groups, injected with different medicaments as following: group A: Propolis; group B: Ca(OH)2; group C: CHX; group D: Propolis + Ca(OH)2; group E: Propolis + CHX, group F: control group with no medicament, teeth sealed with filling material and nail varnish for three times periods 2, 7 and 10 days. After each time period, samples were opened and irrigated with normal saline. H-file applied to the full length to obtain dentine chips, the files collected into test tubes containing sterile BHI and stored for 48 hours under anaerobic conditions. Samples were then removed and culture in Mueller-Hinton agar, then samples incubated for one week at 37 ̊C after which colony forming units counted using colony counter, statistical analysis was done with ANOVA and post hoc use LSD. Results: Propolis, 2% Chlorhexidine gel, and a mixture of Propolis with Calcium hydroxide were effective against E. faecalis, Calcium hydroxide, and a mixture of Propolis Chlorhexidine was not effective against it. Conclusions: Propolis showed a promising role as an intracanal medicament.


Introduction
The endodontic treatment's success is most significantly dependent upon eliminating root canal infection and preventing contamination during treatment [1].
The microbial flora in canals after endodontic failure was limited to predominantly Gram-positive microbial species. Facultative anaerobes, especially E. faecalis, were the most commonly isolated microorganisms [2].
Intracanal medicaments have been thought of as an essential step in killing the bacteria in the root canal, and if multiple-visit endodontics is chosen, an intracanal medicament is strongly recommended [3]. The intracanal medicaments may be used for several purposes; it eliminates any remaining bacteria after canal instrumentation, reduces inflammation of periapical tissues and pulp remnants, renders canal contents inert and neutralizes tissue debris, acts as a barrier against leakage from the temporary filling, and help to dry persistently wet canals [4].
Currently, calcium hydroxide is the most used intracanal dressing [5]. However, its effectiveness against E. faecalis is controversial [6] [7]. Chlorhexidine having an advantage of bactericidal action, substantivity, biocompatibility, low toxicity, and lesser chances of developing resistance [8], as it possesses a wide range of antimicrobial activity; CHX has been used in Endodontics as an irrigating substance or intracanal medicament [9]. However, a recent study shows that Chlorhexidine reduced the success of root canal treatment [10].
Propolis is natural material synthesis by Bee; it has a complex composition such as esters, phenolic compounds, and flavonoids. Propolis has been revealed to possess antibacterial, anti-inflammatory, antiviral, antifungal, antioxidant, and immunomodulatory effects [11]. Propolis exhibits antimicrobial activity against E. faecalis, S. aureus, C. albicans and it could be used as intracanal medicament [12], it has been demonstrated in various in vitro, in vivo and ex vivo studies, as well as in human clinical trials [13]. Several studies revealed that Propolis had good antibacterial activity against E. faecalis in the root canals and suggested that it could be used as an alternative intracanal medicament [12] [14] [15] [16]. This is an ex vivo study aimed to evaluate Propolis, Calcium hydroxide paste, Chlorhexidine gel 2%, and their mixtures as intracanal medicaments against E. faecalis.

Preparation of the Extracts
100 gram of Propolis sample was extracted, extraction was carried out according to method described by Sukhdev et al. [18], and 1000 ml with 80% ethanol (S D Fine Chemicals, India) was used to dissolve the sample. The yield percentage was calculated as followed: Weight of extract obtained/weight of plant sample × 100.
The 100 gms of the Propolis was yield as 38.26 g giving a yield percentage of 38.26% Propolis ether extract.

Preparation of the Samples
The collected single-rooted teeth were stored at formalin solution until used.
The methodology used was adapted from Gomes et al. [19]. Before starting root canal preparation, scaling was done with an ultrasonic scaler to remove tissue remnants and stains around the teeth. To standardized root canal preparation, all teeth crown was cut 14 mm away from the apex using a diamond disc in the micro motor (EMMI VI Sri-Badia Polesine RO, Italy). Then root canals were prepared until size 25 using K-file (Mani, Inc, Tochigi, Japan). After preparation, the apical region sealed by Glass ionomer cement (GIC) (Changshu Shang Chi Dental Materials Co. Ltd) to block bacterial microleakage. The smear layer removed using EDTA 17% (Prep Gel, MIXODENT, Delhi, India). Teeth were kept in sterilizing pouches, then autoclaved at 121˚C for 30 min.

