Iran. Accepted 10 April, 2012

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1 Vol. 7(38), pp , 10 October, 2013 DOI: /JMPR ISSN Academic Journals Journal of Medicinal Plants Research Full Length Research Paper The effect of propolis on bacterial population isolated from necrotizing single canal tooth with chronic apical periodontitis versus chlorhexidine gluconate Maryam Zare Jahromi 1, Arezoo Tahmourespour 2 * and Samane Ziaei 3 1 Department of Endodontics, Dental School, Islamic Azad University, Khorasgan-Isfahan branch, Isfahan, Iran. 2 Department of Basic Medical Science and Biotechnology, Islamic Azad University, Khorasgan-Isfahan branch, Isfahan, Iran. 3 Department of Neurology, Isfahan Universityof Medical Sciences, Isfahan, Iran. Accepted 10 April, 2012 is a sticky resinous material collected from flowers and plants or other botanical sources by honey-bee. It has antibacterial, antifungal, antiviral, antioxidant and anti-inflammatory activities. Effective debridement of the root canal system with chemical irrigants prior to obturation is the key to long-term success of endodontic therapy. An equally effective, but safe irrigant is desirable. The purpose of this study was to compare the antibacterial efficacy of propolis and chlorhexidine 0.12% as root canal irrigants on the bacterial isolates of necrosed teeth with apical chronic periodontitis. Seventeen necrosed single canal teeth with apical chronic periodontitis were used. After cavity preparation, two samples were taken of each canal and transferred to the lab in immediate media. After 3 to 5 days of incubation in presence and absence of irrigants in aerobic or anaerobic condition, the colonies were counted as CFU/ml. According to results, the mean percentages of bacterial growth inhibition in presence of chlorhexidine in aerobic and anaerobic conditions were 82.6 and 79% respectively, while in presence of propolis were and 46.4%, respectively. It is revealed that in each condition chlorhexidin was significantly more effective than propolis also it is more effective in aerobic than anaerobic condition, non-significantly. is significantly more effective in anaerobic than aerobic condition. It can be concluded that propolis and chlorhexidine 0.12% have antimicrobial activity against aerobic and anaerobic bacteria. is a superior root canal antibacterial agent than propolis even though the antibacterial effect of propolis on the growth of aerobic and anaerobic is apparent. Key words: Antibacterial, chlorhexidine 0.12%, propolis, root canal irrigants. INTRODUCTION is a resinous mixture that honey bees collect from tree buds, sap flows, or other botanical sources. It is used as a sealant for unwanted open spaces in the hive. is used for small gaps. Its color varies depending on its botanical source, the most common being dark brown. High temperature, small space and humidity combined cause the honey comb to have the ideal condition for the growth of microorganisms. However, thanks to the antibacterial action of this substance, this will not occur and the honey comb is not contaminated (Sonmez, 2005). Under normal condition, propolis is a solid, soft and flexible compound with a very complex *Corresponding author. atahmoures@khuisf.ac.ir, arezootahmourespour@gmail.com. Tel

2 2874 J. Med. Plants Res. mixture (Parolia et al., 2010). The principal ingredient of all types of propolis is flavonoids recognized as responsible for its biological activities (Al-Shaher, 2004). Many studies have indicated that this substance is antibacterial, antifungal, antiviral, antioxidant and antiinflammatory (Sonmez, 2005; Al-Shaher, 2004; Ozan, 2007). Bacteria are the leading cause of pulp and periapical inflammation (Behnen et al., 2001). Nowadays, studies indicate that anaerobic bacteria play a role in pulp and periapical diseases (Vianna et al., 2004; Seltzer, 1988). However, facultative microorganisms such as Enterococcus faecalis, Staphylococcus aureus, and even Candida albicans are considered to be the most resistant species in the oral cavity, and one possible cause of root canal treatment failure. So, microbial reduction is one of the primary objectives of the endodontic therapy which consecutively promotes the normal healing process of the periodontal tissues (Neelakantan et al., 2011). Although, various irrigants have been indicated for the sanitization process