|Year : 2019 | Volume
| Issue : 2 | Page : 94-98
Antibacterial efficacy of Allium sativum (garlic) and chitosan incorporated into two root canal sealers against Enterococcus faecalis: comparative study
Khaled A Beshr1, Ramy A Abdelrahim2
1 Department of Endodontics, Faculty of Dentistry, Beni Suef University, Beni Suef, Egypt
2 Department of Dental Bio-Materials, Faculty of Dentistry, Al-Azhar University, Cairo, Egypt
|Date of Submission||05-Mar-2019|
|Date of Acceptance||05-Mar-2019|
|Date of Web Publication||23-Sep-2019|
Ramy A Abdelrahim
El Hadba El Wasta, Mokattam, Cairo 1082
Source of Support: None, Conflict of Interest: None
This study evaluated the antibacterial activity of mineral trioxide aggregate (MTA) fillapex and Gutta-flow 2 sealers in the commercial unmodified form and two modified forms with chitosan and Allium sativum against Enterococcus faecalis strain (ATCC 2912).
Materials and Methods
The material tested were MTA fillapex and Gutta-flow 2 sealers in the commercial unmodified form and two modified forms with chitosan and A. sativum at concentrations 5, 10, and 20%. Agar diffusion test was used to evaluate the zone of inhibition of freshly mixed unmodified and modified materials against E. faecalis strain (ATCC 2912).
The incorporation of A. sativum and chitosan into MTA fillapex sealer can significantly enhance its antibacterial activity against E. faecalis at all concentrations. However, the addition of A. sativum and chitosan to Gutta-flow 2 sealer exhibited no antibacterial effect except for Gutta-flow 2 that modified with 20% chitosan.
A. sativum and chitosan incorporated into MTA fillapex sealer can significantly improve its antibacterial efficacy of against E. faecalis. Also the incorporation of 20% of chitosan can enhance the antibacterial activity of Gutta-flow 2 sealer.
Keywords: Allium sativum, antibacterial, chitosan, endodontic sealer, Enterococcus faecalis
|How to cite this article:|
Beshr KA, Abdelrahim RA. Antibacterial efficacy of Allium sativum (garlic) and chitosan incorporated into two root canal sealers against Enterococcus faecalis: comparative study. Tanta Dent J 2019;16:94-8
|How to cite this URL:|
Beshr KA, Abdelrahim RA. Antibacterial efficacy of Allium sativum (garlic) and chitosan incorporated into two root canal sealers against Enterococcus faecalis: comparative study. Tanta Dent J [serial online] 2019 [cited 2020 Feb 17];16:94-8. Available from: http://www.tmj.eg.net/text.asp?2019/16/2/94/267563
| Introduction|| |
The success of endodontic treatment depends on many factors as good mechanical preparation, effective intracanal irrigation, obturation with good hermetic seal ,. Failure of bacterial elimination from root canal system during endodontic treatment consider the essential reason of periapical inflammation, degradation of periapical tissues, periapical lesions and finally endodontic failure ,,. Bacterial persistence after root canal irrigation with effective intracanal irrigant as sodium hypochlorite may be due to the complicated root canal anatomy including accessory canals, isthmus and fines ,. So, the root canal space must obturate effectively to produce hermetic seal to prevent any infection by the persistent bacteria and finally kill them .
Although, the main root canal filling material is Gutta-percha, but, it cannot fill the entire canal space alone or adhere to the canal walls . Therefore, the use of endodontic sealer is a critical item for sealing the three dimension root canal system in combination with the Gutta-percha ,. Unfortunately, there is no any material alone or in combination with other material can provide a complete seal of canal spaces, and there are always submicron spaces remains between the canal walls and the filling material . Clinically, the use of endodontic sealer with antibacterial activity would be effective in elimination of the residual bacteria and also in the avoidance of bacterial recrossing into the root canal after good obturation ,.
Enterococcus faecalis is anaerobic gram-positive cocci, that able to tolerate the alkaline environment and consider the most prevalence bacteria which found in the field of root treatment ,,,,,.