Preparation of the Enterococcus faecalis
The Enterococcus faecalis was obtained from the Department of Microbiology, Faculty of Medical Laboratory, Al Neelain University. The isolate was reconfirmed by morphological appearance, cultural characteristic and biochemical reactions. The average number of viable organisms per ml of the stock suspension was determined by using surface viable counting technique (Miles and Misra technique) [20], the number of developed colonies (CFU) was counted. It was about 6000 × 10 −6 . Brain Heart Infusion Broth (Himedia, Mumbai, India) was prepared with a 37 gm/Liter concentration. Seven bottles with 200 ml each of brain heart infusion broth were prepared. The sterilized teeth were divided into six bottles containing sterile media (18 teeth for every group) and autoclaved at 121˚C for 20 minutes. One bottle containing only sterile media was applied as Control for media sterility.

Inoculation of Samples
After sterilization, the six bottles that contained teeth were removed and wait until it reaches room temperature, then 2 ml of bacterial suspension prepared

Antimicrobial Activity of the Medicaments
After the incubation period (21 days), the teeth were removed divided into six groups the teeth canal were injected with the different intracanal medicament ( it was more than 300 × 10 −2 considered as un countable number of bacteria, then samples were removed and aseptically culture in Mueller-Hinton agar for one week at 37˚C, Colony-forming units were counted using colony counter. All readings were taken after seven days.

Statistical Analysis
Data was manipulated through the statistical software: Statistical package for social science (SPSS) version 20. The data were statistically analyzed with a one-way analysis of variance using the ANOVA and post hoc test with least significant difference (LSD) multiple comparison tests to check the difference between the different medicaments. P-value ≤ 0.05 was considered statistically significant. In this study, CHX showed the best activity for inhibition of the E. faecalis, the number of the inoculated bacteria was dramatically reduced from 6000 × 10 −6 to 1.5 × 10 −2 . Similarly both Propolis and the mixture of Propolis and calcium hydroxide showed apparent reduction of the number of the bacteria to 18.5 × 10 −2 , and 46 × 10 −2 , respectively. While, the teeth that received calcium hydroxide alone, the mixture of Propolis and Chlorhexidine, and control (teeth receiving no treatment), all showed uncountable (more than 300 × 10 −2 ) number of the E. faecalis (Table 2). were all showed an uncountable number of the E. faecalis during these examination, (Table 3). Showed number of the bacteria counting from teeth treated with different medicaments during seven days (CFU 10 −2 ). Table 4, showed the statistical analysis of these groups, the table showed that there is highly significant difference between medicaments.
3) Number of the bacteria counting from teeth treated with different medicaments for the ten days' (group 3).
This group was evaluated after 2 days, 4 days and 7 days. In this group, Chlorhexidine showed the highest activity of inhibition of the E. faecalis after two, four and seven days. The number of the inoculated bacteria was     (Table 5).
Results showed a high significant difference between medicaments, so there is a need for multiple comparisons. Multiple comparison was done use post hock test least square different (LSD), shown in Table 6.
The value of the coefficient of determination (R2) indicates that group and medicament explain 99.5% of CFU variation. There is a highly significant effect of group on CFU, also highly significant effect of medicament on CFU. The interaction between the two independent variables and medicaments is highly significant; each medicament's effect on CFU is not the same for all groups. The final comparison between tested medicaments in different groups (CFU) through different periods is shown in Figure 1.