in apical periodontitis and many of them have shown varied degrees of antimicrobial effectiveness (Gomez et al., 2001), the choice of the ideal irrigant solution and its concentration is difficult. Additionally, studies demonstrated that mechanical canal irrigation alone cannot remove microorganisms from the canal. Hence, there is increasing concern for the antimicrobial action of irrigants (Bystrome and Sandquist, 1985). The use of irrigants to remove microorganisms in endodontic is indispensable. Eventually, a variety of chemicals have been introduced for this purpose (Cohen and Hargreaves, 2010). Among irrigants, chlorhexidine is a bisbiguanide broad spectrum antiseptic with long-lasting effect and low toxicity and side effects (Ferraz et al., 2007). It is widely used as a mouth rinse in the prevention and treatment of periodontal diseases and dental caries, and it has been suggested as an irrigating solution or intracanal dressing in endodontic therapy (Walton and Torabinejad, 2008; Gomez et al., 2003; Sassone et al., 2008). It has unpleasant taste and alters the taste sensation. It also produces brown staining of teeth and affects mucous membrane and tongue. Also, the taste of chlorhexidine mouthwash is not well accepted by the children (Sundas and Rao, 2011). Most researchers have shifted focus on modern natural substances in endodontic treatment with fewer side-effect and greater antibacterial effects, one such modern substance is propolis. There are references made to varied use of the substance, for instance in the intermediary space of avulsed teeth, pulp capping agent, anti decay agent, mouth wash, intra canal medication and canal irrigants (Parolia et al., 2010). The purpose of this study was to compare the antibacterial efficacy of propolis and chlorhexidine 0.12% as root canal irrigants on the bacterial isolates of necrosed teeth with apical chronic periodontitis. MATERIALS AND METHODS In this clinical lab study, 17 necrosed single canal teeth with apical chronic periodontitis of patients referred to the Endodontics Department, School of Dentistry, Islamic Azad University, Khorasgan Branch were used. None of the patients were treated with antibiotics during or prior to sampling. When pulp vitality test was completed anaesthetization and isolation with rubber dam followed. An access cavity was created using a turbine fissure bit. The tooth was dried using sterile absorbent cotton and then two samples of each canal were taken with sterile paper points proportionate to the canal size. Each paper point was dropped into BHI broth or thioglycollate broth for transferring the microorganisms in aerobic or anaerobic condition to the lab. Serial dilutions were made and inoculated on to aerobic 5% sheep blood agar (Biomark, India) and anaerobic 5% sheep blood agar containing L-cycteine HCl (0.25 g/l), Hemin (0.01 g/l) and Vit.K1 (0.001 g/l) (Sigma-Aldrich, USA) supplemented with 100 μl of antimicrobial agents. The antimicrobial agents used in this study were chlorhexidine 0.12% and propolis. (brown) samples were obtained from the beehives of Esfahan, Iran. 300 g of frozen propolis was ground and dissolved in 300 ml 96% ethanol at 37 C to obtain 100% (w/v) extract. The mixture was poured in a bottle and incubated at 30 C for 2 weeks. After incubation, the supernatant mixture was filtered twice with Whatman no. 4 and 1 filter paper. The filtered mixture was concentrated (Eppendorf, Hamburg, Germany) at 30 C for 6 h (1500 rpm). The final extraction of propolis obtained a density of 150 mg/ml. The used antimicrobial agents were UV sterilized and the amount of 100 μl of each were added to the surface of media, thoroughly distributed and kept at ambient temperature till absorbance before inoculation. Of each set of plates, one contained no antibacterial substance taken as positive control and the other plates contained 100 μl of sodium hypochlorite 5.25% and 100 μl of propolis. The aerobic cultures were incubated in normal atmosphere at 37 C while anaerobic cultures were incubated in an anaerobic jar containing gas pack (Anaerocult A Merck, Germany) envelop for providing anaerobic atmosphere at 37 C for 3 to 5 days. After appearance of colonies, they were counted as Colony Forming Unit per milliliter by colony counter (Ziess, Japan). Analysis of variance with a Kruskal-Wallis comparison test was used to evaluate the