Mineral trioxide aggregate (MTA) is calcium silicate-based cement that used as root sealer material especially in roots with open or immature apex, duo to its bioactive properties ,,,. Recently, manufacturer supply in dental market Gutta-flow as endodontic sealer material, that nearly insoluble in body fluid, biocompatible, adhere to Gutta-percha and dentine wall and have excellent flow properties ,. Gutta-flow is a silicon-based sealer that composed mainly of fine Gutta-percha powder ,.
Allium sativum have been used as powerful drug for many centuries all over the world due to its antibacterial activity against a wide variety of fungi and bacteria ,. Chitosan is a natural cationic polymer obtained usually by deacetylation of chitin, which is the main component of the exoskeleton of crustaceans . Chitosan has been established to possess antibacterial activity against a variety of microorganisms .
The aim of the present study is to evaluate the antibacterial activity of MTA fillapex and Gutta-flow 2 as unmodified sealers and modified with chitosan and A. sativum against E. faecalis.
| Materials and Methods|| |
Endodontic sealers tested were MTA fillapex (Angelus, Londrina, Brazil) as calcium silicate-based sealer and Gutta-flow 2 (Colten/Whale dent, Altstatten, Switzerland) as silicon-based sealer. Chitosan and A. sativum solutions at concentrations (5, 10 and 20%) were added into freshly mixed sealers in a vial and it were mixed by vortex for 30 s.
Allium sativum extract preparation
The fresh plant parts of garlic were washed and coarsely chopped into small pieces. About 10 g of the chopped garlic was crushed thoroughly in a blender with 50 ml of ethanol as extraction solvent. Then, kept in a sterile closed glass container for 2 weeks accompanying daily and occasional vigorous hand shaking. The resulting extract was filtered by filter paper (#1) to get ethanol extract of A. sativum. Finally, the extract was kept in sterile glass bottle in refrigerator until used .
Chitosan solution preparation
About 50 mg of chitosan powder (Sigma Aldrich, Darmstadt, Germany) was weighted by using digital (Precisa 205 A; Moosmattstrasse, Dietikon, Switzerland), at the Regional Center of Mycology and Biotechnology, Cairo, Egypt, and then dissolved in 100 ml of 0.3 N acetic acid to get 0.5 mg/ml chitosan solution . The solution is stirred in vortex for 5 min to allow complete dissolving of the chitosan powder in acetic acid and produce homogenous mix.
Unmodified and modified materials were divided into the following groups [Table 1] at different concentrations (5, 10 and 20%).
Agar diffusion test
Freshly mixed modified and unmodified sealers were tested by using agar diffusion technique in Regional Center for Mycology and Biotechnology, Cairo, Egypt. The antibacterial action was assessing by using E. faecalis as a reference strain (ATCC 29212) .
E. faecalis in suspension were spread over Petri dishes containing brain hart infusion agar medium by using a Drigalski loop. The infuse plates were dried for 10 min at 37°C. Four 6 mm wells were prepared in each dish with a sterile paper straw ,. Four Petri dishes were prepared to receive 100 μl of the freshly mixed unmodified and modified tested material as follow:
The wells of the first and second plates were filled with unmodified MTA fillapex as control group, MTA fillapex – chitosan modified groups and MTA fillapex – A. sativum modified groups, respectively. While, the third and fourth plate wells were filled with Gutta-flow 2 as control group, Gutta-flow – chitosan modified groups and Gutta-flow 2 – A. sativum modified groups respectively. Other plate with four wells to receive chitosan solution, A. sativum, acetic acid and ethanol as negative controls.
The Petri dishes were kept at room temperature for 2 h for prediffusion of the tested materials and then incubated at 37°C for 24 h. After 24 h the zone of inhibition around each well were observed and measured using millimeters ruler by a single investigator ,.