Discussion
The causative factors of pulpal and peri-radicular inflammation are bacteria and their byproducts. The cleaning and shaping of the root canal significantly reduce the microbial content; however, the root canal anatomy provides areas in which bacteria can be hidden. Consequently, there is an essential need for the use of an intracanal medicament in addition to the irrigants [21]. The present study focuses on comparing the antibacterial properties of different intracanal medicaments against E. faecalis, which could be considered a limitation of this study as it is better to measure effectiveness against bacterial biofilm. Enterococcus faecalis is the main species that cause endodontic failure [21]. It is known that most of the herbs are safe, readily available, increased shelf life, cost-effective, and lack microbial resistance [22]. Propolis also has these advantages. This study showed that ethanolic extracted Propolis (EEP) was effective against Enterococcus faecalis, it had an apparent effect against E. faecalis in all periods of the study, follow up of bacterial growth show a considerable reduction in the bacterial count, this indicates that Propolis will be useful intracanal medicament especially in the treatment of root canal failure. The present findings agree with others, who concluded that Propolis had potent antibacterial activity against E. faecalis in human teeth [12] [23]. AL-Beitawi et al. also evaluated the efficacy of Propolis and calcium hydroxide in ex vivo as a short-term intracanal medicament. They were found that Propolis is very useful in rapidly eliminating E. faecalis [24]. Other investigations also revealed that Propolis is effective against E. faecalis bacteria [14] [15] [16] [25]. It was indicated that the EEP is effective as an intracanal medicament ex vivo [26] and also in extracted teeth [27]. Another investigation indicated the effectiveness of Propolis in controlling dental infections [28].
CHX has been used in endodontics as an intracanal medicament; present findings showed that 2% CHX gel is patent material against E. faecalis; it reduces bacterial growth after 2 days 7 days after 10 days, the bacterial colonies were increased in number. Zohreh Ahangari et al. found that 2% Chlorhexidine Gluconate (CHX) was influential on Enterococcus faecalis contaminated root canals of human extracted teeth [29]. It was shown that 2% CHX gel was the most effective agent against E. faecalis inside dentinal tubules, while Ca(OH) 2 alone was ineffective [30].  [36]. The mixture between Propolis and calcium hydroxide was done to take the benefits of calcium hydroxide, mainly the high pH, and observe a possible improvement of the calcium hydroxide paste against E. faecalis. Although Ca(OH) 2 alone did not showed activity against E. faecalis, it was surprising that mixing of 1:1 Propolis and Ca(OH) 2 showed evident antibacterial activity in eliminating the tested organism, mostly if left for one week inside the root canal. Attempts to improve the effectiveness of calcium hydroxide pastes have been proposed using chemicals such as p-chlorophenol and Chlorhexidine [37]. Dausage et al. also supported this finding in 2017, who reported that the diffusion ability of calcium hydroxide ions through dentinal tubules is facilitated by the addition of herbs [38]. It has been recognized that long-term exposure to calcium hydroxide alone decreases the strength of root dentin and changes its physical properties, while a mixture of calcium hydroxide with other materials decreases it is an adverse effect on root dentine [39].
This study is in accordance with others who revealed that the bacterial growth of E. faecalis was inhibited by brown Propolis extract alone and when mixed with calcium hydroxide [40]. Previously Calcium hydroxide-Propolis paste was able to diffuse through the dentinal tubules [41] [42].
Regarding the mixture of Propolis with 2% CHX gel, it was not effective against the bacteria after 2 days and 7 days of the insertion inside the root canal, but after insertion of the mixture for 10 days, it reduced the bacterial growth, this may indicate that mixture of Propolis and Chlorhexidine need to stay for a long time in the root canal to be effective. There are no many studies regarding the effect of the mixture between Propolis and CHX against E. faecalis. However, other studies evaluate the combination of CHX and Ca(OH) 2 , and they found that Ca(OH) 2 decreases the antibacterial activity of CHX, possibly to loss of its ability to adhere to the bacterial cell wall [43]. Another study showed an additive antibacterial effect on mixing Ca(OH) 2 powder with 0.5% CHX, where the CHX had a reduced antibacterial action [25]. Still, the usefulness of mixing Ca(OH) 2 with CHX remains unclear. This may also occur when mixing Propolis with Chlorhexidine, the mixture may negatively affect the action of Chlorhexidine and affect its antimicrobial ability, or this mixture may cause difficulty in the diffusion ability of CHX. More researches are needed in this area and to study the effect of Propolis in E. faecalis biofilm.

Conclusion
Within the study's limitations, results showed that both Propolis and Chlorhex-

Ethical Statement
Ethical consent was obtained from Al Neelain University Ethical and Research Committee on December 2016. Verbal consent was also requested by the patient to volunteer in providing their extracted teeth for experimental purposes.

Authors' Contribution
Concept

Conflicts of Interest
The authors deny any conflicts of interest. We have no financial affiliation, or involvement with any commercial organization with direct financial interest in the subject or materials discussed.