difference between the antimicrobial activities of the different irrigants. RESULTS For investigating the effect of irrigants, colonies were counted after their appearance on the described media. The antibacterial effects of studied irrigants in aerobic and anaerobic condition were investigated as CFU/ml and the percentage of inhibition in presence of each irrigants. The results are presented in Tables 1 and 2. In view of the fact that as supported by Kolmogrov-Smirnov Test, the distribution of grown colonies was not normal in the samples. So, Kruskal-Wallis non-parametric test was used to compare the findings. The number of colonies grown in aerobic or anaerobic culture of control groups, was significantly greater than in the other two groups (P = 0.05). The number of colonies in presence of propolis and chlorhexidine were significantly different from each other (P < 0.05). The mean colony numbers grown in the presence of chlorhexidine

3 Jahromi et al Table 1. Percentage of inhibition and the number of bacteria in presence of antimicrobial agents in aerobic culture. S/N Control (CFU/ml*) (% of inhibition) (% of Inhibition) *CFU/ml = CFU 1/dilution 1/volume. Table 2. Percentage of inhibition and the number of bacteria in presence of antimicrobial agents in anaerobic culture. S/N Control (CFU/ml*) (% of inhibition) (% of inhibition) *CFU/ml = CFU 1/dilution 1/volume. was significantly less than propolis. Therefore, chlorhexidine is a superior root canal antibacterial agent than propolis even though the antibacterial effect of propolis on the growth of aerobic and anaerobic is apparent. There are significant differences between the effect of chlorhexidine and propolis in each atmospheric condition. The mean percentages of bacterial growth inhibition in presence of chlorhexidine in aerobic and anaerobic conditions were 82.6 and 79% respectively, while in presence of propolis were and 46.4%, respectively (Table 3, Figure 1).

4 2876 J. Med. Plants Res. Table 3. The mean percentage of inhibition in presence of antimicrobial agents in aerobic and anaerobic condition. Atmospheric condition Aerobic Antimicrobial agents Mean of inhibition (%) Std. Dev. Std. Error Minimum (%) Maximum (%) % Anaerobic % Inhibition (%) Aerobic aerobic anaerobic Anaerobic Figure 1. The comparison of propolis and chlorhexidine antimicrobial activity in aerobic and anaerobic condition. It is revealed that in each condition, chlorhexidin was significantly more effective than propolis also it is more effective in aerobic than anaerobic condition, none significantly. In the case of propolis, it can be mentioned that it is significantly more effective in anaerobic than aerobic condition. DISCUSSION It is assumed that careful debridement of the root canal system is the key to long term success of endodontic therapy. Chemo-mechanical preparation is one of the most important phases of endodontic treatment. The use of irrigants in this process is important for ensuring bacterial elimination (Shingare and Chaugule, 2011). Various results have been reported for irrigant antimicrobial effectiveness. Similar results have been reported for chlorhexidine; however, researches have shown that chlorhexidine has well antimicrobial activity (Gomez et al., 2001, 2003). In a study, Ferraz et al. (2007) concluded that chlorhexidine 2% in comparison with all concentrations of sodium hypochlorite had more antibacterial effect on gram-negative anaerobic bacteria. In another study, Oliveria et al. (2007) demonstrated that chlorhexidine and sodium hypochlorite both were effective in killing E. faecalis. Wang et al., (2007) also discovered that chlorheixdine is an effective disinfectant for root canal. The results of this study also indicate that chlorhexidine 0.12% is more effective than propolis as an antimicrobial endodontic irrigant. There was a significant reduction in the number of bacteria in both groups of treated teeth when compared with the controls. irrigation resulted in more significant positive effects than did propolis irrigation. The results also indicated that propolis have antibacterial effect on the growth of aerobic and anaerobic bacteria when used as an endodontic irrigant and there was a similar significant reduction in the number of colonies grown in both groups when compared with the control group while, it is significantly more effective in anaerobic