The collected data were statistically analyzed by comparing mean inhibition zone for each sealer. The obtained results were evaluated using analysis of variance followed by Tukey's test and nonparametric Spearman's correlation coefficient. Statistical analysis was performed with SPSS software version 20 (Chicago, Illinois, USA); the level of significance was set at P value less than or equal to 0.05.
| Results|| |
The mean zone of inhibition in mm beyond well diameter (6 mm) produced by unmodified and modified sealers against E. faecalis at different concentrations (5, 10 and 20%) are presented in [Figure 1].
|Figure 1: The antibacterial activity against Enterococcus faecalis of unmodified and modified MTA fillapex and Gutta-flow 2 sealers at different concentrations. MTA, mineral trioxide aggregate.|
Click here to view
Our results showed that the addition of A. sativum and chitosan to MTA fillapex sealer improve its antibacterial efficacy against E. faecalis [Figure 2].
|Figure 2: Zone of inhibition against Enterococcus faecalis produced by unmodified and modified sealers.|
Click here to view
For MTA fillapex modified with chitosan (group 2) at 20% chitosan concentration showed the maximum mean diameter of zone of inhibition against E. faecalis (11.67 mm) followed by 9.97 and 9.6 mm for 10 and 5% concentrations, respectively. Therefore, the addition of chitosan improves the antibacterial efficacy of MTA fillapex against E. faecalis and its antibacterial activity will increase with increase in chitosan concentration.
In MTA fillapex modified with A. sativum (group 3) showed the same mean diameter of zone of inhibition against E. faecalis (10.67 mm) at all concentrations. Therefore, the different concentrations of A. sativum have the same antibacterial effect.
Gutta-flow 2 sealer did not show any inhibition zone against E. faecalis. Also, addition of A. sativum to Gutta-flow 2 had no effect to improve its antibacterial activity [Figure 2]. However, the addition of chitosan to Gutta-flow 2 also did not showed any antibacterial activity at concentration 5 and 10%. But at concentration 20% of chitosan, it showed favorable inhibitory activity against E. faecalis with zone of inhibition (9.93 mm) [Figure 1] and [Figure 2].
There is was statistically significant difference (P ≥ 0.05) among different tested groups that showed inhibitory activity. In case of unmodified and modified Gutta-flow sealer, that showed zero inhibitory activity, the statistical analysis could not be performed.
| Discussion|| |
Pathogenic microorganisms in root canal space cannot completely eradicate during mechanico-chemical preparation of root canal . Endodontic sealer with antibacterial activity may help to eliminate the remaining microorganisms unaffected by irrigant solutions .
E. faecalis is the most common resistant microorganism in root canal infection and usually present with persisting periapical lesions in over one third of the canals space of the tooth ,,. Moreover, it was found that E. faecalis is resistant to many root canal irrigants and medications . Therefore, root canal sealers with inhibitory activity against E. faecalis may play important role in prevention and/or decreasing its growth and help the periapical tissues to repair ,.
Antibacterial activity of dental sealer is commonly tested by agar diffusion test (ADT) as one of the most prevalence used test to evaluate nonset sealers ,. ADT allows the tested sealers to be in direct contact with microorganism simulating the clinical cases and test the ability of the sealer to eliminate bacteria in areas such as root canal system . The results of ADT are influenced by solubility and diffusive ability of the tested sealer in the culture media ,.
MTA fillapex endodontic sealer, has resinous component with excellent radio opacity, long setting time and easy handling property ,. The inhibition zone of unmodified MTA fillapex observed in ADT [Figure 2] may be due to its resin content . The antibacterial activity of MTA fillapex against E. faecalis may be also due to the pH elevation due to released calcium hydroxide produced when MTA mixed with water , because E. faecalis cannot survive at pH 11.5 or greater .
Gutta-flow 2 is a silicon-based sealer with dispersed Gutta-percha powder and silver microparticles, and it had no antibacterial activity . Early investigations revealed that the incorporation of microsilver particles did not exhibited an inhibitory effect against E. faecalis ,, and it is only having a role as preservative ingredient . Moreover, it was found that, the antibacterial activity of silver particles depends on its concentration, ionic form and period of contact  and its effect was more noticeable against gram negative microorganisms .
In ADT Gutta-flow 2 sealer cannot produce any antibacterial activity may be due to its lower solubility and diffusive-ability compared to MTA fillapex ,, which has revealed in absence of zone of inhibition [Figure 2]. Therefore, the fortunately results of unmodified and modified Gutta-flow 2 may be due to its lower solubility and diffusive ability .
Many investigations showed that A. sativum and chitosan exhibited antimicrobial action against a wide variety of microorganisms ,,. The antimicrobial activity of chitosan may be due to its cationic 'positively charged' nature, which interact with negatively charged bacterial cell wall, altering its permeability and resulting in cell death due to the leakage of its intracellular component ,.
Recent investigations showed that the addition of chitosan to endodontic sealer can increases its antibacterial activity ,. MTA fillapex was able to maintain it high pH 'alkaline' for long period of time 'about 5 weeks' . The increased antibacterial activity of MTA fillapex modified with chitosan may be due to the hydrophilic nature of chitosan  and its large matrix swilling , which allow its diffusive-ability in agar.
It is also known that, the antibacterial efficacy depends on the concentration and surface area, which can explain the increased antibacterial activity of MTA fillapex modified by chitosan when chitosan concentration increased , and also it can explain the result of antibacterial activity of Gutta-flow 2 modified with 20% chitosan.
The antibacterial activity of A. sativum is due to its allicin compound which consider the main antibacterial constituent of freshly crushed garlic , that produce its effect due to its chemical reaction with thiol groups of various enzymes . Allicin concentration in garlic to exhibited antimicrobial effect should be the same or higher than microorganism concentration . Addition of A. sativum to MTA fillapex significantly increase its antibacterial activity against E. faecalis but the zone of inhibition was the same at all concentrations, this may be due to the allicin concentration in garlic solution not higher than the concentration of E. faecalis .
| Conclusion|| |
Under limitation of the present study, the incorporation of A. sativum and chitosan can significantly improve the antibacterial efficacy of MTA fillapex endodontic sealer against E. faecalis. Also the incorporation of 20% of chitosan can enhance the antibacterial activity of Gutta-flow 2 sealer.
However, more studies about the antibacterial ability of endodontic sealers, A. sativum and chitosan under simulated clinical conditions are necessary.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Ozcan E, Eldeniz AU, Ari H. Bacterial killing by several root filling materials and methods in an ex vivo
infected root canal model. Int Endod J 2011; 44:1102–1109.
Shin J-H, Lee D-Y, Lee S-H. Comparison of antimicrobial activity of traditional and new developed root sealers against pathogens related root canal. J Dent Sci 2018; 13:54–59.
Lakshmi Narayanan L, Vaishnavi C. Endodontic microbiology. J Conserv Dent 2010; 13:233–239.
Kangarlou A, Neshandar R, Matini N, Dianat O. Antibacterial efficacy of AH Plus and AH26 sealers mixed with amoxicillin, triple antibiotic paste and Nano silver. J Dent Res Dent Clin Dent Prospects 2016; 10:220–225.
Ricucci D, Siqueira JFJr. Fate of the tissue in lateral canals and apical rami cations in response to pathologic conditions and treatment procedures. J Endod 2010; 36:1–15.
Tragi S, Mishra P, Tragi P. Evolution of root canal sealers: an insight story. Eur J Gen Dent 2013; 2:199–218.
Kakehashi S, Stanley H, Fitzgerald R. The effects of surgical exposures of dental pulps in germ-free and conventional laboratory rats. Oral Surg Oral Med Oral Pathol 1965; 20:340–349.
Tabrizizadeh M, Rasti M, Ayatollahi F, Mossadegh MH, Zandi H, Dehghan F, et al
. Antimicrobial activity of calcium hydroxide and betamethasone on Enterococcus faecalis
; an in vitro
assessment. Iran Endod J 2015; 10:184–187.
Bhandari S, Ashwini T, Patil CR. An in vitro
evaluation of antimicrobial efficacy of 2% chlorhexidine gel, propolis and calcium hydroxide against Enterococcus faecalis
in human root dentin. J Clin Diagn Res 2014; 8:ZC60–ZC63.
Prabhakar A, Taur S, Hadakar S, Sugandhan S. Comparison of antibacterial efficacy of calcium hydroxide paste, 2% chlorhexidine gel and turmeric extract as an intra-canal medicament and their effect on micro-hardness of root dentin: an in vitro
study. Int J Clin Pediatr Dent 2013; 6:171–177.