than aerobic condition. So, chlorhexidine is a superior root canal antibacterial agent than propolis. In this study, necrotizing single canal tooth with chronic apical periodontitis were used, hence the condition was simulated to clinical situation. Single canal tooth were used because of decreasing the percent of error caused by vitality of other canals in multi canal tooth. This study was the rare research which compared the antimicrobial activity of propolis with that of chlorhexidine in endodontic treatment. The antimicrobial effects of propolis in this study are similar to those obtained by others who have evaluated the inhibitory effect of propolis solution on bacterial growth (Awadeh et al., 2009; Ahangari et al., 2009; Mohammadzade et al., 2007). According to the studies conducted by Awadeh et al. (2009) as well as by Ahangari et al. (2009) and Madhubala et al. (2011), propolis is an effective agent in deactivating the E. faecalis responsible for failure of treatment than other typical intra canal medication including calcium hydroxide. Soley et al. (2011) also concluded that propolis has antimicrobial activity against E. faecalis and C. albicans and is an effective intracanal irrigant in eradicating E. faecalis and C. albicans. In another study focused on the chemical composition, oral toxicity and antibacterial activity of Iranian propolis on male rats, Mohammadzadeh et al. (2007) stated that

5 Jahromi et al this substance has no significant clinical toxicity and is capable of preventing the growth of all the tested microorganisms including bacteria and fungi. Ferreira et al. (2007) found that propolis had an antibacterial effect on selected endodontic anaerobic bacteria (Prevotella nigrescens, Fusobacterium nucleatum, Actinomyces israelii, Clostridium perfringens and E. faecalis). Bruschi et al. (2006) also found that propolis has an inhibitory effect on microorganism of oral importance (E. faecalis, Streptococcus salivarius, Streptococcus sanguinis, Streptococcus mitis, Streptococcus mutans, Streptococcus sobrinus, C. albicans and Latobacillus casei). Kayaoglu et al (2011) investigated the antibacterial activity of propolis against E. faecalis in a dentin block model, and compared its effectiveness with that of chlorhexidine. According to their results, propolis samples were antimicrobially effective; however, their activity did not exceed chlorhexidine. Antibacterial characteristic of propolis had been explained in a number of ways. One study reported that it prevented bacterial cell division and also broke down bacterial walls and cytoplasm similar to the action of some antibiotics (Takaisi-kikuni and Schilcher, 1994). In another study, it is reported that, the antibacterial, antifungal and the antiviral activities of propolis can be due to flavonoids and esters of phenolic acids (Kuiumgiev et al., 1999). On the other hand, chlorhexidine gluconate is a cationic irrigant that seems to act by adsorption onto the cell wall of the microorganism and causing leakage of intracellular components. At low concentration, chlorhexidine shows bacteriostatic effect, causing the leaching of small molecular weight substances from microorganism. At higher concentrations, chlorhexidine has bactericidal effect due to cytoplasmic precipitation and/or coagulation, probably caused by protein cross-linking. Estrela et al. (2003) also concluded that, this substance has the antimicrobial action against S. aureus, E. faecalis, P. aeroginosa, B. subtilis and C. albicans. Sassone et al. (2008) demonstrated that chlorhexidine solution and NaOCl had similar antimicrobial performance to eliminate gram-positive, gram-negative, aerobic and anaerobic bacteria. If antimicrobial activity was the only requirement of an endodontic irrigant, the result of this study would indicate that chlorhexidine is better than propolis, however chlorhexidine possesses other significant attributes that propolis is not known to possess. Further investigation is currently being conducted by the authors to evaluate the effectiveness of propolis in pulp and necrotic tissue debridement. REFERENCES Ahangari Z, Eslami G, Koosedghi H, Ayatollahi S (2009). Comparative study of antibacterial activity of propolis and Ca(OH) 2 against Lactobacillus, Enterococcus faecalis, Pepto Streptococcus, Candida albicans. 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