Mozayeni MA, Haeri A, Dianat O, Jafari AR. Antimicrobial effects of four intra-canal medicaments on Enterococcus faecalis
: an in vitro
study. Iran Endod J 2014; 9:195–198.
Camilleri J, Montesin FE, Brady K, Sweeney R, Curtis RV, Ford TR. The constitution of mineral trioxide aggregate. Dent Mater J 2005; 21:297–303.
Salles LP, Gomes-Cornélio AL, Guimaraes FC, Herrera BS, Bao SN, Rossa-Junior C, et al
. Mineral trioxide aggregate-based endodontic sealer stimulates hydroxyapatite nucleation in human osteoblast-like cell culture. J Endod 2012; 38:971–976.
Maroto M, Barberia E, Planells P, Garcia Godoy F. Dentin bridge formation after mineral trioxide aggregate (MTA) pulpotomies in primary teeth. Am J Dent 2005; 18:151–154.
Aminozarbian MG, Barati M, Salehi I, Mousavi SB. Biocompatibility of mineral trioxide aggregate and three new endodontic cements: an animal study. Dent Res J 2012; 9:54–59.
Wainstein M, Morgental RD, Waltrick SBG, Oliveira SD, Vier-Pelisser FV, Figueiredo JAP, et al
. In vitro
antibacterial activity of a silicone-based endodontic sealer and two conventional sealers. Braz Oral Res 2016; 30 (e18):1–5.
Kapralos V, Koutroulis A, Ørstavik D, Sunde PT, Rukke HV. Antibacterial activity of endodontic sealers against planktonic bacteria and bacteria in biofilms. J Endod 2018; 44:149–154.
Ozcan E, Eldeniz AU, Aydinbelge HA. Assessment of the sealing abilities of several root canal sealers and filling methods. Acta Odontol Scand 2013; 71:1362–1369.
Accardo C, Himel VT, Lallier TE. A novel Gutta-Flow sealer supports cell survival and attachment. J Endod 2014; 40:231–234.
Mathai K, Anand S, Aravind A, Dinatius P, Krishnan AV, Mathai M. Antimicrobial effect of ginger, garlic, honey, and lemon extracts on Streptococcus mutans
. J Contemp Dent Pract 2017; 18:1004–1008.
Ankri S, Mirelman D. Antimicrobial properties of allicin from garlic. Microb Infect 1999; 1:125–129.
Sinha VR, Singla AK, Wadhawan S, Kaushik R, Kumria R, Bansal K, Dhawan S. Chitosan micro-spheresasa potential carrier for drugs. Int J Pharm 2004; 274:1–33.
Rabea EI, Badawy ME, Stevens CV, Smagghe G, Steurbaut W. Chitosan as antimicrobial agent: applications and mode of action. Biomacromolecules 2003; 4:1457–1465.
Mamun MA, Hasan N, Shirin F, Belal MH, Khan MAJ, Tasnin MN, et al.
Anti-hyperglycemic and anti-hyperlipidemic activity of ethanol extract of garlic (Allium sativum
) in streptozotocin-induced diabetic mice. Int J Med Heal Res 2017; 3:63–66.
Karthick A, Kavitha M. Evaluation of micro-shear bond strength of chitosan modified GIC. World J Med Sci 2014; 10:169–173.
Pawińska M, Szczurko G, Kierklo A, Łuczaj-Cepowicz E, Marczuk-Kolada G, Leszczyńska K. In vitro
evaluation of the antibacterial effect of various root canal sealers on selected anaerobic bacteria. J Stoma 2016; 69:521–530.
Pizzo G, Giammanco GM, Cumbo E, Nicolosi G, Gallina G. In vitro
antibacterial activity of endodontic sealers. J Dent 2006; 34:35–40.
Rôças IN, Siqueira JFJr, Santos KR. Association of Enterococcus faecalis
with different forms of peri-radicular diseases. J Endod 2004; 30:315–320.
Omidi S, Hoshyari N, Mirzadeh AR, Hassan-Abadi ME, Ahajan M, Charati JY, et al
. Comparison of antibacterial activity of three endodontic sealers against Enterococcus faecalis
. J Res Med Dent Sci 2018; 6:413–417.
Dornelles-Morgental R, Guerreiro-Tanomaru JM, Faria-Junior NB, Hungaro-Duarte MA, Kuga MC, Tanomaru-Filho M. Antibacterial efficacy of endodontic irrigating solutions and their combinations in root canals contaminated with Enterococcus faecalis
. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2011; 112:396–400.
Poggio C, Lombardini M, Colombo M, Dagna A, Saino E, Arciola CR, et al
. Antibacterial effects of six endodontic sealers. Int J Artif Organs 2011; 34:908–913.
Dornelles-Morgental R, Vier-Pelisser FV, Oliveira SD, Antunes FC, Cogo DM, Kopper PMP. Antibacterial activity of two MTA-based sealers root canal sealers. Int Endod J 2011; 44:1128–1133.
Gomes BPFdA, Pedroso JA, Jacinto RC, Vianna ME, Ferraz CCR, Zaia AA, et al
. In vitro
evaluation of the antimicrobial activity of five root canal sealers. Braz Dent J 2004; 15:30–35.
Jafari F, Kafil HS, Jafari S, Aghazadeh M, Momeni T. Antibacterial activity of MTA fillapex and AH 26 root canal sealers at different time intervals. Iran Endo J 2016; 11:192–197.
Stuart CH, Schwartz AS, Beeson TJ, Owatz CB. Enterococcus faecalis
: its role in root canal treatment failure and current concepts in retreatment. J Endod 2006; 32:93–98.
Farmakis ET, Kontakiotis EG, Tseleni-Kotsovili A, Tsatsas VG. Comparative in vitro
antibacterial activity of six root canal sealers against Enterococcus faecalis
and Proteus vulgaris
. J Investig Clin Dent 2012; 3:271–275.
Nawal RR, Parande M, Sehgal R, Naik A, Rao NR. A comparative evaluation of antimicrobial efficacy and flow properties for Epiphany, Gutta-flow and AH-Plus sealer. Int Endod J 2011; 44:307–313.
Tran QH, Nguyen VQ, Le AT. Silver nanoparticles: synthesis, properties, toxicology, applications and perspectives. Adv Nat Sci Nanosci Nanotechnol 2013; 4:1–20.
Wu D, Fan W, Kishen A, Gutmann JL, Fan B. Evaluation of the antibacterial efficacy of silver nanoparticles against Enterococcus faecalis
biofilm. J Endod 2014; 40:285–290.
Nair N, James B, Devadathan A, Johny MK, Mathew J, Jacob J. Comparative evaluation of antibiofilm efficacy of chitosan nanoparticle- and zinc oxide nanoparticle-incorporated calcium hydroxide-based sealer: an in vitro
study. Contemp Clin Dent 2018; 9:434–439.
Del Carpio-Perochena A, Kishen A, Shrestha A, Bramante CM. Antibacterial properties associated with chitosan nanoparticle treatment on root dentin and 2 types of endodontic sealers. J Endod 2015; 41:1353–1358.
Zhou HM, Shen Y, Zheng W, Li L, Zheng YF, Haapasalo M. Physical properties of 5 root canal sealers. J Endod 2013; 39:1281–1286.
Shrestha A, Hamblin MR, Kishen A. Characterization of a conjugate between rose Bengal and chitosan for targeted antibiofilm and tissue stabilization effects as a potential treatment of infected dentin. Antimicrob Agents Chemother 2012; 56:4876–4884.
Lee HS, Yee MQ, Eckmann YY, Hickok NJ, Eckmann DM, Composto RJ. Reversible swelling of chitosan and quaternary ammonium modified chitosan brush layers: effect of pH and counter anion size and functionality. J Mater Chem 2012; 22:19605–19616.
Stoimenov PK, Klinger RL, Marchin GL, Klabunde KJ. Metal oxide nanoparticles as bactericidal agents. Langmuir 2002; 18:6679–6686.
Zainol N, Rahim SR. Effect of garlic solution to Bacillus sp. removal. Mater Sci Eng 2018; 342:12037–12043.
[Figure 1], [Figure 2]